"Medicine, Faculty of"@en . "Medical Genetics, Department of"@en . "DSpace"@en . "UBCV"@en . "Dahabieh, Matthew Solomon"@en . "2008-04-30T15:10:52Z"@en . "2008"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "During alcoholic fermentation Saccharomyces cerevisiae metabolizes L-arginine to ornithine and urea. S. cerevisiae can metabolize urea through the action of urea amidolyase, encoded by the DUR1,2 gene; however, DUR1,2 is subject to nitrogen catabolite repression (NCR) in the presence of high quality nitrogen sources during fermentation. Being cytotoxic at high concentrations, urea is exported into wine where it spontaneously reacts with ethanol, and forms the carcinogen ethyl carbamate (EC).\n\nUrea degrading yeast strains were created by integrating a linear cassette containing the DUR1,2 gene under the control of the S. cerevisiae PGK1 promoter and terminator signals into the URA3 locus of the Sake yeast strains K7 and K9. The \u00E2\u0080\u0098self-cloned\u00E2\u0080\u0099 strains K7EC- and K9EC- produced Sake wine with 68% less EC. The Sake strains K7EC- and K9EC- did not efficiently reduce EC in Chardonnay wine due to the evolutionary adaptation of said strains to the unique nutrients of rice mash; therefore, the functionality of engineered yeasts must be tested in their niche environments as to correctly characterize new strains.\n\nS. cerevisiae possesses an NCR controlled high affinity urea permease (DUR3). Urea importing yeast strains were created by integrating a linear cassette containing the DUR3 gene under the control of the PGK1 promoter and terminator signals into the TRP1 locus of the yeast strains K7 (Sake) and 522 (wine). In Chardonnay wine, the urea importing strains K7D3 and 522D3 reduced EC by 7% and 81%, respectively; reduction by these strains was equal to reduction by the urea degrading strains K7EC- and 522EC-. In Sake wine, the urea degrading strains K7EC- and 522EC- reduced EC by 87% and 84% respectively, while the urea importing strains K7D3 and 522D3 were significantly less capable of reducing EC (15% and 12% respectively). In Chardonnay and Sake wine, engineered strains that constitutively co-expressed DUR1,2 and DUR3 did not reduce EC more effectively than strains in which either gene was expressed solely. Uptake of 14C-urea under non-inducing conditions was enhanced in urea importing strains; parental strains failed to incorporate any 14C-urea thus confirming the functionality of the urea permease derived from the integrated DUR3 cassette."@en . "https://circle.library.ubc.ca/rest/handle/2429/790?expand=metadata"@en . "1992060 bytes"@en . "application/pdf"@en . "METABOLIC\u00C2\u00A0ENGINEERING\u00C2\u00A0OF\u00C2\u00A0INDUSTRIAL\u00C2\u00A0YEAST\u00C2\u00A0STRAINS\u00C2\u00A0TO\u00C2\u00A0MINIMIZE\u00C2\u00A0THE\u00C2\u00A0 PRODUCTION\u00C2\u00A0OF\u00C2\u00A0ETHYL\u00C2\u00A0CARBAMATE\u00C2\u00A0IN\u00C2\u00A0GRAPE\u00C2\u00A0AND\u00C2\u00A0SAKE\u00C2\u00A0WINE\u00C2\u00A0 \u00C2\u00A0 by\u00C2\u00A0 \u00C2\u00A0 MATTHEW\u00C2\u00A0SOLOMON\u00C2\u00A0DAHABIEH\u00C2\u00A0 B.Sc.\u00C2\u00A0Hon.,\u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia,\u00C2\u00A02006\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0THESIS\u00C2\u00A0SUBMITTED\u00C2\u00A0IN\u00C2\u00A0PARTIAL\u00C2\u00A0FULFILLMENT\u00C2\u00A0OF\u00C2\u00A0\u00C2\u00A0 THE\u00C2\u00A0REQUIREMENTS\u00C2\u00A0FOR\u00C2\u00A0THE\u00C2\u00A0DEGREE\u00C2\u00A0OF\u00C2\u00A0 \u00C2\u00A0 MASTER\u00C2\u00A0OF\u00C2\u00A0SCIENCE\u00C2\u00A0 \u00C2\u00A0 in\u00C2\u00A0 \u00C2\u00A0 THE\u00C2\u00A0FACULTY\u00C2\u00A0OF\u00C2\u00A0GRADUTATE\u00C2\u00A0STUDIES\u00C2\u00A0 (Genetics)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 THE\u00C2\u00A0UNIVERISTY\u00C2\u00A0OF\u00C2\u00A0BRITISH\u00C2\u00A0COLUMBIA\u00C2\u00A0 (Vancouver)\u00C2\u00A0 \u00C2\u00A0 April\u00C2\u00A02008\u00C2\u00A0 \u00C2\u00A9Matthew\u00C2\u00A0Solomon\u00C2\u00A0Dahabieh,\u00C2\u00A02008\u00C2\u00A0 ii\u00C2\u00A0 \u00C2\u00A0 ABSTRACT\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0metabolizes\u00C2\u00A0L\u00E2\u0080\u0090arginine\u00C2\u00A0to\u00C2\u00A0ornithine\u00C2\u00A0and\u00C2\u00A0 urea.\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0can\u00C2\u00A0metabolize\u00C2\u00A0urea\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0amidolyase,\u00C2\u00A0encoded\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0 gene;\u00C2\u00A0however,\u00C2\u00A0DUR1,2\u00C2\u00A0is\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0(NCR)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0high\u00C2\u00A0quality\u00C2\u00A0 nitrogen\u00C2\u00A0sources\u00C2\u00A0during\u00C2\u00A0fermentation.\u00C2\u00A0Being\u00C2\u00A0cytotoxic\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0concentrations,\u00C2\u00A0urea\u00C2\u00A0is\u00C2\u00A0exported\u00C2\u00A0into\u00C2\u00A0wine\u00C2\u00A0 where\u00C2\u00A0it\u00C2\u00A0spontaneously\u00C2\u00A0reacts\u00C2\u00A0with\u00C2\u00A0ethanol,\u00C2\u00A0and\u00C2\u00A0forms\u00C2\u00A0the\u00C2\u00A0carcinogen\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0(EC).\u00C2\u00A0 \u00C2\u00A0 Urea\u00C2\u00A0degrading\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0were\u00C2\u00A0created\u00C2\u00A0by\u00C2\u00A0integrating\u00C2\u00A0a\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0 gene\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9.\u00C2\u00A0The\u00C2\u00A0\u00E2\u0080\u0098self\u00E2\u0080\u0090cloned\u00E2\u0080\u0099\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0produced\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0with\u00C2\u00A068%\u00C2\u00A0 less\u00C2\u00A0EC.\u00C2\u00A0The\u00C2\u00A0Sake\u00C2\u00A0 strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0efficiently\u00C2\u00A0 reduce\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 evolutionary\u00C2\u00A0adaptation\u00C2\u00A0of\u00C2\u00A0said\u00C2\u00A0strains\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0unique\u00C2\u00A0nutrients\u00C2\u00A0of\u00C2\u00A0rice\u00C2\u00A0mash;\u00C2\u00A0therefore,\u00C2\u00A0the\u00C2\u00A0functionality\u00C2\u00A0 of\u00C2\u00A0 engineered\u00C2\u00A0 yeasts\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 tested\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0 niche\u00C2\u00A0 environments\u00C2\u00A0 as\u00C2\u00A0 to\u00C2\u00A0 correctly\u00C2\u00A0 characterize\u00C2\u00A0 new\u00C2\u00A0 strains.\u00C2\u00A0 \u00C2\u00A0 S.\u00C2\u00A0cerevisiae\u00C2\u00A0possesses\u00C2\u00A0an\u00C2\u00A0NCR\u00C2\u00A0controlled\u00C2\u00A0high\u00C2\u00A0affinity\u00C2\u00A0urea\u00C2\u00A0permease\u00C2\u00A0 (DUR3).\u00C2\u00A0Urea\u00C2\u00A0 importing\u00C2\u00A0 yeast\u00C2\u00A0strains\u00C2\u00A0were\u00C2\u00A0created\u00C2\u00A0by\u00C2\u00A0integrating\u00C2\u00A0a\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0gene\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0(Sake)\u00C2\u00A0and\u00C2\u00A0522\u00C2\u00A0 (wine).\u00C2\u00A0 In\u00C2\u00A0 Chardonnay\u00C2\u00A0wine,\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 importing\u00C2\u00A0 strains\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 reduced\u00C2\u00A0 EC\u00C2\u00A0 by\u00C2\u00A0 7%\u00C2\u00A0 and\u00C2\u00A0 81%,\u00C2\u00A0 respectively;\u00C2\u00A0reduction\u00C2\u00A0by\u00C2\u00A0these\u00C2\u00A0strains\u00C2\u00A0was\u00C2\u00A0equal\u00C2\u00A0to\u00C2\u00A0reduction\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 522EC\u00E2\u0080\u0090.\u00C2\u00A0 In\u00C2\u00A0 Sake\u00C2\u00A0 wine,\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 degrading\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0 EC\u00C2\u00A0 by\u00C2\u00A0 87%\u00C2\u00A0 and\u00C2\u00A0 84%\u00C2\u00A0 respectively,\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0importing\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0less\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0reducing\u00C2\u00A0 EC\u00C2\u00A0(15%\u00C2\u00A0and\u00C2\u00A012%\u00C2\u00A0respectively).\u00C2\u00A0In\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine,\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0that\u00C2\u00A0constitutively\u00C2\u00A0co\u00E2\u0080\u0090 expressed\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0more\u00C2\u00A0effectively\u00C2\u00A0than\u00C2\u00A0strains\u00C2\u00A0 in\u00C2\u00A0which\u00C2\u00A0either\u00C2\u00A0gene\u00C2\u00A0was\u00C2\u00A0 expressed\u00C2\u00A0 solely.\u00C2\u00A0Uptake\u00C2\u00A0 of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0under\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0 conditions\u00C2\u00A0was\u00C2\u00A0 enhanced\u00C2\u00A0 in\u00C2\u00A0urea\u00C2\u00A0 importing\u00C2\u00A0 strains;\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0failed\u00C2\u00A0to\u00C2\u00A0 incorporate\u00C2\u00A0any\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0thus\u00C2\u00A0confirming\u00C2\u00A0the\u00C2\u00A0functionality\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0 permease\u00C2\u00A0derived\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette.\u00C2\u00A0 iii\u00C2\u00A0 \u00C2\u00A0 TABLE\u00C2\u00A0OF\u00C2\u00A0CONTENTS\u00C2\u00A0 ABSTRACT\u00C2\u00A0......................................................................................................................................................\u00C2\u00A0ii\u00C2\u00A0 TABLE\u00C2\u00A0OF\u00C2\u00A0CONTENTS\u00C2\u00A0....................................................................................................................................\u00C2\u00A0iii\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0TABLES\u00C2\u00A0..............................................................................................................................................\u00C2\u00A0x\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0FIGURES\u00C2\u00A0..........................................................................................................................................\u00C2\u00A0xii\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0ABBREVIATIONS\u00C2\u00A0..............................................................................................................................\u00C2\u00A0xv\u00C2\u00A0 ACKNOWLEDGEMENTS\u00C2\u00A0..............................................................................................................................\u00C2\u00A0xix\u00C2\u00A0 1\u00C2\u00A0INTRODUCTION\u00C2\u00A0.........................................................................................................................................\u00C2\u00A01\u00C2\u00A0 1.1\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0............................................................................................................\u00C2\u00A01\u00C2\u00A0 1.2\u00C2\u00A0Yeast\u00C2\u00A0strains\u00C2\u00A0from\u00C2\u00A0nature\u00C2\u00A0vs.\u00C2\u00A0laboratory\u00C2\u00A0yeasts\u00C2\u00A0...........................................................................\u00C2\u00A03\u00C2\u00A0 1.3\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0and\u00C2\u00A0industry:\u00C2\u00A0Wine\u00C2\u00A0yeasts\u00C2\u00A0.......................................................................................\u00C2\u00A05\u00C2\u00A0 1.4\u00C2\u00A0Winemaking\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0....................................................................................................\u00C2\u00A06\u00C2\u00A0 1.5\u00C2\u00A0Aspergillus\u00C2\u00A0oryzae\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0.........................................................................\u00C2\u00A010\u00C2\u00A0 1.6\u00C2\u00A0Yeast\u00C2\u00A0nitrogen\u00C2\u00A0metabolism\u00C2\u00A0during\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0.......................................................\u00C2\u00A011\u00C2\u00A0 1.7\u00C2\u00A0Nitrogen\u00C2\u00A0metabolism\u00C2\u00A0and\u00C2\u00A0Nitrogen\u00C2\u00A0Catabolite\u00C2\u00A0Repression\u00C2\u00A0(NCR)\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0....................\u00C2\u00A012\u00C2\u00A0 1.8\u00C2\u00A0Urea\u00C2\u00A0and\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0(EC)\u00C2\u00A0..................................................................................................\u00C2\u00A016\u00C2\u00A0 1.9\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem\u00C2\u00A0..........................................................................................................................\u00C2\u00A017\u00C2\u00A0 1.9.1\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0History\u00C2\u00A0...............................................................................................\u00C2\u00A017\u00C2\u00A0 1.9.2\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0Surveys\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0................................................\u00C2\u00A018\u00C2\u00A0 1.9.3\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0Current\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0lowering\u00C2\u00A0EC\u00C2\u00A0.......................................................\u00C2\u00A019\u00C2\u00A0 1.9.3.1\u00C2\u00A0Agricultural\u00C2\u00A0methods\u00C2\u00A0.......................................................................................\u00C2\u00A019\u00C2\u00A0 1.9.3.2\u00C2\u00A0Additives\u00C2\u00A0(acid\u00C2\u00A0urease)\u00C2\u00A0....................................................................................\u00C2\u00A020\u00C2\u00A0 1.9.3.3\u00C2\u00A0Additives\u00C2\u00A0(DAP)\u00C2\u00A0................................................................................................\u00C2\u00A020\u00C2\u00A0 iv\u00C2\u00A0 \u00C2\u00A0 1.9.3.4\u00C2\u00A0Genetic\u00C2\u00A0engineering\u00C2\u00A0(urease\u00C2\u00A0expression)\u00C2\u00A0........................................................\u00C2\u00A020\u00C2\u00A0 1.9.3.5\u00C2\u00A0Genetic\u00C2\u00A0engineering\u00C2\u00A0(CAR1)\u00C2\u00A0.............................................................................\u00C2\u00A020\u00C2\u00A0 1.9.3.6\u00C2\u00A0Genetic\u00C2\u00A0engineering\u00C2\u00A0(DUR1,2)\u00C2\u00A0.........................................................................\u00C2\u00A021\u00C2\u00A0 1.9.4\u00C2\u00A0Alternative\u00C2\u00A0methods\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0..........................................................................\u00C2\u00A022\u00C2\u00A0 1.10\u00C2\u00A0Introduction\u00C2\u00A0to\u00C2\u00A0DUR3:\u00C2\u00A0Role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0(urea\u00C2\u00A0transport,\u00C2\u00A0polyamines,\u00C2\u00A0boron)\u00C2\u00A0..........................\u00C2\u00A023\u00C2\u00A0 1.11\u00C2\u00A0Proposed\u00C2\u00A0Research\u00C2\u00A0...................................................................................................................\u00C2\u00A025\u00C2\u00A0 1.11.1\u00C2\u00A0Significance\u00C2\u00A0of\u00C2\u00A0Research\u00C2\u00A0.............................................................................................\u00C2\u00A025\u00C2\u00A0 1.11.2\u00C2\u00A0Hypotheses\u00C2\u00A0.................................................................................................................\u00C2\u00A025\u00C2\u00A0 1.11.2.1\u00C2\u00A0 The\u00C2\u00A0 metabolically\u00C2\u00A0 engineered\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 should\u00C2\u00A0 reduce\u00C2\u00A0EC\u00C2\u00A0efficiently\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0trials.\u00C2\u00A0...........................................\u00C2\u00A025\u00C2\u00A0 1.11.2.2\u00C2\u00A0Constitutive\u00C2\u00A0co\u00E2\u0080\u0090expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0 yeasts\u00C2\u00A0should\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0synergistic\u00C2\u00A0EC\u00C2\u00A0reduction.\u00C2\u00A0............................................\u00C2\u00A026\u00C2\u00A0 1.11.3\u00C2\u00A0Main\u00C2\u00A0objectives\u00C2\u00A0...........................................................................................................\u00C2\u00A026\u00C2\u00A0 2\u00C2\u00A0MATERIALS\u00C2\u00A0AND\u00C2\u00A0METHODS\u00C2\u00A0.....................................................................................................................\u00C2\u00A027\u00C2\u00A0 2.1\u00C2\u00A0Strains,\u00C2\u00A0plasmids\u00C2\u00A0and\u00C2\u00A0genetic\u00C2\u00A0cassettes\u00C2\u00A0.....................................................................................\u00C2\u00A027\u00C2\u00A0 2.2\u00C2\u00A0Culture\u00C2\u00A0conditions\u00C2\u00A0......................................................................................................................\u00C2\u00A028\u00C2\u00A0 2.3\u00C2\u00A0Genetic\u00C2\u00A0construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0...........................................\u00C2\u00A029\u00C2\u00A0 2.3.1\u00C2\u00A0Co\u00E2\u0080\u0090transformation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0and\u00C2\u00A0pUT332\u00C2\u00A0..............................................\u00C2\u00A029\u00C2\u00A0 2.3.2\u00C2\u00A0Screening\u00C2\u00A0of\u00C2\u00A0transformants\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0.................................\u00C2\u00A029\u00C2\u00A0 2.3.3\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0...............................................................................................\u00C2\u00A030\u00C2\u00A0 2.3.3.1\u00C2\u00A0Southern\u00C2\u00A0blot\u00C2\u00A0analyses\u00C2\u00A0....................................................................................\u00C2\u00A030\u00C2\u00A0 2.3.3.2\u00C2\u00A0Sequence\u00C2\u00A0analysis\u00C2\u00A0............................................................................................\u00C2\u00A030\u00C2\u00A0 v\u00C2\u00A0 \u00C2\u00A0 2.3.3.3\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR\u00C2\u00A0............................................\u00C2\u00A031\u00C2\u00A0 2.3.3.4\u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0......................................................................\u00C2\u00A032\u00C2\u00A0 2.3.4\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0.........................................................................................\u00C2\u00A033\u00C2\u00A0 2.3.4.1\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0........................................\u00C2\u00A033\u00C2\u00A0 2.3.4.2\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0mash\u00C2\u00A0....................................................\u00C2\u00A033\u00C2\u00A0 2.3.4.3\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0glucose/fructose\u00C2\u00A0utilization\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0production\u00C2\u00A0....................\u00C2\u00A034\u00C2\u00A0 2.3.5\u00C2\u00A0Functionality\u00C2\u00A0analyses\u00C2\u00A0...................................................................................................\u00C2\u00A035\u00C2\u00A0 2.3.5.1\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0..............................................................\u00C2\u00A035\u00C2\u00A0 2.3.5.2\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0..........................................................................\u00C2\u00A035\u00C2\u00A0 2.3.5.3\u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0solid\u00C2\u00A0phase\u00C2\u00A0microextraction\u00C2\u00A0and\u00C2\u00A0GC/MS\u00C2\u00A0......\u00C2\u00A035\u00C2\u00A0 2.4\u00C2\u00A0Genetic\u00C2\u00A0construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0importing\u00C2\u00A0yeasts\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0...............\u00C2\u00A036\u00C2\u00A0 2.4.1\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0.....................................................................\u00C2\u00A036\u00C2\u00A0 2.4.1.1\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pHVX2D3\u00C2\u00A0................................................................................\u00C2\u00A036\u00C2\u00A0 2.4.1.2\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pHVXKD3\u00C2\u00A0................................................................................\u00C2\u00A037\u00C2\u00A0 2.4.1.3\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pUCTRP1\u00C2\u00A0................................................................................\u00C2\u00A038\u00C2\u00A0 2.4.1.4\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pUCMD\u00C2\u00A0..................................................................................\u00C2\u00A039\u00C2\u00A0 2.4.2\u00C2\u00A0Sequence\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0pUCMD\u00C2\u00A0.....................................................\u00C2\u00A040\u00C2\u00A0 2.4.3\u00C2\u00A0 Transformation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 linear\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 and\u00C2\u00A0 selection\u00C2\u00A0 of\u00C2\u00A0 transformants\u00C2\u00A0..............................................................................................................\u00C2\u00A043\u00C2\u00A0 2.4.3.1\u00C2\u00A0Confirmation\u00C2\u00A0of\u00C2\u00A0integration\u00C2\u00A0via\u00C2\u00A0colony\u00C2\u00A0PCR\u00C2\u00A0....................................................\u00C2\u00A043\u00C2\u00A0 2.4.4\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0...............................................................................................\u00C2\u00A043\u00C2\u00A0 vi\u00C2\u00A0 \u00C2\u00A0 2.4.4.1\u00C2\u00A0Southern\u00C2\u00A0blot\u00C2\u00A0analyses\u00C2\u00A0....................................................................................\u00C2\u00A043\u00C2\u00A0 2.4.4.2\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0northern\u00C2\u00A0blotting\u00C2\u00A0...........................................\u00C2\u00A044\u00C2\u00A0 2.4.4.3\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR\u00C2\u00A0...............................................\u00C2\u00A044\u00C2\u00A0 2.4.4.4\u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0......................................................................\u00C2\u00A045\u00C2\u00A0 2.4.5\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0using\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0.........................................................................\u00C2\u00A045\u00C2\u00A0 2.4.6\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0.........................................................................................\u00C2\u00A045\u00C2\u00A0 2.4.6.1\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0........................................\u00C2\u00A045\u00C2\u00A0 2.4.6.2\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0mash\u00C2\u00A0....................................................\u00C2\u00A045\u00C2\u00A0 2.4.6.3\u00C2\u00A0Analysis\u00C2\u00A0for\u00C2\u00A0ethanol\u00C2\u00A0content\u00C2\u00A0\u00C2\u00A0...........................................................................\u00C2\u00A045\u00C2\u00A0 2.4.7\u00C2\u00A0Functionality\u00C2\u00A0of\u00C2\u00A0metabolically\u00C2\u00A0enhanced\u00C2\u00A0yeasts\u00C2\u00A0...........................................................\u00C2\u00A046\u00C2\u00A0 2.4.7.1\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0..............................................................\u00C2\u00A046\u00C2\u00A0 2.4.7.2\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0..........................................................................\u00C2\u00A046\u00C2\u00A0 2.5\u00C2\u00A0Statistical\u00C2\u00A0analyses...........................................................................................................\u00C2\u00A046\u00C2\u00A0 3\u00C2\u00A0RESULTS\u00C2\u00A0...................................................................................................................................................\u00C2\u00A047\u00C2\u00A0 3.1\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0..........................................\u00C2\u00A047\u00C2\u00A0 3.1.1\u00C2\u00A0 Integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 linear\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0 and\u00C2\u00A0K9\u00C2\u00A0..........................................................................................................................\u00C2\u00A047\u00C2\u00A0 3.1.2\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0...................................................................\u00C2\u00A047\u00C2\u00A0 3.1.2.1\u00C2\u00A0Correct\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 linear\u00C2\u00A0cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0..........................................................................................................\u00C2\u00A047\u00C2\u00A0 3.1.2.2\u00C2\u00A0 Sake\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 contain\u00C2\u00A0 the\u00C2\u00A0 bla\u00C2\u00A0 and\u00C2\u00A0 Tn5ble\u00C2\u00A0 antibiotic\u00C2\u00A0 resistance\u00C2\u00A0markers\u00C2\u00A0..........................................................................................\u00C2\u00A050\u00C2\u00A0 vii\u00C2\u00A0 \u00C2\u00A0 3.1.2.3\u00C2\u00A0Sequence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0.................................................................................................................\u00C2\u00A051\u00C2\u00A0 3.1.2.4\u00C2\u00A0Confirmation\u00C2\u00A0of\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090 PCR\u00C2\u00A0..................................................................................................................\u00C2\u00A052\u00C2\u00A0 3.1.2.5\u00C2\u00A0Effect\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 transcriptomes\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0.................................................................................................................\u00C2\u00A053\u00C2\u00A0 3.1.3\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0.............................................................\u00C2\u00A054\u00C2\u00A0 3.1.3.1\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0..............................\u00C2\u00A054\u00C2\u00A0 3.1.3.2\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0mash\u00C2\u00A0..........................................\u00C2\u00A055\u00C2\u00A0 3.1.3.3\u00C2\u00A0Utilization\u00C2\u00A0 of\u00C2\u00A0 glucose\u00C2\u00A0 and\u00C2\u00A0 fructose\u00C2\u00A0 and\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 ethanol\u00C2\u00A0 by\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0............................................................................................\u00C2\u00A056\u00C2\u00A0 3.1.4\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0approximately\u00C2\u00A030%\u00C2\u00A0....................................................................\u00C2\u00A057\u00C2\u00A0 3.1.5\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0approximately\u00C2\u00A068%\u00C2\u00A0................................................................................\u00C2\u00A058\u00C2\u00A0 3.2\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0522\u00C2\u00A0....\u00C2\u00A059\u00C2\u00A0 3.2.1\u00C2\u00A0Sequence\u00C2\u00A0of\u00C2\u00A0pUCMD\u00C2\u00A0.....................................................................................................\u00C2\u00A059\u00C2\u00A0 3.2.2\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0linear\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0 and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0.......................................................................................................................\u00C2\u00A063\u00C2\u00A0 3.2.3\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0.......................................\u00C2\u00A063\u00C2\u00A0 3.2.3.1\u00C2\u00A0Correct\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0..........................................................................................\u00C2\u00A063\u00C2\u00A0 3.2.3.2\u00C2\u00A0 Confirmation\u00C2\u00A0 of\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 by\u00C2\u00A0 northern\u00C2\u00A0blotting\u00C2\u00A0............................................................................................\u00C2\u00A066\u00C2\u00A0 viii\u00C2\u00A0 \u00C2\u00A0 3.2.3.3\u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0constitutive\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR \u00C2\u00A0........................................................................................................................\u00C2\u00A067\u00C2\u00A0 3.2.3.4\u00C2\u00A0Effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcriptome\u00C2\u00A0of\u00C2\u00A0K7D3\u00C2\u00A0............\u00C2\u00A068\u00C2\u00A0 3.2.4\u00C2\u00A0Recombinant\u00C2\u00A0 strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0exhibit\u00C2\u00A0highly\u00C2\u00A0enhanced\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0ability\u00C2\u00A0 in\u00C2\u00A0 conditions\u00C2\u00A0of\u00C2\u00A0strong\u00C2\u00A0NCR\u00C2\u00A0.............................................................................................\u00C2\u00A069\u00C2\u00A0 3.2.5\u00C2\u00A0Recombinant\u00C2\u00A0 strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0exhibit\u00C2\u00A0highly\u00C2\u00A0enhanced\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0ability\u00C2\u00A0 in\u00C2\u00A0 conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0.................................................................................\u00C2\u00A072\u00C2\u00A0 3.2.6\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0.................................\u00C2\u00A075\u00C2\u00A0 3.2.6.1\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0...\u00C2\u00A075\u00C2\u00A0 3.2.6.2\u00C2\u00A0Ethanol\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0.\u00C2\u00A077\u00C2\u00A0 3.2.6.3\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0...............\u00C2\u00A077\u00C2\u00A0 3.2.6.4\u00C2\u00A0Ethanol\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0.............\u00C2\u00A079\u00C2\u00A0 3.2.7\u00C2\u00A0 Constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 in\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 reduces\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A024.97%\u00C2\u00A0and\u00C2\u00A081.38%,\u00C2\u00A0respectively\u00C2\u00A0..............................................\u00C2\u00A079\u00C2\u00A0 3.2.8\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0 reduces\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0 wine\u00C2\u00A0by\u00C2\u00A018.40%\u00C2\u00A0and\u00C2\u00A010.45%,\u00C2\u00A0respectively\u00C2\u00A0..................................................................\u00C2\u00A080\u00C2\u00A0 4\u00C2\u00A0DISCUSSION\u00C2\u00A0.............................................................................................................................................\u00C2\u00A082\u00C2\u00A0 4.1\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0......\u00C2\u00A082\u00C2\u00A0 4.2\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0yielded\u00C2\u00A0 the\u00C2\u00A0functional\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0.........................................................\u00C2\u00A083\u00C2\u00A0 4.2.1\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0...................................................................\u00C2\u00A083\u00C2\u00A0 4.2.2\u00C2\u00A0The\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0conduct\u00C2\u00A0efficient\u00C2\u00A0alcoholic\u00C2\u00A0fermentations\u00C2\u00A0..................\u00C2\u00A085\u00C2\u00A0 4.2.3\u00C2\u00A0The\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0poorly\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0yet\u00C2\u00A0efficiently\u00C2\u00A0in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0.....................................................................................................................\u00C2\u00A085\u00C2\u00A0 ix\u00C2\u00A0 \u00C2\u00A0 4.3\u00C2\u00A0 Constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 permease,\u00C2\u00A0 DUR3,\u00C2\u00A0 in\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 viable\u00C2\u00A0 alternative\u00C2\u00A0 method\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0fermented\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0...........................................................\u00C2\u00A089\u00C2\u00A0 4.3.1\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 linear\u00C2\u00A0PGK1p\u00E2\u0080\u0090DUR3\u00E2\u0080\u0090PGK1t\u00E2\u0080\u0090kanMX\u00C2\u00A0cassette\u00C2\u00A0 for\u00C2\u00A0 integration\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 TRP1\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0............................................................................\u00C2\u00A089\u00C2\u00A0 4.3.2\u00C2\u00A0 Integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 genomes\u00C2\u00A0 of\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 ,\u00C2\u00A0 522,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 yielded\u00C2\u00A0the\u00C2\u00A0functional\u00C2\u00A0urea\u00C2\u00A0transporting\u00C2\u00A0yeasts\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0.....\u00C2\u00A091\u00C2\u00A0 4.3.2.1\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0........................................\u00C2\u00A092\u00C2\u00A0 4.3.2.2\u00C2\u00A0The\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0enhanced\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0....................\u00C2\u00A093\u00C2\u00A0 4.3.2.3\u00C2\u00A0The\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0yeasts\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0ferment\u00C2\u00A0 at\u00C2\u00A0similar\u00C2\u00A0 rates\u00C2\u00A0and\u00C2\u00A0produce\u00C2\u00A0similar\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0........................................................................................................\u00C2\u00A094\u00C2\u00A0 4.3.2.4\u00C2\u00A0 Variability\u00C2\u00A0 of\u00C2\u00A0 metabolically\u00C2\u00A0 engineered\u00C2\u00A0 yeasts\u00C2\u00A0 to\u00C2\u00A0 effectively\u00C2\u00A0 reduce\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0................................................................\u00C2\u00A095\u00C2\u00A0 4.3.2.5\u00C2\u00A0 The\u00C2\u00A0metabolically\u00C2\u00A0 engineered\u00C2\u00A0 yeasts\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 reduce\u00C2\u00A0 EC\u00C2\u00A0 more\u00C2\u00A0effectively\u00C2\u00A0than\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0or\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0in\u00C2\u00A0either\u00C2\u00A0Chardonnay\u00C2\u00A0 or\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0....................................................................................................\u00C2\u00A098\u00C2\u00A0 5\u00C2\u00A0CONCLUSIONS\u00C2\u00A0.......................................................................................................................................\u00C2\u00A0101\u00C2\u00A0 5.1\u00C2\u00A0Future\u00C2\u00A0Directions\u00C2\u00A0......................................................................................................................\u00C2\u00A0103\u00C2\u00A0 REFERENCES\u00C2\u00A0.............................................................................................................................................\u00C2\u00A0104\u00C2\u00A0 \u00C2\u00A0 x\u00C2\u00A0 \u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0TABLES\u00C2\u00A0 Table\u00C2\u00A01.\u00C2\u00A0\u00C2\u00A0 Ranking\u00C2\u00A0of\u00C2\u00A0various\u00C2\u00A0yeast\u00C2\u00A0nitrogen\u00C2\u00A0sources\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0NCR\u00C2\u00A0repression\u00C2\u00A0strength\u00C2\u00A0...............\u00C2\u00A013\u00C2\u00A0 Table\u00C2\u00A02.\u00C2\u00A0\u00C2\u00A0 Maximum\u00C2\u00A0potential\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0detected\u00C2\u00A0by\u00C2\u00A0GC/MS\u00C2\u00A0in\u00C2\u00A020\u00C2\u00A0wines\u00C2\u00A0from\u00C2\u00A0six\u00C2\u00A0countries\u00C2\u00A0..\u00C2\u00A019\u00C2\u00A0 Table\u00C2\u00A03.\u00C2\u00A0\u00C2\u00A0 Strains\u00C2\u00A0 used\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 genetic\u00C2\u00A0 construction\u00C2\u00A0 and\u00C2\u00A0 characterization\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 expressing\u00C2\u00A0 yeast\u00C2\u00A0 strains.\u00C2\u00A0....................................................................................................................................\u00C2\u00A027\u00C2\u00A0 Table\u00C2\u00A04.\u00C2\u00A0\u00C2\u00A0 Plasmids\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0genetic\u00C2\u00A0 construction\u00C2\u00A0and\u00C2\u00A0 characterization\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0expressing\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0.....................................................................................................................................\u00C2\u00A028\u00C2\u00A0 Table\u00C2\u00A05.\u00C2\u00A0\u00C2\u00A0 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Table\u00C2\u00A08.\u00C2\u00A0\u00C2\u00A0 Discrepancies\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0of\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0published\u00C2\u00A0sequences\u00C2\u00A0 51\u00C2\u00A0 Table\u00C2\u00A09.\u00C2\u00A0\u00C2\u00A0 Detailed\u00C2\u00A0description\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassettes\u00C2\u00A0...............\u00C2\u00A051\u00C2\u00A0 Table\u00C2\u00A010\u00C2\u00A0 Effect\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0genome\u00C2\u00A0of\u00C2\u00A0K7\u00C2\u00A0on\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0 patterns\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(\u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0change)\u00C2\u00A0.....................................................................\u00C2\u00A053\u00C2\u00A0 Table\u00C2\u00A011.\u00C2\u00A0\u00C2\u00A0 Utilization\u00C2\u00A0of\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0fructose\u00C2\u00A0and\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0parental\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0(K7\u00C2\u00A0and\u00C2\u00A0K9),\u00C2\u00A0their\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0counterparts\u00C2\u00A0(K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090)\u00C2\u00A0..............\u00C2\u00A057\u00C2\u00A0 Table\u00C2\u00A012.\u00C2\u00A0\u00C2\u00A0 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Table\u00C2\u00A014.\u00C2\u00A0\u00C2\u00A0 Discrepancies\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0pUCMD\u00C2\u00A0and\u00C2\u00A0published\u00C2\u00A0sequences\u00C2\u00A0................\u00C2\u00A060\u00C2\u00A0 Table\u00C2\u00A015.\u00C2\u00A0\u00C2\u00A0 Detailed\u00C2\u00A0description\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0....................\u00C2\u00A060\u00C2\u00A0 xi\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A016.\u00C2\u00A0\u00C2\u00A0 Recombinant\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0created\u00C2\u00A0by\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus 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Ethanol\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0(K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3)\u00C2\u00A0and\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0 (522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3)\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0...........................................................\u00C2\u00A077\u00C2\u00A0 Table\u00C2\u00A019.\u00C2\u00A0\u00C2\u00A0 Ethanol\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0(K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3)\u00C2\u00A0and\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0 (522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3)\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.......................................................................\u00C2\u00A079\u00C2\u00A0 Table\u00C2\u00A020.\u00C2\u00A0\u00C2\u00A0 Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0making\u00C2\u00A0......................\u00C2\u00A080\u00C2\u00A0 Table\u00C2\u00A021.\u00C2\u00A0\u00C2\u00A0 Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0making\u00C2\u00A0......\u00C2\u00A081\u00C2\u00A0 \u00C2\u00A0 xii\u00C2\u00A0 \u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0FIGURES\u00C2\u00A0 Figure\u00C2\u00A01.\u00C2\u00A0\u00C2\u00A0 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Figure\u00C2\u00A010.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pHVX2D3\u00C2\u00A0..............................\u00C2\u00A037\u00C2\u00A0 Figure\u00C2\u00A011.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pHVXKD3\u00C2\u00A0..............................\u00C2\u00A038\u00C2\u00A0 Figure\u00C2\u00A012.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pUCTRP1\u00C2\u00A0...............................\u00C2\u00A038\u00C2\u00A0 Figure\u00C2\u00A013.\u00C2\u00A0\u00C2\u00A0 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generated\u00C2\u00A0 during\u00C2\u00A0 construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0.....................................................................................\u00C2\u00A052\u00C2\u00A0 Figure\u00C2\u00A020.\u00C2\u00A0\u00C2\u00A0 Gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0(qRT\u00E2\u0080\u0090PCR)\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 indicates\u00C2\u00A0functionality\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 DUR1,2\u00C2\u00A0cassette\u00C2\u00A0and\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0(NCR)\u00C2\u00A0conditions\u00C2\u00A0..... 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wine........................................................................................................................................\u00C2\u00A056\u00C2\u00A0 Figure\u00C2\u00A023.\u00C2\u00A0\u00C2\u00A0 DNA\u00C2\u00A0 sequence\u00C2\u00A0 alignment\u00C2\u00A0 of\u00C2\u00A0 S288C\u00C2\u00A0 and\u00C2\u00A0 pUCMD\u00C2\u00A0 revealed\u00C2\u00A0 nine\u00C2\u00A0 discrepancies\u00C2\u00A0 along\u00C2\u00A0 the\u00C2\u00A0 length\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0........................................................................................................\u00C2\u00A062\u00C2\u00A0 Figure\u00C2\u00A024.\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0 schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 new\u00C2\u00A0 ORFs\u00C2\u00A0 of\u00C2\u00A0 more\u00C2\u00A0 than\u00C2\u00A0 100\u00C2\u00A0 codons\u00C2\u00A0 generated\u00C2\u00A0 during\u00C2\u00A0 construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0........................................................................................\u00C2\u00A062\u00C2\u00A0 Figure\u00C2\u00A025.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0linear\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0.........................................................\u00C2\u00A063\u00C2\u00A0 Figure\u00C2\u00A026.\u00C2\u00A0\u00C2\u00A0 Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0522D3,\u00C2\u00A0522EC\u00E2\u0080\u0090D3,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0 confirmed\u00C2\u00A0by\u00C2\u00A0Southern\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0using\u00C2\u00A0DUR3\u00C2\u00A0and\u00C2\u00A0TRP1\u00C2\u00A0probes\u00C2\u00A0.....................................\u00C2\u00A064\u00C2\u00A0 Figure\u00C2\u00A027.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 signals\u00C2\u00A0 expected\u00C2\u00A0 during\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 recombinant\u00C2\u00A0 yeasts\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 TRP1\u00C2\u00A0 locus\u00C2\u00A0.......................................................................................................................................\u00C2\u00A065\u00C2\u00A0 \u00C2\u00A0 xiv\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A028.\u00C2\u00A0\u00C2\u00A0 Alignment\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0of\u00C2\u00A0S288C,\u00C2\u00A0522,\u00C2\u00A0and\u00C2\u00A0K7\u00C2\u00A0confirmed\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0mutant\u00C2\u00A0 EcoR1\u00C2\u00A0site\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0K7.\u00C2\u00A0.........................................................................\u00C2\u00A066\u00C2\u00A0 Figure\u00C2\u00A029.\u00C2\u00A0\u00C2\u00A0 Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0(2208\u00C2\u00A0bp)\u00C2\u00A0was\u00C2\u00A0confirmed\u00C2\u00A0by\u00C2\u00A0northern\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0...................................................................................................................\u00C2\u00A067\u00C2\u00A0 Figure\u00C2\u00A030.\u00C2\u00A0\u00C2\u00A0 Analyses\u00C2\u00A0of\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0(qRT\u00E2\u0080\u0090PCR)\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0confirmed\u00C2\u00A0functionality\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 and\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0 DUR3\u00C2\u00A0 in\u00C2\u00A0 non\u00E2\u0080\u0090inducing\u00C2\u00A0 (NCR)\u00C2\u00A0conditions\u00C2\u00A0....................................................................................................................\u00C2\u00A068\u00C2\u00A0 Figure\u00C2\u00A031.\u00C2\u00A0\u00C2\u00A0 Uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0...........................\u00C2\u00A071\u00C2\u00A0 Figure\u00C2\u00A032.\u00C2\u00A0\u00C2\u00A0 Uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0................................................\u00C2\u00A072\u00C2\u00A0 Figure\u00C2\u00A033.\u00C2\u00A0 Uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0....\u00C2\u00A073\u00C2\u00A0 Figure\u00C2\u00A034.\u00C2\u00A0 Uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0.........................\u00C2\u00A074\u00C2\u00A0 Figure\u00C2\u00A035.\u00C2\u00A0\u00C2\u00A0 Fermentation\u00C2\u00A0profiles\u00C2\u00A0(weight\u00C2\u00A0 loss)\u00C2\u00A0of\u00C2\u00A0(a)\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0and\u00C2\u00A0 (b)\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0.........................\u00C2\u00A076\u00C2\u00A0 Figure\u00C2\u00A036.\u00C2\u00A0\u00C2\u00A0 Fermentation\u00C2\u00A0profiles\u00C2\u00A0(weight\u00C2\u00A0 loss)\u00C2\u00A0of\u00C2\u00A0(a)\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0and\u00C2\u00A0 (b)\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0.....................................\u00C2\u00A078\u00C2\u00A0 Figure\u00C2\u00A037.\u00C2\u00A0\u00C2\u00A0 Schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 inducible\u00C2\u00A0 DUR1,2\u00C2\u00A0 expression\u00C2\u00A0 during\u00C2\u00A0 Chardonnay\u00C2\u00A0 and\u00C2\u00A0 Sake\u00C2\u00A0 wine\u00C2\u00A0fermentation\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0urea\u00C2\u00A0importing\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0.............................................................\u00C2\u00A097\u00C2\u00A0 \u00C2\u00A0 xv\u00C2\u00A0 \u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0ABBREVIATIONS\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0 Microgram\u00C2\u00A0 \u00C2\u00B5M\u00C2\u00A0 Micromolar\u00C2\u00A0 aa\u00C2\u00A0 Amino\u00C2\u00A0acid\u00C2\u00A0 Abs\u00C2\u00A0 Absorbance\u00C2\u00A0 ANOVA\u00C2\u00A0 Analysis\u00C2\u00A0of\u00C2\u00A0variance\u00C2\u00A0 BC\u00C2\u00A0 British\u00C2\u00A0Columbia\u00C2\u00A0\u00C2\u00A0 bp\u00C2\u00A0 Base\u00C2\u00A0pair\u00C2\u00A0 cDNA\u00C2\u00A0 Complementary\u00C2\u00A0deoxyribonucleic\u00C2\u00A0acid\u00C2\u00A0 Ci\u00C2\u00A0 Curie\u00C2\u00A0 cm\u00C2\u00A0\u00C2\u00A0 Centimetre\u00C2\u00A0 cM\u00C2\u00A0 Centimorgan\u00C2\u00A0 Corp.\u00C2\u00A0\u00C2\u00A0 Corporation\u00C2\u00A0 cRNA\u00C2\u00A0 Ribonucleic\u00C2\u00A0acid\u00C2\u00A0derived\u00C2\u00A0from\u00C2\u00A0cDNA\u00C2\u00A0 DAP\u00C2\u00A0 Diammonium\u00C2\u00A0phosphate\u00C2\u00A0 DNA\u00C2\u00A0 Deoxyribonucleic\u00C2\u00A0acid\u00C2\u00A0 DTT\u00C2\u00A0 Dithiothreitol\u00C2\u00A0 EC\u00C2\u00A0 Ethyl\u00C2\u00A0carbamate\u00C2\u00A0 EDTA\u00C2\u00A0 Ethylenediamine\u00C2\u00A0tetraacetic\u00C2\u00A0acid\u00C2\u00A0 ER\u00C2\u00A0 Endoplasmic\u00C2\u00A0reticulum\u00C2\u00A0 EtOH\u00C2\u00A0 Ethanol\u00C2\u00A0 FDA\u00C2\u00A0 Food\u00C2\u00A0and\u00C2\u00A0Drug\u00C2\u00A0Administration\u00C2\u00A0 g\u00C2\u00A0 Gram\u00C2\u00A0 GC\u00C2\u00A0 Gas\u00C2\u00A0chromatograph\u00C2\u00A0 GC/MS\u00C2\u00A0 Gas\u00C2\u00A0chromatograph\u00C2\u00A0coupled\u00C2\u00A0mass\u00C2\u00A0spectrometry\u00C2\u00A0 GM\u00C2\u00A0 Genetically\u00C2\u00A0modified\u00C2\u00A0 GMO\u00C2\u00A0 Genetically\u00C2\u00A0modified\u00C2\u00A0organism\u00C2\u00A0 GO\u00C2\u00A0 Gene\u00C2\u00A0ontology\u00C2\u00A0 GRAS\u00C2\u00A0 Generally\u00C2\u00A0regarded\u00C2\u00A0as\u00C2\u00A0safe\u00C2\u00A0 HPLC\u00C2\u00A0 High\u00C2\u00A0pressure\u00C2\u00A0liquid\u00C2\u00A0chromatography\u00C2\u00A0 kb\u00C2\u00A0 Kilo\u00C2\u00A0base\u00C2\u00A0pair\u00C2\u00A0 xvi\u00C2\u00A0 \u00C2\u00A0 kg\u00C2\u00A0 Kilogram\u00C2\u00A0 L\u00C2\u00A0 Litre\u00C2\u00A0 LB\u00C2\u00A0 Luria\u00E2\u0080\u0090Bertani\u00C2\u00A0medium\u00C2\u00A0 LC\u00C2\u00A0 Liquid\u00C2\u00A0chromatograph\u00C2\u00A0 log\u00C2\u00A0 Logarithm\u00C2\u00A0 LSD\u00C2\u00A0 Least\u00C2\u00A0significant\u00C2\u00A0difference\u00C2\u00A0 m\u00C2\u00A0 Metre\u00C2\u00A0 M\u00C2\u00A0 Molarity\u00C2\u00A0 m/v\u00C2\u00A0 Mass\u00C2\u00A0per\u00C2\u00A0volume\u00C2\u00A0 mCi\u00C2\u00A0 Millicurie\u00C2\u00A0 mg\u00C2\u00A0 Milligram\u00C2\u00A0 min\u00C2\u00A0 Minute\u00C2\u00A0 mL\u00C2\u00A0 Millilitre\u00C2\u00A0 MLF\u00C2\u00A0 Malolactic\u00C2\u00A0fermentation\u00C2\u00A0 mM\u00C2\u00A0 Millimolar\u00C2\u00A0 mRNA\u00C2\u00A0 Messenger\u00C2\u00A0RNA\u00C2\u00A0 MS\u00C2\u00A0 Mass\u00C2\u00A0spectrometry\u00C2\u00A0 NAPS\u00C2\u00A0 Nucleic\u00C2\u00A0Acid\u00C2\u00A0Protein\u00C2\u00A0Service\u00C2\u00A0Unit\u00C2\u00A0at\u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia\u00C2\u00A0 NCR\u00C2\u00A0 Nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0system\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0 ng\u00C2\u00A0 Nanogram\u00C2\u00A0 nM\u00C2\u00A0 Nanomolar\u00C2\u00A0 nt\u00C2\u00A0 Nucleotide\u00C2\u00A0 \u00C2\u00B0C\u00C2\u00A0 Degree\u00C2\u00A0Celsius\u00C2\u00A0 OD\u00C2\u00A0 Optical\u00C2\u00A0density\u00C2\u00A0 O/N\u00C2\u00A0 Overnight\u00C2\u00A0 ORF\u00C2\u00A0 Open\u00C2\u00A0reading\u00C2\u00A0frame\u00C2\u00A0 PB\u00C2\u00A0 Protein\u00C2\u00A0body\u00C2\u00A0 PCR\u00C2\u00A0 Polymerase\u00C2\u00A0Chain\u00C2\u00A0Reaction\u00C2\u00A0 PDM\u00C2\u00A0 Prise\u00C2\u00A0de\u00C2\u00A0Mousse\u00C2\u00A0 pH\u00C2\u00A0 Potential\u00C2\u00A0of\u00C2\u00A0Hydrogen\u00C2\u00A0 ppb\u00C2\u00A0 Parts\u00C2\u00A0per\u00C2\u00A0billion\u00C2\u00A0 xvii\u00C2\u00A0 \u00C2\u00A0 ppm\u00C2\u00A0 Parts\u00C2\u00A0per\u00C2\u00A0million\u00C2\u00A0 qPCR\u00C2\u00A0 Quantitative\u00C2\u00A0PCR\u00C2\u00A0 qRT\u00E2\u0080\u0090PCR\u00C2\u00A0\u00C2\u00A0 Quantitative\u00C2\u00A0Reverse\u00C2\u00A0Transcriptase\u00C2\u00A0PCR\u00C2\u00A0 RFLP\u00C2\u00A0 Restriction\u00C2\u00A0fragment\u00C2\u00A0length\u00C2\u00A0polymorphism\u00C2\u00A0 RNA\u00C2\u00A0 Ribonucleic\u00C2\u00A0acid\u00C2\u00A0 ROX\u00C2\u00A0 Passive\u00C2\u00A0reference\u00C2\u00A0dye\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0qPCR\u00C2\u00A0 rpm\u00C2\u00A0 Revolutions\u00C2\u00A0per\u00C2\u00A0minute\u00C2\u00A0 RQ\u00C2\u00A0 Relative\u00C2\u00A0quantification\u00C2\u00A0 rRNA\u00C2\u00A0 Ribosomal\u00C2\u00A0RNA\u00C2\u00A0 RSD\u00C2\u00A0 Relative\u00C2\u00A0standard\u00C2\u00A0deviation\u00C2\u00A0 RT\u00E2\u0080\u0090PCR\u00C2\u00A0 Reverse\u00C2\u00A0Transcriptase\u00C2\u00A0PCR\u00C2\u00A0 s\u00C2\u00A0 Second\u00C2\u00A0 SAP\u00C2\u00A0 Shrimp\u00C2\u00A0Alkaline\u00C2\u00A0Phosphatase\u00C2\u00A0 STDEV\u00C2\u00A0 Standard\u00C2\u00A0deviation\u00C2\u00A0 SGD\u00C2\u00A0 Saccharomyces\u00C2\u00A0Genome\u00C2\u00A0Database\u00C2\u00A0(www.yeastgenome.org)\u00C2\u00A0 SLR\u00C2\u00A0 Signal\u00C2\u00A0log\u00C2\u00A0ratio\u00C2\u00A0(log\u00C2\u00A0base\u00C2\u00A0=\u00C2\u00A02)\u00C2\u00A0 TBE\u00C2\u00A0 Buffer\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0Tris\u00C2\u00A0base,\u00C2\u00A0boric\u00C2\u00A0acid,\u00C2\u00A0EDTA,\u00C2\u00A0and\u00C2\u00A0water\u00C2\u00A0 TE\u00C2\u00A0 Buffer\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0Tris\u00C2\u00A0base,\u00C2\u00A0EDTA,\u00C2\u00A0and\u00C2\u00A0water\u00C2\u00A0 tRNA\u00C2\u00A0 Transfer\u00C2\u00A0RNA\u00C2\u00A0 TRP\u00C2\u00A0 Tryptophan\u00C2\u00A0 UAS\u00C2\u00A0 Upstream\u00C2\u00A0activation\u00C2\u00A0sequence\u00C2\u00A0 UASNTR\u00C2\u00A0 Upstream\u00C2\u00A0activation\u00C2\u00A0sequence\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0nitrogen\u00C2\u00A0regulated\u00C2\u00A0 UIS\u00C2\u00A0 Upstream\u00C2\u00A0induction\u00C2\u00A0sequence\u00C2\u00A0 UK\u00C2\u00A0\u00C2\u00A0 United\u00C2\u00A0Kingdom\u00C2\u00A0\u00C2\u00A0 URA\u00C2\u00A0 Uracil\u00C2\u00A0 URS\u00C2\u00A0 Upstream\u00C2\u00A0repression\u00C2\u00A0sequence\u00C2\u00A0 USA\u00C2\u00A0\u00C2\u00A0 United\u00C2\u00A0States\u00C2\u00A0of\u00C2\u00A0America\u00C2\u00A0\u00C2\u00A0 v\u00C2\u00A0 Volt\u00C2\u00A0 v/v\u00C2\u00A0 Volume\u00C2\u00A0per\u00C2\u00A0volume\u00C2\u00A0 w/o\u00C2\u00A0 Without\u00C2\u00A0 YAN\u00C2\u00A0 Yeast\u00C2\u00A0assimilable\u00C2\u00A0nitrogen\u00C2\u00A0\u00C2\u00A0 xviii\u00C2\u00A0 \u00C2\u00A0 YEG\u00C2\u00A0 Medium\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0extract\u00C2\u00A0and\u00C2\u00A0dextrose\u00C2\u00A0 YNB\u00C2\u00A0 Yeast\u00C2\u00A0Nitrogen\u00C2\u00A0Base\u00C2\u00A0 YPD\u00C2\u00A0 Medium\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0extract,\u00C2\u00A0peptone,\u00C2\u00A0and\u00C2\u00A0dextrose\u00C2\u00A0 xix\u00C2\u00A0 \u00C2\u00A0 ACKNOWLEDGEMENTS\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 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business.\u00C2\u00A0For\u00C2\u00A0the\u00C2\u00A0opportunities\u00C2\u00A0presented\u00C2\u00A0to\u00C2\u00A0me\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0people\u00C2\u00A0I\u00C2\u00A0have\u00C2\u00A0met\u00C2\u00A0through\u00C2\u00A0Dr.\u00C2\u00A0van\u00C2\u00A0Vuuren,\u00C2\u00A0I\u00C2\u00A0am\u00C2\u00A0 exceptionally\u00C2\u00A0grateful.\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0like\u00C2\u00A0to\u00C2\u00A0thank\u00C2\u00A0the\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0my\u00C2\u00A0supervisory\u00C2\u00A0committee,\u00C2\u00A0Dr.\u00C2\u00A0Ivan\u00C2\u00A0Sadowski\u00C2\u00A0and\u00C2\u00A0Dr.\u00C2\u00A0John\u00C2\u00A0 Smit\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0advice,\u00C2\u00A0criticism,\u00C2\u00A0and\u00C2\u00A0diverse\u00C2\u00A0perspectives\u00C2\u00A0on\u00C2\u00A0this\u00C2\u00A0project.\u00C2\u00A0Working\u00C2\u00A0with\u00C2\u00A0Drs.\u00C2\u00A0Sadowski\u00C2\u00A0and\u00C2\u00A0 Smit\u00C2\u00A0was\u00C2\u00A0an\u00C2\u00A0 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ideas\u00C2\u00A0during\u00C2\u00A0my\u00C2\u00A0early\u00C2\u00A0development\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 scientist.\u00C2\u00A0He\u00C2\u00A0has\u00C2\u00A0 continued\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 generous\u00C2\u00A0with\u00C2\u00A0his\u00C2\u00A0time\u00C2\u00A0and\u00C2\u00A0assistance\u00C2\u00A0despite\u00C2\u00A0living\u00C2\u00A0and\u00C2\u00A0working\u00C2\u00A0halfway\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0country,\u00C2\u00A0and\u00C2\u00A0for\u00C2\u00A0this\u00C2\u00A0 I\u00C2\u00A0am\u00C2\u00A0thankful.\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 thank\u00C2\u00A0my\u00C2\u00A0past\u00C2\u00A0and\u00C2\u00A0present\u00C2\u00A0Wine\u00C2\u00A0Research\u00C2\u00A0Centre\u00C2\u00A0colleagues,\u00C2\u00A0especially\u00C2\u00A0Calvin\u00C2\u00A0 Adams,\u00C2\u00A0Dr.\u00C2\u00A0Zongli\u00C2\u00A0Luo,\u00C2\u00A0and\u00C2\u00A0Lina\u00C2\u00A0Madilao\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0warm\u00C2\u00A0friendship\u00C2\u00A0and\u00C2\u00A0valuable\u00C2\u00A0scientific\u00C2\u00A0collaboration.\u00C2\u00A0 My\u00C2\u00A0special\u00C2\u00A0 thanks\u00C2\u00A0are\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 for\u00C2\u00A0Lina\u00C2\u00A0who\u00C2\u00A0completed\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0GC/MS\u00C2\u00A0analysis\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0study.\u00C2\u00A0To\u00C2\u00A0 the\u00C2\u00A0 members\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 Food,\u00C2\u00A0Nutrition\u00C2\u00A0and\u00C2\u00A0Health\u00C2\u00A0administration\u00C2\u00A0office,\u00C2\u00A0Donna\u00C2\u00A0Bradley,\u00C2\u00A0Tram\u00C2\u00A0Nguyen,\u00C2\u00A0and\u00C2\u00A0 Patrick\u00C2\u00A0Leung,\u00C2\u00A0 I\u00C2\u00A0offer\u00C2\u00A0my\u00C2\u00A0 thanks\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0help\u00C2\u00A0 in\u00C2\u00A0ordering\u00C2\u00A0and\u00C2\u00A0 logistics.\u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 thank\u00C2\u00A0Dr.\u00C2\u00A0 Hugh\u00C2\u00A0Brock\u00C2\u00A0and\u00C2\u00A0Monica\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Genetics\u00C2\u00A0Graduate\u00C2\u00A0program\u00C2\u00A0(GGP)\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0respective\u00C2\u00A0assistance\u00C2\u00A0during\u00C2\u00A0 my\u00C2\u00A0 time\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0program.\u00C2\u00A0Additionally,\u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 thank\u00C2\u00A0my\u00C2\u00A0 fellow\u00C2\u00A0GGP\u00C2\u00A0 student\u00C2\u00A0and\u00C2\u00A0 friend,\u00C2\u00A0Kevin\u00C2\u00A0 Eade.\u00C2\u00A0Kevin\u00E2\u0080\u0099s\u00C2\u00A0friendship\u00C2\u00A0and\u00C2\u00A0coffee\u00C2\u00A0break\u00C2\u00A0companionship\u00C2\u00A0was\u00C2\u00A0much\u00C2\u00A0appreciated.\u00C2\u00A0\u00C2\u00A0 xx\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0am\u00C2\u00A0extremely\u00C2\u00A0appreciative\u00C2\u00A0of\u00C2\u00A0those\u00C2\u00A0who\u00C2\u00A0funded\u00C2\u00A0this\u00C2\u00A0research\u00C2\u00A0and/or\u00C2\u00A0a\u00C2\u00A0portion\u00C2\u00A0of\u00C2\u00A0my\u00C2\u00A0studies:\u00C2\u00A0 National\u00C2\u00A0 Sciences\u00C2\u00A0 and\u00C2\u00A0 Engineering\u00C2\u00A0 Research\u00C2\u00A0 Council\u00C2\u00A0 of\u00C2\u00A0 Canada,\u00C2\u00A0 First\u00C2\u00A0 Venture\u00C2\u00A0 Technology\u00C2\u00A0 Corp.\u00C2\u00A0 (Vancouver,\u00C2\u00A0B.C.)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0Canadian\u00C2\u00A0Vintners\u00C2\u00A0Association.\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0and\u00C2\u00A0most\u00C2\u00A0importantly,\u00C2\u00A0I\u00C2\u00A0thank\u00C2\u00A0my\u00C2\u00A0parents,\u00C2\u00A0Elizabeth\u00C2\u00A0H.\u00C2\u00A0and\u00C2\u00A0Joseph\u00C2\u00A0Dahabieh.\u00C2\u00A0They\u00C2\u00A0bore\u00C2\u00A0 me,\u00C2\u00A0raised\u00C2\u00A0me,\u00C2\u00A0supported\u00C2\u00A0me,\u00C2\u00A0taught\u00C2\u00A0me,\u00C2\u00A0and\u00C2\u00A0loved\u00C2\u00A0me.\u00C2\u00A0Without\u00C2\u00A0them\u00C2\u00A0I\u00C2\u00A0would\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0the\u00C2\u00A0person\u00C2\u00A0I\u00C2\u00A0am\u00C2\u00A0 today. 1\u00C2\u00A0 \u00C2\u00A0 1\u00C2\u00A0\u00C2\u00A0INTRODUCTION\u00C2\u00A0 \u00C2\u00A0 1.1\u00C2\u00A0\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0the\u00C2\u00A0early\u00C2\u00A020th\u00C2\u00A0century\u00C2\u00A0intensive\u00C2\u00A0genetic\u00C2\u00A0research\u00C2\u00A0began\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0budding\u00C2\u00A0yeast\u00C2\u00A0Saccharomyces\u00C2\u00A0 cerevisiae\u00C2\u00A0 and,\u00C2\u00A0 ever\u00C2\u00A0 since,\u00C2\u00A0 scientists\u00C2\u00A0 have\u00C2\u00A0 championed\u00C2\u00A0 yeast\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u009CEscherichia\u00C2\u00A0 coli\u00C2\u00A0 of\u00C2\u00A0 eukaryotes\u00E2\u0080\u009D\u00C2\u00A0 (Miklos\u00C2\u00A0and\u00C2\u00A0Rubin\u00C2\u00A01996).\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0combines\u00C2\u00A0 the\u00C2\u00A0ease\u00C2\u00A0of\u00C2\u00A0use\u00C2\u00A0and\u00C2\u00A0brute\u00C2\u00A0 force\u00C2\u00A0genetics\u00C2\u00A0of\u00C2\u00A0bacteria\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 sophistication\u00C2\u00A0and\u00C2\u00A0elegance\u00C2\u00A0of\u00C2\u00A0higher\u00C2\u00A0eukaryotes,\u00C2\u00A0 thus\u00C2\u00A0making\u00C2\u00A0 it\u00C2\u00A0an\u00C2\u00A0 ideal\u00C2\u00A0organism\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 processes\u00C2\u00A0fundamental\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0eukaryotic\u00C2\u00A0cells.\u00C2\u00A0Such\u00C2\u00A0processes,\u00C2\u00A0many\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0conserved\u00C2\u00A0from\u00C2\u00A0yeast\u00C2\u00A0 to\u00C2\u00A0 humans,\u00C2\u00A0 include\u00C2\u00A0 complex\u00C2\u00A0 cell\u00C2\u00A0 cycle\u00C2\u00A0 control,\u00C2\u00A0 eukaryotic\u00C2\u00A0 meiotic\u00C2\u00A0 recombination,\u00C2\u00A0 mitochondrial\u00C2\u00A0 respiration,\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0fusion\u00C2\u00A0events\u00C2\u00A0(Griffiths,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02005).\u00C2\u00A0 \u00C2\u00A0 S.\u00C2\u00A0cerevisiae\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0unicellular\u00C2\u00A0eukaryotic\u00C2\u00A0fungus\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0ubiquitous\u00C2\u00A0to\u00C2\u00A0wineries\u00C2\u00A0worldwide\u00C2\u00A0(Perez\u00E2\u0080\u0090 Ortin,\u00C2\u00A0 Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0 and\u00C2\u00A0 Alberola\u00C2\u00A0 2002).\u00C2\u00A0While\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 assumed\u00C2\u00A0 that\u00C2\u00A0 its\u00C2\u00A0 natural\u00C2\u00A0 environment\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 winery\u00C2\u00A0itself,\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0found\u00C2\u00A0naturally\u00C2\u00A0in\u00C2\u00A0rotting\u00C2\u00A0grapes\u00C2\u00A0and\u00C2\u00A0fruits,\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0addition,\u00C2\u00A0there\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 some\u00C2\u00A0 yet\u00C2\u00A0 undiscovered\u00C2\u00A0 natural\u00C2\u00A0 habitat\u00C2\u00A0 of\u00C2\u00A0 budding\u00C2\u00A0 yeast\u00C2\u00A0 (Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0 Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0 and\u00C2\u00A0 Alberola\u00C2\u00A02002).\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0 feeds\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0sugars\u00C2\u00A0and\u00C2\u00A0nutrients\u00C2\u00A0of\u00C2\u00A0crushed\u00C2\u00A0and\u00C2\u00A0rotting\u00C2\u00A0 fruit\u00C2\u00A0and,\u00C2\u00A0when\u00C2\u00A0 conditions\u00C2\u00A0are\u00C2\u00A0optimal,\u00C2\u00A0reproduce\u00C2\u00A0approximately\u00C2\u00A0every\u00C2\u00A090\u00C2\u00A0minutes\u00C2\u00A0(Herskowitz\u00C2\u00A01988).\u00C2\u00A0Approximately\u00C2\u00A010\u00C2\u00A0 microns\u00C2\u00A0 in\u00C2\u00A0diameter,\u00C2\u00A0 yeast\u00C2\u00A0 reproduce\u00C2\u00A0 asexually\u00C2\u00A0by\u00C2\u00A0mitotic\u00C2\u00A0budding;\u00C2\u00A0however,\u00C2\u00A0 they\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0undergo\u00C2\u00A0 sexual\u00C2\u00A0reproduction\u00C2\u00A0when\u00C2\u00A0haploid\u00C2\u00A0cells\u00C2\u00A0(created\u00C2\u00A0by\u00C2\u00A0meiotic\u00C2\u00A0division)\u00C2\u00A0of\u00C2\u00A0opposite\u00C2\u00A0mating\u00C2\u00A0type\u00C2\u00A0fuse,\u00C2\u00A0thus\u00C2\u00A0 yielding\u00C2\u00A0a\u00C2\u00A0stable\u00C2\u00A0diploid\u00C2\u00A0cell\u00C2\u00A0 (Herskowitz\u00C2\u00A01988).\u00C2\u00A0The\u00C2\u00A0yeast\u00C2\u00A0mating\u00C2\u00A0 types\u00C2\u00A0 (MATa\u00C2\u00A0and\u00C2\u00A0MATalpha)\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0 likened\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0male\u00C2\u00A0and\u00C2\u00A0female\u00C2\u00A0genders.\u00C2\u00A0 \u00C2\u00A0 Wild\u00C2\u00A0isolates\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0also\u00C2\u00A0possess\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0switch\u00C2\u00A0mating\u00C2\u00A0types\u00C2\u00A0such\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0haploid\u00C2\u00A0 population\u00C2\u00A0 of\u00C2\u00A0 one\u00C2\u00A0 mating\u00C2\u00A0 type\u00C2\u00A0 can\u00C2\u00A0 mate\u00C2\u00A0 with\u00C2\u00A0 itself\u00C2\u00A0 and\u00C2\u00A0 achieve\u00C2\u00A0 diploidy\u00C2\u00A0 (Herskowitz\u00C2\u00A0 1988).\u00C2\u00A0 The\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0mating\u00C2\u00A0 type\u00C2\u00A0 switching\u00C2\u00A0 involves\u00C2\u00A0 the\u00C2\u00A0HO\u00C2\u00A0endonuclease\u00C2\u00A0 and\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0well\u00C2\u00A0 characterized\u00C2\u00A0 (Nasmyth\u00C2\u00A01993).\u00C2\u00A0Yeasts\u00C2\u00A0with\u00C2\u00A0this\u00C2\u00A0ability\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0homothallic\u00C2\u00A0and,\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0Darwinian\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0view,\u00C2\u00A0 achieving\u00C2\u00A0diploidy\u00C2\u00A0 is\u00C2\u00A0desirable.\u00C2\u00A0Having\u00C2\u00A0 two\u00C2\u00A0copies\u00C2\u00A0of\u00C2\u00A0every\u00C2\u00A0gene\u00C2\u00A0makes\u00C2\u00A0an\u00C2\u00A0organism\u00C2\u00A0genetically\u00C2\u00A0more\u00C2\u00A0 stable,\u00C2\u00A0 being\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 tolerate\u00C2\u00A0 loss\u00C2\u00A0 of\u00C2\u00A0 function\u00C2\u00A0 \u00E2\u0080\u009Crecessive\u00E2\u0080\u009D\u00C2\u00A0 mutations\u00C2\u00A0 more\u00C2\u00A0 readily\u00C2\u00A0 than\u00C2\u00A0 a\u00C2\u00A0 haploid\u00C2\u00A0 equivalent\u00C2\u00A0(Greig\u00C2\u00A0and\u00C2\u00A0Travisano\u00C2\u00A02003).\u00C2\u00A0However,\u00C2\u00A0the\u00C2\u00A0haploid\u00C2\u00A0state\u00C2\u00A0can\u00C2\u00A0confer\u00C2\u00A0certain\u00C2\u00A0advantages\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 yeast\u00C2\u00A0cell;\u00C2\u00A0a\u00C2\u00A0second\u00C2\u00A0copy\u00C2\u00A0of\u00C2\u00A0every\u00C2\u00A0gene\u00C2\u00A0buffers\u00C2\u00A0deleterious\u00C2\u00A0mutations,\u00C2\u00A0but\u00C2\u00A0it\u00C2\u00A0also\u00C2\u00A0buffers\u00C2\u00A0advantageous\u00C2\u00A0 2\u00C2\u00A0 \u00C2\u00A0 mutations.\u00C2\u00A0Thus,\u00C2\u00A0a\u00C2\u00A0diploid\u00C2\u00A0organism\u00C2\u00A0may,\u00C2\u00A0under\u00C2\u00A0certain\u00C2\u00A0circumstances,\u00C2\u00A0\u00E2\u0080\u0098evolve\u00E2\u0080\u0099\u00C2\u00A0or\u00C2\u00A0\u00E2\u0080\u0098adapt\u00E2\u0080\u0099\u00C2\u00A0to\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0environment\u00C2\u00A0more\u00C2\u00A0slowly\u00C2\u00A0than\u00C2\u00A0its\u00C2\u00A0haploid\u00C2\u00A0counterpart\u00C2\u00A0(Greig\u00C2\u00A0and\u00C2\u00A0Travisano\u00C2\u00A02003).\u00C2\u00A0Nevertheless,\u00C2\u00A0as\u00C2\u00A0 of\u00C2\u00A0present,\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0has\u00C2\u00A0evolved\u00C2\u00A0into\u00C2\u00A0an\u00C2\u00A0organism\u00C2\u00A0which\u00C2\u00A0seems\u00C2\u00A0to\u00C2\u00A0function\u00C2\u00A0and\u00C2\u00A0prosper\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0life\u00C2\u00A0cycle\u00C2\u00A0 stuck\u00C2\u00A0between\u00C2\u00A0its\u00C2\u00A0haploid\u00C2\u00A0prokaryotic\u00C2\u00A0predecessors\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0diploid\u00C2\u00A0eukaryotic\u00C2\u00A0successors.\u00C2\u00A0 \u00C2\u00A0 Like\u00C2\u00A0most\u00C2\u00A0bacteria\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0microorganisms,\u00C2\u00A0yeast\u00C2\u00A0replicate\u00C2\u00A0rapidly\u00C2\u00A0and\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0easily\u00C2\u00A0cultivated\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 laboratory.\u00C2\u00A0They\u00C2\u00A0grown\u00C2\u00A0well\u00C2\u00A0 in\u00C2\u00A0 liquid\u00C2\u00A0 culture\u00C2\u00A0and\u00C2\u00A0on\u00C2\u00A0 solid\u00C2\u00A0media,\u00C2\u00A0and\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0manipulated\u00C2\u00A0 via\u00C2\u00A0 standard\u00C2\u00A0microbiological\u00C2\u00A0 techniques\u00C2\u00A0 (Ausubel,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A02005).\u00C2\u00A0 In\u00C2\u00A0 addition\u00C2\u00A0 to\u00C2\u00A0other\u00C2\u00A0 common\u00C2\u00A0 techniques,\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 remarkably\u00C2\u00A0 amenable\u00C2\u00A0 to\u00C2\u00A0 chemical\u00C2\u00A0or\u00C2\u00A0UV\u00C2\u00A0mutagenesis,\u00C2\u00A0 genetic\u00C2\u00A0 selection,\u00C2\u00A0 recombinant\u00C2\u00A0 DNA\u00C2\u00A0methods,\u00C2\u00A0 rescue\u00C2\u00A0 cloning,\u00C2\u00A0 complementation,\u00C2\u00A0 and\u00C2\u00A0 high\u00C2\u00A0 efficiency\u00C2\u00A0 transformation\u00C2\u00A0 (Griffiths,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2005).\u00C2\u00A0 However,\u00C2\u00A0 the\u00C2\u00A0 real\u00C2\u00A0 value\u00C2\u00A0 of\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 lies\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 yeasts\u00C2\u00A0 incorporate\u00C2\u00A0 all\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 advantages\u00C2\u00A0into\u00C2\u00A0a\u00C2\u00A0eukaryotic\u00C2\u00A0background.\u00C2\u00A0Thus,\u00C2\u00A0fundamental\u00C2\u00A0eukaryotic\u00C2\u00A0processes\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0dissected\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 molecular\u00C2\u00A0level\u00C2\u00A0when\u00C2\u00A0such\u00C2\u00A0analysis\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0exceedingly\u00C2\u00A0difficult\u00C2\u00A0or\u00C2\u00A0impossible\u00C2\u00A0in\u00C2\u00A0higher\u00C2\u00A0eukaryotes.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0full\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0genome\u00C2\u00A0was\u00C2\u00A0published\u00C2\u00A0in\u00C2\u00A01996\u00C2\u00A0(Goffeau,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01996)\u00C2\u00A0and\u00C2\u00A0 this\u00C2\u00A0has\u00C2\u00A0made\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0even\u00C2\u00A0more\u00C2\u00A0powerful\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0organism.\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0was\u00C2\u00A0 in\u00C2\u00A0fact\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0 eukaryotic\u00C2\u00A0organism\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0completely\u00C2\u00A0sequenced\u00C2\u00A0and\u00C2\u00A0subsequent\u00C2\u00A0analysis\u00C2\u00A0has\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0genome\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0common\u00C2\u00A0laboratory\u00C2\u00A0strain\u00C2\u00A0S288C\u00C2\u00A0is\u00C2\u00A0approximately\u00C2\u00A012\u00C2\u00A0Mb\u00C2\u00A0in\u00C2\u00A0size\u00C2\u00A0and\u00C2\u00A0consists\u00C2\u00A0of\u00C2\u00A016\u00C2\u00A0independently\u00C2\u00A0 assorting\u00C2\u00A0chromosomes\u00C2\u00A0(Goffeau,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01996).\u00C2\u00A0It\u00C2\u00A0contains\u00C2\u00A0approximately\u00C2\u00A06000\u00C2\u00A0genes,\u00C2\u00A0~1000\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0 growth\u00C2\u00A0 on\u00C2\u00A0 rich\u00C2\u00A0media\u00C2\u00A0 (Maftahi,\u00C2\u00A0Gaillardin\u00C2\u00A0 and\u00C2\u00A0Nicaud\u00C2\u00A0 1998),\u00C2\u00A0 and\u00C2\u00A0 ~25%\u00C2\u00A0 of\u00C2\u00A0which\u00C2\u00A0 have\u00C2\u00A0 human\u00C2\u00A0 homologues\u00C2\u00A0 (Griffiths,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2005).\u00C2\u00A0 The\u00C2\u00A0 remaining\u00C2\u00A0 5000\u00C2\u00A0 non\u00E2\u0080\u0090essential\u00C2\u00A0 genes\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 systemically\u00C2\u00A0 knocked\u00C2\u00A0 out\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u0098Saccharomyces\u00C2\u00A0 Genome\u00C2\u00A0 Deletion\u00C2\u00A0 project\u00E2\u0080\u0099\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 knock\u00E2\u0080\u0090out\u00C2\u00A0 collection\u00C2\u00A0(Maftahi,\u00C2\u00A0Gaillardin\u00C2\u00A0and\u00C2\u00A0Nicaud\u00C2\u00A01998)\u00C2\u00A0that\u00C2\u00A0allows\u00C2\u00A0for\u00C2\u00A0streamlined\u00C2\u00A0reverse\u00C2\u00A0genetic\u00C2\u00A0analysis.\u00C2\u00A0In\u00C2\u00A0 addition\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0set\u00C2\u00A0of\u00C2\u00A05000\u00C2\u00A0knockouts\u00C2\u00A0available,\u00C2\u00A0the\u00C2\u00A0remaining\u00C2\u00A01000\u00C2\u00A0essential\u00C2\u00A0genes\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0investigated\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 readily\u00C2\u00A0 available\u00C2\u00A0 temperature\u00C2\u00A0 sensitive\u00C2\u00A0 (TS)\u00C2\u00A0 conditional\u00C2\u00A0 alleles\u00C2\u00A0 (Dohmen\u00C2\u00A0 and\u00C2\u00A0 Varshavsky\u00C2\u00A0 2005).\u00C2\u00A0 Finally,\u00C2\u00A0 both\u00C2\u00A0 expression\u00C2\u00A0 and\u00C2\u00A0 tiling\u00C2\u00A0DNA\u00C2\u00A0microarrays\u00C2\u00A0 exist\u00C2\u00A0 for\u00C2\u00A0 various\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 allowing\u00C2\u00A0complex\u00C2\u00A0global\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0genome,\u00C2\u00A0transcriptome\u00C2\u00A0and\u00C2\u00A0proteome\u00C2\u00A0(Dunn,\u00C2\u00A0Levine\u00C2\u00A0and\u00C2\u00A0 Sherlock\u00C2\u00A0 2005;\u00C2\u00A0Hauser,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2001;\u00C2\u00A0 Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0 and\u00C2\u00A0Alberola\u00C2\u00A0 2002;\u00C2\u00A0Rossignol,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003;\u00C2\u00A0Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 3\u00C2\u00A0 \u00C2\u00A0 Of\u00C2\u00A0 the\u00C2\u00A0 6000\u00C2\u00A0 yeast\u00C2\u00A0 genes,\u00C2\u00A0 the\u00C2\u00A0 Saccharomyces\u00C2\u00A0 Genome\u00C2\u00A0 Database\u00C2\u00A0 (SGD\u00C2\u00A0 \u00E2\u0080\u0093\u00C2\u00A0 http://www.yeastgenome.org)\u00C2\u00A0lists\u00C2\u00A0known\u00C2\u00A0functions\u00C2\u00A0or\u00C2\u00A0GO\u00C2\u00A0(Gene\u00C2\u00A0ontology)\u00C2\u00A0annotations\u00C2\u00A0for\u00C2\u00A0about\u00C2\u00A05000\u00C2\u00A0 genes.\u00C2\u00A0 The\u00C2\u00A0 remaining\u00C2\u00A0 1000\u00C2\u00A0 yeast\u00C2\u00A0 genes\u00C2\u00A0 remain\u00C2\u00A0 uncharacterized\u00C2\u00A0 despite\u00C2\u00A0 70\u00C2\u00A0 years\u00C2\u00A0 of\u00C2\u00A0 yeast\u00C2\u00A0 genetics.\u00C2\u00A0 While\u00C2\u00A0some\u00C2\u00A0speculate\u00C2\u00A0that\u00C2\u00A0many\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A01000\u00C2\u00A0genes\u00C2\u00A0(uncharacterized\u00C2\u00A0ORFs)\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0actually\u00C2\u00A0code\u00C2\u00A0for\u00C2\u00A0 functional\u00C2\u00A0protein,\u00C2\u00A0the\u00C2\u00A0general\u00C2\u00A0consensus\u00C2\u00A0 is\u00C2\u00A0that\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0 indeed\u00C2\u00A0functional\u00C2\u00A0 (Pena\u00E2\u0080\u0090Castillo\u00C2\u00A0and\u00C2\u00A0Hughes\u00C2\u00A0 2007).\u00C2\u00A0Upon\u00C2\u00A0in\u00C2\u00A0silico\u00C2\u00A0analysis,\u00C2\u00A0many\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A01000\u00C2\u00A0uncharacterized\u00C2\u00A0genes\u00C2\u00A0contain\u00C2\u00A0putative\u00C2\u00A0protein\u00C2\u00A0domains\u00C2\u00A0 which\u00C2\u00A0suggest\u00C2\u00A0some\u00C2\u00A0sort\u00C2\u00A0of\u00C2\u00A0metabolic\u00C2\u00A0 function\u00C2\u00A0 (Pena\u00E2\u0080\u0090Castillo\u00C2\u00A0and\u00C2\u00A0Hughes\u00C2\u00A02007).\u00C2\u00A0Furthermore,\u00C2\u00A0a\u00C2\u00A0 large\u00C2\u00A0 proportion\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A01000\u00C2\u00A0uncharacterized\u00C2\u00A0genes\u00C2\u00A0are\u00C2\u00A0homologues\u00C2\u00A0to\u00C2\u00A0genes\u00C2\u00A0found\u00C2\u00A0solely\u00C2\u00A0in\u00C2\u00A0other\u00C2\u00A0species\u00C2\u00A0of\u00C2\u00A0 fungi\u00C2\u00A0 (Pena\u00E2\u0080\u0090Castillo\u00C2\u00A0 and\u00C2\u00A0 Hughes\u00C2\u00A0 2007).\u00C2\u00A0 Thus,\u00C2\u00A0 these\u00C2\u00A0 genes\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 important\u00C2\u00A0 in\u00C2\u00A0 aspects\u00C2\u00A0 of\u00C2\u00A0 fungal\u00C2\u00A0 metabolism/physiology,\u00C2\u00A0 and\u00C2\u00A0 thus\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 assay\u00C2\u00A0well\u00C2\u00A0 under\u00C2\u00A0 standard\u00C2\u00A0 laboratory\u00C2\u00A0 conditions.\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 a\u00C2\u00A0 growing\u00C2\u00A0 consensus\u00C2\u00A0 amongst\u00C2\u00A0 yeast\u00C2\u00A0 biologists\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 1000\u00C2\u00A0 dubious\u00C2\u00A0 genes\u00C2\u00A0 will\u00C2\u00A0 never\u00C2\u00A0 become\u00C2\u00A0 characterized\u00C2\u00A0 until\u00C2\u00A0more\u00C2\u00A0 focus\u00C2\u00A0 is\u00C2\u00A0 placed\u00C2\u00A0 on\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 outside\u00C2\u00A0 the\u00C2\u00A0 laboratory\u00C2\u00A0 (Pena\u00E2\u0080\u0090Castillo\u00C2\u00A0 and\u00C2\u00A0 Hughes\u00C2\u00A02007).\u00C2\u00A0This\u00C2\u00A0requires\u00C2\u00A0doing\u00C2\u00A0away\u00C2\u00A0with\u00C2\u00A0 traditional\u00C2\u00A0 laboratory\u00C2\u00A0screens\u00C2\u00A0and\u00C2\u00A0 looking\u00C2\u00A0at\u00C2\u00A0culturing\u00C2\u00A0S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 under\u00C2\u00A0 natural\u00C2\u00A0 conditions,\u00C2\u00A0 most\u00C2\u00A0 notably\u00C2\u00A0 fermentative\u00C2\u00A0 conditions.\u00C2\u00A0 Under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 fermentation,\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 subjected\u00C2\u00A0 to\u00C2\u00A0 profoundly\u00C2\u00A0 different\u00C2\u00A0 stresses\u00C2\u00A0 than\u00C2\u00A0 under\u00C2\u00A0 laboratory\u00C2\u00A0 conditions.\u00C2\u00A0 These\u00C2\u00A0 stresses\u00C2\u00A0 include,\u00C2\u00A0 but\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 limited\u00C2\u00A0 to,\u00C2\u00A0 osmotic\u00C2\u00A0 stress,\u00C2\u00A0 ethanol\u00C2\u00A0 stress,\u00C2\u00A0 nutrient\u00C2\u00A0 limitation,\u00C2\u00A0oxidative\u00C2\u00A0stress,\u00C2\u00A0and\u00C2\u00A0temperature\u00C2\u00A0stress.\u00C2\u00A0If\u00C2\u00A0some\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A01000\u00C2\u00A0uncharacterized\u00C2\u00A0genes\u00C2\u00A0are\u00C2\u00A0vital\u00C2\u00A0 to\u00C2\u00A0 these\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0stress\u00C2\u00A0 responses,\u00C2\u00A0 their\u00C2\u00A0 functions\u00C2\u00A0will\u00C2\u00A0only\u00C2\u00A0be\u00C2\u00A0 revealed\u00C2\u00A0when\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0cultured\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 that\u00C2\u00A0 create\u00C2\u00A0 such\u00C2\u00A0 stresses.\u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0 a\u00C2\u00A0 recent\u00C2\u00A0 study\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 yeast\u00C2\u00A0 transcriptome\u00C2\u00A0 during\u00C2\u00A0 wine\u00C2\u00A0 fermentation\u00C2\u00A0 identified\u00C2\u00A0 a\u00C2\u00A0 previously\u00C2\u00A0 uncharacterized\u00C2\u00A0 fermentation\u00C2\u00A0 stress\u00C2\u00A0 response\u00C2\u00A0 containing\u00C2\u00A0 approximately\u00C2\u00A0223\u00C2\u00A0 genes,\u00C2\u00A0many\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0 are\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A01000\u00C2\u00A0 remaining\u00C2\u00A0uncharacterized\u00C2\u00A0 yeast\u00C2\u00A0genes\u00C2\u00A0(Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02008).\u00C2\u00A0 \u00C2\u00A0 1.2\u00C2\u00A0\u00C2\u00A0Yeast\u00C2\u00A0strains\u00C2\u00A0from\u00C2\u00A0nature\u00C2\u00A0vs.\u00C2\u00A0laboratory\u00C2\u00A0yeasts\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0laboratory\u00C2\u00A0strains\u00C2\u00A0are\u00C2\u00A0descendents\u00C2\u00A0of\u00C2\u00A0isolates\u00C2\u00A0from\u00C2\u00A0nature,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0common\u00C2\u00A0 laboratory\u00C2\u00A0strain\u00C2\u00A0S288C,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0descendent\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0rotten\u00C2\u00A0fig\u00C2\u00A0in\u00C2\u00A0California\u00C2\u00A0in\u00C2\u00A0 1938\u00C2\u00A0 (Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0 Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0 and\u00C2\u00A0 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laboratory\u00C2\u00A0media,\u00C2\u00A0a\u00C2\u00A0wild\u00C2\u00A0strain\u00C2\u00A0could\u00C2\u00A0evolve\u00C2\u00A0such\u00C2\u00A0that\u00C2\u00A0it\u00C2\u00A0no\u00C2\u00A0longer\u00C2\u00A0requires\u00C2\u00A0much\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0genetic\u00C2\u00A0diversity\u00C2\u00A0 4\u00C2\u00A0 \u00C2\u00A0 and\u00C2\u00A0 robustness\u00C2\u00A0needed\u00C2\u00A0 to\u00C2\u00A0deal\u00C2\u00A0with\u00C2\u00A0constantly\u00C2\u00A0changing\u00C2\u00A0environmental\u00C2\u00A0conditions\u00C2\u00A0 (Dunn,\u00C2\u00A0Levine\u00C2\u00A0and\u00C2\u00A0 Sherlock\u00C2\u00A02005).\u00C2\u00A0Without\u00C2\u00A0environmental\u00C2\u00A0pressures\u00C2\u00A0 to\u00C2\u00A0maintain\u00C2\u00A0 robust\u00C2\u00A0pathways\u00C2\u00A0needed\u00C2\u00A0 for\u00C2\u00A0growth\u00C2\u00A0 in\u00C2\u00A0 nature\u00C2\u00A0 (e.g.\u00C2\u00A0 sporulation,\u00C2\u00A0 pseudohyphal\u00C2\u00A0 growth),\u00C2\u00A0 natural\u00C2\u00A0 isolates\u00C2\u00A0 could\u00C2\u00A0 quickly\u00C2\u00A0 become\u00C2\u00A0 homogenized\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 less\u00C2\u00A0 vigorous\u00C2\u00A0 laboratory\u00C2\u00A0 strains\u00C2\u00A0 we\u00C2\u00A0 see\u00C2\u00A0 today.\u00C2\u00A0 As\u00C2\u00A0 such,\u00C2\u00A0 many\u00C2\u00A0 laboratory\u00C2\u00A0 strains\u00C2\u00A0 exhibit\u00C2\u00A0 dramatically\u00C2\u00A0different\u00C2\u00A0transcriptional\u00C2\u00A0profiles\u00C2\u00A0from\u00C2\u00A0wild\u00C2\u00A0yeasts\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0differ\u00C2\u00A0in\u00C2\u00A0their\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0conduct\u00C2\u00A0 robust\u00C2\u00A0 and\u00C2\u00A0 efficient\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentations\u00C2\u00A0 of\u00C2\u00A0 high\u00C2\u00A0 sugar\u00C2\u00A0 grape\u00C2\u00A0 musts\u00C2\u00A0 (Hauser,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2001).\u00C2\u00A0 Additionally,\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0maintain\u00C2\u00A0 stable\u00C2\u00A0 haploid\u00C2\u00A0 populations,\u00C2\u00A0 the\u00C2\u00A0 HO\u00C2\u00A0 locus\u00C2\u00A0 of\u00C2\u00A0 various\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 laboratory\u00C2\u00A0 strains\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 purposely\u00C2\u00A0 disrupted\u00C2\u00A0 (Nasmyth\u00C2\u00A0 1993).\u00C2\u00A0 Other\u00C2\u00A0 important\u00C2\u00A0 differences\u00C2\u00A0 in\u00C2\u00A0 laboratory\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0include\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0various\u00C2\u00A0auxotrophic\u00C2\u00A0markers\u00C2\u00A0which\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0select\u00C2\u00A0 transformants.\u00C2\u00A0 These\u00C2\u00A0 auxotrophic\u00C2\u00A0 markers\u00C2\u00A0 often\u00C2\u00A0 map\u00C2\u00A0 to\u00C2\u00A0 defects\u00C2\u00A0 in\u00C2\u00A0 amino\u00C2\u00A0 acid\u00C2\u00A0 or\u00C2\u00A0 nucleotide\u00C2\u00A0 biosynthetic\u00C2\u00A0pathways;\u00C2\u00A0common\u00C2\u00A0markers\u00C2\u00A0 include\u00C2\u00A0URA3,\u00C2\u00A0TRP1,\u00C2\u00A0LEU2,\u00C2\u00A0ADE2,\u00C2\u00A0etc.\u00C2\u00A0 (Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995;\u00C2\u00A0 Dohmen\u00C2\u00A0and\u00C2\u00A0Varshavsky\u00C2\u00A02005).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0nature,\u00C2\u00A0many\u00C2\u00A0distinct\u00C2\u00A0strains\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0various\u00C2\u00A0environments\u00C2\u00A0 worldwide\u00C2\u00A0(Dunn,\u00C2\u00A0Levine\u00C2\u00A0and\u00C2\u00A0Sherlock\u00C2\u00A02005).\u00C2\u00A0Environments\u00C2\u00A0can\u00C2\u00A0vary\u00C2\u00A0widely\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0nutrient\u00C2\u00A0(carbon,\u00C2\u00A0 nitrogen,\u00C2\u00A0 and\u00C2\u00A0minerals)\u00C2\u00A0 availability,\u00C2\u00A0 temperature,\u00C2\u00A0 osmolarity,\u00C2\u00A0 etc.\u00C2\u00A0 Thus,\u00C2\u00A0while\u00C2\u00A0 fundamentally\u00C2\u00A0 similar,\u00C2\u00A0 each\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0strains\u00C2\u00A0has\u00C2\u00A0adapted,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0yet\u00C2\u00A0undergone\u00C2\u00A0speciation,\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0various\u00C2\u00A0niches\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 environment.\u00C2\u00A0These\u00C2\u00A0adaptations\u00C2\u00A0manifest\u00C2\u00A0themselves\u00C2\u00A0as\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0growth\u00C2\u00A0rate,\u00C2\u00A0fermentation\u00C2\u00A0rate,\u00C2\u00A0 ethanol\u00C2\u00A0production,\u00C2\u00A0and\u00C2\u00A0various\u00C2\u00A0resistances\u00C2\u00A0when\u00C2\u00A0different\u00C2\u00A0strains\u00C2\u00A0are\u00C2\u00A0grown\u00C2\u00A0in\u00C2\u00A0identical\u00C2\u00A0media\u00C2\u00A0(Dunn,\u00C2\u00A0 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homothallic\u00C2\u00A0and\u00C2\u00A0diploid,\u00C2\u00A0polyploid,\u00C2\u00A0or\u00C2\u00A0aneuploid\u00C2\u00A0 (Bond,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02004;\u00C2\u00A0Dunn,\u00C2\u00A0 Levine\u00C2\u00A0and\u00C2\u00A0Sherlock\u00C2\u00A02005;\u00C2\u00A0 Hauser,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02001;\u00C2\u00A0Hughes,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02000;\u00C2\u00A0Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0and\u00C2\u00A0Alberola\u00C2\u00A02002).\u00C2\u00A0Compared\u00C2\u00A0to\u00C2\u00A0 laboratory\u00C2\u00A0 strains,\u00C2\u00A0 wild\u00C2\u00A0 type\u00C2\u00A0 strains\u00C2\u00A0 also\u00C2\u00A0 differ\u00C2\u00A0 in\u00C2\u00A0 chromosome\u00C2\u00A0 length,\u00C2\u00A0 contain\u00C2\u00A0 large\u00C2\u00A0 scale\u00C2\u00A0 (~50\u00C2\u00A0 kb)\u00C2\u00A0 deletions\u00C2\u00A0or\u00C2\u00A0insertions,\u00C2\u00A0contain\u00C2\u00A0many\u00C2\u00A0more\u00C2\u00A0transposons\u00C2\u00A0(Ty\u00C2\u00A0elements),\u00C2\u00A0differ\u00C2\u00A0in\u00C2\u00A0sporulation\u00C2\u00A0rate\u00C2\u00A0(0\u00E2\u0080\u009075%)\u00C2\u00A0 and\u00C2\u00A0spore\u00C2\u00A0viability\u00C2\u00A0(0\u00E2\u0080\u009098%),\u00C2\u00A0exhibit\u00C2\u00A0variable\u00C2\u00A0pseudohyphal\u00C2\u00A0growth,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0largely\u00C2\u00A0heterozygous\u00C2\u00A0(Perez\u00E2\u0080\u0090 Ortin,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0and\u00C2\u00A0Alberola\u00C2\u00A02002).\u00C2\u00A0 5\u00C2\u00A0 \u00C2\u00A0 1.3\u00C2\u00A0\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0and\u00C2\u00A0industry:\u00C2\u00A0Wine\u00C2\u00A0yeasts\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 interaction\u00C2\u00A0between\u00C2\u00A0Homo\u00C2\u00A0sapiens\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0dates\u00C2\u00A0back\u00C2\u00A0almost\u00C2\u00A08000\u00C2\u00A0years\u00C2\u00A0(Perez\u00E2\u0080\u0090 Ortin,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0 and\u00C2\u00A0Alberola\u00C2\u00A0 2002;\u00C2\u00A0 Vine,\u00C2\u00A0Harkness\u00C2\u00A0 and\u00C2\u00A0 Linton\u00C2\u00A0 2002).\u00C2\u00A0 It\u00C2\u00A0was\u00C2\u00A0 first\u00C2\u00A0 reported\u00C2\u00A0 by\u00C2\u00A0 ancient\u00C2\u00A0Egyptian\u00C2\u00A0civilization\u00C2\u00A0that\u00C2\u00A0crushed\u00C2\u00A0grapes\u00C2\u00A0would\u00C2\u00A0\u00E2\u0080\u0098ferment\u00C2\u00A0spontaneously\u00E2\u0080\u0099\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0 wine\u00C2\u00A0contained\u00C2\u00A0 \u00E2\u0080\u0098magical\u00C2\u00A0and\u00C2\u00A0anesthetic\u00C2\u00A0properties\u00E2\u0080\u0099\u00C2\u00A0 (Vine,\u00C2\u00A0Harkness\u00C2\u00A0and\u00C2\u00A0Linton\u00C2\u00A02002).\u00C2\u00A0Since\u00C2\u00A0 that\u00C2\u00A0 time,\u00C2\u00A0 humans\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 selectively\u00C2\u00A0breeding\u00C2\u00A0 the\u00C2\u00A0 then\u00C2\u00A0unknown\u00C2\u00A0microorganism,\u00C2\u00A0S.\u00C2\u00A0cerevisiae,\u00C2\u00A0 for\u00C2\u00A0desirable\u00C2\u00A0 characteristics\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0tolerance\u00C2\u00A0to\u00C2\u00A0high\u00C2\u00A0sugar\u00C2\u00A0stress,\u00C2\u00A0robust\u00C2\u00A0fermentation,\u00C2\u00A0ethanol\u00C2\u00A0tolerance,\u00C2\u00A0and\u00C2\u00A0good\u00C2\u00A0 flavour\u00C2\u00A0production\u00C2\u00A0(Hauser,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02001).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0an\u00C2\u00A0art\u00C2\u00A0 form,\u00C2\u00A0winemaking\u00C2\u00A0 flourished\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0ancient\u00C2\u00A0Mediterranean\u00C2\u00A0and\u00C2\u00A0was\u00C2\u00A0quickly\u00C2\u00A0adopted\u00C2\u00A0 anywhere\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0climate\u00C2\u00A0was\u00C2\u00A0suitable\u00C2\u00A0for\u00C2\u00A0viticulture\u00C2\u00A0(Goode\u00C2\u00A02005;\u00C2\u00A0Vine,\u00C2\u00A0Harkness\u00C2\u00A0and\u00C2\u00A0Linton\u00C2\u00A02002).\u00C2\u00A0 As\u00C2\u00A0a\u00C2\u00A0science,\u00C2\u00A0however,\u00C2\u00A0winemaking\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0understood\u00C2\u00A0until\u00C2\u00A01863;\u00C2\u00A0Louis\u00C2\u00A0Pasteur\u00C2\u00A0was\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0to\u00C2\u00A0isolate\u00C2\u00A0 S.\u00C2\u00A0cerevisiae\u00C2\u00A0and\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0alcohol\u00C2\u00A0(ethanol)\u00C2\u00A0and\u00C2\u00A0carbon\u00C2\u00A0 dioxide\u00C2\u00A0from\u00C2\u00A0simple\u00C2\u00A0sugars\u00C2\u00A0(glucose)\u00C2\u00A0(Figures\u00C2\u00A01\u00C2\u00A0and\u00C2\u00A02)\u00C2\u00A0(Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez\u00C2\u00A0and\u00C2\u00A0Alberola\u00C2\u00A02002;\u00C2\u00A0 Vine,\u00C2\u00A0Harkness\u00C2\u00A0and\u00C2\u00A0Linton\u00C2\u00A02002).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Sugar (glucose or fructose) \u00E2\u0086\u0092 Ethanol + Carbon dioxide + ATP C6H12O6 + 2Pi + 2ADP \u00E2\u0086\u0092 2CH3CH2OH + 2CO2 + 2 ATP (energy released:118 kJ/mol) Figure\u00C2\u00A01.\u00C2\u00A0Chemical\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0 anaerobic\u00C2\u00A0 fermentation.\u00C2\u00A0Under\u00C2\u00A0 anaerobic\u00C2\u00A0 conditions\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 creates\u00C2\u00A0 energy\u00C2\u00A0(ATP)\u00C2\u00A0for\u00C2\u00A0biomass\u00C2\u00A0by\u00C2\u00A0converting\u00C2\u00A0sugar\u00C2\u00A0into\u00C2\u00A0ethanol\u00C2\u00A0and\u00C2\u00A0carbon\u00C2\u00A0dioxide.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6\u00C2\u00A0 \u00C2\u00A0 Glucose Glycolysis 2 ADP 2 ATP 2 NAD+ 2 NADH 2 Ethanol 2 Acetylaldehyde 2 Pyruvate 2 CO2CH3CH2OH CH3(CO)H CH3(CO)COOH \u00C2\u00A0 Today,\u00C2\u00A0no\u00C2\u00A0other\u00C2\u00A0microorganism\u00C2\u00A0 is\u00C2\u00A0 as\u00C2\u00A0 important\u00C2\u00A0 to\u00C2\u00A0human\u00C2\u00A0diet\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 global\u00C2\u00A0economy\u00C2\u00A0 as\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0(Goode\u00C2\u00A02005;\u00C2\u00A0Vine,\u00C2\u00A0Harkness\u00C2\u00A0and\u00C2\u00A0Linton\u00C2\u00A02002).\u00C2\u00A0Fermentation\u00C2\u00A0by\u00C2\u00A0yeast\u00C2\u00A0is\u00C2\u00A0vital\u00C2\u00A0to\u00C2\u00A0winemaking,\u00C2\u00A0 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final\u00C2\u00A0product.\u00C2\u00A0Some\u00C2\u00A0 strains\u00C2\u00A0produce\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 rich\u00C2\u00A0 in\u00C2\u00A0 fruitiness\u00C2\u00A0 and\u00C2\u00A0 has\u00C2\u00A0 high\u00C2\u00A0 acidity,\u00C2\u00A0 while\u00C2\u00A0 others\u00C2\u00A0 produce\u00C2\u00A0 wine\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 milder\u00C2\u00A0 and\u00C2\u00A0 more\u00C2\u00A0 aromatic\u00C2\u00A0 in\u00C2\u00A0 bouquet.\u00C2\u00A0Sake\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0are\u00C2\u00A0amongst\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0popular\u00C2\u00A0and\u00C2\u00A0widely\u00C2\u00A0used\u00C2\u00A0yeasts\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 industry\u00C2\u00A0 (Kodama\u00C2\u00A0 1993;\u00C2\u00A0 Wu,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 The\u00C2\u00A0 process\u00C2\u00A0 of\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentation\u00C2\u00A0 is\u00C2\u00A0 fundamental\u00C2\u00A0 to\u00C2\u00A0 both\u00C2\u00A0 winemaking\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0and\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0subjected\u00C2\u00A0 to\u00C2\u00A0substantial\u00C2\u00A0 temperature,\u00C2\u00A0acid,\u00C2\u00A0hypoxic\u00C2\u00A0 and\u00C2\u00A0ethanol\u00C2\u00A0stresses\u00C2\u00A0during\u00C2\u00A0both\u00C2\u00A0wine\u00C2\u00A0making\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0(Kodama\u00C2\u00A01993;\u00C2\u00A0Rossignol,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02003;\u00C2\u00A0 Wu,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 However,\u00C2\u00A0 one\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 profound\u00C2\u00A0 differences\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 osmotic\u00C2\u00A0 stress\u00C2\u00A0 experienced\u00C2\u00A0by\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0making.\u00C2\u00A0High\u00C2\u00A0ethanol\u00C2\u00A0levels\u00C2\u00A0produced\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0fermentations\u00C2\u00A0 also\u00C2\u00A0exert\u00C2\u00A0significant\u00C2\u00A0ethanol\u00C2\u00A0stress\u00C2\u00A0on\u00C2\u00A0yeast\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Typical\u00C2\u00A0grape\u00C2\u00A0juice\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0complex\u00C2\u00A0mixture\u00C2\u00A0high\u00C2\u00A0in\u00C2\u00A0carbohydrates,\u00C2\u00A0rich\u00C2\u00A0in\u00C2\u00A0assimilable\u00C2\u00A0nitrogen,\u00C2\u00A0and\u00C2\u00A0 high\u00C2\u00A0 in\u00C2\u00A0 vitamin/mineral\u00C2\u00A0 content\u00C2\u00A0 (Ingledew,\u00C2\u00A0Magnus\u00C2\u00A0 and\u00C2\u00A0 Patterson\u00C2\u00A0 1987;\u00C2\u00A0 Rossignol,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003).\u00C2\u00A0On\u00C2\u00A0 average,\u00C2\u00A0 grape\u00C2\u00A0must\u00C2\u00A0 contains\u00C2\u00A0 approximately\u00C2\u00A0 20%\u00C2\u00A0 w/v\u00C2\u00A0 sugar\u00C2\u00A0 (200\u00C2\u00A0 g/L)\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 form\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0mixture\u00C2\u00A0 of\u00C2\u00A0 sucrose,\u00C2\u00A0 glucose\u00C2\u00A0 and\u00C2\u00A0 fructose\u00C2\u00A0 (Rossignol,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003).\u00C2\u00A0 Consequently,\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 start\u00C2\u00A0 of\u00C2\u00A0 grape\u00C2\u00A0 must\u00C2\u00A0 fermentation\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 subject\u00C2\u00A0 to\u00C2\u00A0 substantial\u00C2\u00A0osmotic\u00C2\u00A0 stress.\u00C2\u00A0Although\u00C2\u00A0 yeast\u00C2\u00A0possess\u00C2\u00A0 a\u00C2\u00A0 cell\u00C2\u00A0wall\u00C2\u00A0 composed\u00C2\u00A0 of\u00C2\u00A0 crosslinked\u00C2\u00A0 1,3\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 1,6\u00E2\u0080\u0090glucans\u00C2\u00A0 that\u00C2\u00A0 protects\u00C2\u00A0 them\u00C2\u00A0 against\u00C2\u00A0 osmotic\u00C2\u00A0 forces,\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0must\u00C2\u00A0 induces\u00C2\u00A0 several\u00C2\u00A0other\u00C2\u00A0metabolic\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0 coping\u00C2\u00A0with\u00C2\u00A0 said\u00C2\u00A0 stress\u00C2\u00A0 (Westfall,\u00C2\u00A0Ballon\u00C2\u00A0and\u00C2\u00A0 Thorner\u00C2\u00A02004).\u00C2\u00A0The\u00C2\u00A0primary\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0dealing\u00C2\u00A0with\u00C2\u00A0osmotic\u00C2\u00A0stress\u00C2\u00A0 is\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0high\u00C2\u00A0osmolarity\u00C2\u00A0 growth\u00C2\u00A0(HOG)\u00C2\u00A0pathway\u00C2\u00A0(Reviewed\u00C2\u00A0in\u00C2\u00A0Westfall,\u00C2\u00A0Ballon\u00C2\u00A0and\u00C2\u00A0Thorner\u00C2\u00A02004;\u00C2\u00A0Han,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01994).\u00C2\u00A0This\u00C2\u00A0pathway,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 highly\u00C2\u00A0 conserved\u00C2\u00A0 between\u00C2\u00A0 most\u00C2\u00A0 eukaryotic\u00C2\u00A0 cells,\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 intracellular\u00C2\u00A0small\u00C2\u00A0molecules\u00C2\u00A0which\u00C2\u00A0help\u00C2\u00A0offset\u00C2\u00A0the\u00C2\u00A0osmotic\u00C2\u00A0pressure\u00C2\u00A0difference.\u00C2\u00A0The\u00C2\u00A0principle\u00C2\u00A0molecules\u00C2\u00A0 produced\u00C2\u00A0 in\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 are\u00C2\u00A0 glycerol\u00C2\u00A0 (1,2,3\u00E2\u0080\u0090propanetriol)\u00C2\u00A0 and\u00C2\u00A0 trehalose\u00C2\u00A0 (\u00CE\u00B11,1\u00C2\u00A0 glucose\u00C2\u00A0 disaccharide)\u00C2\u00A0 (Westfall,\u00C2\u00A0Ballon\u00C2\u00A0and\u00C2\u00A0Thorner\u00C2\u00A02004).\u00C2\u00A0Both\u00C2\u00A0molecules\u00C2\u00A0are\u00C2\u00A0produced\u00C2\u00A0after\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0biosynthetic\u00C2\u00A0 genes\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0MAP\u00C2\u00A0 kinase\u00C2\u00A0mediated\u00C2\u00A0 HOG\u00C2\u00A0 pathway,\u00C2\u00A0 which\u00C2\u00A0 occurs\u00C2\u00A0 within\u00C2\u00A0minutes\u00C2\u00A0 of\u00C2\u00A0 osmotic\u00C2\u00A0 stress\u00C2\u00A0 (Westfall,\u00C2\u00A0Ballon\u00C2\u00A0and\u00C2\u00A0Thorner\u00C2\u00A02004).\u00C2\u00A0In\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0high\u00C2\u00A0osmolarity\u00C2\u00A0of\u00C2\u00A0typical\u00C2\u00A0grape\u00C2\u00A0must,\u00C2\u00A0Sake\u00C2\u00A0rice\u00C2\u00A0 10\u00C2\u00A0 \u00C2\u00A0 mash\u00C2\u00A0contains\u00C2\u00A0much\u00C2\u00A0 less\u00C2\u00A0 initial\u00C2\u00A0free\u00C2\u00A0sugar\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0carbon\u00C2\u00A0 is\u00C2\u00A0tied\u00C2\u00A0up\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0 insoluble\u00C2\u00A0 starch\u00C2\u00A0(Kodama\u00C2\u00A01993;\u00C2\u00A0Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.5\u00C2\u00A0\u00C2\u00A0Aspergillus\u00C2\u00A0oryzae\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 possess\u00C2\u00A0 the\u00C2\u00A0 necessary\u00C2\u00A0 amylases\u00C2\u00A0 needed\u00C2\u00A0 to\u00C2\u00A0 convert\u00C2\u00A0 starch\u00C2\u00A0 (\u00CE\u00B21,4\u00C2\u00A0 glucose\u00C2\u00A0polymer)\u00C2\u00A0to\u00C2\u00A0glucose,\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0consume\u00C2\u00A0starch\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0sole\u00C2\u00A0carbon\u00C2\u00A0source\u00C2\u00A0(Kodama\u00C2\u00A0 1993;\u00C2\u00A0 Shobayashi,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2007;\u00C2\u00A0Wu,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 As\u00C2\u00A0 a\u00C2\u00A0 result,\u00C2\u00A0 steamed\u00C2\u00A0 rice\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0 in\u00C2\u00A0 preparation\u00C2\u00A0 for\u00C2\u00A0alcoholic\u00C2\u00A0 fermentation\u00C2\u00A0 (Figure\u00C2\u00A04).\u00C2\u00A0Sake\u00C2\u00A0brewers\u00C2\u00A0have\u00C2\u00A0 long\u00C2\u00A0used\u00C2\u00A0 the\u00C2\u00A0 fungus\u00C2\u00A0Aspergillus\u00C2\u00A0 oryzae\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0source\u00C2\u00A0of\u00C2\u00A0amylases\u00C2\u00A0(Kodama\u00C2\u00A01993;\u00C2\u00A0Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 At\u00C2\u00A0 the\u00C2\u00A0start\u00C2\u00A0of\u00C2\u00A0 fermentation\u00C2\u00A0steamed\u00C2\u00A0 rice\u00C2\u00A0 is\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A0 rice\u00C2\u00A0 that\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 inoculated\u00C2\u00A0with\u00C2\u00A0A.\u00C2\u00A0 oryzae,\u00C2\u00A0traditionally\u00C2\u00A0referred\u00C2\u00A0to\u00C2\u00A0as\u00C2\u00A0\u00E2\u0080\u0098koji\u00E2\u0080\u0099.\u00C2\u00A0Koji\u00C2\u00A0rice\u00C2\u00A0is\u00C2\u00A0rich\u00C2\u00A0in\u00C2\u00A0free\u00C2\u00A0glucose\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0amylases\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0free\u00C2\u00A0 to\u00C2\u00A0act\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0starch\u00C2\u00A0of\u00C2\u00A0freshly\u00C2\u00A0steamed\u00C2\u00A0rice.\u00C2\u00A0As\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0koji,\u00C2\u00A0glucose\u00C2\u00A0 is\u00C2\u00A0fed\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0 fermentation\u00C2\u00A0mixture\u00C2\u00A0 at\u00C2\u00A0 a\u00C2\u00A0 controlled\u00C2\u00A0 rate\u00C2\u00A0 (amylase\u00C2\u00A0 limited)\u00C2\u00A0which\u00C2\u00A0 substantially\u00C2\u00A0 lowers\u00C2\u00A0 the\u00C2\u00A0 osmotic\u00C2\u00A0 stress\u00C2\u00A0experienced\u00C2\u00A0by\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0(Kodama\u00C2\u00A01993;\u00C2\u00A0Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0This\u00C2\u00A0decrease\u00C2\u00A0 in\u00C2\u00A0osmotic\u00C2\u00A0stress\u00C2\u00A0allows\u00C2\u00A0more\u00C2\u00A0energy\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0devoted\u00C2\u00A0to\u00C2\u00A0biomass\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0enables\u00C2\u00A0Sake\u00C2\u00A0fermentations\u00C2\u00A0to\u00C2\u00A0 reach\u00C2\u00A0higher\u00C2\u00A0titres\u00C2\u00A0and\u00C2\u00A0higher\u00C2\u00A0alcohol\u00C2\u00A0concentrations\u00C2\u00A0(Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Takagi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02005;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A02006).\u00C2\u00A0Consequently,\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0 contains\u00C2\u00A0 the\u00C2\u00A0highest\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0of\u00C2\u00A0any\u00C2\u00A0non\u00E2\u0080\u0090distilled\u00C2\u00A0 alcoholic\u00C2\u00A0beverage\u00C2\u00A0(Kodama\u00C2\u00A01993).\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.\u00C2\u00A0Contrasting\u00C2\u00A0processes\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0making.\u00C2\u00A0During\u00C2\u00A0winemaking,\u00C2\u00A0a\u00C2\u00A0saccharification\u00C2\u00A0 step\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0is\u00C2\u00A0unnecessary\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0carbon\u00C2\u00A0source\u00C2\u00A0in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0is\u00C2\u00A0mostly\u00C2\u00A0 glucose\u00C2\u00A0and\u00C2\u00A0fructose.\u00C2\u00A0During\u00C2\u00A0Sake\u00C2\u00A0brewing,\u00C2\u00A0saccharification\u00C2\u00A0must\u00C2\u00A0precede\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0 as\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0cannot\u00C2\u00A0consume\u00C2\u00A0starch\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0carbon\u00C2\u00A0source.\u00C2\u00A0 \u00C2\u00A0 11\u00C2\u00A0 \u00C2\u00A0 1.6\u00C2\u00A0\u00C2\u00A0Yeast\u00C2\u00A0nitrogen\u00C2\u00A0metabolism\u00C2\u00A0during\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0build\u00C2\u00A0significant\u00C2\u00A0biomass\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0conduct\u00C2\u00A0an\u00C2\u00A0efficient\u00C2\u00A0alcoholic\u00C2\u00A0fermentation,\u00C2\u00A0yeast\u00C2\u00A0 cells\u00C2\u00A0require\u00C2\u00A0significant\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0nitrogen.\u00C2\u00A0 \u00C2\u00A0Free\u00C2\u00A0nitrogen,\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 form\u00C2\u00A0of\u00C2\u00A0ammonia,\u00C2\u00A0 is\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0many\u00C2\u00A0 anabolic\u00C2\u00A0pathways,\u00C2\u00A0while\u00C2\u00A0peptides\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0are\u00C2\u00A0either\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0cellular\u00C2\u00A0processes\u00C2\u00A0directly,\u00C2\u00A0or\u00C2\u00A0 are\u00C2\u00A0broken\u00C2\u00A0down\u00C2\u00A0to\u00C2\u00A0ammonia,\u00C2\u00A0glutamate,\u00C2\u00A0and\u00C2\u00A0glutamine\u00C2\u00A0via\u00C2\u00A0catabolic\u00C2\u00A0pathways\u00C2\u00A0(Cooper\u00C2\u00A02002;\u00C2\u00A0Hofman\u00E2\u0080\u0090 Bang\u00C2\u00A01999).\u00C2\u00A0The\u00C2\u00A0principle\u00C2\u00A0nitrogen\u00C2\u00A0source\u00C2\u00A0present\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0 is\u00C2\u00A0arginine,\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0 the\u00C2\u00A0metabolism\u00C2\u00A0 leads\u00C2\u00A0directly\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0 intracellular\u00C2\u00A0urea\u00C2\u00A0(Figure\u00C2\u00A05)\u00C2\u00A0(Monteiro\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01991).\u00C2\u00A0Urea\u00C2\u00A0forms\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0arginase\u00C2\u00A0 (CAR1\u00E2\u0080\u0090\u00C2\u00A0EC\u00C2\u00A03.5.3.1)\u00C2\u00A0dependent\u00C2\u00A0breakdown\u00C2\u00A0of\u00C2\u00A0arginine\u00C2\u00A0to\u00C2\u00A0ornithine\u00C2\u00A0and\u00C2\u00A0urea\u00C2\u00A0 (Cooper\u00C2\u00A0 1982).\u00C2\u00A0At\u00C2\u00A0high\u00C2\u00A0concentrations,\u00C2\u00A0urea\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0toxic\u00C2\u00A0and\u00C2\u00A0poor\u00C2\u00A0nitrogen\u00C2\u00A0source\u00C2\u00A0for\u00C2\u00A0S.\u00C2\u00A0cerevisiae,\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0therefore\u00C2\u00A0 exported\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0surrounding\u00C2\u00A0medium\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0possesses\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0degrade\u00C2\u00A0 urea\u00C2\u00A0via\u00C2\u00A0urea\u00C2\u00A0amidolyase\u00C2\u00A0(DUR1,2\u00C2\u00A0\u00E2\u0080\u0090\u00C2\u00A0EC\u00C2\u00A03.5.1.54)\u00C2\u00A0(Genbauffe\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A01991);\u00C2\u00A0however,\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0 of\u00C2\u00A0higher\u00C2\u00A0quality\u00C2\u00A0nitrogen\u00C2\u00A0sources,\u00C2\u00A0DUR1,2\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0repressed\u00C2\u00A0while\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0exporter\u00C2\u00A0 (DUR4)\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 (Whitney,\u00C2\u00A0 Cooper\u00C2\u00A0 and\u00C2\u00A0Magasanik\u00C2\u00A0 1973).\u00C2\u00A0 Consequently,\u00C2\u00A0 as\u00C2\u00A0 long\u00C2\u00A0 as\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 starved\u00C2\u00A0 for\u00C2\u00A0 nitrogen,\u00C2\u00A0which\u00C2\u00A0 forces\u00C2\u00A0 them\u00C2\u00A0 to\u00C2\u00A0 degrade\u00C2\u00A0 urea,\u00C2\u00A0 they\u00C2\u00A0will\u00C2\u00A0 preferentially\u00C2\u00A0 export\u00C2\u00A0 urea\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 Figure\u00C2\u00A05\u00C2\u00A0Overview\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0metabolism\u00C2\u00A0 in\u00C2\u00A0S.\u00C2\u00A0cerevisiae.\u00C2\u00A0Arginine\u00C2\u00A0 is\u00C2\u00A0 imported\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0either\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 Arginine\u00C2\u00A0 specific\u00C2\u00A0 transporter\u00C2\u00A0 CAN1\u00C2\u00A0 or\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 general\u00C2\u00A0 amino\u00C2\u00A0 acid\u00C2\u00A0 permease\u00C2\u00A0 GAP1.\u00C2\u00A0 Following\u00C2\u00A0 import\u00C2\u00A0 arginine\u00C2\u00A0is\u00C2\u00A0degraded\u00C2\u00A0to\u00C2\u00A0ornithine\u00C2\u00A0and\u00C2\u00A0urea\u00C2\u00A0by\u00C2\u00A0arginase,\u00C2\u00A0the\u00C2\u00A0product\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0CAR1\u00C2\u00A0gene.\u00C2\u00A0Urea\u00C2\u00A0can\u00C2\u00A0either\u00C2\u00A0be\u00C2\u00A0 exported\u00C2\u00A0by\u00C2\u00A0DUR4\u00C2\u00A0or\u00C2\u00A0degraded\u00C2\u00A0to\u00C2\u00A0ammonia\u00C2\u00A0and\u00C2\u00A0carbon\u00C2\u00A0dioxide\u00C2\u00A0by\u00C2\u00A0DUR1,2.\u00C2\u00A0Cells\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0reabsorb\u00C2\u00A0urea\u00C2\u00A0 through\u00C2\u00A0the\u00C2\u00A0importer\u00C2\u00A0DUR3.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0(2006).\u00C2\u00A0 \u00C2\u00A0 12\u00C2\u00A0 \u00C2\u00A0 extracellular\u00C2\u00A0environment.\u00C2\u00A0 If,\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 later\u00C2\u00A0 stages\u00C2\u00A0of\u00C2\u00A0 fermentation,\u00C2\u00A0 yeast\u00C2\u00A0become\u00C2\u00A0 starved\u00C2\u00A0 for\u00C2\u00A0nitrogen,\u00C2\u00A0 urea\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 reabsorbed\u00C2\u00A0 (DUR3\u00C2\u00A0 \u00E2\u0080\u0090\u00C2\u00A0 TC\u00C2\u00A0 2.A.21.6)\u00C2\u00A0 (Cooper\u00C2\u00A0 and\u00C2\u00A0 Sumrada\u00C2\u00A0 1975;\u00C2\u00A0 ElBerry,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1993)\u00C2\u00A0 and\u00C2\u00A0 metabolized;\u00C2\u00A0however,\u00C2\u00A0finished\u00C2\u00A0wines\u00C2\u00A0made\u00C2\u00A0from\u00C2\u00A0grape\u00C2\u00A0varietals\u00C2\u00A0with\u00C2\u00A0high\u00C2\u00A0assimilable\u00C2\u00A0nitrogen\u00C2\u00A0tend\u00C2\u00A0to\u00C2\u00A0 possess\u00C2\u00A0 significant\u00C2\u00A0 residual\u00C2\u00A0urea\u00C2\u00A0 (Ough,\u00C2\u00A0Crowell\u00C2\u00A0and\u00C2\u00A0Gutlove\u00C2\u00A01988;\u00C2\u00A0Ough,\u00C2\u00A0Crowell\u00C2\u00A0and\u00C2\u00A0Mooney\u00C2\u00A01988;\u00C2\u00A0 Ough,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01990;\u00C2\u00A0Ough,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01991).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.7\u00C2\u00A0Nitrogen\u00C2\u00A0metabolism\u00C2\u00A0and\u00C2\u00A0Nitrogen\u00C2\u00A0Catabolite\u00C2\u00A0Repression\u00C2\u00A0(NCR)\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0discriminate\u00C2\u00A0between\u00C2\u00A0various\u00C2\u00A0nutrient\u00C2\u00A0sources\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0 important\u00C2\u00A0and\u00C2\u00A0evolutionarily\u00C2\u00A0 conserved\u00C2\u00A0theme\u00C2\u00A0 in\u00C2\u00A0biology.\u00C2\u00A0Prokaryotes\u00C2\u00A0and\u00C2\u00A0eukaryotes\u00C2\u00A0alike\u00C2\u00A0exhibit\u00C2\u00A0exquisite\u00C2\u00A0regulatory\u00C2\u00A0control\u00C2\u00A0over\u00C2\u00A0 their\u00C2\u00A0 metabolic\u00C2\u00A0 pathways\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 exist\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 energetically\u00C2\u00A0 efficient\u00C2\u00A0 way\u00C2\u00A0 possible.\u00C2\u00A0 Such\u00C2\u00A0 regulatory\u00C2\u00A0systems\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0referred\u00C2\u00A0to\u00C2\u00A0as\u00C2\u00A0\u00E2\u0080\u0098catabolite\u00C2\u00A0repression\u00E2\u0080\u0099\u00C2\u00A0systems\u00C2\u00A0because\u00C2\u00A0they\u00C2\u00A0repress\u00C2\u00A0genes\u00C2\u00A0 necessary\u00C2\u00A0to\u00C2\u00A0metabolize\u00C2\u00A0a\u00C2\u00A0particular\u00C2\u00A0nutrient\u00C2\u00A0source\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0more\u00C2\u00A0favored\u00C2\u00A0one\u00C2\u00A0(Griffiths,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A02005).\u00C2\u00A0Catabolite\u00C2\u00A0repression\u00C2\u00A0systems\u00C2\u00A0occur\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0various\u00C2\u00A0carbon\u00C2\u00A0(Bruckner\u00C2\u00A0and\u00C2\u00A0Titgemeyer\u00C2\u00A02002;\u00C2\u00A0 Gancedo\u00C2\u00A0 1992)\u00C2\u00A0 and\u00C2\u00A0 nitrogen\u00C2\u00A0 sources\u00C2\u00A0 (Cooper\u00C2\u00A0 2002;\u00C2\u00A0 Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0 1999;\u00C2\u00A0 Salmon\u00C2\u00A0 and\u00C2\u00A0 Barre\u00C2\u00A0 1998).\u00C2\u00A0 Particularly\u00C2\u00A0well\u00C2\u00A0 characterized\u00C2\u00A0 examples\u00C2\u00A0 of\u00C2\u00A0 carbon\u00C2\u00A0 catabolite\u00C2\u00A0 repression\u00C2\u00A0 systems\u00C2\u00A0 include\u00C2\u00A0 the\u00C2\u00A0 lactose\u00C2\u00A0 (lac)\u00C2\u00A0operon\u00C2\u00A0 in\u00C2\u00A0E.\u00C2\u00A0coli\u00C2\u00A0(Griffiths,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02005)\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0galactose\u00C2\u00A0(GAL)\u00C2\u00A0genes\u00C2\u00A0 in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0(Lohr,\u00C2\u00A0 Venkov\u00C2\u00A0 and\u00C2\u00A0 Zlatanova\u00C2\u00A0 1995).\u00C2\u00A0 In\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 nitrogen\u00C2\u00A0 catabolite\u00C2\u00A0 repression\u00C2\u00A0 both\u00C2\u00A0 prokaryotes\u00C2\u00A0 and\u00C2\u00A0 eukaryotes\u00C2\u00A0 utilize\u00C2\u00A0 a\u00C2\u00A0 more\u00C2\u00A0 global\u00C2\u00A0 repression\u00C2\u00A0 system\u00C2\u00A0 that\u00C2\u00A0 encompasses\u00C2\u00A0 many\u00C2\u00A0 different\u00C2\u00A0 catabolic\u00C2\u00A0 pathways.\u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0nitrogen\u00C2\u00A0utilization\u00C2\u00A0 is\u00C2\u00A0centered\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0usage\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0glutamate\u00C2\u00A0and\u00C2\u00A0glutamine\u00C2\u00A0 (Hofman\u00E2\u0080\u0090 Bang\u00C2\u00A01999).\u00C2\u00A0From\u00C2\u00A0glutamate\u00C2\u00A0and\u00C2\u00A0glutamine,\u00C2\u00A0wild\u00C2\u00A0type\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0can\u00C2\u00A0synthesize\u00C2\u00A0any\u00C2\u00A0other\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0 necessary\u00C2\u00A0 and\u00C2\u00A0 thus,\u00C2\u00A0 glutamate\u00C2\u00A0 and\u00C2\u00A0 glutamine,\u00C2\u00A0 along\u00C2\u00A0with\u00C2\u00A0 ammonia,\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 nitrogen\u00C2\u00A0 sources\u00C2\u00A0most\u00C2\u00A0 preferred\u00C2\u00A0by\u00C2\u00A0yeast\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0 \u00C2\u00A0 Due\u00C2\u00A0to\u00C2\u00A0 its\u00C2\u00A0diverse\u00C2\u00A0repertoire\u00C2\u00A0of\u00C2\u00A0nitrogen\u00C2\u00A0catabolic\u00C2\u00A0pathways,\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0can\u00C2\u00A0grow\u00C2\u00A0solely\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 wide\u00C2\u00A0 range\u00C2\u00A0 of\u00C2\u00A0 nitrogenous\u00C2\u00A0 compounds\u00C2\u00A0 (e.g.\u00C2\u00A0 common\u00C2\u00A0 and\u00C2\u00A0 uncommon\u00C2\u00A0 amino\u00C2\u00A0 acids,\u00C2\u00A0 urea,\u00C2\u00A0 GABA,\u00C2\u00A0 allophanate,\u00C2\u00A0allantoin),\u00C2\u00A0as\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0converted\u00C2\u00A0 in\u00C2\u00A0 to\u00C2\u00A0glutamate,\u00C2\u00A0glutamine,\u00C2\u00A0and\u00C2\u00A0ammonia\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0 1999).\u00C2\u00A0However,\u00C2\u00A0 because\u00C2\u00A0 each\u00C2\u00A0 non\u00E2\u0080\u0090optimal\u00C2\u00A0 nitrogenous\u00C2\u00A0 compound\u00C2\u00A0 varies\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 ease\u00C2\u00A0 of\u00C2\u00A0 import\u00C2\u00A0 and\u00C2\u00A0 degradation,\u00C2\u00A0 the\u00C2\u00A0 various\u00C2\u00A0 nitrogen\u00C2\u00A0 sources\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 ranked\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 quality\u00C2\u00A0 or\u00C2\u00A0 13\u00C2\u00A0 \u00C2\u00A0 preference\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0Furthermore,\u00C2\u00A0since\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0advantageous\u00C2\u00A0to\u00C2\u00A0utilize\u00C2\u00A0higher\u00C2\u00A0quality\u00C2\u00A0sources\u00C2\u00A0 preferentially,\u00C2\u00A0and\u00C2\u00A0because\u00C2\u00A0yeast\u00C2\u00A0posses\u00C2\u00A0a\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0system\u00C2\u00A0for\u00C2\u00A0nitrogen\u00C2\u00A0sources,\u00C2\u00A0the\u00C2\u00A0various\u00C2\u00A0 compounds\u00C2\u00A0 can\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0 ranked\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0 strength\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0A\u00C2\u00A0 few\u00C2\u00A0 common\u00C2\u00A0yeast\u00C2\u00A0 nitrogen\u00C2\u00A0sources\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A01.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A01.\u00C2\u00A0Ranking\u00C2\u00A0of\u00C2\u00A0various\u00C2\u00A0yeast\u00C2\u00A0nitrogen\u00C2\u00A0sources\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0NCR\u00C2\u00A0repression\u00C2\u00A0strength.\u00C2\u00A0Low\u00C2\u00A0repression\u00C2\u00A0 strength\u00C2\u00A0indicates\u00C2\u00A0a\u00C2\u00A0poor\u00C2\u00A0nitrogen\u00C2\u00A0source.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0(1999).\u00C2\u00A0 \u00C2\u00A0 NCR\u00C2\u00A0repression\u00C2\u00A0under\u00C2\u00A0 growth\u00C2\u00A0in\u00C2\u00A0listed\u00C2\u00A0media\u00C2\u00A0 (Low\u00C2\u00A0to\u00C2\u00A0High)\u00C2\u00A0 Nitrogen\u00C2\u00A0Source\u00C2\u00A0 1\u00C2\u00A0 Proline\u00C2\u00A0 2\u00C2\u00A0 GABA\u00C2\u00A0 3\u00C2\u00A0 Urea\u00C2\u00A0 4\u00C2\u00A0 Glutamate\u00C2\u00A0 5\u00C2\u00A0 Ammonium\u00C2\u00A0 6\u00C2\u00A0 Asparagine/Glutamine\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0global\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0system\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0well\u00C2\u00A0studied\u00C2\u00A0but\u00C2\u00A0is\u00C2\u00A0far\u00C2\u00A0 from\u00C2\u00A0being\u00C2\u00A0understood\u00C2\u00A0 in\u00C2\u00A0absolute\u00C2\u00A0detail\u00C2\u00A0 (Reviewed\u00C2\u00A0 in\u00C2\u00A0Cooper\u00C2\u00A02002;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0At\u00C2\u00A0 its\u00C2\u00A0core,\u00C2\u00A0 the\u00C2\u00A0NCR\u00C2\u00A0system\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0makes\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0four\u00C2\u00A0known\u00C2\u00A0regulatory\u00C2\u00A0transcription\u00C2\u00A0factors\u00C2\u00A0(GLN3,\u00C2\u00A0GAT1,\u00C2\u00A0 DAL80,\u00C2\u00A0 and\u00C2\u00A0DEH1)\u00C2\u00A0 to\u00C2\u00A0 control\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 all\u00C2\u00A0NCR\u00C2\u00A0 sensitive\u00C2\u00A0 genes\u00C2\u00A0 (Cooper\u00C2\u00A0 2002;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0 1999).\u00C2\u00A0 Two\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 factors,\u00C2\u00A0GLN3\u00C2\u00A0 and\u00C2\u00A0GAT1,\u00C2\u00A0 are\u00C2\u00A0 positive\u00C2\u00A0 regulators\u00C2\u00A0 (activators),\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 two\u00C2\u00A0 factors,\u00C2\u00A0DAL80\u00C2\u00A0and\u00C2\u00A0DEH1,\u00C2\u00A0are\u00C2\u00A0negative\u00C2\u00A0regulators\u00C2\u00A0(repressors)\u00C2\u00A0(Cooper\u00C2\u00A02002;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0The\u00C2\u00A0 complex\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0four\u00C2\u00A0transcription\u00C2\u00A0factors,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0various\u00C2\u00A0 inducers\u00C2\u00A0and\u00C2\u00A0repressors,\u00C2\u00A0at\u00C2\u00A0NCR\u00C2\u00A0 sensitive\u00C2\u00A0promoters\u00C2\u00A0allows\u00C2\u00A0for\u00C2\u00A0highly\u00C2\u00A0regulated\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0(Figures\u00C2\u00A06\u00C2\u00A0and\u00C2\u00A07).\u00C2\u00A0 \u00C2\u00A0 14\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.\u00C2\u00A0Model\u00C2\u00A0of\u00C2\u00A0reciprocal\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0GATA\u00C2\u00A0factor\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0GATA\u00C2\u00A0factor\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0 NCR\u00E2\u0080\u0090sensitive\u00C2\u00A0gene\u00C2\u00A0expression.\u00C2\u00A0Dashed\u00C2\u00A0lines\u00C2\u00A0indicate\u00C2\u00A0weak\u00C2\u00A0association/regulation.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Cooper\u00C2\u00A0 (2002).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 7.\u00C2\u00A0 Permeases\u00C2\u00A0 and\u00C2\u00A0 degradative\u00C2\u00A0 enzymes\u00C2\u00A0 needed\u00C2\u00A0 to\u00C2\u00A0 utilize\u00C2\u00A0 poor\u00C2\u00A0 nitrogen\u00C2\u00A0 sources\u00C2\u00A0 are\u00C2\u00A0 transcriptionally\u00C2\u00A0 silenced\u00C2\u00A0 during\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 abundant\u00C2\u00A0 high\u00C2\u00A0 quality\u00C2\u00A0 nitrogen\u00C2\u00A0 sources.\u00C2\u00A0 Adapted\u00C2\u00A0 from\u00C2\u00A0 Cooper\u00C2\u00A0(2002).\u00C2\u00A0 \u00C2\u00A0 15\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0four\u00C2\u00A0NCR\u00C2\u00A0transcription\u00C2\u00A0factors\u00C2\u00A0are\u00C2\u00A0referred\u00C2\u00A0to\u00C2\u00A0as\u00C2\u00A0GATA\u00C2\u00A0factors\u00C2\u00A0because\u00C2\u00A0they\u00C2\u00A0all\u00C2\u00A0exert\u00C2\u00A0their\u00C2\u00A0 effect\u00C2\u00A0 though\u00C2\u00A0 the\u00C2\u00A0 DNA\u00C2\u00A0 consensus\u00C2\u00A0 sequence\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090GATAA\u00E2\u0080\u00903\u00E2\u0080\u0099.\u00C2\u00A0 Each\u00C2\u00A0 factor\u00C2\u00A0 binds\u00C2\u00A0 DNA\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 GATA\u00C2\u00A0 site\u00C2\u00A0 through\u00C2\u00A0a\u00C2\u00A0conserved\u00C2\u00A0zinc\u00C2\u00A0finger\u00C2\u00A0binding\u00C2\u00A0domain\u00C2\u00A0and\u00C2\u00A0each\u00C2\u00A0factor\u00C2\u00A0is\u00C2\u00A0homologous\u00C2\u00A0to\u00C2\u00A0many\u00C2\u00A0other\u00C2\u00A0zinc\u00C2\u00A0finger\u00C2\u00A0 proteins\u00C2\u00A0found\u00C2\u00A0 in\u00C2\u00A0higher\u00C2\u00A0eukaryotes,\u00C2\u00A0 including\u00C2\u00A0mammals\u00C2\u00A0(Bysani,\u00C2\u00A0Daugherty\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A01991;\u00C2\u00A0Cooper\u00C2\u00A0 2002;\u00C2\u00A0Cox,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02000;\u00C2\u00A0Cox,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02004;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999;\u00C2\u00A0van\u00C2\u00A0Vuuren,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01991).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0four\u00C2\u00A0NCR\u00C2\u00A0GATA\u00C2\u00A0factors\u00C2\u00A0are\u00C2\u00A0controlled\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0upstream\u00C2\u00A0negative\u00C2\u00A0regulator\u00C2\u00A0URE2,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0in\u00C2\u00A0 turn\u00C2\u00A0regulated\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0TOR\u00C2\u00A0pathway\u00C2\u00A0(Target\u00C2\u00A0of\u00C2\u00A0Rapamycin)\u00C2\u00A0(Cooper\u00C2\u00A02002;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0The\u00C2\u00A0TOR\u00C2\u00A0 pathway,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 conserved\u00C2\u00A0 throughout\u00C2\u00A0most\u00C2\u00A0eukaryotic\u00C2\u00A0organisms,\u00C2\u00A0acts\u00C2\u00A0a\u00C2\u00A0master\u00C2\u00A0 regulatory\u00C2\u00A0 sensor\u00C2\u00A0 and\u00C2\u00A0signal\u00C2\u00A0transduction\u00C2\u00A0cascade\u00C2\u00A0that\u00C2\u00A0assesses\u00C2\u00A0and\u00C2\u00A0responds,\u00C2\u00A0with\u00C2\u00A0highly\u00C2\u00A0pleiotropic\u00C2\u00A0effects,\u00C2\u00A0to\u00C2\u00A0general\u00C2\u00A0 cell\u00C2\u00A0 health,\u00C2\u00A0 nutrient\u00C2\u00A0 availability,\u00C2\u00A0 and\u00C2\u00A0 growth\u00C2\u00A0 (Cooper\u00C2\u00A0 2002;\u00C2\u00A0Dann\u00C2\u00A0 and\u00C2\u00A0 Thomas\u00C2\u00A0 2006;\u00C2\u00A0De\u00C2\u00A0 Virgilio\u00C2\u00A0 and\u00C2\u00A0 Loewith\u00C2\u00A02006).\u00C2\u00A0The\u00C2\u00A0TOR\u00C2\u00A0pathway\u00C2\u00A0 \u00C2\u00A0acts\u00C2\u00A0primarily\u00C2\u00A0 through\u00C2\u00A0 two\u00C2\u00A0kinases,\u00C2\u00A0TOR1\u00C2\u00A0and\u00C2\u00A0TOR2,\u00C2\u00A0and\u00C2\u00A0 regulates\u00C2\u00A0 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GLN3p\u00C2\u00A0localization\u00C2\u00A0correlates\u00C2\u00A0highly\u00C2\u00A0with\u00C2\u00A0active\u00C2\u00A0transcription\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0regulated\u00C2\u00A0genes\u00C2\u00A0 b. NCR\u00C2\u00A0regulated\u00C2\u00A0genes\u00C2\u00A0are\u00C2\u00A0largely\u00C2\u00A0activated\u00C2\u00A0when\u00C2\u00A0GLN3p\u00C2\u00A0is\u00C2\u00A0nuclear\u00C2\u00A0 c. URE2p\u00C2\u00A0complexes\u00C2\u00A0with\u00C2\u00A0GLN3p\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0complex\u00C2\u00A0localizes\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cytoplasm\u00C2\u00A0 d. Inhibition\u00C2\u00A0of\u00C2\u00A0TOR1/2p\u00C2\u00A0correlates\u00C2\u00A0highly\u00C2\u00A0with\u00C2\u00A0decreased\u00C2\u00A0GLN3p\u00C2\u00A0phosphorylation,\u00C2\u00A0\u00C2\u00A0which\u00C2\u00A0in\u00C2\u00A0turn\u00C2\u00A0 correlates\u00C2\u00A0with\u00C2\u00A0GLN3p\u00C2\u00A0being\u00C2\u00A0nuclear\u00C2\u00A0and\u00C2\u00A0NCR\u00C2\u00A0regulated\u00C2\u00A0genes\u00C2\u00A0being\u00C2\u00A0expressed\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0most\u00C2\u00A0commonly\u00C2\u00A0accepted\u00C2\u00A0model\u00C2\u00A0of\u00C2\u00A0TOR1/2\u00C2\u00A0control\u00C2\u00A0on\u00C2\u00A0NCR\u00C2\u00A0 is\u00C2\u00A0depicted\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A08.\u00C2\u00A0 In\u00C2\u00A0this\u00C2\u00A0 model\u00C2\u00A0 TOR1/2\u00C2\u00A0 keep\u00C2\u00A0NCR\u00C2\u00A0 genes\u00C2\u00A0 repressed\u00C2\u00A0by\u00C2\u00A0 inhibiting\u00C2\u00A0 a\u00C2\u00A0phosphatase\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0necessary\u00C2\u00A0 for\u00C2\u00A0GLN3p\u00C2\u00A0 dephosphorylation\u00C2\u00A0and\u00C2\u00A0nuclear\u00C2\u00A0import.\u00C2\u00A0 \u00C2\u00A0 16\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 8.\u00C2\u00A0Model\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 regulatory\u00C2\u00A0 pathway\u00C2\u00A0 by\u00C2\u00A0which\u00C2\u00A0 rapamycin\u00C2\u00A0 and\u00C2\u00A0 nitrogen\u00C2\u00A0 starvation\u00C2\u00A0 induce\u00C2\u00A0NCR\u00C2\u00A0 regulated\u00C2\u00A0gene\u00C2\u00A0expression.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Cooper\u00C2\u00A0(2002).\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.8\u00C2\u00A0\u00C2\u00A0Urea\u00C2\u00A0and\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0(EC)\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0excretion\u00C2\u00A0of\u00C2\u00A0non\u00E2\u0080\u0090metabolized\u00C2\u00A0urea\u00C2\u00A0into\u00C2\u00A0wine\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0major\u00C2\u00A0factor\u00C2\u00A0involved\u00C2\u00A0in\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 compound\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0(EC)\u00C2\u00A0(Monteiro\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01991).\u00C2\u00A0\u00C2\u00A0Under\u00C2\u00A0ambient\u00C2\u00A0conditions\u00C2\u00A0(wine\u00C2\u00A0storage),\u00C2\u00A0 ethanol\u00C2\u00A0 reacts\u00C2\u00A0 with\u00C2\u00A0 carbamyl\u00C2\u00A0 compounds\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 fermented\u00C2\u00A0 beverages\u00C2\u00A0 to\u00C2\u00A0 form\u00C2\u00A0 EC\u00C2\u00A0 (Ingledew,\u00C2\u00A0 Magnus\u00C2\u00A0 and\u00C2\u00A0 Patterson\u00C2\u00A0 1987;\u00C2\u00A0 Kodama,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1994;\u00C2\u00A0Ough,\u00C2\u00A0 Crowell\u00C2\u00A0 and\u00C2\u00A0 Gutlove\u00C2\u00A0 1988)\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 time\u00C2\u00A0 and\u00C2\u00A0 temperature\u00C2\u00A0dependent\u00C2\u00A0manner\u00C2\u00A0 (Figure\u00C2\u00A09).\u00C2\u00A0Potentially\u00C2\u00A0reactive\u00C2\u00A0carbamyl\u00C2\u00A0compounds\u00C2\u00A0 include\u00C2\u00A0citrulline\u00C2\u00A0 and\u00C2\u00A0carbamyl\u00C2\u00A0phosphate,\u00C2\u00A0which\u00C2\u00A0result\u00C2\u00A0from\u00C2\u00A0arginine\u00C2\u00A0and\u00C2\u00A0nucleotide\u00C2\u00A0metabolism,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0urea\u00C2\u00A0(Ough,\u00C2\u00A0 Crowell\u00C2\u00A0and\u00C2\u00A0Gutlove\u00C2\u00A01988).\u00C2\u00A0\u00C2\u00A0 17\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.9\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem\u00C2\u00A0 \u00C2\u00A0 1.9.1\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0History\u00C2\u00A0 \u00C2\u00A0 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liver,\u00C2\u00A0 lung,\u00C2\u00A0and\u00C2\u00A0harderian\u00C2\u00A0gland\u00C2\u00A0 cancers\u00C2\u00A0 in\u00C2\u00A0 male\u00C2\u00A0and\u00C2\u00A0female\u00C2\u00A0mice.\u00C2\u00A0Moreover,\u00C2\u00A0 incidence\u00C2\u00A0of\u00C2\u00A0mammary\u00C2\u00A0and\u00C2\u00A0ovarian\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0skin\u00C2\u00A0and\u00C2\u00A0forestomach\u00C2\u00A0 cancers\u00C2\u00A0was\u00C2\u00A0substantially\u00C2\u00A0increased\u00C2\u00A0in\u00C2\u00A0female\u00C2\u00A0and\u00C2\u00A0male\u00C2\u00A0mice,\u00C2\u00A0respectively\u00C2\u00A0(National\u00C2\u00A0Institutes\u00C2\u00A0of\u00C2\u00A0Health\u00C2\u00A0 National\u00C2\u00A0Toxicology\u00C2\u00A0Program\u00C2\u00A02004).\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A09.\u00C2\u00A0Synthesis\u00C2\u00A0reaction\u00C2\u00A0and\u00C2\u00A0bioactivation\u00C2\u00A0pathway\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0carbamate.\u00C2\u00A0a)\u00C2\u00A0The\u00C2\u00A0synthesis\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0wines\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spontaneous\u00C2\u00A0reaction\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0and\u00C2\u00A0urea.\u00C2\u00A0b)\u00C2\u00A0The\u00C2\u00A0epoxide\u00C2\u00A0degradation\u00C2\u00A0 product\u00C2\u00A0 of\u00C2\u00A0 EC\u00C2\u00A0 binds\u00C2\u00A0 DNA,\u00C2\u00A0 causing\u00C2\u00A0 damage\u00C2\u00A0 and\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 increased\u00C2\u00A0 rates\u00C2\u00A0 of\u00C2\u00A0 cancers\u00C2\u00A0 in\u00C2\u00A0 test\u00C2\u00A0 animals.\u00C2\u00A0 Vinyl Carbamate Epoxide Vinyl CarbamateEthyl Carbamate a)\u00C2\u00A0 b)\u00C2\u00A0 18\u00C2\u00A0 \u00C2\u00A0 Known\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 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that\u00C2\u00A0most\u00C2\u00A0wines\u00C2\u00A0and\u00C2\u00A0spirits\u00C2\u00A0contain\u00C2\u00A0high\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0EC,\u00C2\u00A0during\u00C2\u00A0the\u00C2\u00A0 mid\u00C2\u00A0 1980\u00E2\u0080\u0099s\u00C2\u00A0 Canada\u00C2\u00A0 set\u00C2\u00A0 a\u00C2\u00A0 legal\u00C2\u00A0 limit\u00C2\u00A0 of\u00C2\u00A0 30\u00C2\u00A0 \u00C2\u00B5g/L\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 allowable\u00C2\u00A0 EC\u00C2\u00A0 content\u00C2\u00A0 in\u00C2\u00A0wines;\u00C2\u00A0 the\u00C2\u00A0US\u00C2\u00A0 set\u00C2\u00A0 a\u00C2\u00A0 voluntary\u00C2\u00A0limit\u00C2\u00A0of\u00C2\u00A015\u00C2\u00A0\u00C2\u00B5g/L.\u00C2\u00A0Long\u00C2\u00A0term\u00C2\u00A0toxicology\u00C2\u00A0studies\u00C2\u00A0eventually\u00C2\u00A0gave\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A01997\u00C2\u00A0action\u00C2\u00A0manual\u00C2\u00A0 on\u00C2\u00A0the\u00C2\u00A0prevention\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0published\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0FDA\u00C2\u00A0in\u00C2\u00A0conjunction\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0Department\u00C2\u00A0of\u00C2\u00A0Viticulture\u00C2\u00A0 and\u00C2\u00A0 Enology\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0University\u00C2\u00A0 of\u00C2\u00A0 California\u00C2\u00A0Davis\u00C2\u00A0 (http://vm.cfsan.fda.gov/~frf/ecaction.html)\u00C2\u00A0 (Butzke\u00C2\u00A0 and\u00C2\u00A0Bisson\u00C2\u00A01998).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Exposure\u00C2\u00A0 to\u00C2\u00A0EC,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 significantly\u00C2\u00A0 increased\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 regular\u00C2\u00A0consumption\u00C2\u00A0of\u00C2\u00A0alcoholic\u00C2\u00A0 beverages\u00C2\u00A0 (Zimmerli\u00C2\u00A0 and\u00C2\u00A0 Schlatter\u00C2\u00A0 1991),\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 a\u00C2\u00A0 significant\u00C2\u00A0 factor\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 human\u00C2\u00A0 cellular\u00C2\u00A0 mutagenesis\u00C2\u00A0and\u00C2\u00A0resultant\u00C2\u00A0tumorigenesis.\u00C2\u00A0As\u00C2\u00A0a\u00C2\u00A0result,\u00C2\u00A0winemakers\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0actively\u00C2\u00A0reducing\u00C2\u00A0EC\u00C2\u00A0levels\u00C2\u00A0 in\u00C2\u00A0wines\u00C2\u00A0 both\u00C2\u00A0 by\u00C2\u00A0 agricultural\u00C2\u00A0 practices\u00C2\u00A0 and,\u00C2\u00A0more\u00C2\u00A0 recently,\u00C2\u00A0molecular\u00C2\u00A0 biological\u00C2\u00A0means\u00C2\u00A0 (Butzke\u00C2\u00A0 and\u00C2\u00A0\u00C2\u00A0 Bisson\u00C2\u00A01998).\u00C2\u00A0 \u00C2\u00A0 1.9.2\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0Surveys\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0 \u00C2\u00A0 Despite\u00C2\u00A0 the\u00C2\u00A0 government\u00C2\u00A0 imposed\u00C2\u00A0 limits\u00C2\u00A0 on\u00C2\u00A0 EC\u00C2\u00A0 levels,\u00C2\u00A0 table\u00C2\u00A0wines,\u00C2\u00A0 Sake\u00C2\u00A0 and\u00C2\u00A0 other\u00C2\u00A0 alcoholic\u00C2\u00A0 beverages\u00C2\u00A0currently\u00C2\u00A0available\u00C2\u00A0to\u00C2\u00A0consumers\u00C2\u00A0contain\u00C2\u00A0far\u00C2\u00A0more\u00C2\u00A0EC\u00C2\u00A0than\u00C2\u00A0was\u00C2\u00A0previously\u00C2\u00A0suggested.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0 the\u00C2\u00A0 scope\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0EC\u00C2\u00A0problem,\u00C2\u00A0numerous\u00C2\u00A0 stuides\u00C2\u00A0have\u00C2\u00A0 assayed\u00C2\u00A0EC\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0 various\u00C2\u00A0 foods\u00C2\u00A0 and\u00C2\u00A0beverages.\u00C2\u00A0The\u00C2\u00A0 EC\u00C2\u00A0 content\u00C2\u00A0of\u00C2\u00A020\u00C2\u00A0 randomly\u00C2\u00A0 chosen\u00C2\u00A0wines\u00C2\u00A0 from\u00C2\u00A0 six\u00C2\u00A0wine\u00C2\u00A0producing\u00C2\u00A0 countries\u00C2\u00A0(five\u00C2\u00A0whites\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Riesling,\u00C2\u00A0Pinot\u00C2\u00A0Gris,\u00C2\u00A0and\u00C2\u00A0Chenin\u00C2\u00A0Blanc;\u00C2\u00A015\u00C2\u00A0reds\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Pinot\u00C2\u00A0Noir,\u00C2\u00A0Cabernet\u00C2\u00A0Sauvignon,\u00C2\u00A0 Shiraz,\u00C2\u00A0Zinfandel,\u00C2\u00A0and\u00C2\u00A0Nebbiolo)\u00C2\u00A0 is\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0Table\u00C2\u00A02\u00C2\u00A0 (Reproduced\u00C2\u00A0 from\u00C2\u00A0Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 (2006)).\u00C2\u00A0As\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0indicates,\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A020\u00C2\u00A0wines,\u00C2\u00A014\u00C2\u00A0exceeded\u00C2\u00A0the\u00C2\u00A0Canadian\u00C2\u00A0EC\u00C2\u00A0legal\u00C2\u00A0limit\u00C2\u00A0(30\u00C2\u00A0\u00C2\u00B5g/L)\u00C2\u00A0and\u00C2\u00A017\u00C2\u00A0exceeded\u00C2\u00A0 the\u00C2\u00A0US\u00C2\u00A0voluntary\u00C2\u00A0limit\u00C2\u00A0(15\u00C2\u00A0\u00C2\u00B5g/L)\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 19\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A02.\u00C2\u00A0Maximum\u00C2\u00A0potential\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0detected\u00C2\u00A0by\u00C2\u00A0GC/MS\u00C2\u00A0in\u00C2\u00A020\u00C2\u00A0wines\u00C2\u00A0from\u00C2\u00A0six\u00C2\u00A0countries.\u00C2\u00A0Wines\u00C2\u00A0 were\u00C2\u00A0heated\u00C2\u00A0at\u00C2\u00A070\u00C2\u00B0C\u00C2\u00A0for\u00C2\u00A048\u00C2\u00A0hours\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0analysis.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Coulon\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0(2006).\u00C2\u00A0 Wine Concentration EC (\u00C2\u00B5g/L) Area 1 Area 2 Area 3 Mean peak area SD RSDa (%) 1 11.83 4274 4542 4185 4334 186 4.29 2 9.12 3649 3586 3887 3707 159 4.28 3 14.63 5043 4959 4948 4983 52 1.04 4 38.64 10262 10112 11269 10548 629 5.97 5 57.57 14342 15833 14632 14936 791 5.29 6 66.15 16610 16734 17430 16925 442 2.61 7 59.52 15890 15015 15260 15388 451 2.93 8 46.39 11208 13611 12213 12344 1207 9.78 9 55.41 13865 14820 14618 14434 503 3.49 10 24.36 7012 6808 7895 7238 578 7.98 11 44.76 11650 12576 11677 11968 527 4.40 12 41.32 11186 10952 11368 11169 209 1.87 13 27.5 7664 8366 7868 7966 361 4.53 14 38.22 10375 10471 10508 10451 69 0.66 15 57.68 14069 14839 15974 14961 958 6.41 16 33.82 9034 9428 9832 9431 399 4.23 17 51.99 14146 12880 13899 13642 671 4.92 18 18.61 5734 5935 6047 5905 159 2.69 19 34.99 9396 9295 10417 9703 621 6.40 20 36.57 9635 9756 10811 10067 647 6.43 \u00C2\u00A0 a\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Relative\u00C2\u00A0standard\u00C2\u00A0deviation\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 addition\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 goal\u00C2\u00A0of\u00C2\u00A0 reducing\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0 table\u00C2\u00A0wines,\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0has\u00C2\u00A0 some\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0highest\u00C2\u00A0EC\u00C2\u00A0 contents\u00C2\u00A0amongst\u00C2\u00A0fermented\u00C2\u00A0beverages,\u00C2\u00A0thus\u00C2\u00A0making\u00C2\u00A0the\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0an\u00C2\u00A0 intensely\u00C2\u00A0relevant\u00C2\u00A0 and\u00C2\u00A0important\u00C2\u00A0pursuit.\u00C2\u00A0Typical\u00C2\u00A0Sake\u00C2\u00A0wines\u00C2\u00A0have\u00C2\u00A0anywhere\u00C2\u00A0from\u00C2\u00A0100\u00E2\u0080\u0090250\u00C2\u00A0\u00C2\u00B5g/L\u00C2\u00A0EC\u00C2\u00A0(Canas,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01989)\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0pasteurization\u00C2\u00A0process\u00C2\u00A0that\u00C2\u00A0all\u00C2\u00A0Sakes\u00C2\u00A0undergo\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0bottling.\u00C2\u00A0 \u00C2\u00A0 1.9.3\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0EC\u00C2\u00A0problem:\u00C2\u00A0Current\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0lowering\u00C2\u00A0EC\u00C2\u00A0 \u00C2\u00A0 Current\u00C2\u00A0strategies\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0generally\u00C2\u00A0fall\u00C2\u00A0into\u00C2\u00A0three\u00C2\u00A0categories:\u00C2\u00A0agricultural\u00C2\u00A0practices,\u00C2\u00A0the\u00C2\u00A0 use\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0additives,\u00C2\u00A0and\u00C2\u00A0genetic\u00C2\u00A0engineering\u00C2\u00A0of\u00C2\u00A0yeasts.\u00C2\u00A0 \u00C2\u00A0 1.9.3.1\u00C2\u00A0 \u00C2\u00A0 Agricultural\u00C2\u00A0 methods.\u00C2\u00A0 The\u00C2\u00A0 yeast\u00C2\u00A0 assimilable\u00C2\u00A0 nitrogen\u00C2\u00A0 (YAN)\u00C2\u00A0 and,\u00C2\u00A0more\u00C2\u00A0 specifically,\u00C2\u00A0 arginine\u00C2\u00A0 content\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0fermentation\u00C2\u00A0substrate\u00C2\u00A0directly\u00C2\u00A0influences\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0final\u00C2\u00A0product\u00C2\u00A0 (Butzke\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01998).\u00C2\u00A0Thus,\u00C2\u00A0a\u00C2\u00A0reasonable\u00C2\u00A0approach\u00C2\u00A0to\u00C2\u00A0controlling\u00C2\u00A0EC\u00C2\u00A0levels\u00C2\u00A0is\u00C2\u00A0to\u00C2\u00A0limit\u00C2\u00A0arginine\u00C2\u00A0levels\u00C2\u00A0 20\u00C2\u00A0 \u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0and\u00C2\u00A0rice\u00C2\u00A0mash.\u00C2\u00A0It\u00C2\u00A0 is\u00C2\u00A0possible\u00C2\u00A0to\u00C2\u00A0regulate\u00C2\u00A0the\u00C2\u00A0type\u00C2\u00A0and\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0nitrogen\u00C2\u00A0 in\u00C2\u00A0fertilizers\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0minimize\u00C2\u00A0 urea\u00C2\u00A0 production.\u00C2\u00A0 Furthermore,\u00C2\u00A0management\u00C2\u00A0 of\u00C2\u00A0 legume\u00C2\u00A0 ground\u00C2\u00A0 cover\u00C2\u00A0 foliage\u00C2\u00A0 and\u00C2\u00A0 nitrogen\u00C2\u00A0fixing\u00C2\u00A0bacteria\u00C2\u00A0can\u00C2\u00A0keep\u00C2\u00A0juice\u00C2\u00A0arginine\u00C2\u00A0content\u00C2\u00A0below\u00C2\u00A01000\u00C2\u00A0mg/L,\u00C2\u00A0however\u00C2\u00A0additional\u00C2\u00A0methods\u00C2\u00A0 are\u00C2\u00A0needed\u00C2\u00A0to\u00C2\u00A0further\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0wines\u00C2\u00A0(Butzke\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01998).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.9.3.2\u00C2\u00A0\u00C2\u00A0Additives\u00C2\u00A0(acid\u00C2\u00A0urease).\u00C2\u00A0Alternative\u00C2\u00A0methodologies\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0include\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0post\u00E2\u0080\u0090\u00C2\u00A0or\u00C2\u00A0 peri\u00E2\u0080\u0090fermentation\u00C2\u00A0additives\u00C2\u00A0 (Ough\u00C2\u00A0and\u00C2\u00A0Trioli\u00C2\u00A01988).\u00C2\u00A0These\u00C2\u00A0additives,\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0most\u00C2\u00A0often\u00C2\u00A0 lyophilized\u00C2\u00A0 preparations\u00C2\u00A0of\u00C2\u00A0urease\u00C2\u00A0from\u00C2\u00A0Lactobacillus\u00C2\u00A0fermentum,\u00C2\u00A0degrade\u00C2\u00A0urea\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0wine\u00C2\u00A0before\u00C2\u00A0 it\u00C2\u00A0has\u00C2\u00A0a\u00C2\u00A0chance\u00C2\u00A0 to\u00C2\u00A0form\u00C2\u00A0EC;\u00C2\u00A0however,\u00C2\u00A0urease\u00C2\u00A0additives\u00C2\u00A0yield\u00C2\u00A0variable\u00C2\u00A0results\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0pH\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0sensitivity\u00C2\u00A0(Kodama,\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A01994)\u00C2\u00A0which,\u00C2\u00A0can\u00C2\u00A0significantly\u00C2\u00A0lengthen\u00C2\u00A0wine\u00C2\u00A0processing\u00C2\u00A0time\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0necessary\u00C2\u00A0enzyme\u00C2\u00A0incubation.\u00C2\u00A0 The\u00C2\u00A0enzyme\u00C2\u00A0is\u00C2\u00A0also\u00C2\u00A0expensive\u00C2\u00A0for\u00C2\u00A0winemakers\u00C2\u00A0to\u00C2\u00A0use.\u00C2\u00A0 \u00C2\u00A0 1.9.3.3\u00C2\u00A0 \u00C2\u00A0 Additives\u00C2\u00A0 (DAP).\u00C2\u00A0 Supplementation\u00C2\u00A0 of\u00C2\u00A0 grape\u00C2\u00A0musts\u00C2\u00A0with\u00C2\u00A0 nitrogen\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 common\u00C2\u00A0winemaking\u00C2\u00A0 practice.\u00C2\u00A0 In\u00C2\u00A0musts\u00C2\u00A0with\u00C2\u00A0 low\u00C2\u00A0 assimilable\u00C2\u00A0 nitrogen,\u00C2\u00A0 supplementation\u00C2\u00A0 is\u00C2\u00A0 crucial\u00C2\u00A0 for\u00C2\u00A0 preventing\u00C2\u00A0 stuck\u00C2\u00A0 or\u00C2\u00A0 sluggish\u00C2\u00A0 fermentations\u00C2\u00A0 and\u00C2\u00A0 subsequent\u00C2\u00A0 spoilage.\u00C2\u00A0 Given\u00C2\u00A0 the\u00C2\u00A0 obvious\u00C2\u00A0 inappropriateness\u00C2\u00A0 of\u00C2\u00A0 supplementation\u00C2\u00A0with\u00C2\u00A0urea\u00C2\u00A0or\u00C2\u00A0arginine,\u00C2\u00A0the\u00C2\u00A0 industry\u00C2\u00A0makes\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cheap\u00C2\u00A0and\u00C2\u00A0efficient\u00C2\u00A0supplement\u00C2\u00A0 diammonium\u00C2\u00A0phosphate\u00C2\u00A0 (DAP);\u00C2\u00A0DAP\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0 source\u00C2\u00A0of\u00C2\u00A0 free\u00C2\u00A0ammonia\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0highly\u00C2\u00A0 favorable\u00C2\u00A0nitrogen\u00C2\u00A0 source,\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 downregulate\u00C2\u00A0 CAR1\u00C2\u00A0 during\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentation\u00C2\u00A0 (Marks,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003).\u00C2\u00A0 Lessening\u00C2\u00A0the\u00C2\u00A0cell\u00E2\u0080\u0099s\u00C2\u00A0dependence\u00C2\u00A0on\u00C2\u00A0arginine\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0nitrogen\u00C2\u00A0source\u00C2\u00A0could\u00C2\u00A0prove\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0useful\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0 lowering\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0wines.\u00C2\u00A0 \u00C2\u00A0 1.9.3.4\u00C2\u00A0\u00C2\u00A0Genetic\u00C2\u00A0engineering\u00C2\u00A0(urease\u00C2\u00A0expression).\u00C2\u00A0Another\u00C2\u00A0possible\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0control\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0 engineering\u00C2\u00A0 of\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 which\u00C2\u00A0 express\u00C2\u00A0 bacterial\u00C2\u00A0 or\u00C2\u00A0 fungal\u00C2\u00A0 ureases\u00C2\u00A0 (Ough\u00C2\u00A0 and\u00C2\u00A0 Trioli\u00C2\u00A0 1988).\u00C2\u00A0 This\u00C2\u00A0 approach,\u00C2\u00A0whether\u00C2\u00A0 integrated\u00C2\u00A0 or\u00C2\u00A0 plasmid\u00C2\u00A0 borne,\u00C2\u00A0would\u00C2\u00A0 allow\u00C2\u00A0 yeast\u00C2\u00A0 to\u00C2\u00A0 directly\u00C2\u00A0 degrade\u00C2\u00A0 urea\u00C2\u00A0 during\u00C2\u00A0 fermentation,\u00C2\u00A0 and\u00C2\u00A0 eliminates\u00C2\u00A0 the\u00C2\u00A0 need\u00C2\u00A0 for\u00C2\u00A0 urease\u00C2\u00A0 additives\u00C2\u00A0 post\u00E2\u0080\u0090fermentation.\u00C2\u00A0However,\u00C2\u00A0 because\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0possess\u00C2\u00A0such\u00C2\u00A0an\u00C2\u00A0enzyme,\u00C2\u00A0a\u00C2\u00A0urease\u00C2\u00A0expressing\u00C2\u00A0strain\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0classified\u00C2\u00A0as\u00C2\u00A0transgenic\u00C2\u00A0 thus\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 difficulty\u00C2\u00A0 obtaining\u00C2\u00A0 regulatory\u00C2\u00A0 approval\u00C2\u00A0 for\u00C2\u00A0 use.\u00C2\u00A0 Furthermore,\u00C2\u00A0 transgenic\u00C2\u00A0 organisms\u00C2\u00A0 carry\u00C2\u00A0a\u00C2\u00A0strong\u00C2\u00A0negative\u00C2\u00A0connotation\u00C2\u00A0in\u00C2\u00A0today\u00E2\u0080\u0099s\u00C2\u00A0society\u00C2\u00A0thus\u00C2\u00A0making\u00C2\u00A0their\u00C2\u00A0universal\u00C2\u00A0acceptance\u00C2\u00A0unlikely.\u00C2\u00A0 \u00C2\u00A0 1.9.3.5\u00C2\u00A0 \u00C2\u00A0 Genetic\u00C2\u00A0 engineering\u00C2\u00A0 (CAR1).\u00C2\u00A0More\u00C2\u00A0 central\u00C2\u00A0 to\u00C2\u00A0 this\u00C2\u00A0work\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 genetic\u00C2\u00A0 engineering\u00C2\u00A0 of\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0disrupted\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0CAR1\u00C2\u00A0 locus\u00C2\u00A0(Kitamoto,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01991).\u00C2\u00A0By\u00C2\u00A0disrupting\u00C2\u00A0CAR1,\u00C2\u00A0arginase\u00C2\u00A0will\u00C2\u00A0 no\u00C2\u00A0 longer\u00C2\u00A0be\u00C2\u00A0available\u00C2\u00A0to\u00C2\u00A0degrade\u00C2\u00A0arginine\u00C2\u00A0to\u00C2\u00A0urea\u00C2\u00A0thus\u00C2\u00A0preventing\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0wine.\u00C2\u00A0While\u00C2\u00A0 21\u00C2\u00A0 \u00C2\u00A0 this\u00C2\u00A0method\u00C2\u00A0does\u00C2\u00A0work\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 laboratory\u00C2\u00A0 (Kitamoto,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01991;\u00C2\u00A0Yoshiuchi,\u00C2\u00A0Watanabe\u00C2\u00A0and\u00C2\u00A0Nishimura\u00C2\u00A0 2000),\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0widely\u00C2\u00A0adopted\u00C2\u00A0in\u00C2\u00A0practice\u00C2\u00A0because\u00C2\u00A0arginine\u00C2\u00A0is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0major\u00C2\u00A0nitrogen\u00C2\u00A0sources\u00C2\u00A0 for\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 (Ingledew,\u00C2\u00A0Magnus\u00C2\u00A0 and\u00C2\u00A0 Patterson\u00C2\u00A0 1987;\u00C2\u00A0Rossignol,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003).\u00C2\u00A0Moreover,\u00C2\u00A0 usage\u00C2\u00A0 of\u00C2\u00A0 \u00CE\u0094car1\u00C2\u00A0strains\u00C2\u00A0is\u00C2\u00A0difficult\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0diploid\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0industrial\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0yeasts,\u00C2\u00A0and\u00C2\u00A0because\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 high\u00C2\u00A0 risk\u00C2\u00A0 of\u00C2\u00A0 contamination\u00C2\u00A0 from\u00C2\u00A0 wild\u00C2\u00A0 type\u00C2\u00A0 CAR1/CAR1\u00C2\u00A0 yeasts;\u00C2\u00A0 however,\u00C2\u00A0 the\u00C2\u00A0 problem\u00C2\u00A0 of\u00C2\u00A0 contamination\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0dealt\u00C2\u00A0with\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0by\u00C2\u00A0engineering\u00C2\u00A0\u00CE\u0094car1\u00C2\u00A0with\u00C2\u00A0killer\u00C2\u00A0character\u00C2\u00A0 (Yoshiuchi,\u00C2\u00A0Watanabe\u00C2\u00A0 and\u00C2\u00A0Nishimura\u00C2\u00A02000).\u00C2\u00A0 It\u00C2\u00A0 should\u00C2\u00A0be\u00C2\u00A0noted\u00C2\u00A0 that\u00C2\u00A0CAR1\u00C2\u00A0 knockouts\u00C2\u00A0have\u00C2\u00A0only\u00C2\u00A0been\u00C2\u00A0 attempted\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0since\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0generally\u00C2\u00A0regarded\u00C2\u00A0that\u00C2\u00A0yeast\u00E2\u0080\u0099s\u00C2\u00A0inability\u00C2\u00A0to\u00C2\u00A0metabolize\u00C2\u00A0arginine\u00C2\u00A0would,\u00C2\u00A0 in\u00C2\u00A0most\u00C2\u00A0cases,\u00C2\u00A0lead\u00C2\u00A0to\u00C2\u00A0stuck\u00C2\u00A0fermentations\u00C2\u00A0and\u00C2\u00A0possible\u00C2\u00A0spoilage.\u00C2\u00A0An\u00C2\u00A0antisense\u00C2\u00A0mediated\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 knockdown\u00C2\u00A0of\u00C2\u00A0CAR1\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0successful\u00C2\u00A0in\u00C2\u00A0reducing\u00C2\u00A0Arginase\u00C2\u00A0activity\u00C2\u00A0in\u00C2\u00A0laboratory\u00C2\u00A0yeasts\u00C2\u00A0(Park,\u00C2\u00A0Shin\u00C2\u00A0and\u00C2\u00A0 Woo\u00C2\u00A02001);\u00C2\u00A0however,\u00C2\u00A0this\u00C2\u00A0methodology\u00C2\u00A0has\u00C2\u00A0yet\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0examined\u00C2\u00A0in\u00C2\u00A0industrial\u00C2\u00A0yeasts.\u00C2\u00A0 \u00C2\u00A0 1.9.3.6\u00C2\u00A0 \u00C2\u00A0Genetic\u00C2\u00A0 engineering\u00C2\u00A0 (DUR1,2).\u00C2\u00A0 Yeast\u00C2\u00A0 possess\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 degrade\u00C2\u00A0 urea\u00C2\u00A0 via\u00C2\u00A0DUR1,2\u00C2\u00A0which\u00C2\u00A0 encodes\u00C2\u00A0a\u00C2\u00A0bi\u00E2\u0080\u0090functional\u00C2\u00A0adenosine\u00C2\u00A0tri\u00E2\u0080\u0090phosphate\u00C2\u00A0(ATP)\u00C2\u00A0and\u00C2\u00A0biotin\u00C2\u00A0dependent\u00C2\u00A0enzyme\u00C2\u00A0(urea\u00C2\u00A0amidolyase)\u00C2\u00A0 that\u00C2\u00A0degrades\u00C2\u00A0urea\u00C2\u00A0to\u00C2\u00A0two\u00C2\u00A0molecules\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0CO2\u00C2\u00A0and\u00C2\u00A0NH3\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0two\u00C2\u00A0step\u00C2\u00A0reaction\u00C2\u00A0(Genbauffe\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A0 1986;\u00C2\u00A0Genbauffe\u00C2\u00A0 and\u00C2\u00A0Cooper\u00C2\u00A0 1991;\u00C2\u00A0Whitney\u00C2\u00A0 and\u00C2\u00A0Cooper\u00C2\u00A0 1972;\u00C2\u00A0Whitney\u00C2\u00A0 and\u00C2\u00A0Cooper\u00C2\u00A0 1973;\u00C2\u00A0Whitney,\u00C2\u00A0 Cooper\u00C2\u00A0and\u00C2\u00A0Magasanik\u00C2\u00A01973).\u00C2\u00A0Urea\u00C2\u00A0is\u00C2\u00A0first\u00C2\u00A0carboxylated\u00C2\u00A0using\u00C2\u00A0ATP\u00C2\u00A0and\u00C2\u00A0biotin\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0allophanate,\u00C2\u00A0after\u00C2\u00A0 which\u00C2\u00A0allophanate\u00C2\u00A0is\u00C2\u00A0hydrolyzed\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0CO2\u00C2\u00A0and\u00C2\u00A0NH3.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0DUR1,2\u00C2\u00A0 is\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0regulation\u00C2\u00A0by\u00C2\u00A0NCR,\u00C2\u00A0and\u00C2\u00A0because\u00C2\u00A0regulatory\u00C2\u00A0mechanisms\u00C2\u00A0exist\u00C2\u00A0which\u00C2\u00A0 allow\u00C2\u00A0for\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0wines\u00C2\u00A0with\u00C2\u00A0high\u00C2\u00A0residual\u00C2\u00A0urea\u00C2\u00A0content,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0reasonable\u00C2\u00A0to\u00C2\u00A0expect\u00C2\u00A0that\u00C2\u00A0constitutive\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0should\u00C2\u00A0result\u00C2\u00A0 in\u00C2\u00A0substantially\u00C2\u00A0 lowered\u00C2\u00A0EC\u00C2\u00A0 levels.\u00C2\u00A0 Indeed,\u00C2\u00A0our\u00C2\u00A0group\u00C2\u00A0has\u00C2\u00A0explored\u00C2\u00A0 this\u00C2\u00A0approach,\u00C2\u00A0and\u00C2\u00A0such\u00C2\u00A0yeasts\u00C2\u00A0are\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0producing\u00C2\u00A0wines\u00C2\u00A0which\u00C2\u00A0contain\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A089%\u00C2\u00A0less\u00C2\u00A0EC\u00C2\u00A0(Coulon,\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0More\u00C2\u00A0specifically,\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0ORF\u00C2\u00A0was\u00C2\u00A0placed\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0PGK1\u00C2\u00A0 promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0single\u00C2\u00A0copy\u00C2\u00A0was\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0industrial\u00C2\u00A0 strain\u00C2\u00A0UC\u00C2\u00A0Davis\u00C2\u00A0522,\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0created\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0strain\u00C2\u00A0(522EC\u00E2\u0080\u0090)\u00C2\u00A0contained\u00C2\u00A0 89.1%\u00C2\u00A0 less\u00C2\u00A0EC\u00C2\u00A0 (Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0genotype,\u00C2\u00A0phenotype\u00C2\u00A0and\u00C2\u00A0 transcriptome\u00C2\u00A0of\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0metabolically\u00C2\u00A0 engineered\u00C2\u00A0 strain\u00C2\u00A0 was\u00C2\u00A0 substantially\u00C2\u00A0 equivalent\u00C2\u00A0 to\u00C2\u00A0 its\u00C2\u00A0 parent,\u00C2\u00A0 thus\u00C2\u00A0 making\u00C2\u00A0 it\u00C2\u00A0 suitable\u00C2\u00A0 for\u00C2\u00A0commercialization\u00C2\u00A0 (Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Furthermore,\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0 strain\u00C2\u00A0 (522EC\u00E2\u0080\u0090)\u00C2\u00A0contains\u00C2\u00A0no\u00C2\u00A0 foreign\u00C2\u00A0DNA\u00C2\u00A0sequences,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0classified\u00C2\u00A0as\u00C2\u00A0 transgenic\u00C2\u00A0and\u00C2\u00A0 thus\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 22\u00C2\u00A0 \u00C2\u00A0 given\u00C2\u00A0FDA\u00C2\u00A0GRAS\u00C2\u00A0approval\u00C2\u00A0which\u00C2\u00A0should\u00C2\u00A0make\u00C2\u00A0it\u00C2\u00A0much\u00C2\u00A0more\u00C2\u00A0readily\u00C2\u00A0accepted\u00C2\u00A0by\u00C2\u00A0industry\u00C2\u00A0and\u00C2\u00A0consumers\u00C2\u00A0 (Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0 \u00C2\u00A0 1.9.4\u00C2\u00A0\u00C2\u00A0Alternative\u00C2\u00A0methods\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0 \u00C2\u00A0 Realistically,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 only\u00C2\u00A0 four\u00C2\u00A0 genes\u00C2\u00A0 that\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0manipulated\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0wine:\u00C2\u00A0 urea\u00C2\u00A0 production\u00C2\u00A0(CAR1),\u00C2\u00A0urea\u00C2\u00A0degradation\u00C2\u00A0(DUR1,2),\u00C2\u00A0urea\u00C2\u00A0export\u00C2\u00A0(DUR4),\u00C2\u00A0and\u00C2\u00A0urea\u00C2\u00A0import\u00C2\u00A0(DUR3).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Based\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 literature\u00C2\u00A0and\u00C2\u00A0published\u00C2\u00A0data,\u00C2\u00A0we\u00C2\u00A0understand\u00C2\u00A0 the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0manipulating\u00C2\u00A0CAR1\u00C2\u00A0 and\u00C2\u00A0DUR1,2\u00C2\u00A0expression\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006;\u00C2\u00A0Yoshiuchi,\u00C2\u00A0Watanabe\u00C2\u00A0and\u00C2\u00A0Nishimura\u00C2\u00A02000),\u00C2\u00A0and\u00C2\u00A0while\u00C2\u00A0CAR1\u00C2\u00A0 mutants\u00C2\u00A0produce\u00C2\u00A0almost\u00C2\u00A0no\u00C2\u00A0EC,\u00C2\u00A0CAR1\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0a\u00C2\u00A0practical\u00C2\u00A0industrial\u00C2\u00A0target\u00C2\u00A0for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0problems\u00C2\u00A0 with\u00C2\u00A0stuck\u00C2\u00A0fermentations.\u00C2\u00A0Thus,\u00C2\u00A0manipulation\u00C2\u00A0of\u00C2\u00A0DUR4\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0remain\u00C2\u00A0as\u00C2\u00A0potential\u00C2\u00A0targets\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0 Sake.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0option\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0 to\u00C2\u00A0 knockout\u00C2\u00A0 the\u00C2\u00A0urea\u00C2\u00A0exporter\u00C2\u00A0DUR4,\u00C2\u00A0 thus\u00C2\u00A0disabling\u00C2\u00A0 the\u00C2\u00A0 yeast\u00E2\u0080\u0099s\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0export\u00C2\u00A0urea\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0wine.\u00C2\u00A0Although\u00C2\u00A0this\u00C2\u00A0sounds\u00C2\u00A0like\u00C2\u00A0an\u00C2\u00A0attractive\u00C2\u00A0option,\u00C2\u00A0this\u00C2\u00A0strategy\u00C2\u00A0 would\u00C2\u00A0 likely\u00C2\u00A0cause\u00C2\u00A0serious\u00C2\u00A0problems\u00C2\u00A0for\u00C2\u00A0winemakers.\u00C2\u00A0Urea\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0toxic\u00C2\u00A0byproduct\u00C2\u00A0of\u00C2\u00A0arginine\u00C2\u00A0metabolism\u00C2\u00A0 and\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0export\u00C2\u00A0urea\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0stay\u00C2\u00A0healthy.\u00C2\u00A0By\u00C2\u00A0knocking\u00C2\u00A0out\u00C2\u00A0DUR4,\u00C2\u00A0urea\u00C2\u00A0will\u00C2\u00A0accumulate\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 and\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 an\u00C2\u00A0 adverse\u00C2\u00A0 physiological\u00C2\u00A0 state\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 stuck\u00C2\u00A0 fermentations.\u00C2\u00A0 The\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 existing\u00C2\u00A0 recombinant\u00C2\u00A0DUR1,2\u00C2\u00A0 yeasts\u00C2\u00A0 produce\u00C2\u00A0 any\u00C2\u00A0 EC\u00C2\u00A0 at\u00C2\u00A0 all\u00C2\u00A0 (Coulon,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006)\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 under\u00C2\u00A0 typical\u00C2\u00A0winemaking\u00C2\u00A0 conditions\u00C2\u00A0more\u00C2\u00A0urea\u00C2\u00A0 is\u00C2\u00A0produced\u00C2\u00A0 than\u00C2\u00A0DUR1,2p,\u00C2\u00A0 from\u00C2\u00A0a\u00C2\u00A0 constitutively\u00C2\u00A0expressed\u00C2\u00A0 copy\u00C2\u00A0of\u00C2\u00A0DUR1,2,\u00C2\u00A0can\u00C2\u00A0degrade.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Given\u00C2\u00A0the\u00C2\u00A0potential\u00C2\u00A0problems\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0DUR4\u00C2\u00A0knockouts,\u00C2\u00A0we\u00C2\u00A0focused\u00C2\u00A0our\u00C2\u00A0attention\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 urea\u00C2\u00A0permease,\u00C2\u00A0DUR3.\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0should\u00C2\u00A0result\u00C2\u00A0 in\u00C2\u00A0yeasts\u00C2\u00A0which\u00C2\u00A0will\u00C2\u00A0act\u00C2\u00A0 like\u00C2\u00A0urea\u00C2\u00A0 sponges;\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 will\u00C2\u00A0 these\u00C2\u00A0 yeasts\u00C2\u00A0 reabsorb\u00C2\u00A0 urea\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0 excreted\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 byproduct\u00C2\u00A0 of\u00C2\u00A0 arginine\u00C2\u00A0 metabolism,\u00C2\u00A0 but\u00C2\u00A0 they\u00C2\u00A0 should\u00C2\u00A0 also\u00C2\u00A0 absorb\u00C2\u00A0 a\u00C2\u00A0 significant\u00C2\u00A0 amount\u00C2\u00A0 of\u00C2\u00A0 urea\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 naturally\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 fermentation\u00C2\u00A0substrate.\u00C2\u00A0By\u00C2\u00A0combining\u00C2\u00A0DUR1,2\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0with\u00C2\u00A0 that\u00C2\u00A0of\u00C2\u00A0DUR3,\u00C2\u00A0 it\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0 possible\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0recombinant\u00C2\u00A0yeasts\u00C2\u00A0which\u00C2\u00A0can\u00C2\u00A0conduct\u00C2\u00A0efficient\u00C2\u00A0and\u00C2\u00A0substantially\u00C2\u00A0equivalent\u00C2\u00A0alcoholic\u00C2\u00A0 fermentations\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0little\u00C2\u00A0or\u00C2\u00A0no\u00C2\u00A0EC.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 23\u00C2\u00A0 \u00C2\u00A0 1.10\u00C2\u00A0\u00C2\u00A0Introduction\u00C2\u00A0to\u00C2\u00A0DUR3:\u00C2\u00A0Role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0(urea\u00C2\u00A0transport,\u00C2\u00A0polyamines,\u00C2\u00A0boron)\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0the\u00C2\u00A0early\u00C2\u00A01970\u00E2\u0080\u0099s\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0known\u00C2\u00A0that\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0metabolizing\u00C2\u00A0urea\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0sole\u00C2\u00A0 nitrogen\u00C2\u00A0source\u00C2\u00A0(Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975);\u00C2\u00A0however,\u00C2\u00A0there\u00C2\u00A0was\u00C2\u00A0no\u00C2\u00A0detailed\u00C2\u00A0knowledge\u00C2\u00A0of\u00C2\u00A0how\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0 brought\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cell.\u00C2\u00A0Studies\u00C2\u00A0using\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0entry\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0is\u00C2\u00A0bimodal\u00C2\u00A0(Cooper\u00C2\u00A0 and\u00C2\u00A0Sumrada\u00C2\u00A01975;\u00C2\u00A0Sumrada,\u00C2\u00A0Gorski\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A01976).\u00C2\u00A0A\u00C2\u00A0 facilitated\u00C2\u00A0diffusion\u00C2\u00A0system\u00C2\u00A0brings\u00C2\u00A0urea\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0energy\u00C2\u00A0independent\u00C2\u00A0fashion\u00C2\u00A0when\u00C2\u00A0urea\u00C2\u00A0is\u00C2\u00A0present\u00C2\u00A0at\u00C2\u00A0concentrations\u00C2\u00A0greater\u00C2\u00A0than\u00C2\u00A00.5\u00C2\u00A0mM.\u00C2\u00A0 More\u00C2\u00A0interestingly\u00C2\u00A0however,\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0energy\u00C2\u00A0(ATP)\u00C2\u00A0dependent\u00C2\u00A0active\u00C2\u00A0transport\u00C2\u00A0system\u00C2\u00A0(Km\u00C2\u00A0=\u00C2\u00A0 14\u00C2\u00A0\u00CE\u00BCM)\u00C2\u00A0which\u00C2\u00A0functions\u00C2\u00A0at\u00C2\u00A0low\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0 (Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975;\u00C2\u00A0Sumrada,\u00C2\u00A0Gorski\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A01976).\u00C2\u00A0 \u00C2\u00A0 First\u00C2\u00A0purified\u00C2\u00A0and\u00C2\u00A0characterized\u00C2\u00A0in\u00C2\u00A01993,\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0(Degradation\u00C2\u00A0of\u00C2\u00A0URea)\u00C2\u00A0ORF\u00C2\u00A0encodes\u00C2\u00A0a\u00C2\u00A0735\u00C2\u00A0aa\u00C2\u00A0 integral\u00C2\u00A0membrane\u00C2\u00A0protein\u00C2\u00A0which\u00C2\u00A0contains\u00C2\u00A015\u00C2\u00A0predicted\u00C2\u00A0transmembrane\u00C2\u00A0domains\u00C2\u00A0(ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01993).\u00C2\u00A0 The\u00C2\u00A0protein,\u00C2\u00A0which\u00C2\u00A0localizes\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0utilizes\u00C2\u00A0ATP\u00C2\u00A0to\u00C2\u00A0transport\u00C2\u00A0urea\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0at\u00C2\u00A0low\u00C2\u00A0 extracellular\u00C2\u00A0concentrations.\u00C2\u00A0There\u00C2\u00A0is\u00C2\u00A0some\u00C2\u00A0evidence\u00C2\u00A0to\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0physiological\u00C2\u00A0functioning\u00C2\u00A0state\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0urea\u00C2\u00A0 transporter\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0multimeric\u00C2\u00A0complex,\u00C2\u00A0however\u00C2\u00A0 this\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0confirmed\u00C2\u00A0 (ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 1993).\u00C2\u00A0 \u00C2\u00A0 Expression\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 is\u00C2\u00A0 regulated\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0manner\u00C2\u00A0 highly\u00C2\u00A0 similar\u00C2\u00A0 to\u00C2\u00A0 other\u00C2\u00A0 genes\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 and\u00C2\u00A0 allantoin\u00C2\u00A0degradative\u00C2\u00A0pathways\u00C2\u00A0(ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01993).\u00C2\u00A0Being\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0NCR,\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0promoter\u00C2\u00A0contains\u00C2\u00A0 two\u00C2\u00A0 sets\u00C2\u00A0 of\u00C2\u00A0 tandem\u00C2\u00A0 GATAA\u00C2\u00A0 consensus\u00C2\u00A0 sequences;\u00C2\u00A0 however,\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 promoter\u00C2\u00A0 contains\u00C2\u00A0 efficient\u00C2\u00A0 GATAA\u00C2\u00A0 transcription\u00C2\u00A0 factor\u00C2\u00A0binding\u00C2\u00A0 sites\u00C2\u00A0 (UASNTR),\u00C2\u00A0high\u00C2\u00A0 level\u00C2\u00A0expression\u00C2\u00A0 is\u00C2\u00A0 strongly\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0 two\u00C2\u00A0 upstream\u00C2\u00A0 induction\u00C2\u00A0 sequences\u00C2\u00A0 (UIS)\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0During\u00C2\u00A0growth\u00C2\u00A0on\u00C2\u00A0proline\u00C2\u00A0media\u00C2\u00A0 (no\u00C2\u00A0NCR),\u00C2\u00A0 little\u00C2\u00A0 DUR3\u00C2\u00A0 (and\u00C2\u00A0 DUR1,2)\u00C2\u00A0 mRNA\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 detected\u00C2\u00A0 by\u00C2\u00A0 northern\u00C2\u00A0 blotting\u00C2\u00A0 without\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 gratuitous\u00C2\u00A0inducer\u00C2\u00A0(oxalurate,\u00C2\u00A0an\u00C2\u00A0allophanate\u00C2\u00A0analogue)\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0activating\u00C2\u00A0transcription\u00C2\u00A0factors\u00C2\u00A0DAL81\u00C2\u00A0and\u00C2\u00A0DAL82\u00C2\u00A0act\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0UIS\u00C2\u00A0sequences\u00C2\u00A0contained\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0promoter.\u00C2\u00A0The\u00C2\u00A0consensus\u00C2\u00A0sequence,\u00C2\u00A05\u00E2\u0080\u0099\u00C2\u00A0(G/C)\u00C2\u00A0AAA\u00C2\u00A0(A/T)\u00C2\u00A0NTGCG\u00C2\u00A0(T/C)\u00C2\u00A0T\u00C2\u00A0(T/G/C)\u00C2\u00A0(T/G/C)\u00C2\u00A03\u00E2\u0080\u0099,\u00C2\u00A0 for\u00C2\u00A0DAL81\u00C2\u00A0and\u00C2\u00A0DAL82\u00C2\u00A0binding\u00C2\u00A0 is\u00C2\u00A0shared\u00C2\u00A0between\u00C2\u00A0other\u00C2\u00A0allophanate\u00C2\u00A0 induced\u00C2\u00A0genes\u00C2\u00A0(CAR2,\u00C2\u00A0DAL2,\u00C2\u00A0DAL4,\u00C2\u00A0 DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 24\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0 times\u00C2\u00A0of\u00C2\u00A0 strong\u00C2\u00A0NCR,\u00C2\u00A0DUR3\u00C2\u00A0 is\u00C2\u00A0 actively\u00C2\u00A0 repressed\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0negative\u00C2\u00A0GATAA\u00C2\u00A0 transcription\u00C2\u00A0 factor\u00C2\u00A0DAL80,\u00C2\u00A0and\u00C2\u00A0deletion\u00C2\u00A0of\u00C2\u00A0DAL80\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0even\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0 inducer\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0Conversely,\u00C2\u00A0during\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression,\u00C2\u00A0DUR3\u00C2\u00A0is\u00C2\u00A0actively\u00C2\u00A0transcribed,\u00C2\u00A0if\u00C2\u00A0an\u00C2\u00A0inducer\u00C2\u00A0is\u00C2\u00A0 present,\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0actions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0positive\u00C2\u00A0GATAA\u00C2\u00A0transcription\u00C2\u00A0factor\u00C2\u00A0GLN3\u00C2\u00A0(Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0addition\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0obvious\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0urea\u00C2\u00A0uptake,\u00C2\u00A0DUR3\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0other\u00C2\u00A0 important\u00C2\u00A0cellular\u00C2\u00A0processes.\u00C2\u00A0DUR3\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0an\u00C2\u00A0important\u00C2\u00A0regulator\u00C2\u00A0of\u00C2\u00A0intracellular\u00C2\u00A0boron\u00C2\u00A0 concentration\u00C2\u00A0 (Nozawa,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 Cells\u00C2\u00A0 lacking\u00C2\u00A0 DUR3\u00C2\u00A0 show\u00C2\u00A0 decreased\u00C2\u00A0 intracellular\u00C2\u00A0 boron\u00C2\u00A0 concentration;\u00C2\u00A0thus,\u00C2\u00A0DUR3\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0function\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0active\u00C2\u00A0transporter\u00C2\u00A0of\u00C2\u00A0boron\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0(Nozawa,\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Although\u00C2\u00A0evidence\u00C2\u00A0suggests\u00C2\u00A0DUR3\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0boron\u00C2\u00A0 transport\u00C2\u00A0and\u00C2\u00A0 regulation,\u00C2\u00A0a\u00C2\u00A0clear\u00C2\u00A0 physiological\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0boron\u00C2\u00A0utilization\u00C2\u00A0has\u00C2\u00A0yet\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0defined.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 other\u00C2\u00A0 important\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 is\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 uptake\u00C2\u00A0 of\u00C2\u00A0 polyamines\u00C2\u00A0 (Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0 and\u00C2\u00A0 Igarashi\u00C2\u00A02007).\u00C2\u00A0Polyamines,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0putrescine,\u00C2\u00A0spermidine,\u00C2\u00A0and\u00C2\u00A0spermine,\u00C2\u00A0are\u00C2\u00A0highly\u00C2\u00A0regulated\u00C2\u00A0peptides\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0 cell\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 proliferation\u00C2\u00A0 (Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0 and\u00C2\u00A0 Igarashi\u00C2\u00A0 2007).\u00C2\u00A0 Their\u00C2\u00A0 function\u00C2\u00A0 is\u00C2\u00A0 ubiquitous\u00C2\u00A0 to\u00C2\u00A0 both\u00C2\u00A0 pro\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 eukaryotes.\u00C2\u00A0 Like\u00C2\u00A0 E.\u00C2\u00A0 coli,\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 possesses\u00C2\u00A0 general\u00C2\u00A0 polyamine\u00C2\u00A0 transporters\u00C2\u00A0 (TPO1\u00E2\u0080\u00904,\u00C2\u00A0 UGA4,\u00C2\u00A0 TPO5,\u00C2\u00A0 GAP1),\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 polyamine\u00C2\u00A0 specific\u00C2\u00A0 transporters\u00C2\u00A0 (AGP2).\u00C2\u00A0 Interestingly,\u00C2\u00A0DUR3\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0specifically\u00C2\u00A0uptake\u00C2\u00A0polyamines\u00C2\u00A0concurrently\u00C2\u00A0with\u00C2\u00A0urea\u00C2\u00A0(Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0and\u00C2\u00A0 Igarashi\u00C2\u00A02007).\u00C2\u00A0As\u00C2\u00A0urea\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0very\u00C2\u00A0poor\u00C2\u00A0nitrogen\u00C2\u00A0source,\u00C2\u00A0and\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0normally\u00C2\u00A0occur\u00C2\u00A0 in\u00C2\u00A0 significant\u00C2\u00A0 quantities\u00C2\u00A0 outside\u00C2\u00A0 the\u00C2\u00A0 cell,\u00C2\u00A0 the\u00C2\u00A0main\u00C2\u00A0 physiological\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0may\u00C2\u00A0 well\u00C2\u00A0 be\u00C2\u00A0 polyamine\u00C2\u00A0 uptake;\u00C2\u00A0 in\u00C2\u00A0 fact,\u00C2\u00A0DUR3\u00C2\u00A0mRNA\u00C2\u00A0 is\u00C2\u00A0 repressed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 large\u00C2\u00A0quantities\u00C2\u00A0of\u00C2\u00A0polyamines\u00C2\u00A0 (Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0and\u00C2\u00A0Igarashi\u00C2\u00A02007).\u00C2\u00A0 \u00C2\u00A0 Most\u00C2\u00A0interesting\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0apparent\u00C2\u00A0post\u00E2\u0080\u0090translational\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0polyamine\u00C2\u00A0uptake\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0 serine/threonine\u00C2\u00A0kinase\u00C2\u00A0PTK2\u00C2\u00A0(Uemura,\u00C2\u00A0Kashiwagi\u00C2\u00A0and\u00C2\u00A0Igarashi\u00C2\u00A02007).\u00C2\u00A0PTK2\u00C2\u00A0seems\u00C2\u00A0to\u00C2\u00A0positively\u00C2\u00A0regulate\u00C2\u00A0 DUR3\u00C2\u00A0polyamine\u00C2\u00A0uptake\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0phosphorylation\u00C2\u00A0of\u00C2\u00A0cytoplasmic\u00C2\u00A0residues\u00C2\u00A0Thr\u00E2\u0080\u0090250,\u00C2\u00A0Ser\u00E2\u0080\u0090251,\u00C2\u00A0and\u00C2\u00A0Thr\u00E2\u0080\u0090684\u00C2\u00A0 (Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0 and\u00C2\u00A0 Igarashi\u00C2\u00A0 2007).\u00C2\u00A0 Although\u00C2\u00A0 DUR3\u00C2\u00A0 polyamine\u00C2\u00A0 activity\u00C2\u00A0 and\u00C2\u00A0 subsequent\u00C2\u00A0 PTK2\u00C2\u00A0 regulation\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0preliminarily\u00C2\u00A0 investigated\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 laboratory\u00C2\u00A0yeast\u00C2\u00A0YPH499\u00C2\u00A0(Uemura,\u00C2\u00A0Kashiwagi\u00C2\u00A0and\u00C2\u00A0 Igarashi\u00C2\u00A02007),\u00C2\u00A0there\u00C2\u00A0are\u00C2\u00A0no\u00C2\u00A0known\u00C2\u00A0studies\u00C2\u00A0which\u00C2\u00A0have\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0mediated\u00C2\u00A0urea\u00C2\u00A0or\u00C2\u00A0 polyamine\u00C2\u00A0 uptake\u00C2\u00A0 during\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentation.\u00C2\u00A0However,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 that\u00C2\u00A0 different\u00C2\u00A0 strains\u00C2\u00A0 of\u00C2\u00A0wine\u00C2\u00A0 25\u00C2\u00A0 \u00C2\u00A0 yeast\u00C2\u00A0react\u00C2\u00A0differentially\u00C2\u00A0 in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0and\u00C2\u00A0biomass\u00C2\u00A0production\u00C2\u00A0 in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0varying\u00C2\u00A0 polyamine\u00C2\u00A0concentrations\u00C2\u00A0(Uemura,\u00C2\u00A0Kashiwagi\u00C2\u00A0and\u00C2\u00A0Igarashi\u00C2\u00A02007).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0with\u00C2\u00A0polyamine\u00C2\u00A0uptake,\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0mediated\u00C2\u00A0uptake\u00C2\u00A0of\u00C2\u00A0boron\u00C2\u00A0seems\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0post\u00E2\u0080\u0090translationally\u00C2\u00A0 regulated.\u00C2\u00A0Although\u00C2\u00A0cells\u00C2\u00A0lacking\u00C2\u00A0DUR3\u00C2\u00A0exhibit\u00C2\u00A0lower\u00C2\u00A0boron\u00C2\u00A0concentrations,\u00C2\u00A0the\u00C2\u00A0converse\u00C2\u00A0situation\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0 true\u00C2\u00A0i.e.\u00C2\u00A0cells\u00C2\u00A0overexpressing\u00C2\u00A0DUR3\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0show\u00C2\u00A0significant\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0boron\u00C2\u00A0concentration\u00C2\u00A0(Nozawa,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 the\u00C2\u00A0 cases\u00C2\u00A0 of\u00C2\u00A0 polyamine\u00C2\u00A0 and\u00C2\u00A0 boron\u00C2\u00A0 uptake\u00C2\u00A0 provide\u00C2\u00A0 good\u00C2\u00A0 evidence\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 existence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0DUR3\u00C2\u00A0regulatory\u00C2\u00A0protein,\u00C2\u00A0presumably\u00C2\u00A0PTK2.\u00C2\u00A0 \u00C2\u00A0 1.11\u00C2\u00A0\u00C2\u00A0Proposed\u00C2\u00A0Research\u00C2\u00A0 1.11.1\u00C2\u00A0\u00C2\u00A0Significance\u00C2\u00A0of\u00C2\u00A0Research\u00C2\u00A0 \u00C2\u00A0 Given\u00C2\u00A0 the\u00C2\u00A0obvious\u00C2\u00A0 governmental\u00C2\u00A0health\u00C2\u00A0 concern\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 EC\u00C2\u00A0 content\u00C2\u00A0of\u00C2\u00A0wines,\u00C2\u00A0 it\u00C2\u00A0 seems\u00C2\u00A0 logical\u00C2\u00A0 to\u00C2\u00A0 pursue\u00C2\u00A0 the\u00C2\u00A0 goal\u00C2\u00A0 of\u00C2\u00A0 producing\u00C2\u00A0wines\u00C2\u00A0 that\u00C2\u00A0 contain\u00C2\u00A0 no\u00C2\u00A0 EC.\u00C2\u00A0Until\u00C2\u00A0 recently\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 advent\u00C2\u00A0 of\u00C2\u00A0 constitutive\u00C2\u00A0 DUR1,2\u00C2\u00A0 expression\u00C2\u00A0 (Coulon,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006),\u00C2\u00A0 existing\u00C2\u00A0 methods\u00C2\u00A0 of\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 were\u00C2\u00A0 cumbersome,\u00C2\u00A0ineffective,\u00C2\u00A0expensive,\u00C2\u00A0and/or\u00C2\u00A0impractical.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0non\u00E2\u0080\u0090transgenic\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0producing\u00C2\u00A0little\u00C2\u00A0or\u00C2\u00A0no\u00C2\u00A0EC\u00C2\u00A0 during\u00C2\u00A0 an\u00C2\u00A0 efficient\u00C2\u00A0 and\u00C2\u00A0 substantially\u00C2\u00A0 equivalent\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentation\u00C2\u00A0would\u00C2\u00A0 be\u00C2\u00A0 of\u00C2\u00A0 direct\u00C2\u00A0 benefit\u00C2\u00A0 to\u00C2\u00A0 industry\u00C2\u00A0and\u00C2\u00A0consumers\u00C2\u00A0alike.\u00C2\u00A0As\u00C2\u00A0such,\u00C2\u00A0the\u00C2\u00A0research\u00C2\u00A0described\u00C2\u00A0herein\u00C2\u00A0 is\u00C2\u00A0both\u00C2\u00A0an\u00C2\u00A0application\u00C2\u00A0of\u00C2\u00A0existing\u00C2\u00A0 technology\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0target,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0proof\u00C2\u00A0of\u00C2\u00A0concept\u00C2\u00A0exploration\u00C2\u00A0of\u00C2\u00A0one\u00C2\u00A0possible\u00C2\u00A0new\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0 EC\u00C2\u00A0reduction.\u00C2\u00A0 \u00C2\u00A0 1.11.2\u00C2\u00A0\u00C2\u00A0Hypotheses\u00C2\u00A0 \u00C2\u00A0 1.11.2.1\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0should\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0efficiently\u00C2\u00A0 during\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0trials.\u00C2\u00A0Given\u00C2\u00A0the\u00C2\u00A0substantially\u00C2\u00A0different\u00C2\u00A0environment\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0mash,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0reasonable\u00C2\u00A0 that\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0highly\u00C2\u00A0superior\u00C2\u00A0when\u00C2\u00A0fermenting\u00C2\u00A0rice\u00C2\u00A0 mash\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0 grape\u00C2\u00A0 must.\u00C2\u00A0 Sake\u00C2\u00A0 yeasts\u00C2\u00A0 have\u00C2\u00A0 evolved\u00C2\u00A0 to\u00C2\u00A0 function\u00C2\u00A0 optimally\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 nutrient\u00C2\u00A0 composition\u00C2\u00A0of\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0\u00E2\u0080\u0098koji\u00E2\u0080\u0099,\u00C2\u00A0thus\u00C2\u00A0the\u00C2\u00A0efficiency\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0should\u00C2\u00A0 also\u00C2\u00A0function\u00C2\u00A0optimally.\u00C2\u00A0Such\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0should\u00C2\u00A0reveal\u00C2\u00A0the\u00C2\u00A0true\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0potential\u00C2\u00A0of\u00C2\u00A0our\u00C2\u00A0recombinant\u00C2\u00A0 26\u00C2\u00A0 \u00C2\u00A0 yeasts\u00C2\u00A0 and\u00C2\u00A0 would\u00C2\u00A0 affirm\u00C2\u00A0 our\u00C2\u00A0 belief\u00C2\u00A0 that\u00C2\u00A0 each\u00C2\u00A0 specific\u00C2\u00A0 yeast\u00C2\u00A0 strain\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 tested\u00C2\u00A0 in\u00C2\u00A0 its\u00C2\u00A0 native\u00C2\u00A0 environment\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0yield\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0accurate\u00C2\u00A0results.\u00C2\u00A0 1.11.2.2\u00C2\u00A0 \u00C2\u00A0Constitutive\u00C2\u00A0co\u00E2\u0080\u0090expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0yeasts\u00C2\u00A0should\u00C2\u00A0 result\u00C2\u00A0in\u00C2\u00A0synergistic\u00C2\u00A0EC\u00C2\u00A0reduction.\u00C2\u00A0DUR1,2/DUR3\u00C2\u00A0yeasts\u00C2\u00A0should\u00C2\u00A0produce\u00C2\u00A0substantially\u00C2\u00A0 less\u00C2\u00A0EC\u00C2\u00A0than\u00C2\u00A0both\u00C2\u00A0 DUR1,2\u00C2\u00A0and\u00C2\u00A0parental\u00C2\u00A0yeasts\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0ability\u00C2\u00A0 to\u00C2\u00A0absorb\u00C2\u00A0native\u00C2\u00A0urea\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0musts\u00C2\u00A0and\u00C2\u00A0 to\u00C2\u00A0reabsorb\u00C2\u00A0 excreted\u00C2\u00A0 urea.\u00C2\u00A0Moreover,\u00C2\u00A0 these\u00C2\u00A0 yeasts\u00C2\u00A0 should\u00C2\u00A0 behave\u00C2\u00A0 like\u00C2\u00A0 their\u00C2\u00A0 parental\u00C2\u00A0 counterparts\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 other\u00C2\u00A0 aspects\u00C2\u00A0of\u00C2\u00A0 fermentation\u00C2\u00A0 i.e.\u00C2\u00A0growth\u00C2\u00A0 rate,\u00C2\u00A0ethanol\u00C2\u00A0production,\u00C2\u00A0CO2\u00C2\u00A0production,\u00C2\u00A0kinetics,\u00C2\u00A0etc.\u00C2\u00A0Obtaining\u00C2\u00A0 these\u00C2\u00A0results\u00C2\u00A0will\u00C2\u00A0affirm\u00C2\u00A0our\u00C2\u00A0belief\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0problem\u00C2\u00A0with\u00C2\u00A0current\u00C2\u00A0DUR1,2\u00C2\u00A0clones\u00C2\u00A0is\u00C2\u00A0their\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0export\u00C2\u00A0 metabolic\u00C2\u00A0urea\u00C2\u00A0before\u00C2\u00A0 it\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0 completely\u00C2\u00A0degraded.\u00C2\u00A0Additionally\u00C2\u00A0DUR1,2\u00C2\u00A0 yeasts\u00C2\u00A0 cannot\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0 degrade\u00C2\u00A0any\u00C2\u00A0urea\u00C2\u00A0natively\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0must/mash\u00C2\u00A0while\u00C2\u00A0DUR1,2/DUR3\u00C2\u00A0yeasts\u00C2\u00A0could.\u00C2\u00A0 \u00C2\u00A0 1.11.3\u00C2\u00A0\u00C2\u00A0Main\u00C2\u00A0objectives\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0main\u00C2\u00A0objectives\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0study\u00C2\u00A0are\u00C2\u00A0to:\u00C2\u00A0 \u00C2\u00A0 1. 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(Gatignol\u00C2\u00A01987)\u00C2\u00A0 pUC18\u00C2\u00A0 High\u00C2\u00A0copy\u00C2\u00A0number\u00C2\u00A0E.\u00C2\u00A0coli\u00C2\u00A0plasmid\u00C2\u00A0that\u00C2\u00A0contains\u00C2\u00A0AmpR,\u00C2\u00A0ori,\u00C2\u00A0 and\u00C2\u00A0an\u00C2\u00A0MCS\u00C2\u00A0located\u00C2\u00A0within\u00C2\u00A0a\u00C2\u00A0lacZ\u00C2\u00A0coding\u00C2\u00A0sequence\u00C2\u00A0thus\u00C2\u00A0 facilitating\u00C2\u00A0cloning\u00C2\u00A0via\u00C2\u00A0blue/white\u00C2\u00A0X\u00E2\u0080\u0090Gal\u00C2\u00A0selection.\u00C2\u00A0 (Yanisch\u00E2\u0080\u0090Perron,\u00C2\u00A0Vieira\u00C2\u00A0 and\u00C2\u00A0Messing\u00C2\u00A01985)\u00C2\u00A0 pUG6\u00C2\u00A0 High\u00C2\u00A0copy\u00C2\u00A0number\u00C2\u00A0E.\u00C2\u00A0coli\u00C2\u00A0plasmid\u00C2\u00A0that\u00C2\u00A0contains\u00C2\u00A0AmpR,\u00C2\u00A0 kanMXR,\u00C2\u00A0and\u00C2\u00A0ori.\u00C2\u00A0 (Guldener,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01996)\u00C2\u00A0 pHVX2\u00C2\u00A0 A\u00C2\u00A0YEplac181\u00C2\u00A0based\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0expression\u00C2\u00A0vector\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0 gene\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0driven\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0constitutive\u00C2\u00A0PGK1\u00C2\u00A0 promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0 (Volschenk,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01997)\u00C2\u00A0 pUCTRP1\u00C2\u00A0 pUC18\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0was\u00C2\u00A0inserted\u00C2\u00A0into\u00C2\u00A0 the\u00C2\u00A0BamH1\u00C2\u00A0site\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0MCS\u00C2\u00A0 This\u00C2\u00A0study\u00C2\u00A0 pHVX2D3\u00C2\u00A0 pHVX2\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0was\u00C2\u00A0inserted\u00C2\u00A0 between\u00C2\u00A0the\u00C2\u00A0PGKp\u00C2\u00A0and\u00C2\u00A0PGKt\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0Xho1\u00C2\u00A0cloning\u00C2\u00A0site\u00C2\u00A0 This\u00C2\u00A0study\u00C2\u00A0 pHVXKD3\u00C2\u00A0 pHVX2D3\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0a\u00C2\u00A0kanMX\u00C2\u00A0resistance\u00C2\u00A0marker\u00C2\u00A0(from\u00C2\u00A0pUG6)\u00C2\u00A0 was\u00C2\u00A0inserted\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0Sal1\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0pHVX2D3\u00C2\u00A0 This\u00C2\u00A0study\u00C2\u00A0 pUCMD\u00C2\u00A0 pUCTRP1\u00C2\u00A0based\u00C2\u00A0plasmid\u00C2\u00A0into\u00C2\u00A0which\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0 cassette\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090PGKp\u00E2\u0080\u0090DUR3\u00E2\u0080\u0090PGKt\u00E2\u0080\u0090kanMX\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0was\u00C2\u00A0PCR\u00C2\u00A0blunt\u00C2\u00A0 end\u00C2\u00A0cloned\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0middle\u00C2\u00A0of\u00C2\u00A0TRP1\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0EcoRV\u00C2\u00A0site\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0study\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A05.\u00C2\u00A0Genetic\u00C2\u00A0cassettes\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0genetic\u00C2\u00A0construction\u00C2\u00A0and\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0expressing\u00C2\u00A0 yeast\u00C2\u00A0strains.\u00C2\u00A0 Cassette\u00C2\u00A0 Description\u00C2\u00A0 Reference\u00C2\u00A0 DUR1,2\u00C2\u00A0 Linear\u00C2\u00A0expression\u00C2\u00A0cassette\u00C2\u00A0containing\u00C2\u00A05\u00E2\u0080\u0099\u00E2\u0080\u0090URA3\u00E2\u0080\u0090PGKp\u00E2\u0080\u0090 DUR1,2\u00E2\u0080\u0090PGKt\u00E2\u0080\u0090URA3\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 (Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006)\u00C2\u00A0 DUR3\u00C2\u00A0 Linear\u00C2\u00A0expression\u00C2\u00A0cassette\u00C2\u00A0containing\u00C2\u00A05\u00E2\u0080\u0099\u00E2\u0080\u0090TRP1\u00E2\u0080\u0090PGKp\u00E2\u0080\u0090 DUR3\u00E2\u0080\u0090PGKt\u00E2\u0080\u0090kanMX\u00E2\u0080\u0090TRP1\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 This\u00C2\u00A0study\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.2\u00C2\u00A0\u00C2\u00A0Culture\u00C2\u00A0conditions\u00C2\u00A0 \u00C2\u00A0 E.\u00C2\u00A0 coli\u00C2\u00A0 DH5\u00CE\u00B1\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0molecular\u00C2\u00A0 cloning\u00C2\u00A0 and\u00C2\u00A0 propagation\u00C2\u00A0 of\u00C2\u00A0 plasmids;\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 standard\u00C2\u00A0 methods\u00C2\u00A0 (Ausubel,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1995).\u00C2\u00A0 Unless\u00C2\u00A0 otherwise\u00C2\u00A0 indicated,\u00C2\u00A0 all\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 strains\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0 aerobically\u00C2\u00A0with\u00C2\u00A0 shaking\u00C2\u00A0 at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0 in\u00C2\u00A0 either\u00C2\u00A0 liquid\u00C2\u00A0 YPD\u00C2\u00A0medium\u00C2\u00A0 (Difco,\u00C2\u00A0 Becton\u00C2\u00A0Dickinson\u00C2\u00A0 and\u00C2\u00A0 Co.,\u00C2\u00A0USA),\u00C2\u00A0 or\u00C2\u00A0 on\u00C2\u00A0 YPD\u00C2\u00A0 +\u00C2\u00A0 2%\u00C2\u00A0 (w/v)\u00C2\u00A0 agar\u00C2\u00A0 (Difco,\u00C2\u00A0 Becton\u00C2\u00A0Dickinson\u00C2\u00A0 and\u00C2\u00A0 Co.,\u00C2\u00A0USA)\u00C2\u00A0 29\u00C2\u00A0 \u00C2\u00A0 plates.\u00C2\u00A0YPD\u00C2\u00A0plates\u00C2\u00A0supplemented\u00C2\u00A0with\u00C2\u00A0300\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0G418\u00C2\u00A0(Sigma,\u00C2\u00A0USA)\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0select\u00C2\u00A0for\u00C2\u00A0positive\u00C2\u00A0S.\u00C2\u00A0 cerevisiae\u00C2\u00A0transformants\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0cassette.\u00C2\u00A0 \u00C2\u00A0 2.3\u00C2\u00A0\u00C2\u00A0Genetic\u00C2\u00A0construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 2.3.1\u00C2\u00A0\u00C2\u00A0Co\u00E2\u0080\u0090transformation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0and\u00C2\u00A0pUT332\u00C2\u00A0 \u00C2\u00A0 S.\u00C2\u00A0cerevisiae\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090transformed\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A09191\u00C2\u00A0bp\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0(Table\u00C2\u00A05)\u00C2\u00A0 and\u00C2\u00A0 pUT332\u00E2\u0088\u0086ura3\u00C2\u00A0 (Gatignol\u00C2\u00A0 1987)\u00C2\u00A0 combined\u00C2\u00A0 at\u00C2\u00A0 a\u00C2\u00A0 10:1\u00C2\u00A0 (DUR1,2\u00C2\u00A0 cassette:pUT332)\u00C2\u00A0molar\u00C2\u00A0 ratio.\u00C2\u00A0 Yeast\u00C2\u00A0 strains\u00C2\u00A0 were\u00C2\u00A0 transformed\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 lithium\u00C2\u00A0 acetate/polyethylene\u00C2\u00A0 glycol/ssDNA\u00C2\u00A0 method\u00C2\u00A0 (Gietz\u00C2\u00A0 and\u00C2\u00A0 Woods\u00C2\u00A02002).\u00C2\u00A0 Following\u00C2\u00A0 transformation,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 left\u00C2\u00A0 to\u00C2\u00A0 recover\u00C2\u00A0 in\u00C2\u00A0YEG\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0 for\u00C2\u00A02\u00C2\u00A0hours\u00C2\u00A0before\u00C2\u00A0 plating\u00C2\u00A0 on\u00C2\u00A0 YEG\u00C2\u00A0 plates\u00C2\u00A0 supplemented\u00C2\u00A0 with\u00C2\u00A0 100\u00C2\u00A0 \u00C2\u00B5g/mL\u00C2\u00A0 phleomycin\u00C2\u00A0 (Invitrogen,\u00C2\u00A0 USA).\u00C2\u00A0 Plates\u00C2\u00A0 were\u00C2\u00A0 incubated\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0colonies\u00C2\u00A0appeared.\u00C2\u00A0 \u00C2\u00A0 2.3.2\u00C2\u00A0\u00C2\u00A0Screening\u00C2\u00A0of\u00C2\u00A0transformants\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 \u00C2\u00A0 Colony\u00C2\u00A0PCR\u00C2\u00A0 (Ward\u00C2\u00A01992)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0 as\u00C2\u00A0described\u00C2\u00A0 to\u00C2\u00A0detect\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 linear\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0yeast\u00C2\u00A0genome\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0URA3\u00C2\u00A0 locus.\u00C2\u00A0Zymolyase\u00C2\u00A0100\u00C2\u00A0U/mL\u00C2\u00A0 (Seikagaku\u00C2\u00A0Corp.,\u00C2\u00A0 Japan)\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 lyse\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0 (30\u00C2\u00A0 \u00C2\u00B5l\u00C2\u00A0 zymolyase\u00C2\u00A0 solution).\u00C2\u00A0 Primers\u00C2\u00A0 InDURURA\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 TGGTGATATGGTTGATTCTGGTGACATA\u00C2\u00A0 \u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 and\u00C2\u00A0 OutURAb\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 TTCCAGCCCATATCCAACTTCCAATTTA\u00C2\u00A0 \u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 were\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0generate\u00C2\u00A0an\u00C2\u00A0approximately\u00C2\u00A01500\u00C2\u00A0bp\u00C2\u00A0fragment\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A03\u00E2\u0080\u0099\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette.\u00C2\u00A0The\u00C2\u00A0primers\u00C2\u00A0 are\u00C2\u00A0 specific\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 inside\u00C2\u00A0 and\u00C2\u00A0 outside\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0 cassette\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 detect\u00C2\u00A0 integration\u00C2\u00A0 and\u00C2\u00A0 correct\u00C2\u00A0orientation.\u00C2\u00A0The\u00C2\u00A0yeasts\u00C2\u00A0522\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0negative\u00C2\u00A0and\u00C2\u00A0positive\u00C2\u00A0controls,\u00C2\u00A0respectively.\u00C2\u00A0 PCR\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0with\u00C2\u00A0iProofTM\u00C2\u00A0High\u00C2\u00A0Fidelity\u00C2\u00A0DNA\u00C2\u00A0polymerase\u00C2\u00A0(BioRad,\u00C2\u00A0USA)\u00C2\u00A0using\u00C2\u00A0suggested\u00C2\u00A0reagent\u00C2\u00A0 concentrations\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5l\u00C2\u00A0of\u00C2\u00A0Zymolyase\u00C2\u00A0treated\u00C2\u00A0cell\u00C2\u00A0suspension\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 template.\u00C2\u00A0The\u00C2\u00A0PCR\u00C2\u00A0program\u00C2\u00A0was\u00C2\u00A0as\u00C2\u00A0 follows:\u00C2\u00A01.\u00C2\u00A0Initial\u00C2\u00A0denaturation\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A03\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A098\u00C2\u00B0C.\u00C2\u00A02.\u00C2\u00A0Denaturation\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A010\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A098\u00C2\u00B0C.\u00C2\u00A03.\u00C2\u00A0Annealing\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A030\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A0 58.5\u00C2\u00B0C.\u00C2\u00A04.\u00C2\u00A0Extension\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A045\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A072\u00C2\u00B0C.\u00C2\u00A05.\u00C2\u00A0Cycle\u00C2\u00A0to\u00C2\u00A0step\u00C2\u00A02,\u00C2\u00A030\u00C2\u00A0times.\u00C2\u00A06.\u00C2\u00A0Final\u00C2\u00A0extension\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A072\u00C2\u00B0C.\u00C2\u00A0\u00C2\u00A0 Colony\u00C2\u00A0PCR\u00C2\u00A0reactions\u00C2\u00A0were\u00C2\u00A0visualized\u00C2\u00A0on\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gels\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0SYBR\u00E2\u0084\u00A2\u00C2\u00A0Safe\u00C2\u00A0(Invitrogen,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0 After\u00C2\u00A0 identification\u00C2\u00A0of\u00C2\u00A0positive\u00C2\u00A0transformants,\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0for\u00C2\u00A0~10\u00C2\u00A0generations\u00C2\u00A0on\u00C2\u00A0 non\u00E2\u0080\u0090selective\u00C2\u00A0media\u00C2\u00A0(YPD)\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0ensure\u00C2\u00A0loss\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0co\u00E2\u0080\u0090transforming\u00C2\u00A0plasmid.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 30\u00C2\u00A0 \u00C2\u00A0 2.3.3\u00C2\u00A0\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0 \u00C2\u00A0 2.3.3.1\u00C2\u00A0 \u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analyses.\u00C2\u00A0 Southern\u00C2\u00A0 blotting\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 confirm\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0URA3\u00C2\u00A0 locus.\u00C2\u00A0Genomic\u00C2\u00A0DNA\u00C2\u00A0 from\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 their\u00C2\u00A0 respective\u00C2\u00A0parent\u00C2\u00A0strains\u00C2\u00A0was\u00C2\u00A0digested\u00C2\u00A0with\u00C2\u00A0BglII\u00C2\u00A0(Roche,\u00C2\u00A0Germany)\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995),\u00C2\u00A0and\u00C2\u00A0separated\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 0.8%\u00C2\u00A0 agarose\u00C2\u00A0 gel.\u00C2\u00A0 Following\u00C2\u00A0 gel\u00C2\u00A0 preparation,\u00C2\u00A0 transfer\u00C2\u00A0 and\u00C2\u00A0 fixing\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 positively\u00C2\u00A0 charged\u00C2\u00A0 Nylon\u00C2\u00A0 membrane\u00C2\u00A0 (Roche\u00C2\u00A0 Diagnostics,\u00C2\u00A0 Germany)\u00C2\u00A0 (Ausubel,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1995),\u00C2\u00A0 the\u00C2\u00A0 blots\u00C2\u00A0 were\u00C2\u00A0 probed\u00C2\u00A0 with\u00C2\u00A0 PCR\u00C2\u00A0 generated\u00C2\u00A0fragments\u00C2\u00A0specific\u00C2\u00A0for\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0URA3.\u00C2\u00A0The\u00C2\u00A0AlkPhosTM\u00C2\u00A0Direct\u00C2\u00A0Nucleic\u00C2\u00A0Acid\u00C2\u00A0Labeling\u00C2\u00A0and\u00C2\u00A0CDP\u00E2\u0080\u0090 Star\u00C2\u00A0Detection\u00C2\u00A0system\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0recommended\u00C2\u00A0for\u00C2\u00A0probe\u00C2\u00A0detection\u00C2\u00A0(Amersham\u00C2\u00A0Biosciences,\u00C2\u00A0England).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0736\u00C2\u00A0bp\u00C2\u00A0DUR1,2\u00C2\u00A0probe\u00C2\u00A0was\u00C2\u00A0generated\u00C2\u00A0by\u00C2\u00A0PCR\u00C2\u00A0using\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0 from\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0strain\u00C2\u00A0 522\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0template\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0primers\u00C2\u00A0DUR1,2probe5\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090TTAGACTGCGTCTCCATCTTTG\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0and\u00C2\u00A0DUR1,2F\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090 TGCTGGCTTTACTGAAGAAGAG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0The\u00C2\u00A0927\u00C2\u00A0bp\u00C2\u00A0URA3\u00C2\u00A0probe\u00C2\u00A0was\u00C2\u00A0generated\u00C2\u00A0by\u00C2\u00A0PCR\u00C2\u00A0using\u00C2\u00A0522\u00C2\u00A0genomic\u00C2\u00A0 DNA\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 primers\u00C2\u00A0 3\u00E2\u0080\u0099URA3\u00C2\u00A0 (5'\u00E2\u0080\u0090TGGGAAGCATATTTGAGAAGATG\u00E2\u0080\u00903')\u00C2\u00A0 and\u00C2\u00A0 OutURAb\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 TTCCAGCCCATATCCAACTTCCAATTTA\u00C2\u00A0\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0 \u00C2\u00A0 2.3.3.2\u00C2\u00A0\u00C2\u00A0Sequence\u00C2\u00A0analysis.\u00C2\u00A0Genomic\u00C2\u00A0DNA\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0amplify\u00C2\u00A0two\u00C2\u00A0 separate\u00C2\u00A0 fragments\u00C2\u00A0which\u00C2\u00A0 together\u00C2\u00A0encompassed\u00C2\u00A0 the\u00C2\u00A0entire\u00C2\u00A0~10\u00C2\u00A0kb\u00C2\u00A0 linear\u00C2\u00A0ura3\u00E2\u0080\u0090PGK1p\u00E2\u0080\u0090DUR1,2\u00E2\u0080\u0090PGK1t\u00E2\u0080\u0090 ura3\u00C2\u00A0 cassette.\u00C2\u00A0 Primers\u00C2\u00A0 5\u00E2\u0080\u0099OUTURA3Cas\u00C2\u00A0 (5'\u00E2\u0080\u0090AACTAATGAGATGGAATCGGTAG\u00E2\u0080\u00903')\u00C2\u00A0 and\u00C2\u00A0 DUR12rev1\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090 TCCTGGAATGCTGTGATGG\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0amplified\u00C2\u00A0a\u00C2\u00A07900\u00C2\u00A0bp\u00C2\u00A0fragment\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A05\u00E2\u0080\u0099\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette,\u00C2\u00A0while\u00C2\u00A0primers\u00C2\u00A0 PGK1forDUR\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090TGGTTTAGTTTAGTAGAACCTCGTGAAACTTAC\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 and\u00C2\u00A0 OutURAb\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 TTCCAGCCCATATCCAACTTCCAATTTA\u00C2\u00A0\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0amplified\u00C2\u00A0a\u00C2\u00A07100\u00C2\u00A0bp\u00C2\u00A0fragment\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A03\u00E2\u0080\u0099\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Sequencing\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0Nucleic\u00C2\u00A0Acid\u00C2\u00A0Protein\u00C2\u00A0Service\u00C2\u00A0Unit\u00C2\u00A0(NAPS)\u00C2\u00A0at\u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0 British\u00C2\u00A0Columbia\u00C2\u00A0using\u00C2\u00A0an\u00C2\u00A0Applied\u00C2\u00A0Biosystems\u00C2\u00A0PRISM\u00C2\u00A0377\u00C2\u00A0sequencer\u00C2\u00A0and\u00C2\u00A0Applied\u00C2\u00A0Biosystems\u00C2\u00A0BigDye\u00C2\u00A0v3.1\u00C2\u00A0 sequencing\u00C2\u00A0chemistry.\u00C2\u00A0Primers\u00C2\u00A0and\u00C2\u00A0template\u00C2\u00A0were\u00C2\u00A0supplied\u00C2\u00A0to\u00C2\u00A0NAPS\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0concentrations\u00C2\u00A0specified\u00C2\u00A0by\u00C2\u00A0 their\u00C2\u00A0 sample\u00C2\u00A0 submission\u00C2\u00A0 requirements.\u00C2\u00A0 The\u00C2\u00A0 entire\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0was\u00C2\u00A0 sequenced\u00C2\u00A0 via\u00C2\u00A0 19\u00C2\u00A0 different\u00C2\u00A0sequencing\u00C2\u00A0reads\u00C2\u00A0(Table\u00C2\u00A06)\u00C2\u00A0and\u00C2\u00A0later\u00C2\u00A0assembled\u00C2\u00A0in\u00C2\u00A0silico\u00C2\u00A0using\u00C2\u00A0Accelrys\u00C2\u00A0DS\u00C2\u00A0Gene\u00C2\u00A0v1.1\u00C2\u00A0software.\u00C2\u00A0 The\u00C2\u00A0assembled\u00C2\u00A0 sequences\u00C2\u00A0were\u00C2\u00A0aligned\u00C2\u00A0against\u00C2\u00A0previously\u00C2\u00A0published\u00C2\u00A0 sequences\u00C2\u00A0and\u00C2\u00A0 those\u00C2\u00A0of\u00C2\u00A0DUR1,2,\u00C2\u00A0 URA3,\u00C2\u00A0PGK1p,\u00C2\u00A0and\u00C2\u00A0PGK1t\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0SGD.\u00C2\u00A0 If\u00C2\u00A0any\u00C2\u00A0discrepancies\u00C2\u00A0were\u00C2\u00A0 found,\u00C2\u00A0 the\u00C2\u00A0specific\u00C2\u00A0read\u00C2\u00A0which\u00C2\u00A0 gave\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0discrepancy\u00C2\u00A0was\u00C2\u00A0repeated\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0bona\u00C2\u00A0fide\u00C2\u00A0mutations.\u00C2\u00A0\u00C2\u00A0 31\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A06.\u00C2\u00A0\u00C2\u00A0Oligonucleotide\u00C2\u00A0primers\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0sequencing\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette.\u00C2\u00A0\u00C2\u00A0 Primer\u00C2\u00A0 Primer\u00C2\u00A0Name\u00C2\u00A0 Sequence\u00C2\u00A0(5\u00E2\u0080\u0099\u00C3\u00863\u00E2\u0080\u0099)\u00C2\u00A0 P1\u00C2\u00A0 5'OUTURA3Cas\u00C2\u00A0 5'\u00E2\u0080\u0090AACTAATGAGATGGAATCGGTAG\u00E2\u0080\u00903'\u00C2\u00A0 P2\u00C2\u00A0 5'URA3Flank\u00C2\u00A0 5'\u00E2\u0080\u0090AGTATTCTTAACCCAACTGCACAGA\u00E2\u0080\u00903'\u00C2\u00A0 P3\u00C2\u00A0 5'PGK1pro1\u00C2\u00A0 5'\u00E2\u0080\u0090ACAAAATCTTCTTGACAAACGTCACAA3'\u00C2\u00A0 P4\u00C2\u00A0 5'PGK1pro2\u00C2\u00A0 5'\u00E2\u0080\u0090AATTGATGTTACCCTCATAAAGCACGT\u00E2\u0080\u00903'\u00C2\u00A0 P5\u00C2\u00A0 PGK1forDUR\u00C2\u00A0 5'\u00E2\u0080\u0090TGGTTTAGTTTAGTAGAACCTCGTGAAACTTAC\u00E2\u0080\u00903'\u00C2\u00A0 P6\u00C2\u00A0 DUR12G\u00C2\u00A0 5'\u00E2\u0080\u0090TACCAGAACCTGCTGTATCAG\u00E2\u0080\u00903'\u00C2\u00A0 P7\u00C2\u00A0 DUR12rev6\u00C2\u00A0 5'\u00E2\u0080\u0090TCATCCGCAACTTGTTGCATAG\u00E2\u0080\u00903'\u00C2\u00A0 P8\u00C2\u00A0 DUR12F\u00C2\u00A0 5'\u00E2\u0080\u0090TGCTGGCTTTACTGAAGAAGAG\u00E2\u0080\u00903'\u00C2\u00A0 P9\u00C2\u00A0 DUR12rev5\u00C2\u00A0 5'\u00E2\u0080\u0090TCGGAATAAACTGCAACTGATC\u00E2\u0080\u00903'\u00C2\u00A0 P10\u00C2\u00A0 DUR12E\u00C2\u00A0 5'\u00E2\u0080\u0090ACCTCTGATAATATCTCCCGAAG\u00E2\u0080\u00903'\u00C2\u00A0 P11\u00C2\u00A0 DUR12D\u00C2\u00A0 5'\u00E2\u0080\u0090TTTTGGCCAATGTTGGATCATATTC\u00E2\u0080\u00903'\u00C2\u00A0 P12\u00C2\u00A0 DUR12rev3\u00C2\u00A0 5'\u00E2\u0080\u0090TGTCAACTTGCCAATGGATAAAGTAG\u00E2\u0080\u00903'\u00C2\u00A0 P13\u00C2\u00A0 DUR12C\u00C2\u00A0 5'\u00E2\u0080\u0090TTGTAATGAACCTTCCACTTCTC\u00E2\u0080\u00903'\u00C2\u00A0 P14\u00C2\u00A0 DUR12B\u00C2\u00A0 5'\u00E2\u0080\u0090ACACATGCCAAAGTCTTCGAG3'\u00C2\u00A0 P15\u00C2\u00A0 DUR12A\u00C2\u00A0 5'\u00E2\u0080\u0090ATTTCCAAAAACGCCCAGAATAC\u00E2\u0080\u00903'\u00C2\u00A0 P16\u00C2\u00A0 DUR12for3end\u00C2\u00A0 5'\u00E2\u0080\u0090TCATCAAGAATACTTGAGATGGATC\u00E2\u0080\u00903'\u00C2\u00A0 P17\u00C2\u00A0 InDURURA\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TGGTGATATGGTTGATTCTGGTGACATA\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P18\u00C2\u00A0 3'URA3\u00C2\u00A0 5'\u00E2\u0080\u0090TGGGAAGCATATTTGAGAAGATG\u00E2\u0080\u00903'\u00C2\u00A0 P19\u00C2\u00A0 OutURAb\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TTCCAGCCCATATCCAACTTCCAATTTA\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.3.3\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR.\u00C2\u00A0Single\u00C2\u00A0colonies\u00C2\u00A0of\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0 engineered\u00C2\u00A0strains\u00C2\u00A0from\u00C2\u00A0freshly\u00C2\u00A0streaked\u00C2\u00A0YPD\u00C2\u00A0plates\u00C2\u00A0were\u00C2\u00A0inoculated\u00C2\u00A0into\u00C2\u00A05\u00C2\u00A0mL\u00C2\u00A0YPD\u00C2\u00A0and\u00C2\u00A0grown\u00C2\u00A0overnight\u00C2\u00A0 at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0rotary\u00C2\u00A0wheel.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0subcultured\u00C2\u00A0 into\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0YPD\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.05)\u00C2\u00A0and\u00C2\u00A0again\u00C2\u00A0grown\u00C2\u00A0 overnight\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0water\u00C2\u00A0shaker\u00C2\u00A0bath\u00C2\u00A0(180\u00C2\u00A0rpm).\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0(5000\u00C2\u00A0rpm,\u00C2\u00A0 4\u00C2\u00B0C,\u00C2\u00A05\u00C2\u00A0min)\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0once\u00C2\u00A0with\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0 sterile\u00C2\u00A0water.\u00C2\u00A0Cell\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A05\u00C2\u00A0mL\u00C2\u00A0 sterile\u00C2\u00A0 water\u00C2\u00A0and\u00C2\u00A0OD600\u00C2\u00A0measured.\u00C2\u00A0Cell\u00C2\u00A0suspensions\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0inoculate\u00C2\u00A0sterile\u00C2\u00A0250\u00C2\u00A0mL\u00C2\u00A0Schott\u00C2\u00A0bottles\u00C2\u00A0filled\u00C2\u00A0 with\u00C2\u00A0200\u00C2\u00A0mL\u00C2\u00A0 filter\u00C2\u00A0 sterilized\u00C2\u00A0 (0.22\u00C2\u00A0\u00C2\u00B5m,\u00C2\u00A0Millipore,\u00C2\u00A0USA)\u00C2\u00A0Calona\u00C2\u00A0Chardonnay\u00C2\u00A0 juice\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0 final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1.\u00C2\u00A0 Bottles\u00C2\u00A0were\u00C2\u00A0 aseptically\u00C2\u00A0 sealed\u00C2\u00A0with\u00C2\u00A0 vapour\u00C2\u00A0 locks\u00C2\u00A0 (sterilized\u00C2\u00A0with\u00C2\u00A0 70%\u00C2\u00A0 v/v\u00C2\u00A0 ethanol)\u00C2\u00A0 filled\u00C2\u00A0with\u00C2\u00A0 sterile\u00C2\u00A0 water.\u00C2\u00A0Sealed\u00C2\u00A0bottles\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A020\u00C2\u00B0C\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0hours.\u00C2\u00A0 \u00C2\u00A0 Total\u00C2\u00A0RNA\u00C2\u00A0from\u00C2\u00A024\u00C2\u00A0hour\u00C2\u00A0fermentations\u00C2\u00A0was\u00C2\u00A0extracted\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0hot\u00C2\u00A0phenol\u00C2\u00A0method\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A01995);\u00C2\u00A0RNA\u00C2\u00A0was\u00C2\u00A0cleaned\u00C2\u00A0up\u00C2\u00A0post\u00C2\u00A0extraction\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0total\u00C2\u00A0RNeasy\u00C2\u00A0Midi\u00C2\u00A0Kit\u00C2\u00A0(Qiagen),\u00C2\u00A0and\u00C2\u00A0quantified\u00C2\u00A0on\u00C2\u00A0 a\u00C2\u00A0 Pharmacia\u00C2\u00A0Ultrospec\u00C2\u00A0 3000\u00C2\u00A0UV/Vis\u00C2\u00A0 spectrophotometer.\u00C2\u00A0 Clean\u00C2\u00A0 total\u00C2\u00A0 RNA\u00C2\u00A0 (1\u00C2\u00A0 \u00C2\u00B5g)\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0 cDNA\u00C2\u00A0 synthesis\u00C2\u00A0 (iScriptTM,\u00C2\u00A0BioRad,\u00C2\u00A0USA)\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions.\u00C2\u00A0 \u00C2\u00A0 iTAQ\u00E2\u0084\u00A2\u00C2\u00A0 SYBR\u00C2\u00AE\u00C2\u00A0Green\u00C2\u00A0 32\u00C2\u00A0 \u00C2\u00A0 Supermix\u00C2\u00A0with\u00C2\u00A0ROX\u00C2\u00A0(BioRad,\u00C2\u00A0USA)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0conjunction\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0Applied\u00C2\u00A0Biosystems\u00C2\u00A07500\u00C2\u00A0Real\u00C2\u00A0Time\u00C2\u00A0 PCR\u00C2\u00A0machine\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 strains\u00C2\u00A0 studied.\u00C2\u00A0Real\u00C2\u00A0 time\u00C2\u00A0 primers\u00C2\u00A0 for\u00C2\u00A0 both\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0 ACT1\u00C2\u00A0were\u00C2\u00A0 automatically\u00C2\u00A0 optimized\u00C2\u00A0 and\u00C2\u00A0 designed\u00C2\u00A0 using\u00C2\u00A0 Applied\u00C2\u00A0 Biosystem\u00E2\u0080\u0099s\u00C2\u00A0Primer\u00C2\u00A0ExpressTM\u00C2\u00A0v2.0\u00C2\u00A0software.\u00C2\u00A0The\u00C2\u00A0DUR1,2\u00C2\u00A0amplification\u00C2\u00A0product\u00C2\u00A0was\u00C2\u00A0amplified\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0 primers\u00C2\u00A0 DUR12RTfwd\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090CTCTGGTCCAATGGACGCATA\u00C2\u00A0 \u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 DUR12RTrev\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090 GATGGATGGACCAGTCAACGTT\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0 ACT1\u00C2\u00A0 was\u00C2\u00A0 amplified\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 primers\u00C2\u00A0 act1forward\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090 GTTTCCATCCAAGCCGTTTTG\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0and\u00C2\u00A0act1reverse\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090GCGTAAATTGGAACGACGTGAG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0 strain,\u00C2\u00A0 DUR1,2\u00C2\u00A0and\u00C2\u00A0ACT1\u00C2\u00A0expression\u00C2\u00A0was\u00C2\u00A0analyzed\u00C2\u00A0six\u00C2\u00A0times\u00C2\u00A0and\u00C2\u00A0results\u00C2\u00A0were\u00C2\u00A0averaged.\u00C2\u00A0RQ\u00C2\u00A0data\u00C2\u00A0were\u00C2\u00A0analyzed\u00C2\u00A0 using\u00C2\u00A0the\u00C2\u00A0Applied\u00C2\u00A0Biosystems\u00C2\u00A0RQ\u00C2\u00A0Study\u00C2\u00A0software\u00C2\u00A0v1.2.2.\u00C2\u00A0 \u00C2\u00A0 2.3.3.4\u00C2\u00A0 \u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0 strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0was\u00C2\u00A0extracted\u00C2\u00A0after\u00C2\u00A024\u00C2\u00A0 hours\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0(Section\u00C2\u00A02.3.3.3),\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0hot\u00C2\u00A0phenol\u00C2\u00A0method\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995),\u00C2\u00A0quantified\u00C2\u00A0on\u00C2\u00A0 a\u00C2\u00A0NanoDrop\u00C2\u00A0ND\u00E2\u0080\u00901000\u00C2\u00A0spectrophotometer\u00C2\u00A0and\u00C2\u00A0visualized\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gel.\u00C2\u00A0A\u00C2\u00A0\u00E2\u0080\u0098GeneChip\u00C2\u00AE\u00C2\u00A0One\u00E2\u0080\u0090Cycle\u00C2\u00A0 Target\u00C2\u00A0Labeling\u00C2\u00A0and\u00C2\u00A0Control\u00E2\u0080\u0099\u00C2\u00A0kit\u00C2\u00A0(Affymetrix,\u00C2\u00A0USA)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0instructions\u00C2\u00A0for\u00C2\u00A0 synthesis\u00C2\u00A0and\u00C2\u00A0clean\u00C2\u00A0up\u00C2\u00A0of\u00C2\u00A0cDNA\u00C2\u00A0and\u00C2\u00A0for\u00C2\u00A0synthesis,\u00C2\u00A0cleanup\u00C2\u00A0and\u00C2\u00A0fragmentation\u00C2\u00A0of\u00C2\u00A0biotinylated\u00C2\u00A0cRNA\u00C2\u00A0from\u00C2\u00A0 10\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0high\u00C2\u00A0quality\u00C2\u00A0total\u00C2\u00A0RNA.\u00C2\u00A0Microarray\u00C2\u00A0analyses\u00C2\u00A0were\u00C2\u00A0done\u00C2\u00A0 in\u00C2\u00A0duplicate,\u00C2\u00A0each\u00C2\u00A0with\u00C2\u00A0 independently\u00C2\u00A0 grown\u00C2\u00A0cell\u00C2\u00A0cultures.\u00C2\u00A0 \u00C2\u00A0 Four\u00C2\u00A0oligonucleotide\u00C2\u00A0yeast\u00C2\u00A0genome\u00C2\u00A0arrays,\u00C2\u00A0two\u00C2\u00A0per\u00C2\u00A0strain,\u00C2\u00A0(YGS98;\u00C2\u00A0Affymetrix,\u00C2\u00A0USA)\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0 for\u00C2\u00A0 hybridization\u00C2\u00A0 of\u00C2\u00A0 fragmented\u00C2\u00A0 labelled\u00C2\u00A0 cRNA.\u00C2\u00A0 Preparation\u00C2\u00A0 of\u00C2\u00A0 hybridization\u00C2\u00A0 solution,\u00C2\u00A0 hybridization,\u00C2\u00A0 washing,\u00C2\u00A0 staining,\u00C2\u00A0 and\u00C2\u00A0 scanning\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 microarrays\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 as\u00C2\u00A0 per\u00C2\u00A0 the\u00C2\u00A0 manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions\u00C2\u00A0 (Eukaryotic\u00C2\u00A0Array\u00C2\u00A0Gene\u00C2\u00A0Chip\u00C2\u00A0Expression\u00C2\u00A0Analysis\u00C2\u00A0and\u00C2\u00A0Technical\u00C2\u00A0Manual;\u00C2\u00A0Affymetrix,\u00C2\u00A0USA).\u00C2\u00A0 The\u00C2\u00A0 EukGE\u00E2\u0080\u0090WS2v4\u00C2\u00A0 fluidics\u00C2\u00A0 protocol\u00C2\u00A0 of\u00C2\u00A0Affymetrix\u00C2\u00A0MASv5.0\u00C2\u00A0 software\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0 array\u00C2\u00A0 staining\u00C2\u00A0 and\u00C2\u00A0 washing\u00C2\u00A0 procedures\u00C2\u00A0 while\u00C2\u00A0 arrays\u00C2\u00A0 were\u00C2\u00A0 scanned\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 G2500A\u00C2\u00A0 GeneArray\u00C2\u00A0 Scanner\u00C2\u00A0 (Agilent\u00C2\u00A0 Technologies,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 were\u00C2\u00A0 analyzed\u00C2\u00A0 with\u00C2\u00A0MASv5.0\u00C2\u00A0 and\u00C2\u00A0 DMT\u00C2\u00A0 software\u00C2\u00A0 (Affymetrix,\u00C2\u00A0 USA)\u00C2\u00A0 running\u00C2\u00A0 on\u00C2\u00A0 default\u00C2\u00A0 settings\u00C2\u00A0 (Affymetrix\u00C2\u00A0 Statistical\u00C2\u00A0 Algorithm\u00C2\u00A0 Reference\u00C2\u00A0 Guide).\u00C2\u00A0 Statistically\u00C2\u00A0 significant\u00C2\u00A0 and\u00C2\u00A0 reproducible\u00C2\u00A0 results\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0only\u00C2\u00A0 including\u00C2\u00A0genes\u00C2\u00A0which\u00C2\u00A0responded\u00C2\u00A0similarly\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0 four\u00C2\u00A0cross\u00C2\u00A0comparisons\u00C2\u00A0 and\u00C2\u00A0with\u00C2\u00A0change\u00C2\u00A0p\u00E2\u0080\u0090values\u00C2\u00A0of\u00C2\u00A0\u00E2\u0089\u00A40.005\u00C2\u00A0(increasers)\u00C2\u00A0or\u00C2\u00A0\u00E2\u0089\u00A50.995\u00C2\u00A0(decreasers).\u00C2\u00A0Reported\u00C2\u00A0fold\u00C2\u00A0change\u00C2\u00A0values\u00C2\u00A0are\u00C2\u00A0 derived\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0average\u00C2\u00A0(n=4)\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Signal\u00C2\u00A0Log\u00C2\u00A0(base\u00C2\u00A02)\u00C2\u00A0Ratio\u00C2\u00A0(SLR).\u00C2\u00A0Array\u00C2\u00A0annotations\u00C2\u00A0were\u00C2\u00A0linked\u00C2\u00A0to\u00C2\u00A0 33\u00C2\u00A0 \u00C2\u00A0 their\u00C2\u00A0 gene\u00C2\u00A0 ontology\u00C2\u00A0 (GO)\u00C2\u00A0 annotations\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u0098gene_association.sgd.tab\u00E2\u0080\u0099\u00C2\u00A0 table\u00C2\u00A0 (http://www.yeastgenome.org/gene_list.shtml).\u00C2\u00A0 \u00C2\u00A0 2.3.4\u00C2\u00A0\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0 \u00C2\u00A0 2.3.4.1\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0must.\u00C2\u00A0Single\u00C2\u00A0colonies\u00C2\u00A0of\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0(K7\u00C2\u00A0and\u00C2\u00A0 K9)\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0appropriate\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0from\u00C2\u00A0freshly\u00C2\u00A0streaked\u00C2\u00A0YPD\u00C2\u00A0plates,\u00C2\u00A0were\u00C2\u00A0 inoculated\u00C2\u00A0 into\u00C2\u00A05\u00C2\u00A0 mL\u00C2\u00A0YPD\u00C2\u00A0and\u00C2\u00A0grown\u00C2\u00A0overnight\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0on\u00C2\u00A0 \u00C2\u00A0a\u00C2\u00A0rotary\u00C2\u00A0wheel.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0subcultured\u00C2\u00A0 into\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0YPD\u00C2\u00A0(final\u00C2\u00A0 OD600\u00C2\u00A0=\u00C2\u00A00.05)\u00C2\u00A0and\u00C2\u00A0again\u00C2\u00A0grown\u00C2\u00A0overnight\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0water\u00C2\u00A0shaker\u00C2\u00A0bath\u00C2\u00A0(180\u00C2\u00A0rpm).\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0 by\u00C2\u00A0centrifugation\u00C2\u00A0 (5000\u00C2\u00A0rpm,\u00C2\u00A04\u00C2\u00B0C,\u00C2\u00A05\u00C2\u00A0min)\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0once\u00C2\u00A0with\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0sterile\u00C2\u00A0water.\u00C2\u00A0Cell\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0in\u00C2\u00A05\u00C2\u00A0mL\u00C2\u00A0sterile\u00C2\u00A0water\u00C2\u00A0and\u00C2\u00A0OD600\u00C2\u00A0measured.\u00C2\u00A0Cell\u00C2\u00A0suspensions\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0inoculate\u00C2\u00A0sterile\u00C2\u00A0 250\u00C2\u00A0mL\u00C2\u00A0Schott\u00C2\u00A0bottles\u00C2\u00A0filled\u00C2\u00A0with\u00C2\u00A0200\u00C2\u00A0mL\u00C2\u00A0unfiltered\u00C2\u00A0Chardonnay\u00C2\u00A0 juice\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Calona\u00C2\u00A0Vineyards,\u00C2\u00A0 Kelowna,\u00C2\u00A0 BC,\u00C2\u00A0 Canada\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 final\u00C2\u00A0OD600\u00C2\u00A0 =\u00C2\u00A0 0.1.\u00C2\u00A0 Bottles\u00C2\u00A0were\u00C2\u00A0 aseptically\u00C2\u00A0 sealed\u00C2\u00A0with\u00C2\u00A0 sterilized\u00C2\u00A0 (70%\u00C2\u00A0 v/v\u00C2\u00A0 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sieve\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0placed\u00C2\u00A0over\u00C2\u00A0a\u00C2\u00A0pot\u00C2\u00A0of\u00C2\u00A0boiling\u00C2\u00A0water\u00C2\u00A0and\u00C2\u00A0covered\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0bamboo\u00C2\u00A0steamer\u00C2\u00A0lid.\u00C2\u00A0The\u00C2\u00A0sieve\u00C2\u00A0was\u00C2\u00A0 arranged\u00C2\u00A0 in\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0way\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0rice\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0 in\u00C2\u00A0direct\u00C2\u00A0contact\u00C2\u00A0with\u00C2\u00A0boiling\u00C2\u00A0water.\u00C2\u00A0After\u00C2\u00A0steaming\u00C2\u00A0until\u00C2\u00A0 soft\u00C2\u00A0and\u00C2\u00A0slightly\u00C2\u00A0transparent,\u00C2\u00A0the\u00C2\u00A0cooked\u00C2\u00A0rice\u00C2\u00A0was\u00C2\u00A0placed\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0stainless\u00C2\u00A0steel\u00C2\u00A0bowl\u00C2\u00A0and\u00C2\u00A0cooled\u00C2\u00A0to\u00C2\u00A0~30\u00C2\u00B0C.\u00C2\u00A0 Upon\u00C2\u00A0 reaching\u00C2\u00A0 30\u00C2\u00B0C,\u00C2\u00A0 the\u00C2\u00A0 cooled\u00C2\u00A0 rice\u00C2\u00A0 was\u00C2\u00A0 inoculated\u00C2\u00A0 with\u00C2\u00A0 1.5\u00C2\u00A0 g\u00C2\u00A0 of\u00C2\u00A0 koji\u00C2\u00A0 seeds\u00C2\u00A0 (Vision\u00C2\u00A0 Brewing,\u00C2\u00A0 Washington,\u00C2\u00A0USA)\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0teaspoon\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0purpose\u00C2\u00A0white\u00C2\u00A0flour.\u00C2\u00A0The\u00C2\u00A0rice\u00C2\u00A0was\u00C2\u00A0mixed\u00C2\u00A0well,\u00C2\u00A0covered\u00C2\u00A0 with\u00C2\u00A0a\u00C2\u00A0piece\u00C2\u00A0of\u00C2\u00A0moist\u00C2\u00A0Whatman\u00C2\u00A0No.\u00C2\u00A03\u00C2\u00A0paper,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0bowl\u00C2\u00A0was\u00C2\u00A0 sealed\u00C2\u00A0with\u00C2\u00A0plastic\u00C2\u00A0 film.\u00C2\u00A0The\u00C2\u00A0covered\u00C2\u00A0 bowl\u00C2\u00A0was\u00C2\u00A0incubated,\u00C2\u00A0with\u00C2\u00A0occasional\u00C2\u00A0mixing,\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0for\u00C2\u00A048\u00C2\u00A0hours,\u00C2\u00A0or\u00C2\u00A0until\u00C2\u00A0the\u00C2\u00A0rice\u00C2\u00A0grains\u00C2\u00A0were\u00C2\u00A0covered\u00C2\u00A0 with\u00C2\u00A0fine\u00C2\u00A0white\u00C2\u00A0fibres\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0entire\u00C2\u00A0mixture\u00C2\u00A0had\u00C2\u00A0a\u00C2\u00A0cheese\u00E2\u0080\u0090like\u00C2\u00A0aroma.\u00C2\u00A0The\u00C2\u00A0koji\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0 sterile\u00C2\u00A0500\u00C2\u00A0mL\u00C2\u00A0centrifuge\u00C2\u00A0bottles\u00C2\u00A0and\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A0\u00E2\u0080\u009030\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0use.\u00C2\u00A0 \u00C2\u00A0 Single\u00C2\u00A0colonies\u00C2\u00A0of\u00C2\u00A0parental\u00C2\u00A0 strains\u00C2\u00A0 (K7\u00C2\u00A0and\u00C2\u00A0K9)\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0appropriate\u00C2\u00A0engineered\u00C2\u00A0 strains\u00C2\u00A0 from\u00C2\u00A0 freshly\u00C2\u00A0 streaked\u00C2\u00A0YPD\u00C2\u00A0plates,\u00C2\u00A0 \u00C2\u00A0were\u00C2\u00A0 inoculated\u00C2\u00A0 into\u00C2\u00A05mL\u00C2\u00A0YPD\u00C2\u00A0and\u00C2\u00A0grown\u00C2\u00A0overnight\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 rotary\u00C2\u00A0 34\u00C2\u00A0 \u00C2\u00A0 wheel.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0subcultured\u00C2\u00A0into\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0YPD\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.05)\u00C2\u00A0and\u00C2\u00A0again\u00C2\u00A0grown\u00C2\u00A0overnight\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0 water\u00C2\u00A0shaker\u00C2\u00A0bath\u00C2\u00A0(180\u00C2\u00A0rpm).\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0(5000\u00C2\u00A0rpm,\u00C2\u00A04\u00C2\u00B0C,\u00C2\u00A05\u00C2\u00A0min)\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0 once\u00C2\u00A0with\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0sterile\u00C2\u00A0water.\u00C2\u00A0Cell\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A05\u00C2\u00A0mL\u00C2\u00A0sterile\u00C2\u00A0water\u00C2\u00A0and\u00C2\u00A0OD600\u00C2\u00A0measured.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0cell\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0inoculate\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1)\u00C2\u00A0sterile\u00C2\u00A0250\u00C2\u00A0mL\u00C2\u00A0Schott\u00C2\u00A0bottles\u00C2\u00A0filled\u00C2\u00A0 with\u00C2\u00A013\u00C2\u00A0g\u00C2\u00A0\u00E2\u0080\u0098koji\u00E2\u0080\u0099\u00C2\u00A0rice,\u00C2\u00A048\u00C2\u00A0g\u00C2\u00A0freshly\u00C2\u00A0steamed\u00C2\u00A0rice\u00C2\u00A0(steamed\u00C2\u00A0as\u00C2\u00A0per\u00C2\u00A0\u00E2\u0080\u0098koji\u00E2\u0080\u0099\u00C2\u00A0preparation\u00C2\u00A0above),\u00C2\u00A0and\u00C2\u00A0100\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0 water\u00C2\u00A0 containing\u00C2\u00A0 0.125\u00C2\u00A0 g/L\u00C2\u00A0 citric\u00C2\u00A0 acid.\u00C2\u00A0 Bottles\u00C2\u00A0were\u00C2\u00A0 aseptically\u00C2\u00A0 sealed\u00C2\u00A0with\u00C2\u00A0 sterilized\u00C2\u00A0 (70%\u00C2\u00A0 ethanol)\u00C2\u00A0 vapour\u00C2\u00A0 locks\u00C2\u00A0 filled\u00C2\u00A0with\u00C2\u00A0 sterile\u00C2\u00A0water.\u00C2\u00A0 Sealed\u00C2\u00A0 bottles\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 at\u00C2\u00A0 18\u00C2\u00B0C,\u00C2\u00A0 and\u00C2\u00A0weighed\u00C2\u00A0 daily\u00C2\u00A0 to\u00C2\u00A0 monitor\u00C2\u00A0CO2\u00C2\u00A0production.\u00C2\u00A0Data\u00C2\u00A0were\u00C2\u00A0plotted\u00C2\u00A0in\u00C2\u00A0Excel\u00C2\u00A0to\u00C2\u00A0generate\u00C2\u00A0fermentation\u00C2\u00A0profiles.\u00C2\u00A0 \u00C2\u00A0 2.3.4.3\u00C2\u00A0\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0glucose/fructose\u00C2\u00A0utilization\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0production.\u00C2\u00A0Following\u00C2\u00A0fermentation,\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0 of\u00C2\u00A0fresh\u00C2\u00A0unheated\u00C2\u00A0Sake\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0sterile\u00C2\u00A01.5\u00C2\u00A0mL\u00C2\u00A0microcentrifuge\u00C2\u00A0tube\u00C2\u00A0and\u00C2\u00A0centrifuged\u00C2\u00A0for\u00C2\u00A010\u00C2\u00A0 min\u00C2\u00A0at\u00C2\u00A0max\u00C2\u00A0speed\u00C2\u00A0(13K\u00C2\u00A0RPM)\u00C2\u00A0to\u00C2\u00A0pellet\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0any\u00C2\u00A0particulate.\u00C2\u00A0Supernatant\u00C2\u00A0(500\u00C2\u00A0\u00CE\u00BCL)\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0 a\u00C2\u00A0new\u00C2\u00A0autosampler\u00C2\u00A0screw\u00C2\u00A0cap\u00C2\u00A0vial\u00C2\u00A0(Agilent,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A020%\u00C2\u00A0 (v/v)\u00C2\u00A0EtOH\u00C2\u00A0standard\u00C2\u00A0was\u00C2\u00A0made\u00C2\u00A0 from\u00C2\u00A0100%\u00C2\u00A0EtOH\u00C2\u00A0 (Sigma,\u00C2\u00A0USA)\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A0sterile\u00C2\u00A0MilliQ\u00C2\u00A0 water\u00C2\u00A0(Millipore,\u00C2\u00A0USA),\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0standard\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0new\u00C2\u00A0autosampler\u00C2\u00A0screw\u00C2\u00A0cap\u00C2\u00A0vial.\u00C2\u00A0A\u00C2\u00A0 standard\u00C2\u00A0curve\u00C2\u00A0corresponding\u00C2\u00A0to\u00C2\u00A04,\u00C2\u00A08,\u00C2\u00A012,\u00C2\u00A016,\u00C2\u00A0and\u00C2\u00A020%\u00C2\u00A0(v/v)\u00C2\u00A0EtOH\u00C2\u00A0was\u00C2\u00A0plotted\u00C2\u00A0after\u00C2\u00A0duplicate\u00C2\u00A0injections\u00C2\u00A0 of\u00C2\u00A02,\u00C2\u00A04,\u00C2\u00A06,\u00C2\u00A08,\u00C2\u00A0and\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A020%\u00C2\u00A0standard\u00C2\u00A0as\u00C2\u00A0outlined\u00C2\u00A0below.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 3\u00C2\u00A0 g/L\u00C2\u00A0 glucose/fructose\u00C2\u00A0 standard\u00C2\u00A0was\u00C2\u00A0made\u00C2\u00A0 from\u00C2\u00A0 D\u00E2\u0080\u0090Glucose\u00C2\u00A0 (Fisher\u00C2\u00A0 Scientific,\u00C2\u00A0USA)\u00C2\u00A0 and\u00C2\u00A0 D\u00E2\u0080\u0090 Fructose\u00C2\u00A0 (Fisher\u00C2\u00A0 Scientific,\u00C2\u00A0 USA)\u00C2\u00A0mixed\u00C2\u00A0 with\u00C2\u00A0 sterile\u00C2\u00A0MilliQ\u00C2\u00A0 water\u00C2\u00A0 (Millipore,\u00C2\u00A0 USA),\u00C2\u00A0 and\u00C2\u00A0 1\u00C2\u00A0mL\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 standard\u00C2\u00A0was\u00C2\u00A0 transferred\u00C2\u00A0 to\u00C2\u00A0new\u00C2\u00A0autosampler\u00C2\u00A0 screw\u00C2\u00A0cap\u00C2\u00A0vial.\u00C2\u00A0A\u00C2\u00A0 standard\u00C2\u00A0curve\u00C2\u00A0corresponding\u00C2\u00A0 to\u00C2\u00A00.6,\u00C2\u00A0 1.2,\u00C2\u00A01.8,\u00C2\u00A02.4,\u00C2\u00A0and\u00C2\u00A03.0\u00C2\u00A0g/L\u00C2\u00A0glucose/fructose\u00C2\u00A0was\u00C2\u00A0plotted\u00C2\u00A0after\u00C2\u00A0duplicate\u00C2\u00A0injections\u00C2\u00A0of\u00C2\u00A02,\u00C2\u00A04,\u00C2\u00A06,\u00C2\u00A08,\u00C2\u00A0and\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A020%\u00C2\u00A0standard\u00C2\u00A0as\u00C2\u00A0outlined\u00C2\u00A0below.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0were\u00C2\u00A0analyzed\u00C2\u00A0on\u00C2\u00A0an\u00C2\u00A0Agilent\u00C2\u00A01100\u00C2\u00A0 series\u00C2\u00A0 liquid\u00C2\u00A0 chromatograph\u00C2\u00A0 running\u00C2\u00A0Chemstation\u00C2\u00A0 Rev\u00C2\u00A0A.09.03\u00C2\u00A0 [1417]\u00C2\u00A0 software\u00C2\u00A0 (Agilent\u00C2\u00A0Technologies,\u00C2\u00A0USA).\u00C2\u00A0The\u00C2\u00A0LC\u00C2\u00A0was\u00C2\u00A0 fitted\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0Supelcogel\u00C2\u00A0C\u00E2\u0080\u0090610H\u00C2\u00A0 main\u00C2\u00A0column\u00C2\u00A0[column\u00C2\u00A0temperature:\u00C2\u00A050\u00C2\u00B0C,\u00C2\u00A030\u00C2\u00A0cm\u00C2\u00A0x\u00C2\u00A07.8\u00C2\u00A0mm\u00C2\u00A0 ID]\u00C2\u00A0(Supelco,\u00C2\u00A0USA)\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0protected\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0 Supelguard\u00C2\u00A0C\u00E2\u0080\u0090610H\u00C2\u00A0[5cm\u00C2\u00A0x\u00C2\u00A04.6\u00C2\u00A0mm\u00C2\u00A0ID]\u00C2\u00A0(Supelco,\u00C2\u00A0USA)\u00C2\u00A0guard\u00C2\u00A0column.\u00C2\u00A0A\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0sample\u00C2\u00A0was\u00C2\u00A0run\u00C2\u00A0isocratically\u00C2\u00A0 with\u00C2\u00A00.1%\u00C2\u00A0(v/v)\u00C2\u00A0H3PO4/H20\u00C2\u00A0buffer\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0flow\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A00.75\u00C2\u00A0mL/min.\u00C2\u00A0Ethanol\u00C2\u00A0was\u00C2\u00A0eluted\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0at\u00C2\u00A0 35\u00C2\u00A0 \u00C2\u00A0 ~19\u00C2\u00A0min,\u00C2\u00A0 fructose\u00C2\u00A0was\u00C2\u00A0eluted\u00C2\u00A0at\u00C2\u00A0~8\u00C2\u00A0min\u00C2\u00A0and\u00C2\u00A0glucose\u00C2\u00A0was\u00C2\u00A0eluted\u00C2\u00A0at\u00C2\u00A0~9.5\u00C2\u00A0min,\u00C2\u00A0and\u00C2\u00A0all\u00C2\u00A0compounds\u00C2\u00A0were\u00C2\u00A0 detected\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0refractive\u00C2\u00A0index\u00C2\u00A0detector\u00C2\u00A0running\u00C2\u00A0in\u00C2\u00A0positive\u00C2\u00A0mode.\u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0compound\u00C2\u00A0 was\u00C2\u00A0determined\u00C2\u00A0automatically\u00C2\u00A0by\u00C2\u00A0Chemstation\u00C2\u00A0software\u00C2\u00A0as\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0standard\u00C2\u00A0curves.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.3.5\u00C2\u00A0Functionality\u00C2\u00A0analyses\u00C2\u00A0 \u00C2\u00A0 2.3.5.1\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.4.1.\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0fermentation,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0removed\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0(5000\u00C2\u00A0rpm,\u00C2\u00A0 4\u00C2\u00B0C,\u00C2\u00A05\u00C2\u00A0min),\u00C2\u00A0and\u00C2\u00A0~\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0decanted\u00C2\u00A0into\u00C2\u00A0sterile\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0Schott\u00C2\u00A0bottles.\u00C2\u00A0Bottles\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A070\u00C2\u00B0C\u00C2\u00A0water\u00C2\u00A0bath\u00C2\u00A0for\u00C2\u00A0exactly\u00C2\u00A048\u00C2\u00A0hours\u00C2\u00A0to\u00C2\u00A0maximize\u00C2\u00A0EC\u00C2\u00A0production,\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A04\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0GC/MS\u00C2\u00A0 analysis\u00C2\u00A0(Section\u00C2\u00A02.3.5.3).\u00C2\u00A0 \u00C2\u00A0 2.3.5.2\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A0 2.3.4.2.\u00C2\u00A0At\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation,\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0 rice\u00C2\u00A0were\u00C2\u00A0 removed\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0 (5000\u00C2\u00A0 rpm,\u00C2\u00A04\u00C2\u00B0C,\u00C2\u00A05\u00C2\u00A0 min),\u00C2\u00A0and\u00C2\u00A0~\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0decanted\u00C2\u00A0 into\u00C2\u00A0sterile\u00C2\u00A050\u00C2\u00A0mL\u00C2\u00A0Schott\u00C2\u00A0bottles.\u00C2\u00A0Bottles\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0 70\u00C2\u00B0C\u00C2\u00A0water\u00C2\u00A0bath\u00C2\u00A0 for\u00C2\u00A0exactly\u00C2\u00A048\u00C2\u00A0hours\u00C2\u00A0 to\u00C2\u00A0maximize\u00C2\u00A0EC\u00C2\u00A0production,\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A04\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0GC/MS\u00C2\u00A0 analysis\u00C2\u00A0(Section\u00C2\u00A02.3.5.3).\u00C2\u00A0 \u00C2\u00A0 2.3.5.3\u00C2\u00A0 \u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 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otherwise\u00C2\u00A0 stated,\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 standard\u00C2\u00A0 molecular\u00C2\u00A0 biology\u00C2\u00A0 standards\u00C2\u00A0 methods\u00C2\u00A0 (Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995).\u00C2\u00A0All\u00C2\u00A0PCR\u00C2\u00A0reactions,\u00C2\u00A0unless\u00C2\u00A0otherwise\u00C2\u00A0indicated,\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0using\u00C2\u00A0iProofTM\u00C2\u00A0High\u00C2\u00A0 Fidelity\u00C2\u00A0DNA\u00C2\u00A0polymerase\u00C2\u00A0(BioRad,\u00C2\u00A0USA)\u00C2\u00A0as\u00C2\u00A0per\u00C2\u00A0the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0instructions.\u00C2\u00A0 \u00C2\u00A0 2.4.1.1\u00C2\u00A0 \u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pHVX2D3.\u00C2\u00A0 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1.\u00C2\u00A0DUR3forXho1\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAACTCGAGATGGGAGAATTTAAACCTCCGCTAC\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0\u00C2\u00A0 2.\u00C2\u00A0DUR3revXho1\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAACTCGAGCTAAATTATTTCATCAACTTGTCCGAAATGTG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0 \u00C2\u00A0 Following\u00C2\u00A0PCR,\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gel\u00C2\u00A0visualization,\u00C2\u00A0and\u00C2\u00A0PCR\u00C2\u00A0cleanup\u00C2\u00A0(Qiagen,\u00C2\u00A0USA\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0PCR\u00C2\u00A0Purification\u00C2\u00A0 Kit),\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0PCR\u00C2\u00A0product\u00C2\u00A0(insert)\u00C2\u00A0and\u00C2\u00A0pHVX2\u00C2\u00A0(vector)\u00C2\u00A0were\u00C2\u00A0digested\u00C2\u00A0with\u00C2\u00A0Xho1\u00C2\u00A0(Roche,\u00C2\u00A0Germany).\u00C2\u00A0After\u00C2\u00A0 the\u00C2\u00A0digested\u00C2\u00A0vector\u00C2\u00A0was\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0SAP\u00C2\u00A0(Fermentas,\u00C2\u00A0USA)\u00C2\u00A0to\u00C2\u00A0prevent\u00C2\u00A0re\u00E2\u0080\u0090circularization,\u00C2\u00A0the\u00C2\u00A0insert\u00C2\u00A0and\u00C2\u00A0 linearized\u00E2\u0080\u0090SAP\u00C2\u00A0 treated\u00C2\u00A0 vector\u00C2\u00A0were\u00C2\u00A0 ligated\u00C2\u00A0overnight\u00C2\u00A0 at\u00C2\u00A0 22\u00C2\u00B0C\u00C2\u00A0 (T4\u00C2\u00A0DNA\u00C2\u00A0 Ligase\u00C2\u00A0 \u00E2\u0080\u0093\u00C2\u00A0 Fermentas,\u00C2\u00A0USA);\u00C2\u00A0 the\u00C2\u00A0 37\u00C2\u00A0 \u00C2\u00A0 ligation\u00C2\u00A0mixture\u00C2\u00A0 (5\u00C2\u00A0 \u00CE\u00BCL)\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 transform\u00C2\u00A0 DH5\u00CE\u00B1\u00E2\u0084\u00A2\u00C2\u00A0 competent\u00C2\u00A0 cells\u00C2\u00A0 (Invitrogen,\u00C2\u00A0 USA)\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 subsequently\u00C2\u00A0 grown\u00C2\u00A0 on\u00C2\u00A0 100\u00C2\u00A0 \u00C2\u00B5g/mL\u00C2\u00A0 Ampicillin\u00C2\u00A0 (Fisher,\u00C2\u00A0 USA)\u00C2\u00A0 supplemented\u00C2\u00A0 LB\u00C2\u00A0 (Difco,\u00C2\u00A0 USA)\u00C2\u00A0 plates.\u00C2\u00A0 Plasmids\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0random\u00C2\u00A0selection\u00C2\u00A0of\u00C2\u00A0transformant\u00C2\u00A0colonies\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0(Qiagen,\u00C2\u00A0USA\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0QIAprep\u00C2\u00A0Spin\u00C2\u00A0 Miniprep\u00C2\u00A0kit)\u00C2\u00A0and\u00C2\u00A0digested\u00C2\u00A0by\u00C2\u00A0EcoR1\u00C2\u00A0(Roche,\u00C2\u00A0Germany);\u00C2\u00A0PCR,\u00C2\u00A0using\u00C2\u00A0inside\u00E2\u0080\u0090outside\u00C2\u00A0primers,\u00C2\u00A0was\u00C2\u00A0done\u00C2\u00A0to\u00C2\u00A0 identify\u00C2\u00A0plasmids\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0desired\u00C2\u00A0insert.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A010.\u00C2\u00A0Schematic\u00C2\u00A0 representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0 strategy\u00C2\u00A0 for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pHVX2D3.\u00C2\u00A0The\u00C2\u00A0DUR3\u00C2\u00A0ORF\u00C2\u00A0was\u00C2\u00A0 PCR\u00C2\u00A0amplified\u00C2\u00A0from\u00C2\u00A0522\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0and\u00C2\u00A0ligated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0Xho1\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0pHVX2.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.1.2\u00C2\u00A0\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pHVXKD3.\u00C2\u00A0A\u00C2\u00A0kanMX\u00C2\u00A0marker\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0pUG6\u00C2\u00A0(Guldener,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01996)\u00C2\u00A0via\u00C2\u00A0 double\u00C2\u00A0digestion\u00C2\u00A0with\u00C2\u00A0Xho1\u00C2\u00A0and\u00C2\u00A0Sal1\u00C2\u00A0 (Fermentas,\u00C2\u00A0USA).\u00C2\u00A0Following\u00C2\u00A0digestion,\u00C2\u00A0the\u00C2\u00A01500\u00C2\u00A0bp\u00C2\u00A0kanMX\u00C2\u00A0band\u00C2\u00A0 was\u00C2\u00A0gel\u00C2\u00A0purified\u00C2\u00A0(Qiagen,\u00C2\u00A0USA\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Gel\u00C2\u00A0Extraction\u00C2\u00A0Kit)\u00C2\u00A0and\u00C2\u00A0ligated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0Sal1\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0linearized\u00E2\u0080\u0090SAP\u00C2\u00A0treated\u00C2\u00A0 pHVX2D3.\u00C2\u00A0The\u00C2\u00A0ligation\u00C2\u00A0mixture\u00C2\u00A0(5\u00C2\u00A0\u00CE\u00BCL)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0transform\u00C2\u00A0DH5\u00CE\u00B1\u00E2\u0084\u00A2\u00C2\u00A0competent\u00C2\u00A0cells\u00C2\u00A0which\u00C2\u00A0were\u00C2\u00A0grown\u00C2\u00A0 on\u00C2\u00A0 LB\u00E2\u0080\u0090Ampicillin\u00C2\u00A0 (100\u00C2\u00A0 \u00C2\u00B5g/mL).\u00C2\u00A0 Recombinant\u00C2\u00A0 plasmids\u00C2\u00A0 (Figure\u00C2\u00A0 11)\u00C2\u00A0 were\u00C2\u00A0 identified\u00C2\u00A0 by\u00C2\u00A0 HindIII\u00C2\u00A0 (Roche,\u00C2\u00A0 Germany)\u00C2\u00A0digestion\u00C2\u00A0of\u00C2\u00A0plasmids\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A024\u00C2\u00A0randomly\u00C2\u00A0chosen\u00C2\u00A0colonies.\u00C2\u00A0 \u00C2\u00A0 38\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A011.\u00C2\u00A0Schematic\u00C2\u00A0 representation\u00C2\u00A0of\u00C2\u00A0 cloning\u00C2\u00A0 strategy\u00C2\u00A0 for\u00C2\u00A0 creation\u00C2\u00A0of\u00C2\u00A0pHVXKD3.\u00C2\u00A0The\u00C2\u00A0kanMX\u00C2\u00A0marker\u00C2\u00A0 was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Xho1/Sal1\u00C2\u00A0digestion\u00C2\u00A0of\u00C2\u00A0pUG6\u00C2\u00A0and\u00C2\u00A0ligated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0Sal1\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0pHVXKD3.\u00C2\u00A0 \u00C2\u00A0 2.4.1.3\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pUCTRP1.\u00C2\u00A0The\u00C2\u00A0TRP1\u00C2\u00A0coding\u00C2\u00A0 region\u00C2\u00A0was\u00C2\u00A0PCR\u00C2\u00A0amplified\u00C2\u00A0 from\u00C2\u00A0522\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0 using\u00C2\u00A0TRP1\u00C2\u00A0specific\u00C2\u00A0primers,\u00C2\u00A0each\u00C2\u00A0containing\u00C2\u00A0BamH1\u00C2\u00A0 (bold)\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0Apa1\u00C2\u00A0 (underline)\u00C2\u00A0sites\u00C2\u00A0at\u00C2\u00A0 their\u00C2\u00A05\u00E2\u0080\u0099\u00C2\u00A0 ends:\u00C2\u00A0BamH1Apa1TRP1ORFfwd\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAAAAGGATCCAAAAAAGGGCCCATGTCTGTTATTAATTTCACAGG\u00E2\u0080\u00903\u00E2\u0080\u0099);\u00C2\u00A0 BamH1Apa1TRP1ORFrev\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAAAAGGATCCAAAAAAGGGCCCCTATTTCTTAGCATTTTTGACG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Following\u00C2\u00A0amplification,\u00C2\u00A0cleanup,\u00C2\u00A0and\u00C2\u00A0quantification,\u00C2\u00A0the\u00C2\u00A0~750\u00C2\u00A0bp\u00C2\u00A0fragment\u00C2\u00A0was\u00C2\u00A0ligated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0 BamH1\u00C2\u00A0(Roche,\u00C2\u00A0Germany)\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0linearized\u00E2\u0080\u0090SAP\u00C2\u00A0treated\u00C2\u00A0pUC18\u00C2\u00A0(Figure\u00C2\u00A012).\u00C2\u00A0Recombinant\u00C2\u00A0plasmids\u00C2\u00A0were\u00C2\u00A0 identified\u00C2\u00A0primarily\u00C2\u00A0through\u00C2\u00A0blue/white\u00C2\u00A0screening\u00C2\u00A0(growth\u00C2\u00A0on\u00C2\u00A0LB\u00E2\u0080\u0090Ampicillin\u00C2\u00A0supplemented\u00C2\u00A0with\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCg/mL\u00C2\u00A0 Xgal)\u00C2\u00A0and\u00C2\u00A0subsequently\u00C2\u00A0confirmed\u00C2\u00A0through\u00C2\u00A0HindIII/EcoR1\u00C2\u00A0digestion.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A012.\u00C2\u00A0Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pUCTRP1.\u00C2\u00A0The\u00C2\u00A0TRP1\u00C2\u00A0ORF\u00C2\u00A0was\u00C2\u00A0PCR\u00C2\u00A0 amplified\u00C2\u00A0from\u00C2\u00A0522\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0and\u00C2\u00A0ligated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0BamH1\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0pUC18.\u00C2\u00A0 \u00C2\u00A0 39\u00C2\u00A0 \u00C2\u00A0 2.4.1.4\u00C2\u00A0\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0pUCMD.\u00C2\u00A0The\u00C2\u00A0PGK1p\u00E2\u0080\u0090DUR3\u00E2\u0080\u0090PGK1t\u00E2\u0080\u0090kanMX\u00C2\u00A0cassette\u00C2\u00A0located\u00C2\u00A0within\u00C2\u00A0pHVXKD3\u00C2\u00A0was\u00C2\u00A0 amplified\u00C2\u00A0from\u00C2\u00A0pHVXKD3\u00C2\u00A0plasmid\u00C2\u00A0DNA\u00C2\u00A0using\u00C2\u00A0cassette\u00C2\u00A0specific\u00C2\u00A0primers:\u00C2\u00A01.\u00C2\u00A0pHVXKfwdlong\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0CTGGCACG\u00C2\u00A0 ACAGGTTTCCCGACTGGAAAGCGGGCAGTGAG\u00E2\u0080\u00903\u00E2\u0080\u0099);\u00C2\u00A0pHVXKrevlong\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0CTGGCGAAAGGGGGATGTGCTGCAA\u00C2\u00A0 GGCGATTAAGTTGGG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0Following\u00C2\u00A0amplification,\u00C2\u00A0cleanup,\u00C2\u00A0and\u00C2\u00A0quantification,\u00C2\u00A0 the\u00C2\u00A0~6500\u00C2\u00A0bp\u00C2\u00A0blunt\u00C2\u00A0end\u00C2\u00A0 PCR\u00C2\u00A0generated\u00C2\u00A0fragment\u00C2\u00A0was\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0polynucleotide\u00C2\u00A0kinase\u00C2\u00A0(New\u00C2\u00A0England\u00C2\u00A0Biolabs,\u00C2\u00A0USA)\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0 facilitate\u00C2\u00A0 ligation\u00C2\u00A0 (O/N\u00C2\u00A0 at\u00C2\u00A0 22\u00C2\u00B0C)\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0blunt\u00C2\u00A0 EcoRV\u00C2\u00A0 (Fermentas,\u00C2\u00A0USA)\u00C2\u00A0 site\u00C2\u00A0 of\u00C2\u00A0 linearized\u00E2\u0080\u0090SAP\u00C2\u00A0 treated\u00C2\u00A0 pUCTRP1.\u00C2\u00A0 \u00C2\u00A0 Recombinant\u00C2\u00A0plasmids\u00C2\u00A0(Figure\u00C2\u00A013)\u00C2\u00A0were\u00C2\u00A0 initially\u00C2\u00A0 identified\u00C2\u00A0using\u00C2\u00A0E\u00E2\u0080\u0090lyse\u00C2\u00A0analysis\u00C2\u00A0(Eckhardt\u00C2\u00A01978)\u00C2\u00A0 and\u00C2\u00A0 later\u00C2\u00A0confirmed\u00C2\u00A0via\u00C2\u00A0Apa1\u00C2\u00A0 (Stratagene,\u00C2\u00A0USA)\u00C2\u00A0/Sal1\u00C2\u00A0digestion.\u00C2\u00A0Briefly,\u00C2\u00A0E\u00E2\u0080\u0090lyse\u00C2\u00A0efficiently\u00C2\u00A0screens\u00C2\u00A0 large\u00C2\u00A0 numbers\u00C2\u00A0 of\u00C2\u00A0 colonies\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 plasmid\u00C2\u00A0DNA\u00C2\u00A0 by\u00C2\u00A0 lysing\u00C2\u00A0 the\u00C2\u00A0 colonies\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0wells\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 agarose\u00C2\u00A0gel,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0electrophoresis\u00C2\u00A0(Eckhardt\u00C2\u00A01978).\u00C2\u00A0More\u00C2\u00A0specifically,\u00C2\u00A0after\u00C2\u00A0patching\u00C2\u00A0onto\u00C2\u00A0selective\u00C2\u00A0 media,\u00C2\u00A0 small\u00C2\u00A0aliquots\u00C2\u00A0of\u00C2\u00A0 colonies\u00C2\u00A0were\u00C2\u00A0 suspended\u00C2\u00A0 in\u00C2\u00A05\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0TBE\u00C2\u00A0buffer\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0SRL\u00C2\u00A0 buffer\u00C2\u00A0 (25%\u00C2\u00A0 v/v\u00C2\u00A0 sucrose,\u00C2\u00A0 50\u00C2\u00A0 \u00C2\u00B5g/mL\u00C2\u00A0 RNaseA,\u00C2\u00A0 1\u00C2\u00A0 mg/mL\u00C2\u00A0 lysozyme).\u00C2\u00A0 After\u00C2\u00A0 mixing\u00C2\u00A0 by\u00C2\u00A0 pipetting,\u00C2\u00A0 cell\u00C2\u00A0 suspensions\u00C2\u00A0were\u00C2\u00A0 loaded\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0wells\u00C2\u00A0of\u00C2\u00A0 a\u00C2\u00A0 0.2%\u00C2\u00A0 (w/v)\u00C2\u00A0 SDS\u00C2\u00A0 \u00E2\u0080\u0090\u00C2\u00A0 0.8%\u00C2\u00A0 (w/v)\u00C2\u00A0 agarose\u00C2\u00A0 gel.\u00C2\u00A0After\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 suspension\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0wells\u00C2\u00A0had\u00C2\u00A0become\u00C2\u00A0clear\u00C2\u00A0indicating\u00C2\u00A0cell\u00C2\u00A0lysis\u00C2\u00A0(~\u00C2\u00A030\u00C2\u00A0min),\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0was\u00C2\u00A0electrophoresed\u00C2\u00A0 at\u00C2\u00A020\u00C2\u00A0V\u00C2\u00A0 for\u00C2\u00A045\u00C2\u00A0min,\u00C2\u00A0 then\u00C2\u00A0at\u00C2\u00A080\u00C2\u00A0V\u00C2\u00A0 for\u00C2\u00A045\u00C2\u00A0min.\u00C2\u00A0Finally\u00C2\u00A0 the\u00C2\u00A0gel\u00C2\u00A0was\u00C2\u00A0stained\u00C2\u00A0as\u00C2\u00A0 required\u00C2\u00A0with\u00C2\u00A0SYBR\u00E2\u0084\u00A2\u00C2\u00A0Safe\u00C2\u00A0 (Invitrogen,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0 40\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A013.\u00C2\u00A0Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0for\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0pUCMD.\u00C2\u00A0The\u00C2\u00A0linear\u00C2\u00A0PGK1p\u00E2\u0080\u0090DUR3\u00E2\u0080\u0090 PGK1t\u00E2\u0080\u0090kanMX\u00C2\u00A0construction\u00C2\u00A0was\u00C2\u00A0PCR\u00C2\u00A0amplified\u00C2\u00A0from\u00C2\u00A0pHVXKD3\u00C2\u00A0and\u00C2\u00A0blunt\u00C2\u00A0end\u00C2\u00A0cloned\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0EcoRV\u00C2\u00A0site\u00C2\u00A0 of\u00C2\u00A0pUCTRP1\u00C2\u00A0to\u00C2\u00A0yield\u00C2\u00A0pUCMD.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.2\u00C2\u00A0\u00C2\u00A0Sequence\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0pUCMD\u00C2\u00A0 \u00C2\u00A0 Plasmid\u00C2\u00A0pUCMD\u00C2\u00A0(Figure\u00C2\u00A014)\u00C2\u00A0was\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0E.\u00C2\u00A0coli\u00C2\u00A0(Qiagen,\u00C2\u00A0USA\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0QIAprep\u00C2\u00A0Spin\u00C2\u00A0Miniprep\u00C2\u00A0kit)\u00C2\u00A0 and\u00C2\u00A0 used\u00C2\u00A0 directly\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 sequencing\u00C2\u00A0 template.\u00C2\u00A0 Sequencing\u00C2\u00A0was\u00C2\u00A0 performed\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0Nucleic\u00C2\u00A0Acid\u00C2\u00A0 Protein\u00C2\u00A0 Service\u00C2\u00A0 Unit\u00C2\u00A0 (NAPS)\u00C2\u00A0 at\u00C2\u00A0 The\u00C2\u00A0 University\u00C2\u00A0 of\u00C2\u00A0 British\u00C2\u00A0 Columbia\u00C2\u00A0 using\u00C2\u00A0 an\u00C2\u00A0 Applied\u00C2\u00A0 Biosystems\u00C2\u00A0 PRISM\u00C2\u00A0 377\u00C2\u00A0 sequencer\u00C2\u00A0 and\u00C2\u00A0 Applied\u00C2\u00A0 Biosystems\u00C2\u00A0 BigDye\u00C2\u00A0 v3.1\u00C2\u00A0 sequencing\u00C2\u00A0 chemistry.\u00C2\u00A0 Primers\u00C2\u00A0 and\u00C2\u00A0 template\u00C2\u00A0 were\u00C2\u00A0 supplied\u00C2\u00A0to\u00C2\u00A0NAPS\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0concentrations\u00C2\u00A0specified\u00C2\u00A0by\u00C2\u00A0their\u00C2\u00A0sample\u00C2\u00A0submission\u00C2\u00A0requirements.\u00C2\u00A0The\u00C2\u00A0entire\u00C2\u00A0 DUR3\u00C2\u00A0 cassette,\u00C2\u00A0 beginning\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0 TRP1\u00C2\u00A0 flanking\u00C2\u00A0 sequence\u00C2\u00A0 and\u00C2\u00A0 ending\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0 TRP1\u00C2\u00A0 flanking\u00C2\u00A0 sequence,\u00C2\u00A0was\u00C2\u00A0sequenced\u00C2\u00A0via\u00C2\u00A016\u00C2\u00A0different\u00C2\u00A0sequencing\u00C2\u00A0reads\u00C2\u00A0(Table\u00C2\u00A06)\u00C2\u00A0and\u00C2\u00A0later\u00C2\u00A0assembled\u00C2\u00A0in\u00C2\u00A0silico\u00C2\u00A0using\u00C2\u00A0 Accelrys\u00C2\u00A0DS\u00C2\u00A0Gene\u00C2\u00A0v1.1\u00C2\u00A0 software.\u00C2\u00A0The\u00C2\u00A0assembled\u00C2\u00A0 sequences\u00C2\u00A0were\u00C2\u00A0aligned\u00C2\u00A0against\u00C2\u00A0previously\u00C2\u00A0published\u00C2\u00A0 sequences\u00C2\u00A0and\u00C2\u00A0 those\u00C2\u00A0of\u00C2\u00A0DUR3,\u00C2\u00A0TRP1,\u00C2\u00A0PGK1p,\u00C2\u00A0and\u00C2\u00A0PGK1t\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0SGD.\u00C2\u00A0 If\u00C2\u00A0any\u00C2\u00A0discrepancies\u00C2\u00A0were\u00C2\u00A0 found,\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0read\u00C2\u00A0which\u00C2\u00A0gave\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0discrepancy\u00C2\u00A0was\u00C2\u00A0repeated\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0bona\u00C2\u00A0fide\u00C2\u00A0 mutations.\u00C2\u00A0\u00C2\u00A0 41\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A07.\u00C2\u00A0Oligonucleotide\u00C2\u00A0primers\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0sequencing\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0pUCMD.\u00C2\u00A0\u00C2\u00A0 Primer\u00C2\u00A0 Primer\u00C2\u00A0Name\u00C2\u00A0 Sequence\u00C2\u00A0(5\u00E2\u0080\u0099\u00C3\u00863\u00E2\u0080\u0099)\u00C2\u00A0 P1\u00C2\u00A0 BamH1TRP1Apa1Fwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAAAAGGATCCAAAAAAGGGCCCATGTCTGTTATTAATTTCACAGG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P2\u00C2\u00A0 pHVXKlongfwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P3\u00C2\u00A0 pHVXKfwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090CTGGCACGACAGGTTTCCCGACTGG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P4\u00C2\u00A0 pDUR3tfwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TTTCCGCGGAGCTTTCTAACTGATCTATCC\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P5\u00C2\u00A0 DUR3Xho1fwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAACTCGAGATGGGAGAATTTAAACCTCCGCTAC\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P6\u00C2\u00A0 DUR3Xho1rev\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAACTCGAGCTAAATTATTTCATCAACTTGTCCGAAATGTG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P7\u00C2\u00A0 pDUR3trev\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TTTCCGCGGTGCGGTGTGAAATACC\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P8\u00C2\u00A0 kanMXORFrev\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TTAGAAAAACTCACTGAGCATCAAATGAAACTGC\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P9\u00C2\u00A0 kanMXORFfwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090ATGGGTAAGGAAAAGACTCACGTTTCGAGG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P10\u00C2\u00A0 pHVXKlongrev\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090CTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P11\u00C2\u00A0 BamH1TRP1Apa1rev\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090AAAAAAGGATCCAAAAAAGGGCCCCTATTTCTTAGCATTTTTGACG\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P12\u00C2\u00A0 DUR3RTfwd\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090GATCGGCCATGGTTGCTACTT\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P13\u00C2\u00A0 RevPGKtPst1\u00C2\u00A0 5\u00E2\u0080\u0099\u00E2\u0080\u0090TTTTCTGCAGAAGCTTTAACGAACGCAGAATT\u00E2\u0080\u00903\u00E2\u0080\u0099\u00C2\u00A0 P14\u00C2\u00A0 PGKpro1\u00C2\u00A0 5'\u00E2\u0080\u0090ACAAAATCTTCTTGACAAACGTCACAA3'\u00C2\u00A0 P15\u00C2\u00A0 PGKpro2\u00C2\u00A0 5'\u00E2\u0080\u0090AATTGATGTTACCCTCATAAAGCACGT\u00E2\u0080\u00903'\u00C2\u00A0 P16\u00C2\u00A0 PGKforDUR\u00C2\u00A0 5'\u00E2\u0080\u0090TGGTTTAGTTTAGTAGAACCTCGTGAAACTTAC\u00E2\u0080\u00903'\u00C2\u00A0 42\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A014.\u00C2\u00A0Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0pUCMD.\u00C2\u00A0The\u00C2\u00A0DUR3\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0stretches\u00C2\u00A0between\u00C2\u00A0Apa1\u00C2\u00A0sites\u00C2\u00A0encompassing\u00C2\u00A05\u00E2\u0080\u00991/2TRP1\u00E2\u0080\u0090PGK1p\u00E2\u0080\u0090 DUR3\u00E2\u0080\u0090PGK1t\u00E2\u0080\u0090kanMXp\u00E2\u0080\u0090kanMX\u00E2\u0080\u0090kanMXt\u00E2\u0080\u00903\u00E2\u0080\u00991/2TRP1.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0\u00C2\u00BD\u00C2\u00A0TRP1\u00C2\u00A0 PGK1\u00C2\u00A0Promoter\u00C2\u00A0 PGK1\u00C2\u00A0Terminator\u00C2\u00A0 DUR3\u00C2\u00A0 kanMX\u00C2\u00A0Promoter\u00C2\u00A0 kanMX\u00C2\u00A0Terminator\u00C2\u00A0 kanMX\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0\u00C2\u00BD\u00C2\u00A0TRP1\u00C2\u00A0 AmpR\u00C2\u00A0 rep\u00C2\u00A0(pMB1)\u00C2\u00A0 pUCMD\u00C2\u00A0(9221bp) \u00C2\u00A0 \u00C2\u00A0 43\u00C2\u00A0 \u00C2\u00A0 2.4.3\u00C2\u00A0\u00C2\u00A0Transformation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0linear\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0and\u00C2\u00A0selection\u00C2\u00A0of\u00C2\u00A0transformants\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A06536\u00C2\u00A0bp\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0was\u00C2\u00A0cut\u00C2\u00A0from\u00C2\u00A0pUCMD\u00C2\u00A0using\u00C2\u00A0Apa1\u00C2\u00A0(Stratagene,\u00C2\u00A0USA)\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 1995)\u00C2\u00A0and\u00C2\u00A0visualized\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gel.\u00C2\u00A0From\u00C2\u00A0the\u00C2\u00A0gel,\u00C2\u00A0the\u00C2\u00A0expected\u00C2\u00A06536\u00C2\u00A0bp\u00C2\u00A0band\u00C2\u00A0was\u00C2\u00A0resolved\u00C2\u00A0and\u00C2\u00A0 extracted\u00C2\u00A0 (Qiagen,\u00C2\u00A0 USA\u00C2\u00A0 \u00E2\u0080\u0093\u00C2\u00A0 Gel\u00C2\u00A0 extraction\u00C2\u00A0 kit).\u00C2\u00A0 After\u00C2\u00A0 extraction,\u00C2\u00A0 clean\u00C2\u00A0 up,\u00C2\u00A0 and\u00C2\u00A0 quantification\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 Nanodrop\u00C2\u00A0 ND\u00E2\u0080\u00901000\u00C2\u00A0 spectrophotometer\u00C2\u00A0 (Nanodrop,\u00C2\u00A0 USA),\u00C2\u00A0 250\u00C2\u00A0 ng\u00C2\u00A0 of\u00C2\u00A0 linear\u00C2\u00A0 cassette\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 transform\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 strains\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 522,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090.\u00C2\u00A0 Yeast\u00C2\u00A0 strains\u00C2\u00A0 were\u00C2\u00A0 transformed\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 lithium\u00C2\u00A0acetate/polyethylene\u00C2\u00A0glycol/ssDNA\u00C2\u00A0method\u00C2\u00A0(Gietz\u00C2\u00A0and\u00C2\u00A0Woods\u00C2\u00A02002).\u00C2\u00A0Following\u00C2\u00A0transformation,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0left\u00C2\u00A0to\u00C2\u00A0recover\u00C2\u00A0in\u00C2\u00A0YPD\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0for\u00C2\u00A02\u00C2\u00A0hours\u00C2\u00A0before\u00C2\u00A0plating\u00C2\u00A0on\u00C2\u00A0to\u00C2\u00A0YPD\u00C2\u00A0plates\u00C2\u00A0supplemented\u00C2\u00A0with\u00C2\u00A0 300\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0G418\u00C2\u00A0(Sigma,\u00C2\u00A0USA).\u00C2\u00A0Plates\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A030\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0colonies\u00C2\u00A0appeared.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.4.3.1\u00C2\u00A0 \u00C2\u00A0Confirmation\u00C2\u00A0of\u00C2\u00A0 integration\u00C2\u00A0via\u00C2\u00A0colony\u00C2\u00A0PCR.\u00C2\u00A0Colony\u00C2\u00A0PCR\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0previously\u00C2\u00A0described\u00C2\u00A0 to\u00C2\u00A0 detect\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 linear\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0yeast\u00C2\u00A0genome\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0TRP1\u00C2\u00A0 locus\u00C2\u00A0 (Ward\u00C2\u00A01992).\u00C2\u00A0Zymolyase\u00C2\u00A0100\u00C2\u00A0U/mL\u00C2\u00A0(Seikagaku\u00C2\u00A0Corp.,\u00C2\u00A0Japan)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0lyse\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0(30\u00C2\u00A0\u00C2\u00B5l\u00C2\u00A0zymolyase\u00C2\u00A0 solution).\u00C2\u00A0 Primers\u00C2\u00A0 kanMXORFfwd\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090ATGGGTAAGGAAAAGACTCACGTTTCGAGG\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 and\u00C2\u00A0 kanMXORFrev\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090TTAGAAAAACTCATCGAGCATCAAATGAAACTGC\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0generate\u00C2\u00A0an\u00C2\u00A0809\u00C2\u00A0bp\u00C2\u00A0 fragment\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A03\u00E2\u0080\u0099\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette.\u00C2\u00A0522\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A0pUCMD\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0positive\u00C2\u00A0 control.\u00C2\u00A0PCR\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0with\u00C2\u00A0iProofTM\u00C2\u00A0High\u00C2\u00A0Fidelity\u00C2\u00A0DNA\u00C2\u00A0polymerase\u00C2\u00A0(BioRad,\u00C2\u00A0USA)\u00C2\u00A0using\u00C2\u00A0suggested\u00C2\u00A0 reagent\u00C2\u00A0concentrations\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5l\u00C2\u00A0of\u00C2\u00A0Zymolyase\u00C2\u00A0 treated\u00C2\u00A0cell\u00C2\u00A0supernatant\u00C2\u00A0as\u00C2\u00A0 template.\u00C2\u00A0The\u00C2\u00A0PCR\u00C2\u00A0program\u00C2\u00A0 was\u00C2\u00A0as\u00C2\u00A0follows:\u00C2\u00A01.\u00C2\u00A0Initial\u00C2\u00A0denaturation\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A03\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A098\u00C2\u00B0C.\u00C2\u00A02.\u00C2\u00A0Denaturation\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A010\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A098\u00C2\u00B0C.\u00C2\u00A03.\u00C2\u00A0Annealing\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0 20\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A062.4\u00C2\u00B0C.\u00C2\u00A04.\u00C2\u00A0Extension\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A030\u00C2\u00A0sec\u00C2\u00A0at\u00C2\u00A072\u00C2\u00B0C.\u00C2\u00A05.\u00C2\u00A0Cycle\u00C2\u00A0to\u00C2\u00A0step\u00C2\u00A02,\u00C2\u00A030\u00C2\u00A0times.\u00C2\u00A06.\u00C2\u00A0Final\u00C2\u00A0extension\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A0 72\u00C2\u00B0C.\u00C2\u00A0 \u00C2\u00A0Colony\u00C2\u00A0PCR\u00C2\u00A0reactions\u00C2\u00A0were\u00C2\u00A0visualized\u00C2\u00A0on\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gels\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0SYBR\u00E2\u0084\u00A2\u00C2\u00A0Safe\u00C2\u00A0(Invitrogen,\u00C2\u00A0 USA).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.4.4\u00C2\u00A0\u00C2\u00A0Genetic\u00C2\u00A0characterization\u00C2\u00A0 \u00C2\u00A0 2.4.4.1\u00C2\u00A0\u00C2\u00A0Southern\u00C2\u00A0blot\u00C2\u00A0analyses.\u00C2\u00A0Southern\u00C2\u00A0blotting\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0confirm\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0TRP1\u00C2\u00A0 locus.\u00C2\u00A0Genomic\u00C2\u00A0DNA\u00C2\u00A0 from\u00C2\u00A0engineered\u00C2\u00A0 strains\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3,\u00C2\u00A0as\u00C2\u00A0 well\u00C2\u00A0as\u00C2\u00A0their\u00C2\u00A0respective\u00C2\u00A0parent\u00C2\u00A0strains,\u00C2\u00A0was\u00C2\u00A0digested\u00C2\u00A0with\u00C2\u00A0EcoRI\u00C2\u00A0(Roche,\u00C2\u00A0Germany)\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995),\u00C2\u00A0 and\u00C2\u00A0 separated\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 0.8%\u00C2\u00A0 agarose\u00C2\u00A0 gel.\u00C2\u00A0 Following\u00C2\u00A0 gel\u00C2\u00A0 preparation,\u00C2\u00A0 transfer\u00C2\u00A0 and\u00C2\u00A0 fixing\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 positively\u00C2\u00A0 charged\u00C2\u00A0Nylon\u00C2\u00A0membrane\u00C2\u00A0 (Roche\u00C2\u00A0Diagnostics,\u00C2\u00A0Germany)\u00C2\u00A0 (Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995),\u00C2\u00A0the\u00C2\u00A0blots\u00C2\u00A0were\u00C2\u00A0probed\u00C2\u00A0 with\u00C2\u00A0PCR\u00C2\u00A0generated\u00C2\u00A0fragments\u00C2\u00A0specific\u00C2\u00A0for\u00C2\u00A0DUR3\u00C2\u00A0and\u00C2\u00A0TRP1.\u00C2\u00A0The\u00C2\u00A0AlkPhosTM\u00C2\u00A0Direct\u00C2\u00A0Nucleic\u00C2\u00A0Acid\u00C2\u00A0Labeling\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 44\u00C2\u00A0 \u00C2\u00A0 and\u00C2\u00A0CDP\u00E2\u0080\u0090Star\u00C2\u00A0Detection\u00C2\u00A0system\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0recommended\u00C2\u00A0for\u00C2\u00A0probe\u00C2\u00A0detection\u00C2\u00A0(Amersham\u00C2\u00A0Biosciences,\u00C2\u00A0 England).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0661\u00C2\u00A0bp\u00C2\u00A0DUR3\u00C2\u00A0probe\u00C2\u00A0was\u00C2\u00A0generated\u00C2\u00A0using\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0 from\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0strain\u00C2\u00A0522\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 template\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 primers\u00C2\u00A0 DUR3probefwd\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 CAGCAGAAGAATTCACCACCGCCGGTAGATC\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0 and\u00C2\u00A0 DUR3proberev\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 CAATCAGGTTAATAATTAATAAAATACCAGCGG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0 The\u00C2\u00A0 461\u00C2\u00A0 bp\u00C2\u00A0 TRP1\u00C2\u00A0 probe\u00C2\u00A0 was\u00C2\u00A0 generated\u00C2\u00A0 using\u00C2\u00A0 genomic\u00C2\u00A0 DNA\u00C2\u00A0 from\u00C2\u00A0 522\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 template\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 primers\u00C2\u00A0 TRP1probefwd\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0 TTAATTTCACAGGTAGTTCTGGTCCATTGG\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0and\u00C2\u00A0TRP1proberev\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090\u00C2\u00A0CAATCCAAAAGTTCACCTGTCCCACCT\u00C2\u00A0 GCTTCTG\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.4.4.2\u00C2\u00A0\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0northern\u00C2\u00A0blotting.\u00C2\u00A0\u00C2\u00A0Fermentations\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 in\u00C2\u00A0 filter\u00C2\u00A0sterilized\u00C2\u00A0Calona\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0as\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A02.3.3.3.\u00C2\u00A0After\u00C2\u00A024\u00C2\u00A0hours,\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0harvested\u00C2\u00A0by\u00C2\u00A0 centrifugation\u00C2\u00A0 (5000\u00C2\u00A0 rpm,\u00C2\u00A04\u00C2\u00B0C,\u00C2\u00A04\u00C2\u00A0min),\u00C2\u00A0 snap\u00C2\u00A0 frozen\u00C2\u00A0 in\u00C2\u00A0 liquid\u00C2\u00A0nitrogen\u00C2\u00A0 (3\u00C2\u00A0min),\u00C2\u00A0and\u00C2\u00A0 stored\u00C2\u00A0at\u00C2\u00A0\u00E2\u0080\u009080\u00C2\u00B0C\u00C2\u00A0until\u00C2\u00A0RNA\u00C2\u00A0extraction.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0was\u00C2\u00A0extracted\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0hot\u00C2\u00A0phenol\u00C2\u00A0method\u00C2\u00A0(Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 1995),\u00C2\u00A0quantified\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0NanoDrop\u00C2\u00A0ND\u00E2\u0080\u00901000\u00C2\u00A0spectrophotometer\u00C2\u00A0and\u00C2\u00A0visualized\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A00.8%\u00C2\u00A0agarose\u00C2\u00A0gel.\u00C2\u00A0 \u00C2\u00A0 Northern\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 (Ausubel,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01995).\u00C2\u00A0Briefly,\u00C2\u00A030\u00C2\u00A0 \u00CE\u00BCg\u00C2\u00A0 total\u00C2\u00A0 RNA\u00C2\u00A0 was\u00C2\u00A0 separated\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 1%\u00C2\u00A0 agarose\u00E2\u0080\u0090formaldehyde\u00C2\u00A0 denaturing\u00C2\u00A0 gel\u00C2\u00A0 and\u00C2\u00A0 transferred\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 positively\u00C2\u00A0 charged\u00C2\u00A0 Nylon\u00C2\u00A0 membrane\u00C2\u00A0 (Roche\u00C2\u00A0 Diagnostics,\u00C2\u00A0 Germany).\u00C2\u00A0 Blots\u00C2\u00A0 were\u00C2\u00A0 probed\u00C2\u00A0 with\u00C2\u00A0 PCR\u00C2\u00A0 generated\u00C2\u00A0 fragments\u00C2\u00A0specific\u00C2\u00A0 for\u00C2\u00A0DUR3\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 loading\u00C2\u00A0control\u00C2\u00A0HHF1.\u00C2\u00A0The\u00C2\u00A0AlkPhosTM\u00C2\u00A0Direct\u00C2\u00A0Nucleic\u00C2\u00A0Acid\u00C2\u00A0Labeling\u00C2\u00A0 and\u00C2\u00A0CDP\u00E2\u0080\u0090Star\u00C2\u00A0Detection\u00C2\u00A0system\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0recommended\u00C2\u00A0for\u00C2\u00A0probe\u00C2\u00A0detection\u00C2\u00A0(Amersham\u00C2\u00A0Biosciences,\u00C2\u00A0 England).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0661\u00C2\u00A0bp\u00C2\u00A0DUR3\u00C2\u00A0probe\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0northern\u00C2\u00A0blotting\u00C2\u00A0was\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0probe\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0Southern\u00C2\u00A0 blotting\u00C2\u00A0(Section\u00C2\u00A02.4.4.1).\u00C2\u00A0The\u00C2\u00A0~500\u00C2\u00A0bp\u00C2\u00A0HHF1\u00C2\u00A0probe\u00C2\u00A0was\u00C2\u00A0supplied\u00C2\u00A0by\u00C2\u00A0another\u00C2\u00A0member\u00C2\u00A0of\u00C2\u00A0our\u00C2\u00A0lab\u00C2\u00A0(Coulon,\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0 \u00C2\u00A0 2.4.4.3\u00C2\u00A0\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR.\u00C2\u00A0Gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 2.3.3.3\u00C2\u00A0 using\u00C2\u00A0 total\u00C2\u00A0 RNA\u00C2\u00A0 from\u00C2\u00A0 24\u00C2\u00A0 hour\u00C2\u00A0 fermentations\u00C2\u00A0 (Section\u00C2\u00A02.4.4.2).\u00C2\u00A0 The\u00C2\u00A0DUR3\u00C2\u00A0 real\u00C2\u00A0 time\u00C2\u00A0PCR\u00C2\u00A0product\u00C2\u00A0was\u00C2\u00A0 generated\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0primers\u00C2\u00A0DUR3RTfwd\u00C2\u00A0 (5\u00E2\u0080\u0099\u00E2\u0080\u0090 GATCGGCCATGGTTGCTACTT\u00E2\u0080\u00903\u00E2\u0080\u0099)\u00C2\u00A0and\u00C2\u00A0DUR3RTrev\u00C2\u00A0(5\u00E2\u0080\u0099\u00E2\u0080\u0090GCGATAGTGTTCATCCCGGTT\u00E2\u0080\u00903\u00E2\u0080\u0099).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 45\u00C2\u00A0 \u00C2\u00A0 2.4.4.4\u00C2\u00A0 \u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis.\u00C2\u00A0Following\u00C2\u00A024\u00C2\u00A0hour\u00C2\u00A0fermentations\u00C2\u00A0(Section\u00C2\u00A02.4.4.2),\u00C2\u00A0total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7D3\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0transcriptome\u00C2\u00A0analysis\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.3.4.\u00C2\u00A0 \u00C2\u00A0 2.4.5\u00C2\u00A0\u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0using\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0on\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0activity,\u00C2\u00A0a\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0 uptake\u00C2\u00A0assay\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0previously\u00C2\u00A0described\u00C2\u00A0 (Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975).\u00C2\u00A0Briefly,\u00C2\u00A0appropriate\u00C2\u00A0 strains\u00C2\u00A0were\u00C2\u00A0grown\u00C2\u00A0(30\u00C2\u00B0C\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0180\u00C2\u00A0RPM)\u00C2\u00A0 in\u00C2\u00A0minimal\u00C2\u00A0media\u00C2\u00A0(1.7\u00C2\u00A0g/L\u00C2\u00A0YNB\u00C2\u00A0w/o\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0w/o\u00C2\u00A0ammonium\u00C2\u00A0 sulfate,\u00C2\u00A020\u00C2\u00A0g/L\u00C2\u00A0glucose,\u00C2\u00A01\u00C2\u00A0g/L\u00C2\u00A0ammonium\u00C2\u00A0sulfate\u00C2\u00A0or\u00C2\u00A01\u00C2\u00A0g/L\u00C2\u00A0L\u00E2\u0080\u0090proline)\u00C2\u00A0to\u00C2\u00A0approximately\u00C2\u00A01x107\u00C2\u00A0cells/mL.\u00C2\u00A0An\u00C2\u00A0 11\u00C2\u00A0mL\u00C2\u00A0sample\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00A0culture\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0250\u00C2\u00A0mL\u00C2\u00A0Erlenmeyer\u00C2\u00A0flask\u00C2\u00A0containing\u00C2\u00A080\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0of\u00C2\u00A036.6\u00C2\u00A0mM\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0(Sigma,\u00C2\u00A0USA\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A06.8\u00C2\u00A0mCi/mmol).\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0cultured\u00C2\u00A0(30\u00C2\u00B0C\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0180\u00C2\u00A0RPM)\u00C2\u00A0for\u00C2\u00A020\u00C2\u00A0min\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0 samples\u00C2\u00A0were\u00C2\u00A0 taken\u00C2\u00A0every\u00C2\u00A02\u00C2\u00A0min.\u00C2\u00A0The\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0 samples\u00C2\u00A0were\u00C2\u00A0applied\u00C2\u00A0 to\u00C2\u00A00.22\u00C2\u00A0 \u00CE\u00BCm\u00C2\u00A0nylon\u00C2\u00A0 filters\u00C2\u00A0 (Millipore,\u00C2\u00A0 USA)\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A025\u00C2\u00A0mL\u00C2\u00A0aliquots\u00C2\u00A0of\u00C2\u00A0cold\u00C2\u00A0minimal\u00C2\u00A0media\u00C2\u00A0 to\u00C2\u00A0which\u00C2\u00A010\u00C2\u00A0mM\u00C2\u00A0urea\u00C2\u00A0 (Fisher,\u00C2\u00A0USA)\u00C2\u00A0had\u00C2\u00A0 been\u00C2\u00A0added.\u00C2\u00A0Filters\u00C2\u00A0were\u00C2\u00A0placed\u00C2\u00A0 in\u00C2\u00A0scintillation\u00C2\u00A0vials,\u00C2\u00A0filled\u00C2\u00A0with\u00C2\u00A0scintillation\u00C2\u00A0fluid\u00C2\u00A0 (Fisher,\u00C2\u00A0USA)\u00C2\u00A0and\u00C2\u00A0 left\u00C2\u00A0 overnight\u00C2\u00A0to\u00C2\u00A0equilibrate.\u00C2\u00A0Samples\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0counted\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0recently\u00C2\u00A0calibrated\u00C2\u00A0Beckman\u00C2\u00A0LS6000IC\u00C2\u00A0 liquid\u00C2\u00A0 scintillation\u00C2\u00A0counter\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0counter\u00E2\u0080\u0099s\u00C2\u00A0 factory\u00C2\u00A0 \u00E2\u0080\u009814C\u00C2\u00A0quench\u00E2\u0080\u0099\u00C2\u00A0mode.\u00C2\u00A0DPM\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0converted\u00C2\u00A0 into\u00C2\u00A0nano\u00E2\u0080\u0090mole\u00C2\u00A0urea\u00C2\u00A0transported\u00C2\u00A0(Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975)\u00C2\u00A0and\u00C2\u00A0plotted\u00C2\u00A0against\u00C2\u00A0time\u00C2\u00A0in\u00C2\u00A0Excel.\u00C2\u00A0 \u00C2\u00A0 2.4.6\u00C2\u00A0\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0 \u00C2\u00A0 2.4.6.1\u00C2\u00A0 \u00C2\u00A0 Analysis\u00C2\u00A0 of\u00C2\u00A0 fermentation\u00C2\u00A0 rate\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0 must.\u00C2\u00A0 Fermentations\u00C2\u00A0 of\u00C2\u00A0 unfiltered\u00C2\u00A0 Calona\u00C2\u00A0 Chardonnay\u00C2\u00A0 must\u00C2\u00A0 with\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 522,\u00C2\u00A0 522EC\u00E2\u0080\u0090,\u00C2\u00A0 522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.4.1.\u00C2\u00A0 \u00C2\u00A0 2.4.6.2\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0 fermentation\u00C2\u00A0 rate\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0mash.\u00C2\u00A0Fermentations\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0 rice\u00C2\u00A0mash\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.4.2.\u00C2\u00A0 \u00C2\u00A0 2.4.6.3\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0for\u00C2\u00A0ethanol\u00C2\u00A0content.\u00C2\u00A0Ethanol\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0 and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0quantified\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.4.3.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 46\u00C2\u00A0 \u00C2\u00A0 2.4.7\u00C2\u00A0\u00C2\u00A0Functionality\u00C2\u00A0of\u00C2\u00A0metabolically\u00C2\u00A0enhanced\u00C2\u00A0yeasts\u00C2\u00A0 \u00C2\u00A0 2.4.7.1\u00C2\u00A0\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090 D3,\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A02.3.4.1.\u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0 as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.5.1.\u00C2\u00A0 \u00C2\u00A0 2.4.7.2\u00C2\u00A0\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0with\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0 522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A02.3.4.2.\u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0 Section\u00C2\u00A02.3.5.2.\u00C2\u00A0 \u00C2\u00A0 2.5\u00C2\u00A0Statistical\u00C2\u00A0analyses\u00C2\u00A0 \u00C2\u00A0 Two\u00C2\u00A0 factor\u00C2\u00A0 ANOVA\u00C2\u00A0 analyses\u00C2\u00A0 were\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 evaluate\u00C2\u00A0 the\u00C2\u00A0 variations\u00C2\u00A0 in\u00C2\u00A0 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the\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0were\u00C2\u00A0 transformed\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 linear\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 (Figure\u00C2\u00A015).\u00C2\u00A0After\u00C2\u00A0 colony\u00C2\u00A0PCR\u00C2\u00A0 screening\u00C2\u00A0of\u00C2\u00A0approximately\u00C2\u00A01500\u00C2\u00A0 yeast\u00C2\u00A0 transformants\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette,\u00C2\u00A0two\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0strains,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 K9EC\u00E2\u0080\u0090,\u00C2\u00A0were\u00C2\u00A0obtained.\u00C2\u00A0The\u00C2\u00A0designation\u00C2\u00A0 \u00E2\u0080\u0098EC\u00E2\u0080\u0090\u00E2\u0080\u0098\u00C2\u00A0denotes\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 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generated\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0URA3\u00C2\u00A0probes\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0BglII\u00C2\u00A0digested\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0probed\u00C2\u00A0with\u00C2\u00A0DUR1,2,\u00C2\u00A0two\u00C2\u00A0signals\u00C2\u00A0were\u00C2\u00A0detected\u00C2\u00A0for\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0corresponding\u00C2\u00A0to\u00C2\u00A05.0\u00C2\u00A0 kb\u00C2\u00A0and\u00C2\u00A09.0\u00C2\u00A0kb\u00C2\u00A0DNA\u00C2\u00A0fragments,\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0signal\u00C2\u00A0was\u00C2\u00A0detected\u00C2\u00A0for\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0corresponding\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A05.0\u00C2\u00A0kb\u00C2\u00A0DNA\u00C2\u00A0 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Probing\u00C2\u00A0with\u00C2\u00A0URA3\u00C2\u00A0gave\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0two\u00C2\u00A0signals\u00C2\u00A0for\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090,\u00C2\u00A0corresponding\u00C2\u00A0to\u00C2\u00A03.8\u00C2\u00A0kb\u00C2\u00A0and\u00C2\u00A04.4\u00C2\u00A0kb\u00C2\u00A0 DNA\u00C2\u00A0fragments,\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0signal\u00C2\u00A0for\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0corresponding\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A04.4\u00C2\u00A0kb\u00C2\u00A0fragment\u00C2\u00A0(Figure\u00C2\u00A017a).\u00C2\u00A0The\u00C2\u00A03.8\u00C2\u00A0 kb\u00C2\u00A0 fragment\u00C2\u00A0matches\u00C2\u00A0 the\u00C2\u00A0 expected\u00C2\u00A0 fragment\u00C2\u00A0 size\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0 URA3\u00C2\u00A0 locus,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 4.4\u00C2\u00A0 kb\u00C2\u00A0 fragment\u00C2\u00A0matches\u00C2\u00A0the\u00C2\u00A0expected\u00C2\u00A0fragment\u00C2\u00A0size\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090disrupted\u00C2\u00A0URA3\u00C2\u00A0locus\u00C2\u00A0(Figure\u00C2\u00A017b).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 16.\u00C2\u00A0 Integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 URA3\u00C2\u00A0 locus\u00C2\u00A0 of\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090 was\u00C2\u00A0 confirmed\u00C2\u00A0 by\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analyses\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 DUR1,2\u00C2\u00A0 probe\u00C2\u00A0 (a).\u00C2\u00A0 All\u00C2\u00A0 faint\u00C2\u00A0 bands\u00C2\u00A0 that\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 correspond\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0size\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0schematic\u00C2\u00A0representation\u00C2\u00A0were\u00C2\u00A0deemed\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0non\u00E2\u0080\u0090specific.\u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0 sample,\u00C2\u00A0the\u00C2\u00A0agarose\u00C2\u00A0gel\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0left\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0exposed\u00C2\u00A0film\u00C2\u00A0is\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0right.\u00C2\u00A0b)\u00C2\u00A0Schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 signals\u00C2\u00A0 expected\u00C2\u00A0 during\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analyses\u00C2\u00A0 (DUR1,2\u00C2\u00A0 probe)\u00C2\u00A0 of\u00C2\u00A0 recombinant\u00C2\u00A0yeasts\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0recombinant\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0locus.\u00C2\u00A0 The\u00C2\u00A0DUR1,2\u00C2\u00A0probe\u00C2\u00A0is\u00C2\u00A0illustrated\u00C2\u00A0with\u00C2\u00A0blue\u00C2\u00A0hatched\u00C2\u00A0lines\u00C2\u00A0(b).\u00C2\u00A0 \u00C2\u00A0 BglII DUR1,2PGK1p PGK1t ura3ura3 BglII 9085 bp DUR1,2 BglII 5084 bp BglII a)\u00C2\u00A0 b)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 49\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A017.\u00C2\u00A0Disruption\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0locus\u00C2\u00A0by\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090 \u00C2\u00A0 was\u00C2\u00A0confirmed\u00C2\u00A0by\u00C2\u00A0Southern\u00C2\u00A0blot\u00C2\u00A0analyses\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0URA3\u00C2\u00A0probe\u00C2\u00A0 (a).\u00C2\u00A0All\u00C2\u00A0 faint\u00C2\u00A0bands\u00C2\u00A0 that\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 correspond\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0size\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0schematic\u00C2\u00A0representation\u00C2\u00A0were\u00C2\u00A0deemed\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0non\u00E2\u0080\u0090specific.\u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0 sample,\u00C2\u00A0the\u00C2\u00A0agarose\u00C2\u00A0gel\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0left\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0exposed\u00C2\u00A0film\u00C2\u00A0is\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0right.\u00C2\u00A0b)\u00C2\u00A0Schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 signals\u00C2\u00A0 expected\u00C2\u00A0 during\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 (URA3\u00C2\u00A0 probe)\u00C2\u00A0 of\u00C2\u00A0 recombinant\u00C2\u00A0 yeasts\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 URA3\u00C2\u00A0 locus.\u00C2\u00A0The\u00C2\u00A0URA3\u00C2\u00A0probe\u00C2\u00A0is\u00C2\u00A0illustrated\u00C2\u00A0with\u00C2\u00A0green\u00C2\u00A0hatched\u00C2\u00A0lines\u00C2\u00A0(b).\u00C2\u00A0 BglII DUR1,2PGK1p PGK1t ura3ura3 BglII 3757 bp URA3 BglII 4455 bp BglII a)\u00C2\u00A0 b)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 50\u00C2\u00A0 \u00C2\u00A0 3.1.2.2\u00C2\u00A0 \u00C2\u00A0Sake\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0contain\u00C2\u00A0the\u00C2\u00A0bla\u00C2\u00A0and\u00C2\u00A0Tn5ble\u00C2\u00A0antibiotic\u00C2\u00A0resistance\u00C2\u00A0markers.\u00C2\u00A0 After\u00C2\u00A0transformation,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0successively\u00C2\u00A0sub\u00E2\u0080\u0090cultured\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090selective\u00C2\u00A0medium\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0eliminate\u00C2\u00A0pUT332,\u00C2\u00A0whose\u00C2\u00A0only\u00C2\u00A0purpose\u00C2\u00A0was\u00C2\u00A0to\u00C2\u00A0facilitate\u00C2\u00A0early\u00C2\u00A0screening\u00C2\u00A0for\u00C2\u00A0transformants.\u00C2\u00A0A\u00C2\u00A0Southern\u00C2\u00A0 blot\u00C2\u00A0using\u00C2\u00A0probes\u00C2\u00A0 specific\u00C2\u00A0 for\u00C2\u00A0bla\u00C2\u00A0 (Ampicillin\u00C2\u00A0 resistance)\u00C2\u00A0 and\u00C2\u00A0 Tn5ble\u00C2\u00A0 (Phleomycin\u00C2\u00A0 resistance)\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0these\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0absent\u00C2\u00A0from\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0(Figure\u00C2\u00A018).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A018.\u00C2\u00A0The\u00C2\u00A0genetically\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0contain\u00C2\u00A0the\u00C2\u00A0bla\u00C2\u00A0and\u00C2\u00A0Tn5ble\u00C2\u00A0antibiotic\u00C2\u00A0 resistance\u00C2\u00A0markers.\u00C2\u00A0The\u00C2\u00A0plasmid\u00C2\u00A0pUT332\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0bla\u00C2\u00A0and\u00C2\u00A0Tn5ble.\u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0 sample,\u00C2\u00A0the\u00C2\u00A0agarose\u00C2\u00A0gel\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0left\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0exposed\u00C2\u00A0film\u00C2\u00A0is\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0right.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 51\u00C2\u00A0 \u00C2\u00A0 3.1.2.3\u00C2\u00A0\u00C2\u00A0Sequence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090.\u00C2\u00A0To\u00C2\u00A0verify\u00C2\u00A0the\u00C2\u00A0 sequence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0 locus,\u00C2\u00A0single\u00C2\u00A0strand\u00C2\u00A0DNA\u00C2\u00A0sequencing\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 cassette\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0was\u00C2\u00A0completed.\u00C2\u00A0In\u00C2\u00A0silico\u00C2\u00A0sequence\u00C2\u00A0assembly\u00C2\u00A0and\u00C2\u00A0subsequent\u00C2\u00A0analysis\u00C2\u00A0revealed\u00C2\u00A0 one\u00C2\u00A0nucleotide\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0cassette\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0that\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0match\u00C2\u00A0the\u00C2\u00A0previously\u00C2\u00A0reported\u00C2\u00A0recombinant\u00C2\u00A0 DUR1,2\u00C2\u00A0 sequences\u00C2\u00A0and/or\u00C2\u00A0SGD\u00E2\u0080\u0099s\u00C2\u00A0S288C\u00C2\u00A0DUR1,2\u00C2\u00A0 sequence\u00C2\u00A0 (Table\u00C2\u00A08).\u00C2\u00A0The\u00C2\u00A0C\u00C2\u00A0 to\u00C2\u00A0T\u00C2\u00A0 switch\u00C2\u00A0 (theoretical\u00C2\u00A0 to\u00C2\u00A0 sequenced\u00C2\u00A0data)\u00C2\u00A0at\u00C2\u00A0nucleotide\u00C2\u00A0position\u00C2\u00A0821\u00C2\u00A0is\u00C2\u00A0located\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A05\u00E2\u0080\u0099URA3\u00C2\u00A0flanking\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette\u00C2\u00A0 and\u00C2\u00A0is\u00C2\u00A0likely\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0genetic\u00C2\u00A0polymorphism\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0Sake\u00C2\u00A0strain\u00C2\u00A0K9\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0laboratory\u00C2\u00A0strain\u00C2\u00A0S288C.\u00C2\u00A0 Comparison\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0DUR1,2\u00C2\u00A0ORF\u00C2\u00A0sequence\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 that\u00C2\u00A0of\u00C2\u00A0SGD\u00C2\u00A0 revealed\u00C2\u00A0no\u00C2\u00A0 nucleotide\u00C2\u00A0 changes\u00C2\u00A0 or\u00C2\u00A0 amino\u00C2\u00A0 acid\u00C2\u00A0 substitutions.\u00C2\u00A0 A\u00C2\u00A0 detailed\u00C2\u00A0 description\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DNA\u00C2\u00A0 sequences\u00C2\u00A0 that\u00C2\u00A0 comprise\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0is\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A09.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A08.\u00C2\u00A0Discrepancies\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0of\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0published\u00C2\u00A0sequences.\u00C2\u00A0 \u00C2\u00A0 Nucleotide\u00C2\u00A0 position\u00C2\u00A0 Description\u00C2\u00A0 821\u00C2\u00A0 Difference\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A05\u00E2\u0080\u0099\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0open\u00C2\u00A0reading\u00C2\u00A0frame\u00C2\u00A0of\u00C2\u00A0K9EC\u00E2\u0080\u0090 C\u00C3\u0086T\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A09.\u00C2\u00A0Detailed\u00C2\u00A0description\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette.\u00C2\u00A0 Nucleotide\u00C2\u00A0 position\u00C2\u00A0 Description\u00C2\u00A0 1\u00E2\u0080\u00904\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0SRF1\u00C2\u00A0\u00C2\u00BD\u00C2\u00A0site\u00C2\u00A0 5\u00E2\u0080\u0090950\u00C2\u00A0 URA3\u00C2\u00A0sequence\u00C2\u00A0 5\u00E2\u0080\u0090508\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0non\u00C2\u00A0coding\u00C2\u00A0sequence\u00C2\u00A0 509\u00E2\u0080\u0090950\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0URA3\u00C2\u00A0ORF\u00C2\u00A0 951\u00E2\u0080\u00902445\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 2446\u00E2\u0080\u00907953\u00C2\u00A0 DUR1,2\u00C2\u00A0ORF\u00C2\u00A0 7954\u00E2\u0080\u00908235\u00C2\u00A0 PGK1\u00C2\u00A0terminator\u00C2\u00A0 8236\u00E2\u0080\u00909187\u00C2\u00A0 URA3\u00C2\u00A0sequence\u00C2\u00A0 8236\u00E2\u0080\u00908640\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0URA3\u00C2\u00A0ORF\u00C2\u00A0 8641\u00E2\u0080\u00909187\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0non\u00C2\u00A0coding\u00C2\u00A0sequence\u00C2\u00A0 9188\u00E2\u0080\u00909191\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0SRF1\u00C2\u00A0\u00C2\u00BD\u00C2\u00A0site\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0silico\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 two\u00C2\u00A0new\u00C2\u00A0ORFs\u00C2\u00A0were\u00C2\u00A0created\u00C2\u00A0 during\u00C2\u00A0 construction\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette;\u00C2\u00A0 these\u00C2\u00A0 ORFs\u00C2\u00A0 were\u00C2\u00A0 composed\u00C2\u00A0 entirely\u00C2\u00A0 of\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 sequences\u00C2\u00A0(Figure\u00C2\u00A019).\u00C2\u00A0Novel\u00C2\u00A0ORF1\u00C2\u00A0(447\u00C2\u00A0bp)\u00C2\u00A0is\u00C2\u00A0located\u00C2\u00A0at\u00C2\u00A0nucleotide\u00C2\u00A0position\u00C2\u00A0509\u00E2\u0080\u0090955\u00C2\u00A0while\u00C2\u00A0novel\u00C2\u00A0ORF2\u00C2\u00A0 (792\u00C2\u00A0bp)\u00C2\u00A0is\u00C2\u00A0located\u00C2\u00A0at\u00C2\u00A0position\u00C2\u00A0767\u00E2\u0080\u00901558.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 52\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.1.2.4\u00C2\u00A0 \u00C2\u00A0Confirmation\u00C2\u00A0of\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 24\u00C2\u00A0 hour\u00C2\u00A0 fermentations\u00C2\u00A0 of\u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 confirm\u00C2\u00A0 and\u00C2\u00A0 quantify\u00C2\u00A0 constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090.\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 up\u00C2\u00A0 regulated\u00C2\u00A0DUR1,2\u00C2\u00A0 expression\u00C2\u00A0 by\u00C2\u00A0 9.13\u00E2\u0080\u0090fold\u00C2\u00A0 and\u00C2\u00A0 12.77\u00E2\u0080\u0090fold,\u00C2\u00A0 respectively,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 parental\u00C2\u00A0 strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0(Figure\u00C2\u00A020).\u00C2\u00A0Expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0was\u00C2\u00A0detected\u00C2\u00A0in\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0(NCR)\u00C2\u00A0 conditions\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 and\u00C2\u00A0 terminator\u00C2\u00A0 signals\u00C2\u00A0 are\u00C2\u00A0 effective\u00C2\u00A0 at\u00C2\u00A0 overcoming\u00C2\u00A0 repression\u00C2\u00A0by\u00C2\u00A0NCR\u00C2\u00A0during\u00C2\u00A0fermentation.\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A019.\u00C2\u00A0A\u00C2\u00A0 schematic\u00C2\u00A0 representation\u00C2\u00A0of\u00C2\u00A0new\u00C2\u00A0ORFs\u00C2\u00A0of\u00C2\u00A0more\u00C2\u00A0 than\u00C2\u00A0100\u00C2\u00A0 codons\u00C2\u00A0generated\u00C2\u00A0during\u00C2\u00A0 construction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette.\u00C2\u00A0Two\u00C2\u00A0new\u00C2\u00A0ORFs,\u00C2\u00A0entirely\u00C2\u00A0composed\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0sequences,\u00C2\u00A0 were\u00C2\u00A0created.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Novel\u00C2\u00A0ORF2 (792\u00C2\u00A0bp)\u00C2\u00A0 Novel\u00C2\u00A0ORF1\u00C2\u00A0 (447\u00C2\u00A0bp)\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0URA3\u00C2\u00A0 PGK1p\u00C2\u00A0 DUR1,2 5\u00E2\u0080\u0099\u00C2\u00A0SRF1\u00C2\u00A0\u00C2\u00BD\u00C2\u00A0site\u00C2\u00A0 PGK1t\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0URA3 \u00C2\u00A0 \u00C2\u00A0 53\u00C2\u00A0 \u00C2\u00A0 1.00 9.13 1.00 12.77 0 2 4 6 8 10 12 14 16 18 K7 K7EC- K9 K9EC- Strain R el at iv e qu an tif ic at io n (fo ld ) R el at iv e qu an tif ic at io n (fo ld ) \u00C2\u00A0 Figure\u00C2\u00A020.\u00C2\u00A0Gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0 (qRT\u00E2\u0080\u0090PCR)\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 indicates\u00C2\u00A0 functionality\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0cassette\u00C2\u00A0and\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0(NCR)\u00C2\u00A0conditions.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0was\u00C2\u00A0 extracted\u00C2\u00A0 from\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 harvested\u00C2\u00A0 after\u00C2\u00A0 24\u00C2\u00A0 hour\u00C2\u00A0 fermentation\u00C2\u00A0 (20\u00C2\u00B0C)\u00C2\u00A0 in\u00C2\u00A0 filter\u00C2\u00A0 sterilized\u00C2\u00A0 Calona\u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0 that\u00C2\u00A0 was\u00C2\u00A0 inoculated\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 final\u00C2\u00A0 OD600\u00C2\u00A0 =\u00C2\u00A0 0.1.\u00C2\u00A0 Total\u00C2\u00A0 RNA\u00C2\u00A0 was\u00C2\u00A0 subsequently\u00C2\u00A0 reverse\u00C2\u00A0 transcribed,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 resultant\u00C2\u00A0 cDNA\u00C2\u00A0was\u00C2\u00A0 amplified\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 SYBR\u00C2\u00A0 green\u00C2\u00A0dye.\u00C2\u00A0DUR1,2\u00C2\u00A0gene\u00C2\u00A0 expression\u00C2\u00A0was\u00C2\u00A0standardized\u00C2\u00A0to\u00C2\u00A0ACT1\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0for\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0calibrated\u00C2\u00A0to\u00C2\u00A0 their\u00C2\u00A0 respective\u00C2\u00A0 parental\u00C2\u00A0 strain\u00C2\u00A0 K7\u00C2\u00A0 and\u00C2\u00A0 K9.\u00C2\u00A0 Fermentations\u00C2\u00A0were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0represent\u00C2\u00A095%\u00C2\u00A0confidence\u00C2\u00A0intervals.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 3.1.2.5\u00C2\u00A0 \u00C2\u00A0Effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcriptomes\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 24\u00C2\u00A0 hour\u00C2\u00A0 fermentations\u00C2\u00A0 of\u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 analyze\u00C2\u00A0 the\u00C2\u00A0 impact\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0on\u00C2\u00A0 global\u00C2\u00A0 gene\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090.\u00C2\u00A0Reported\u00C2\u00A0 changes\u00C2\u00A0 in\u00C2\u00A0 gene\u00C2\u00A0 expression\u00C2\u00A0 were\u00C2\u00A0cut\u00C2\u00A0off\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0minimum\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0change\u00C2\u00A0in\u00C2\u00A0expression\u00C2\u00A0(SLRAvg\u00C2\u00A0\u00E2\u0089\u00A5\u00C2\u00A02\u00C2\u00A0or\u00C2\u00A0SLRAvg\u00C2\u00A0\u00E2\u0089\u00A4\u00C2\u00A0\u00E2\u0080\u00902)\u00C2\u00A0to\u00C2\u00A0ensure\u00C2\u00A0elimination\u00C2\u00A0of\u00C2\u00A0 experimental\u00C2\u00A0noise\u00C2\u00A0inherent\u00C2\u00A0in\u00C2\u00A0microarray\u00C2\u00A0analysis;\u00C2\u00A0this\u00C2\u00A0cut\u00E2\u0080\u0090off\u00C2\u00A0is\u00C2\u00A0supported\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0previously\u00C2\u00A0published\u00C2\u00A0 statistical\u00C2\u00A0examination\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0wine\u00C2\u00A0yeast\u00E2\u0080\u0099s\u00C2\u00A0transcriptome\u00C2\u00A0during\u00C2\u00A0fermentation\u00C2\u00A0(Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02008).\u00C2\u00A0 \u00C2\u00A0 Integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0URA3\u00C2\u00A0 locus\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0had\u00C2\u00A0 a\u00C2\u00A0minimal\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 yeast\u00E2\u0080\u0099s\u00C2\u00A0transcriptome.\u00C2\u00A0DUR1,2\u00C2\u00A0was\u00C2\u00A0upregulated\u00C2\u00A0by\u00C2\u00A06.35\u00E2\u0080\u0090fold\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090;\u00C2\u00A0URA3\u00C2\u00A0was\u00C2\u00A0downregulated\u00C2\u00A0by\u00C2\u00A02.35\u00E2\u0080\u0090 \u00C2\u00A0 \u00C2\u00A0 54\u00C2\u00A0 \u00C2\u00A0 fold\u00C2\u00A0but\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0included\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A010\u00C2\u00A0as\u00C2\u00A0it\u00C2\u00A0fell\u00C2\u00A0below\u00C2\u00A0the\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0cut\u00C2\u00A0off.\u00C2\u00A0Besides\u00C2\u00A0DUR1,2,\u00C2\u00A0two\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0 upregulated\u00C2\u00A0greater\u00C2\u00A0than\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(Table\u00C2\u00A010);\u00C2\u00A0seven\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0downregulated\u00C2\u00A0more\u00C2\u00A0than\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0in\u00C2\u00A0 K7EC\u00E2\u0080\u0090.\u00C2\u00A0 No\u00C2\u00A0 metabolic\u00C2\u00A0 pathways\u00C2\u00A0 were\u00C2\u00A0 affected\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette;\u00C2\u00A0 however,\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 downregulated\u00C2\u00A0 three\u00C2\u00A0 unrelated\u00C2\u00A0 genes\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 meiosis/sporulation\u00C2\u00A0 (RME1,\u00C2\u00A0SSP1,\u00C2\u00A0SDS3)\u00C2\u00A0 indicating\u00C2\u00A0a\u00C2\u00A0possible\u00C2\u00A0negative\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0 sporulation\u00C2\u00A0efficiency\u00C2\u00A0 (Table\u00C2\u00A010).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A0 10.\u00C2\u00A0 Effect\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 genome\u00C2\u00A0 of\u00C2\u00A0 K7\u00C2\u00A0 on\u00C2\u00A0 global\u00C2\u00A0 gene\u00C2\u00A0 expression\u00C2\u00A0 patterns\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(\u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0change).\u00C2\u00A0Reported\u00C2\u00A0changes\u00C2\u00A0are\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strain\u00C2\u00A0K7.\u00C2\u00A0 Total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0harvested\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0hours\u00C2\u00A0 into\u00C2\u00A0 fermentation\u00C2\u00A0of\u00C2\u00A0 filter\u00C2\u00A0 sterilized\u00C2\u00A0Chardonnay\u00C2\u00A0 must,\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0 for\u00C2\u00A0hybridization\u00C2\u00A0 to\u00C2\u00A0microarray.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0 in\u00C2\u00A0duplicate\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0were\u00C2\u00A0averaged\u00C2\u00A0(p\u00E2\u0089\u00A40.005).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Genes expressed at higher levels in K7EC- Fold Change Gene Symbol Biological Process 6.60 RTG1 Transcription factor (bHLH) involved in interorganelle communication 6.35 DUR1,2 Urea amidolyase 4.23 HAC1 bZIP (basic-leucine zipper) protein involved in unfolded protein response Genes expressed at lower levels in K7EC- Fold Change Gene Symbol Biological Process -5.64 RME1 Zinc finger protein involved in control of meiosis -4.97 SEO1 Permease involved in methionine transport -4.67 MF(Alpha)1 Mating factor alpha -4.64 SSP1 Protein involved in the control of meiotic nuclear divisions and spore formation -4.21 SDS3 Protein involved in deactylase complex and transcriptional silencing during sporulation -4.16 CBP1 Protein required for Cytochrome B mRNA stability or 5\u00E2\u0080\u0099 processing -4.12 RUD3 Protein involved in organization of Golgi \u00C2\u00A0 3.1.3\u00C2\u00A0\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 3.1.3.1\u00C2\u00A0\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0must.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0 and\u00C2\u00A0metabolically\u00C2\u00A0 engineered\u00C2\u00A0 Sake\u00C2\u00A0 yeasts\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figures\u00C2\u00A0 21a,b.\u00C2\u00A0As\u00C2\u00A0 is\u00C2\u00A0 common\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0must,\u00C2\u00A0 fermentations\u00C2\u00A0were\u00C2\u00A0 robust\u00C2\u00A0and\u00C2\u00A0 rapid\u00C2\u00A0 (~400\u00C2\u00A0hours).\u00C2\u00A0The\u00C2\u00A0 fermentation\u00C2\u00A0profiles\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A021a,b\u00C2\u00A0 indicate\u00C2\u00A0substantial\u00C2\u00A0equivalence\u00C2\u00A0amongst\u00C2\u00A0the\u00C2\u00A0parent\u00C2\u00A0and\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0as\u00C2\u00A0far\u00C2\u00A0as\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0 is\u00C2\u00A0concerned.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 55\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 0 100 200 300 400 500 T im e \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 7 K 7E C \u00E2\u0080\u0090 \u00C2\u00A0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 0 100 200 300 400 500 T im e \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 9 K 9E C \u00E2\u0080\u0090 \u00C2\u00A0 Figure\u00C2\u00A0 21.\u00C2\u00A0 Fermentation\u00C2\u00A0 profiles\u00C2\u00A0 (weight\u00C2\u00A0 loss)\u00C2\u00A0 of\u00C2\u00A0 parental\u00C2\u00A0 and\u00C2\u00A0 DUR1,2\u00C2\u00A0 engineered\u00C2\u00A0 Sake\u00C2\u00A0 strains\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0inoculating\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0(a)\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 (b)\u00C2\u00A0 K9\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 into\u00C2\u00A0 unfiltered\u00C2\u00A0 Calona\u00C2\u00A0 Chardonnay\u00C2\u00A0 must\u00C2\u00A0 (final\u00C2\u00A0 OD600\u00C2\u00A0 =\u00C2\u00A0 0.1).\u00C2\u00A0 Fermentations\u00C2\u00A0 were\u00C2\u00A0 incubated\u00C2\u00A0to\u00C2\u00A0completion\u00C2\u00A0(~400\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A020\u00C2\u00B0C.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0in\u00C2\u00A0triplicate\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0 were\u00C2\u00A0averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0indicate\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.1.3.2\u00C2\u00A0 \u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0mash.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0and\u00C2\u00A0 metabolically\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A022a,b.\u00C2\u00A0 In\u00C2\u00A0contrast\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 fermentations\u00C2\u00A0of\u00C2\u00A0 a)\u00C2\u00A0 b)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 56\u00C2\u00A0 \u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A03.1.3.1,\u00C2\u00A0Sake\u00C2\u00A0 fermentations\u00C2\u00A0were\u00C2\u00A0 less\u00C2\u00A0 robust,\u00C2\u00A0and\u00C2\u00A0 slower\u00C2\u00A0 (~600\u00C2\u00A0hours\u00C2\u00A0 to\u00C2\u00A0 completion).\u00C2\u00A0The\u00C2\u00A0fermentation\u00C2\u00A0profiles\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A022a,b\u00C2\u00A0indicate\u00C2\u00A0substantial\u00C2\u00A0equivalence\u00C2\u00A0amongst\u00C2\u00A0 the\u00C2\u00A0parent\u00C2\u00A0and\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0as\u00C2\u00A0far\u00C2\u00A0as\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0is\u00C2\u00A0concerned.\u00C2\u00A0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 100 200 300 400 500 600 T im e \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 7 K 7E C \u00E2\u0080\u0090 \u00C2\u00A0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 100 200 300 400 500 600 T ime \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 9 K 9E C \u00E2\u0080\u0090 \u00C2\u00A0 Figure\u00C2\u00A022.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0 (weight\u00C2\u00A0 loss)\u00C2\u00A0of\u00C2\u00A0parental\u00C2\u00A0and\u00C2\u00A0DUR1,2\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0strains\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0 wine.\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0inoculation\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1)\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0(a)\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7 EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 (b)\u00C2\u00A0 K9\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 into\u00C2\u00A0white\u00C2\u00A0 rice\u00C2\u00A0 (Kokako\u00C2\u00A0 Rose)\u00C2\u00A0 and\u00C2\u00A0 koji\u00C2\u00A0mash\u00C2\u00A0 (Vision\u00C2\u00A0 Brewing).\u00C2\u00A0 Fermentations\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 to\u00C2\u00A0 completion\u00C2\u00A0 (~600\u00C2\u00A0 hours)\u00C2\u00A0 at\u00C2\u00A0 18\u00C2\u00B0C.\u00C2\u00A0 Fermentations\u00C2\u00A0were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 and\u00C2\u00A0 data\u00C2\u00A0 averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0indicate\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 3.1.3.3\u00C2\u00A0\u00C2\u00A0Utilization\u00C2\u00A0of\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0fructose\u00C2\u00A0and\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0by\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0 The\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0 fructose\u00C2\u00A0utilization\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0ethanol\u00C2\u00A0production\u00C2\u00A0 in\u00C2\u00A0 a)\u00C2\u00A0 b)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 57\u00C2\u00A0 \u00C2\u00A0 strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0was\u00C2\u00A0investigated.\u00C2\u00A0Glucose,\u00C2\u00A0fructose\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0were\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0LC\u00C2\u00A0analysis\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation.\u00C2\u00A0Compared\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0respective\u00C2\u00A0parental\u00C2\u00A0strains,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0produced\u00C2\u00A0Sake\u00C2\u00A0 wine\u00C2\u00A0with\u00C2\u00A0substantially\u00C2\u00A0equivalent\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0residual\u00C2\u00A0glucose,\u00C2\u00A0residual\u00C2\u00A0fructose\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0(Table\u00C2\u00A011).\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A011.\u00C2\u00A0Utilization\u00C2\u00A0of\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0fructose\u00C2\u00A0and\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0parental\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 (K7\u00C2\u00A0 and\u00C2\u00A0 K9),\u00C2\u00A0 their\u00C2\u00A0 metabolically\u00C2\u00A0 engineered\u00C2\u00A0 counterparts\u00C2\u00A0 (K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090).\u00C2\u00A0 Sake\u00C2\u00A0 wine\u00C2\u00A0 was\u00C2\u00A0 produced\u00C2\u00A0by\u00C2\u00A0inoculation\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains,\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0white\u00C2\u00A0rice\u00C2\u00A0(Kokako\u00C2\u00A0Rose)\u00C2\u00A0and\u00C2\u00A0koji\u00C2\u00A0 mash\u00C2\u00A0 (Vision\u00C2\u00A0Brewing).\u00C2\u00A0 Fermentations\u00C2\u00A0 (Figures\u00C2\u00A0 22a,b)\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 to\u00C2\u00A0 completion\u00C2\u00A0 (~600\u00C2\u00A0hours)\u00C2\u00A0 at\u00C2\u00A0 18\u00C2\u00B0C.\u00C2\u00A0Glucose\u00C2\u00A0and\u00C2\u00A0 fructose\u00C2\u00A0 (g/L)\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0 (v/v)\u00C2\u00A0were\u00C2\u00A0quantified\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation.\u00C2\u00A0Data\u00C2\u00A0 were\u00C2\u00A0analyzed\u00C2\u00A0for\u00C2\u00A0statistical\u00C2\u00A0significance\u00C2\u00A0(p\u00E2\u0089\u00A40.05)\u00C2\u00A0using\u00C2\u00A0two\u00C2\u00A0factor\u00C2\u00A0ANOVA\u00C2\u00A0analysis.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.1.4\u00C2\u00A0\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0 wine\u00C2\u00A0by\u00C2\u00A0approximately\u00C2\u00A030%\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0ferment\u00C2\u00A0Chardonnay\u00C2\u00A0must,\u00C2\u00A0and\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0wine.\u00C2\u00A0 \u00C2\u00A0 Glucose\u00C2\u00A0 \u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 p* K9\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 0.059\u00C2\u00A0 0.056\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.057\u00C2\u00A0 0.102\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 0.066\u00C2\u00A0 0.056\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.114\u00C2\u00A0 0.103\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 0.059\u00C2\u00A0 0.051\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.072\u00C2\u00A0 0.090\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Residual\u00C2\u00A0glucose\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 0.06\u00C2\u00A0 0.05\u00C2\u00A0 ns 0.08\u00C2\u00A0 0.10\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.00\u00C2\u00A0 0.00\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.03\u00C2\u00A0 0.01\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Fructose\u00C2\u00A0 \u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 p* K9\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 0.736\u00C2\u00A0 0.733\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.333\u00C2\u00A0 0.242\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 0.841\u00C2\u00A0 0.752\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.271\u00C2\u00A0 0.361\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 0.374\u00C2\u00A0 0.665\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.338\u00C2\u00A0 0.303\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Residual\u00C2\u00A0fructose\u00C2\u00A0average\u00C2\u00A0(n=3) 0.65\u00C2\u00A0 0.72\u00C2\u00A0 ns 0.31\u00C2\u00A0 0.30\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.25\u00C2\u00A0 0.05\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.04\u00C2\u00A0 0.06\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Ethanol\u00C2\u00A0 \u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 p* K9\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 12.23\u00C2\u00A0 12.49\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 12.71\u00C2\u00A0 13.22\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 12.27\u00C2\u00A0 12.64\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 13.59\u00C2\u00A0 12.71\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 11.95\u00C2\u00A0 11.93\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 13.05\u00C2\u00A0 12.17\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Ethanol\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 12.15\u00C2\u00A0 12.35 ns 13.12\u00C2\u00A0 12.70\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.17\u00C2\u00A0 0.37\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 0.44\u00C2\u00A0 0.53\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 *\u00C2\u00A0si,\u00C2\u00A0ns:\u00C2\u00A0significant\u00C2\u00A0at\u00C2\u00A0p\u00E2\u0089\u00A40.05,\u00C2\u00A0or\u00C2\u00A0non\u00E2\u0080\u0090significant\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 58\u00C2\u00A0 \u00C2\u00A0 Fermentation\u00C2\u00A0profiles\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0and\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A0 21a,b.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0usually\u00C2\u00A0high\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0found\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine,\u00C2\u00A0during\u00C2\u00A0 laboratory\u00C2\u00A0scale\u00C2\u00A0wine\u00C2\u00A0 fermentations\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0Sake\u00C2\u00A0 strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0produced\u00C2\u00A0 relatively\u00C2\u00A0 low\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0EC,\u00C2\u00A064.87\u00C2\u00A0and\u00C2\u00A0 56.16\u00C2\u00A0 ppm,\u00C2\u00A0 respectively\u00C2\u00A0 (Table\u00C2\u00A0 12);\u00C2\u00A0 the\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0 by\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A029.88%\u00C2\u00A0and\u00C2\u00A00%,\u00C2\u00A0respectively.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A012.\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0making.\u00C2\u00A0The\u00C2\u00A0 concentration\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 (\u00C2\u00B5g/L)\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K9,\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 was\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0GC/MS.\u00C2\u00A0Strains\u00C2\u00A0were\u00C2\u00A0inoculated\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1)\u00C2\u00A0into\u00C2\u00A0unfiltered\u00C2\u00A0Calona\u00C2\u00A0Chardonnay\u00C2\u00A0 must\u00C2\u00A0and\u00C2\u00A0fermentations\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0to\u00C2\u00A0completion\u00C2\u00A0(~350\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A020\u00C2\u00B0C.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0 given\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A021.\u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K9\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 69.05\u00C2\u00A0 46.3\u00C2\u00A0 56.13\u00C2\u00A0 55.18\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 63.62\u00C2\u00A0 42.94\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 61.93\u00C2\u00A0 47.21\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Average\u00C2\u00A0(n=3)\u00C2\u00A0 64.87\u00C2\u00A0 45.48\u00C2\u00A0 56.16\u00C2\u00A0 55.18\u00C2\u00A0 STDEV\u00C2\u00A0 3.72\u00C2\u00A0 2.25\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 RSD\u00C2\u00A0(%)\u00C2\u00A0 5.73\u00C2\u00A0 4.94\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Reduction\u00C2\u00A0(%)\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 29.88\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 ~0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.1.5\u00C2\u00A0 \u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0 by\u00C2\u00A0approximately\u00C2\u00A068%\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0 used\u00C2\u00A0to\u00C2\u00A0ferment\u00C2\u00A0Sake\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0(rice\u00C2\u00A0and\u00C2\u00A0koji),\u00C2\u00A0and\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0 Fermentation\u00C2\u00A0profiles\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0and\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A0 22a,b.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0laboratory\u00C2\u00A0scale\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0fermentations\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0Sake\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0produced\u00C2\u00A0 significantly\u00C2\u00A0more\u00C2\u00A0EC\u00C2\u00A0(211.19\u00C2\u00A0and\u00C2\u00A0344.16\u00C2\u00A0ppm,\u00C2\u00A0respectively\u00C2\u00A0\u00E2\u0080\u0090\u00C2\u00A0Table\u00C2\u00A013)\u00C2\u00A0than\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0fermentations\u00C2\u00A0 (Table\u00C2\u00A012).\u00C2\u00A0 In\u00C2\u00A0Sake\u00C2\u00A0wine,\u00C2\u00A0 the\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0by\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A067.54%\u00C2\u00A0and\u00C2\u00A068.33%,\u00C2\u00A0 respectively\u00C2\u00A0(Table\u00C2\u00A013),\u00C2\u00A0making\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0much\u00C2\u00A0more\u00C2\u00A0effective\u00C2\u00A0at\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0than\u00C2\u00A0in\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 59\u00C2\u00A0 \u00C2\u00A0 Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Given\u00C2\u00A0the\u00C2\u00A0substantial\u00C2\u00A0difference\u00C2\u00A0in\u00C2\u00A0EC\u00C2\u00A0production\u00C2\u00A0and\u00C2\u00A0reduction\u00C2\u00A0by\u00C2\u00A0identical\u00C2\u00A0strains,\u00C2\u00A0it\u00C2\u00A0 seems\u00C2\u00A0 imperative\u00C2\u00A0 that\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0be\u00C2\u00A0evaluated\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0native\u00C2\u00A0environment\u00C2\u00A0 to\u00C2\u00A0accurately\u00C2\u00A0assess\u00C2\u00A0 the\u00C2\u00A0 efficacy\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A013.\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0 functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0brewing.\u00C2\u00A0 The\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 (\u00C2\u00B5g/L)\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine,\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0 inoculation\u00C2\u00A0 (final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1)\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K9,\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 into\u00C2\u00A0 white\u00C2\u00A0 rice\u00C2\u00A0 (Kokako\u00C2\u00A0 Rose)\u00C2\u00A0 and\u00C2\u00A0 koji\u00C2\u00A0mash\u00C2\u00A0 (Vision\u00C2\u00A0 Brewing),\u00C2\u00A0 was\u00C2\u00A0 quantified\u00C2\u00A0 by\u00C2\u00A0 GC/MS.\u00C2\u00A0 Fermentations\u00C2\u00A0 were\u00C2\u00A0 incubated\u00C2\u00A0 to\u00C2\u00A0 completion\u00C2\u00A0 (~600\u00C2\u00A0 hours)\u00C2\u00A0 at\u00C2\u00A0 18\u00C2\u00B0C\u00C2\u00A0 and\u00C2\u00A0 fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A022.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2\u00C2\u00A0\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0522\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 3.2.1\u00C2\u00A0\u00C2\u00A0Sequence\u00C2\u00A0of\u00C2\u00A0pUCMD\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0multicopy\u00C2\u00A0episomal\u00C2\u00A0plasmid\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0ORF\u00C2\u00A0 inserted\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0 and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0flanked\u00C2\u00A0by\u00C2\u00A0TRP1\u00C2\u00A0sequences\u00C2\u00A0was\u00C2\u00A0constructed;\u00C2\u00A0kanMX\u00C2\u00A0served\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0selective\u00C2\u00A0marker\u00C2\u00A0 in\u00C2\u00A0 pUCMD\u00C2\u00A0 (Figure\u00C2\u00A0 14).\u00C2\u00A0 Single\u00C2\u00A0 strand\u00C2\u00A0 sequencing\u00C2\u00A0 revealed\u00C2\u00A0 this\u00C2\u00A0 plasmid\u00C2\u00A0 contained\u00C2\u00A0 the\u00C2\u00A0 desired\u00C2\u00A0 DNA\u00C2\u00A0 fragments\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 correct\u00C2\u00A0 order\u00C2\u00A0 and\u00C2\u00A0 orientation.\u00C2\u00A0 Furthermore,\u00C2\u00A0 in\u00C2\u00A0 silico\u00C2\u00A0 assembly\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 coding\u00C2\u00A0 region\u00C2\u00A0in\u00C2\u00A0pUCMD\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0ORF\u00C2\u00A0was\u00C2\u00A0identical\u00C2\u00A0in\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0sequence\u00C2\u00A0and\u00C2\u00A0length\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0 published\u00C2\u00A0on\u00C2\u00A0SGD.\u00C2\u00A0 \u00C2\u00A0 Aligning\u00C2\u00A0 the\u00C2\u00A0 sequence\u00C2\u00A0 data\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 expected\u00C2\u00A0 sequence\u00C2\u00A0 revealed\u00C2\u00A0 nine\u00C2\u00A0 single\u00C2\u00A0nucleotide\u00C2\u00A0changes\u00C2\u00A0along\u00C2\u00A0 the\u00C2\u00A0 length\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0cassette\u00C2\u00A0 (Table\u00C2\u00A014),\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0highlighted\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0DNA\u00C2\u00A0 sequence\u00C2\u00A0 alignment\u00C2\u00A0 between\u00C2\u00A0 of\u00C2\u00A0 S288C\u00C2\u00A0 and\u00C2\u00A0 pUCMD\u00C2\u00A0 (Figure\u00C2\u00A0 23).\u00C2\u00A0 A\u00C2\u00A0 detailed\u00C2\u00A0 description\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DNA\u00C2\u00A0 sequences\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0is\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A015.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K9\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 224.11\u00C2\u00A0 72.5\u00C2\u00A0 241.73\u00C2\u00A0 108.92\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 198.7\u00C2\u00A0 79.28\u00C2\u00A0 265.01\u00C2\u00A0 105.96\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 210.75\u00C2\u00A0 53.85\u00C2\u00A0 525.74\u00C2\u00A0 112.15\u00C2\u00A0 Average\u00C2\u00A0(n=3)\u00C2\u00A0 211.19\u00C2\u00A0 68.54\u00C2\u00A0 344.16\u00C2\u00A0 109.01\u00C2\u00A0 STDEV\u00C2\u00A0 12.71\u00C2\u00A0 13.17\u00C2\u00A0 157.68\u00C2\u00A0 3.10\u00C2\u00A0 RSD\u00C2\u00A0(%)\u00C2\u00A0 6.02\u00C2\u00A0 19.22\u00C2\u00A0 45.82\u00C2\u00A0 2.84\u00C2\u00A0 Reduction\u00C2\u00A0(%)\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 67.54\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 68.33\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 60\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A014.\u00C2\u00A0Discrepancies\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 in\u00C2\u00A0pUCMD\u00C2\u00A0 and\u00C2\u00A0published\u00C2\u00A0 sequences.\u00C2\u00A0Nucleotide\u00C2\u00A0 mismatches\u00C2\u00A0are\u00C2\u00A0designated\u00C2\u00A0as\u00C2\u00A0\u00E2\u0080\u0098X\u00C3\u0086\u00C2\u00A0Y\u00E2\u0080\u0099,\u00C2\u00A0meaning\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0predicted\u00C2\u00A0nucleotide\u00C2\u00A0\u00E2\u0080\u0098X\u00E2\u0080\u0099\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0sequenced\u00C2\u00A0as\u00C2\u00A0 \u00E2\u0080\u0098Y\u00E2\u0080\u0099.\u00C2\u00A0The\u00C2\u00A0DNA\u00C2\u00A0alignment\u00C2\u00A0of\u00C2\u00A0S288C\u00C2\u00A0and\u00C2\u00A0pUCMD\u00C2\u00A0is\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A023.\u00C2\u00A0 \u00C2\u00A0 Nucleotide\u00C2\u00A0 position\u00C2\u00A0 Region\u00C2\u00A0of\u00C2\u00A0cassette\u00C2\u00A0 Description\u00C2\u00A0 928\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 C\u00C3\u0086T\u00C2\u00A0\u00C2\u00A0 999\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 C\u00C3\u0086T\u00C2\u00A0 1230\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 C\u00C3\u0086T\u00C2\u00A0 1491\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 C\u00C3\u0086T\u00C2\u00A0 1494\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 G\u00C3\u0086A\u00C2\u00A0 3524\u00C2\u00A0 DUR3\u00C2\u00A0ORF\u00C2\u00A0 G\u00C3\u0086C:\u00C2\u00A0Silent\u00C2\u00A0 3821\u00C2\u00A0 DUR3\u00C2\u00A0ORF\u00C2\u00A0 T\u00C3\u0086C:\u00C2\u00A0Silent\u00C2\u00A0 3970\u00C2\u00A0 DUR3\u00C2\u00A0ORF\u00C2\u00A0 G\u00C3\u0086A:\u00C2\u00A0Silent\u00C2\u00A0 4160\u00C2\u00A0 DUR3\u00C2\u00A0ORF\u00C2\u00A0 A\u00C3\u0086C:\u00C2\u00A0Silent\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A015.\u00C2\u00A0\u00C2\u00A0Detailed\u00C2\u00A0description\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette.\u00C2\u00A0 \u00C2\u00A0 Nucleotide\u00C2\u00A0 position\u00C2\u00A0 Description\u00C2\u00A0 1\u00E2\u0080\u00906\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0Apa1\u00C2\u00A0restriction\u00C2\u00A0site\u00C2\u00A0 7\u00E2\u0080\u0090291\u00C2\u00A0 5\u00E2\u0080\u0099\u00C2\u00A0TRP1\u00C2\u00A0sequence\u00C2\u00A0 292\u00E2\u0080\u0090475\u00C2\u00A0 pHVXKD3\u00C2\u00A0vector\u00C2\u00A0sequence\u00C2\u00A0from\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0 476\u00E2\u0080\u00901976\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0 1977\u00E2\u0080\u00904184\u00C2\u00A0 DUR3\u00C2\u00A0ORF\u00C2\u00A0 4185\u00E2\u0080\u00904467\u00C2\u00A0 PGK1\u00C2\u00A0terminator\u00C2\u00A0 4468\u00E2\u0080\u00904535\u00C2\u00A0 pHVXKD3\u00C2\u00A0vector\u00C2\u00A0sequence\u00C2\u00A0from\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0 4536\u00E2\u0080\u00904933\u00C2\u00A0 kanMX\u00C2\u00A0promoter\u00C2\u00A0 4934\u00E2\u0080\u00905743\u00C2\u00A0 kanMX\u00C2\u00A0ORF\u00C2\u00A0 5744\u00E2\u0080\u00905992\u00C2\u00A0 kanMX\u00C2\u00A0terminator\u00C2\u00A0 5993\u00E2\u0080\u00906120\u00C2\u00A0 pHVXKD3\u00C2\u00A0vector\u00C2\u00A0sequence\u00C2\u00A0from\u00C2\u00A0cloning\u00C2\u00A0strategy\u00C2\u00A0 6121\u00E2\u0080\u00906510\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0TRP1\u00C2\u00A0sequence\u00C2\u00A0 6511\u00E2\u0080\u00906516\u00C2\u00A0 3\u00E2\u0080\u0099\u00C2\u00A0Apa1\u00C2\u00A0restriction\u00C2\u00A0site\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 61\u00C2\u00A0 \u00C2\u00A0 Alignment\u00C2\u00A0of\u00C2\u00A0DNA\u00C2\u00A0sequences:\u00C2\u00A0S288C\u00C2\u00A0and\u00C2\u00A0pUCMD\u00C2\u00A0 \u00C2\u00A0 Upper\u00C2\u00A0line:\u00C2\u00A0S288C,\u00C2\u00A0from\u00C2\u00A01\u00C2\u00A0to\u00C2\u00A06515\u00C2\u00A0 Lower\u00C2\u00A0line:\u00C2\u00A0pUCMD,\u00C2\u00A0from\u00C2\u00A01\u00C2\u00A0to\u00C2\u00A06515\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0identity=\u00C2\u00A099%\u00C2\u00A0 901 TAGCATACAATTAAAACATGGCGGGCACGTATCATTGCCCTTATCTTGTGCAGTTAGACG ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| 901 TAGCATACAATTAAAACATGGCGGGCATGTATCATTGCCCTTATCTTGTGCAGTTAGACG 961 CGAATTTTTCGAAGAAGTACCTTCAAAGAATGGGGTCTCATCTTGTTTTGCAAGTACCAC |||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| 961 CGAATTTTTCGAAGAAGTACCTTCAAAGAATGGGGTCTTATCTTGTTTTGCAAGTACCAC ------------------------------------------------------------ ---------------CONTINUATION OF PGK1 PROMOTER---------------- ------------------------------------------------------------ 1201 TCAAGACGCACAGATATTATAACATCTGCACAATAGGCATTTGCAAGAATTACTCGTGAG |||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||| 1201 TCAAGACGCACAGATATTATAACATCTGCATAATAGGCATTTGCAAGAATTACTCGTGAG ------------------------------------------------------------ ---------------CONTINUATION OF PGK1 PROMOTER---------------- ------------------------------------------------------------ 1441 CCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGAC |||||||||||||||||||||||||||||||||||||||||||||||||| || |||||| 1441 CCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGATACACTCGAC 1501 TTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCGACG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 1501 TTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCGACG ------------------------------------------------------------ ----CONTINUATION OF PGK1 PROMOTER AND START OF DUR3 ORF----- ------------------------------------------------------------ 3481 CTTTGCTATCACCAGCCATTTTTATTCCTATTTTAACGTATGTGTTTAAGCCACAAAATT ||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||| 3481 CTTTGCTATCACCAGCCATTTTTATTCCTATTTTAACGTATGTCTTTAAGCCACAAAATT ------------------------------------------------------------ ---------------CONTINUATION OF DUR3 ORF--------------------- ------------------------------------------------------------ 3781 TACAAAATGAATTAGACGAAGAACAAAGAGAACTAGCACGTGGTTTAAAAATTGCATACT |||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| 3781 TACAAAATGAATTAGACGAAGAACAAAGAGAACTAGCACGCGGTTTAAAAATTGCATACT 3841 TCCTATGTGTTTTTTTCGCTTTGGCATTTTTGGTAGTTTGGCCCATGCCCATGTATGGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 3841 TCCTATGTGTTTTTTTCGCTTTGGCATTTTTGGTAGTTTGGCCCATGCCCATGTATGGTT 3901 CCAAATATATCTTCAGTAAAAAATTCTTTACCGGTTGGGTTGTTGTGATGATCATCTGGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 3901 CCAAATATATCTTCAGTAAAAAATTCTTTACCGGTTGGGTTGTTGTGATGATCATCTGGC 3961 TTTTTTTCAGTGCGTTTGCCGTTTGTATTTATCCACTCTGGGAAGGTAGGCATGGTATAT \u00C2\u00A0 \u00C2\u00A0 62\u00C2\u00A0 \u00C2\u00A0 ||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||| 3961 TTTTTTTCAGTGCATTTGCCGTTTGTATTTATCCACTCTGGGAAGGTAGGCATGGTATAT 4021 ATACCACTTTGCGAGGCCTTTACTGGGATCTATCTGGTCAAACTTATAAATTAAGGGAAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 4021 ATACCACTTTGCGAGGCCTTTACTGGGATCTATCTGGTCAAACTTATAAATTAAGGGAAT 4081 GGCAAAATTCGAACCCACAAGATCTGCATGTAGTAACAAGCCAAATTAGTGCGAGAGCAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 4081 GGCAAAATTCGAACCCACAAGATCTGCATGTAGTAACAAGCCAAATTAGTGCGAGAGCAC 4141 ATAGACAATCATCACATTTCGGACAAGTTGATGAAATAATTTAGCTCGAGGATTGAATTG |||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||| 4141 ATAGACAATCATCACATTTCGGCCAAGTTGATGAAATAATTTAGCTCGAGGATTGAATTG 4201 AATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 4201 AATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATA Figure\u00C2\u00A023.\u00C2\u00A0\u00C2\u00A0DNA\u00C2\u00A0sequence\u00C2\u00A0alignment\u00C2\u00A0of\u00C2\u00A0S288C\u00C2\u00A0and\u00C2\u00A0pUCMD\u00C2\u00A0revealed\u00C2\u00A0nine\u00C2\u00A0discrepancies\u00C2\u00A0along\u00C2\u00A0the\u00C2\u00A0length\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0cassette.\u00C2\u00A0The\u00C2\u00A0sequence\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0S288C\u00C2\u00A0 is\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0upper\u00C2\u00A0 line\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0pUCMD\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0lower\u00C2\u00A0line.\u00C2\u00A0Only\u00C2\u00A0a\u00C2\u00A0partial\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0is\u00C2\u00A0 displayed.\u00C2\u00A0 The\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 is\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 green,\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 ORF\u00C2\u00A0 is\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 black,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 terminator\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0blue.\u00C2\u00A0Nucleotide\u00C2\u00A0mismatches\u00C2\u00A0are\u00C2\u00A0highlighted\u00C2\u00A0in\u00C2\u00A0bold\u00C2\u00A0and\u00C2\u00A0red,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0underlined.\u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0ORF,\u00C2\u00A0mismatches\u00C2\u00A0 are\u00C2\u00A0 oriented\u00C2\u00A0within\u00C2\u00A0 their\u00C2\u00A0 respective\u00C2\u00A0 codons\u00C2\u00A0 by\u00C2\u00A0 underlining;\u00C2\u00A0 the\u00C2\u00A0 stop\u00C2\u00A0 codon\u00C2\u00A0is\u00C2\u00A0italicized.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 silico\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 two\u00C2\u00A0 new\u00C2\u00A0ORFs\u00C2\u00A0were\u00C2\u00A0 created\u00C2\u00A0 during\u00C2\u00A0 construction\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette;\u00C2\u00A0 these\u00C2\u00A0ORFs\u00C2\u00A0were\u00C2\u00A0 composed\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0mixture\u00C2\u00A0 of\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 sequence\u00C2\u00A0and\u00C2\u00A0pHVXK\u00C2\u00A0vector\u00C2\u00A0sequence\u00C2\u00A0(Figure\u00C2\u00A024).\u00C2\u00A0Novel\u00C2\u00A0ORF1\u00C2\u00A0(324\u00C2\u00A0bp)\u00C2\u00A0is\u00C2\u00A0located\u00C2\u00A0at\u00C2\u00A0nucleotide\u00C2\u00A0position\u00C2\u00A0 7\u00E2\u0080\u0090330\u00C2\u00A0while\u00C2\u00A0novel\u00C2\u00A0ORF2\u00C2\u00A0(621\u00C2\u00A0bp)\u00C2\u00A0is\u00C2\u00A0located\u00C2\u00A0at\u00C2\u00A0position\u00C2\u00A0463\u00E2\u0080\u00901083.\u00C2\u00A0 Figure\u00C2\u00A0 24.\u00C2\u00A0 A\u00C2\u00A0 schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 new\u00C2\u00A0 ORFs\u00C2\u00A0 of\u00C2\u00A0 more\u00C2\u00A0 than\u00C2\u00A0 100\u00C2\u00A0 codons\u00C2\u00A0 generated\u00C2\u00A0 during\u00C2\u00A0 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native\u00C2\u00A0DUR3\u00C2\u00A0 locus.\u00C2\u00A0 This\u00C2\u00A0 result\u00C2\u00A0was\u00C2\u00A0 confirmed\u00C2\u00A0 by\u00C2\u00A0 sequencing\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 caused\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0restriction\u00C2\u00A0fragment\u00C2\u00A0length\u00C2\u00A0polymorphism\u00C2\u00A0(RFLP)\u00C2\u00A0between\u00C2\u00A0522\u00C2\u00A0and\u00C2\u00A0K7.\u00C2\u00A0The\u00C2\u00A0DUR3\u00C2\u00A0locus\u00C2\u00A0in\u00C2\u00A0K7\u00C2\u00A0 is\u00C2\u00A0mutated\u00C2\u00A0such\u00C2\u00A0that\u00C2\u00A0it\u00C2\u00A0no\u00C2\u00A0longer\u00C2\u00A0contains\u00C2\u00A0the\u00C2\u00A0EcoR1\u00C2\u00A0site\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0(Figure\u00C2\u00A028)\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 Figure\u00C2\u00A026.\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0522D3,\u00C2\u00A0522EC\u00E2\u0080\u0090D3,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0 confirmed\u00C2\u00A0 by\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 using\u00C2\u00A0 DUR3\u00C2\u00A0 and\u00C2\u00A0 TRP1\u00C2\u00A0 probes.\u00C2\u00A0 Approximately\u00C2\u00A0 1\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0 of\u00C2\u00A0 EcoR1\u00C2\u00A0 digested\u00C2\u00A0genomic\u00C2\u00A0DNA\u00C2\u00A0from\u00C2\u00A0522\u00C2\u00A0(lane\u00C2\u00A0#1),\u00C2\u00A0522D3\u00C2\u00A0(lane\u00C2\u00A0#2),\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0(lane\u00C2\u00A0#3),\u00C2\u00A0K7\u00C2\u00A0(lane\u00C2\u00A0#4),\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(lane\u00C2\u00A0 #5),\u00C2\u00A0K7D3\u00C2\u00A0(lane\u00C2\u00A0#6),\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0(lane\u00C2\u00A0#7)\u00C2\u00A0was\u00C2\u00A0probed\u00C2\u00A0with\u00C2\u00A0either\u00C2\u00A0DUR3\u00C2\u00A0or\u00C2\u00A0TRP1.\u00C2\u00A0In\u00C2\u00A0lane\u00C2\u00A0#5\u00C2\u00A0less\u00C2\u00A0DNA\u00C2\u00A0was\u00C2\u00A0 accidentally\u00C2\u00A0loaded\u00C2\u00A0thus\u00C2\u00A0accounting\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0faint\u00C2\u00A0band\u00C2\u00A0pattern.\u00C2\u00A0All\u00C2\u00A0faint\u00C2\u00A0bands\u00C2\u00A0that\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0correspond\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0size\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0schematic\u00C2\u00A0representation\u00C2\u00A0were\u00C2\u00A0deemed\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0non\u00E2\u0080\u0090specific\u00C2\u00A0binding.\u00C2\u00A0 \u00C2\u00A0 4680 bp DUR3 probe TRP1 probe 1452 bp 3088 bp 4680 bp 2444 bp ~ 3500 bp 1 2 3 4 5 6 7 1 2 3 4 5 6 7 \u00C2\u00A0 \u00C2\u00A0 65\u00C2\u00A0 \u00C2\u00A0 EcoR1 a)\u00C2\u00A0 DUR3 PGK1p trp1 trp1 PGK1t DUR3 EcoR1 2444 bp EcoR1 EcoR1 4680 bp \u00C2\u00A0 EcoR1 3088 bp EcoR1 DUR3 PGK1p trp1 trp1 PGK1t TRP1 EcoR1 1452 bp EcoR1 EcoR1 4680 bp \u00C2\u00A0 b)\u00C2\u00A0 result,\u00C2\u00A0the\u00C2\u00A0native\u00C2\u00A0DUR3\u00C2\u00A0 locus\u00C2\u00A0 in\u00C2\u00A0K7\u00C2\u00A0produced\u00C2\u00A0a\u00C2\u00A0 larger\u00C2\u00A0signal\u00C2\u00A0(~3500\u00C2\u00A0bp).\u00C2\u00A0By\u00C2\u00A0 integrating\u00C2\u00A0a\u00C2\u00A0copy\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 derived\u00C2\u00A0from\u00C2\u00A0522,\u00C2\u00A0which\u00C2\u00A0contains\u00C2\u00A0the\u00C2\u00A0internal\u00C2\u00A0EcoR1\u00C2\u00A0site,\u00C2\u00A0a\u00C2\u00A0recombinant\u00C2\u00A0locus\u00C2\u00A0was\u00C2\u00A0created\u00C2\u00A0which\u00C2\u00A0gives\u00C2\u00A0 rise\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0band\u00C2\u00A0pattern\u00C2\u00A0seen\u00C2\u00A0 in\u00C2\u00A0522.\u00C2\u00A0Although\u00C2\u00A0this\u00C2\u00A0RFLP\u00C2\u00A0should\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 it\u00C2\u00A0 was\u00C2\u00A0not\u00C2\u00A0and\u00C2\u00A0therefore\u00C2\u00A0warrants\u00C2\u00A0further\u00C2\u00A0investigation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 27.\u00C2\u00A0 Schematic\u00C2\u00A0 representation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 signals\u00C2\u00A0 expected\u00C2\u00A0 during\u00C2\u00A0 Southern\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 recombinant\u00C2\u00A0 yeasts\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 TRP1\u00C2\u00A0 locus.\u00C2\u00A0 Expected\u00C2\u00A0 signals\u00C2\u00A0 during\u00C2\u00A0 Southern\u00C2\u00A0 blotting\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 probe\u00C2\u00A0 (drawn\u00C2\u00A0 in\u00C2\u00A0 green)\u00C2\u00A0 (a).\u00C2\u00A0 Expected\u00C2\u00A0 signals\u00C2\u00A0during\u00C2\u00A0Southern\u00C2\u00A0blotting\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0probe\u00C2\u00A0(drawn\u00C2\u00A0in\u00C2\u00A0red)\u00C2\u00A0(b).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 66\u00C2\u00A0 \u00C2\u00A0 Alignment\u00C2\u00A0of\u00C2\u00A0DNA\u00C2\u00A0sequences:\u00C2\u00A0S288C,\u00C2\u00A0522,\u00C2\u00A0K7\u00C2\u00A0(1\u00E2\u0080\u0090240\u00C2\u00A0bp\u00C2\u00A0DUR3)\u00C2\u00A0 S288C ATGGGAGAATTTAAACCTCCGCTACCTCAAGGCGCTGGGT 40 522 ......................cAtCcaggGGCGCTGGGa 18 K7 ............cAACaaaCcCcctactcAGGCGCTGGGT 28 S288C ACGCTATTGTATTGGGCCTAGGGGCCGTATTTGCAGGAAT 80 522 .CGCTATTGTATTGGGCCTAGGGGCCGTATTTGCAGGAAT 57 K7 .CGCTATTGTATTGGGCCTAGGGGCCGTATTTGCAGGAAT 67 S288C GATGGTTTTGACCACTTATTTACTGAAACGTTATCAAAAG 120 522 GATGGTTTTGACCACTTATTTACTGAAACGTTATCAAAAG 97 K7 GATGGTTTTGACCACTTATTTACTGAAACGTTATCAAAAG 107 S288C GAAATCATCACAGCAGAAGAATTCACCACCGCCGGTAGAT 160 522 GAAATCATCACAGCAGAAGAATTCACCACCGCCGGTAGAT 137 K7 GAAATCATCACAGCAGAAGAATTtACCACCGCCGGcAGAT 147 S288C CTGTAAAAACCGGCTTAGTGGCTGCAGCCGTGGTTTCTAG 200 522 CTGTAAAAACCGGCTTAGTGGCTGCAGCCGTGGTTTCTAG 177 K7 CTGTAAAAACCGGCTTAGTGGCTGCcGCCGTGGTTTCTAG 187 S288C TTGGATCTGGTGTTCTACATTGTTAACGTCGTCAACAAAG 240 522 TTGGATCTGGTGTTCTACATTGTTAACGTCGTCAACAAAG 217 K7 TTGGATCTGGTGTTCTACATTGTTAACGTCGTCAACAAAG 227 \u00C2\u00A0 Figure\u00C2\u00A028.\u00C2\u00A0Alignment\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DNA\u00C2\u00A0sequences\u00C2\u00A0of\u00C2\u00A0S288C,\u00C2\u00A0522,\u00C2\u00A0and\u00C2\u00A0K7\u00C2\u00A0confirmed\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0mutant\u00C2\u00A0 EcoR1\u00C2\u00A0site\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0K7.\u00C2\u00A0Only\u00C2\u00A0a\u00C2\u00A0partial\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0is\u00C2\u00A0displayed.\u00C2\u00A0The\u00C2\u00A0EcoR1\u00C2\u00A0site\u00C2\u00A0 in\u00C2\u00A0question\u00C2\u00A0is\u00C2\u00A0displayed\u00C2\u00A0in\u00C2\u00A0red;\u00C2\u00A0other\u00C2\u00A0mismatches\u00C2\u00A0revealed\u00C2\u00A0by\u00C2\u00A0sequencing\u00C2\u00A0are\u00C2\u00A0highlighted\u00C2\u00A0in\u00C2\u00A0blue.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.3.2\u00C2\u00A0 \u00C2\u00A0Confirmation\u00C2\u00A0of\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0by\u00C2\u00A0northern\u00C2\u00A0blotting.\u00C2\u00A0 In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 a\u00C2\u00A0 northern\u00C2\u00A0blot\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0using\u00C2\u00A0PCR\u00C2\u00A0generated\u00C2\u00A0probes\u00C2\u00A0for\u00C2\u00A0HHF1\u00C2\u00A0(Histone\u00C2\u00A0H4)\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0on\u00C2\u00A0freshly\u00C2\u00A0 harvested\u00C2\u00A0total\u00C2\u00A0RNA\u00C2\u00A0from\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3.\u00C2\u00A0Identical\u00C2\u00A0signals\u00C2\u00A0matching\u00C2\u00A0the\u00C2\u00A0predicted\u00C2\u00A0transcript\u00C2\u00A0 size\u00C2\u00A0for\u00C2\u00A0HHF1\u00C2\u00A0(312\u00C2\u00A0bp)\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0strains\u00C2\u00A0tested\u00C2\u00A0(Figure\u00C2\u00A029),\u00C2\u00A0indicating\u00C2\u00A0an\u00C2\u00A0abundance\u00C2\u00A0of\u00C2\u00A0 un\u00E2\u0080\u0090degraded\u00C2\u00A0mRNA\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0samples.\u00C2\u00A0Signals,\u00C2\u00A0which\u00C2\u00A0matched\u00C2\u00A0the\u00C2\u00A0predicted\u00C2\u00A0transcript\u00C2\u00A0size\u00C2\u00A0 (2208\u00C2\u00A0bp)\u00C2\u00A0for\u00C2\u00A0 DUR3,\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0 strains\u00C2\u00A0K7D3\u00C2\u00A0 and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0when\u00C2\u00A0probed\u00C2\u00A0 for\u00C2\u00A0DUR3\u00C2\u00A0 (Figure\u00C2\u00A029),\u00C2\u00A0 thus\u00C2\u00A0 confirming\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 in\u00C2\u00A0 non\u00E2\u0080\u0090inducing\u00C2\u00A0 conditions\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 result\u00C2\u00A0 of\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette.\u00C2\u00A0No\u00C2\u00A0DUR3\u00C2\u00A0 signal\u00C2\u00A0was\u00C2\u00A0 detected\u00C2\u00A0 in\u00C2\u00A0 K7\u00C2\u00A0 or\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 confirming\u00C2\u00A0 that\u00C2\u00A0DUR3\u00C2\u00A0mRNA\u00C2\u00A0 is\u00C2\u00A0 absent\u00C2\u00A0 during\u00C2\u00A0 fermentation\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0repression\u00C2\u00A0by\u00C2\u00A0NCR.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 67\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.3.3\u00C2\u00A0 \u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0constitutive\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0 in\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR.\u00C2\u00A0Expression\u00C2\u00A0of\u00C2\u00A0 DUR3\u00C2\u00A0and\u00C2\u00A0DUR1,2\u00C2\u00A0was\u00C2\u00A0verified\u00C2\u00A0and\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0cDNA\u00C2\u00A0reverse\u00C2\u00A0transcribed\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 total\u00C2\u00A0 RNA\u00C2\u00A0 of\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3.\u00C2\u00A0 \u00C2\u00A0Analyses\u00C2\u00A0were\u00C2\u00A0 performed\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 RNA\u00C2\u00A0 samples\u00C2\u00A0 as\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A03.2.3.2.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0high\u00C2\u00A0level\u00C2\u00A0expression,\u00C2\u00A0under\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0 (NCR)\u00C2\u00A0conditions,\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0genes\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 integrated\u00C2\u00A0cassettes\u00C2\u00A0(Figure\u00C2\u00A030);\u00C2\u00A0DUR1,2\u00C2\u00A0 was\u00C2\u00A0upregulated\u00C2\u00A011.8\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0while\u00C2\u00A0DUR3\u00C2\u00A0was\u00C2\u00A0upregulated\u00C2\u00A022.1\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0K7D3.\u00C2\u00A0High\u00C2\u00A0 level\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0both\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0 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the\u00C2\u00A0loading\u00C2\u00A0control\u00C2\u00A0HHF1\u00C2\u00A0and\u00C2\u00A0DUR3.\u00C2\u00A0 \u00C2\u00A0 K7 \u00C2\u00A0 K7 EC \u00E2\u0080\u0090 \u00C2\u00A0 K7 D 3 \u00C2\u00A0 K7 EC \u00E2\u0080\u0090D 3 \u00C2\u00A0 K7 \u00C2\u00A0 K7 EC \u00E2\u0080\u0090 \u00C2\u00A0 K7 D 3 \u00C2\u00A0 K7 EC \u00E2\u0080\u0090D 3 \u00C2\u00A0 DUR3HHF1 312bp\u00C2\u00A0 2208bp\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 68\u00C2\u00A0 \u00C2\u00A0 induced\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 by\u00C2\u00A0 3.78\u00E2\u0080\u0090fold.\u00C2\u00A0 Likewise,\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 induced\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0by\u00C2\u00A03.61\u00E2\u0080\u0090fold.\u00C2\u00A0\u00C2\u00A0 1.00 9.26 3.61 11.82 1.00 3.78 14.19 7.10 0 2 4 6 8 10 12 14 16 18 K7 K7EC- K7D3 K7EC-D3 Strain R el at iv e qu an tif ic at io n (fo ld ) DUR1,2 DUR3 R 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that\u00C2\u00A0 was\u00C2\u00A0 inoculated\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 final\u00C2\u00A0 OD600\u00C2\u00A0 =\u00C2\u00A0 0.1.\u00C2\u00A0 Total\u00C2\u00A0 RNA\u00C2\u00A0 was\u00C2\u00A0 subsequently\u00C2\u00A0 reverse\u00C2\u00A0 transcribed,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0cDNA\u00C2\u00A0was\u00C2\u00A0amplified\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0SYBR\u00C2\u00A0green\u00C2\u00A0dye.\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0 gene\u00C2\u00A0expression\u00C2\u00A0was\u00C2\u00A0standardized\u00C2\u00A0to\u00C2\u00A0ACT1\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0for\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0calibrated\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0 strain\u00C2\u00A0K7.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0 represent\u00C2\u00A095%\u00C2\u00A0confidence\u00C2\u00A0intervals.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.3.4\u00C2\u00A0\u00C2\u00A0Effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcriptome\u00C2\u00A0of\u00C2\u00A0K7D3.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0from\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A024\u00C2\u00A0hour\u00C2\u00A0fermentations\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0analyze\u00C2\u00A0the\u00C2\u00A0impact\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0 DUR3\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0K7D3.\u00C2\u00A0Reported\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0were\u00C2\u00A0cut\u00C2\u00A0off\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0 minimum\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0change\u00C2\u00A0 in\u00C2\u00A0expression\u00C2\u00A0 (SLRAvg\u00C2\u00A0\u00E2\u0089\u00A5\u00C2\u00A02\u00C2\u00A0or\u00C2\u00A0SLRAvg\u00C2\u00A0\u00E2\u0089\u00A4\u00C2\u00A0 \u00E2\u0080\u00902)\u00C2\u00A0 to\u00C2\u00A0ensure\u00C2\u00A0elimination\u00C2\u00A0of\u00C2\u00A0experimental\u00C2\u00A0 noise\u00C2\u00A0 inherent\u00C2\u00A0 in\u00C2\u00A0microarray\u00C2\u00A0 analysis;\u00C2\u00A0 this\u00C2\u00A0 cut\u00E2\u0080\u0090off\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0previously\u00C2\u00A0published\u00C2\u00A0 statistical\u00C2\u00A0 examination\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0wine\u00C2\u00A0yeast\u00E2\u0080\u0099s\u00C2\u00A0transcriptome\u00C2\u00A0during\u00C2\u00A0fermentation\u00C2\u00A0(Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02008).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 69\u00C2\u00A0 \u00C2\u00A0 Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0of\u00C2\u00A0K7D3\u00C2\u00A0had\u00C2\u00A0a\u00C2\u00A0minimal\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0yeast\u00E2\u0080\u0099s\u00C2\u00A0 transcriptome.\u00C2\u00A0DUR3\u00C2\u00A0was\u00C2\u00A0upregulated\u00C2\u00A0by\u00C2\u00A036.95\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0K7D3;\u00C2\u00A0TRP1\u00C2\u00A0was\u00C2\u00A0downregulated\u00C2\u00A0by\u00C2\u00A01.25\u00E2\u0080\u0090fold\u00C2\u00A0but\u00C2\u00A0 was\u00C2\u00A0 not\u00C2\u00A0 included\u00C2\u00A0 in\u00C2\u00A0 Table\u00C2\u00A0 17\u00C2\u00A0 as\u00C2\u00A0 it\u00C2\u00A0 fell\u00C2\u00A0 below\u00C2\u00A0 the\u00C2\u00A0 4\u00E2\u0080\u0090fold\u00C2\u00A0 cut\u00C2\u00A0 off.\u00C2\u00A0 Besides\u00C2\u00A0 DUR3,\u00C2\u00A0 three\u00C2\u00A0 genes\u00C2\u00A0 were\u00C2\u00A0 upregulated\u00C2\u00A0 greater\u00C2\u00A0 than\u00C2\u00A0 4\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 (Table\u00C2\u00A0 17);\u00C2\u00A0 one\u00C2\u00A0 gene\u00C2\u00A0 (HAC1)\u00C2\u00A0 was\u00C2\u00A0 common\u00C2\u00A0 to\u00C2\u00A0 both\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 engineered\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K7D3\u00C2\u00A0(Tables\u00C2\u00A010\u00C2\u00A0and\u00C2\u00A017).\u00C2\u00A0Four\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0downregulated\u00C2\u00A0more\u00C2\u00A0than\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0K7D3.\u00C2\u00A0Despite\u00C2\u00A0falling\u00C2\u00A0below\u00C2\u00A0the\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0cut\u00C2\u00A0off,\u00C2\u00A0one\u00C2\u00A0gene\u00C2\u00A0(RUD3)\u00C2\u00A0was\u00C2\u00A0included\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A017\u00C2\u00A0because\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0 common\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0list\u00C2\u00A0of\u00C2\u00A0genes\u00C2\u00A0downregulated\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K7D3\u00C2\u00A0(Tables\u00C2\u00A010\u00C2\u00A0and\u00C2\u00A017)\u00C2\u00A0and\u00C2\u00A0because\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 very\u00C2\u00A0close\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0cut\u00C2\u00A0off.\u00C2\u00A0No\u00C2\u00A0metabolic\u00C2\u00A0pathways\u00C2\u00A0were\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0 DUR3\u00C2\u00A0 cassette;\u00C2\u00A0 however,\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 upregulated\u00C2\u00A0 one\u00C2\u00A0 gene\u00C2\u00A0 (FIG1)\u00C2\u00A0 and\u00C2\u00A0 downregulated\u00C2\u00A0 one\u00C2\u00A0 gene\u00C2\u00A0 (TID3)\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 meiosis/sporulation\u00C2\u00A0 indicating\u00C2\u00A0 a\u00C2\u00A0 possible\u00C2\u00A0 effect\u00C2\u00A0 on\u00C2\u00A0 meiosis/sporulation\u00C2\u00A0efficiency\u00C2\u00A0(Table\u00C2\u00A017).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A017.\u00C2\u00A0Effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0genome\u00C2\u00A0of\u00C2\u00A0K7\u00C2\u00A0on\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0patterns\u00C2\u00A0 in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0K7D3\u00C2\u00A0(\u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0change).\u00C2\u00A0Reported\u00C2\u00A0changes\u00C2\u00A0are\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strain\u00C2\u00A0K7.\u00C2\u00A0Total\u00C2\u00A0RNA\u00C2\u00A0 from\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7D3,\u00C2\u00A0harvested\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0hours\u00C2\u00A0 into\u00C2\u00A0 fermentation\u00C2\u00A0of\u00C2\u00A0 filter\u00C2\u00A0sterilized\u00C2\u00A0Chardonnay\u00C2\u00A0must,\u00C2\u00A0were\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0hybridization\u00C2\u00A0 to\u00C2\u00A0microarrays.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0 in\u00C2\u00A0duplicate\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0were\u00C2\u00A0 averaged\u00C2\u00A0(p\u00E2\u0089\u00A40.005).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Genes expressed at higher levels in K7D3 Fold Change Gene Symbol Biological Process 36.95 DUR3 Urea permease 5.67 HAC1 bZIP (basic-leucine zipper) protein involved in unfolded protein response 4.93 BRN1 Protein required for chromosome condensation 4.24 FIG1 Integral membrane protein required for efficient mating and low affinity Ca2+ transport Genes expressed at lower levels in K7D3 Fold Change Gene Symbol Biological Process -8.18 TID3 Meiotic protein required for synapsis and meiotic recombination; interaction partner with DMC1p -7.31 SNT309 Splicing factor protein -5.98 TOA1 Transcription factor IIA, large chain -3.98 RUD3 Protein involved in organization of Golgi \u00C2\u00A0 3.2.4\u00C2\u00A0\u00C2\u00A0Recombinant\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0exhibit\u00C2\u00A0highly\u00C2\u00A0enhanced\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0ability\u00C2\u00A0in\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0 strong\u00C2\u00A0NCR\u00C2\u00A0 \u00C2\u00A0 Radiolabelled\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0uptake\u00C2\u00A0assays\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0confirm\u00C2\u00A0constitutive\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 result\u00C2\u00A0of\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 70\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0parental\u00C2\u00A0Sake\u00C2\u00A0 strain\u00C2\u00A0K7,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0 yeast\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0 locus,\u00C2\u00A0failed\u00C2\u00A0to\u00C2\u00A0 incorporate\u00C2\u00A0any\u00C2\u00A0significant\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0radiolabelled\u00C2\u00A0urea\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0 minimal\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A01%\u00C2\u00A0(w/v)\u00C2\u00A0ammonium\u00C2\u00A0sulphate\u00C2\u00A0(Figure\u00C2\u00A031).\u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0NCR\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR\u00C2\u00A0genes\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0preferred\u00C2\u00A0nitrogen\u00C2\u00A0source\u00C2\u00A0(ammonium\u00C2\u00A0sulphate).\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0K7D3\u00C2\u00A0 and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0highly\u00C2\u00A0 efficient\u00C2\u00A0 at\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0 (Figure\u00C2\u00A031),\u00C2\u00A0 confirming\u00C2\u00A0 that\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0functional\u00C2\u00A0protein\u00C2\u00A0that\u00C2\u00A0 localizes\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0 and\u00C2\u00A0that\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0is\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0overcoming\u00C2\u00A0native\u00C2\u00A0 repression\u00C2\u00A0by\u00C2\u00A0NCR.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 significant\u00C2\u00A0difference\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0 rates\u00C2\u00A0of\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0 (Figure\u00C2\u00A031),\u00C2\u00A0 despite\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 identical\u00C2\u00A0DUR3\u00C2\u00A0cassettes\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0strains.\u00C2\u00A0The\u00C2\u00A0observed\u00C2\u00A0difference\u00C2\u00A0 in\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0 between\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0explained\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0need\u00C2\u00A0for\u00C2\u00A0urea\u00C2\u00A0degradation\u00C2\u00A0(DUR1,2p)\u00C2\u00A0after\u00C2\u00A0its\u00C2\u00A0import\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 cell.\u00C2\u00A0While\u00C2\u00A0 DUR1,2\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 induced\u00C2\u00A0 and\u00C2\u00A0 then\u00C2\u00A0 synthesized\u00C2\u00A0 in\u00C2\u00A0 K7D3,\u00C2\u00A0 DUR1,2p\u00C2\u00A0 is\u00C2\u00A0 constitutively\u00C2\u00A0 expressed\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0rapid\u00C2\u00A0degradation\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0might\u00C2\u00A0mask\u00C2\u00A0an\u00C2\u00A0increased\u00C2\u00A0uptake;\u00C2\u00A0the\u00C2\u00A0 radiolabel\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0quickly\u00C2\u00A0lost\u00C2\u00A0as\u00C2\u00A014CO2.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 71\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 31.\u00C2\u00A0 \u00C2\u00A0Uptake\u00C2\u00A0 of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 by\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0NCR.\u00C2\u00A0 \u00C2\u00A0 Strains\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A01)\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A01%\u00C2\u00A0(w/v)\u00C2\u00A0ammonium\u00C2\u00A0sulphate\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A00.27\u00C2\u00A0 mM\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 (6.8\u00C2\u00A0mCi/mmol).\u00C2\u00A0Assays\u00C2\u00A0were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0 averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0 represent\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0either\u00C2\u00A0K7D3\u00C2\u00A0or\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0both\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0exhibited\u00C2\u00A0relatively\u00C2\u00A0poor\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0 under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0tested\u00C2\u00A0 (Figure\u00C2\u00A031).\u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0differentiate\u00C2\u00A0between\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0Figure\u00C2\u00A031\u00C2\u00A0was\u00C2\u00A0 modified\u00C2\u00A0by\u00C2\u00A0significantly\u00C2\u00A0reducing\u00C2\u00A0the\u00C2\u00A0scale\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Y\u00E2\u0080\u0090axis;\u00C2\u00A0the\u00C2\u00A0maximum\u00C2\u00A0value\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Y\u00E2\u0080\u0090axis\u00C2\u00A0was\u00C2\u00A0reduced\u00C2\u00A0 from\u00C2\u00A07.0\u00C2\u00A0nmole\u00C2\u00A0(Figure\u00C2\u00A031)\u00C2\u00A0to\u00C2\u00A00.1\u00C2\u00A0nmole\u00C2\u00A0(Figure\u00C2\u00A032).\u00C2\u00A0K7\u00C2\u00A0was\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0accumulating\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0 urea\u00C2\u00A0 than\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 (Figure\u00C2\u00A0 32),\u00C2\u00A0 probably\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0DUR1,2\u00C2\u00A0 in\u00C2\u00A0 K7EC\u00E2\u0080\u0090;\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 might\u00C2\u00A0be\u00C2\u00A0actively\u00C2\u00A0degraded\u00C2\u00A0as\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 imported\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0 thus\u00C2\u00A0giving\u00C2\u00A0 the\u00C2\u00A0 illusion\u00C2\u00A0 that\u00C2\u00A0 transport\u00C2\u00A0 is\u00C2\u00A0 less\u00C2\u00A0 efficient.\u00C2\u00A0This\u00C2\u00A0phenomenon\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0observed\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 to\u00C2\u00A0 transport\u00C2\u00A0urea\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0were\u00C2\u00A0compared\u00C2\u00A0(Figure\u00C2\u00A031).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 72\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A032.\u00C2\u00A0Uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR. Strains\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0 =\u00C2\u00A01)\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A01%\u00C2\u00A0 (w/v)\u00C2\u00A0ammonium\u00C2\u00A0 sulphate\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0prior\u00C2\u00A0 to\u00C2\u00A0exposure\u00C2\u00A0 to\u00C2\u00A00.27\u00C2\u00A0mM\u00C2\u00A0 14C\u00C2\u00A0urea\u00C2\u00A0 (6.8\u00C2\u00A0 mCi/mmol).\u00C2\u00A0 Assays\u00C2\u00A0 were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 averaged;\u00C2\u00A0 error\u00C2\u00A0 bars\u00C2\u00A0 represent\u00C2\u00A0 one\u00C2\u00A0 standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.5\u00C2\u00A0\u00C2\u00A0Recombinant\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0exhibit\u00C2\u00A0highly\u00C2\u00A0enhanced\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0ability\u00C2\u00A0in\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0 NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0 \u00C2\u00A0 Radiolabelled\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 uptake\u00C2\u00A0 assays\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 to\u00C2\u00A0 uptake\u00C2\u00A0 urea\u00C2\u00A0 under\u00C2\u00A0 non\u00E2\u0080\u0090repressive\u00C2\u00A0 conditions.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 73\u00C2\u00A0 \u00C2\u00A0 Both\u00C2\u00A0 the\u00C2\u00A0 parental\u00C2\u00A0 Sake\u00C2\u00A0 strain\u00C2\u00A0 K7,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 recombinant\u00C2\u00A0 yeast\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0URA3\u00C2\u00A0 locus,\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 incorporate\u00C2\u00A0 any\u00C2\u00A0 significant\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 minimal\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A01%\u00C2\u00A0(w/v)\u00C2\u00A0L\u00E2\u0080\u0090proline\u00C2\u00A0(Figure\u00C2\u00A033).\u00C2\u00A0This\u00C2\u00A0result,\u00C2\u00A0which\u00C2\u00A0parallels\u00C2\u00A0the\u00C2\u00A0inability\u00C2\u00A0of\u00C2\u00A0K7\u00C2\u00A0 and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0to\u00C2\u00A0incorporate\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0ammonium\u00C2\u00A0sulphate\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0(Figure\u00C2\u00A031),\u00C2\u00A0is\u00C2\u00A0likely\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0 slow\u00C2\u00A0 induction\u00C2\u00A0and\u00C2\u00A0membrane\u00C2\u00A0 trafficking\u00C2\u00A0of\u00C2\u00A0 functional\u00C2\u00A0DUR3p.\u00C2\u00A0 In\u00C2\u00A0 contrast,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0 highly\u00C2\u00A0efficient\u00C2\u00A0at\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0(Figure\u00C2\u00A033),\u00C2\u00A0confirming\u00C2\u00A0that\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 functional\u00C2\u00A0 protein,\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 transcription\u00C2\u00A0 of\u00C2\u00A0 DUR3\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 and\u00C2\u00A0 terminator\u00C2\u00A0signals\u00C2\u00A0is\u00C2\u00A0strong\u00C2\u00A0regardless\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0state.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 33.\u00C2\u00A0 Uptake\u00C2\u00A0 of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 by\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 NCR\u00C2\u00A0 de\u00E2\u0080\u0090repression. Strains\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A01)\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A01%\u00C2\u00A0(w/v)\u00C2\u00A0L\u00E2\u0080\u0090proline\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A00.27\u00C2\u00A0 mM\u00C2\u00A0 14C\u00C2\u00A0urea\u00C2\u00A0 (6.8\u00C2\u00A0mCi/mmol).\u00C2\u00A0Assays\u00C2\u00A0were\u00C2\u00A0 conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0 represent\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 74\u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0either\u00C2\u00A0K7D3\u00C2\u00A0or\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0both\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0exhibited\u00C2\u00A0relatively\u00C2\u00A0poor\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0 under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0 (Figure\u00C2\u00A033),\u00C2\u00A0K7\u00C2\u00A0was\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0accumulating\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0 urea\u00C2\u00A0than\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(Figure\u00C2\u00A034).\u00C2\u00A0To\u00C2\u00A0differentiate\u00C2\u00A0between\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0Figure\u00C2\u00A033\u00C2\u00A0was\u00C2\u00A0modified\u00C2\u00A0by\u00C2\u00A0reducing\u00C2\u00A0 the\u00C2\u00A0scale\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Y\u00E2\u0080\u0090axis;\u00C2\u00A0the\u00C2\u00A0maximum\u00C2\u00A0value\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Y\u00E2\u0080\u0090axis\u00C2\u00A0was\u00C2\u00A0reduced\u00C2\u00A0from\u00C2\u00A07.0\u00C2\u00A0nmole\u00C2\u00A0(Figure\u00C2\u00A033)\u00C2\u00A0to\u00C2\u00A00.1\u00C2\u00A0 nmole\u00C2\u00A0 (Figure\u00C2\u00A034).\u00C2\u00A0K7\u00C2\u00A0was\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0accumulating\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0urea\u00C2\u00A0 than\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 (Figure\u00C2\u00A034);\u00C2\u00A0 14C\u00E2\u0080\u0090 urea\u00C2\u00A0might\u00C2\u00A0be\u00C2\u00A0actively\u00C2\u00A0degraded\u00C2\u00A0as\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 imported\u00C2\u00A0 into\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0where\u00C2\u00A0DUR1,2\u00C2\u00A0 is\u00C2\u00A0constitutively\u00C2\u00A0expressed,\u00C2\u00A0 thus\u00C2\u00A0masking\u00C2\u00A0the\u00C2\u00A0strain\u00E2\u0080\u0099s\u00C2\u00A0true\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0import\u00C2\u00A0urea.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 34.\u00C2\u00A0 Uptake\u00C2\u00A0 of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 by\u00C2\u00A0 K7\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 NCR\u00C2\u00A0 de\u00E2\u0080\u0090repression. Strains\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0(final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A01)\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A01%\u00C2\u00A0(w/v)\u00C2\u00A0L\u00E2\u0080\u0090proline\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A00.27\u00C2\u00A0mM\u00C2\u00A0 14C\u00C2\u00A0urea\u00C2\u00A0 (6.8\u00C2\u00A0mCi/mmol).\u00C2\u00A0Assays\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0represent\u00C2\u00A0one\u00C2\u00A0 standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 75\u00C2\u00A0 \u00C2\u00A0 3.2.6\u00C2\u00A0\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0 \u00C2\u00A0 3.2.6.1\u00C2\u00A0 \u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0The\u00C2\u00A0 fermentation\u00C2\u00A0 profiles\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0 and\u00C2\u00A0metabolically\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figures\u00C2\u00A035a,b.\u00C2\u00A0 The\u00C2\u00A0 robust\u00C2\u00A0 fermentations\u00C2\u00A0were\u00C2\u00A0completed\u00C2\u00A0within\u00C2\u00A0300\u00C2\u00A0hours,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0fermentation\u00C2\u00A0rates\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0but\u00C2\u00A0one\u00C2\u00A0engineered\u00C2\u00A0 strain\u00C2\u00A0closely\u00C2\u00A0matched\u00C2\u00A0those\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0respective\u00C2\u00A0parental\u00C2\u00A0strains,\u00C2\u00A0thus\u00C2\u00A0indicating\u00C2\u00A0substantial\u00C2\u00A0equivalence.\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0engineered\u00C2\u00A0 strain\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 completed\u00C2\u00A0 the\u00C2\u00A0 fermentation\u00C2\u00A0by\u00C2\u00A0approximately\u00C2\u00A0200\u00E2\u0080\u0090250\u00C2\u00A0hours\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0 strains\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0and\u00C2\u00A0K7D3\u00C2\u00A0 required\u00C2\u00A0300\u00C2\u00A0hours\u00C2\u00A0 to\u00C2\u00A0 finish;\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0 strain\u00C2\u00A0K7\u00C2\u00A0also\u00C2\u00A0 required\u00C2\u00A0300\u00C2\u00A0hours\u00C2\u00A0 to\u00C2\u00A0 complete\u00C2\u00A0the\u00C2\u00A0fermentation.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 76\u00C2\u00A0 \u00C2\u00A0 0.00 5.00 10.00 15.00 20.00 25.00 0 50 100 150 200 250 300 350 T im e \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 7 K 7 \u00C2\u00A0E C \u00E2\u0080\u0090 K 7 \u00C2\u00A0D3 K 7 \u00C2\u00A0E C \u00E2\u0080\u0090D3 \u00C2\u00A0 0.00 5.00 10.00 15.00 20.00 25.00 0 50 100 150 200 250 300 350 T im e \u00C2\u00A0 (Hours) W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) 522 522 \u00C2\u00A0E C \u00E2\u0080\u0090 522 \u00C2\u00A0D3 522 \u00C2\u00A0E C \u00E2\u0080\u0090D3 \u00C2\u00A0 Figure\u00C2\u00A035.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0(weight\u00C2\u00A0loss)\u00C2\u00A0of\u00C2\u00A0(a)\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0and\u00C2\u00A0(b)\u00C2\u00A0 wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0 from\u00C2\u00A0unfiltered\u00C2\u00A0Calona\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0inoculated\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0OD600\u00C2\u00A0=\u00C2\u00A00.1\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0to\u00C2\u00A0completion\u00C2\u00A0 (~300\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A020\u00C2\u00B0C.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0were\u00C2\u00A0averaged;\u00C2\u00A0error\u00C2\u00A0bars\u00C2\u00A0 indicate\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 b)\u00C2\u00A0 a)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 77\u00C2\u00A0 \u00C2\u00A0 3.2.6.2\u00C2\u00A0 \u00C2\u00A0Ethanol\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0The\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 DUR3\u00C2\u00A0cassette\u00C2\u00A0on\u00C2\u00A0ethanol\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0 investigated\u00C2\u00A0by\u00C2\u00A0LC\u00C2\u00A0analysis\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation.\u00C2\u00A0Compared\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0 respective\u00C2\u00A0parental\u00C2\u00A0strains,\u00C2\u00A0 K7D3,\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 produced\u00C2\u00A0 Chardonnay\u00C2\u00A0 wine\u00C2\u00A0 with\u00C2\u00A0 substantially\u00C2\u00A0 equivalent\u00C2\u00A0 ethanol\u00C2\u00A0 content\u00C2\u00A0(Table\u00C2\u00A018).\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A018.\u00C2\u00A0Ethanol\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 (K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3)\u00C2\u00A0and\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 (522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3)\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine.\u00C2\u00A0Ethanol\u00C2\u00A0content\u00C2\u00A0(%v/v)\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0LC\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0 end\u00C2\u00A0of\u00C2\u00A0fermentation.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A035a,b.\u00C2\u00A0Data\u00C2\u00A0were\u00C2\u00A0analyzed\u00C2\u00A0for\u00C2\u00A0statistical\u00C2\u00A0 significance\u00C2\u00A0(p\u00E2\u0089\u00A40.05)\u00C2\u00A0using\u00C2\u00A0two\u00C2\u00A0factor\u00C2\u00A0ANOVA\u00C2\u00A0analysis.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.6.3\u00C2\u00A0\u00C2\u00A0Fermentation\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0The\u00C2\u00A0fermentation\u00C2\u00A0profiles\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 parental\u00C2\u00A0 and\u00C2\u00A0 metabolically\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figures\u00C2\u00A0 36a,b.\u00C2\u00A0 In\u00C2\u00A0 all\u00C2\u00A0 cases,\u00C2\u00A0 the\u00C2\u00A0 fermentations\u00C2\u00A0proceeded\u00C2\u00A0 at\u00C2\u00A0 a\u00C2\u00A0 slower\u00C2\u00A0 rate\u00C2\u00A0 than\u00C2\u00A0 those\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0must;\u00C2\u00A0 fermentations\u00C2\u00A0were\u00C2\u00A0 completed\u00C2\u00A0 within\u00C2\u00A0450\u00C2\u00A0hours\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0300\u00C2\u00A0hours\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0(Figure\u00C2\u00A035).\u00C2\u00A0The\u00C2\u00A0fermentation\u00C2\u00A0rates\u00C2\u00A0 of\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 closely\u00C2\u00A0 matched\u00C2\u00A0 those\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 respective\u00C2\u00A0 parental\u00C2\u00A0 strains,\u00C2\u00A0 thus\u00C2\u00A0 indicating\u00C2\u00A0 substantial\u00C2\u00A0equivalence\u00C2\u00A0in\u00C2\u00A0fermentation\u00C2\u00A0rate.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K7D3\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 12.89\u00C2\u00A0 12.84\u00C2\u00A0 12.89\u00C2\u00A0 12.91\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 12.90\u00C2\u00A0 12.75\u00C2\u00A0 13.01\u00C2\u00A0 12.95\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 12.96\u00C2\u00A0 12.97\u00C2\u00A0 12.97\u00C2\u00A0 12.89\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Ethanol\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 12.92\u00C2\u00A0 12.85\u00C2\u00A0 12.96\u00C2\u00A0 12.92\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.04\u00C2\u00A0 0.11\u00C2\u00A0 0.06\u00C2\u00A0 0.03\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 522\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 522D3\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 13.65\u00C2\u00A0 13.71\u00C2\u00A0 13.74\u00C2\u00A0 13.54\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 13.60\u00C2\u00A0 13.65\u00C2\u00A0 13.71\u00C2\u00A0 13.62\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 13.71\u00C2\u00A0 13.66\u00C2\u00A0 13.55\u00C2\u00A0 13.58\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Ethanol\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 13.65\u00C2\u00A0 13.67\u00C2\u00A0 13.67\u00C2\u00A0 13.58\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.06\u00C2\u00A0 0.03\u00C2\u00A0 0.10\u00C2\u00A0 0.04\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 *\u00C2\u00A0si,\u00C2\u00A0ns:\u00C2\u00A0significant\u00C2\u00A0at\u00C2\u00A0p\u00E2\u0089\u00A40.05,\u00C2\u00A0or\u00C2\u00A0non\u00E2\u0080\u0090significant\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 78\u00C2\u00A0 \u00C2\u00A0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 100 200 300 400 500 Hours W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) K 7 K 7 \u00C2\u00A0E C \u00E2\u0080\u0090 K 7 \u00C2\u00A0D3 K 7 \u00C2\u00A0E C \u00E2\u0080\u0090D3 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 100 200 300 400 500 Hours W ei g h t\u00C2\u00A0 lo ss \u00C2\u00A0( g ) 522 522\u00C2\u00A0E C \u00E2\u0080\u0090 522\u00C2\u00A0D3 522\u00C2\u00A0E C \u00E2\u0080\u0090D3 \u00C2\u00A0 Figure\u00C2\u00A036.\u00C2\u00A0Fermentation\u00C2\u00A0profiles\u00C2\u00A0(weight\u00C2\u00A0loss)\u00C2\u00A0of\u00C2\u00A0(a)\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0and\u00C2\u00A0(b)\u00C2\u00A0 wine\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 522,\u00C2\u00A0 522EC\u00E2\u0080\u0090,\u00C2\u00A0 522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 in\u00C2\u00A0 Sake. Sake\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0 produced\u00C2\u00A0 from\u00C2\u00A0white\u00C2\u00A0 rice\u00C2\u00A0 (Kokako\u00C2\u00A0 Rose)\u00C2\u00A0 and\u00C2\u00A0 koji\u00C2\u00A0 mash\u00C2\u00A0 (Vision\u00C2\u00A0 Brewing)\u00C2\u00A0 inoculated\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 final\u00C2\u00A0 OD600\u00C2\u00A0 =\u00C2\u00A0 0.1\u00C2\u00A0 and\u00C2\u00A0 incubated\u00C2\u00A0 to\u00C2\u00A0 completion\u00C2\u00A0(~450\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A018\u00C2\u00B0C.\u00C2\u00A0Fermentations\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0 in\u00C2\u00A0triplicate\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0were\u00C2\u00A0averaged;\u00C2\u00A0 error\u00C2\u00A0bars\u00C2\u00A0indicate\u00C2\u00A0one\u00C2\u00A0standard\u00C2\u00A0deviation.\u00C2\u00A0 \u00C2\u00A0 a)\u00C2\u00A0 b)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 79\u00C2\u00A0 \u00C2\u00A0 3.2.6.4\u00C2\u00A0 \u00C2\u00A0Ethanol\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0The\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0on\u00C2\u00A0ethanol\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0investigated\u00C2\u00A0 by\u00C2\u00A0LC\u00C2\u00A0analysis\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation.\u00C2\u00A0Compared\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0respective\u00C2\u00A0parental\u00C2\u00A0strains,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0produced\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0with\u00C2\u00A0substantially\u00C2\u00A0equivalent\u00C2\u00A0ethanol\u00C2\u00A0content\u00C2\u00A0(Table\u00C2\u00A019).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A019.\u00C2\u00A0Ethanol\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 (K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3)\u00C2\u00A0and\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 (522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3)\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Ethanol\u00C2\u00A0content\u00C2\u00A0(%\u00C2\u00A0v/v)\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0LC\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 fermentation.\u00C2\u00A0 Fermentation\u00C2\u00A0 profiles\u00C2\u00A0 are\u00C2\u00A0 given\u00C2\u00A0 in\u00C2\u00A0 Figures\u00C2\u00A0 36a,b.\u00C2\u00A0 Data\u00C2\u00A0 were\u00C2\u00A0 analyzed\u00C2\u00A0 for\u00C2\u00A0 statistical\u00C2\u00A0 significance\u00C2\u00A0(p\u00E2\u0089\u00A40.05)\u00C2\u00A0using\u00C2\u00A0two\u00C2\u00A0factor\u00C2\u00A0ANOVA\u00C2\u00A0analysis.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K7D3\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 9.33\u00C2\u00A0 9.22\u00C2\u00A0 8.90\u00C2\u00A0 8.63\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 9.20\u00C2\u00A0 8.14\u00C2\u00A0 9.58\u00C2\u00A0 8.96\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 10.01\u00C2\u00A0 8.14\u00C2\u00A0 9.64\u00C2\u00A0 9.01\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Ethanol\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 9.51\u00C2\u00A0 8.50\u00C2\u00A0 9.37\u00C2\u00A0 8.87\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.44\u00C2\u00A0 0.62\u00C2\u00A0 0.41\u00C2\u00A0 0.21\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 522\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 522D3\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 p*\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 8.78\u00C2\u00A0 7.46\u00C2\u00A0 8.77\u00C2\u00A0 8.99\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A02\u00C2\u00A0 8.80\u00C2\u00A0 7.81\u00C2\u00A0 8.99\u00C2\u00A0 8.86\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Replicate\u00C2\u00A03\u00C2\u00A0 8.61\u00C2\u00A0 9.22\u00C2\u00A0 8.98\u00C2\u00A0 8.37\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 Ethanol\u00C2\u00A0average\u00C2\u00A0(n=3)\u00C2\u00A0 8.73\u00C2\u00A0 8.16\u00C2\u00A0 8.91\u00C2\u00A0 8.74\u00C2\u00A0 ns\u00C2\u00A0 STDEV\u00C2\u00A0 0.10\u00C2\u00A0 0.93\u00C2\u00A0 0.12\u00C2\u00A0 0.33\u00C2\u00A0 \u00E2\u0080\u0090\u00E2\u0080\u0090\u00C2\u00A0 *\u00C2\u00A0si,\u00C2\u00A0ns:\u00C2\u00A0significant\u00C2\u00A0at\u00C2\u00A0p\u00E2\u0089\u00A40.05,\u00C2\u00A0or\u00C2\u00A0non\u00E2\u0080\u0090significant\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.7\u00C2\u00A0\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0 by\u00C2\u00A024.97%\u00C2\u00A0and\u00C2\u00A081.38%,\u00C2\u00A0respectively.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains,\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0 was\u00C2\u00A0made\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0522\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0recombinant\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0 522D3,\u00C2\u00A0 \u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 EC\u00C2\u00A0 content\u00C2\u00A0 quantified\u00C2\u00A0 by\u00C2\u00A0 GC/MS\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 end\u00C2\u00A0 of\u00C2\u00A0 fermentation.\u00C2\u00A0 The\u00C2\u00A0 fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A035a,b.\u00C2\u00A0 During\u00C2\u00A0wine\u00C2\u00A0making,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0 reduced\u00C2\u00A0EC\u00C2\u00A0as\u00C2\u00A0efficiently\u00C2\u00A0as\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0 respectively\u00C2\u00A0 (Table\u00C2\u00A020).\u00C2\u00A0 In\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0 K7D3\u00C2\u00A0 reduced\u00C2\u00A0 EC\u00C2\u00A0by\u00C2\u00A012.64%\u00C2\u00A0while\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0 EC\u00C2\u00A0by\u00C2\u00A0 6.85%;\u00C2\u00A0522D3\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 80\u00C2\u00A0 \u00C2\u00A0 reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A082.96%\u00C2\u00A0while\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A081.48%.\u00C2\u00A0Constitutive\u00C2\u00A0 co\u00E2\u0080\u0090expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0 DUR3\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0synergistic\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0or\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0(Table\u00C2\u00A020).\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A020.\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0making.\u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0 of\u00C2\u00A0EC\u00C2\u00A0 (\u00C2\u00B5g/L)\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0and\u00C2\u00A0wine\u00C2\u00A0 yeast\u00C2\u00A0strains\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0from\u00C2\u00A0unfiltered\u00C2\u00A0Calona\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0was\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0 GC/MS.\u00C2\u00A0Triplicate\u00C2\u00A0fermentations\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0to\u00C2\u00A0completion\u00C2\u00A0(~300\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A020\u00C2\u00B0C\u00C2\u00A0and\u00C2\u00A0fermentation\u00C2\u00A0 profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A035a,b.\u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K7D3\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 34.19 34.25 29.39 29.74 Replicate\u00C2\u00A02\u00C2\u00A0 40.31 30.98 29.98 31.29 Replicate\u00C2\u00A03\u00C2\u00A0 28.69 30.89 30.78 30.06 Average\u00C2\u00A0(n=3)\u00C2\u00A0 34.40 32.04 30.05 30.36 STDEV\u00C2\u00A0 5.81 1.91 0.70 0.82 RSD\u00C2\u00A0(%)\u00C2\u00A0 16.89 5.96 2.33 2.70 Reduction\u00C2\u00A0(%)\u00C2\u00A0 -- 6.85 12.64 11.73 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 522\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 522D3\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 199.3 31.32 28.76 36.63 Replicate\u00C2\u00A02\u00C2\u00A0 169.95 37.32 29.89 38.61 Replicate\u00C2\u00A03\u00C2\u00A0 177.61 32.66 34.56 31.3 Average\u00C2\u00A0(n=3)\u00C2\u00A0 182.29 33.77 31.07 35.51 STDEV\u00C2\u00A0 15.22 3.15 3.07 3.78 RSD\u00C2\u00A0(%)\u00C2\u00A0 8.34 9.33 9.88 10.64 Reduction\u00C2\u00A0(%)\u00C2\u00A0 -- 81.48 82.96 80.52 \u00C2\u00A0 \u00C2\u00A0 3.2.8\u00C2\u00A0 \u00C2\u00A0Constitutive\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0K7D3\u00C2\u00A0 and\u00C2\u00A0522D3\u00C2\u00A0 reduces\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0 18.40%\u00C2\u00A0and\u00C2\u00A010.45%,\u00C2\u00A0respectively.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0assay\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0making,\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0 was\u00C2\u00A0brewed\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0522\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0recombinant\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0 522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 EC\u00C2\u00A0 content\u00C2\u00A0 quantified\u00C2\u00A0 by\u00C2\u00A0 GC/MS\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 end\u00C2\u00A0 of\u00C2\u00A0 fermentation.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A036a,b.\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0observed\u00C2\u00A0before\u00C2\u00A0 (Section\u00C2\u00A03.1.5),\u00C2\u00A0engineered\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 reduce\u00C2\u00A0EC\u00C2\u00A0more\u00C2\u00A0effectively\u00C2\u00A0 when\u00C2\u00A0assessed\u00C2\u00A0by\u00C2\u00A0brewing\u00C2\u00A0 Sake;\u00C2\u00A0 the\u00C2\u00A0 Sake\u00C2\u00A0 yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0both\u00C2\u00A0 reduced\u00C2\u00A0EC\u00C2\u00A0 content\u00C2\u00A0by\u00C2\u00A0~84%\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 81\u00C2\u00A0 \u00C2\u00A0 (Table\u00C2\u00A021).\u00C2\u00A0During\u00C2\u00A0the\u00C2\u00A0wine\u00C2\u00A0making\u00C2\u00A0trial\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A03.2.7,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0both\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0 only\u00C2\u00A0~15%\u00C2\u00A0(Table\u00C2\u00A020).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A03.2.7\u00C2\u00A0for\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0as\u00C2\u00A0effectively\u00C2\u00A0as\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0(Tables\u00C2\u00A020\u00C2\u00A0and\u00C2\u00A021).\u00C2\u00A0In\u00C2\u00A0Sake\u00C2\u00A0 wine,\u00C2\u00A0K7D3\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A014.97%\u00C2\u00A0while\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A087.07%;\u00C2\u00A0522D3\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A011.49%\u00C2\u00A0while\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 reduced\u00C2\u00A0 EC\u00C2\u00A0 by\u00C2\u00A0 84.30%\u00C2\u00A0 (Table\u00C2\u00A0 21).\u00C2\u00A0 Constitutive\u00C2\u00A0 co\u00E2\u0080\u0090expression\u00C2\u00A0 of\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0 DUR3\u00C2\u00A0 had\u00C2\u00A0 no\u00C2\u00A0 synergistic\u00C2\u00A0 effect\u00C2\u00A0 on\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 during\u00C2\u00A0 Sake\u00C2\u00A0 production\u00C2\u00A0 (Table\u00C2\u00A0 21);\u00C2\u00A0 there\u00C2\u00A0 was\u00C2\u00A0 no\u00C2\u00A0 appreciable\u00C2\u00A0 difference\u00C2\u00A0 in\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 between\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 expressing\u00C2\u00A0 strains\u00C2\u00A0 and\u00C2\u00A0 those\u00C2\u00A0 which\u00C2\u00A0 constitutively\u00C2\u00A0 expressed\u00C2\u00A0both\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A021.\u00C2\u00A0Reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0making.\u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0 of\u00C2\u00A0 EC\u00C2\u00A0 (\u00C2\u00B5g/L)\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0wine\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0522,\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0from\u00C2\u00A0white\u00C2\u00A0rice\u00C2\u00A0(Kokako\u00C2\u00A0Rose)\u00C2\u00A0and\u00C2\u00A0koji\u00C2\u00A0mash\u00C2\u00A0(Vision\u00C2\u00A0Brewing)\u00C2\u00A0was\u00C2\u00A0 quantified\u00C2\u00A0by\u00C2\u00A0GC/MS.\u00C2\u00A0Triplicate\u00C2\u00A0fermentations\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0to\u00C2\u00A0completion\u00C2\u00A0(~450\u00C2\u00A0hours)\u00C2\u00A0at\u00C2\u00A018\u00C2\u00B0C\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0fermentation\u00C2\u00A0profiles\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A036a,b.\u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 K7\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 K7D3\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 109.83 13.37 93.42 15.72 Replicate\u00C2\u00A02\u00C2\u00A0 80.68 12.3 79.86 18.6 Replicate\u00C2\u00A03\u00C2\u00A0 108.26 12.96 80.77 17.79 Average\u00C2\u00A0(n=3)\u00C2\u00A0 99.59 12.88 84.68 17.37 STDEV\u00C2\u00A0 16.40 0.54 7.58 1.49 RSD\u00C2\u00A0(%)\u00C2\u00A0 16.47 4.19 8.95 8.58 Reduction\u00C2\u00A0(%)\u00C2\u00A0 -- 87.07 14.97 82.56 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Yeast\u00C2\u00A0strain\u00C2\u00A0 522\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 522D3\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 Replicate\u00C2\u00A01\u00C2\u00A0 77.05 11.6 85.85 16.12 Replicate\u00C2\u00A02\u00C2\u00A0 104.01 12.17 82.17 13.07 Replicate\u00C2\u00A03\u00C2\u00A0 85.11 18.03 67.57 13.51 Average\u00C2\u00A0(n=3)\u00C2\u00A0 88.72 13.93 78.53 14.23 STDEV\u00C2\u00A0 13.84 3.56 9.67 1.65 RSD\u00C2\u00A0(%)\u00C2\u00A0 15.60 25.56 12.31 11.60 Reduction\u00C2\u00A0(%)\u00C2\u00A0 -- 84.30 11.49 83.96 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 82\u00C2\u00A0 \u00C2\u00A0 4\u00C2\u00A0\u00C2\u00A0DISCUSSION\u00C2\u00A0 \u00C2\u00A0 4.1\u00C2\u00A0\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0reduces\u00C2\u00A0EC\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0common\u00C2\u00A0theme\u00C2\u00A0throughout\u00C2\u00A0the\u00C2\u00A0history\u00C2\u00A0of\u00C2\u00A0mankind\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0the\u00C2\u00A0lack\u00C2\u00A0of\u00C2\u00A0effective\u00C2\u00A0solutions\u00C2\u00A0to\u00C2\u00A0 problems\u00C2\u00A0until\u00C2\u00A0 the\u00C2\u00A0 advent\u00C2\u00A0of\u00C2\u00A0 certain\u00C2\u00A0 technologies.\u00C2\u00A0 Such\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 the\u00C2\u00A0 case\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0problem\u00C2\u00A0of\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 fermented\u00C2\u00A0 foods\u00C2\u00A0and\u00C2\u00A0beverages,\u00C2\u00A0more\u00C2\u00A0 specifically\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Since\u00C2\u00A0 its\u00C2\u00A0discovery\u00C2\u00A0and\u00C2\u00A0 subsequent\u00C2\u00A0characterization\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0carcinogen\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0mid\u00C2\u00A020th\u00C2\u00A0century\u00C2\u00A0 (Nettleship,\u00C2\u00A0Henshaw\u00C2\u00A0and\u00C2\u00A0Meyer\u00C2\u00A0 1943),\u00C2\u00A0EC\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0found\u00C2\u00A0ubiquitously\u00C2\u00A0 in\u00C2\u00A0almost\u00C2\u00A0all\u00C2\u00A0wines\u00C2\u00A0and\u00C2\u00A0spirits\u00C2\u00A0(Canas,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01989;\u00C2\u00A0Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 2006).\u00C2\u00A0Although\u00C2\u00A0the\u00C2\u00A0actual\u00C2\u00A0health\u00C2\u00A0 implications\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0consumption\u00C2\u00A0by\u00C2\u00A0humans\u00C2\u00A0has\u00C2\u00A0yet\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0definitively\u00C2\u00A0 established,\u00C2\u00A0the\u00C2\u00A0Canadian\u00C2\u00A0government\u00C2\u00A0has\u00C2\u00A0imposed\u00C2\u00A0a\u00C2\u00A0legal\u00C2\u00A0limit\u00C2\u00A0(30\u00C2\u00A0\u00C2\u00B5g/L)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0US\u00C2\u00A0FDA\u00C2\u00A0has\u00C2\u00A0imposed\u00C2\u00A0a\u00C2\u00A0 voluntary\u00C2\u00A0 limit\u00C2\u00A0 (15\u00C2\u00A0\u00C2\u00B5g/L)\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0EC\u00C2\u00A0 content\u00C2\u00A0of\u00C2\u00A0wines\u00C2\u00A0 (Butzke\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01998).\u00C2\u00A0 Furthermore,\u00C2\u00A0 several\u00C2\u00A0 agencies,\u00C2\u00A0including\u00C2\u00A0the\u00C2\u00A0National\u00C2\u00A0Institute\u00C2\u00A0of\u00C2\u00A0Health\u00E2\u0080\u0099s\u00C2\u00A0National\u00C2\u00A0Toxicology\u00C2\u00A0Programme\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0University\u00C2\u00A0 of\u00C2\u00A0California\u00C2\u00A0Davis\u00C2\u00A0Department\u00C2\u00A0of\u00C2\u00A0Enology\u00C2\u00A0and\u00C2\u00A0Viticulture,\u00C2\u00A0have\u00C2\u00A0published\u00C2\u00A0preventative\u00C2\u00A0action\u00C2\u00A0manuals\u00C2\u00A0 for\u00C2\u00A0limiting\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0wines\u00C2\u00A0and\u00C2\u00A0spirits\u00C2\u00A0(Butzke\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01998).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Despite\u00C2\u00A0the\u00C2\u00A0mandatory\u00C2\u00A0and\u00C2\u00A0voluntary\u00C2\u00A0 limits\u00C2\u00A0imposed\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0food\u00C2\u00A0and\u00C2\u00A0beverages,\u00C2\u00A0 its\u00C2\u00A0 presence\u00C2\u00A0 continues\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 a\u00C2\u00A0 pervasive\u00C2\u00A0 problem.\u00C2\u00A0 A\u00C2\u00A0 recent\u00C2\u00A0 survey\u00C2\u00A0 by\u00C2\u00A0 our\u00C2\u00A0 group\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 of\u00C2\u00A0 20\u00C2\u00A0 randomly\u00C2\u00A0chosen,\u00C2\u00A0commercially\u00C2\u00A0available\u00C2\u00A0wines\u00C2\u00A0from\u00C2\u00A0six\u00C2\u00A0wine\u00C2\u00A0producing\u00C2\u00A0countries,\u00C2\u00A014\u00C2\u00A0and\u00C2\u00A017\u00C2\u00A0exceeded\u00C2\u00A0 the\u00C2\u00A0 Canadian\u00C2\u00A0 and\u00C2\u00A0 US\u00C2\u00A0 limits,\u00C2\u00A0 respectively\u00C2\u00A0 (Coulon,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0 obvious\u00C2\u00A0 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1991;\u00C2\u00A0Monteiro,\u00C2\u00A0Trousdale\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01989;\u00C2\u00A0Ough,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01990;\u00C2\u00A0Ough,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01991;\u00C2\u00A0Stevens\u00C2\u00A0and\u00C2\u00A0Ough\u00C2\u00A0 1993).\u00C2\u00A0The\u00C2\u00A0successful\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0(Figure\u00C2\u00A015)\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006)\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0 locus\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0popular\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0yielded\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090.\u00C2\u00A0 In\u00C2\u00A0 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As\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0contain\u00C2\u00A0any\u00C2\u00A0antibiotic\u00C2\u00A0resistance\u00C2\u00A0markers,\u00C2\u00A0no\u00C2\u00A0positive\u00C2\u00A0selection\u00C2\u00A0 method\u00C2\u00A0was\u00C2\u00A0available\u00C2\u00A0 to\u00C2\u00A0 identify\u00C2\u00A0 transformants\u00C2\u00A0 that\u00C2\u00A0carried\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0cassette;\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 result,\u00C2\u00A0urea\u00C2\u00A0 degrading\u00C2\u00A0yeasts\u00C2\u00A0were\u00C2\u00A0initially\u00C2\u00A0identified\u00C2\u00A0by\u00C2\u00A0colony\u00C2\u00A0PCR.\u00C2\u00A0Screening\u00C2\u00A0was\u00C2\u00A0completed\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0assistance\u00C2\u00A0of\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 84\u00C2\u00A0 \u00C2\u00A0 the\u00C2\u00A0 co\u00E2\u0080\u0090transforming\u00C2\u00A0 plasmid\u00C2\u00A0 pUT332\u00C2\u00A0 which\u00C2\u00A0 contains\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 bla\u00C2\u00A0 and\u00C2\u00A0 Tn5ble\u00C2\u00A0 resistance\u00C2\u00A0 markers\u00C2\u00A0 (Ampicillin\u00C2\u00A0 and\u00C2\u00A0 phleomycin,\u00C2\u00A0 respectively);\u00C2\u00A0 pUT332\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 transformants\u00C2\u00A0 that\u00C2\u00A0 had\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 screened\u00C2\u00A0 by\u00C2\u00A0 colony\u00C2\u00A0 PCR.\u00C2\u00A0 By\u00C2\u00A0 successive\u00C2\u00A0 subculturing\u00C2\u00A0 on\u00C2\u00A0 non\u00E2\u0080\u0090selective\u00C2\u00A0 media,\u00C2\u00A0 the\u00C2\u00A0 plasmid\u00C2\u00A0was\u00C2\u00A0 lost\u00C2\u00A0 and\u00C2\u00A0 this\u00C2\u00A0was\u00C2\u00A0 confirmed\u00C2\u00A0 on\u00C2\u00A0 Southern\u00C2\u00A0 blots\u00C2\u00A0 hybridized\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 bla\u00C2\u00A0 and\u00C2\u00A0 Tn5ble\u00C2\u00A0genes\u00C2\u00A0 (Figure\u00C2\u00A018).\u00C2\u00A0Sake\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0 first\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0 strains\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 constructed\u00C2\u00A0 without\u00C2\u00A0 the\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 antibiotic\u00C2\u00A0 resistance\u00C2\u00A0markers\u00C2\u00A0 or\u00C2\u00A0 E.\u00C2\u00A0 coli\u00C2\u00A0 vector\u00C2\u00A0 sequences,\u00C2\u00A0 thus\u00C2\u00A0making\u00C2\u00A0 them\u00C2\u00A0 suitable\u00C2\u00A0 for\u00C2\u00A0 commercialization;\u00C2\u00A0 these\u00C2\u00A0 strains\u00C2\u00A0will\u00C2\u00A0 be\u00C2\u00A0more\u00C2\u00A0 likely\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 accepted\u00C2\u00A0by\u00C2\u00A0consumers\u00C2\u00A0than\u00C2\u00A0strains\u00C2\u00A0containing\u00C2\u00A0antibiotic\u00C2\u00A0resistance\u00C2\u00A0marker\u00C2\u00A0genes\u00C2\u00A0or\u00C2\u00A0foreign\u00C2\u00A0DNA.\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 strand\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassettes\u00C2\u00A0 in\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 was\u00C2\u00A0 sequenced.\u00C2\u00A0 Upon\u00C2\u00A0 comparison\u00C2\u00A0to\u00C2\u00A0previously\u00C2\u00A0published\u00C2\u00A0sequences\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006),\u00C2\u00A0a\u00C2\u00A0single\u00C2\u00A0nucleotide\u00C2\u00A0difference\u00C2\u00A0was\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 sequence\u00C2\u00A0of\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 (Table\u00C2\u00A08).\u00C2\u00A0 This\u00C2\u00A0 single\u00C2\u00A0nucleotide\u00C2\u00A0difference\u00C2\u00A0was\u00C2\u00A0 localized\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A05\u00E2\u0080\u0099\u00C2\u00A0 URA3\u00C2\u00A0 flanking\u00C2\u00A0 region\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 cassette\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 genetic\u00C2\u00A0polymorphism\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 Sake\u00C2\u00A0 strain\u00C2\u00A0K9\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0wine\u00C2\u00A0 strain\u00C2\u00A0522.\u00C2\u00A0No\u00C2\u00A0differences\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 coding\u00C2\u00A0 region\u00C2\u00A0or\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Analysis\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0expression\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0fermentation\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0indeed\u00C2\u00A0relieves\u00C2\u00A0repression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0by\u00C2\u00A0NCR,\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0is\u00C2\u00A0much\u00C2\u00A0stronger\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0 inducible/repressible,\u00C2\u00A0NCR\u00C2\u00A0sensitive\u00C2\u00A0DUR1,2\u00C2\u00A0promoter.\u00C2\u00A0DUR1,2\u00C2\u00A0mRNA\u00C2\u00A0was\u00C2\u00A0approximately\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0more\u00C2\u00A0 abundant\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0than\u00C2\u00A0in\u00C2\u00A0their\u00C2\u00A0respective\u00C2\u00A0parent\u00C2\u00A0strains\u00C2\u00A0(Figure\u00C2\u00A020).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0pattern\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0engineered\u00C2\u00A0strain\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0as\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0pattern\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0parent\u00C2\u00A0strain\u00C2\u00A0K7\u00C2\u00A0was\u00C2\u00A0studied\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0hours\u00C2\u00A0into\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0fermentation.\u00C2\u00A0Besides\u00C2\u00A0DUR1,2\u00C2\u00A0(6.35\u00E2\u0080\u0090 fold\u00C2\u00A0overexpression),\u00C2\u00A0nine\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0affected\u00C2\u00A0\u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold;\u00C2\u00A0thus,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0evident\u00C2\u00A0that\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genome\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0K7\u00C2\u00A0had\u00C2\u00A0a\u00C2\u00A0minimal\u00C2\u00A0effect\u00C2\u00A0(0.15%\u00C2\u00A0change)\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcription\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A05795\u00C2\u00A0ORFs\u00C2\u00A0(4692\u00C2\u00A0verified\u00C2\u00A0and\u00C2\u00A01103\u00C2\u00A0uncharacterized,\u00C2\u00A0SGD,\u00C2\u00A0March,\u00C2\u00A02008)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0cell.\u00C2\u00A0One\u00C2\u00A0gene\u00C2\u00A0 of\u00C2\u00A0interest\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0upregulated\u00C2\u00A0\u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(Table\u00C2\u00A010)\u00C2\u00A0was\u00C2\u00A0HAC1\u00C2\u00A0(4.23\u00E2\u0080\u0090fold),\u00C2\u00A0a\u00C2\u00A0transcription\u00C2\u00A0factor\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0unfolded\u00C2\u00A0protein\u00C2\u00A0response\u00C2\u00A0(Cox\u00C2\u00A0and\u00C2\u00A0Walter\u00C2\u00A01996,\u00C2\u00A0Mori\u00C2\u00A01996,\u00C2\u00A0Nikawa,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01996);\u00C2\u00A0this\u00C2\u00A0 response\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0 needed\u00C2\u00A0 to\u00C2\u00A0 support\u00C2\u00A0 the\u00C2\u00A0 increased\u00C2\u00A0 translation\u00C2\u00A0 and\u00C2\u00A0 folding\u00C2\u00A0 of\u00C2\u00A0DUR1,2p\u00C2\u00A0 constitutively\u00C2\u00A0 expressed\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 strong\u00C2\u00A0 PGK1\u00C2\u00A0 promoter;\u00C2\u00A0 indeed,\u00C2\u00A0 HAC1\u00C2\u00A0 was\u00C2\u00A0 also\u00C2\u00A0 upregulated\u00C2\u00A0 (5.67\u00E2\u0080\u0090fold)\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 engineered\u00C2\u00A0 strain\u00C2\u00A0 K7D3\u00C2\u00A0which\u00C2\u00A0 contains\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 gene\u00C2\u00A0 under\u00C2\u00A0 the\u00C2\u00A0 control\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 and\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 85\u00C2\u00A0 \u00C2\u00A0 terminator\u00C2\u00A0signals\u00C2\u00A0(Table\u00C2\u00A017).\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0also\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0no\u00C2\u00A0metabolic\u00C2\u00A0pathways\u00C2\u00A0were\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0 presence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0K7EC\u00E2\u0080\u0090;\u00C2\u00A0however,\u00C2\u00A0three\u00C2\u00A0(RME1,\u00C2\u00A0SSP1,\u00C2\u00A0SDS3)\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0seven\u00C2\u00A0 genes\u00C2\u00A0 downregulated\u00C2\u00A0 in\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 are\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 different\u00C2\u00A0 aspects\u00C2\u00A0 of\u00C2\u00A0meiosis/sporulation\u00C2\u00A0 (Table\u00C2\u00A0 10).\u00C2\u00A0 As\u00C2\u00A0 sporulation\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0triggered\u00C2\u00A0by\u00C2\u00A0nutrient\u00C2\u00A0deficiency\u00C2\u00A0(Malone\u00C2\u00A01990),\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0reasonable\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0DUR1,2\u00C2\u00A0cassette,\u00C2\u00A0which\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0constitutive\u00C2\u00A0utilization\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0nitrogen\u00C2\u00A0source,\u00C2\u00A0may\u00C2\u00A0cause\u00C2\u00A0 yeast\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0alter\u00C2\u00A0or\u00C2\u00A0delay\u00C2\u00A0their\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0nutrient\u00C2\u00A0limitation.\u00C2\u00A0 \u00C2\u00A0 4.2.2\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0conduct\u00C2\u00A0efficient\u00C2\u00A0alcoholic\u00C2\u00A0fermentations\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0measures\u00C2\u00A0 of\u00C2\u00A0 substantial\u00C2\u00A0 equivalence,\u00C2\u00A0 fermentation\u00C2\u00A0 rates,\u00C2\u00A0 glucose/fructose\u00C2\u00A0 utilization\u00C2\u00A0 and\u00C2\u00A0 ethanol\u00C2\u00A0 production\u00C2\u00A0 was\u00C2\u00A0 evaluated\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 metabolically\u00C2\u00A0 engineered\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090.\u00C2\u00A0 Prior\u00C2\u00A0 to\u00C2\u00A0 commercialization,\u00C2\u00A0engineered\u00C2\u00A0yeasts\u00C2\u00A0must\u00C2\u00A0obtain\u00C2\u00A0approval\u00C2\u00A0from\u00C2\u00A0regulatory\u00C2\u00A0agencies\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0FDA,\u00C2\u00A0 Health\u00C2\u00A0 Canada,\u00C2\u00A0 and\u00C2\u00A0 Environment\u00C2\u00A0 Canada.\u00C2\u00A0 In\u00C2\u00A0 North\u00C2\u00A0 America,\u00C2\u00A0 approval\u00C2\u00A0 is\u00C2\u00A0 granted\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 basis\u00C2\u00A0 of\u00C2\u00A0 substantial\u00C2\u00A0 equivalence,\u00C2\u00A0which\u00C2\u00A0means\u00C2\u00A0 that\u00C2\u00A0 should\u00C2\u00A0 a\u00C2\u00A0 foodstuff\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 genetically\u00C2\u00A0modified\u00C2\u00A0 organism\u00C2\u00A0 (GMO)\u00C2\u00A0be\u00C2\u00A0proven\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0\u00E2\u0080\u0098as\u00C2\u00A0safe\u00C2\u00A0as\u00E2\u0080\u0099\u00C2\u00A0the\u00C2\u00A0food\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0traditional\u00C2\u00A0means,\u00C2\u00A0then\u00C2\u00A0both\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0treated\u00C2\u00A0 equally\u00C2\u00A0(Kessler\u00C2\u00A01992).\u00C2\u00A0Proof\u00C2\u00A0of\u00C2\u00A0substantial\u00C2\u00A0equivalence\u00C2\u00A0and\u00C2\u00A0safety\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0genetically\u00C2\u00A0engineered\u00C2\u00A0organism\u00C2\u00A0 generally\u00C2\u00A0 requires\u00C2\u00A0 detailed\u00C2\u00A0 data\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 organism\u00E2\u0080\u0099s\u00C2\u00A0 genotype,\u00C2\u00A0 phenotype,\u00C2\u00A0 transcriptome,\u00C2\u00A0 proteome,\u00C2\u00A0and\u00C2\u00A0metabolome.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 both\u00C2\u00A0 Chardonnay\u00C2\u00A0 must\u00C2\u00A0 and\u00C2\u00A0 Sake\u00C2\u00A0 mash,\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 conducted\u00C2\u00A0 efficient\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentations\u00C2\u00A0(Figures\u00C2\u00A021\u00C2\u00A0and\u00C2\u00A022),\u00C2\u00A0comparable\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9;\u00C2\u00A0fermentations\u00C2\u00A0 were\u00C2\u00A0 completed\u00C2\u00A0 in\u00C2\u00A0 ~300\u00C2\u00A0 hours\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0 and\u00C2\u00A0 ~500\u00E2\u0080\u0090600\u00C2\u00A0hours\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine.\u00C2\u00A0 Furthermore,\u00C2\u00A0 residual\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0 fructose\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0produced\u00C2\u00A0with\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0 (0.05,\u00C2\u00A00.010\u00C2\u00A0g/L\u00C2\u00A0glucose\u00C2\u00A0and\u00C2\u00A0 0.72,\u00C2\u00A00.30\u00C2\u00A0g/L\u00C2\u00A0fructose,\u00C2\u00A0respectively)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strains\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K9\u00C2\u00A0were\u00C2\u00A0comparable\u00C2\u00A0(0.06,\u00C2\u00A00.08\u00C2\u00A0g/L\u00C2\u00A0 glucose\u00C2\u00A0and\u00C2\u00A00.65,\u00C2\u00A00.31\u00C2\u00A0g/L\u00C2\u00A0fructose,\u00C2\u00A0respectively).\u00C2\u00A0All\u00C2\u00A0four\u00C2\u00A0yeasts\u00C2\u00A0produced\u00C2\u00A0similar\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0(Table\u00C2\u00A011).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.2.3\u00C2\u00A0The\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0poorly\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0yet\u00C2\u00A0efficiently\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 two\u00C2\u00A0 functionally\u00C2\u00A0 enhanced\u00C2\u00A0 Sake\u00C2\u00A0 yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0 evaluated\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0 In\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 86\u00C2\u00A0 \u00C2\u00A0 ineffective\u00C2\u00A0in\u00C2\u00A0reducing\u00C2\u00A0EC;\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A030%\u00C2\u00A0and\u00C2\u00A00%,\u00C2\u00A0respectively\u00C2\u00A0(Table\u00C2\u00A012).\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0both\u00C2\u00A0effectively\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A068%\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0(Table\u00C2\u00A013).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0fermentations,\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0exposed\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0substantially\u00C2\u00A0different\u00C2\u00A0set\u00C2\u00A0 of\u00C2\u00A0environmental\u00C2\u00A0conditions\u00C2\u00A0and\u00C2\u00A0stresses\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0experienced\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0fermentation.\u00C2\u00A0\u00C2\u00A0Specialized\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 selected\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0 (Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007)\u00C2\u00A0and\u00C2\u00A0 fermentation\u00C2\u00A0conditions\u00C2\u00A0undoubtedly\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0metabolism.\u00C2\u00A0While\u00C2\u00A0several\u00C2\u00A0environmental\u00C2\u00A0 parameters\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0 a\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 explaining\u00C2\u00A0 the\u00C2\u00A0 ineffective\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0of\u00C2\u00A0 Sake\u00C2\u00A0 strains\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0 during\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0making\u00C2\u00A0(Tables\u00C2\u00A012\u00C2\u00A0and\u00C2\u00A013),\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0available\u00C2\u00A0nitrogen\u00C2\u00A0on\u00C2\u00A0native\u00C2\u00A0NCR\u00C2\u00A0 controlled\u00C2\u00A0genes\u00C2\u00A0is\u00C2\u00A0likely\u00C2\u00A0the\u00C2\u00A0predominant\u00C2\u00A0factor.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0type\u00C2\u00A0and\u00C2\u00A0quantity\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0assimilable nitrogen\u00C2\u00A0(YAN)\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0mash\u00C2\u00A0is\u00C2\u00A0substantially\u00C2\u00A0different\u00C2\u00A0 from\u00C2\u00A0grape\u00C2\u00A0must.\u00C2\u00A0Grape\u00C2\u00A0must\u00C2\u00A0of\u00C2\u00A0almost\u00C2\u00A0all\u00C2\u00A0varietals\u00C2\u00A0is\u00C2\u00A0usually\u00C2\u00A0high\u00C2\u00A0in\u00C2\u00A0free\u00C2\u00A0arginine\u00C2\u00A0and\u00C2\u00A0proline\u00C2\u00A0(Kliewer\u00C2\u00A0 1970).\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0Sake\u00C2\u00A0mash\u00C2\u00A0is\u00C2\u00A0rich\u00C2\u00A0in\u00C2\u00A0polypeptides\u00C2\u00A0that\u00C2\u00A0form\u00C2\u00A0structures\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0protein\u00C2\u00A0bodies\u00C2\u00A0(PB)\u00C2\u00A0 (Kizaki,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A01991).\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0protein\u00C2\u00A0bodies,\u00C2\u00A0which\u00C2\u00A0 fall\u00C2\u00A0 into\u00C2\u00A0 two\u00C2\u00A0major\u00C2\u00A0 categories\u00C2\u00A0 (PB\u00E2\u0080\u0090I\u00C2\u00A0 and\u00C2\u00A0PB\u00E2\u0080\u0090II),\u00C2\u00A0 are\u00C2\u00A0 primarily\u00C2\u00A0composed\u00C2\u00A0of\u00C2\u00A0prolamins\u00C2\u00A0and\u00C2\u00A0glutelins,\u00C2\u00A0respectively\u00C2\u00A0(Hashizume,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0During\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0fermentation\u00C2\u00A0yeast\u00C2\u00A0draw\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0pool\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0replenished\u00C2\u00A0by\u00C2\u00A0degradation\u00C2\u00A0of\u00C2\u00A0 PB\u00C2\u00A0by\u00C2\u00A0koji\u00E2\u0080\u0090provided\u00C2\u00A0acid\u00C2\u00A0carboxypeptidases\u00C2\u00A0and\u00C2\u00A0acid\u00C2\u00A0proteases\u00C2\u00A0 (Hashizume,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006;\u00C2\u00A0 Iemura,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 1999a;\u00C2\u00A0Iemura,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01999b).\u00C2\u00A0This\u00C2\u00A0controlled\u00C2\u00A0release\u00C2\u00A0process,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0 limit\u00C2\u00A0the\u00C2\u00A0 large\u00C2\u00A0scale\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0certain\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0catabolic\u00C2\u00A0enzymes,\u00C2\u00A0functions\u00C2\u00A0to\u00C2\u00A0prevent\u00C2\u00A0nitrogen\u00C2\u00A0exhaustion\u00C2\u00A0(Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006)\u00C2\u00A0and\u00C2\u00A0 subsequent\u00C2\u00A0TOR\u00C2\u00A0mediated\u00C2\u00A0transcriptional\u00C2\u00A0reprogramming\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0sensitive\u00C2\u00A0 genes\u00C2\u00A0(Cooper\u00C2\u00A02002;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0nitrogen\u00C2\u00A0available\u00C2\u00A0in\u00C2\u00A0Sake\u00C2\u00A0mash,\u00C2\u00A0yeasts\u00C2\u00A0draw\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0large\u00C2\u00A0pool\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0amino\u00C2\u00A0 acids\u00C2\u00A0in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0biomass\u00C2\u00A0during\u00C2\u00A0fermentation.\u00C2\u00A0However,\u00C2\u00A0at\u00C2\u00A0approximately\u00C2\u00A02\u00E2\u0080\u00904\u00C2\u00A0days\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0 fermentation,\u00C2\u00A0cells\u00C2\u00A0 stop\u00C2\u00A0dividing\u00C2\u00A0and\u00C2\u00A0enter\u00C2\u00A0a\u00C2\u00A0 stationary\u00C2\u00A0growth\u00C2\u00A0phase\u00C2\u00A0 in\u00C2\u00A0which\u00C2\u00A0 they\u00C2\u00A0 ferment\u00C2\u00A0actively.\u00C2\u00A0 While\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0new\u00C2\u00A0evidence\u00C2\u00A0to\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0ethanol\u00C2\u00A0stress\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0transitioning\u00C2\u00A0into\u00C2\u00A0stationary\u00C2\u00A0 phase\u00C2\u00A0(Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02003;\u00C2\u00A0Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02008),\u00C2\u00A0the\u00C2\u00A0general\u00C2\u00A0consensus\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0literature,\u00C2\u00A0however,\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0 nitrogen\u00C2\u00A0exhaustion\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0primary\u00C2\u00A0 reason\u00C2\u00A0why\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0enter\u00C2\u00A0 into\u00C2\u00A0 stationary\u00C2\u00A0phase\u00C2\u00A0 (Hauser,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 2001;\u00C2\u00A0Rossignol,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02003;\u00C2\u00A0Shobayashi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Nitrogen\u00C2\u00A0exhaustion,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 detected\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0TOR\u00C2\u00A0pathway,\u00C2\u00A0is\u00C2\u00A0accompanied\u00C2\u00A0by\u00C2\u00A0large\u00C2\u00A0scale\u00C2\u00A0transcriptional\u00C2\u00A0changes\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0de\u00E2\u0080\u0090 \u00C2\u00A0 \u00C2\u00A0 87\u00C2\u00A0 \u00C2\u00A0 repression\u00C2\u00A0of\u00C2\u00A0genes\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0catabolism\u00C2\u00A0of\u00C2\u00A0poor\u00C2\u00A0nitrogen\u00C2\u00A0sources,\u00C2\u00A0 including\u00C2\u00A0urea\u00C2\u00A0(Cooper\u00C2\u00A02002;\u00C2\u00A0 Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0Interestingly,\u00C2\u00A0the\u00C2\u00A0second\u00C2\u00A0most\u00C2\u00A0abundant\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0(Kliewer\u00C2\u00A01970),\u00C2\u00A0 proline,\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0metabolized\u00C2\u00A0during\u00C2\u00A0fermentation.\u00C2\u00A0Proline\u00C2\u00A0catabolism\u00C2\u00A0requires\u00C2\u00A0molecular\u00C2\u00A0oxygen\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0 fermentations\u00C2\u00A0 are\u00C2\u00A0 anaerobic,\u00C2\u00A0proline\u00C2\u00A0 cannot\u00C2\u00A0be\u00C2\u00A0utilized\u00C2\u00A0 (Ingledew,\u00C2\u00A0Magnus\u00C2\u00A0 and\u00C2\u00A0 Sosulski\u00C2\u00A01987).\u00C2\u00A0Thus,\u00C2\u00A0 after\u00C2\u00A0 nitrogen\u00C2\u00A0 exhaustion\u00C2\u00A0 shifts\u00C2\u00A0 cells\u00C2\u00A0 into\u00C2\u00A0 stationary\u00C2\u00A0 phase,\u00C2\u00A0 the\u00C2\u00A0 high\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 proline\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 ferment\u00C2\u00A0 likely\u00C2\u00A0 reinforces,\u00C2\u00A0 sustains\u00C2\u00A0and\u00C2\u00A0 strengthens\u00C2\u00A0 the\u00C2\u00A0 transcriptional\u00C2\u00A0 reprogramming\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0 NCR\u00C2\u00A0de\u00E2\u0080\u0090repression.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Of\u00C2\u00A0particular\u00C2\u00A0interest\u00C2\u00A0to\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0NCR\u00C2\u00A0sensitive\u00C2\u00A0urea\u00C2\u00A0amidolyase\u00C2\u00A0 encoding\u00C2\u00A0gene\u00C2\u00A0DUR1,2.\u00C2\u00A0 Induction\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0allows\u00C2\u00A0yeast\u00C2\u00A0 to\u00C2\u00A0degrade\u00C2\u00A0urea\u00C2\u00A0 in\u00C2\u00A0an\u00C2\u00A0 increasingly\u00C2\u00A0nitrogen\u00C2\u00A0 scarce\u00C2\u00A0environment.\u00C2\u00A0Thus,\u00C2\u00A0during\u00C2\u00A0 the\u00C2\u00A0 later\u00C2\u00A0 stages\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0 fermentation,\u00C2\u00A0DUR1,2\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0 induced\u00C2\u00A0and\u00C2\u00A0 maintained\u00C2\u00A0throughout\u00C2\u00A0the\u00C2\u00A0fermentation\u00C2\u00A0as\u00C2\u00A0long\u00C2\u00A0as\u00C2\u00A0yeast\u00C2\u00A0are\u00C2\u00A0starved\u00C2\u00A0for\u00C2\u00A0nitrogen.\u00C2\u00A0As\u00C2\u00A0a\u00C2\u00A0result,\u00C2\u00A0parental\u00C2\u00A0 strains\u00C2\u00A0produce\u00C2\u00A0less\u00C2\u00A0EC\u00C2\u00A0overall\u00C2\u00A0since\u00C2\u00A0they\u00C2\u00A0tend\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0DUR1,2\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0middle/end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0fermentation,\u00C2\u00A0 thereby\u00C2\u00A0 reducing\u00C2\u00A0 the\u00C2\u00A0 effectiveness\u00C2\u00A0 of\u00C2\u00A0 functionally\u00C2\u00A0 enhanced\u00C2\u00A0 clones\u00C2\u00A0 (Table\u00C2\u00A0 12).\u00C2\u00A0During\u00C2\u00A0 Sake\u00C2\u00A0 brewing\u00C2\u00A0 however,\u00C2\u00A0no\u00C2\u00A0nitrogen\u00C2\u00A0exhaustion\u00C2\u00A0 is\u00C2\u00A0experienced\u00C2\u00A0by\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0DUR1,2,\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0NCR\u00C2\u00A0 sensitive\u00C2\u00A0genes,\u00C2\u00A0remains\u00C2\u00A0largely\u00C2\u00A0repressed,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0higher\u00C2\u00A0absolute\u00C2\u00A0EC\u00C2\u00A0values\u00C2\u00A0and\u00C2\u00A0an\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 effectiveness\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0metabolically\u00C2\u00A0enhanced\u00C2\u00A0yeasts\u00C2\u00A0containing\u00C2\u00A0 the\u00C2\u00A0constitutive\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0 (Table\u00C2\u00A0 13).\u00C2\u00A0Data\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0studies\u00C2\u00A0during\u00C2\u00A0grape\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0fermentations\u00C2\u00A0 confirm\u00C2\u00A0that,\u00C2\u00A0during\u00C2\u00A0grape\u00C2\u00A0wine\u00C2\u00A0fermentation,\u00C2\u00A0the\u00C2\u00A0native\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0genes\u00C2\u00A0are\u00C2\u00A0both\u00C2\u00A0 induced\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0transition\u00C2\u00A0 into\u00C2\u00A0stationary\u00C2\u00A0phase\u00C2\u00A0(Rossignol,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02003);\u00C2\u00A0this\u00C2\u00A0 is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0nitrogen\u00C2\u00A0exhaustion\u00C2\u00A0 and\u00C2\u00A0subsequent\u00C2\u00A0transcriptional\u00C2\u00A0reprogramming\u00C2\u00A0inducing\u00C2\u00A0stationary\u00C2\u00A0phase.\u00C2\u00A0In\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0making,\u00C2\u00A0DUR1,2\u00C2\u00A0 and\u00C2\u00A0DUR3\u00C2\u00A0are\u00C2\u00A0transcriptionally\u00C2\u00A0inactive\u00C2\u00A0throughout\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0fermentation\u00C2\u00A0(Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006),\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0adequate\u00C2\u00A0nitrogen\u00C2\u00A0supply.\u00C2\u00A0 \u00C2\u00A0 Another\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0and\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0concerns\u00C2\u00A0osmotic\u00C2\u00A0stress.\u00C2\u00A0As\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 continuous\u00C2\u00A0 saccharification\u00C2\u00A0 process\u00C2\u00A0 during\u00C2\u00A0 Sake\u00C2\u00A0 brewing,\u00C2\u00A0 (Figure\u00C2\u00A0 4),\u00C2\u00A0 yeasts\u00C2\u00A0 are\u00C2\u00A0 subjected\u00C2\u00A0 to\u00C2\u00A0 substantially\u00C2\u00A0 less\u00C2\u00A0osmotic\u00C2\u00A0stress\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0 fermentation\u00C2\u00A0than\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0 fermentation\u00C2\u00A0where\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0sugars\u00C2\u00A0are\u00C2\u00A0present\u00C2\u00A0at\u00C2\u00A0 inoculation\u00C2\u00A0 (Takagi,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02005;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0This\u00C2\u00A0reduced\u00C2\u00A0stress\u00C2\u00A0during\u00C2\u00A0\u00C2\u00A0 Sake\u00C2\u00A0 fermentations\u00C2\u00A0 is\u00C2\u00A0 thought\u00C2\u00A0 to\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0 to\u00C2\u00A0produce\u00C2\u00A0up\u00C2\u00A0 to\u00C2\u00A020%\u00C2\u00A0 (w/v)\u00C2\u00A0 ethanol\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0 (Shobayashi,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2007).\u00C2\u00A0 During\u00C2\u00A0 osmotic\u00C2\u00A0 stress,\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 activate\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u0098high\u00C2\u00A0 osmolarity\u00C2\u00A0growth\u00E2\u0080\u0099\u00C2\u00A0(HOG)\u00C2\u00A0pathway\u00C2\u00A0which\u00C2\u00A0allows\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0survive\u00C2\u00A0osmotic\u00C2\u00A0stress\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0biosynthesis\u00C2\u00A0and\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 88\u00C2\u00A0 \u00C2\u00A0 intracellular\u00C2\u00A0retention\u00C2\u00A0of\u00C2\u00A0small\u00C2\u00A0molecules\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0 trehalose\u00C2\u00A0and\u00C2\u00A0glycerol\u00C2\u00A0 (Westfall,\u00C2\u00A0Ballon\u00C2\u00A0and\u00C2\u00A0Thorner\u00C2\u00A0 2004).\u00C2\u00A0 These\u00C2\u00A0 molecules\u00C2\u00A0 help\u00C2\u00A0 to\u00C2\u00A0 offset\u00C2\u00A0 the\u00C2\u00A0 osmotic\u00C2\u00A0 pressure\u00C2\u00A0 created\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 high\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 extracellular\u00C2\u00A0 sugars\u00C2\u00A0 (often\u00C2\u00A0 20\u00E2\u0080\u009030%\u00C2\u00A0w/v,\u00C2\u00A0 equimolar\u00C2\u00A0 amounts\u00C2\u00A0 of\u00C2\u00A0 glucose\u00C2\u00A0 and\u00C2\u00A0 fructose\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0must),\u00C2\u00A0 which\u00C2\u00A0would\u00C2\u00A0 otherwise\u00C2\u00A0 crenate\u00C2\u00A0 the\u00C2\u00A0 cells.\u00C2\u00A0 Although\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 has\u00C2\u00A0 evolved\u00C2\u00A0 to\u00C2\u00A0 tolerate\u00C2\u00A0 substantial\u00C2\u00A0 osmotic\u00C2\u00A0 stress,\u00C2\u00A0 induction\u00C2\u00A0 and\u00C2\u00A0 maintenance\u00C2\u00A0 of\u00C2\u00A0 tolerance\u00C2\u00A0 mechanisms\u00C2\u00A0 requires\u00C2\u00A0 significant\u00C2\u00A0 energy\u00C2\u00A0 expenditure\u00C2\u00A0that\u00C2\u00A0could\u00C2\u00A0otherwise\u00C2\u00A0be\u00C2\u00A0devoted\u00C2\u00A0to\u00C2\u00A0biomass\u00C2\u00A0creation\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0metabolic\u00C2\u00A0processes\u00C2\u00A0(Wu,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 For\u00C2\u00A0 Sake\u00C2\u00A0 yeast,\u00C2\u00A0which\u00C2\u00A0 has\u00C2\u00A0 evolved\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 niche\u00C2\u00A0 of\u00C2\u00A0markedly\u00C2\u00A0 reduced\u00C2\u00A0 osmotic\u00C2\u00A0 stress,\u00C2\u00A0 the\u00C2\u00A0 osmotic\u00C2\u00A0shock\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0 factor\u00C2\u00A0 in\u00C2\u00A0explaining\u00C2\u00A0 the\u00C2\u00A0poor\u00C2\u00A0EC\u00C2\u00A0 reduction\u00C2\u00A0of\u00C2\u00A0engineered\u00C2\u00A0Sake\u00C2\u00A0 strains\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0making.\u00C2\u00A0Specifically,\u00C2\u00A0 the\u00C2\u00A0yeast\u00C2\u00A0 stress\u00C2\u00A0 response\u00C2\u00A0 tends\u00C2\u00A0 to\u00C2\u00A0down\u00E2\u0080\u0090regulate\u00C2\u00A0 translation\u00C2\u00A0 globally,\u00C2\u00A0 which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0 could\u00C2\u00A0 decrease\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 DUR1,2p\u00C2\u00A0 (Cooper\u00C2\u00A0 2002;\u00C2\u00A0 Hauser,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2001;\u00C2\u00A0 Rossignol,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02003).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0koji\u00C2\u00A0to\u00C2\u00A0Sake\u00C2\u00A0fermentations\u00C2\u00A0plays\u00C2\u00A0an\u00C2\u00A0 important\u00C2\u00A0role\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0availability\u00C2\u00A0of\u00C2\u00A0 some\u00C2\u00A0vital\u00C2\u00A0auxiliary\u00C2\u00A0factors.\u00C2\u00A0One\u00C2\u00A0such\u00C2\u00A0factor\u00C2\u00A0is\u00C2\u00A0ergosterol,\u00C2\u00A0a\u00C2\u00A0cholesterol\u00C2\u00A0derivative\u00C2\u00A0compound\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0 yeast\u00C2\u00A0cell\u00C2\u00A0membranes\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0vital\u00C2\u00A0for\u00C2\u00A0ethanol\u00C2\u00A0tolerance\u00C2\u00A0(Inoue\u00C2\u00A02000).\u00C2\u00A0Ethanol\u00C2\u00A0causes\u00C2\u00A0rigidity\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0 membrane\u00C2\u00A0 and\u00C2\u00A0 induces\u00C2\u00A0 fatal\u00C2\u00A0 cracks;\u00C2\u00A0 ergosterol,\u00C2\u00A0 like\u00C2\u00A0 cholesterol,\u00C2\u00A0 functions\u00C2\u00A0 to\u00C2\u00A0 decrease\u00C2\u00A0 the\u00C2\u00A0 packing\u00C2\u00A0 density\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0phospholipids\u00C2\u00A0and\u00C2\u00A0 increase\u00C2\u00A0membrane\u00C2\u00A0fluidity,\u00C2\u00A0thus\u00C2\u00A0counteracting\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0 ethanol\u00C2\u00A0 (Inoue\u00C2\u00A0 2000).\u00C2\u00A0 Yeast\u00C2\u00A0 cells\u00C2\u00A0 require\u00C2\u00A0 oxygen\u00C2\u00A0 to\u00C2\u00A0 synthesize\u00C2\u00A0 ergosterol\u00C2\u00A0 (Jahnke\u00C2\u00A0 1983),\u00C2\u00A0 and\u00C2\u00A0 during\u00C2\u00A0 anaerobic\u00C2\u00A0 fermentations\u00C2\u00A0 this\u00C2\u00A0dependency\u00C2\u00A0on\u00C2\u00A0ergosterol\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 a\u00C2\u00A0 limiting\u00C2\u00A0 factor\u00C2\u00A0on\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 and\u00C2\u00A0 ethanol\u00C2\u00A0 production.\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 wine\u00C2\u00A0 fermentations\u00C2\u00A0 are\u00C2\u00A0 often\u00C2\u00A0 oxygenated\u00C2\u00A0 briefly\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 or\u00C2\u00A0 during\u00C2\u00A0 fermentation\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 facilitate\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 ergosterol\u00C2\u00A0 (Rossignol,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2003).\u00C2\u00A0 In\u00C2\u00A0 Sake\u00C2\u00A0 fermentations\u00C2\u00A0however,\u00C2\u00A0ergosterol\u00C2\u00A0is\u00C2\u00A0supplied\u00C2\u00A0primarily\u00C2\u00A0from\u00C2\u00A0koji\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0cultured\u00C2\u00A0aerobically\u00C2\u00A0(Wu,\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A0 2006),\u00C2\u00A0 and\u00C2\u00A0 as\u00C2\u00A0 such\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 may\u00C2\u00A0 have\u00C2\u00A0 evolved\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 dependent\u00C2\u00A0 on\u00C2\u00A0 this\u00C2\u00A0 external\u00C2\u00A0 source\u00C2\u00A0 of\u00C2\u00A0 ergosterol.\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 Sake\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0were\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 all\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ergosterol\u00C2\u00A0 biosynthetic\u00C2\u00A0 genes\u00C2\u00A0 during\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0fermentation\u00C2\u00A0thus\u00C2\u00A0indicating\u00C2\u00A0an\u00C2\u00A0unexpected\u00C2\u00A0shortage\u00C2\u00A0of\u00C2\u00A0ergosterol\u00C2\u00A0despite\u00C2\u00A0the\u00C2\u00A0 presence\u00C2\u00A0of\u00C2\u00A0koji\u00C2\u00A0derived\u00C2\u00A0ergosterol\u00C2\u00A0(Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0Thus,\u00C2\u00A0when\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0are\u00C2\u00A0 used\u00C2\u00A0to\u00C2\u00A0ferment\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0oxygenated,\u00C2\u00A0the\u00C2\u00A0lack\u00C2\u00A0of\u00C2\u00A0ergosterol\u00C2\u00A0contribution\u00C2\u00A0from\u00C2\u00A0koji\u00C2\u00A0may\u00C2\u00A0 play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0reduction\u00C2\u00A0in\u00C2\u00A0cell\u00C2\u00A0health,\u00C2\u00A0viability\u00C2\u00A0and\u00C2\u00A0EC\u00C2\u00A0reduction.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Given\u00C2\u00A0the\u00C2\u00A0observed\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0in\u00C2\u00A0grape\u00C2\u00A0must\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0mash,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0imperative\u00C2\u00A0that,\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0gather\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0relevant\u00C2\u00A0data,\u00C2\u00A0the\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 89\u00C2\u00A0 \u00C2\u00A0 functionality\u00C2\u00A0 of\u00C2\u00A0 engineered\u00C2\u00A0 yeasts\u00C2\u00A0 be\u00C2\u00A0 tested\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0 niche\u00C2\u00A0 environments;\u00C2\u00A0 this\u00C2\u00A0 should\u00C2\u00A0 prevent\u00C2\u00A0 the\u00C2\u00A0 identification\u00C2\u00A0of\u00C2\u00A0false\u00C2\u00A0negatives\u00C2\u00A0and\u00C2\u00A0will\u00C2\u00A0expedite\u00C2\u00A0commercialization\u00C2\u00A0of\u00C2\u00A0new\u00C2\u00A0strains.\u00C2\u00A0 \u00C2\u00A0 4.3\u00C2\u00A0\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0permease,\u00C2\u00A0DUR3,\u00C2\u00A0in\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0viable\u00C2\u00A0alternative\u00C2\u00A0method\u00C2\u00A0 to\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0fermented\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 most\u00C2\u00A0 efficient\u00C2\u00A0 metabolically\u00C2\u00A0 enhanced\u00C2\u00A0 wine\u00C2\u00A0 yeast\u00C2\u00A0 522EC\u00E2\u0080\u0090,\u00C2\u00A0 which\u00C2\u00A0 contains\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0URA3\u00C2\u00A0locus,\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A089%\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 further\u00C2\u00A0 improve\u00C2\u00A0 EC\u00C2\u00A0 reduction,\u00C2\u00A0 we\u00C2\u00A0 created\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 that\u00C2\u00A0 acted\u00C2\u00A0 as\u00C2\u00A0 \u00E2\u0080\u0098urea\u00C2\u00A0 sponges\u00E2\u0080\u0099,\u00C2\u00A0 reabsorbing\u00C2\u00A0any\u00C2\u00A0urea\u00C2\u00A0secreted\u00C2\u00A0during\u00C2\u00A0fermentation\u00C2\u00A0or\u00C2\u00A0urea\u00C2\u00A0native\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0must.\u00C2\u00A0This\u00C2\u00A0goal\u00C2\u00A0was\u00C2\u00A0completed\u00C2\u00A0 through\u00C2\u00A0the\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0permease\u00C2\u00A0DUR3\u00C2\u00A0(YHL016C),\u00C2\u00A0a\u00C2\u00A0NCR\u00C2\u00A0sensitive\u00C2\u00A0gene\u00C2\u00A0native\u00C2\u00A0 to\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0(Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975;\u00C2\u00A0ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01993;\u00C2\u00A0Sumrada,\u00C2\u00A0Gorski\u00C2\u00A0and\u00C2\u00A0Cooper\u00C2\u00A01976).\u00C2\u00A0Due\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0abundance\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0high\u00C2\u00A0quality\u00C2\u00A0nitrogen\u00C2\u00A0sources\u00C2\u00A0during\u00C2\u00A0primary\u00C2\u00A0fermentation,\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0no\u00C2\u00A0need\u00C2\u00A0 for\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0actively\u00C2\u00A0 import\u00C2\u00A0urea,\u00C2\u00A0thus\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0transporter\u00C2\u00A0 is\u00C2\u00A0transcriptionally\u00C2\u00A0silenced.\u00C2\u00A0Given\u00C2\u00A0the\u00C2\u00A0 success\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0constitutively\u00C2\u00A0expressed\u00C2\u00A0DUR1,2\u00C2\u00A0cassette\u00C2\u00A0previously\u00C2\u00A0characterized\u00C2\u00A0by\u00C2\u00A0our\u00C2\u00A0group\u00C2\u00A0(Coulon,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006),\u00C2\u00A0 we\u00C2\u00A0 chose\u00C2\u00A0 to\u00C2\u00A0 utilize\u00C2\u00A0 similar\u00C2\u00A0 methodology\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 transcriptionally\u00C2\u00A0 activate\u00C2\u00A0 and\u00C2\u00A0 constitutively\u00C2\u00A0express\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0yeasts.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.3.1\u00C2\u00A0\u00C2\u00A0Construction\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0linear\u00C2\u00A0PGK1p\u00E2\u0080\u0090DUR3\u00E2\u0080\u0090PGK1t\u00E2\u0080\u0090kanMX\u00C2\u00A0cassette\u00C2\u00A0for\u00C2\u00A0integration\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0 of\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0yeasts\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 could\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0more\u00C2\u00A0 easily\u00C2\u00A0 achieved\u00C2\u00A0 through\u00C2\u00A0 an\u00C2\u00A0 episomal\u00C2\u00A0plasmid\u00C2\u00A0expression\u00C2\u00A0system,\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0for\u00C2\u00A0integration\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0genome\u00C2\u00A0 was\u00C2\u00A0a\u00C2\u00A0more\u00C2\u00A0attractive\u00C2\u00A0option.\u00C2\u00A0The\u00C2\u00A0main\u00C2\u00A0problem\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0plasmid\u00C2\u00A0borne\u00C2\u00A0system\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0without\u00C2\u00A0a\u00C2\u00A0positive\u00C2\u00A0 selection\u00C2\u00A0pressure,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0antibiotics\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0must,\u00C2\u00A0plasmid\u00C2\u00A0loss\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0generally\u00C2\u00A0 occurs\u00C2\u00A0within\u00C2\u00A03\u00E2\u0080\u00905\u00C2\u00A0generations\u00C2\u00A0(Jones,\u00C2\u00A0Pringle\u00C2\u00A0and\u00C2\u00A0Broach\u00C2\u00A01992)\u00C2\u00A0thereby\u00C2\u00A0reverting\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0to\u00C2\u00A0 wild\u00C2\u00A0 type.\u00C2\u00A0 Coupled\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 nutrient\u00C2\u00A0 prototrophy\u00C2\u00A0 of\u00C2\u00A0 industrial\u00C2\u00A0 yeasts,\u00C2\u00A0 positive\u00C2\u00A0 selection\u00C2\u00A0 during\u00C2\u00A0 winemaking\u00C2\u00A0 is\u00C2\u00A0extremely\u00C2\u00A0 impractical\u00C2\u00A0as\u00C2\u00A0grape\u00C2\u00A0must/rice\u00C2\u00A0mash\u00C2\u00A0 is\u00C2\u00A0essentially\u00C2\u00A0a\u00C2\u00A0 \u00E2\u0080\u0098rich\u00E2\u0080\u0099\u00C2\u00A0medium\u00C2\u00A0 for\u00C2\u00A0yeast\u00C2\u00A0 growth.\u00C2\u00A0Furthermore,\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0antibiotics\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 fermentation\u00C2\u00A0substrate\u00C2\u00A0 is\u00C2\u00A0undesirable\u00C2\u00A0and\u00C2\u00A0negates\u00C2\u00A0 the\u00C2\u00A0 goal\u00C2\u00A0 of\u00C2\u00A0 creating\u00C2\u00A0 \u00E2\u0080\u0098self\u00E2\u0080\u0090cloned\u00E2\u0080\u0099\u00C2\u00A0 yeast\u00C2\u00A0 strains.\u00C2\u00A0Additionally,\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 is\u00C2\u00A0 highly\u00C2\u00A0 amenable\u00C2\u00A0 to\u00C2\u00A0 gene\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 90\u00C2\u00A0 \u00C2\u00A0 uptake\u00C2\u00A0 and\u00C2\u00A0 integration\u00C2\u00A0 via\u00C2\u00A0 homologous\u00C2\u00A0 recombination\u00C2\u00A0 (Ausubel,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1995;\u00C2\u00A0 Griffiths\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005),\u00C2\u00A0 making\u00C2\u00A0precise\u00C2\u00A0manipulation,\u00C2\u00A0replacement,\u00C2\u00A0and\u00C2\u00A0deletion\u00C2\u00A0of\u00C2\u00A0genes\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0genome\u00C2\u00A0possible.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0a\u00C2\u00A0linear\u00C2\u00A0cassette\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0(Figure\u00C2\u00A025),\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0 ORF\u00C2\u00A0was\u00C2\u00A0placed\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals\u00C2\u00A0(Figure\u00C2\u00A010).\u00C2\u00A0 PGK1\u00C2\u00A0(YCR012W)\u00C2\u00A0encodes\u00C2\u00A03\u00E2\u0080\u0090phosphoglycerate\u00C2\u00A0kinase\u00C2\u00A0(EC\u00C2\u00A02.7.2.3)\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0enzyme\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0glycolytic\u00C2\u00A0 pathway\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0transferring\u00C2\u00A0the\u00C2\u00A0acyl\u00C2\u00A0phosphate\u00C2\u00A0from\u00C2\u00A01,3\u00E2\u0080\u0090bisphosphoglycerate\u00C2\u00A0to\u00C2\u00A0ADP\u00C2\u00A0thereby\u00C2\u00A0 creating\u00C2\u00A0one\u00C2\u00A0molecule\u00C2\u00A0of\u00C2\u00A0energy\u00C2\u00A0rich\u00C2\u00A0ATP\u00C2\u00A0(Blake\u00C2\u00A01981;\u00C2\u00A0Hitzeman\u00C2\u00A01980;\u00C2\u00A0Lam\u00C2\u00A01977).\u00C2\u00A0Being\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0enzyme\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0ubiquitous\u00C2\u00A0process\u00C2\u00A0of\u00C2\u00A0glycolysis,\u00C2\u00A0PGK1,\u00C2\u00A0while\u00C2\u00A0technically\u00C2\u00A0inducible,\u00C2\u00A0is\u00C2\u00A0constitutively\u00C2\u00A0expressed\u00C2\u00A0so\u00C2\u00A0long\u00C2\u00A0 as\u00C2\u00A0 yeast\u00C2\u00A0are\u00C2\u00A0 grown\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0glucose\u00C2\u00A0 (Lam\u00C2\u00A01977).\u00C2\u00A0During\u00C2\u00A0both\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0 Sake\u00C2\u00A0 fermentation\u00C2\u00A0 glucose\u00C2\u00A0is\u00C2\u00A0abundant\u00C2\u00A0and\u00C2\u00A0cells\u00C2\u00A0never\u00C2\u00A0experience\u00C2\u00A0carbon\u00C2\u00A0exhaustion\u00C2\u00A0(Marks,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02008;\u00C2\u00A0Wu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0 Additionally,\u00C2\u00A0as\u00C2\u00A0glycolysis\u00C2\u00A0is\u00C2\u00A0essential\u00C2\u00A0during\u00C2\u00A0fermentation\u00C2\u00A0(Lam\u00C2\u00A01977),\u00C2\u00A0placing\u00C2\u00A0DUR3\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0 PGK1\u00C2\u00A0ensured\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0throughout\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0fermentation.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0this\u00C2\u00A0section\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0research\u00C2\u00A0was\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0proof\u00C2\u00A0of\u00C2\u00A0concept\u00C2\u00A0study\u00C2\u00A0only,\u00C2\u00A0the\u00C2\u00A0antibiotic\u00C2\u00A0resistance\u00C2\u00A0 marker\u00C2\u00A0 kanMX\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 simplify\u00C2\u00A0 the\u00C2\u00A0 selection\u00C2\u00A0 of\u00C2\u00A0 positive\u00C2\u00A0 clones\u00C2\u00A0 (Figure\u00C2\u00A0 11).\u00C2\u00A0A\u00C2\u00A0 positive\u00C2\u00A0 selection\u00C2\u00A0marker\u00C2\u00A0allowed\u00C2\u00A0us\u00C2\u00A0to\u00C2\u00A0distinguish\u00C2\u00A0DUR3\u00C2\u00A0clones\u00C2\u00A0from\u00C2\u00A0untransformed\u00C2\u00A0cells\u00C2\u00A0thus\u00C2\u00A0eliminating\u00C2\u00A0the\u00C2\u00A0 need\u00C2\u00A0 for\u00C2\u00A0 costly\u00C2\u00A0 and\u00C2\u00A0 time\u00C2\u00A0 consuming\u00C2\u00A0 colony\u00C2\u00A0 PCR\u00C2\u00A0 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cassette\u00C2\u00A0was\u00C2\u00A0 flanked\u00C2\u00A0 on\u00C2\u00A0 either\u00C2\u00A0 side\u00C2\u00A0with\u00C2\u00A0 300\u00C2\u00A0 bp\u00C2\u00A0 of\u00C2\u00A0 TRP1\u00C2\u00A0 homology\u00C2\u00A0 and\u00C2\u00A0 successfully\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0(Figure\u00C2\u00A013).\u00C2\u00A0While\u00C2\u00A0homologous\u00C2\u00A0recombination\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 in\u00C2\u00A0 laboratory\u00C2\u00A0 strains\u00C2\u00A0with\u00C2\u00A0 as\u00C2\u00A0 few\u00C2\u00A0 as\u00C2\u00A0 40\u00C2\u00A0 nucleotides\u00C2\u00A0 of\u00C2\u00A0 flanking\u00C2\u00A0 homology\u00C2\u00A0 (Baudin\u00C2\u00A0 1993;\u00C2\u00A0 Manivasakam\u00C2\u00A0 1995),\u00C2\u00A0 we\u00C2\u00A0 used\u00C2\u00A0 300\u00C2\u00A0 homologous\u00C2\u00A0 nucleotides\u00C2\u00A0 on\u00C2\u00A0 either\u00C2\u00A0 end\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 to\u00C2\u00A0 ensure\u00C2\u00A0efficient\u00C2\u00A0and\u00C2\u00A0specific\u00C2\u00A0integration\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus.\u00C2\u00A0Shorter\u00C2\u00A0regions\u00C2\u00A0of\u00C2\u00A0homology\u00C2\u00A0increases\u00C2\u00A0the\u00C2\u00A0 likelihood\u00C2\u00A0 that\u00C2\u00A0 strain\u00C2\u00A0 sequence\u00C2\u00A0polymorphisms\u00C2\u00A0will\u00C2\u00A0 reduce\u00C2\u00A0 integration\u00C2\u00A0efficiency.\u00C2\u00A0Efficiencies\u00C2\u00A0 increase\u00C2\u00A0 30\u00E2\u0080\u0090\u00C2\u00A0to\u00C2\u00A050\u00E2\u0080\u0090fold\u00C2\u00A0when\u00C2\u00A0flanking\u00C2\u00A0sequences\u00C2\u00A0of\u00C2\u00A0several\u00C2\u00A0hundred\u00C2\u00A0base\u00C2\u00A0pairs\u00C2\u00A0in\u00C2\u00A0length\u00C2\u00A0are\u00C2\u00A0used\u00C2\u00A0(Wach\u00C2\u00A01996).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 91\u00C2\u00A0 \u00C2\u00A0 While\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0could\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0integrated\u00C2\u00A0into\u00C2\u00A0any\u00C2\u00A0locus,\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0locus\u00C2\u00A0was\u00C2\u00A0chosen\u00C2\u00A0 for\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 reasons.\u00C2\u00A0 Firstly,\u00C2\u00A0 TRP1\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 well\u00C2\u00A0 characterized\u00C2\u00A0 and\u00C2\u00A0 common\u00C2\u00A0 auxotrophic\u00C2\u00A0 marker\u00C2\u00A0 (Hampsey\u00C2\u00A01997;\u00C2\u00A0Mortimer\u00C2\u00A01966;\u00C2\u00A0Stolz\u00C2\u00A01998).\u00C2\u00A0Secondly,\u00C2\u00A0TRP1\u00C2\u00A0is\u00C2\u00A0closely\u00C2\u00A0located\u00C2\u00A0(1\u00C2\u00A0cM)\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0centromere\u00C2\u00A0 of\u00C2\u00A0chromosome\u00C2\u00A0four\u00C2\u00A0(Mortimer\u00C2\u00A01966)\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0thus\u00C2\u00A0highly\u00C2\u00A0stable,\u00C2\u00A0as\u00C2\u00A0genetic\u00C2\u00A0recombination\u00C2\u00A0during\u00C2\u00A0meiosis\u00C2\u00A0 is\u00C2\u00A0 proportional\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 locus\u00E2\u0080\u0099\u00C2\u00A0 distance\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 centromere\u00C2\u00A0 (Griffiths,\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005).\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 a\u00C2\u00A0 result\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 genetic\u00C2\u00A0stability,\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0suitable\u00C2\u00A0for\u00C2\u00A0active\u00C2\u00A0dry\u00C2\u00A0yeast\u00C2\u00A0production\u00C2\u00A0and\u00C2\u00A0industrial\u00C2\u00A0use.\u00C2\u00A0 \u00C2\u00A0 Prior\u00C2\u00A0to\u00C2\u00A0 integration\u00C2\u00A0 into\u00C2\u00A0test\u00C2\u00A0strains,\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasmid\u00C2\u00A0pUCMD\u00C2\u00A0was\u00C2\u00A0sequenced\u00C2\u00A0 (single\u00C2\u00A0strand)\u00C2\u00A0to\u00C2\u00A0confirm\u00C2\u00A0its\u00C2\u00A0structure;\u00C2\u00A0analysis\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0plasmid\u00C2\u00A0contained\u00C2\u00A0all\u00C2\u00A0the\u00C2\u00A0desired\u00C2\u00A0DNA\u00C2\u00A0 fragments\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0correct\u00C2\u00A0order\u00C2\u00A0and\u00C2\u00A0orientation\u00C2\u00A0(Figure\u00C2\u00A014).\u00C2\u00A0Furthermore,\u00C2\u00A0sequencing\u00C2\u00A0data\u00C2\u00A0revealed\u00C2\u00A0nine\u00C2\u00A0 single\u00C2\u00A0 nucleotide\u00C2\u00A0 changes\u00C2\u00A0 along\u00C2\u00A0 the\u00C2\u00A0 length\u00C2\u00A0 of\u00C2\u00A0 cassette\u00C2\u00A0 (Table\u00C2\u00A0 14\u00C2\u00A0 and\u00C2\u00A0 Figure\u00C2\u00A0 23);\u00C2\u00A0 however,\u00C2\u00A0 all\u00C2\u00A0 four\u00C2\u00A0 mismatches\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0coding\u00C2\u00A0region\u00C2\u00A0were\u00C2\u00A0silent\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0deduced\u00C2\u00A0DUR3p\u00C2\u00A0was\u00C2\u00A0identical\u00C2\u00A0in\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0 sequence\u00C2\u00A0 and\u00C2\u00A0 length\u00C2\u00A0 to\u00C2\u00A0 that\u00C2\u00A0 published\u00C2\u00A0 for\u00C2\u00A0 DUR3p\u00C2\u00A0 on\u00C2\u00A0 SGD.\u00C2\u00A0 Five\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 nine\u00C2\u00A0 base\u00C2\u00A0 pair\u00C2\u00A0mismatches\u00C2\u00A0 localized\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0PGK1\u00C2\u00A0promoter;\u00C2\u00A0 four\u00C2\u00A0were\u00C2\u00A0C\u00C2\u00A0 to\u00C2\u00A0T\u00C2\u00A0 conversions.\u00C2\u00A0Given\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 silico\u00C2\u00A0 sequence\u00C2\u00A0was\u00C2\u00A0 assembled\u00C2\u00A0from\u00C2\u00A0single\u00C2\u00A0pass,\u00C2\u00A0single\u00C2\u00A0strand\u00C2\u00A0sequencing,\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0logical\u00C2\u00A0explanation\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0mismatches\u00C2\u00A0is\u00C2\u00A0 sequencing\u00C2\u00A0error.\u00C2\u00A0This\u00C2\u00A0conclusion\u00C2\u00A0is\u00C2\u00A0supported\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0none\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0identified\u00C2\u00A0mismatches\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 were\u00C2\u00A0 previously\u00C2\u00A0 reported\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 utilization\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0 for\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 (Coulon,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006;\u00C2\u00A0 Husnik,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006;\u00C2\u00A0 Volschenk,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1997).\u00C2\u00A0 Furthermore,\u00C2\u00A0 during\u00C2\u00A0 characterization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0ML01\u00C2\u00A0(Husnik,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006),\u00C2\u00A0two\u00C2\u00A0mismatches\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0found\u00C2\u00A0 in\u00C2\u00A0one\u00C2\u00A0 copy\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 PGK1\u00C2\u00A0 promoter\u00C2\u00A0were\u00C2\u00A0 absent\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 copy\u00C2\u00A0 (Husnik,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006),\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 sequence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0prone\u00C2\u00A0 to\u00C2\u00A0 sequencing\u00C2\u00A0errors.\u00C2\u00A0Given\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0was\u00C2\u00A0 fully\u00C2\u00A0functional,\u00C2\u00A0further\u00C2\u00A0sequencing\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cassette\u00C2\u00A0to\u00C2\u00A0 identify\u00C2\u00A0bona\u00C2\u00A0fide\u00C2\u00A0mismatches\u00C2\u00A0was\u00C2\u00A0 deemed\u00C2\u00A0non\u00E2\u0080\u0090essential.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.3.2\u00C2\u00A0 \u00C2\u00A0 Integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 genomes\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 ,\u00C2\u00A0522,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0 yielded\u00C2\u00A0 the\u00C2\u00A0 functional\u00C2\u00A0urea\u00C2\u00A0transporting\u00C2\u00A0yeasts\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Like\u00C2\u00A0DUR1,2,\u00C2\u00A0DUR3\u00C2\u00A0is\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0transcriptional\u00C2\u00A0repression\u00C2\u00A0by\u00C2\u00A0NCR\u00C2\u00A0during\u00C2\u00A0fermentation,\u00C2\u00A0resulting\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 inability\u00C2\u00A0 of\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 to\u00C2\u00A0 re\u00E2\u0080\u0090absorb\u00C2\u00A0 excreted\u00C2\u00A0 urea\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 good\u00C2\u00A0 nitrogen\u00C2\u00A0 sources\u00C2\u00A0 (ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01993;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0This\u00C2\u00A0inability\u00C2\u00A0to\u00C2\u00A0absorb\u00C2\u00A0excreted\u00C2\u00A0urea\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0contributing\u00C2\u00A0factor\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0wines\u00C2\u00A0with\u00C2\u00A0 high\u00C2\u00A0 residual\u00C2\u00A0 urea,\u00C2\u00A0 thus\u00C2\u00A0 leading\u00C2\u00A0 to\u00C2\u00A0 high\u00C2\u00A0 EC.\u00C2\u00A0 In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 constitutively\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 92\u00C2\u00A0 \u00C2\u00A0 express\u00C2\u00A0DUR3\u00C2\u00A0 throughout\u00C2\u00A0 fermentation,\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 (Figure\u00C2\u00A025)\u00C2\u00A0was\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 TRP1\u00C2\u00A0 locus\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 ,\u00C2\u00A0522,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0which\u00C2\u00A0yielded\u00C2\u00A0the\u00C2\u00A0functionally\u00C2\u00A0enhanced\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 (Table\u00C2\u00A0 16).\u00C2\u00A0 The\u00C2\u00A0 enhanced\u00C2\u00A0 strains\u00C2\u00A0 were\u00C2\u00A0 genetically,\u00C2\u00A0 phenotypically,\u00C2\u00A0 and\u00C2\u00A0 functionally\u00C2\u00A0 characterized.\u00C2\u00A0 \u00C2\u00A0 4.3.2.1\u00C2\u00A0\u00C2\u00A0Integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genomes\u00C2\u00A0of\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3.\u00C2\u00A0Correct\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0TRP1\u00C2\u00A0 locus\u00C2\u00A0was\u00C2\u00A0 confirmed\u00C2\u00A0by\u00C2\u00A0Southern\u00C2\u00A0blots\u00C2\u00A0probed\u00C2\u00A0with\u00C2\u00A0both\u00C2\u00A0DUR3\u00C2\u00A0and\u00C2\u00A0TRP1\u00C2\u00A0fragments.\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090 D3,\u00C2\u00A0 522D3,\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0 all\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 contain\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 copy\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ~6.5\u00C2\u00A0 kb\u00C2\u00A0 linear\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0TRP1\u00C2\u00A0 loci\u00C2\u00A0 (Figure\u00C2\u00A026).\u00C2\u00A0Blotting\u00C2\u00A0also\u00C2\u00A0 confirmed\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0diploid\u00C2\u00A0 strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0retained\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090disrupted\u00C2\u00A0TRP1\u00C2\u00A0locus,\u00C2\u00A0thus\u00C2\u00A0maintaining\u00C2\u00A0their\u00C2\u00A0tryptophan\u00C2\u00A0prototrophy\u00C2\u00A0 and\u00C2\u00A0wild\u00C2\u00A0type\u00C2\u00A0phenotype.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0cassette,\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 is\u00C2\u00A0controlled\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0 terminator\u00C2\u00A0 signals.\u00C2\u00A0 As\u00C2\u00A0 was\u00C2\u00A0 previously\u00C2\u00A0 observed\u00C2\u00A0 (Section\u00C2\u00A0 4.2.1)\u00C2\u00A0 during\u00C2\u00A0 the\u00C2\u00A0 characterization\u00C2\u00A0 of\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0 clones,\u00C2\u00A0PGK1\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0strong\u00C2\u00A0promoter\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0driving\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0under\u00C2\u00A0NCR\u00C2\u00A0conditions.\u00C2\u00A0To\u00C2\u00A0 confirm\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette,\u00C2\u00A0a\u00C2\u00A0northern\u00C2\u00A0blot\u00C2\u00A0was\u00C2\u00A0 hybridized\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0DUR3\u00C2\u00A0probe;\u00C2\u00A0blots\u00C2\u00A0of\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0total\u00C2\u00A0RNA\u00C2\u00A0revealed\u00C2\u00A0strong\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0in\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0(Figure\u00C2\u00A029).\u00C2\u00A0RNA\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0northern\u00C2\u00A0blot\u00C2\u00A0was\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0cells\u00C2\u00A024\u00C2\u00A0hours\u00C2\u00A0into\u00C2\u00A0a\u00C2\u00A0 fermentation\u00C2\u00A0of\u00C2\u00A0Chardonnay\u00C2\u00A0must;\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0hours,\u00C2\u00A0quality\u00C2\u00A0nitrogen\u00C2\u00A0sources\u00C2\u00A0are\u00C2\u00A0still\u00C2\u00A0abundant\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0NCR\u00C2\u00A0 is\u00C2\u00A0still\u00C2\u00A0strong.\u00C2\u00A0Thus,\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0and\u00C2\u00A0terminator\u00C2\u00A0signals,\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0 high\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0despite\u00C2\u00A0non\u00E2\u0080\u0090inducing\u00C2\u00A0conditions.\u00C2\u00A0Quantification\u00C2\u00A0of\u00C2\u00A0 constitutive\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090PCR\u00C2\u00A0revealed\u00C2\u00A0a\u00C2\u00A014\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A07\u00E2\u0080\u0090fold\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0DUR3\u00C2\u00A0mRNA\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0hours\u00C2\u00A0 into\u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0 fermentations\u00C2\u00A0 in\u00C2\u00A0K7D3\u00C2\u00A0 and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 respectively\u00C2\u00A0 (Figure\u00C2\u00A030).\u00C2\u00A0 Furthermore,\u00C2\u00A0high\u00C2\u00A0 level\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0was\u00C2\u00A0maintained\u00C2\u00A0in\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0(12\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A07\u00E2\u0080\u0090fold,\u00C2\u00A0respectively),\u00C2\u00A0which\u00C2\u00A0had\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0 DUR3\u00C2\u00A0 cassettes\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 its\u00C2\u00A0 genome.\u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 K7,\u00C2\u00A0 and\u00C2\u00A0 presumably\u00C2\u00A0 other\u00C2\u00A0 strains\u00C2\u00A0 of\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae,\u00C2\u00A0 is\u00C2\u00A0 tolerant\u00C2\u00A0 to\u00C2\u00A0 altered,\u00C2\u00A0 high\u00C2\u00A0 level\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 multiple\u00C2\u00A0 proteins.\u00C2\u00A0Coupled\u00C2\u00A0with\u00C2\u00A0data\u00C2\u00A0confirming\u00C2\u00A0the\u00C2\u00A0functionality\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0DUR1,2p\u00C2\u00A0(Table\u00C2\u00A021)\u00C2\u00A0and\u00C2\u00A0DUR3p\u00C2\u00A0(Figure\u00C2\u00A0 31),\u00C2\u00A0this\u00C2\u00A0result\u00C2\u00A0validates\u00C2\u00A0the\u00C2\u00A0metabolic\u00C2\u00A0engineering\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0for\u00C2\u00A0polygenic\u00C2\u00A0traits.\u00C2\u00A0In\u00C2\u00A0strains\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0 only\u00C2\u00A0 one\u00C2\u00A0 cassette\u00C2\u00A0 (DUR1,2\u00C2\u00A0 or\u00C2\u00A0 DUR3)\u00C2\u00A0 was\u00C2\u00A0 integrated,\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 that\u00C2\u00A0 gene\u00C2\u00A0 induced\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0gene\u00C2\u00A0(Figure\u00C2\u00A030).\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0indicate\u00C2\u00A0a\u00C2\u00A0certain\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0cross\u00C2\u00A0talk\u00C2\u00A0between\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 93\u00C2\u00A0 \u00C2\u00A0 the\u00C2\u00A0 regulatory\u00C2\u00A0mechanisms\u00C2\u00A0 for\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3.\u00C2\u00A0Presumably\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0actively\u00C2\u00A0degrading\u00C2\u00A0urea\u00C2\u00A0 instead\u00C2\u00A0 of\u00C2\u00A0 exporting\u00C2\u00A0 it\u00C2\u00A0 (constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR1,2),\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 degradation\u00C2\u00A0 intermediate,\u00C2\u00A0 allophanate,\u00C2\u00A0causes\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0DUR3.\u00C2\u00A0Allophanate\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0known\u00C2\u00A0inducer\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0DUR\u00C2\u00A0genes\u00C2\u00A0(Cooper\u00C2\u00A01982;\u00C2\u00A0 Cooper\u00C2\u00A02002;\u00C2\u00A0Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975;\u00C2\u00A0ElBerry,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01993;\u00C2\u00A0Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999;\u00C2\u00A0Uemura,\u00C2\u00A0Kashiwagi\u00C2\u00A0 and\u00C2\u00A0 Igarashi\u00C2\u00A0 2007;\u00C2\u00A0Whitney\u00C2\u00A0 and\u00C2\u00A0 Cooper\u00C2\u00A0 1972;\u00C2\u00A0Whitney,\u00C2\u00A0 Cooper\u00C2\u00A0 and\u00C2\u00A0Magasanik\u00C2\u00A0 1973)\u00C2\u00A0 and\u00C2\u00A0 exerts\u00C2\u00A0 its\u00C2\u00A0 effect\u00C2\u00A0 through\u00C2\u00A0 an\u00C2\u00A0 upstream\u00C2\u00A0 induction\u00C2\u00A0 sequence\u00C2\u00A0 that\u00C2\u00A0 binds\u00C2\u00A0 the\u00C2\u00A0 transcriptional\u00C2\u00A0 activators\u00C2\u00A0 DAL81\u00C2\u00A0 and\u00C2\u00A0 DAL82,\u00C2\u00A0and\u00C2\u00A0through\u00C2\u00A0an\u00C2\u00A0upstream\u00C2\u00A0activation\u00C2\u00A0sequence\u00C2\u00A0that\u00C2\u00A0binds\u00C2\u00A0the\u00C2\u00A0NCR\u00C2\u00A0GATA\u00C2\u00A0factor\u00C2\u00A0GLN3\u00C2\u00A0(Hofman\u00E2\u0080\u0090 Bang\u00C2\u00A0 1999).\u00C2\u00A0 Similarly,\u00C2\u00A0when\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 actively\u00C2\u00A0 importing\u00C2\u00A0 urea\u00C2\u00A0 (constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR3),\u00C2\u00A0 the\u00C2\u00A0 increased\u00C2\u00A0 intracellular\u00C2\u00A0 urea\u00C2\u00A0 concentration\u00C2\u00A0 induces\u00C2\u00A0 DUR1,2\u00C2\u00A0 expression\u00C2\u00A0 such\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 intracellular\u00C2\u00A0 concentration\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0lowered\u00C2\u00A0before\u00C2\u00A0it\u00C2\u00A0becomes\u00C2\u00A0toxic.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0patterns\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0strain\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0parent\u00C2\u00A0 strain\u00C2\u00A0 K7\u00C2\u00A0 were\u00C2\u00A0 studied\u00C2\u00A0 at\u00C2\u00A0 24\u00C2\u00A0 hours\u00C2\u00A0 into\u00C2\u00A0 Chardonnay\u00C2\u00A0must\u00C2\u00A0 fermentation.\u00C2\u00A0 Besides\u00C2\u00A0 DUR3\u00C2\u00A0 (36.95\u00E2\u0080\u0090fold\u00C2\u00A0 overexpression),\u00C2\u00A0 seven\u00C2\u00A0 genes\u00C2\u00A0were\u00C2\u00A0affected\u00C2\u00A0 \u00E2\u0089\u00A5\u00C2\u00A04\u00E2\u0080\u0090fold;\u00C2\u00A0 thus,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0evident\u00C2\u00A0 that\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0genome\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0K7\u00C2\u00A0had\u00C2\u00A0a\u00C2\u00A0minimal\u00C2\u00A0effect\u00C2\u00A0(0.1%\u00C2\u00A0change)\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcription\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A05795\u00C2\u00A0ORFs\u00C2\u00A0(4692\u00C2\u00A0verified\u00C2\u00A0and\u00C2\u00A01103\u00C2\u00A0uncharacterized,\u00C2\u00A0SGD,\u00C2\u00A0March,\u00C2\u00A02008)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0cell.\u00C2\u00A0One\u00C2\u00A0gene\u00C2\u00A0 that\u00C2\u00A0was\u00C2\u00A0 upregulated\u00C2\u00A0 \u00E2\u0089\u00A5\u00C2\u00A0 4\u00E2\u0080\u0090fold\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 (Table\u00C2\u00A0 17)\u00C2\u00A0was\u00C2\u00A0 common\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0 upregulated\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette\u00C2\u00A0containing\u00C2\u00A0engineered\u00C2\u00A0 strain\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 (Table\u00C2\u00A010);\u00C2\u00A0HAC1\u00C2\u00A0 (5.67\u00E2\u0080\u0090fold)\u00C2\u00A0encodes\u00C2\u00A0a\u00C2\u00A0 transcription\u00C2\u00A0 factor\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0unfolded\u00C2\u00A0protein\u00C2\u00A0response\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0needed\u00C2\u00A0for\u00C2\u00A0 increased\u00C2\u00A0translation\u00C2\u00A0and\u00C2\u00A0folding\u00C2\u00A0of\u00C2\u00A0 DUR3\u00C2\u00A0constitutively\u00C2\u00A0expressed\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0strong\u00C2\u00A0PGK1\u00C2\u00A0promoter.\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0also\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0no\u00C2\u00A0metabolic\u00C2\u00A0 pathways\u00C2\u00A0 were\u00C2\u00A0 affected\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 K7EC\u00E2\u0080\u0090.\u00C2\u00A0 However,\u00C2\u00A0 integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 into\u00C2\u00A0 K7D3\u00C2\u00A0 appears\u00C2\u00A0 to\u00C2\u00A0 have\u00C2\u00A0 had\u00C2\u00A0 some\u00C2\u00A0 effect\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 meiosis/sporulation,\u00C2\u00A0processes\u00C2\u00A0 controlled\u00C2\u00A0by\u00C2\u00A0nutrient\u00C2\u00A0deficiency\u00C2\u00A0 (Malone\u00C2\u00A01990).\u00C2\u00A0One\u00C2\u00A0gene\u00C2\u00A0 (FIG1)\u00C2\u00A0was\u00C2\u00A0 upregulated\u00C2\u00A0 and\u00C2\u00A0 one\u00C2\u00A0 gene\u00C2\u00A0 (TID3)\u00C2\u00A0 was\u00C2\u00A0 downregulated\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 (Table\u00C2\u00A0 17);\u00C2\u00A0 both\u00C2\u00A0 are\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 meiosis/sporulation.\u00C2\u00A0As\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 increased\u00C2\u00A0nitrogen\u00C2\u00A0 availability\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 reasonable\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0would\u00C2\u00A0exert\u00C2\u00A0some\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0sporulation\u00C2\u00A0gene\u00C2\u00A0regulation.\u00C2\u00A0 \u00C2\u00A0 4.3.2.2\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 integrated\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 enhanced\u00C2\u00A0 urea\u00C2\u00A0 uptake.\u00C2\u00A0 In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 confirm\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 functional\u00C2\u00A0urea\u00C2\u00A0permease\u00C2\u00A0encoded\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette,\u00C2\u00A0and\u00C2\u00A0 to\u00C2\u00A0correlate\u00C2\u00A0 DUR3\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0with\u00C2\u00A0increased\u00C2\u00A0urea\u00C2\u00A0uptake,\u00C2\u00A0the\u00C2\u00A0uptake\u00C2\u00A0of\u00C2\u00A0radiolabelled\u00C2\u00A0urea\u00C2\u00A0by\u00C2\u00A0K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0studied.\u00C2\u00A0Under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0tested,\u00C2\u00A0the\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0both\u00C2\u00A0highly\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 94\u00C2\u00A0 \u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0importing\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0while\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0incorporate\u00C2\u00A0any\u00C2\u00A0appreciable\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 (Figure\u00C2\u00A031).\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 integration\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DUR3\u00C2\u00A0 cassette\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0active\u00C2\u00A0urea\u00C2\u00A0permease\u00C2\u00A0(DUR3p)\u00C2\u00A0under\u00C2\u00A0NCR\u00C2\u00A0conditions.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0In\u00C2\u00A0 the\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 uptake\u00C2\u00A0 assay,\u00C2\u00A0 the\u00C2\u00A0 strains\u00C2\u00A0 K7,\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0 K7D3,\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 in\u00C2\u00A0 an\u00C2\u00A0 ammonium\u00C2\u00A0sulphate\u00C2\u00A0minimal\u00C2\u00A0medium\u00C2\u00A0that\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0repression\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0NCR\u00C2\u00A0sensitive\u00C2\u00A0genes\u00C2\u00A0(Cooper\u00C2\u00A01982;\u00C2\u00A0 Hofman\u00E2\u0080\u0090Bang\u00C2\u00A01999).\u00C2\u00A0In\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0strains\u00C2\u00A0(K7,\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3)\u00C2\u00A0transcription\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0from\u00C2\u00A0its\u00C2\u00A0native\u00C2\u00A0 promoter\u00C2\u00A0was\u00C2\u00A0repressed\u00C2\u00A0due\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0ammonium\u00C2\u00A0sulphate;\u00C2\u00A0however\u00C2\u00A0in\u00C2\u00A0strains\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0 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capable\u00C2\u00A0of\u00C2\u00A0degrading\u00C2\u00A0urea\u00C2\u00A0as\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 incorporated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 cell.\u00C2\u00A0 Thus,\u00C2\u00A0both\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0likely\u00C2\u00A0incorporate\u00C2\u00A0urea\u00C2\u00A0with\u00C2\u00A0equivalent\u00C2\u00A0efficiencies,\u00C2\u00A0however,\u00C2\u00A0this\u00C2\u00A0conclusion\u00C2\u00A0 was\u00C2\u00A0 not\u00C2\u00A0 confirmed\u00C2\u00A0 by\u00C2\u00A0 experimental\u00C2\u00A0 data.\u00C2\u00A0 The\u00C2\u00A0 same\u00C2\u00A0 masking\u00C2\u00A0 phenomenon\u00C2\u00A0 was\u00C2\u00A0 observed\u00C2\u00A0 when\u00C2\u00A0 comparing\u00C2\u00A0the\u00C2\u00A0uptake\u00C2\u00A0of\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0by\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(Figure\u00C2\u00A032).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.3.2.3\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0yeasts\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0ferment\u00C2\u00A0at\u00C2\u00A0similar\u00C2\u00A0rates\u00C2\u00A0 and\u00C2\u00A0produce\u00C2\u00A0 similar\u00C2\u00A0 amounts\u00C2\u00A0of\u00C2\u00A0 ethanol\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0 and\u00C2\u00A0 Sake\u00C2\u00A0wine.\u00C2\u00A0As\u00C2\u00A0measures\u00C2\u00A0of\u00C2\u00A0 substantial\u00C2\u00A0 equivalence,\u00C2\u00A0fermentation\u00C2\u00A0rate\u00C2\u00A0and\u00C2\u00A0ethanol\u00C2\u00A0production\u00C2\u00A0was\u00C2\u00A0evaluated\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0 strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3.\u00C2\u00A0 In\u00C2\u00A0both\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0mash,\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 95\u00C2\u00A0 \u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0 conducted\u00C2\u00A0 substantially\u00C2\u00A0 equivalent\u00C2\u00A0 alcoholic\u00C2\u00A0 fermentations\u00C2\u00A0 (Figures\u00C2\u00A0 35\u00C2\u00A0 and\u00C2\u00A0 36).\u00C2\u00A0 Furthermore,\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0ethanol\u00C2\u00A0produced\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0 522EC\u00E2\u0080\u0090D3\u00C2\u00A0was\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0similar\u00C2\u00A0(Tables\u00C2\u00A018\u00C2\u00A0and\u00C2\u00A019).\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0 and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0strains\u00C2\u00A0are\u00C2\u00A0suitable\u00C2\u00A0for\u00C2\u00A0commercialization\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0fermentation\u00C2\u00A0standpoint.\u00C2\u00A0 \u00C2\u00A0 4.3.2.4\u00C2\u00A0\u00C2\u00A0Variability\u00C2\u00A0of\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0yeasts\u00C2\u00A0to\u00C2\u00A0effectively\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0potential\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0 and\u00C2\u00A0522EC\u00E2\u0080\u0090D3,\u00C2\u00A0fermentations\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0Chardonnay\u00C2\u00A0must\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0were\u00C2\u00A0conducted\u00C2\u00A0after\u00C2\u00A0which\u00C2\u00A0 the\u00C2\u00A0EC\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0wines\u00C2\u00A0was\u00C2\u00A0quantified.\u00C2\u00A0Consistent\u00C2\u00A0with\u00C2\u00A0prior\u00C2\u00A0observations\u00C2\u00A0of\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0(Section\u00C2\u00A0 4.2.2),\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 by\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0was\u00C2\u00A0 inefficient\u00C2\u00A0 (6.85%\u00C2\u00A0 \u00E2\u0080\u0090\u00C2\u00A0 Table\u00C2\u00A0 20)\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 low\u00C2\u00A0 EC\u00C2\u00A0 production\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0parental\u00C2\u00A0 strain\u00C2\u00A0K7.\u00C2\u00A0As\u00C2\u00A0expected,\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0with\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0 yielded\u00C2\u00A0highly\u00C2\u00A0 efficient\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 (87%\u00C2\u00A0 \u00E2\u0080\u0090\u00C2\u00A0 Table\u00C2\u00A0 21).\u00C2\u00A0 Furthermore,\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 was\u00C2\u00A0 effective\u00C2\u00A0 at\u00C2\u00A0 reducing\u00C2\u00A0 EC\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 Chardonnay\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0(81%\u00C2\u00A0and\u00C2\u00A084%,\u00C2\u00A0respectively\u00C2\u00A0\u00E2\u0080\u0090\u00C2\u00A0Tables\u00C2\u00A020\u00C2\u00A0and\u00C2\u00A021).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0Chardonnay\u00C2\u00A0wine,\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0 reduced\u00C2\u00A0EC\u00C2\u00A0as\u00C2\u00A0efficiently\u00C2\u00A0as\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090,\u00C2\u00A0 respectively\u00C2\u00A0 (Table\u00C2\u00A020);\u00C2\u00A0both\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0~10%\u00C2\u00A0while\u00C2\u00A0522D3\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0both\u00C2\u00A0reduced\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A0~81%.\u00C2\u00A0The\u00C2\u00A0 observed\u00C2\u00A0equivalency\u00C2\u00A0in\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0is\u00C2\u00A0likely\u00C2\u00A0a\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0need\u00C2\u00A0for\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0degrade\u00C2\u00A0urea\u00C2\u00A0once\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 internalized,\u00C2\u00A0 as\u00C2\u00A0 urea\u00C2\u00A0 is\u00C2\u00A0 toxic\u00C2\u00A0 to\u00C2\u00A0 cells\u00C2\u00A0 at\u00C2\u00A0 high\u00C2\u00A0 concentrations.\u00C2\u00A0 Presumably,\u00C2\u00A0 the\u00C2\u00A0 3.6\u00E2\u0080\u0090fold\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 DUR1,2\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 (Figure\u00C2\u00A0 30),\u00C2\u00A0 coupled\u00C2\u00A0 with\u00C2\u00A0 constitutive\u00C2\u00A0 urea\u00C2\u00A0 import,\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 strain\u00E2\u0080\u0099s\u00C2\u00A0ability\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0EC\u00C2\u00A0as\u00C2\u00A0efficiently\u00C2\u00A0as\u00C2\u00A0K7EC\u00E2\u0080\u0090.\u00C2\u00A0Despite\u00C2\u00A0 the\u00C2\u00A0 lack\u00C2\u00A0of\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0data,\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0 rationale\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A0522D3.\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0 that\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0522\u00C2\u00A0are\u00C2\u00A0highly\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0genes\u00C2\u00A0involved\u00C2\u00A0in\u00C2\u00A0urea\u00C2\u00A0metabolism\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 engineering\u00C2\u00A0 yeasts\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 large\u00C2\u00A0 scale\u00C2\u00A0 expression\u00C2\u00A0 changes\u00C2\u00A0 (greater\u00C2\u00A0 than\u00C2\u00A0 5\u00E2\u0080\u0090fold)\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 unnecessary.\u00C2\u00A0 Furthermore,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 are\u00C2\u00A0 important\u00C2\u00A0 because\u00C2\u00A0 they\u00C2\u00A0 validate\u00C2\u00A0 the\u00C2\u00A0 application\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 constitutive\u00C2\u00A0expression\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0wine,\u00C2\u00A0 specifically\u00C2\u00A0 in\u00C2\u00A0wine\u00C2\u00A0derived\u00C2\u00A0 from\u00C2\u00A0musts\u00C2\u00A0 with\u00C2\u00A0high\u00C2\u00A0endogenous\u00C2\u00A0urea.\u00C2\u00A0In\u00C2\u00A0such\u00C2\u00A0cases,\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0would\u00C2\u00A0 likely\u00C2\u00A0be\u00C2\u00A0greatest\u00C2\u00A0 in\u00C2\u00A0wine\u00C2\u00A0fermented\u00C2\u00A0by\u00C2\u00A0 urea\u00C2\u00A0importing\u00C2\u00A0yeasts\u00C2\u00A0(DUR3\u00C2\u00A0cassette)\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0yeasts\u00C2\u00A0(DUR1,2\u00C2\u00A0cassette).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 Sake\u00C2\u00A0 wine,\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 were\u00C2\u00A0 much\u00C2\u00A0 less\u00C2\u00A0 effective\u00C2\u00A0 in\u00C2\u00A0 reducing\u00C2\u00A0 EC\u00C2\u00A0 than\u00C2\u00A0 their\u00C2\u00A0 DUR1,2\u00C2\u00A0 counterparts\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0(Table\u00C2\u00A021).\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0explained\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0requirements\u00C2\u00A0for\u00C2\u00A0DUR1,2\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0(Figure\u00C2\u00A037).\u00C2\u00A0In\u00C2\u00A0a\u00C2\u00A0system\u00C2\u00A0analogous\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0well\u00C2\u00A0characterized\u00C2\u00A0 lac\u00C2\u00A0operon\u00C2\u00A0 in\u00C2\u00A0E.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 96\u00C2\u00A0 \u00C2\u00A0 coli,\u00C2\u00A0 NCR\u00C2\u00A0 in\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 functions\u00C2\u00A0 to\u00C2\u00A0 force\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 metabolize\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 energetically\u00C2\u00A0 favourable\u00C2\u00A0 nutrients.\u00C2\u00A0As\u00C2\u00A0such,\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0regulated\u00C2\u00A0genes\u00C2\u00A0is\u00C2\u00A0highly\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0two\u00C2\u00A0conditions\u00C2\u00A0being\u00C2\u00A0met:\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0 inducer\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0NCR\u00C2\u00A0associated\u00C2\u00A0transcriptional\u00C2\u00A0activator\u00C2\u00A0 like\u00C2\u00A0GLN3.\u00C2\u00A0 Indeed,\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0was\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0highly\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0GLN3\u00C2\u00A0under\u00C2\u00A0 induced\u00C2\u00A0 conditions\u00C2\u00A0 (Cooper,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01990).\u00C2\u00A0Likewise,\u00C2\u00A0DUR1,2\u00C2\u00A0expression\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0proline\u00C2\u00A0medium\u00C2\u00A0(no\u00C2\u00A0NCR\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0GLN3\u00C2\u00A0active)\u00C2\u00A0was\u00C2\u00A0very\u00C2\u00A0 low\u00C2\u00A0 without\u00C2\u00A0 an\u00C2\u00A0 inducer\u00C2\u00A0 present\u00C2\u00A0 (Cooper,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1990).\u00C2\u00A0 As\u00C2\u00A0 discussed\u00C2\u00A0 previously\u00C2\u00A0 (Section\u00C2\u00A0 4.2.3),\u00C2\u00A0 fermentations\u00C2\u00A0 of\u00C2\u00A0 grape\u00C2\u00A0must\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 subject\u00C2\u00A0 to\u00C2\u00A0 nitrogen\u00C2\u00A0 exhaustion\u00C2\u00A0 and\u00C2\u00A0 thus\u00C2\u00A0 yeasts\u00C2\u00A0may\u00C2\u00A0 undergo\u00C2\u00A0 transcriptional\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0 sensitive\u00C2\u00A0genes\u00C2\u00A0 i.e.\u00C2\u00A0GLN3\u00C2\u00A0becomes\u00C2\u00A0activated.\u00C2\u00A0During\u00C2\u00A0Chardonnay\u00C2\u00A0 must\u00C2\u00A0 fermentation\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0constitutively\u00C2\u00A0 imported\u00C2\u00A0urea\u00C2\u00A0 thus\u00C2\u00A0 fulfilling\u00C2\u00A0 the\u00C2\u00A0 inducer\u00C2\u00A0 requirement\u00C2\u00A0 for\u00C2\u00A0DUR1,2\u00C2\u00A0expression.\u00C2\u00A0Upon\u00C2\u00A0nitrogen\u00C2\u00A0 limitation,\u00C2\u00A0 transcriptional\u00C2\u00A0 reprogramming\u00C2\u00A0 likely\u00C2\u00A0activated\u00C2\u00A0GLN3\u00C2\u00A0 and,\u00C2\u00A0 because\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 urea,\u00C2\u00A0 DUR1,2\u00C2\u00A0was\u00C2\u00A0 induced\u00C2\u00A0 enabling\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 to\u00C2\u00A0 degrade\u00C2\u00A0urea\u00C2\u00A0and\u00C2\u00A0reduce\u00C2\u00A0EC.\u00C2\u00A0 In\u00C2\u00A0contrast,\u00C2\u00A0 fermentations\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0nitrogen\u00C2\u00A0 exhaustion\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0koji\u00C2\u00A0enzymes\u00C2\u00A0and\u00C2\u00A0 rice\u00C2\u00A0protein\u00C2\u00A0bodies.\u00C2\u00A0Although\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0still\u00C2\u00A0 constitutively\u00C2\u00A0 imported\u00C2\u00A0 urea\u00C2\u00A0 during\u00C2\u00A0 Sake\u00C2\u00A0 fermentations,\u00C2\u00A0 GLN3\u00C2\u00A0 remained\u00C2\u00A0 inactive\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 plentiful\u00C2\u00A0 supply\u00C2\u00A0 of\u00C2\u00A0 good\u00C2\u00A0 nitrogen.\u00C2\u00A0 Thus,\u00C2\u00A0without\u00C2\u00A0 satisfying\u00C2\u00A0 both\u00C2\u00A0 conditions\u00C2\u00A0 for\u00C2\u00A0 transcriptional\u00C2\u00A0 activation,\u00C2\u00A0 the\u00C2\u00A0 native\u00C2\u00A0 DUR1,2\u00C2\u00A0 genes\u00C2\u00A0 remained\u00C2\u00A0 repressed\u00C2\u00A0 in\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 and\u00C2\u00A0 constitutively\u00C2\u00A0 absorbed\u00C2\u00A0 urea\u00C2\u00A0 likely\u00C2\u00A0 diffused\u00C2\u00A0back\u00C2\u00A0out\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0where\u00C2\u00A0 it\u00C2\u00A0could\u00C2\u00A0form\u00C2\u00A0EC\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Indeed,\u00C2\u00A0the\u00C2\u00A0facilitated\u00C2\u00A0 diffusion\u00C2\u00A0system\u00C2\u00A0for\u00C2\u00A0urea\u00C2\u00A0(DUR4),\u00C2\u00A0which\u00C2\u00A0allows\u00C2\u00A0leakage\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0is\u00C2\u00A0energy\u00C2\u00A0independent,\u00C2\u00A0insensitive\u00C2\u00A0 to\u00C2\u00A0NCR,\u00C2\u00A0and\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0growing\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0any\u00C2\u00A0inducer\u00C2\u00A0(Cooper\u00C2\u00A0and\u00C2\u00A0Sumrada\u00C2\u00A01975).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 97\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A037.\u00C2\u00A0Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0 inducible\u00C2\u00A0DUR1,2\u00C2\u00A0expression\u00C2\u00A0during\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0 fermentation\u00C2\u00A0by\u00C2\u00A0 a\u00C2\u00A0urea\u00C2\u00A0 importing\u00C2\u00A0 yeast\u00C2\u00A0 strain.\u00C2\u00A0During\u00C2\u00A0Chardonnay\u00C2\u00A0 fermentation\u00C2\u00A0partial\u00C2\u00A0 activation\u00C2\u00A0of\u00C2\u00A0 GLN3\u00C2\u00A0 coupled\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 inducer\u00C2\u00A0 allows\u00C2\u00A0 for\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0 reduction\u00C2\u00A0of\u00C2\u00A0EC.\u00C2\u00A0Despite\u00C2\u00A0the\u00C2\u00A0constitutive\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0inducer,\u00C2\u00A0constant\u00C2\u00A0NCR\u00C2\u00A0maintains\u00C2\u00A0repression\u00C2\u00A0of\u00C2\u00A0 DUR1,2\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0fermentations\u00C2\u00A0and\u00C2\u00A0constitutively\u00C2\u00A0imported\u00C2\u00A0urea\u00C2\u00A0is\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0leak\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0and\u00C2\u00A0 form\u00C2\u00A0EC.\u00C2\u00A0 \u00C2\u00A0 While\u00C2\u00A0Figure\u00C2\u00A037\u00C2\u00A0explains\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0 likely\u00C2\u00A0reason\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 inability\u00C2\u00A0of\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0 efficiently\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0 wine,\u00C2\u00A0 there\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 other\u00C2\u00A0minor\u00C2\u00A0 issues.\u00C2\u00A0 Firstly,\u00C2\u00A0 yeast\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 rice\u00C2\u00A0mash\u00C2\u00A0may\u00C2\u00A0 influence\u00C2\u00A0 the\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 protein\u00C2\u00A0 mistargeting,\u00C2\u00A0 a\u00C2\u00A0 common\u00C2\u00A0 problem\u00C2\u00A0 for\u00C2\u00A0 membrane\u00C2\u00A0 protein\u00C2\u00A0 over\u00C2\u00A0 expression.\u00C2\u00A0Unlike\u00C2\u00A0 soluble\u00C2\u00A0proteins,\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0 translated\u00C2\u00A0and\u00C2\u00A0kept\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0cytosol,\u00C2\u00A0membrane\u00C2\u00A0proteins\u00C2\u00A0 must\u00C2\u00A0navigate\u00C2\u00A0the\u00C2\u00A0protein\u00C2\u00A0trafficking\u00C2\u00A0system\u00C2\u00A0which,\u00C2\u00A0consequently,\u00C2\u00A0is\u00C2\u00A0subject\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0rate\u00C2\u00A0limiting\u00C2\u00A0 steps\u00C2\u00A0 (Reviewed\u00C2\u00A0 in\u00C2\u00A0Wagner,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0 In\u00C2\u00A0eukaryotes,\u00C2\u00A0membrane\u00C2\u00A0protein\u00C2\u00A0production\u00C2\u00A0begins\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 rough\u00C2\u00A0 endoplasmic\u00C2\u00A0 reticulum\u00C2\u00A0 (ER)\u00C2\u00A0 where\u00C2\u00A0 ribosomes\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0 ER\u00C2\u00A0 translate\u00C2\u00A0 proteins\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 immediately\u00C2\u00A0 translocated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0ER\u00C2\u00A0 lumen\u00C2\u00A0 (Wagner,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0From\u00C2\u00A0 there,\u00C2\u00A0proteins\u00C2\u00A0can\u00C2\u00A0begin\u00C2\u00A0 to\u00C2\u00A0 undergo\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0post\u00E2\u0080\u0090translational\u00C2\u00A0modifications\u00C2\u00A0(most\u00C2\u00A0commonly\u00C2\u00A0glycosylation)\u00C2\u00A0and\u00C2\u00A0quality\u00C2\u00A0control\u00C2\u00A0 checks\u00C2\u00A0(Wagner,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Proteins\u00C2\u00A0 leave\u00C2\u00A0the\u00C2\u00A0ER\u00C2\u00A0by\u00C2\u00A0means\u00C2\u00A0of\u00C2\u00A0COPII\u00C2\u00A0coated\u00C2\u00A0vesicles\u00C2\u00A0that\u00C2\u00A0travel\u00C2\u00A0along\u00C2\u00A0 microtubules\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0cis\u00E2\u0080\u0090,\u00C2\u00A0mid\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 trans\u00E2\u0080\u0090\u00C2\u00A0Golgi\u00C2\u00A0apparatus\u00C2\u00A0before\u00C2\u00A0being\u00C2\u00A0excreted\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0via\u00C2\u00A0additional\u00C2\u00A0vesicles\u00C2\u00A0(Wagner,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006).\u00C2\u00A0Since\u00C2\u00A0protein\u00C2\u00A0trafficking\u00C2\u00A0machinery\u00C2\u00A0is\u00C2\u00A0limited\u00C2\u00A0in\u00C2\u00A0 its\u00C2\u00A0 speed\u00C2\u00A0 and\u00C2\u00A0 efficiency,\u00C2\u00A0 and\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 shared\u00C2\u00A0 with\u00C2\u00A0 other\u00C2\u00A0 essential\u00C2\u00A0 membrane\u00C2\u00A0 proteins,\u00C2\u00A0 excess\u00C2\u00A0 Minimal\u00C2\u00A0leakage\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 98\u00C2\u00A0 \u00C2\u00A0 overexpressed\u00C2\u00A0 protein\u00C2\u00A0 often\u00C2\u00A0 backs\u00C2\u00A0 up\u00C2\u00A0 (Wagner,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006).\u00C2\u00A0 Such\u00C2\u00A0 backups\u00C2\u00A0 are\u00C2\u00A0 often\u00C2\u00A0 dealt\u00C2\u00A0with\u00C2\u00A0 via\u00C2\u00A0 ubiquitin\u00E2\u0080\u0090proteasome\u00C2\u00A0mediated\u00C2\u00A0 degradation\u00C2\u00A0 (Wagner,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 2006),\u00C2\u00A0 or,\u00C2\u00A0 as\u00C2\u00A0more\u00C2\u00A0 often\u00C2\u00A0 the\u00C2\u00A0 case\u00C2\u00A0 in\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae,\u00C2\u00A0excess\u00C2\u00A0protein\u00C2\u00A0is\u00C2\u00A0diverted\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0vacuole\u00C2\u00A0where\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0either\u00C2\u00A0stored\u00C2\u00A0or\u00C2\u00A0degraded\u00C2\u00A0(Wagner,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 2006).\u00C2\u00A0Although\u00C2\u00A0expressed\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0high\u00C2\u00A0copy\u00C2\u00A0plasmid\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0an\u00C2\u00A0integrated\u00C2\u00A0cassette,\u00C2\u00A0a\u00C2\u00A0recent\u00C2\u00A0study\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0polyamine\u00C2\u00A0 transport\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0DUR3p\u00C2\u00A0reported\u00C2\u00A0significant\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0over\u00C2\u00A0expressed\u00C2\u00A0DUR3p\u00C2\u00A0 localizing\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 vacuole\u00C2\u00A0 suggesting\u00C2\u00A0 a\u00C2\u00A0 high\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 degradation\u00C2\u00A0 (Uemura,\u00C2\u00A0 Kashiwagi\u00C2\u00A0 and\u00C2\u00A0 Igarashi\u00C2\u00A0 2007).\u00C2\u00A0 This\u00C2\u00A0 result\u00C2\u00A0was\u00C2\u00A0 subsequently\u00C2\u00A0 confirmed\u00C2\u00A0 by\u00C2\u00A0 another\u00C2\u00A0 study\u00C2\u00A0 looking\u00C2\u00A0 at\u00C2\u00A0 optimizing\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae\u00C2\u00A0 membrane\u00C2\u00A0protein\u00C2\u00A0over\u00C2\u00A0expression\u00C2\u00A0(Newstead,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02007).\u00C2\u00A0Thus,\u00C2\u00A0 if\u00C2\u00A0cell\u00C2\u00A0growth\u00C2\u00A0 in\u00C2\u00A0rice\u00C2\u00A0mash\u00C2\u00A0 increases\u00C2\u00A0 DUR3p\u00C2\u00A0mistargeting\u00C2\u00A0 relative\u00C2\u00A0 to\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0must,\u00C2\u00A0 or,\u00C2\u00A0more\u00C2\u00A0 likely,\u00C2\u00A0 if\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0 wine\u00C2\u00A0 (perhaps\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0high\u00C2\u00A0ethanol\u00C2\u00A0stress\u00C2\u00A0and\u00C2\u00A0low\u00C2\u00A0osmotic\u00C2\u00A0stress)\u00C2\u00A0requires\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0greater\u00C2\u00A0number\u00C2\u00A0 of\u00C2\u00A0membrane\u00C2\u00A0proteins,\u00C2\u00A0 the\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0 functional\u00C2\u00A0DUR3p\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0diminished,\u00C2\u00A0 thus\u00C2\u00A0 lowering\u00C2\u00A0 the\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0ability.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0may\u00C2\u00A0change\u00C2\u00A0the\u00C2\u00A0primary\u00C2\u00A0physiological\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0DUR3p.\u00C2\u00A0As\u00C2\u00A0noted\u00C2\u00A0previously\u00C2\u00A0 DUR3p\u00C2\u00A0 is\u00C2\u00A0primarily\u00C2\u00A0 an\u00C2\u00A0 active\u00C2\u00A0 transporter\u00C2\u00A0of\u00C2\u00A0urea,\u00C2\u00A0however\u00C2\u00A0 it\u00C2\u00A0has\u00C2\u00A0 also\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 boron\u00C2\u00A0 transport\u00C2\u00A0 (Nozawa,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006)\u00C2\u00A0and\u00C2\u00A0polyamine\u00C2\u00A0uptake\u00C2\u00A0 (Uemura,\u00C2\u00A0Kashiwagi\u00C2\u00A0and\u00C2\u00A0 Igarashi\u00C2\u00A02007).\u00C2\u00A0 While\u00C2\u00A0little\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0about\u00C2\u00A0boron\u00E2\u0080\u0099s\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0fermentation,\u00C2\u00A0polyamines\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0crucial\u00C2\u00A0for\u00C2\u00A0 cell\u00C2\u00A0growth\u00C2\u00A0 (Tabor\u00C2\u00A0and\u00C2\u00A0Tabor\u00C2\u00A01999);\u00C2\u00A0polyamine\u00C2\u00A0 levels\u00C2\u00A0are\u00C2\u00A0elevated\u00C2\u00A0 in\u00C2\u00A0actively\u00C2\u00A0growing\u00C2\u00A0cells\u00C2\u00A0 (Monteiro\u00C2\u00A0 and\u00C2\u00A0 Bisson\u00C2\u00A0 1992).\u00C2\u00A0 Polyamines\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 especially\u00C2\u00A0 important\u00C2\u00A0 for\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 substantially\u00C2\u00A0 different\u00C2\u00A0 nutrient\u00C2\u00A0environment\u00C2\u00A0of\u00C2\u00A0rice\u00C2\u00A0mash;\u00C2\u00A0thus,\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0brewing,\u00C2\u00A0an\u00C2\u00A0 increased\u00C2\u00A0need\u00C2\u00A0 for\u00C2\u00A0polyamines,\u00C2\u00A0and\u00C2\u00A0 perhaps\u00C2\u00A0boron,\u00C2\u00A0may\u00C2\u00A0decrease\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0over\u00C2\u00A0expressed\u00C2\u00A0DUR3p\u00C2\u00A0available\u00C2\u00A0for\u00C2\u00A0urea\u00C2\u00A0uptake,\u00C2\u00A0thereby\u00C2\u00A0 lowering\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0ability.\u00C2\u00A0 \u00C2\u00A0 4.3.2.5\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0metabolically\u00C2\u00A0 engineered\u00C2\u00A0 yeasts\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 reduce\u00C2\u00A0 EC\u00C2\u00A0more\u00C2\u00A0 effectively\u00C2\u00A0 than\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 522EC\u00E2\u0080\u0090\u00C2\u00A0 or\u00C2\u00A0 K7D3\u00C2\u00A0 and\u00C2\u00A0 522D3\u00C2\u00A0 in\u00C2\u00A0 either\u00C2\u00A0 Chardonnay\u00C2\u00A0 or\u00C2\u00A0 Sake\u00C2\u00A0wine.\u00C2\u00A0 The\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0cassettes\u00C2\u00A0on\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0was\u00C2\u00A0evaluated\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0wine.\u00C2\u00A0Strains\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0producing\u00C2\u00A0Chardonnay\u00C2\u00A0or\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0with\u00C2\u00A0less\u00C2\u00A0EC\u00C2\u00A0than\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0or\u00C2\u00A0 K7D3\u00C2\u00A0and\u00C2\u00A0522D3\u00C2\u00A0(Tables\u00C2\u00A020\u00C2\u00A0and\u00C2\u00A021).\u00C2\u00A0Thus,\u00C2\u00A0it\u00C2\u00A0seems\u00C2\u00A0that\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0DUR1,2\u00C2\u00A0and\u00C2\u00A0DUR3\u00C2\u00A0 offers\u00C2\u00A0no\u00C2\u00A0 synergistic\u00C2\u00A0advantage\u00C2\u00A0over\u00C2\u00A0 the\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0either\u00C2\u00A0gene\u00C2\u00A0alone\u00C2\u00A0and\u00C2\u00A0 that,\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 case\u00C2\u00A0of\u00C2\u00A0metabolic\u00C2\u00A0engineering\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0wine,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0prudent\u00C2\u00A0to\u00C2\u00A0limit\u00C2\u00A0engineering\u00C2\u00A0to\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 99\u00C2\u00A0 \u00C2\u00A0 one\u00C2\u00A0 gene.\u00C2\u00A0 Yeast\u00C2\u00A0 strains\u00C2\u00A0 constitutively\u00C2\u00A0 expressing\u00C2\u00A0 only\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 gene\u00C2\u00A0 are\u00C2\u00A0 logistically\u00C2\u00A0 and\u00C2\u00A0 functionally\u00C2\u00A0 simpler,\u00C2\u00A0more\u00C2\u00A0cost\u00C2\u00A0effective,\u00C2\u00A0and\u00C2\u00A0can\u00C2\u00A0more\u00C2\u00A0expediently\u00C2\u00A0receive\u00C2\u00A0regulatory\u00C2\u00A0approval.\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 from\u00C2\u00A0 this\u00C2\u00A0study\u00C2\u00A0suggested\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0which\u00C2\u00A0has\u00C2\u00A0both\u00C2\u00A0cassettes\u00C2\u00A0 integrated,\u00C2\u00A0high\u00C2\u00A0 level\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0DUR1,2\u00C2\u00A0 and\u00C2\u00A0DUR3\u00C2\u00A0was\u00C2\u00A0maintained\u00C2\u00A0 (Figure\u00C2\u00A0 30),\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 both\u00C2\u00A0 cassettes\u00C2\u00A0 produce\u00C2\u00A0 functional\u00C2\u00A0enzymes\u00C2\u00A0(Figure\u00C2\u00A031).\u00C2\u00A0Moreover,\u00C2\u00A0urea\u00C2\u00A0degrading\u00C2\u00A0and\u00C2\u00A0urea\u00C2\u00A0importing\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0(integration\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0DUR1,2\u00C2\u00A0 and\u00C2\u00A0DUR3\u00C2\u00A0 cassettes,\u00C2\u00A0 respectively)\u00C2\u00A0 each\u00C2\u00A0 reduce\u00C2\u00A0 EC\u00C2\u00A0 by\u00C2\u00A0 up\u00C2\u00A0 to\u00C2\u00A0 90%\u00C2\u00A0 (Tables\u00C2\u00A0 20\u00C2\u00A0 and\u00C2\u00A0 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DUR3\u00C2\u00A0cassette\u00C2\u00A0should\u00C2\u00A0not\u00C2\u00A0confer\u00C2\u00A0any\u00C2\u00A0advantage\u00C2\u00A0 in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0uptake\u00C2\u00A0and\u00C2\u00A0EC\u00C2\u00A0reduction.\u00C2\u00A0To\u00C2\u00A0test\u00C2\u00A0this\u00C2\u00A0 hypothesis,\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0for\u00C2\u00A0yeasts\u00C2\u00A0to\u00C2\u00A0uptake\u00C2\u00A0urea\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression\u00C2\u00A0was\u00C2\u00A0examined\u00C2\u00A0 (Figures\u00C2\u00A0 33\u00C2\u00A0 and\u00C2\u00A0 34).\u00C2\u00A0 These\u00C2\u00A0 14C\u00E2\u0080\u0090uptake\u00C2\u00A0 assays\u00C2\u00A0 were\u00C2\u00A0 completed\u00C2\u00A0 in\u00C2\u00A0 an\u00C2\u00A0 L\u00E2\u0080\u0090proline\u00C2\u00A0 minimal\u00C2\u00A0 medium\u00C2\u00A0 to\u00C2\u00A0 simulate\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0NCR\u00C2\u00A0de\u00E2\u0080\u0090repression.\u00C2\u00A0Proline\u00C2\u00A0has\u00C2\u00A0 long\u00C2\u00A0been\u00C2\u00A0recognized\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0poor\u00C2\u00A0quality\u00C2\u00A0nitrogen\u00C2\u00A0 source\u00C2\u00A0 for\u00C2\u00A0 yeast\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0 1999;\u00C2\u00A0 Ingledew,\u00C2\u00A0 Magnus\u00C2\u00A0 and\u00C2\u00A0 Sosulski\u00C2\u00A0 1987).\u00C2\u00A0 Under\u00C2\u00A0 de\u00E2\u0080\u0090repressive\u00C2\u00A0 conditions,\u00C2\u00A0uptake\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0parental\u00C2\u00A0strain\u00C2\u00A0K7\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0engineered\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090,\u00C2\u00A0K7D3,\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3\u00C2\u00A0 largely\u00C2\u00A0mimicked\u00C2\u00A0uptake\u00C2\u00A0under\u00C2\u00A0repressive\u00C2\u00A0conditions\u00C2\u00A0(Figures\u00C2\u00A031\u00C2\u00A0and\u00C2\u00A033).\u00C2\u00A0Both\u00C2\u00A0K7D3\u00C2\u00A0and\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0which\u00C2\u00A0 contained\u00C2\u00A0the\u00C2\u00A0integrated\u00C2\u00A0DUR3\u00C2\u00A0cassette,\u00C2\u00A0were\u00C2\u00A0highly\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0incorporating\u00C2\u00A014C\u00E2\u0080\u0090urea\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0strains\u00C2\u00A0 that\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 contain\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette,\u00C2\u00A0 K7\u00C2\u00A0 and\u00C2\u00A0 K7EC\u00E2\u0080\u0090,\u00C2\u00A0were\u00C2\u00A0 unable\u00C2\u00A0 to\u00C2\u00A0 incorporate\u00C2\u00A0 any\u00C2\u00A0 appreciable\u00C2\u00A0 amounts\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0 (Figure\u00C2\u00A033)\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0 and\u00C2\u00A0 terminator\u00C2\u00A0 signals\u00C2\u00A0 does\u00C2\u00A0 indeed\u00C2\u00A0 confer\u00C2\u00A0 an\u00C2\u00A0 advantage\u00C2\u00A0 to\u00C2\u00A0 engineered\u00C2\u00A0 yeast\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 urea\u00C2\u00A0 uptake.\u00C2\u00A0Furthermore,\u00C2\u00A0data\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0 studies\u00C2\u00A0during\u00C2\u00A0grape\u00C2\u00A0wine\u00C2\u00A0and\u00C2\u00A0Sake\u00C2\u00A0 wine\u00C2\u00A0fermentations\u00C2\u00A0confirm\u00C2\u00A0that\u00C2\u00A0during\u00C2\u00A0grape\u00C2\u00A0wine\u00C2\u00A0fermentation\u00C2\u00A0DUR3\u00C2\u00A0is\u00C2\u00A0only\u00C2\u00A0induced\u00C2\u00A0during\u00C2\u00A0nitrogen\u00C2\u00A0 exhaustion\u00C2\u00A0 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native\u00C2\u00A0 promoter,\u00C2\u00A0 both\u00C2\u00A0 an\u00C2\u00A0 inducer\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 present\u00C2\u00A0 (urea)\u00C2\u00A0 and\u00C2\u00A0 NCR\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 de\u00E2\u0080\u0090repressed\u00C2\u00A0 (Hofman\u00E2\u0080\u0090Bang\u00C2\u00A0 1999).\u00C2\u00A0Despite\u00C2\u00A0 the\u00C2\u00A0 de\u00E2\u0080\u0090repression\u00C2\u00A0 of\u00C2\u00A0NCR,\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 grown\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 inducer\u00C2\u00A0 thus\u00C2\u00A0maintaining\u00C2\u00A0 low\u00E2\u0080\u0090level\u00C2\u00A0DUR3\u00C2\u00A0expression;\u00C2\u00A0only\u00C2\u00A0when\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0exposed\u00C2\u00A0 to\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0were\u00C2\u00A0 both\u00C2\u00A0conditions\u00C2\u00A0for\u00C2\u00A0DUR3\u00C2\u00A0expression\u00C2\u00A0met.\u00C2\u00A0\u00C2\u00A0 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no\u00C2\u00A0 detectable\u00C2\u00A0 urea\u00C2\u00A0 or\u00C2\u00A0 EC\u00C2\u00A0 (Kitamoto,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 1991;\u00C2\u00A0 Yoshiuchi,\u00C2\u00A0 Watanabe\u00C2\u00A0 and\u00C2\u00A0 Nishimura\u00C2\u00A0 2000).\u00C2\u00A0 These\u00C2\u00A0 studies\u00C2\u00A0 provide\u00C2\u00A0 strong\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 important\u00C2\u00A0 precursor\u00C2\u00A0 for\u00C2\u00A0 EC\u00C2\u00A0 is\u00C2\u00A0 urea\u00C2\u00A0 derived\u00C2\u00A0 from\u00C2\u00A0 CAR1\u00C2\u00A0 mediated\u00C2\u00A0 arginine\u00C2\u00A0 degradation;\u00C2\u00A0 however,\u00C2\u00A0 in\u00C2\u00A0 certain\u00C2\u00A0 circumstances,\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0wine\u00C2\u00A0 that\u00C2\u00A0has\u00C2\u00A0undergone\u00C2\u00A0a\u00C2\u00A0MLF,\u00C2\u00A0a\u00C2\u00A0 certain\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0derived\u00C2\u00A0 from\u00C2\u00A0alternate\u00C2\u00A0sources.\u00C2\u00A0While\u00C2\u00A0 this\u00C2\u00A0rationale\u00C2\u00A0would\u00C2\u00A0explain\u00C2\u00A0a\u00C2\u00A0 lack\u00C2\u00A0of\u00C2\u00A0synergistic\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0wine\u00C2\u00A0 that\u00C2\u00A0had\u00C2\u00A0undergone\u00C2\u00A0MLF,\u00C2\u00A0it\u00C2\u00A0fails\u00C2\u00A0to\u00C2\u00A0explain\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0study.\u00C2\u00A0 \u00C2\u00A0 Besides\u00C2\u00A0 urea,\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 other\u00C2\u00A0 compounds\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 implicated\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 EC.\u00C2\u00A0 Carbamyl\u00C2\u00A0phosphate\u00C2\u00A0and\u00C2\u00A0citrulline\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0react\u00C2\u00A0with\u00C2\u00A0ethanol\u00C2\u00A0and\u00C2\u00A0form\u00C2\u00A0EC\u00C2\u00A0(Matsudo\u00C2\u00A01993;\u00C2\u00A0Ough\u00C2\u00A0 1976b);\u00C2\u00A0 however,\u00C2\u00A0while\u00C2\u00A0 they\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 detected,\u00C2\u00A0 carbamyl\u00C2\u00A0 phosphate\u00C2\u00A0 and\u00C2\u00A0 citrulline\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 usually\u00C2\u00A0 found\u00C2\u00A0 in\u00C2\u00A0grape\u00C2\u00A0or\u00C2\u00A0Sake\u00C2\u00A0wine\u00C2\u00A0(Ough,\u00C2\u00A0Crowell\u00C2\u00A0and\u00C2\u00A0Gutlove\u00C2\u00A01988).\u00C2\u00A0Citrulline\u00C2\u00A0and\u00C2\u00A0carbamyl\u00C2\u00A0phosphate\u00C2\u00A0are\u00C2\u00A0 by\u00E2\u0080\u0090products\u00C2\u00A0 of\u00C2\u00A0 arginine\u00C2\u00A0 metabolism\u00C2\u00A0 in\u00C2\u00A0 lactic\u00C2\u00A0 acid\u00C2\u00A0 bacteria\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 only\u00C2\u00A0 found\u00C2\u00A0 in\u00C2\u00A0 wines\u00C2\u00A0 that\u00C2\u00A0 have\u00C2\u00A0 undergone\u00C2\u00A0MLF;\u00C2\u00A0 in\u00C2\u00A0 lactic\u00C2\u00A0acid\u00C2\u00A0bacteria\u00C2\u00A0arginine\u00C2\u00A0 is\u00C2\u00A0metabolized\u00C2\u00A0via\u00C2\u00A0 the\u00C2\u00A0arginine\u00C2\u00A0deiminase\u00C2\u00A0pathway\u00C2\u00A0 in\u00C2\u00A0 which\u00C2\u00A0arginine\u00C2\u00A0 is\u00C2\u00A0degraded\u00C2\u00A0 to\u00C2\u00A0citrulline\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 then\u00C2\u00A0degraded\u00C2\u00A0 to\u00C2\u00A0ornithine\u00C2\u00A0and\u00C2\u00A0carbamyl\u00C2\u00A0phosphate\u00C2\u00A0 (Liu,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A01994).\u00C2\u00A0Utilization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0metabolically\u00C2\u00A0engineered\u00C2\u00A0yeast\u00C2\u00A0strain\u00C2\u00A0ML01,\u00C2\u00A0which\u00C2\u00A0conducts\u00C2\u00A0parallel\u00C2\u00A0 alcoholic\u00C2\u00A0and\u00C2\u00A0malolactic\u00C2\u00A0fermentations\u00C2\u00A0without\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0lactic\u00C2\u00A0acid\u00C2\u00A0bacteria\u00C2\u00A0(Husnik,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006),\u00C2\u00A0 could\u00C2\u00A0help\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0certain\u00C2\u00A0applications.\u00C2\u00A0In\u00C2\u00A0stone\u00C2\u00A0fruit\u00C2\u00A0spirits\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0fruit\u00C2\u00A0brandies,\u00C2\u00A0amygdalin\u00C2\u00A0from\u00C2\u00A0 the\u00C2\u00A0stone\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0degrade\u00C2\u00A0into\u00C2\u00A0cyanide\u00C2\u00A0during\u00C2\u00A0fermentation,\u00C2\u00A0the\u00C2\u00A0oxidized\u00C2\u00A0form\u00C2\u00A0(cyannate)\u00C2\u00A0of\u00C2\u00A0 which\u00C2\u00A0reacts\u00C2\u00A0with\u00C2\u00A0ethanol\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0EC\u00C2\u00A0(Schehl\u00C2\u00A02007).\u00C2\u00A0Finally,\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0compounds,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0N\u00E2\u0080\u0090 carbamyl\u00C2\u00A0 \u00CE\u00B1\u00E2\u0080\u0090amino\u00C2\u00A0 acids,\u00C2\u00A0N\u00E2\u0080\u0090carbamyl\u00C2\u00A0 \u00CE\u00B2\u00E2\u0080\u0090amino\u00C2\u00A0 acids,\u00C2\u00A0 and\u00C2\u00A0 allantoin\u00C2\u00A0 (by\u00E2\u0080\u0090product\u00C2\u00A0of\u00C2\u00A0purine\u00C2\u00A0degradation)\u00C2\u00A0 have\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0react\u00C2\u00A0with\u00C2\u00A0ethanol\u00C2\u00A0and\u00C2\u00A0 form\u00C2\u00A0EC\u00C2\u00A0 (Ough,\u00C2\u00A0Crowell\u00C2\u00A0and\u00C2\u00A0Gutlove\u00C2\u00A01988);\u00C2\u00A0however,\u00C2\u00A0the\u00C2\u00A0 contribution\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0compounds\u00C2\u00A0to\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0has\u00C2\u00A0yet\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0substantiated.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 101\u00C2\u00A0 \u00C2\u00A0 5\u00C2\u00A0\u00C2\u00A0CONCLUSIONS\u00C2\u00A0 \u00C2\u00A0 Amongst\u00C2\u00A0 other\u00C2\u00A0 reasons,\u00C2\u00A0 increased\u00C2\u00A0 awareness\u00C2\u00A0 surrounding\u00C2\u00A0 the\u00C2\u00A0 health\u00C2\u00A0 benefits\u00C2\u00A0 of\u00C2\u00A0 wine\u00C2\u00A0 consumption\u00C2\u00A0has\u00C2\u00A0caused\u00C2\u00A0 the\u00C2\u00A0popularity\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0 to\u00C2\u00A0grow\u00C2\u00A0worldwide.\u00C2\u00A0Wine,\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0particular\u00C2\u00A0 red\u00C2\u00A0wine,\u00C2\u00A0 has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0help\u00C2\u00A0prevent\u00C2\u00A0cardiovascular\u00C2\u00A0disease,\u00C2\u00A0dietary\u00C2\u00A0cancers,\u00C2\u00A0diabetes,\u00C2\u00A0hypertension,\u00C2\u00A0peptic\u00C2\u00A0 users,\u00C2\u00A0and\u00C2\u00A0macular\u00C2\u00A0degeneration,\u00C2\u00A0amongst\u00C2\u00A0others\u00C2\u00A0(Bisson\u00C2\u00A02002).\u00C2\u00A0Along\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0growing\u00C2\u00A0appreciation\u00C2\u00A0for\u00C2\u00A0 the\u00C2\u00A0 benefits\u00C2\u00A0 of\u00C2\u00A0 alcoholic\u00C2\u00A0 beverage\u00C2\u00A0 consumption,\u00C2\u00A0 consumers\u00C2\u00A0 have\u00C2\u00A0 become\u00C2\u00A0 increasingly\u00C2\u00A0 conscious\u00C2\u00A0 of\u00C2\u00A0 consumption\u00C2\u00A0 risks\u00C2\u00A0 and\u00C2\u00A0 therefore\u00C2\u00A0 have\u00C2\u00A0 begun\u00C2\u00A0 to\u00C2\u00A0 demand\u00C2\u00A0 increasingly\u00C2\u00A0 safe\u00C2\u00A0 products.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0mindset\u00C2\u00A0 echoes\u00C2\u00A0beyond\u00C2\u00A0alcoholic\u00C2\u00A0beverages\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0highlighted\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0prevalence\u00C2\u00A0of\u00C2\u00A0 recent\u00C2\u00A0 food\u00C2\u00A0 safety\u00C2\u00A0 issues\u00C2\u00A0 including\u00C2\u00A0mad\u00C2\u00A0cow\u00C2\u00A0disease,\u00C2\u00A0pesticide\u00C2\u00A0usage,\u00C2\u00A0heavy\u00C2\u00A0metal\u00C2\u00A0contamination,\u00C2\u00A0E.\u00C2\u00A0coli\u00C2\u00A0O157:H7,\u00C2\u00A0GMO\u00C2\u00A0corn,\u00C2\u00A0and\u00C2\u00A0 other\u00C2\u00A0GMO\u00C2\u00A0 crops.\u00C2\u00A0Of\u00C2\u00A0particular\u00C2\u00A0 concern\u00C2\u00A0 to\u00C2\u00A0 this\u00C2\u00A0 study\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 increasing\u00C2\u00A0 recognition\u00C2\u00A0of\u00C2\u00A0 EC,\u00C2\u00A0 a\u00C2\u00A0naturally\u00C2\u00A0 occurring\u00C2\u00A0and\u00C2\u00A0well\u00C2\u00A0characterized\u00C2\u00A0carcinogen\u00C2\u00A0found\u00C2\u00A0in\u00C2\u00A0wine,\u00C2\u00A0Sake,\u00C2\u00A0and\u00C2\u00A0many\u00C2\u00A0other\u00C2\u00A0yeast\u00C2\u00A0fermented\u00C2\u00A0foods\u00C2\u00A0 and\u00C2\u00A0beverages.\u00C2\u00A0Indeed,\u00C2\u00A0winemakers\u00C2\u00A0and\u00C2\u00A0governments\u00C2\u00A0have\u00C2\u00A0become\u00C2\u00A0progressively\u00C2\u00A0more\u00C2\u00A0cognisant\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 widespread\u00C2\u00A0prevalence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0EC\u00C2\u00A0problem.\u00C2\u00A0Spearheaded\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0FDA\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0California\u00C2\u00A0at\u00C2\u00A0 Davis\u00E2\u0080\u0099\u00C2\u00A0Department\u00C2\u00A0of\u00C2\u00A0Enology\u00C2\u00A0and\u00C2\u00A0Viticulture\u00C2\u00A0(Butzke\u00C2\u00A0and\u00C2\u00A0Bisson\u00C2\u00A01998),\u00C2\u00A0action\u00C2\u00A0manuals\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0have\u00C2\u00A0 been\u00C2\u00A0 complied\u00C2\u00A0 concerning\u00C2\u00A0 a\u00C2\u00A0 wide\u00C2\u00A0 range\u00C2\u00A0 of\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 techniques.\u00C2\u00A0 Until\u00C2\u00A0 recently,\u00C2\u00A0 none\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 techniques\u00C2\u00A0 employed\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 highly\u00C2\u00A0 effective\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 low\u00C2\u00A0 efficacy,\u00C2\u00A0 high\u00C2\u00A0 cost,\u00C2\u00A0 or\u00C2\u00A0 lengthened\u00C2\u00A0 production\u00C2\u00A0 time.\u00C2\u00A0However,\u00C2\u00A0 in\u00C2\u00A0 2006\u00C2\u00A0 our\u00C2\u00A0 group\u00C2\u00A0 developed\u00C2\u00A0 a\u00C2\u00A0 substantially\u00C2\u00A0 equivalent,\u00C2\u00A0 self\u00E2\u0080\u0090cloned,\u00C2\u00A0 FDA\u00C2\u00A0 approved,\u00C2\u00A0industrial\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0(522EC\u00E2\u0080\u0090)\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0reducing\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0by\u00C2\u00A089%.\u00C2\u00A0The\u00C2\u00A0amount\u00C2\u00A0 of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0wines\u00C2\u00A0is\u00C2\u00A0proportional\u00C2\u00A0to\u00C2\u00A0residual\u00C2\u00A0urea,\u00C2\u00A0thus\u00C2\u00A0making\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0a\u00C2\u00A0matter\u00C2\u00A0of\u00C2\u00A0eliminating\u00C2\u00A0residual\u00C2\u00A0 urea\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0alcoholic\u00C2\u00A0fermentation.\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0strain\u00C2\u00A0522EC\u00E2\u0080\u0090\u00C2\u00A0is\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0reducing\u00C2\u00A0EC\u00C2\u00A0by\u00C2\u00A089%\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0amidolyase\u00C2\u00A0 (DUR1,2)\u00C2\u00A0which\u00C2\u00A0degrades\u00C2\u00A0urea\u00C2\u00A0 to\u00C2\u00A0ammonia\u00C2\u00A0and\u00C2\u00A0carbon\u00C2\u00A0 dioxide;\u00C2\u00A0 the\u00C2\u00A0 native\u00C2\u00A0 copy\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 gene\u00C2\u00A0 is\u00C2\u00A0 normally\u00C2\u00A0 transcriptionally\u00C2\u00A0 silent\u00C2\u00A0 in\u00C2\u00A0 fermentations\u00C2\u00A0 with\u00C2\u00A0 rich\u00C2\u00A0 nitrogen\u00C2\u00A0supplies\u00C2\u00A0thus\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0wines\u00C2\u00A0with\u00C2\u00A0high\u00C2\u00A0residual\u00C2\u00A0urea\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0high\u00C2\u00A0EC\u00C2\u00A0.\u00C2\u00A0 \u00C2\u00A0 Numerous\u00C2\u00A0industrial\u00C2\u00A0strains\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0exist\u00C2\u00A0around\u00C2\u00A0the\u00C2\u00A0world,\u00C2\u00A0each\u00C2\u00A0filling\u00C2\u00A0an\u00C2\u00A0environmental\u00C2\u00A0 niche.\u00C2\u00A0Arising\u00C2\u00A0from\u00C2\u00A0growth\u00C2\u00A0on\u00C2\u00A0different\u00C2\u00A0nutrient\u00C2\u00A0sources,\u00C2\u00A0strains\u00C2\u00A0have\u00C2\u00A0developed\u00C2\u00A0different\u00C2\u00A0fermentative\u00C2\u00A0 properties\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0such,\u00C2\u00A0are\u00C2\u00A0uniquely\u00C2\u00A0suited\u00C2\u00A0 to\u00C2\u00A0 fermentation\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0substrates.\u00C2\u00A0However,\u00C2\u00A0EC\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0 pervasive\u00C2\u00A0problem\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0alcoholic\u00C2\u00A0fermentation\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result,\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0is\u00C2\u00A0important\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0 yeast\u00C2\u00A0strains.\u00C2\u00A0The\u00C2\u00A0self\u00E2\u0080\u0090cloned\u00C2\u00A0Sake\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0K7EC\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0K9EC\u00E2\u0080\u0090\u00C2\u00A0containing\u00C2\u00A0 the\u00C2\u00A0constitutively\u00C2\u00A0expressed\u00C2\u00A0 DUR1,2\u00C2\u00A0 cassette,\u00C2\u00A0were\u00C2\u00A0 ineffective\u00C2\u00A0when\u00C2\u00A0 tested\u00C2\u00A0 in\u00C2\u00A0wine\u00C2\u00A0making\u00C2\u00A0 trials;\u00C2\u00A0 however,\u00C2\u00A0 K7EC\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 K9EC\u00E2\u0080\u0090\u00C2\u00A0were\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 102\u00C2\u00A0 \u00C2\u00A0 highly\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0during\u00C2\u00A0Sake\u00C2\u00A0brewing\u00C2\u00A0trials.\u00C2\u00A0Thus,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0clear\u00C2\u00A0that\u00C2\u00A0certain\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0are\u00C2\u00A0 uniquely\u00C2\u00A0 adapted\u00C2\u00A0 to\u00C2\u00A0 specific\u00C2\u00A0 niches\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 optimally\u00C2\u00A0 functional\u00C2\u00A0 in\u00C2\u00A0 other\u00C2\u00A0 niches.\u00C2\u00A0 The\u00C2\u00A0 functionality\u00C2\u00A0of\u00C2\u00A0enhanced\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0must\u00C2\u00A0therefore\u00C2\u00A0be\u00C2\u00A0assessed\u00C2\u00A0in\u00C2\u00A0their\u00C2\u00A0native\u00C2\u00A0environment\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0most\u00C2\u00A0accurately\u00C2\u00A0gauge\u00C2\u00A0performance\u00C2\u00A0and\u00C2\u00A0avoid\u00C2\u00A0the\u00C2\u00A0identification\u00C2\u00A0of\u00C2\u00A0false\u00C2\u00A0negatives.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0to\u00C2\u00A0further\u00C2\u00A0reduce\u00C2\u00A0EC\u00C2\u00A0 levels\u00C2\u00A0beyond\u00C2\u00A0the\u00C2\u00A0capability\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0technology,\u00C2\u00A0this\u00C2\u00A0study\u00C2\u00A0 was\u00C2\u00A0also\u00C2\u00A0focused\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0substantially\u00C2\u00A0equivalent,\u00C2\u00A0self\u00E2\u0080\u0090cloned,\u00C2\u00A0industrial\u00C2\u00A0wine\u00C2\u00A0yeasts\u00C2\u00A0capable\u00C2\u00A0 of\u00C2\u00A0constitutive\u00C2\u00A0urea\u00C2\u00A0import.\u00C2\u00A0In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0the\u00C2\u00A0industrial\u00C2\u00A0strains\u00C2\u00A0K7D3,\u00C2\u00A0K7EC\u00E2\u0080\u0090D3,\u00C2\u00A0522D3,\u00C2\u00A0and\u00C2\u00A0522EC\u00E2\u0080\u0090D3\u00C2\u00A0the\u00C2\u00A0 S.\u00C2\u00A0cerevisiae\u00C2\u00A0urea\u00C2\u00A0permease\u00C2\u00A0(DUR3)\u00C2\u00A0was\u00C2\u00A0expressed\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0control\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0PGK1\u00C2\u00A0promoter\u00C2\u00A0 and\u00C2\u00A0 terminator\u00C2\u00A0 signals\u00C2\u00A0 and\u00C2\u00A0 integrated\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 TRP1\u00C2\u00A0 locus\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 relevant\u00C2\u00A0 parental\u00C2\u00A0 strains.\u00C2\u00A0 DNA\u00C2\u00A0 sequencing\u00C2\u00A0confirmed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0recombinant\u00C2\u00A0cassette\u00C2\u00A0contained\u00C2\u00A0no\u00C2\u00A0deleterious\u00C2\u00A0mutations\u00C2\u00A0or\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0 substitutions.\u00C2\u00A0 Furthermore,\u00C2\u00A0 Southern\u00C2\u00A0 blotting\u00C2\u00A0 confirmed\u00C2\u00A0 proper\u00C2\u00A0 cassette\u00C2\u00A0 integration\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 TRP1\u00C2\u00A0locus.\u00C2\u00A0Constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0was\u00C2\u00A0confirmed\u00C2\u00A0by\u00C2\u00A0northern\u00C2\u00A0blotting\u00C2\u00A0and\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0qRT\u00E2\u0080\u0090 PCR.\u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0indicated\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0integration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0cassette\u00C2\u00A0had\u00C2\u00A0a\u00C2\u00A0minimal\u00C2\u00A0 impact\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0transcriptome\u00C2\u00A0of\u00C2\u00A0S.\u00C2\u00A0cerevisiae\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0no\u00C2\u00A0major\u00C2\u00A0metabolic\u00C2\u00A0pathways\u00C2\u00A0were\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 DUR3\u00C2\u00A0 cassette.\u00C2\u00A0 Urea\u00C2\u00A0 import\u00C2\u00A0 studies\u00C2\u00A0 using\u00C2\u00A0 14C\u00E2\u0080\u0090urea\u00C2\u00A0 indicated\u00C2\u00A0 that\u00C2\u00A0 DUR3\u00C2\u00A0 cassette\u00C2\u00A0 containing\u00C2\u00A0 strains\u00C2\u00A0 indeed\u00C2\u00A0 express\u00C2\u00A0 functional\u00C2\u00A0 DUR3p\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 this\u00C2\u00A0 functionality\u00C2\u00A0 is\u00C2\u00A0 preserved\u00C2\u00A0 when\u00C2\u00A0 DUR1,2\u00C2\u00A0 and\u00C2\u00A0DUR3\u00C2\u00A0 are\u00C2\u00A0 constitutively\u00C2\u00A0 co\u00E2\u0080\u0090expressed.\u00C2\u00A0 Comparisons\u00C2\u00A0 of\u00C2\u00A0 small\u00C2\u00A0 scale\u00C2\u00A0 Chardonnay\u00C2\u00A0 and\u00C2\u00A0 Sake\u00C2\u00A0 wine\u00C2\u00A0making\u00C2\u00A0 demonstrated\u00C2\u00A0 that,\u00C2\u00A0 although\u00C2\u00A0 constitutive\u00C2\u00A0 co\u00E2\u0080\u0090expression\u00C2\u00A0 of\u00C2\u00A0DUR1,2\u00C2\u00A0 and\u00C2\u00A0DUR3\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 yield\u00C2\u00A0 synergistic\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0 or\u00C2\u00A0 Sake\u00C2\u00A0wine,\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 alone\u00C2\u00A0 is\u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0reducing\u00C2\u00A0EC\u00C2\u00A0as\u00C2\u00A0efficiently\u00C2\u00A0as\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR1,2\u00C2\u00A0in\u00C2\u00A0Chardonnay\u00C2\u00A0wine\u00C2\u00A0yet\u00C2\u00A0not\u00C2\u00A0in\u00C2\u00A0 Sake\u00C2\u00A0 wine.\u00C2\u00A0 Thus,\u00C2\u00A0 in\u00C2\u00A0 grape\u00C2\u00A0 wine\u00C2\u00A0 making\u00C2\u00A0 applications,\u00C2\u00A0 and\u00C2\u00A0 specifically\u00C2\u00A0 in\u00C2\u00A0 musts\u00C2\u00A0 which\u00C2\u00A0 contain\u00C2\u00A0 high\u00C2\u00A0 endogenous\u00C2\u00A0urea,\u00C2\u00A0DUR3\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0 important,\u00C2\u00A0valuable,\u00C2\u00A0and\u00C2\u00A0alternative\u00C2\u00A0strategy\u00C2\u00A0 for\u00C2\u00A0 EC\u00C2\u00A0reduction.\u00C2\u00A0A\u00C2\u00A0provisional\u00C2\u00A0patent\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0filed\u00C2\u00A0concerning\u00C2\u00A0the\u00C2\u00A0constitutive\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0 in\u00C2\u00A0S.\u00C2\u00A0 cerevisiae\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0reduction\u00C2\u00A0of\u00C2\u00A0EC\u00C2\u00A0in\u00C2\u00A0grape\u00C2\u00A0wine.\u00C2\u00A0Furthermore,\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DUR3\u00C2\u00A0expressing\u00C2\u00A0strains\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0 constructed\u00C2\u00A0by\u00C2\u00A0self\u00E2\u0080\u0090cloned\u00C2\u00A0means,\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0acceptable\u00C2\u00A0both\u00C2\u00A0worldwide\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0non\u00E2\u0080\u0090 transgenic\u00C2\u00A0nature,\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0 countries\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0Germany\u00C2\u00A0and\u00C2\u00A0 Japan\u00C2\u00A0where\u00C2\u00A0 self\u00E2\u0080\u0090cloned\u00C2\u00A0organisms\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0 considered\u00C2\u00A0as\u00C2\u00A0GMO\u00E2\u0080\u0099s.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 103\u00C2\u00A0 \u00C2\u00A0 5.1\u00C2\u00A0\u00C2\u00A0Future\u00C2\u00A0Directions\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0study\u00C2\u00A0and\u00C2\u00A0previous\u00C2\u00A0work\u00C2\u00A0by\u00C2\u00A0our\u00C2\u00A0group\u00C2\u00A0(Coulon,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A02006),\u00C2\u00A0winemakers\u00C2\u00A0now\u00C2\u00A0 have\u00C2\u00A0highly\u00C2\u00A0effective\u00C2\u00A0methods\u00C2\u00A0 for\u00C2\u00A0EC\u00C2\u00A0reduction\u00C2\u00A0at\u00C2\u00A0their\u00C2\u00A0 fingertips;\u00C2\u00A0however,\u00C2\u00A0several\u00C2\u00A0topics\u00C2\u00A0still\u00C2\u00A0require\u00C2\u00A0 investigation.\u00C2\u00A0 Firstly,\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 facilitate\u00C2\u00A0 the\u00C2\u00A0 commercialization\u00C2\u00A0 of\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 constitutively\u00C2\u00A0 expressing\u00C2\u00A0 DUR3,\u00C2\u00A0 a\u00C2\u00A0 cassette\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 developed\u00C2\u00A0 which\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 contain\u00C2\u00A0 any\u00C2\u00A0 antibiotic\u00C2\u00A0 resistance\u00C2\u00A0 markers.\u00C2\u00A0 Secondly,\u00C2\u00A0 experiments\u00C2\u00A0 should\u00C2\u00A0 be\u00C2\u00A0 done\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 confirm\u00C2\u00A0 which\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 potential\u00C2\u00A0 causes\u00C2\u00A0 discussed\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A04.3.2.4\u00C2\u00A0 are\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 constitutive\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0DUR3\u00C2\u00A0not\u00C2\u00A0 reducing\u00C2\u00A0 EC\u00C2\u00A0 efficiently\u00C2\u00A0 in\u00C2\u00A0 Sake\u00C2\u00A0wine\u00C2\u00A0 as\u00C2\u00A0 it\u00C2\u00A0 does\u00C2\u00A0 in\u00C2\u00A0 Chardonnay\u00C2\u00A0wine;\u00C2\u00A0 answering\u00C2\u00A0 this\u00C2\u00A0 question\u00C2\u00A0may\u00C2\u00A0 allow\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 subsequent\u00C2\u00A0 adaptation\u00C2\u00A0 of\u00C2\u00A0DUR3\u00C2\u00A0 technology\u00C2\u00A0 to\u00C2\u00A0 Sake\u00C2\u00A0making.\u00C2\u00A0 Thirdly,\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 alternative\u00C2\u00A0 methods\u00C2\u00A0 of\u00C2\u00A0 EC\u00C2\u00A0 reduction\u00C2\u00A0would\u00C2\u00A0 prove\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 useful\u00C2\u00A0 in\u00C2\u00A0 further\u00C2\u00A0 reducing\u00C2\u00A0 EC\u00C2\u00A0 beyond\u00C2\u00A0 90%\u00C2\u00A0 thus\u00C2\u00A0making\u00C2\u00A0 wines\u00C2\u00A0and\u00C2\u00A0spirits\u00C2\u00A0safer\u00C2\u00A0for\u00C2\u00A0consumers.\u00C2\u00A0Moreover,\u00C2\u00A0as\u00C2\u00A0urea\u00C2\u00A0and\u00C2\u00A0EC\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0strains\u00C2\u00A0is\u00C2\u00A0highly\u00C2\u00A0 variable,\u00C2\u00A0 studies\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 molecular\u00C2\u00A0 mechanisms\u00C2\u00A0 behind\u00C2\u00A0 these\u00C2\u00A0 variations\u00C2\u00A0 will\u00C2\u00A0 aid\u00C2\u00A0 scientists\u00C2\u00A0 and\u00C2\u00A0 winemakers\u00C2\u00A0in\u00C2\u00A0engineering\u00C2\u00A0new\u00C2\u00A0strains\u00C2\u00A0or\u00C2\u00A0selecting\u00C2\u00A0natural\u00C2\u00A0strains\u00C2\u00A0which\u00C2\u00A0produce\u00C2\u00A0little\u00C2\u00A0or\u00C2\u00A0no\u00C2\u00A0EC.\u00C2\u00A0Finally,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 imperative\u00C2\u00A0 that,\u00C2\u00A0before\u00C2\u00A0 the\u00C2\u00A0yeasts\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0commercialized,\u00C2\u00A0 the\u00C2\u00A0proteome\u00C2\u00A0and\u00C2\u00A0metabolome\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 metabolically\u00C2\u00A0 enhanced\u00C2\u00A0 yeast\u00C2\u00A0 strains\u00C2\u00A0 described\u00C2\u00A0 here\u00C2\u00A0 be\u00C2\u00A0 characterized\u00C2\u00A0 as\u00C2\u00A0 to\u00C2\u00A0 establish\u00C2\u00A0 substantial\u00C2\u00A0 equivalence.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 104\u00C2\u00A0 \u00C2\u00A0 REFERENCES\u00C2\u00A0 Ashby,\u00C2\u00A0 J.\u00C2\u00A0 (1991)\u00C2\u00A0Genotoxicity\u00C2\u00A0 data\u00C2\u00A0 supporting\u00C2\u00A0 the\u00C2\u00A0 proposed\u00C2\u00A0metabolic\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 ethyl\u00C2\u00A0 carbamate\u00C2\u00A0 (urethane)\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0carcinogen:\u00C2\u00A0the\u00C2\u00A0problem\u00C2\u00A0now\u00C2\u00A0posed\u00C2\u00A0by\u00C2\u00A0methyl\u00C2\u00A0carbamate.\u00C2\u00A0Mutat.\u00C2\u00A0Res.\u00C2\u00A0260,\u00C2\u00A0307\u00E2\u0080\u0090 308.\u00C2\u00A0 Ausubel,\u00C2\u00A0 F.M.,\u00C2\u00A0Brent,\u00C2\u00A0R.,\u00C2\u00A0 Kingston,\u00C2\u00A0R.E.,\u00C2\u00A0Moore,\u00C2\u00A0D.D.,\u00C2\u00A0 Seidman,\u00C2\u00A0 J.G.,\u00C2\u00A0 Smith,\u00C2\u00A0 J.A.\u00C2\u00A0 and\u00C2\u00A0 Struhl,\u00C2\u00A0 K.\u00C2\u00A0 (1995)\u00C2\u00A0 Short\u00C2\u00A0Protocols\u00C2\u00A0in\u00C2\u00A0Molecular\u00C2\u00A0Biology,\u00C2\u00A03rd\u00C2\u00A0ed.,\u00C2\u00A0John\u00C2\u00A0Wiley\u00C2\u00A0&\u00C2\u00A0Sons,\u00C2\u00A0Inc.,\u00C2\u00A0Boston,\u00C2\u00A0USA.\u00C2\u00A0 Baudin,\u00C2\u00A0A.\u00C2\u00A0 (1993)\u00C2\u00A0A\u00C2\u00A0simple\u00C2\u00A0and\u00C2\u00A0efficient\u00C2\u00A0method\u00C2\u00A0 for\u00C2\u00A0direct\u00C2\u00A0gene\u00C2\u00A0deletion\u00C2\u00A0 in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0 Nucl.\u00C2\u00A0Acids\u00C2\u00A0Res.\u00C2\u00A021,\u00C2\u00A03329\u00E2\u0080\u00903330.\u00C2\u00A0 Bisson,\u00C2\u00A0L.F.\u00C2\u00A0(2002)\u00C2\u00A0The\u00C2\u00A0present\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0international\u00C2\u00A0wine\u00C2\u00A0industry.\u00C2\u00A0Nature\u00C2\u00A0418,\u00C2\u00A0696\u00E2\u0080\u0090699.\u00C2\u00A0 Blake,\u00C2\u00A0C.\u00C2\u00A0C.\u00C2\u00A0(1981)\u00C2\u00A0Phosphoglycerate\u00C2\u00A0kinase.\u00C2\u00A0Philos.\u00C2\u00A0Trans.\u00C2\u00A0R.\u00C2\u00A0Soc.\u00C2\u00A0Lond.\u00C2\u00A0B.\u00C2\u00A0Biol.\u00C2\u00A0Sci.\u00C2\u00A0293,\u00C2\u00A093\u00E2\u0080\u0090104.\u00C2\u00A0 Bond,\u00C2\u00A0U.,\u00C2\u00A0Neal,\u00C2\u00A0C.,\u00C2\u00A0Donnelly,\u00C2\u00A0D.\u00C2\u00A0and\u00C2\u00A0 James,\u00C2\u00A0T.\u00C2\u00A0 (2004)\u00C2\u00A0Aneuploidy\u00C2\u00A0and\u00C2\u00A0copy\u00C2\u00A0number\u00C2\u00A0breakpoints\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 genome\u00C2\u00A0of\u00C2\u00A0lager\u00C2\u00A0yeasts\u00C2\u00A0mapped\u00C2\u00A0by\u00C2\u00A0microarray\u00C2\u00A0hybridisation.\u00C2\u00A0Curr.\u00C2\u00A0Genet.\u00C2\u00A045,\u00C2\u00A0360\u00E2\u0080\u0090370.\u00C2\u00A0 Bruckner,\u00C2\u00A0R.\u00C2\u00A0and\u00C2\u00A0Titgemeyer,\u00C2\u00A0F.\u00C2\u00A0 (2002)\u00C2\u00A0Carbon\u00C2\u00A0catabolite\u00C2\u00A0 repression\u00C2\u00A0 in\u00C2\u00A0bacteria:\u00C2\u00A0choice\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0carbon\u00C2\u00A0 source\u00C2\u00A0and\u00C2\u00A0autoregulatory\u00C2\u00A0limitation\u00C2\u00A0of\u00C2\u00A0sugar\u00C2\u00A0utilization.\u00C2\u00A0FEMS\u00C2\u00A0Microbiol.\u00C2\u00A0Lett.\u00C2\u00A0209,\u00C2\u00A0141\u00E2\u0080\u0090148.\u00C2\u00A0 Butzke,\u00C2\u00A0 C.E.\u00C2\u00A0 and\u00C2\u00A0 Bisson,\u00C2\u00A0 L.F.\u00C2\u00A0 (1998)\u00C2\u00A0 Ethyl\u00C2\u00A0 Carbamate\u00C2\u00A0 Preventative\u00C2\u00A0 Action\u00C2\u00A0 Manual.\u00C2\u00A0 2008.\u00C2\u00A0 (http://www.cfsan.fda.gov/~frf/ecaction.html).\u00C2\u00A0 Bysani,\u00C2\u00A0N.,\u00C2\u00A0Daugherty,\u00C2\u00A0 J.\u00C2\u00A0R.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0 (1991)\u00C2\u00A0Saturation\u00C2\u00A0mutagenesis\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0UASNTR\u00C2\u00A0 (GATAA)\u00C2\u00A0 responsible\u00C2\u00A0for\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00E2\u0080\u0090sensitive\u00C2\u00A0transcriptional\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0allantoin\u00C2\u00A0 pathway\u00C2\u00A0genes\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Bacteriol.\u00C2\u00A0173,\u00C2\u00A04977\u00E2\u0080\u00904982.\u00C2\u00A0 Canas,\u00C2\u00A0B.\u00C2\u00A0J.,\u00C2\u00A0Havery,\u00C2\u00A0D.\u00C2\u00A0C.,\u00C2\u00A0Robinson,\u00C2\u00A0L.\u00C2\u00A0R.,\u00C2\u00A0Sullivan,\u00C2\u00A0M.\u00C2\u00A0P.,\u00C2\u00A0Joe,\u00C2\u00A0F.\u00C2\u00A0L.,\u00C2\u00A0Jr\u00C2\u00A0and\u00C2\u00A0Diachenko,\u00C2\u00A0G.\u00C2\u00A0W.\u00C2\u00A0(1989)\u00C2\u00A0Ethyl\u00C2\u00A0 carbamate\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0selected\u00C2\u00A0fermented\u00C2\u00A0foods\u00C2\u00A0and\u00C2\u00A0beverages.\u00C2\u00A0J.\u00C2\u00A0Assoc.\u00C2\u00A0Off.\u00C2\u00A0Anal.\u00C2\u00A0Chem.\u00C2\u00A072,\u00C2\u00A0873\u00E2\u0080\u0090 876.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 105\u00C2\u00A0 \u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 G.\u00C2\u00A0 (1982)\u00C2\u00A0 Nitrogen\u00C2\u00A0 metabolism\u00C2\u00A0 in\u00C2\u00A0 S.\u00C2\u00A0 cerevisiae;\u00C2\u00A0 in\u00C2\u00A0 The\u00C2\u00A0 molecular\u00C2\u00A0 biology\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 yeast:\u00C2\u00A0 metabolism\u00C2\u00A0and\u00C2\u00A0gene\u00C2\u00A0expression.\u00C2\u00A0J.N.\u00C2\u00A0Strathern,\u00C2\u00A0E.W.\u00C2\u00A0Jones\u00C2\u00A0(ed.),\u00C2\u00A0pp.\u00C2\u00A039\u00E2\u0080\u009099,\u00C2\u00A0Cold\u00C2\u00A0Spring\u00C2\u00A0Harbor\u00C2\u00A0 Press,\u00C2\u00A0New\u00C2\u00A0York.\u00C2\u00A0 Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0(2002)\u00C2\u00A0Transmitting\u00C2\u00A0the\u00C2\u00A0signal\u00C2\u00A0of\u00C2\u00A0excess\u00C2\u00A0nitrogen\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0Tor\u00C2\u00A0 proteins\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0GATA\u00C2\u00A0factors:\u00C2\u00A0connecting\u00C2\u00A0the\u00C2\u00A0dots.\u00C2\u00A0FEMS\u00C2\u00A0Microbiol.\u00C2\u00A0Rev.\u00C2\u00A026,\u00C2\u00A0223\u00E2\u0080\u0090238.\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 G.,\u00C2\u00A0 Ferguson,\u00C2\u00A0 D.,\u00C2\u00A0 Rai,\u00C2\u00A0 R.\u00C2\u00A0 and\u00C2\u00A0 Bysani,\u00C2\u00A0 N.\u00C2\u00A0 (1990)\u00C2\u00A0 The\u00C2\u00A0 GLN3\u00C2\u00A0 gene\u00C2\u00A0 product\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 transcriptional\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 allantoin\u00C2\u00A0 system\u00C2\u00A0 gene\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0 J.\u00C2\u00A0 Bacteriol.\u00C2\u00A0172,\u00C2\u00A01014\u00E2\u0080\u00901018.\u00C2\u00A0 Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0and\u00C2\u00A0Sumrada,\u00C2\u00A0R.\u00C2\u00A0(1975)\u00C2\u00A0Urea\u00C2\u00A0transport\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Bacteriol.\u00C2\u00A0121,\u00C2\u00A0571\u00E2\u0080\u0090 576.\u00C2\u00A0 Coulon,\u00C2\u00A0J.,\u00C2\u00A0Husnik,\u00C2\u00A0J.\u00C2\u00A0I.,\u00C2\u00A0Inglis,\u00C2\u00A0D.\u00C2\u00A0L.,\u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Merwe,\u00C2\u00A0G.\u00C2\u00A0K.,\u00C2\u00A0Lonvaud,\u00C2\u00A0A.,\u00C2\u00A0Erasmus,\u00C2\u00A0D.\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0van\u00C2\u00A0Vuuren,\u00C2\u00A0H.\u00C2\u00A0 J.\u00C2\u00A0 J.\u00C2\u00A0 (2006)\u00C2\u00A0Metabolic\u00C2\u00A0 Engineering\u00C2\u00A0of\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae\u00C2\u00A0 to\u00C2\u00A0Minimize\u00C2\u00A0 the\u00C2\u00A0Production\u00C2\u00A0of\u00C2\u00A0 Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0in\u00C2\u00A0Wine.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A057,\u00C2\u00A0113\u00E2\u0080\u0090124.\u00C2\u00A0 Cox,\u00C2\u00A0J.\u00C2\u00A0S.\u00C2\u00A0and\u00C2\u00A0Walter,\u00C2\u00A0P.\u00C2\u00A0(1996)\u00C2\u00A0A\u00C2\u00A0Novel\u00C2\u00A0Mechanism\u00C2\u00A0for\u00C2\u00A0Regulating\u00C2\u00A0Activity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0Transcription\u00C2\u00A0Factor\u00C2\u00A0That\u00C2\u00A0 Controls\u00C2\u00A0the\u00C2\u00A0Unfolded\u00C2\u00A0Protein\u00C2\u00A0Response.\u00C2\u00A0Cell,\u00C2\u00A087,\u00C2\u00A0391\u00E2\u0080\u0090404.\u00C2\u00A0 Cox,\u00C2\u00A0K.\u00C2\u00A0H.,\u00C2\u00A0Rai,\u00C2\u00A0R.,\u00C2\u00A0Distler,\u00C2\u00A0M.,\u00C2\u00A0Daugherty,\u00C2\u00A0J.\u00C2\u00A0R.,\u00C2\u00A0Coffman,\u00C2\u00A0J.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0(2000)\u00C2\u00A0Saccharomyces\u00C2\u00A0 cerevisiae\u00C2\u00A0GATA\u00C2\u00A0sequences\u00C2\u00A0function\u00C2\u00A0as\u00C2\u00A0TATA\u00C2\u00A0elements\u00C2\u00A0during\u00C2\u00A0nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0and\u00C2\u00A0 when\u00C2\u00A0Gln3p\u00C2\u00A0is\u00C2\u00A0excluded\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0nucleus\u00C2\u00A0by\u00C2\u00A0overproduction\u00C2\u00A0of\u00C2\u00A0Ure2p.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0275,\u00C2\u00A017611\u00E2\u0080\u0090 17618.\u00C2\u00A0 Cox,\u00C2\u00A0 K.\u00C2\u00A0 H.,\u00C2\u00A0 Kulkarni,\u00C2\u00A0 A.,\u00C2\u00A0 Tate,\u00C2\u00A0 J.\u00C2\u00A0 J.\u00C2\u00A0 and\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 G.\u00C2\u00A0 (2004)\u00C2\u00A0 Gln3\u00C2\u00A0 Phosphorylation\u00C2\u00A0 and\u00C2\u00A0 Intracellular\u00C2\u00A0 Localization\u00C2\u00A0 in\u00C2\u00A0Nutrient\u00C2\u00A0 Limitation\u00C2\u00A0 and\u00C2\u00A0 Starvation\u00C2\u00A0Differ\u00C2\u00A0 from\u00C2\u00A0 Those\u00C2\u00A0Generated\u00C2\u00A0 by\u00C2\u00A0Rapamycin\u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0Tor1/2\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0279,\u00C2\u00A010270\u00E2\u0080\u009010278.\u00C2\u00A0 Dahl,\u00C2\u00A0G.,\u00C2\u00A0Miller,\u00C2\u00A0 J.\u00C2\u00A0 and\u00C2\u00A0Miller,\u00C2\u00A0 E.\u00C2\u00A0 (1978)\u00C2\u00A0 Vinyl\u00C2\u00A0 carbamate\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 promutagen\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0more\u00C2\u00A0 carcinogenic\u00C2\u00A0 analog\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0carbamate.\u00C2\u00A0Cancer\u00C2\u00A0Res.\u00C2\u00A038,\u00C2\u00A03793\u00E2\u0080\u00903804.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 106\u00C2\u00A0 \u00C2\u00A0 Dann,\u00C2\u00A0S.\u00C2\u00A0G.\u00C2\u00A0and\u00C2\u00A0Thomas,\u00C2\u00A0G.\u00C2\u00A0(2006)\u00C2\u00A0The\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0sensitive\u00C2\u00A0TOR\u00C2\u00A0pathway\u00C2\u00A0from\u00C2\u00A0yeast\u00C2\u00A0to\u00C2\u00A0mammals.\u00C2\u00A0FEBS\u00C2\u00A0 Letters\u00C2\u00A0580,\u00C2\u00A02821\u00E2\u0080\u00902829.\u00C2\u00A0 De\u00C2\u00A0Virgilio,\u00C2\u00A0C.\u00C2\u00A0and\u00C2\u00A0Loewith,\u00C2\u00A0R.\u00C2\u00A0(2006)\u00C2\u00A0The\u00C2\u00A0TOR\u00C2\u00A0signalling\u00C2\u00A0network\u00C2\u00A0from\u00C2\u00A0yeast\u00C2\u00A0to\u00C2\u00A0man.\u00C2\u00A0Int.\u00C2\u00A0J.\u00C2\u00A0Biochem.\u00C2\u00A0Cell\u00C2\u00A0 Bio.\u00C2\u00A038,\u00C2\u00A01476\u00E2\u0080\u00901481.\u00C2\u00A0 Dohmen,\u00C2\u00A0R.\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0Varshavsky,\u00C2\u00A0A.\u00C2\u00A0(2005)\u00C2\u00A0Heat\u00E2\u0080\u0090inducible\u00C2\u00A0degron\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0making\u00C2\u00A0of\u00C2\u00A0conditional\u00C2\u00A0mutants.\u00C2\u00A0 Methods\u00C2\u00A0Enzymol.\u00C2\u00A0399,\u00C2\u00A0799\u00E2\u0080\u0090822.\u00C2\u00A0 Dunn,\u00C2\u00A0B.,\u00C2\u00A0Levine,\u00C2\u00A0R.\u00C2\u00A0P.\u00C2\u00A0and\u00C2\u00A0Sherlock,\u00C2\u00A0G.\u00C2\u00A0(2005)\u00C2\u00A0Microarray\u00C2\u00A0karyotyping\u00C2\u00A0of\u00C2\u00A0commercial\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strains\u00C2\u00A0 reveals\u00C2\u00A0shared,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0unique,\u00C2\u00A0genomic\u00C2\u00A0signatures.\u00C2\u00A0BMC\u00C2\u00A0Genomics\u00C2\u00A06,\u00C2\u00A053\u00E2\u0080\u009074.\u00C2\u00A0 Eckhardt,\u00C2\u00A0T.\u00C2\u00A0(1978)\u00C2\u00A0A\u00C2\u00A0rapid\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 identification\u00C2\u00A0of\u00C2\u00A0plasmid\u00C2\u00A0desoxyribonucleic\u00C2\u00A0acid\u00C2\u00A0 in\u00C2\u00A0bacteria.\u00C2\u00A0 Plasmid\u00C2\u00A01,\u00C2\u00A0584\u00E2\u0080\u0090588.\u00C2\u00A0 ElBerry,\u00C2\u00A0H.\u00C2\u00A0M.,\u00C2\u00A0Majumdar,\u00C2\u00A0M.\u00C2\u00A0L.,\u00C2\u00A0Cunningham,\u00C2\u00A0T.\u00C2\u00A0S.,\u00C2\u00A0Sumrada,\u00C2\u00A0R.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0(1993)\u00C2\u00A0Regulation\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0urea\u00C2\u00A0active\u00C2\u00A0transporter\u00C2\u00A0gene\u00C2\u00A0(DUR3)\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Bacteriol.\u00C2\u00A0175,\u00C2\u00A04688\u00E2\u0080\u0090 4698.\u00C2\u00A0 Farrell\u00C2\u00A0A.,\u00C2\u00A0E.,\u00C2\u00A0Plevin\u00C2\u00A0R.,\u00C2\u00A0J.,\u00C2\u00A0Turner\u00C2\u00A0B.,\u00C2\u00A0T.,\u00C2\u00A0Jones\u00C2\u00A0A.,\u00C2\u00A0D.,\u00C2\u00A0O'Hare,\u00C2\u00A0M.\u00C2\u00A0and\u00C2\u00A0Kammen\u00C2\u00A0D.,\u00C2\u00A0M.\u00C2\u00A0(2006)\u00C2\u00A0Ethanol\u00C2\u00A0Can\u00C2\u00A0 Contribute\u00C2\u00A0to\u00C2\u00A0Energy\u00C2\u00A0and\u00C2\u00A0Environmental\u00C2\u00A0Goals.\u00C2\u00A0Science\u00C2\u00A0311,\u00C2\u00A0506\u00E2\u0080\u0090508.\u00C2\u00A0 Gancedo\u00C2\u00A0,\u00C2\u00A0J.\u00C2\u00A0M.\u00C2\u00A0(1992)\u00C2\u00A0Carbon\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0in\u00C2\u00A0yeast.\u00C2\u00A0Eur.\u00C2\u00A0J.\u00C2\u00A0Biochem.\u00C2\u00A0\u00C2\u00A0206,\u00C2\u00A0297\u00E2\u0080\u0090313.\u00C2\u00A0 Gatignol,\u00C2\u00A0A.\u00C2\u00A0(1987)\u00C2\u00A0Phleomycin\u00C2\u00A0resistance\u00C2\u00A0encoded\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0ble\u00C2\u00A0gene\u00C2\u00A0from\u00C2\u00A0transposon\u00C2\u00A0Tn\u00C2\u00A05\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0dominant\u00C2\u00A0 selectable\u00C2\u00A0marker\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Mol.\u00C2\u00A0Gen.\u00C2\u00A0Genetics\u00C2\u00A0207,\u00C2\u00A0342\u00E2\u0080\u0090348.\u00C2\u00A0 Genbauffe,\u00C2\u00A0 F.\u00C2\u00A0 S.\u00C2\u00A0 and\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 G.\u00C2\u00A0 (1991)\u00C2\u00A0 The\u00C2\u00A0 urea\u00C2\u00A0 amidolyase\u00C2\u00A0 (DUR1,2)\u00C2\u00A0 gene\u00C2\u00A0 of\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0DNA\u00C2\u00A0Seq.\u00C2\u00A02,\u00C2\u00A019\u00E2\u0080\u009032.\u00C2\u00A0 Genbauffe,\u00C2\u00A0 F.\u00C2\u00A0 S.\u00C2\u00A0 and\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0G.\u00C2\u00A0 (1986)\u00C2\u00A0 Induction\u00C2\u00A0 and\u00C2\u00A0 repression\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 urea\u00C2\u00A0 amidolyase\u00C2\u00A0 gene\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Mol.\u00C2\u00A0Cell.\u00C2\u00A0Biol.\u00C2\u00A06,\u00C2\u00A03954\u00E2\u0080\u00903964.\u00C2\u00A0 Gietz,\u00C2\u00A0R.\u00C2\u00A0D.\u00C2\u00A0and\u00C2\u00A0Woods,\u00C2\u00A0R.\u00C2\u00A0A.\u00C2\u00A0(2002)\u00C2\u00A0Transformation\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0by\u00C2\u00A0 lithium\u00C2\u00A0acetate/single\u00E2\u0080\u0090stranded\u00C2\u00A0carrier\u00C2\u00A0 DNA/polyethylene\u00C2\u00A0glycol\u00C2\u00A0method.\u00C2\u00A0Methods\u00C2\u00A0Enzymol.\u00C2\u00A0350,\u00C2\u00A087\u00E2\u0080\u009096.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 107\u00C2\u00A0 \u00C2\u00A0 Goffeau,\u00C2\u00A0A.,\u00C2\u00A0Barrell,\u00C2\u00A0B.\u00C2\u00A0G.,\u00C2\u00A0Bussey,\u00C2\u00A0H.,\u00C2\u00A0Davis,\u00C2\u00A0R.\u00C2\u00A0W.,\u00C2\u00A0Dujon,\u00C2\u00A0B.,\u00C2\u00A0Feldmann,\u00C2\u00A0H.,\u00C2\u00A0Galibert,\u00C2\u00A0F.,\u00C2\u00A0Hoheisel,\u00C2\u00A0J.\u00C2\u00A0D.,\u00C2\u00A0 Jacq,\u00C2\u00A0C.,\u00C2\u00A0 Johnston,\u00C2\u00A0M.,\u00C2\u00A0Louis,\u00C2\u00A0E.\u00C2\u00A0 J.,\u00C2\u00A0Mewes,\u00C2\u00A0H.\u00C2\u00A0W.,\u00C2\u00A0Murakami,\u00C2\u00A0Y.,\u00C2\u00A0Philippsen,\u00C2\u00A0P.,\u00C2\u00A0Tettelin,\u00C2\u00A0H.\u00C2\u00A0and\u00C2\u00A0 Oliver,\u00C2\u00A0S.\u00C2\u00A0G.\u00C2\u00A0(1996)\u00C2\u00A0Life\u00C2\u00A0with\u00C2\u00A06000\u00C2\u00A0Genes.\u00C2\u00A0Science\u00C2\u00A0274,\u00C2\u00A0546\u00E2\u0080\u0090567.\u00C2\u00A0 Goode,\u00C2\u00A0 J.\u00C2\u00A0 (2005)\u00C2\u00A0 The\u00C2\u00A0 Science\u00C2\u00A0 of\u00C2\u00A0Wine:\u00C2\u00A0 From\u00C2\u00A0 Vine\u00C2\u00A0 to\u00C2\u00A0 Glass,\u00C2\u00A0 University\u00C2\u00A0 of\u00C2\u00A0 California\u00C2\u00A0 Press,\u00C2\u00A0 Berkley,\u00C2\u00A0 California.\u00C2\u00A0 Greig,\u00C2\u00A0D.\u00C2\u00A0and\u00C2\u00A0Travisano,\u00C2\u00A0M.\u00C2\u00A0(2003)\u00C2\u00A0Evolution:\u00C2\u00A0Haploid\u00C2\u00A0Superiority.\u00C2\u00A0Science\u00C2\u00A0299,\u00C2\u00A0524\u00E2\u0080\u0090525.\u00C2\u00A0 Griffiths,\u00C2\u00A0 A.\u00C2\u00A0 J.\u00C2\u00A0 F.,\u00C2\u00A0 Miller,\u00C2\u00A0 J.\u00C2\u00A0 H.,\u00C2\u00A0 Suzuki,\u00C2\u00A0 D.\u00C2\u00A0 T.,\u00C2\u00A0 Lewontin,\u00C2\u00A0 R.\u00C2\u00A0 C.,\u00C2\u00A0 and\u00C2\u00A0 Gelbart,\u00C2\u00A0 W.\u00C2\u00A0 M.\u00C2\u00A0 (2005)\u00C2\u00A0\u00C2\u00A0 An\u00C2\u00A0Introduction\u00C2\u00A0to\u00C2\u00A0Genetic\u00C2\u00A0Analysis.\u00C2\u00A0Eight\u00C2\u00A0ed.,\u00C2\u00A0W.\u00C2\u00A0H.\u00C2\u00A0Freeman,\u00C2\u00A0New\u00C2\u00A0York,\u00C2\u00A0USA.\u00C2\u00A0 Guengerich,\u00C2\u00A0F.\u00C2\u00A0P.\u00C2\u00A0and\u00C2\u00A0Kim,\u00C2\u00A0D.\u00C2\u00A0H.\u00C2\u00A0(1991)\u00C2\u00A0Enzymatic\u00C2\u00A0oxidation\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0to\u00C2\u00A0vinyl\u00C2\u00A0carbamate\u00C2\u00A0and\u00C2\u00A0 its\u00C2\u00A0role\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0intermediate\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A01,N6\u00E2\u0080\u0090ethenoadenosine.\u00C2\u00A0Chem.\u00C2\u00A0Res.\u00C2\u00A0Toxicol.\u00C2\u00A04,\u00C2\u00A0413\u00E2\u0080\u0090 421.\u00C2\u00A0 Guldener,\u00C2\u00A0U.,\u00C2\u00A0Heck,\u00C2\u00A0S.,\u00C2\u00A0Fielder,\u00C2\u00A0T.,\u00C2\u00A0Beinhauer,\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0Hegemann,\u00C2\u00A0J.\u00C2\u00A0(1996)\u00C2\u00A0A\u00C2\u00A0new\u00C2\u00A0efficient\u00C2\u00A0gene\u00C2\u00A0disruption\u00C2\u00A0 cassette\u00C2\u00A0for\u00C2\u00A0repeated\u00C2\u00A0use\u00C2\u00A0in\u00C2\u00A0budding\u00C2\u00A0yeast.\u00C2\u00A0Nucl.\u00C2\u00A0Acids\u00C2\u00A0Res.\u00C2\u00A024,\u00C2\u00A02519\u00E2\u0080\u00902524.\u00C2\u00A0 Hampsey,\u00C2\u00A0M.\u00C2\u00A0(1997)\u00C2\u00A0A\u00C2\u00A0review\u00C2\u00A0of\u00C2\u00A0phenotypes\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Yeast\u00C2\u00A013,\u00C2\u00A01099\u00E2\u0080\u00901133.\u00C2\u00A0 Han,\u00C2\u00A0 J.,\u00C2\u00A0 Lee,\u00C2\u00A0 J.,\u00C2\u00A0 Bibbs,\u00C2\u00A0 L.\u00C2\u00A0 and\u00C2\u00A0 Ulevitch,\u00C2\u00A0 R.\u00C2\u00A0 (1994)\u00C2\u00A0 A\u00C2\u00A0 MAP\u00C2\u00A0 kinase\u00C2\u00A0 targeted\u00C2\u00A0 by\u00C2\u00A0 endotoxin\u00C2\u00A0 and\u00C2\u00A0 hyperosmolarity\u00C2\u00A0in\u00C2\u00A0mammalian\u00C2\u00A0cells.\u00C2\u00A0Science\u00C2\u00A0265,\u00C2\u00A0808\u00E2\u0080\u0090811.\u00C2\u00A0 Hashizume,\u00C2\u00A0K.,\u00C2\u00A0Okuda,\u00C2\u00A0M.,\u00C2\u00A0Sakurao,\u00C2\u00A0S.,\u00C2\u00A0Numata,\u00C2\u00A0M.,\u00C2\u00A0Koseki,\u00C2\u00A0T.,\u00C2\u00A0Aramaki,\u00C2\u00A0 I.,\u00C2\u00A0Kumamaru,\u00C2\u00A0T.\u00C2\u00A0and\u00C2\u00A0Sato,\u00C2\u00A0H.\u00C2\u00A0 (2006)\u00C2\u00A0Rice\u00C2\u00A0protein\u00C2\u00A0digestion\u00C2\u00A0by\u00C2\u00A0sake\u00C2\u00A0koji\u00C2\u00A0enzymes:\u00C2\u00A0comparison\u00C2\u00A0between\u00C2\u00A0steamed\u00C2\u00A0rice\u00C2\u00A0grains\u00C2\u00A0and\u00C2\u00A0 isolated\u00C2\u00A0protein\u00C2\u00A0bodies\u00C2\u00A0from\u00C2\u00A0rice\u00C2\u00A0endosperm.\u00C2\u00A0J.\u00C2\u00A0Biosci.\u00C2\u00A0Bioeng.\u00C2\u00A0102,\u00C2\u00A0340\u00E2\u0080\u0090345.\u00C2\u00A0 Hauser,\u00C2\u00A0 N.,\u00C2\u00A0 Fellenberg,\u00C2\u00A0 R.,\u00C2\u00A0 Gil,\u00C2\u00A0 R.,\u00C2\u00A0 Bastuk,\u00C2\u00A0 S.,\u00C2\u00A0 Hoheisel,\u00C2\u00A0 J.\u00C2\u00A0 and\u00C2\u00A0 Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0 J.\u00C2\u00A0 (2001)\u00C2\u00A0Whole\u00C2\u00A0 genome\u00C2\u00A0 analysis\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0strain.\u00C2\u00A0Comp.\u00C2\u00A0Funct.\u00C2\u00A0Genomics\u00C2\u00A02,\u00C2\u00A069\u00E2\u0080\u009079.\u00C2\u00A0 Herskowitz,\u00C2\u00A0I.\u00C2\u00A0(1988)\u00C2\u00A0Life\u00C2\u00A0cycle\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0budding\u00C2\u00A0yeast\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Microbiol.\u00C2\u00A0Rev.\u00C2\u00A052,\u00C2\u00A0536\u00E2\u0080\u0090 553.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 108\u00C2\u00A0 \u00C2\u00A0 Hitzeman,\u00C2\u00A0R.\u00C2\u00A0A.\u00C2\u00A0(1980)\u00C2\u00A0Isolation\u00C2\u00A0and\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A03\u00E2\u0080\u0090phosphoglycerokinase\u00C2\u00A0gene\u00C2\u00A0(PGK)\u00C2\u00A0by\u00C2\u00A0 an\u00C2\u00A0immunological\u00C2\u00A0screening\u00C2\u00A0technique.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0255,\u00C2\u00A012073\u00E2\u0080\u009012080.\u00C2\u00A0 Hofman\u00E2\u0080\u0090Bang,\u00C2\u00A0J.\u00C2\u00A0(1999)\u00C2\u00A0Nitrogen\u00C2\u00A0catabolite\u00C2\u00A0repression\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Mol.\u00C2\u00A0Biotechnol.\u00C2\u00A012,\u00C2\u00A0 35\u00E2\u0080\u009073.\u00C2\u00A0 Hughes,\u00C2\u00A0T.,\u00C2\u00A0Roberts,\u00C2\u00A0C.,\u00C2\u00A0Dai,\u00C2\u00A0H.,\u00C2\u00A0Jones,\u00C2\u00A0A.,\u00C2\u00A0Meyer,\u00C2\u00A0M.,\u00C2\u00A0Slade,\u00C2\u00A0D.,\u00C2\u00A0Burchard,\u00C2\u00A0J.,\u00C2\u00A0Dow,\u00C2\u00A0S.,\u00C2\u00A0Ward,\u00C2\u00A0T.,\u00C2\u00A0Kidd,\u00C2\u00A0M.,\u00C2\u00A0 Friend,\u00C2\u00A0S.\u00C2\u00A0and\u00C2\u00A0Marton,\u00C2\u00A0M.\u00C2\u00A0(2000)\u00C2\u00A0Widespread\u00C2\u00A0aneuploidy\u00C2\u00A0revealed\u00C2\u00A0by\u00C2\u00A0DNA\u00C2\u00A0microarray\u00C2\u00A0expression\u00C2\u00A0 profiling.\u00C2\u00A0Nat.\u00C2\u00A0Genet.\u00C2\u00A025,\u00C2\u00A0333\u00E2\u0080\u0090337.\u00C2\u00A0 Husnik,\u00C2\u00A0 J.\u00C2\u00A0 I.,\u00C2\u00A0Volschenk,\u00C2\u00A0H.,\u00C2\u00A0Bauer,\u00C2\u00A0 J.,\u00C2\u00A0Colavizza,\u00C2\u00A0D.,\u00C2\u00A0 Luo,\u00C2\u00A0 Z.\u00C2\u00A0 and\u00C2\u00A0 van\u00C2\u00A0Vuuren,\u00C2\u00A0H.\u00C2\u00A0 J.\u00C2\u00A0 J.\u00C2\u00A0 (2006)\u00C2\u00A0Metabolic\u00C2\u00A0 engineering\u00C2\u00A0of\u00C2\u00A0malolactic\u00C2\u00A0wine\u00C2\u00A0yeast.\u00C2\u00A0Metabol.\u00C2\u00A0Eng.\u00C2\u00A08,\u00C2\u00A0315\u00E2\u0080\u0090323.\u00C2\u00A0 Iemura,\u00C2\u00A0 Y.,\u00C2\u00A0 Takahashi,\u00C2\u00A0 T.,\u00C2\u00A0 Yamada,\u00C2\u00A0 T.,\u00C2\u00A0 Furukawa,\u00C2\u00A0 K.\u00C2\u00A0 and\u00C2\u00A0Hara,\u00C2\u00A0 S.\u00C2\u00A0 (1999a)\u00C2\u00A0 Properties\u00C2\u00A0 of\u00C2\u00A0 TCA\u00E2\u0080\u0090Insoluble\u00C2\u00A0 peptides\u00C2\u00A0in\u00C2\u00A0Kimoto\u00C2\u00A0(traditional\u00C2\u00A0seed\u00C2\u00A0mash\u00C2\u00A0for\u00C2\u00A0sake\u00C2\u00A0brewing)\u00C2\u00A0and\u00C2\u00A0conditions\u00C2\u00A0for\u00C2\u00A0liberation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 peptides\u00C2\u00A0from\u00C2\u00A0rice\u00C2\u00A0protein.\u00C2\u00A0J.\u00C2\u00A0Biosci.\u00C2\u00A0Bioeng.\u00C2\u00A088,\u00C2\u00A0531\u00E2\u0080\u0090535.\u00C2\u00A0 Iemura,\u00C2\u00A0 Y.,\u00C2\u00A0 Yamada,\u00C2\u00A0 T.,\u00C2\u00A0 Takahashi,\u00C2\u00A0 T.,\u00C2\u00A0 Furukawa,\u00C2\u00A0 K.\u00C2\u00A0 and\u00C2\u00A0Hara,\u00C2\u00A0 S.\u00C2\u00A0 (1999b)\u00C2\u00A0 Properties\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 peptides\u00C2\u00A0 liberated\u00C2\u00A0from\u00C2\u00A0rice\u00C2\u00A0protein\u00C2\u00A0in\u00C2\u00A0Sokujo\u00E2\u0080\u0090moto.\u00C2\u00A0J.\u00C2\u00A0Biosci.\u00C2\u00A0Bioeng.\u00C2\u00A088,\u00C2\u00A0276\u00E2\u0080\u0090280.\u00C2\u00A0 Ingledew,\u00C2\u00A0W.\u00C2\u00A0M.,\u00C2\u00A0Magnus,\u00C2\u00A0C.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Patterson,\u00C2\u00A0J.\u00C2\u00A0R.\u00C2\u00A0(1987)\u00C2\u00A0Yeast\u00C2\u00A0Foods\u00C2\u00A0and\u00C2\u00A0Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0Formation\u00C2\u00A0 in\u00C2\u00A0Wine.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A038,\u00C2\u00A0332\u00E2\u0080\u0090335.\u00C2\u00A0 Ingledew,\u00C2\u00A0W.\u00C2\u00A0M.,\u00C2\u00A0Magnus,\u00C2\u00A0C.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Sosulski,\u00C2\u00A0F.\u00C2\u00A0W.\u00C2\u00A0 (1987)\u00C2\u00A0 Influence\u00C2\u00A0of\u00C2\u00A0Oxygen\u00C2\u00A0on\u00C2\u00A0Proline\u00C2\u00A0Utilization\u00C2\u00A0 During\u00C2\u00A0the\u00C2\u00A0Wine\u00C2\u00A0Fermentation.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A038,\u00C2\u00A0246\u00E2\u0080\u0090248.\u00C2\u00A0 Inoue,T\u00C2\u00A0 .\u00C2\u00A0 (2000)\u00C2\u00A0Cloning\u00C2\u00A0and\u00C2\u00A0Characterization\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0Gene\u00C2\u00A0Complementing\u00C2\u00A0 the\u00C2\u00A0Mutation\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0Ethanol\u00E2\u0080\u0090 sensitive\u00C2\u00A0Mutant\u00C2\u00A0of\u00C2\u00A0Sake\u00C2\u00A0Yeast.\u00C2\u00A0Biosci.\u00C2\u00A0Biotech.\u00C2\u00A0Biochem.\u00C2\u00A064,\u00C2\u00A0229\u00E2\u0080\u0090236.\u00C2\u00A0 Jacobs,\u00C2\u00A0E.,\u00C2\u00A0Dubois,\u00C2\u00A0E.\u00C2\u00A0and\u00C2\u00A0Wiame,\u00C2\u00A0J.\u00C2\u00A0M.\u00C2\u00A0(1985)\u00C2\u00A0Regulation\u00C2\u00A0of\u00C2\u00A0urea\u00C2\u00A0amidolyase\u00C2\u00A0synthesis\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0 cerevisiae,\u00C2\u00A0RNA\u00C2\u00A0analysis,\u00C2\u00A0and\u00C2\u00A0cloning\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0positive\u00C2\u00A0regulatory\u00C2\u00A0gene\u00C2\u00A0DURM.\u00C2\u00A0Curr.\u00C2\u00A0Genet.\u00C2\u00A09,\u00C2\u00A0333\u00E2\u0080\u0090 339.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 109\u00C2\u00A0 \u00C2\u00A0 Jahnke,\u00C2\u00A0 L.\u00C2\u00A0 (1983)\u00C2\u00A0 Oxygen\u00C2\u00A0 requirements\u00C2\u00A0 for\u00C2\u00A0 formation\u00C2\u00A0 and\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 squalene\u00C2\u00A0 epoxidase\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Bacteriol\u00C2\u00A0\u00C2\u00A0155,\u00C2\u00A0488\u00E2\u0080\u0090492.\u00C2\u00A0 Jones\u00C2\u00A0 E.\u00C2\u00A0 W.,\u00C2\u00A0 Pringle\u00C2\u00A0 J.\u00C2\u00A0 R.,\u00C2\u00A0 Broach\u00C2\u00A0 J.\u00C2\u00A0 R.\u00C2\u00A0 (1992)\u00C2\u00A0 The\u00C2\u00A0 Molecular\u00C2\u00A0 and\u00C2\u00A0 Cellular\u00C2\u00A0 Biology\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Yeast\u00C2\u00A0 Saccharomyces,\u00C2\u00A0Cold\u00C2\u00A0Spring\u00C2\u00A0Harbor\u00C2\u00A0Laboratory\u00C2\u00A0Press,\u00C2\u00A0Cold\u00C2\u00A0Spring\u00C2\u00A0Harbor,\u00C2\u00A0New\u00C2\u00A0York,\u00C2\u00A0USA.\u00C2\u00A0\u00C2\u00A0 Kessler,\u00C2\u00A0D.\u00C2\u00A0A.\u00C2\u00A0(1992)\u00C2\u00A0The\u00C2\u00A0safety\u00C2\u00A0of\u00C2\u00A0foods\u00C2\u00A0developed\u00C2\u00A0by\u00C2\u00A0biotechnology.\u00C2\u00A0Science\u00C2\u00A0256,\u00C2\u00A01747\u00E2\u0080\u00901749.\u00C2\u00A0 Kinzy,\u00C2\u00A0 T.\u00C2\u00A0 G.\u00C2\u00A0 (1994)\u00C2\u00A0 Multiple\u00C2\u00A0 genes\u00C2\u00A0 encode\u00C2\u00A0 the\u00C2\u00A0 translation\u00C2\u00A0 elongation\u00C2\u00A0 factor\u00C2\u00A0 EF\u00E2\u0080\u00901\u00C2\u00A0 gamma\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Nucl.\u00C2\u00A0Acids\u00C2\u00A0Res.\u00C2\u00A022,\u00C2\u00A02703\u00E2\u0080\u00902707.\u00C2\u00A0 Kitamoto,\u00C2\u00A0K.,\u00C2\u00A0Oda,\u00C2\u00A0K.,\u00C2\u00A0Gomi,\u00C2\u00A0K.\u00C2\u00A0and\u00C2\u00A0Takahashi,\u00C2\u00A0K.\u00C2\u00A0(1991)\u00C2\u00A0Genetic\u00C2\u00A0engineering\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0sake\u00C2\u00A0yeast\u00C2\u00A0producing\u00C2\u00A0 no\u00C2\u00A0urea\u00C2\u00A0by\u00C2\u00A0successive\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0arginase\u00C2\u00A0gene.\u00C2\u00A0Appl.\u00C2\u00A0Environ.\u00C2\u00A0Microbiol.\u00C2\u00A057,\u00C2\u00A0301\u00E2\u0080\u0090306.\u00C2\u00A0 Kizaki,\u00C2\u00A0Y.,\u00C2\u00A0Inoue,\u00C2\u00A0Y.,\u00C2\u00A0Okazaki,\u00C2\u00A0N.\u00C2\u00A0and\u00C2\u00A0Kobayashi,\u00C2\u00A0S.\u00C2\u00A0(1991)\u00C2\u00A0Isolation\u00C2\u00A0and\u00C2\u00A0determination\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0bodies\u00C2\u00A0 (PB\u00E2\u0080\u0090I,\u00C2\u00A0PB\u00E2\u0080\u0090II)\u00C2\u00A0in\u00C2\u00A0polished\u00C2\u00A0rice\u00C2\u00A0endosperm.\u00C2\u00A0J.\u00C2\u00A0Brew.\u00C2\u00A0Soc.\u00C2\u00A0Jpn.\u00C2\u00A086,\u00C2\u00A0293\u00E2\u0080\u0090298.\u00C2\u00A0 Kliewer,\u00C2\u00A0W.M.\u00C2\u00A0(1970)\u00C2\u00A0Free\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0nitrogenous\u00C2\u00A0fractions\u00C2\u00A0 in\u00C2\u00A0wine\u00C2\u00A0grapes.\u00C2\u00A0J.\u00C2\u00A0Food\u00C2\u00A0Sci.\u00C2\u00A035,\u00C2\u00A0 17\u00E2\u0080\u009021.\u00C2\u00A0 Kodama,\u00C2\u00A0K.\u00C2\u00A0(1993)\u00C2\u00A0Sake\u00E2\u0080\u0090brewing\u00C2\u00A0yeast;\u00C2\u00A0in\u00C2\u00A0The\u00C2\u00A0Yeasts,\u00C2\u00A0Rose,\u00C2\u00A0A.\u00C2\u00A0H.\u00C2\u00A0and\u00C2\u00A0Harrison,\u00C2\u00A0J.\u00C2\u00A0S.\u00C2\u00A0(eds.),\u00C2\u00A0pp.\u00C2\u00A0129\u00E2\u0080\u0090168,\u00C2\u00A0 Academic\u00C2\u00A0Press,\u00C2\u00A0London,\u00C2\u00A0United\u00C2\u00A0Kingdom.\u00C2\u00A0 Kodama,\u00C2\u00A0S.,\u00C2\u00A0Suzuki,\u00C2\u00A0T.,\u00C2\u00A0Fujinawa,\u00C2\u00A0S.,\u00C2\u00A0de\u00C2\u00A0 la\u00C2\u00A0Teja,\u00C2\u00A0P.\u00C2\u00A0and\u00C2\u00A0Yotsuzuka,\u00C2\u00A0F.\u00C2\u00A0 (1994)\u00C2\u00A0Urea\u00C2\u00A0Contribution\u00C2\u00A0 to\u00C2\u00A0Ethyl\u00C2\u00A0 Carbamate\u00C2\u00A0Formation\u00C2\u00A0in\u00C2\u00A0Commercial\u00C2\u00A0Wines\u00C2\u00A0During\u00C2\u00A0Storage.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A045,\u00C2\u00A017\u00E2\u0080\u009024.\u00C2\u00A0 Lam,\u00C2\u00A0K.\u00C2\u00A0B.\u00C2\u00A0(1977)\u00C2\u00A0Isolation\u00C2\u00A0and\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0glycolytic\u00C2\u00A0pathway\u00C2\u00A0mutants.\u00C2\u00A0 J.\u00C2\u00A0Bacteriol\u00C2\u00A0130,\u00C2\u00A0746\u00E2\u0080\u0090749.\u00C2\u00A0 Leithauser,\u00C2\u00A0M.\u00C2\u00A0T.,\u00C2\u00A0 Liem,\u00C2\u00A0A.,\u00C2\u00A0 Stewart,\u00C2\u00A0B.\u00C2\u00A0C.,\u00C2\u00A0Miller,\u00C2\u00A0E.\u00C2\u00A0C.\u00C2\u00A0 and\u00C2\u00A0Miller,\u00C2\u00A0 J.\u00C2\u00A0A.\u00C2\u00A0 (1990)\u00C2\u00A01,N6\u00E2\u0080\u0090ethenoadenosine\u00C2\u00A0 formation,\u00C2\u00A0 mutagenicity\u00C2\u00A0 and\u00C2\u00A0 murine\u00C2\u00A0 tumor\u00C2\u00A0 induction\u00C2\u00A0 as\u00C2\u00A0 indicators\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 electrophilic\u00C2\u00A0epoxide\u00C2\u00A0metabolite\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0closely\u00C2\u00A0 related\u00C2\u00A0carcinogens\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0 (urethane)\u00C2\u00A0 and\u00C2\u00A0vinyl\u00C2\u00A0carbamate.\u00C2\u00A0Carcinogenesis\u00C2\u00A011,\u00C2\u00A0463\u00E2\u0080\u0090473.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 110\u00C2\u00A0 \u00C2\u00A0 Liu,\u00C2\u00A0S.,\u00C2\u00A0Pritchard,\u00C2\u00A0G.\u00C2\u00A0G.,\u00C2\u00A0Hardman,\u00C2\u00A0M.\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0Pilone,\u00C2\u00A0G.\u00C2\u00A0J.\u00C2\u00A0(1994)\u00C2\u00A0Citrulline\u00C2\u00A0Production\u00C2\u00A0and\u00C2\u00A0Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0 (Urethane)\u00C2\u00A0 Precursor\u00C2\u00A0 Formation\u00C2\u00A0 From\u00C2\u00A0 Arginine\u00C2\u00A0 Degradation\u00C2\u00A0 by\u00C2\u00A0 Wine\u00C2\u00A0 Lactic\u00C2\u00A0 Acid\u00C2\u00A0 Bacteria\u00C2\u00A0 Leuconostoc\u00C2\u00A0oenos\u00C2\u00A0and\u00C2\u00A0Lactobacillus\u00C2\u00A0buchneri.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A045,\u00C2\u00A0235\u00E2\u0080\u0090242.\u00C2\u00A0 Lohr,\u00C2\u00A0D.,\u00C2\u00A0Venkov,\u00C2\u00A0P.\u00C2\u00A0and\u00C2\u00A0Zlatanova,\u00C2\u00A0J.\u00C2\u00A0(1995)\u00C2\u00A0Transcriptional\u00C2\u00A0regulation\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0GAL\u00C2\u00A0gene\u00C2\u00A0family:\u00C2\u00A0a\u00C2\u00A0 complex\u00C2\u00A0genetic\u00C2\u00A0network.\u00C2\u00A0FASEB\u00C2\u00A0J.\u00C2\u00A09,\u00C2\u00A0777\u00E2\u0080\u0090787.\u00C2\u00A0 Maftahi,\u00C2\u00A0M.,\u00C2\u00A0Gaillardin,\u00C2\u00A0C.\u00C2\u00A0and\u00C2\u00A0Nicaud,\u00C2\u00A0 J.\u00C2\u00A0M.\u00C2\u00A0 (1998)\u00C2\u00A0Generation\u00C2\u00A0of\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0deletants\u00C2\u00A0 and\u00C2\u00A0basic\u00C2\u00A0phenotypic\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0eight\u00C2\u00A0novel\u00C2\u00A0genes\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 left\u00C2\u00A0arm\u00C2\u00A0of\u00C2\u00A0chromosome\u00C2\u00A0XIV.\u00C2\u00A0Yeast\u00C2\u00A0 14,\u00C2\u00A0271\u00E2\u0080\u0090280.\u00C2\u00A0 Malone,\u00C2\u00A0R.\u00C2\u00A0E.\u00C2\u00A0(1990)\u00C2\u00A0Dual\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0meiosis\u00C2\u00A0in\u00C2\u00A0yeast.\u00C2\u00A0Cell,\u00C2\u00A061,\u00C2\u00A0375\u00E2\u0080\u0090378.\u00C2\u00A0 Manivasakam,\u00C2\u00A0 P.\u00C2\u00A0 (1995)\u00C2\u00A0Micro\u00E2\u0080\u0090homology\u00C2\u00A0mediated\u00C2\u00A0 PCR\u00C2\u00A0 targeting\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0 Nuc.\u00C2\u00A0 Acids\u00C2\u00A0Res.\u00C2\u00A023,\u00C2\u00A02799\u00E2\u0080\u00902800.\u00C2\u00A0 Marks,\u00C2\u00A0V.\u00C2\u00A0D.,\u00C2\u00A0Sui,\u00C2\u00A0S.\u00C2\u00A0 J.,\u00C2\u00A0Erasmus,\u00C2\u00A0D.,\u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Merwe,\u00C2\u00A0G.,\u00C2\u00A0Brumm,\u00C2\u00A0 J.,\u00C2\u00A0Wasserman,\u00C2\u00A0W.\u00C2\u00A0W.,\u00C2\u00A0Bryan,\u00C2\u00A0 J.,\u00C2\u00A0van\u00C2\u00A0 Vuuren,\u00C2\u00A0H.\u00C2\u00A0J.\u00C2\u00A0J.\u00C2\u00A0(2008)\u00C2\u00A0Dynamics\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0yeast\u00C2\u00A0transcriptome\u00C2\u00A0during\u00C2\u00A0wine\u00C2\u00A0fermentation\u00C2\u00A0reveals\u00C2\u00A0a\u00C2\u00A0 novel\u00C2\u00A0fermentation\u00C2\u00A0stress\u00C2\u00A0response.\u00C2\u00A0FEMS\u00C2\u00A0Yeast\u00C2\u00A0Research\u00C2\u00A08,\u00C2\u00A035\u00E2\u0080\u009052.\u00C2\u00A0 Marks,\u00C2\u00A0V.\u00C2\u00A0D.,\u00C2\u00A0 \u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Merwe,\u00C2\u00A0G.,\u00C2\u00A0van\u00C2\u00A0Vuuren,\u00C2\u00A0H.\u00C2\u00A0 J.\u00C2\u00A0 J.\u00C2\u00A0 (2003)\u00C2\u00A0Transcriptional\u00C2\u00A0profiling\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0 in\u00C2\u00A0 fermenting\u00C2\u00A0 grape\u00C2\u00A0 juice:\u00C2\u00A0 regulatory\u00C2\u00A0 effect\u00C2\u00A0of\u00C2\u00A0diammonium\u00C2\u00A0phosphate.\u00C2\u00A0 FEMS\u00C2\u00A0 Yeast\u00C2\u00A0Research\u00C2\u00A03,\u00C2\u00A0 269\u00E2\u0080\u0090287.\u00C2\u00A0 Matsudo,\u00C2\u00A0T.\u00C2\u00A0(1993)\u00C2\u00A0Determination\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0carbamate\u00C2\u00A0in\u00C2\u00A0soy\u00C2\u00A0sauce\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0possible\u00C2\u00A0precursor.\u00C2\u00A0J.\u00C2\u00A0Agricult.\u00C2\u00A0 Food\u00C2\u00A0Chem.\u00C2\u00A041,\u00C2\u00A0352\u00E2\u0080\u0090356.\u00C2\u00A0 Miklos,\u00C2\u00A0G.\u00C2\u00A0 G.\u00C2\u00A0 and\u00C2\u00A0 Rubin,\u00C2\u00A0G.\u00C2\u00A0 (1996)\u00C2\u00A0 The\u00C2\u00A0 Role\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0Genome\u00C2\u00A0 Project\u00C2\u00A0 in\u00C2\u00A0Determining\u00C2\u00A0Gene\u00C2\u00A0 Function:\u00C2\u00A0 Insights\u00C2\u00A0from\u00C2\u00A0Model\u00C2\u00A0Organisms.\u00C2\u00A0Cell,\u00C2\u00A086,\u00C2\u00A0521\u00E2\u0080\u0090529.\u00C2\u00A0 Monteiro,\u00C2\u00A0F.\u00C2\u00A0F.\u00C2\u00A0and\u00C2\u00A0Bisson,\u00C2\u00A0L.\u00C2\u00A0F.\u00C2\u00A0 (1991)\u00C2\u00A0Amino\u00C2\u00A0Acid\u00C2\u00A0Utilization\u00C2\u00A0and\u00C2\u00A0Urea\u00C2\u00A0Formation\u00C2\u00A0During\u00C2\u00A0Vinification\u00C2\u00A0 Fermentations.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A042,\u00C2\u00A0199\u00E2\u0080\u0090208.\u00C2\u00A0 Monteiro,\u00C2\u00A0F.\u00C2\u00A0F.\u00C2\u00A0and\u00C2\u00A0Bisson,\u00C2\u00A0L.\u00C2\u00A0F.\u00C2\u00A0(1992)\u00C2\u00A0Nitrogen\u00C2\u00A0Supplementation\u00C2\u00A0of\u00C2\u00A0Grape\u00C2\u00A0Juice.\u00C2\u00A0I.\u00C2\u00A0Effect\u00C2\u00A0on\u00C2\u00A0Amino\u00C2\u00A0Acid\u00C2\u00A0 Utilization\u00C2\u00A0During\u00C2\u00A0Fermentation.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A043,\u00C2\u00A01\u00E2\u0080\u009010.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 111\u00C2\u00A0 \u00C2\u00A0 Monteiro,\u00C2\u00A0 F.\u00C2\u00A0 F.,\u00C2\u00A0Trousdale,\u00C2\u00A0E.\u00C2\u00A0K.\u00C2\u00A0 and\u00C2\u00A0Bisson,\u00C2\u00A0 L.\u00C2\u00A0 F.\u00C2\u00A0 (1989)\u00C2\u00A0Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0 Formation\u00C2\u00A0 in\u00C2\u00A0Wine:\u00C2\u00A0Use\u00C2\u00A0of\u00C2\u00A0 Radioactively\u00C2\u00A0Labeled\u00C2\u00A0Precursors\u00C2\u00A0to\u00C2\u00A0Demonstrate\u00C2\u00A0the\u00C2\u00A0Involvement\u00C2\u00A0of\u00C2\u00A0Urea.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A040,\u00C2\u00A0 1\u00E2\u0080\u00908.\u00C2\u00A0 Mori,\u00C2\u00A0 K.\u00C2\u00A0 (1996)\u00C2\u00A0 Signalling\u00C2\u00A0 from\u00C2\u00A0 endoplasmic\u00C2\u00A0 reticulum\u00C2\u00A0 to\u00C2\u00A0 nucleus:\u00C2\u00A0 transcription\u00C2\u00A0 factor\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A0 basic\u00E2\u0080\u0090 leucine\u00C2\u00A0 zipper\u00C2\u00A0motif\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0unfolded\u00C2\u00A0protein\u00E2\u0080\u0090response\u00C2\u00A0pathway.\u00C2\u00A0Genes\u00C2\u00A0 to\u00C2\u00A0Cells\u00C2\u00A01,\u00C2\u00A0 803\u00E2\u0080\u0090817.\u00C2\u00A0 Mortimer,\u00C2\u00A0R.\u00C2\u00A0K.\u00C2\u00A0(1966)\u00C2\u00A0Genetic\u00C2\u00A0mapping\u00C2\u00A0in\u00C2\u00A0Saccharomyces.\u00C2\u00A0Genetics\u00C2\u00A053,\u00C2\u00A0165\u00E2\u0080\u0090173.\u00C2\u00A0 Nasmyth,\u00C2\u00A0K.\u00C2\u00A0(1993)\u00C2\u00A0Regulating\u00C2\u00A0the\u00C2\u00A0HO\u00C2\u00A0endonuclease\u00C2\u00A0in\u00C2\u00A0yeast.\u00C2\u00A0Curr.\u00C2\u00A0Opin.\u00C2\u00A0Genet.\u00C2\u00A0Dev.\u00C2\u00A03,\u00C2\u00A0286\u00E2\u0080\u0090294.\u00C2\u00A0 National\u00C2\u00A0 Institutes\u00C2\u00A0 of\u00C2\u00A0 Health\u00C2\u00A0 National\u00C2\u00A0 Toxicology\u00C2\u00A0 Program.\u00C2\u00A0 (2004)\u00C2\u00A0 NTP\u00C2\u00A0 Technical\u00C2\u00A0 Report\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 Toxicology\u00C2\u00A0 and\u00C2\u00A0 Carcinogenesis\u00C2\u00A0 Studies\u00C2\u00A0 of\u00C2\u00A0Urethane,\u00C2\u00A0 Ethanol,\u00C2\u00A0 and\u00C2\u00A0Urethane/Ethanol\u00C2\u00A0 in\u00C2\u00A0B6C3F1\u00C2\u00A0 Mice\u00C2\u00A0(Drinking\u00C2\u00A0Water\u00C2\u00A0Studies).\u00C2\u00A0TR\u00C2\u00A0510,\u00C2\u00A01\u00E2\u0080\u0090351.\u00C2\u00A0 Newstead,\u00C2\u00A0S.,\u00C2\u00A0Kim,\u00C2\u00A0H.,\u00C2\u00A0von\u00C2\u00A0Heijne,\u00C2\u00A0G.,\u00C2\u00A0Iwata,\u00C2\u00A0S.\u00C2\u00A0and\u00C2\u00A0Drew,\u00C2\u00A0D.\u00C2\u00A0(2007)\u00C2\u00A0High\u00E2\u0080\u0090throughput\u00C2\u00A0fluorescent\u00E2\u0080\u0090based\u00C2\u00A0 optimization\u00C2\u00A0of\u00C2\u00A0eukaryotic\u00C2\u00A0membrane\u00C2\u00A0protein\u00C2\u00A0overexpression\u00C2\u00A0and\u00C2\u00A0purification\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0Proc.\u00C2\u00A0Natl.\u00C2\u00A0Acad.\u00C2\u00A0Sci.\u00C2\u00A0USA\u00C2\u00A0104,\u00C2\u00A013936\u00E2\u0080\u009013941.\u00C2\u00A0 Nettleship,\u00C2\u00A0A.,\u00C2\u00A0Henshaw,\u00C2\u00A0PS.\u00C2\u00A0and\u00C2\u00A0Meyer,\u00C2\u00A0HL.\u00C2\u00A0 (1943)\u00C2\u00A0 Induction\u00C2\u00A0of\u00C2\u00A0pulmonary\u00C2\u00A0 tumors\u00C2\u00A0 in\u00C2\u00A0mice\u00C2\u00A0with\u00C2\u00A0ethyl\u00C2\u00A0 carbamate\u00C2\u00A0(urethane).\u00C2\u00A0J.\u00C2\u00A0Natl.\u00C2\u00A0Cancer\u00C2\u00A0Inst.\u00C2\u00A04,\u00C2\u00A0309\u00E2\u0080\u0090319.\u00C2\u00A0 Nikawa,\u00C2\u00A0J.,\u00C2\u00A0Akiyoshi,\u00C2\u00A0M.,\u00C2\u00A0Hirata,\u00C2\u00A0S.\u00C2\u00A0and\u00C2\u00A0Fukuda,\u00C2\u00A0T.\u00C2\u00A0(1996)\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0IRE2/HAC1\u00C2\u00A0is\u00C2\u00A0involved\u00C2\u00A0 in\u00C2\u00A0IRE1\u00E2\u0080\u0090mediated\u00C2\u00A0KAR2\u00C2\u00A0expression.\u00C2\u00A0Nucl.\u00C2\u00A0Acids\u00C2\u00A0Res.\u00C2\u00A024,\u00C2\u00A04222\u00E2\u0080\u00904226.\u00C2\u00A0 Nozawa,\u00C2\u00A0A.,\u00C2\u00A0Takano,\u00C2\u00A0J.,\u00C2\u00A0Kobayashi,\u00C2\u00A0M.,\u00C2\u00A0von\u00C2\u00A0Wiren,\u00C2\u00A0N.\u00C2\u00A0and\u00C2\u00A0Fujiwara,\u00C2\u00A0T.\u00C2\u00A0(2006)\u00C2\u00A0Roles\u00C2\u00A0of\u00C2\u00A0BOR1,\u00C2\u00A0DUR3,\u00C2\u00A0and\u00C2\u00A0 FPS1\u00C2\u00A0 in\u00C2\u00A0boron\u00C2\u00A0 transport\u00C2\u00A0and\u00C2\u00A0 tolerance\u00C2\u00A0 in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0FEMS\u00C2\u00A0Microbiol.\u00C2\u00A0Lett.\u00C2\u00A0262,\u00C2\u00A0 216\u00E2\u0080\u0090222.\u00C2\u00A0 Ough,\u00C2\u00A0 C.\u00C2\u00A0 S.\u00C2\u00A0 (1976a)\u00C2\u00A0 Ethyl\u00C2\u00A0 carbamate\u00C2\u00A0 in\u00C2\u00A0 fermented\u00C2\u00A0 beverages\u00C2\u00A0 and\u00C2\u00A0 foods.\u00C2\u00A0 I.\u00C2\u00A0Naturally\u00C2\u00A0 occurring\u00C2\u00A0 ethyl\u00C2\u00A0 carbamate.\u00C2\u00A0J.\u00C2\u00A0Agric.\u00C2\u00A0Food\u00C2\u00A0Chem.\u00C2\u00A024,\u00C2\u00A0323\u00E2\u0080\u0090328.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 112\u00C2\u00A0 \u00C2\u00A0 Ough,\u00C2\u00A0C.\u00C2\u00A0S.\u00C2\u00A0 (1976b)\u00C2\u00A0Ethyl\u00C2\u00A0carbamate\u00C2\u00A0 in\u00C2\u00A0fermented\u00C2\u00A0beverages\u00C2\u00A0and\u00C2\u00A0foods.\u00C2\u00A0 II.\u00C2\u00A0Possible\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0ethyl\u00C2\u00A0 carbamate\u00C2\u00A0from\u00C2\u00A0diethyl\u00C2\u00A0dicarbonate\u00C2\u00A0addition\u00C2\u00A0to\u00C2\u00A0wine.\u00C2\u00A0J.\u00C2\u00A0Agric.\u00C2\u00A0Food\u00C2\u00A0Chem.\u00C2\u00A024,\u00C2\u00A0328\u00E2\u0080\u0090331.\u00C2\u00A0 Ough,\u00C2\u00A0C.\u00C2\u00A0S.,\u00C2\u00A0Crowell,\u00C2\u00A0E.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Gutlove,\u00C2\u00A0B.\u00C2\u00A0R.\u00C2\u00A0(1988)\u00C2\u00A0Carbamyl\u00C2\u00A0Compound\u00C2\u00A0Reactions\u00C2\u00A0with\u00C2\u00A0Ethanol.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0 Enol.\u00C2\u00A0Vitic.\u00C2\u00A039,\u00C2\u00A0239\u00E2\u0080\u0090242.\u00C2\u00A0 Ough,\u00C2\u00A0C.\u00C2\u00A0S.,\u00C2\u00A0Crowell,\u00C2\u00A0E.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Mooney,\u00C2\u00A0L.\u00C2\u00A0A.\u00C2\u00A0 (1988)\u00C2\u00A0Formation\u00C2\u00A0of\u00C2\u00A0Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0Precursors\u00C2\u00A0During\u00C2\u00A0 Grape\u00C2\u00A0Juice\u00C2\u00A0(Chardonnay)\u00C2\u00A0Fermentation.\u00C2\u00A0I.\u00C2\u00A0Addition\u00C2\u00A0of\u00C2\u00A0Amino\u00C2\u00A0Acids,\u00C2\u00A0Urea,\u00C2\u00A0and\u00C2\u00A0Ammonia:\u00C2\u00A0Effects\u00C2\u00A0 of\u00C2\u00A0Fortification\u00C2\u00A0on\u00C2\u00A0Intracellular\u00C2\u00A0and\u00C2\u00A0Extracellular\u00C2\u00A0Precursors.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A039,\u00C2\u00A0243\u00E2\u0080\u0090249.\u00C2\u00A0 Ough,\u00C2\u00A0C.\u00C2\u00A0S.,\u00C2\u00A0Huang,\u00C2\u00A0Z.,\u00C2\u00A0An,\u00C2\u00A0D.\u00C2\u00A0and\u00C2\u00A0Stevens,\u00C2\u00A0D.\u00C2\u00A0(1991)\u00C2\u00A0Amino\u00C2\u00A0Acid\u00C2\u00A0Uptake\u00C2\u00A0by\u00C2\u00A0Four\u00C2\u00A0Commercial\u00C2\u00A0Yeasts\u00C2\u00A0at\u00C2\u00A0 Two\u00C2\u00A0 Different\u00C2\u00A0 Temperatures\u00C2\u00A0 of\u00C2\u00A0 Growth\u00C2\u00A0 and\u00C2\u00A0 Fermentation:\u00C2\u00A0 Effects\u00C2\u00A0 on\u00C2\u00A0 Urea\u00C2\u00A0 Excretion\u00C2\u00A0 and\u00C2\u00A0 Reabsorption.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A042,\u00C2\u00A026\u00E2\u0080\u009040.\u00C2\u00A0 Ough,\u00C2\u00A0 C.\u00C2\u00A0 S.,\u00C2\u00A0 Stevens,\u00C2\u00A0 D.,\u00C2\u00A0 Sendovski,\u00C2\u00A0 T.,\u00C2\u00A0 Huang,\u00C2\u00A0 Z.\u00C2\u00A0 and\u00C2\u00A0 An,\u00C2\u00A0 D.\u00C2\u00A0 (1990)\u00C2\u00A0 Factors\u00C2\u00A0 Contributing\u00C2\u00A0 to\u00C2\u00A0 Urea\u00C2\u00A0 Formation\u00C2\u00A0in\u00C2\u00A0Commercially\u00C2\u00A0Fermented\u00C2\u00A0Wines.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A041,\u00C2\u00A068\u00E2\u0080\u009073.\u00C2\u00A0 Ough,\u00C2\u00A0C.\u00C2\u00A0S.\u00C2\u00A0and\u00C2\u00A0Trioli,\u00C2\u00A0G.\u00C2\u00A0(1988)\u00C2\u00A0Urea\u00C2\u00A0Removal\u00C2\u00A0from\u00C2\u00A0Wine\u00C2\u00A0by\u00C2\u00A0an\u00C2\u00A0Acid\u00C2\u00A0Urease.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A039,\u00C2\u00A0303\u00E2\u0080\u0090 307.\u00C2\u00A0 Park,\u00C2\u00A0H.,\u00C2\u00A0Shin,\u00C2\u00A0M.\u00C2\u00A0and\u00C2\u00A0Woo,\u00C2\u00A0I.\u00C2\u00A0(2001)\u00C2\u00A0Antisense\u00E2\u0080\u0090mediated\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0arginase\u00C2\u00A0(CAR1)\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0 in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Biosci.\u00C2\u00A0Bioeng.\u00C2\u00A092,\u00C2\u00A0481\u00E2\u0080\u0090484.\u00C2\u00A0 Park,\u00C2\u00A0 K.,\u00C2\u00A0 Liem,\u00C2\u00A0 A.,\u00C2\u00A0 Stewart,\u00C2\u00A0 B.\u00C2\u00A0 C.\u00C2\u00A0 and\u00C2\u00A0Miller,\u00C2\u00A0 J.\u00C2\u00A0 A.\u00C2\u00A0 (1993)\u00C2\u00A0 Vinyl\u00C2\u00A0 carbamate\u00C2\u00A0 epoxide,\u00C2\u00A0 a\u00C2\u00A0major\u00C2\u00A0 strong\u00C2\u00A0 electrophilic,\u00C2\u00A0mutagenic\u00C2\u00A0 and\u00C2\u00A0 carcinogenic\u00C2\u00A0metabolite\u00C2\u00A0 of\u00C2\u00A0 vinyl\u00C2\u00A0 carbamate\u00C2\u00A0 and\u00C2\u00A0 ethyl\u00C2\u00A0 carbamate\u00C2\u00A0 (urethane).\u00C2\u00A0Carcinogenesis\u00C2\u00A014,\u00C2\u00A0441\u00E2\u0080\u0090450.\u00C2\u00A0 Pena\u00E2\u0080\u0090Castillo,\u00C2\u00A0 L.\u00C2\u00A0 and\u00C2\u00A0Hughes,\u00C2\u00A0T.\u00C2\u00A0 (2007)\u00C2\u00A0Why\u00C2\u00A0Are\u00C2\u00A0There\u00C2\u00A0 Still\u00C2\u00A0Over\u00C2\u00A01000\u00C2\u00A0Uncharacterized\u00C2\u00A0Yeast\u00C2\u00A0Genes?\u00C2\u00A0 Genetics\u00C2\u00A0176,\u00C2\u00A07\u00E2\u0080\u009014.\u00C2\u00A0 Perez\u00E2\u0080\u0090Ortin,\u00C2\u00A0J.,\u00C2\u00A0Garcia\u00E2\u0080\u0090Martinez,\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0Alberola,\u00C2\u00A0T.\u00C2\u00A0(2002)\u00C2\u00A0DNA\u00C2\u00A0chips\u00C2\u00A0for\u00C2\u00A0yeast\u00C2\u00A0biotechnology.\u00C2\u00A0The\u00C2\u00A0case\u00C2\u00A0of\u00C2\u00A0 wine\u00C2\u00A0yeasts.\u00C2\u00A0J.\u00C2\u00A0Biotechnol.\u00C2\u00A098,\u00C2\u00A0227\u00E2\u0080\u0090241.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 113\u00C2\u00A0 \u00C2\u00A0 Rossignol,\u00C2\u00A0T.,\u00C2\u00A0Dulau,\u00C2\u00A0L.,\u00C2\u00A0 Julien,\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Blondin,\u00C2\u00A0B.\u00C2\u00A0 (2003)\u00C2\u00A0Genome\u00E2\u0080\u0090wide\u00C2\u00A0monitoring\u00C2\u00A0of\u00C2\u00A0wine\u00C2\u00A0yeast\u00C2\u00A0gene\u00C2\u00A0 expression\u00C2\u00A0during\u00C2\u00A0alcoholic\u00C2\u00A0fermentation.\u00C2\u00A0Yeast\u00C2\u00A020,\u00C2\u00A01369\u00E2\u0080\u00901385.\u00C2\u00A0 Salmon,\u00C2\u00A0J.\u00C2\u00A0and\u00C2\u00A0Barre,\u00C2\u00A0P.\u00C2\u00A0(1998)\u00C2\u00A0Improvement\u00C2\u00A0of\u00C2\u00A0Nitrogen\u00C2\u00A0Assimilation\u00C2\u00A0and\u00C2\u00A0Fermentation\u00C2\u00A0Kinetics\u00C2\u00A0under\u00C2\u00A0 Enological\u00C2\u00A0 Conditions\u00C2\u00A0 by\u00C2\u00A0 Derepression\u00C2\u00A0 of\u00C2\u00A0 Alternative\u00C2\u00A0 Nitrogen\u00E2\u0080\u0090Assimilatory\u00C2\u00A0 Pathways\u00C2\u00A0 in\u00C2\u00A0 an\u00C2\u00A0 Industrial\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae\u00C2\u00A0Strain.\u00C2\u00A0Appl.\u00C2\u00A0Environ.\u00C2\u00A0Microbiol.\u00C2\u00A064,\u00C2\u00A03831\u00E2\u0080\u00903837.\u00C2\u00A0 Schehl,\u00C2\u00A0B.\u00C2\u00A0 (2007)\u00C2\u00A0Contribution\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 fermenting\u00C2\u00A0 yeast\u00C2\u00A0 strain\u00C2\u00A0 to\u00C2\u00A0ethyl\u00C2\u00A0 carbamate\u00C2\u00A0generation\u00C2\u00A0 in\u00C2\u00A0 stone\u00C2\u00A0 fruit\u00C2\u00A0spirits.\u00C2\u00A0Appl.\u00C2\u00A0Microbiol.\u00C2\u00A0Biotech.\u00C2\u00A074,\u00C2\u00A0843\u00E2\u0080\u0090850.\u00C2\u00A0 Schlatter,\u00C2\u00A0 J.\u00C2\u00A0 and\u00C2\u00A0 Lutz,\u00C2\u00A0W.\u00C2\u00A0 K.\u00C2\u00A0 (1990)\u00C2\u00A0 The\u00C2\u00A0 carcinogenic\u00C2\u00A0 potential\u00C2\u00A0 of\u00C2\u00A0 ethyl\u00C2\u00A0 carbamate\u00C2\u00A0 (urethane):\u00C2\u00A0 risk\u00C2\u00A0 assessment\u00C2\u00A0at\u00C2\u00A0human\u00C2\u00A0dietary\u00C2\u00A0exposure\u00C2\u00A0levels.\u00C2\u00A0Food\u00C2\u00A0Chem.\u00C2\u00A0Toxicol.\u00C2\u00A028,\u00C2\u00A0205\u00E2\u0080\u0090211.\u00C2\u00A0 Shobayashi,\u00C2\u00A0M.,\u00C2\u00A0Ukena,\u00C2\u00A0E.,\u00C2\u00A0Fujii,\u00C2\u00A0T.\u00C2\u00A0and\u00C2\u00A0Iefuji,\u00C2\u00A0H.\u00C2\u00A0(2007)\u00C2\u00A0Genome\u00E2\u0080\u0090wide\u00C2\u00A0expression\u00C2\u00A0profile\u00C2\u00A0of\u00C2\u00A0sake\u00C2\u00A0brewing\u00C2\u00A0 yeast\u00C2\u00A0under\u00C2\u00A0shaking\u00C2\u00A0and\u00C2\u00A0static\u00C2\u00A0conditions.\u00C2\u00A0Biosci.\u00C2\u00A0Biotechnol.\u00C2\u00A0Biochem.\u00C2\u00A071,\u00C2\u00A0323\u00E2\u0080\u0090335.\u00C2\u00A0 Smart,\u00C2\u00A0W.\u00C2\u00A0C.,\u00C2\u00A0Coffman,\u00C2\u00A0 J.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0 (1996)\u00C2\u00A0Combinatorial\u00C2\u00A0 regulation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Saccharomyces\u00C2\u00A0 cerevisiae\u00C2\u00A0CAR1\u00C2\u00A0 (arginase)\u00C2\u00A0promoter\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0multiple\u00C2\u00A0 environmental\u00C2\u00A0 signals.\u00C2\u00A0Mol.\u00C2\u00A0Cell.\u00C2\u00A0 Biol.\u00C2\u00A016,\u00C2\u00A05876\u00E2\u0080\u00905887.\u00C2\u00A0 Stevens,\u00C2\u00A0D.\u00C2\u00A0F.\u00C2\u00A0and\u00C2\u00A0Ough,\u00C2\u00A0C.\u00C2\u00A0S.\u00C2\u00A0 (1993)\u00C2\u00A0Ethyl\u00C2\u00A0Carbamate\u00C2\u00A0Formation:\u00C2\u00A0Reaction\u00C2\u00A0of\u00C2\u00A0Urea\u00C2\u00A0and\u00C2\u00A0Citrulline\u00C2\u00A0with\u00C2\u00A0 Ethanol\u00C2\u00A0in\u00C2\u00A0Wine\u00C2\u00A0Under\u00C2\u00A0Low\u00C2\u00A0to\u00C2\u00A0Normal\u00C2\u00A0Temperature\u00C2\u00A0Conditions.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A044,\u00C2\u00A0309\u00E2\u0080\u0090312.\u00C2\u00A0 Stolz,\u00C2\u00A0L.\u00C2\u00A0E.\u00C2\u00A0(1998)\u00C2\u00A0INP51,\u00C2\u00A0a\u00C2\u00A0yeast\u00C2\u00A0inositol\u00C2\u00A0polyphosphate\u00C2\u00A05\u00E2\u0080\u0090phosphatase\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0phosphatidylinositol\u00C2\u00A0 4,5\u00E2\u0080\u0090bisphosphate\u00C2\u00A0homeostasis\u00C2\u00A0 and\u00C2\u00A0whose\u00C2\u00A0 absence\u00C2\u00A0 confers\u00C2\u00A0 a\u00C2\u00A0 cold\u00E2\u0080\u0090resistant\u00C2\u00A0phenotype.\u00C2\u00A0 J.\u00C2\u00A0Biol.\u00C2\u00A0 Chem.\u00C2\u00A0273,\u00C2\u00A011852\u00E2\u0080\u009011861.\u00C2\u00A0 Sumrada,\u00C2\u00A0 R.,\u00C2\u00A0 Gorski,\u00C2\u00A0 M.\u00C2\u00A0 and\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 (1976)\u00C2\u00A0 Urea\u00C2\u00A0 transport\u00E2\u0080\u0090defective\u00C2\u00A0 strains\u00C2\u00A0 of\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Bacteriol.\u00C2\u00A0125,\u00C2\u00A01048\u00E2\u0080\u00901056.\u00C2\u00A0 Tabor,\u00C2\u00A0H.\u00C2\u00A0and\u00C2\u00A0Tabor,\u00C2\u00A0C.\u00C2\u00A0W.\u00C2\u00A0 (1999)\u00C2\u00A0A\u00C2\u00A0Guide\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0Polyamines.\u00C2\u00A0Seymour\u00C2\u00A0S.\u00C2\u00A0Cohen.\u00C2\u00A0Analytical\u00C2\u00A0Biochem.\u00C2\u00A0 274,\u00C2\u00A0150.\u00C2\u00A0Oxford\u00C2\u00A0University\u00C2\u00A0Press,\u00C2\u00A0New\u00C2\u00A0York,\u00C2\u00A0USA.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 114\u00C2\u00A0 \u00C2\u00A0 Takagi,\u00C2\u00A0H.,\u00C2\u00A0 Takaoka,\u00C2\u00A0M.,\u00C2\u00A0 Kawaguchi,\u00C2\u00A0A.\u00C2\u00A0 and\u00C2\u00A0 Kubo,\u00C2\u00A0 Y.\u00C2\u00A0 (2005)\u00C2\u00A0 Effect\u00C2\u00A0 of\u00C2\u00A0 L\u00E2\u0080\u0090Proline\u00C2\u00A0 on\u00C2\u00A0 Sake\u00C2\u00A0 Brewing\u00C2\u00A0 and\u00C2\u00A0 Ethanol\u00C2\u00A0Stress\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Appl.\u00C2\u00A0Environ.\u00C2\u00A0Microbiol.\u00C2\u00A071,\u00C2\u00A08656\u00E2\u0080\u00908662.\u00C2\u00A0 Uemura,\u00C2\u00A0 T.,\u00C2\u00A0 Kashiwagi,\u00C2\u00A0 K.\u00C2\u00A0 and\u00C2\u00A0 Igarashi,\u00C2\u00A0 K.\u00C2\u00A0 (2007)\u00C2\u00A0 Polyamine\u00C2\u00A0 Uptake\u00C2\u00A0 by\u00C2\u00A0 DUR3\u00C2\u00A0 and\u00C2\u00A0 SAM3\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0282,\u00C2\u00A07733\u00E2\u0080\u00907741.\u00C2\u00A0 van\u00C2\u00A0Vuuren,\u00C2\u00A0H.\u00C2\u00A0J.\u00C2\u00A0J.,\u00C2\u00A0Daugherty,\u00C2\u00A0J.\u00C2\u00A0R.,\u00C2\u00A0Rai,\u00C2\u00A0R.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0(1991)\u00C2\u00A0Upstream\u00C2\u00A0induction\u00C2\u00A0sequence,\u00C2\u00A0the\u00C2\u00A0 cis\u00E2\u0080\u0090acting\u00C2\u00A0element\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0allantoin\u00C2\u00A0pathway\u00C2\u00A0 inducer\u00C2\u00A0and\u00C2\u00A0enhancement\u00C2\u00A0of\u00C2\u00A0 operation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0nitrogen\u00E2\u0080\u0090regulated\u00C2\u00A0upstream\u00C2\u00A0activation\u00C2\u00A0sequence\u00C2\u00A0 in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0 J.\u00C2\u00A0Bacteriol.\u00C2\u00A0173,\u00C2\u00A07186\u00E2\u0080\u00907195.\u00C2\u00A0 Vine,\u00C2\u00A0R.\u00C2\u00A0P.,\u00C2\u00A0Harkness,\u00C2\u00A0E.\u00C2\u00A0M.\u00C2\u00A0and\u00C2\u00A0Linton,\u00C2\u00A0S.\u00C2\u00A0J.\u00C2\u00A0(2002)\u00C2\u00A0Winemaking:\u00C2\u00A0From\u00C2\u00A0Grape\u00C2\u00A0Growing\u00C2\u00A0to\u00C2\u00A0Marketplace,\u00C2\u00A0 Second\u00C2\u00A0ed.,\u00C2\u00A0Kluwer\u00C2\u00A0Academic/Plenum,\u00C2\u00A0New\u00C2\u00A0York.\u00C2\u00A0 Volschenk,\u00C2\u00A0H.,\u00C2\u00A0Viljoen,\u00C2\u00A0M.,\u00C2\u00A0Grobler,\u00C2\u00A0J.,\u00C2\u00A0Bauer,\u00C2\u00A0F.,\u00C2\u00A0Lonvaud\u00E2\u0080\u0090Funel,\u00C2\u00A0A.,\u00C2\u00A0Denayrolles,\u00C2\u00A0M.,\u00C2\u00A0Subden,\u00C2\u00A0R.\u00C2\u00A0E.\u00C2\u00A0and\u00C2\u00A0 Van\u00C2\u00A0Vuuren,\u00C2\u00A0H.\u00C2\u00A0J.\u00C2\u00A0J.\u00C2\u00A0(1997)\u00C2\u00A0Malolactic\u00C2\u00A0Fermentation\u00C2\u00A0in\u00C2\u00A0Grape\u00C2\u00A0Musts\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0Genetically\u00C2\u00A0Engineered\u00C2\u00A0 Strain\u00C2\u00A0of\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Am.\u00C2\u00A0J.\u00C2\u00A0Enol.\u00C2\u00A0Vitic.\u00C2\u00A048,\u00C2\u00A0193\u00E2\u0080\u0090197.\u00C2\u00A0 Wach,\u00C2\u00A0 A.\u00C2\u00A0 (1996)\u00C2\u00A0 PCR\u00E2\u0080\u0090synthesis\u00C2\u00A0 of\u00C2\u00A0 marker\u00C2\u00A0 cassettes\u00C2\u00A0 with\u00C2\u00A0 long\u00C2\u00A0 flanking\u00C2\u00A0 homology\u00C2\u00A0 regions\u00C2\u00A0 for\u00C2\u00A0 gene\u00C2\u00A0 disruptions\u00C2\u00A0in\u00C2\u00A0S.\u00C2\u00A0cerevisiae.\u00C2\u00A0Yeast\u00C2\u00A012,\u00C2\u00A0259\u00E2\u0080\u0090265.\u00C2\u00A0 Ward,\u00C2\u00A0A.\u00C2\u00A0(1992)\u00C2\u00A0Rapid\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0yeast\u00C2\u00A0transformants\u00C2\u00A0using\u00C2\u00A0colony\u00E2\u0080\u0090PCR.\u00C2\u00A0Biotechniques,\u00C2\u00A013,\u00C2\u00A0350.\u00C2\u00A0\u00C2\u00A0 Wagner,\u00C2\u00A0 S.,\u00C2\u00A0 Bader,\u00C2\u00A0 M.\u00C2\u00A0 L.,\u00C2\u00A0 Drew,\u00C2\u00A0 D.\u00C2\u00A0 and\u00C2\u00A0 de\u00C2\u00A0 Gier,\u00C2\u00A0 J.\u00C2\u00A0 (2006)\u00C2\u00A0 Rationalizing\u00C2\u00A0 membrane\u00C2\u00A0 protein\u00C2\u00A0 overexpression.\u00C2\u00A0Trends\u00C2\u00A0in\u00C2\u00A0Biotech.\u00C2\u00A024,\u00C2\u00A0364\u00E2\u0080\u0090371.\u00C2\u00A0 Westfall,\u00C2\u00A0P.\u00C2\u00A0J.,\u00C2\u00A0Ballon,\u00C2\u00A0D.\u00C2\u00A0R.\u00C2\u00A0and\u00C2\u00A0Thorner,\u00C2\u00A0J.\u00C2\u00A0(2004)\u00C2\u00A0When\u00C2\u00A0the\u00C2\u00A0stress\u00C2\u00A0of\u00C2\u00A0your\u00C2\u00A0environment\u00C2\u00A0makes\u00C2\u00A0you\u00C2\u00A0go\u00C2\u00A0 HOG\u00C2\u00A0wild.\u00C2\u00A0Science\u00C2\u00A0306,\u00C2\u00A01511\u00E2\u0080\u00901512.\u00C2\u00A0 Whitney,\u00C2\u00A0P.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0G.\u00C2\u00A0(1972)\u00C2\u00A0Urea\u00C2\u00A0Carboxylase\u00C2\u00A0and\u00C2\u00A0Allophanate\u00C2\u00A0Hydrolase.\u00C2\u00A0Two\u00C2\u00A0components\u00C2\u00A0 of\u00C2\u00A0 adenosine\u00C2\u00A0 triphosphate:\u00C2\u00A0 urea\u00C2\u00A0 amidolyase\u00C2\u00A0 in\u00C2\u00A0 Saccharomyces\u00C2\u00A0 cerevisiae.\u00C2\u00A0 J.\u00C2\u00A0 Biol.\u00C2\u00A0 Chem.\u00C2\u00A0 247,\u00C2\u00A0 1349\u00E2\u0080\u00901353.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 115\u00C2\u00A0 \u00C2\u00A0 Whitney,\u00C2\u00A0P.\u00C2\u00A0A.\u00C2\u00A0and\u00C2\u00A0Cooper,\u00C2\u00A0T.\u00C2\u00A0 (1973)\u00C2\u00A0Urea\u00C2\u00A0carboxylase\u00C2\u00A0 from\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0Evidence\u00C2\u00A0 for\u00C2\u00A0a\u00C2\u00A0 minimal\u00C2\u00A0two\u00E2\u0080\u0090step\u00C2\u00A0reaction\u00C2\u00A0sequence.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0248,\u00C2\u00A0325\u00E2\u0080\u0090330.\u00C2\u00A0 Whitney,\u00C2\u00A0 P.\u00C2\u00A0 A.,\u00C2\u00A0 Cooper,\u00C2\u00A0 T.\u00C2\u00A0 G.\u00C2\u00A0 and\u00C2\u00A0 Magasanik,\u00C2\u00A0 B.\u00C2\u00A0 (1973)\u00C2\u00A0 The\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 urea\u00C2\u00A0 carboxylase\u00C2\u00A0 and\u00C2\u00A0 allophanate\u00C2\u00A0hydrolase\u00C2\u00A0in\u00C2\u00A0Saccharomyces\u00C2\u00A0cerevisiae.\u00C2\u00A0J.\u00C2\u00A0Biol.\u00C2\u00A0Chem.\u00C2\u00A0248,\u00C2\u00A06203\u00E2\u0080\u00906209.\u00C2\u00A0 Wu,\u00C2\u00A0H.,\u00C2\u00A0Zheng,\u00C2\u00A0X.,\u00C2\u00A0Araki,\u00C2\u00A0Y.,\u00C2\u00A0Sahara,\u00C2\u00A0H.,\u00C2\u00A0Takagi,\u00C2\u00A0H.\u00C2\u00A0and\u00C2\u00A0Shimoi,\u00C2\u00A0H.\u00C2\u00A0(2006)\u00C2\u00A0Global\u00C2\u00A0gene\u00C2\u00A0expression\u00C2\u00A0analysis\u00C2\u00A0 of\u00C2\u00A0yeast\u00C2\u00A0cells\u00C2\u00A0during\u00C2\u00A0sake\u00C2\u00A0brewing.\u00C2\u00A0Appl.\u00C2\u00A0Environ.\u00C2\u00A0Microbiol.\u00C2\u00A072,\u00C2\u00A07353\u00E2\u0080\u00907358.\u00C2\u00A0 Yanisch\u00E2\u0080\u0090Perron,\u00C2\u00A0 C.,\u00C2\u00A0 Vieira,\u00C2\u00A0 J.\u00C2\u00A0 and\u00C2\u00A0Messing,\u00C2\u00A0 J.\u00C2\u00A0 (1985)\u00C2\u00A0 Improved\u00C2\u00A0M13\u00C2\u00A0 phage\u00C2\u00A0 cloning\u00C2\u00A0 vectors\u00C2\u00A0 and\u00C2\u00A0 host\u00C2\u00A0 strains:\u00C2\u00A0nucleotide\u00C2\u00A0sequences\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0M13mp18\u00C2\u00A0and\u00C2\u00A0pUC19\u00C2\u00A0vectors.\u00C2\u00A0Gene\u00C2\u00A033,\u00C2\u00A0103\u00E2\u0080\u0090119.\u00C2\u00A0 Yoshiuchi,\u00C2\u00A0K.,\u00C2\u00A0Watanabe,\u00C2\u00A0M.\u00C2\u00A0and\u00C2\u00A0Nishimura,\u00C2\u00A0A.\u00C2\u00A0(2000)\u00C2\u00A0Breeding\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090urea\u00C2\u00A0producing\u00C2\u00A0sake\u00C2\u00A0yeast\u00C2\u00A0with\u00C2\u00A0 killer\u00C2\u00A0character\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0kar1\u00E2\u0080\u00901\u00C2\u00A0mutant\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0killer\u00C2\u00A0donor.\u00C2\u00A0J.\u00C2\u00A0Indust.\u00C2\u00A0Microbiol.\u00C2\u00A0Biotech.\u00C2\u00A024,\u00C2\u00A0203\u00E2\u0080\u0090209.\u00C2\u00A0 Zimmerli,\u00C2\u00A0B.\u00C2\u00A0and\u00C2\u00A0Schlatter,\u00C2\u00A0 J.\u00C2\u00A0 (1991)\u00C2\u00A0Ethyl\u00C2\u00A0 carbamate:\u00C2\u00A0analytical\u00C2\u00A0methodology,\u00C2\u00A0occurrence,\u00C2\u00A0 formation,\u00C2\u00A0 biological\u00C2\u00A0activity\u00C2\u00A0and\u00C2\u00A0risk\u00C2\u00A0assessment.\u00C2\u00A0Mutat.\u00C2\u00A0Res.\u00C2\u00A0259,\u00C2\u00A0325\u00E2\u0080\u0090350.\u00C2\u00A0"@en . "Thesis/Dissertation"@en . "2008-11"@en . "10.14288/1.0066389"@en . "eng"@en . "Genetics"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial-NoDerivatives 4.0 International"@en . "http://creativecommons.org/licenses/by-nc-nd/4.0/"@en . "Graduate"@en . "Metabolic engineering of industrial yeast strains to minimize the production of ethyl carbamate in grape and Sake wine"@en . "Text"@en . "http://hdl.handle.net/2429/790"@en .