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Characterization of a vanillate non-oxidative decaroxylation gene cluster from streptomyces sp.d7 Chow, Kevin Toshio 2000

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C H A R A C T E R I Z A T I O N OF A V A N I L L A T E N O N - O X I D A T I V E D E C A R B O X Y L A T I O N G E N E C L U S T E R F R O M STREPTOMYCES SP. D7 by K E V I N TOSHIO C H O W  M . S c , The University of British Columbia, 1996 B.Sc., The University of British Columbia, 1992 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A December 1999 © Kevin Toshio Chow, 1999  in presenting  this  thesis  in  degree at the University of  partial  fulfilment  of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of  this thesis for scholarly purposes may be granted  department  or  by  his  or  her  representatives.  It  publication of this thesis for financial gain shall not  is  understood  A//C&&I'QlOfr/ fflfJ* /MMtf^0Lcl 6rY  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  that  head of  my  copying  or  be allowed without my written  permission.  Department of  by the  ii  ABSTRACT The genetics of non-oxidative decarboxylation of aromatic acids to phenolic compounds are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous microorganisms acting on. a variety of substrates, genetic analyses of these processes have not, to my knowledge, been reported in the literature. Previously, I isolated a streptomycete from soil (Streptomyces sp. D7), which efficiently converts 4-hydroxy-3-methoxybenzoic (vanillic) acid to 2-methoxyphenol (guaiacol). Protein two-dimensional gel electrophoresis revealed that several proteins are synthesized in response to vanillic acid, one of which was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic lambda phage library of Streptomyces sp. D7.  This cluster consists of four open reading frames, vdcA  (sequencing in progress), vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp). Protein sequence comparisons suggest that the product of vdcB (201 aa) is similar to phenylacrylate decarboxylase of yeast; the putative products of vdcC (475 aa) and vdcD (80 aa) are similar to hypothetical proteins of unknown function from various microorganisms, and are found in a similar gene cluster in Bacillus subtilis. VdcA is a putative transcriptional regulatory gene.  VdcB, vdcC and vdcD homologues are also  clustered, along with putative />-cresol methylhydroxylase and vanillin oxidoreductase genes, on the 184 kb catabolic plasmid p N L l of Sphingomonas aromaticivorans F199. Northern blot analysis revealed the synthesis of a 2.5 kb mRNA transcript, which hybridized strongly to a vdcC gene probe, in vanillic acid-induced cells, suggesting that the cluster is under the control of a single inducible promoter. Expression of the entire vdc gene cluster in Streptomyces lividans 1326, as a heterologous host, resulted in that  Ill  strain acquiring the ability to decarboxylate vanillic acid to guaiacol non-oxidatively. Both Streptomyces strain D7 and recombinant S. lividans 1326 expressing the vdc gene cluster do not, however, decarboxylate structurally similar aromatic acids, suggesting that the system is specific for vanillic acid. By Southern blot hybridization, we detected the presence of the vdc gene cluster in several streptomycetes, including Streptomyces setonii 75Vi2, which has been previously shown to decarboxylate vanillic acid in a nonoxidative reaction. The vanillate decarboxylase catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural byproducts.  TABLE OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F FIGURES  viii  LIST O F ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xii  Chapter 1: I N T R O D U C T I O N  1  1.1 Non-oxidative decarboxylation: an industrially useful but poorly characterized process  4  1.2 Non-oxidative decarboxylation in prokaryotes  7  1.3 Structure and function studies of non-oxidative decarboxylases  9  1.4 Metabolic engineering for the production of chemicals from renewable resources 10 1.5 Streptomyces: versatile soil microbes  11  1.6 Isolation and identification of Streptomyces sp. D7  15  1.7 Catabolic tests and 2D-PAGE analysis  17  1.8 Objectives  22  Chapter 2: M A T E R I A L S A N D M E T H O D S  2.1 Bacterial strains and plasmids  23  23  2.1.1 Bacterial strains  23  2.1.2 Isolation and cloning of plasmids  23  2.1.3 16s rDNA sequence-based strain identification  28  2.2 Media and growth conditions  28  2.3 Library construction and gene cloning  31  2.3.1 Lambda phage D N A preparation  31  2.4 D N A sequencing and analysis  32  2.5 Southern blotting and hybridization  33  2.6 Chemical analyses  34  2.7 R N A isolation and analysis  34  2.8 Gene expression  38  2.8.1 pET22b(+) - E. coli BL21  38  2.8.2 pIJ702 - S. lividans 1326  38  2.8.3 pIJ680 - S. lividans 1326  40  2.9 Enzyme assays  42  2.10 Recombinant protein purification from E. coli BL21(DE3)  42  2.10.1 Ly sate preparation  43  2.10.2 Inclusion body protein purification, solubilization and refolding procedure 2.10.3 Soluble protein purification 2.11 Construction of a knockout allele of the vdcC gene 2.12 Streptomyces sp. D7 protoplast preparation and transformation 2.13 E. coli-Streptomyces interspecies conjugation  43 44 44 49 49  2.13.1 E. coli S17-1 /pPM801 preparation  49  2.13.2 Streptomyces sp. D7 preparation and mating  50  2.14 Reagents and enzymes Chapter 3: R E S U L T S  50 51  3.1 16s rDNA sequence identification  51  3.1 Cloning of the V D C gene cluster  51  3.2 Detection of a putative transcriptional regulatory gene  69  3.3 Messenger R N A analyses  72  3.4 Detection of vdc genes in S. setonii 75Vi2  74  3.5 Gene knockout studies  76  3.6 Gene expression  77  3.6.1 pET22b(+) - E. coli BL21  77  3.6.2 pIJ702 - S. lividans 1326  81  3.6.3 pIJ680 - S. lividans 1326  81  3.7 Substrate specificity  82  C h a p t e r 4: C O N C L U S I O N S  88  Chapter 5: D I S C U S S I O N  89  5.1 Aromatic acid non-oxidative decarboxylases are multi-subunit enzymes  89  5.2 Distribution of the V D C gene cluster among streptomycetes  91  5.3 Substrate specificity  92  5.4 Primary structure motifs  93  5.5 Transcriptional activation  93  5.6 Biodegradation - a result of the microbial community gene pool  94  5.7 Sphingomonas aromaticivorans F199 catabolic plasmid p N L l  96  5.8 Metabolic engineering applications for vanillate decarboxylase  103  5.9 Future directions  106  C h a p t e r 6: L I T E R A T U R E C I T E D  109  vii  LIST OF TABLES Table  Page  1  Bacterial strains and plasmids  24  2  PCR primers and hybridization oligonucleotides  36  3  Variations in subunit size and configuration - characteristics of  90  some microbial aromatic acid non-oxidative decarboxylases  viii  LIST OF FIGURES Figure  Page  1  Lignin backbone structure  2  2  Solubilization of lignin yields a mixture of low-molecular weight aromatic compounds  3  3  Reaction scheme for non-oxidative decarboxylation of vanillate to guaiacol  6  4  The Streptomyces growth cycle  14  5  A possible niche for Streptomyces in lignin degradation  15  6  Decarboxylation of vanillic acid to guaiacol by Streptomyces sp. D7  19  7  Experimental scheme for growth and vanillic acid induction of Streptomyces sp. D7 cells for 2D-PAGE analyses  20  8  Synthesis of protein 3717 in response to vanillic acid  21  9  Map of expression vector pIJ680  29  10  Map of cloning/expression vector pi J702  30  11  pIJ702 and the 4 kb BamHl Streptomyces sp. D7 genomic D N A fragment inserted in both orientations in the tyrosinase gene (mei) promoter  12  pIJ680 and the PCR-generated gene combinations inserted downstream of the promoter for the aminoglycoside phosphotransferase (aph) gene  13  Strategy for a knockout of the vdcC gene using the thiostrepton (tsr) resistance gene  14  Amino terminal sequence alignment of Streptomyces sp. D7 protein 3717 and Clostridium hydroxybenzoicum /(-hydroxybenzoate carboxy-lyase  15  Lambda D A S H II phage clone carrying the putative vanillate decarboxylase gene  55  16  D N A purified from phage clone 3717C(+) and digested with BamHl  56  17  p S U B l (Lane A), pSUB3 (Lane B), pSUB4 (Lane C), pSUB5 (Lane D) and pSUB6 (Lane E) purified plasmid D N A , digested with  57  BamHl  39  41  45 54  18  Plasmids carrying subfragments of the BamHI genomic D N A fragment from Streptomyces sp. D7, which was cloned in lambda D A S H II phage clone 3717C(+)  58  19  Nucleotide sequence of the vdc gene cluster, featuring vdcB, vdcC and vdcD  59  20  Schematic diagram of the 4.4 kb BamHI genomic D N A fragment from Streptomyces sp. D7, containing the V D C gene cluster  60  21  Dendrograms depicting the sequence-based relationships between proteins and hypothetical proteins similar to the product of the vdcB gene and vdcC gene  61  22  Location of the putative divergent regulatory gene in relation to the V D C gene cluster  70  23  B L A S T - X result indicating that the nucleotide region immediately upstream of vdcB encodes a polypeptide similar to a putative transcriptional regulator from S. coelicolor A3 (2)  71  24  Northern blot hybridization of PCR-amplified, radiolabeled vdcC D N A against mRNA isolated from uninduced Streptomyces sp. D7 (Lane 1) and Streptomyces sp. D7 induced with 3.6 m M vanillic acid (Lane 2)  73  25  Southern blot hybridization of Sa/I-digested chromosomal D N A from Streptomyces sp. D7 (Lane 1) and Streptomyces setonii 75Vi2 (Lane 2) with radiolabeled vdcB, vdcC and vdcD PCR-amplified D N A probes at 65 °C  75  26  SDS-PAGE (12.5% acrylamide) of cell extracts showing the expression product of the vdcC gene, produced using pET22b(+) in E. co//BL21(DE3)  79  27  SDS-PAGE (12.5% acrylamide) of cell extracts showing the expression product of the vdcC gene, produced using pET22b(+) in E. coli BL21 (DE3) at 25°C and 30°C with 0.1 m M IPTG for induction  80  28  Decarboxylation of vanillic acid to guaiacol by recombinant Streptomyces lividans 1326 strains  84  29  Results of incubating the insoluble and soluble fractions of sonicated cell extracts of S. lividans 1326 - pKCS8 with 1 m M vanillic acid for fifteen minutes at 25°C under both aerobic (O2) and anaerobic (N2) conditions  85  X  30  Expression of the vdc genes under control of the aph promoter in pIJ680  86  31  Chemical structures of vanillic acid and similar aromatic acids used to test the substrate specificity of vanillate decarboxylase  87  32  Amino acid sequence comparisons between the Streptomyces sp. D7 vdcB translation product (strPAD) and its Sphingomonas p N L l homologue (sphPAD) (a), and the Streptomyces sp. D7 vdcC translation product (strC) and its Sphingomonas p N L l homologue (sphC)(b)  33  Physical map of p N L l and the location of the V D C gene cluster homologues  102  34  Vanillate decarboxylase, Cytochrome P-450 and catechol 1,2dioxygenase can be used in combinations to produce various industrially useful chemicals from vanillic acid as a starting material  105  35  Non-oxidative decarboxylases represent connections between the two major branches of aromatic acid catabolism, characterized by either catechol or protocatechuate central intermediates  107  97  LIST OF ABBREVIATIONS Abbreviation  Definition  2D-PAGE  Protein two-dimensional polyacrylamide gel electrophoresis  aph  Aminoglycoside phosphotransferase gene  BLAST  Basic Alignment and Search Tool  DNA  Deoxyribonucleic acid  DTT  Dithiothreitol  EDTA  Ethylenediaminetetraacetate  FAD  Flavin adenine dinucleotide  FAME  Fatty acid methyl ester  HPLC  High pressure liquid chromatography  Kb  Kilobase  KDa  Kilodalton  mei  Melanin synthesis gene (tyrosinase)  MRNA  Messenger ribonucleic acid  MSMYE  Mineral salts medium with yeast extract  PAD  Phenyl acrylate decarboxylase  PCR  Polymerase chain reaction  PEG  Polyethylene glycol  pi  Isoelectric point  NMR  Nuclear magnetic resonance  RBS  Ribosome binding (Shine-Delgarno) site  RNA  Ribonucleic acid  SSC  Sodium saline citrate  SDS  Sodium dodecyl sulfate  TSB  Trypic soy broth  UV  Ultraviolet  VDC  Vanillate decarboxylase  YEME  Yeast extract malt extract  Xll  ACKNOWLEDGEMENTS I thank all the members, past and present, of the Julian Davies Laboratory for their support, encouragement, and friendship.  In particular, I thank Rumi Asano, Dr. Jeff  Rogers, Sakura Iwagami-Hayward, Grace Law and Richard Kao for being great graduate school companions.  In addition, I gratefully acknowledge Dr. Margaret K . Pope for  providing essential guidance and technical expertise for the project.  I thank Dr. Vera  Webb for making it possible to prove myself in the laboratory. Vincent Martin, Professor William Mohn, Professor Douglas Kilburn, and Professor James Kronstad provided valuable technical advice for the project.  I am grateful to my parents, who always  believed in me, and Alisa Chan, who provided much appreciated support to get me through the most trying stages of graduate school life.  Canadian Forest Products  (Canfor) Research and Development Centre staff provided valuable expertise and resources. Most importantly, Professor Julian Davies (who said I would be rich and/or famous  someday) provided me with the encouragement,  support, finances, and  worldwide network of contacts that made my efforts worthwhile. Finally, there are many others who helped me in one way or another, and to them I am sincerely thankful.  This research was funded by the G R E A T Scholarship program provided by the Science Council of British Columbia (SCBC), Forest Renewal British Columbia (FRBC), and the Natural Sciences and Engineering Research Council of Canada (NSERC).  1  1 . INTRODUCTION Chemical manufacture of benzenoid compounds from petroleum relies on abiotic, chemical catalysts. The use of petroleum poses a number of problems, it being a nonrenewable resource, a geopolitically volatile commodity, and a source of many environmentally toxic compounds. Therefore, there is growing interest in developing processes for enzymatic conversion of renewable resources such as plant biomass for the production of chemicals traditionally derived from petroleum.  The abundance of  phenylmethylether motifs in natural compounds such as lignin (Figure 1) and their release as a result of lignin solubilization (Figure 2) has resulted in the evolution of mechanisms  for  the  degradation  of  phenolic  structures  by  microorganisms.  Microorganisms exhibiting such enzymatic biotransformation potential could be harnessed for industrial use to supplement or replace traditional chemical synthesis methods, should the need arise (Frost & Draths, 1995).  Vanillic acid is an abundant component of solubilized lignin biomass, and degradative mechanisms by which this compound is catabolized have been elucidated in several prokaryotic organisms.  The genes responsible for vanillate demethylation have been  cloned and sequenced from Pseudomonas sp. strain A T C C 19151 (Brunei & Davison, 1988), Acinetobacter sp. ADP1 (Segura & Ornston, 1997) and, most recently, Sphingomonas paucimobilis (Nishikawa et al, 1998). In these microbes, vanillate is converted to protocatechuate, which is in turn degraded by enzymes of the P-ketoadipate pathway.  CH OH 2  I CH OH 2  Figure 1: The lignin backbone structure.  H  C  " ° "  3  CH OH 2  Figure 2: Solubilization of lignin yields a mixture of low-molecular weight aromatic compounds, some of which are shown here.  4 However, in some strains of Streptomyces and Bacillus (Crawford & Olson, 1978), vanillate  is  catabolized  via  an  alternative  pathway  involving  non-oxidative  decarboxylation to guaiacol (Figure 3), with further catabolism via cytochrome P-450 mediated demethylation and mineralization through the intermediate catechol. In fact, it was demonstrated that individual Streptomyces isolates degraded vanillate by both routes, that is, through both catechol and protocatechuate as central intermediates. There are a number of reports in the literature of aromatic acid non-oxidative decarboxylases from various microorganisms (Grant & Patel, 1969; Yoshida & Yamada, 1985; Nakajima et al, 1992; Huang et al, 1993; Santha et al, 1995; He & Wiegel, 1995; He & Wiegel, 1996; Zeida et al, 1998) but thus far there have been no molecular studies of these processes.  1.1  N o n - o x i d a t i v e  d e c a r b o x y l a t i o n :  c h a r a c t e r i z e d  process  a n  i n d u s t r i a l l y  u s e f u l  b u t  p o o r l y  Non-oxidative decarboxylation of aromatic acids involves the removal of the carboxyl moiety from the benzene nucleus via a reaction that requires neither oxygen, nor cofactors such as N A D and FAD, typical elements of the oxidative process. The nonoxidative process results in a "clean" removal of the carboxyl group, in contrast to the oxidative reaction, which substitutes a hydroxyl group at the relevant carbon atom. In nature, non-oxidative decarboxylation is not only observed for biodegradative pathways, but also for anabolic pathways, such as in the biosynthesis of naphthoquinones (Santha et al, 1995). Similarly, for biotransformation and metabolic engineering applications, both oxidative and non-oxidative processes are valuable as components of hybrid pathways for  the production of various industrially useful compounds. In fact, a Klebsiella  5  aerogenes protocatechuate non-oxidative decarboxylase has been engineered into a hybrid pathway to produce catechol, a useful building block for pharmaceuticals, from glucose as a renewable starting material (Frost & Draths, 1995).  6  COOH  Vanillic Acid  Guaiacol  Figure 3: Reaction scheme for the non-oxidative decarboxylation of vanillate to guaiacol.  7  1.2 N o n - o x i d a t i v e  d e c a r b o x y l a t i o n i n p r o k a r y o t e s  Reports on microbial non-oxidative decarboxylation of aromatic acids date back as far as 1924, but the first published work that was of significance towards modern studies of these enzymes was in 1969, by Grant and Patel, then at the University of Nottingham. Their thorough physiological study of Klebsiella aerogenes and its abilities to nonoxidatively decarboxylate />-hydroxybenzoate,  gentisate, protocatechuate and gallate  revealed an organism that was likely responsible for decarboxylations of phenolic acids found in intestinal microflora. The study suggested that the aforementioned aromatic acids were decarboxylated by different enzymes, and that all of these enzymes were membrane associated, according to activity localization after ultrasonication and debris fractionation.  The researchers noted that the non-oxidative decarboxylation of p-  hydroxybenzoate was likely an injurious side reaction to the bacterium due to the toxic characteristics of the product, phenol.  As  already mentioned, and relevant to this study, Streptomyces is one of the  microorganisms in which non-oxidative decarboxylation has been biochemically well characterized. Crawford and Olson published data that would become the first in a series of  reports  detailing  a  streptomycete's  capability  to  catabolize  vanillate  by  decarboxylation to guaiacol (Crawford & Olson, 1978). Streptomyces strain 179 was isolated from Idaho forest soils, and exhibited what was then considered a novel catabolic reaction for the utilization of vanillic acid. Further research on another microorganism, S. setonii 75Vi2, revealed that, in addition to non-oxidative decarboxylation of vanillate to guaiacol, a cytochrome P-450 system is involved in demethylating guaiacol to  8 catechol.  Catechol is then mineralized (presumably, though not demonstrated) by  catechol 1,2-dioxygenase and other associated lower pathway enzymes (Sutherland, 1986). Although non-oxidative decarboxylation of vanillate by S. setonii 75Vi2 was well-documented biochemically, the genetic basis for the reaction was not investigated.  The purification and partial sequencing of two non-oxidative decarboxylase enzymes from the strict anaerobe Clostridium hydroxybenzoicum has been accomplished (He and Weigel, 1995). C. hydroxybenzoicum, an novel anaerobe, was isolated from freshwater sediments.  The microorganism decarboxylates both 4-hydroxybenzoic acid and 3,4-  dihydroxybenzoic acid under anaerobic conditions but does not metabolize the products further. The enzymes responsible for these reactions, />-hydroxybenzoate carboxy-lyase and 3,4-dihydroxybenzoate carboxy-lyase, showed no significant similarity to any protein sequences in the databases. The authors indicated that cloning of the genes encoding these proteins was in progress, but at the time of writing of this thesis, no such gene sequences have been reported.  From an industrial standpoint, non-oxidative decarboxylases that convert gallate to pyrogallol are particularly interesting.  There are numerous reports in the literature  detailing whole cell bioconversions of gallate using various microorganisms, both prokaryotic and eukaryotic. Yoshida and Yamada (1982) described the optimization of whole cell bioconversions using a Citrobacter sp., in which a yield of 97.4% pyrogallol was obtained from gallate as an initial substrate.  A recent development with great  industrial potential was a report of the purification and characterization of gallate  9  decarboxylase from Pantoea agglomeransTll  (Zeida et al, 1998). P.  agglomeransTll  expresses not only a gallate non-oxidative decarboxylase, but also a tannase, which allows the organism to produce pyrogallol from tannic acid, an abundant waste product of the forest industry. The gallate decarboxylase was highly specific for gallate. As in the other studies mentioned in this survey, the genes encoding the decarboxylase were not cloned; however, the study emphasized the industrial relevance of this seemingly simple, yet poorly characterized, reaction.  1.3 S t r u c t u r e a n d f u n c t i o n s t u d i e s o f n o n - o x i d a t i v e d e c a r b o x y l a s e s  Several reports  have  described the  instability of aromatic  acid non-oxidative  decarboxylases during purification procedures. Perhaps it is this characteristic of these enzyme systems that has resulted in limited studies of the proteins and their corresponding genes. Nevertheless, several research groups have succeeded in purifying, partially sequencing, and characterizing various non-oxidative decarboxylases. Santha et al.  (1995)  reported  the  structure  and  function  of 2,3-dihydroxybenzoic acid  decarboxylase from Aspergillus niger. In this work, the active site peptide of the enzyme was determined, and a partial primary structure map was created based upon sequences derived from enzymic cleavage of the protein. The enzyme system did not require any cofactors, emphasizing the importance of multiple active site residues (yet to be characterized) in the reaction mechanism.  Huang et al, in a report describing the  mechanism of action of ferulate decarboxylase in the yeast, Rhodotorula rubra (the enzyme does not decarboxylate the aromatic nucleus, but rather a side chain of the compound), also observed vanillate decarboxylation to guaiacol as a downstream  10  catabolic step (Huang et al, 1993). The researchers included a N M R analysis of the chemical mechanism of vanillate non-oxidative decarboxylation in their studies. He et al, in their study of the non-oxidative decarboxylases from C. hydroxybenzoicum, found that the enzyme did not require any cofactors or metal ions for activity (He et al, 1995). The  enzymes were observed to be reversible, depending on the reaction conditions.  Finally, the aforementioned study of gallate decarboxylase in P. agglomerans T71 (Zeida et al, 1998) details the stabilization of the enzyme and sequencing of its amino-terminus. The authors found that gallate decarboxylase from P. agglomerans T71 is unique among similar decarboxylases in that it requires iron as a cofactor. Information on non-oxidative decarboxylases from microorganisms is accumulating, from both fundamental and applied research, and thus far it can be generalized that these proteins form a class of enzymes which are fairly substrate specific, unstable, and for the most part function independently of cofactors.  1.4  M e t a b o l i c  e n g i n e e r i n g  r e n e w a b l e  r e s o u r c e s  f o r t h e  p r o d u c t i o n  o f  c h e m i c a l s  f r o m  Vast amounts of aromatic carboxylic acids are available as natural products from plant and wood residues. However, exploitation of such substances as starting material for chemical syntheses or as fermentation substrates has not attracted the sustained attention of molecular biologists. Much remains to be learned about the microbiological systems that offer potential avenues for recovering the major biochemical resource that the plantderived aromatic carboxylic acids represent. To my knowledge, there are no published studies of metabolically engineered organisms for the biotransformation of lignin residues.  However, E. coli has been modified, in an elaborate genetic engineering  11 scheme, to bioconvert D-glucose to adipic acid, the building block of nylon (Draths & Frost, 1994). Briefly, E. coli AB2834, a mutant lacking shikimate dehydrogenase, was transformed with the plasmids pKD136 (encoding a transketolase, D A H P synthase, and DHQ  synthase),  pKD8.243A  (encoding  DHS dehydratase  and  protocatechuate  decarboxylase), and pKD8.292 (encoding catechol 1,2-dioxygenase). The bacterial strain was kept stable due to plasmid compatibility and the use of drug resistance selections for each plasmid.  E. coli AB2834/pKD136/pKD8.243A/pKD8.292 utilized its suite of  aromatic amino acid biosynthetic genes as well as aromatic acid catabolic genes to effectively convert D-glucose, derived from many agricultural sources, to adipic acid. The process provides an effective alternative to conventional adipic acid synthesis from benzene, a process that results in the production of high amounts of nitric oxide, the "greenhouse" gas implicated in the depletion of the ozone layer of the Earth. It should be noted that almost all the genes used in the engineered E. coli strain have been patented, and the D N A sequences of these genes, if known, are not freely available to the public.  Vanillate decarboxylase from Streptomyces sp. D7 could be useful in the future as a critical link in a multi-step metabolic engineering regime similar to that described for E. coli AB2834. The utility of vanillate decarboxylase in value-added biomass conversions will be described in more detail in the Discussion section of this report.  1.5  Streptomyces:  versatile soil  m i c r o b e s  Streptomyces is a genus of gram positive, filamentous, sporulating bacteria that are mainly native to soil, but are also found in aquatic environments. Being predominantly  12  soil-borne microbes (Atlas & Bartha, 1993), these organisms have developed diverse metabolic capabilities that allow for the production of a diverse array of chemical compounds. These secondary metabolites (produced during the late (stationary) phases of growth) have found many applications for humans, ranging from anti-inflammatory therapeutics to potent anti-microbial agents.  The high industrial value of certain  streptomycete strains for their secondary metabolites has resulted in the development of a detailed understanding of the genetics and physiology of these microorganisms (Hopwood et al, 1985). Starting from spores, streptomycetes undergo germination to form substrate mycelial (hyphal) growth. Subsequently, the microorganism shifts to a secondary phase of growth, in which aerial mycelia are formed on the substrate mycelial base. It is during this phase that most of the industrially useful compounds are formed. Upon the induction of signaling responses (such as two-component serine-threonine phosphorylation cascades) due to environmental stimuli such as starvation, the organisms shift to a sporulation cycle, in which chains of uni-nucleate spores are produced at the  end of the aerial mycelial tips. The streptomycete growth cycle is illustrated in Figure 4. At a genetic level, streptomycetes possess linear chromosomes, and may contain both circular and linear plasmids. As members of the Actinomycetes, they characteristically possess D N A with a high guanosine and cytosine (G+C). content, typically in the range of 70-75%.  Currently, the Streptomyces  coelicolor  A3(2) genome sequencing project is  well underway, and thus far, of the 8 Mb, >7000 gene (estimated) genome, just over 3 Mb have been sequenced. The data accumulated provides the following statistics (D. Hopwood, J. Davies, personal communication). The average G+C content is 71.72%. Of the open reading frames (ORFs), 3.5% have been previously sequenced, 50.5% resemble  13  those of known function, 19.2% resemble those of unknown function (hypothetical proteins), and 26.8% have no database match. There is an average of 1.14 kb per ORF, suggesting that the genome is tightly packed ~ by comparison, the yeast Saccharomyces cerevisiae has 13 Mb and <6000 genes, with an average of 1.2 kb per ORF. Streptomycetes are renowned for their secondary metabolite production, but their abundance in soil, particularly in environments rich in humic matter and lignocellulose, have adapted them to degrade a wide variety of natural substances.  These  microorganisms provide a major contribution to the global carbon cycle by assisting fungi in the mineralization of cellulose and lignin.  Surprisingly, this aspect of  streptomycete biology has been little investigated; Streptomyces viridosporus T7A, which secretes a powerful lignin peroxidase is, thus far, the most well characterized lignin-degrading  streptomycete  (Thomas  & Crawford, 1998).  However,  most  streptomycetes isolated from soils do not degrade lignin, but are very efficient at the catabolism of lignin-related, low molecular weight aromatic compounds such as vanillic acid. A possible scenario (Kirk, 1987) is that in the natural consortia of microorganisms present in forest soils, fungi such as the basidiomycetes perform depolymerization of intact lignin, leading to the release of more soluble fragments which can be transformed and mineralized by other microorganisms such as streptomycetes (Figure 5). The nonoxidative decarboxylation of vanillic acid to guaiacol is one such transformation.  Figure 4: The Streptomyces growth cycle.  15  It is the intent of these studies to shed light on the process of non-oxidative decarboxylation of aromatic acids by utilizing a proteomics and functional genomics approach. By studying gene expression patterns during microbial catabolic processes, I have been able to partially characterize the molecular basis by which non-oxidative decarboxylation occurs in Streptomyces, and perhaps in other organisms with similar functions.  The following information provides background details regarding the  isolation of Streptomyces sp. D7, catabolic phenotyping, proteomic analysis of gene expression, and isolation and partial sequencing of a putative vanillate catabolic enzyme, leading to the work described in this thesis (Chow, 1996).  1.6 Isolation and identification of Streptomyces sp. D 7 The organism used for this study, Streptomyces sp. D7, was isolated from a soil sample taken from forest land on the University of British Columbia campus in Vancouver, B.C., Canada (Chow, 1996). The organism produces abundant gray spores when grown on a mannitol soya agar medium, and produces a bright yellow diffusable, water-soluble pigment during growth in various solid and liquid media.  Figure 5 (following page): A possible niche for Streptomyces in lignin degradation.  16  Fungal lignin solubilization  17  1.7  C a t a b o l i c  tests a n d 2 D - P A G E  analysis  Streptomyces sp. D7 was determined, by U V spectrophotometry and H P L C analyses of culture supernatants, to efficiently bioconvert vanillate to guaiacol (Figure 6), suggestive of the activity of a non-oxidative decarboxylase (Chow et al, 1999). While Streptomyces sp. D7 was apparently capable of limited growth using the carboxylic acid moiety of vanillate as a sole carbon source, no further degradation of guaiacol was observed. As mentioned previously, other microorganisms and strains of Streptomyces have been shown to perform this enzymatic reaction (Crawford & Olson, 1978; Pometto III et al, 1981;  Sutherland et al, 1981), but thus far, no genetic information has been published  regarding these enzyme systems.  In order to identify proteins synthesized during  vanillate catabolism, high-resolution 2D-PAGE technology (the "Investigator System", Genomic Solutions Inc.) was used to visualize "genetic snapshots" of cellular activity when growing cells of Streptomyces sp. D7 were induced with non-inhibitory amounts of vanillate (Chow et al, 1999; Chow, 1996). Streptomyces sp. D7 was grown in a mineral salts medium supplemented with 0.5% yeast extract until mycelia were in early logarithmic growth phase. At this point in the growth cycle, the culture was divided and 3.6 m M vanillic acid was added to one of the cultures to induce a response. Aliquots of both induced and uninduced cultures were pulse labeled with S-methionine/cysteine at 33  1, 2, 5, 12, and 15 hours post-induction. A diagram depicting this experiment is shown in Figure 7. The labeled mycelium samples were sonicated to extract total cell protein and separated by 2D-PAGE.  Compilation and analysis, by PDQUEST software (PDI, Inc.)  using a SparcStation 5 workstation (Sun Microsystems), of numerous 2D-PAGE gels from several time course experiments resulted in the identification of at least six major  18  proteins that were synthesized in response to vanillate.  The most prominent and  abundant protein, of 52 kDa in molecular mass with a pi of 4.9 (Figure 8), was pooled from replicate gels, blotted to PVDF membrane and Edman-degradation sequenced, yielding sufficient and reliable amino-terminal data ( A Y D D L R Y F L D T L E K E G Q L L R I T ) to allow synthesis of a degenerate oligonucleotide probe. This probe allowed me to proceed with the cloning of the gene encoding the 52 kDa, vanillate-induced protein in order to isolate and characterize the genetic elements forming the basis for vanillate decarboxylation in this organism. Such experiments form the basis for this thesis and are described herein.  19  3.5  _ •  3.0  0  10  20  30  40  50  60  Time (h)  Figure 6: Decarboxylation of vanillic acid to guaiacol by Streptomyces sp. D7. Concentration of aromatic compounds in culture supernatants was measured using H P L C and known concentration standard solutions. Vanillic acid concentration is represented by squares ( • ); guaiacol concentration is represented by circles ( • ) . Cells were grown to late log phase in Y E M E liquid medium, harvested, washed and resuspended in M S M Y E minimal medium with approximately 3.6 m M vanillic acid. Each time point represents a concentration as measured by H P L C of culture supernatant samples.  20  t  +5  hours  +12 hours  t  +18 hours  Cell Density Substrate addition (induction)  +2 hours +1 hour 0 (pre-induction) Time  Figure 7 : Experimental scheme for growth and vanillic acid induction by Streptomyces sp. D 7 cells for 2 D - P A G E analyses. 1 ml aliquots of mycelia were radioactively pulse labeled at the times indicated. The panels in Figure 6 (following page) correspond to these time points. (Chow, 1996)  21  t= 1 h  t = 2h  t = 5h  t = 12.5h  t=18h  Figure 8: Protein 2D-PAGE profile of the synthesis of protein 3717 by Streptomyces sp. D7 in response to 3.6 m M vanillic acid. 2D-PAGE profiles of an uninduced culture are also shown for comparison. Time values are measured as hours post induction. Arrows highlight the area in which protein 3717 appears. The panels are enlargements of the 52 kDa, pi 4.9 region from eleven different 2D-PAGE gels representing eleven time point samples and two treatments, (from Chow et al, 1999)  22  1.8 Objectives The objectives for this study are as follows: 1. Clone and sequence the gene encoding the Streptomyces sp. D7 52 kDa protein induced by vanillic acid (the putative vanillate decarboxylase gene). 2. Sequence regions up- and downstream of the gene encoding the 52 kDa protein, and locate other open reading frames in the immediate vicinity. 3. Compare the putative vanillate decarboxylase gene and its translation product to similar genes and proteins from other microorganisms. 4. Determine i f the putative vanillate decarboxylase gene is present in other streptomycetes. 5. Confirm the function of the putative vanillate decarboxylase gene by obtaining a gene knockout mutant of Streptomyces sp. D7, expressing the cloned gene in S. lividans 1326 as a recombinant host, or by expression of the gene in E. coli. 6. Characterize purified vanillate decarboxylase enzyme, i f sufficient quantities can be obtained.  23  2. M A T E R I A L S A N D  METHODS  2.1 B a c t e r i a l s t r a i n s a n d p l a s m i d s 2.1.1 B a c t e r i a l strains  Bacterial strains used in this study are shown in Table 1. Streptomyces sp. D7 was isolated from forest soil on the University of British Columbia campus, as described above. Streptomyces lividans 1326, which was used for gene expression experiments, was obtained from the John Innes Collection in Norwich, United Kingdom. Escherichia coli DH5a was obtained from Gibco B R L and used for general D N A cloning and sequencing procedures. Escherichia coli BL21(DE3), which served as host for the T7 R N A polymerase gene expression system, was obtained from Novagen as a component of the pET22b(+) gene expression kit.  2.1.2 P l a s m i d s - cloning and isolation  Plasmids used in this study are listed in Table 1. Subcloning of Streptomyces D N A (to be described in detail in Section 2.3) was performed in Escherichia coli D H 5 a with pUC19. Transformation of E. coli DH5a was achieved using a heat shock protocol, in which plasmid D N A was incubated with competent cells for 30 minutes on ice, then placed at 37°C for 30 seconds, then another 2 minutes on ice. One milliliter of SOC liquid medium (20 g of Bacto-Tryptone, 5 g of yeast extract, 0.5 g of NaCI, 10 ml of 250 m M KC1, 950 ml of distilled water, pH to 7.0; before use, add 5 ml of 2 M M g C l and 20 2  ml/L of sterile 1 M glucose) was added to the transformation mixture followed by one hour incubation at 37°C. Aliquots were plated on L B agar supplemented with an  24  antibiotic appropriate for selection of the plasmid being transformed (for example, 100 ug/ml ampicillin for pUC19).  Expression studies of the V D C genes in Streptomyces lividans 1326 (Section 2.8) were performed using pIJ680 (Figure 9) (Hopwood et al, 1985), a vector that places target genes under the control of the aminoglycoside phosphotransferase promoter, and pIJ702 (Figure 10) (Katz et al,  (aph) constitutive  1983), which provides the weaker  constitutive tyrosinase (mei) promoter. S. lividans 1326 was converted to protoplasts and transformed according to published methods (Bibb et al, 1978; Thompson et al, 1982). Protoplasts were plated on R5 solid medium (Thompson et al, 1980) and allowed to regenerate for 14 hours before transformants were selected by an overlay of soft nutrient agar containing thiostrepton to achieve a final concentration of 50 pg ml" thiostrepton 1  per plate.  Chromosomal D N A was extracted from Streptomyces strains by the method of Fisher (Hopwood et al, 1985).  Streptomyces plasmids were isolated by an alkaline lysis  procedure (Hopwood et al, 1985) and E. coli plasmid D N A was routinely isolated using the Qiaprep Spin miniprep kit (Qiagen) or the NucleoSpin miniprep kit (Clontech) for sequencing and routine manipulations.  Table 1 (following 2 pages): Bacterial strains and plasmids used in this study. Construction of gene expression plasmids is described in detail in a subsequent section of Materials and Methods.  25  Relevant properties  Reference/source  Host for pUC19and derivatives  Gibco B R L  Streptomyces sp. D7  Wild-type vanillate decarboxylase isolate  Chow, 1996  Streptomyces lividans 1326  Wild-type Streptomyces heterologous expression host  John Innes Collection, Norwich  pUC19  2.7 kb A p E. coli cloning vector  Gibco B R L  pKCEl  This study; Chow et al., pUC19 carrying 4.4 kb 1999 BamHI sub-fragment of the ~13 kb Streptomyces sp. D7 genomic D N A piece cloned from the phage library; contains vdcB, vdcC, vdcD  pKCE2  pUC19 carrying 527 bp Sail sub-fragment of p K C E l insert (see above)  This study  pKCE3  pUC19 - 3 kb BamHI subfragment of phage clone  This study  pKCE4  pUC19-2.2kb£amHI sub-fragment of phage clone  This study  pKCE5  p U C 1 9 - 1.9 kb BamHI sub-fragment of phage clone  This study  pKCE6  pUC19 - 0.8 kb BamHI sub-fragment of phage clone  This study  Strain or plasmid  E. coli DH5a M C R  Streptomyces  P l a s m i d vectors r  (  26  T a b l e 1 (continued): Strain or plasmid  Relevant properties  Reference/source  pIJ702  7.2 kb Ts Streptomyces cloning vector with mei promoter  Katzetal., 1983/ TerraGen Discovery, Inc.  pIJ680  5.3 kb Ts Streptomyces expression vector with aph promoter  Hopwood et al, 1985/ Dr. L . Sandercock, U B C Biotechnology Laboratory  pKCSl  pIJ702 carrying 4.4 kb BamHl insert from p K C E l inserted in same orientation as mei promoter  This study; Chow et al., 1999  pKCS2  pIJ702 carrying 4.4 kb BamHl insert from p K C E l inserted in opposite orientation as mei promoter  This study; Chow et al, 1999  pKCS4  pIJ680 carrying P C R amplified vdcB inserted downstream of aph promoter  This study  pKCS5  pIJ680 carrying P C R amplified vdcC inserted downstream of aph promoter  This study  pKCS6  pIJ680 carrying P C R amplified vdcD inserted downstream of aph promoter  This study  pKCS7  pIJ680 carrying P C R amplified vdcBC inserted downstream of aph promoter  This study  r  r  27 T a b l e 1 (continued): Strain or plasmid  Relevant properties  Reference/source  pKCS8  pIJ680 carrying PCR amplified vdcCD inserted downstream of aph promoter  This study  pKCS3  pIJ680 carrying PCR amplified vdcBCD inserted downstream of aph promoter  This study; Chow et al., 1999  28 2.1.3 16s r D N A sequence-based strain identification  Streptomyces sp. D7 was characterized by sequencing a 505 base pair 16S ribosomal D N A fragment produced using streptomycete specific P C R primers.  The primers  consisted of the following sequences: forward: 5 ' - G A G A T T T G A T C C T G G C T C A G - 3 ' ; reverse: 5 ' - C G G A C T G G T T G T T A C G A C T T C - 3 ' .  Thermocycling was performed as  follows: 1 minute denaturation at 95°C, 2 minutes annealing at 55°C and 2 minutes extension at 72°C. The cycle was repeated 30 times, with a final extension of 10 minutes at 72°C.  2.2 M e d i a a n d g r o w t h c o n d i t i o n s  Streptomyces sp. D7 and S. lividans 1326 were routinely cultivated in tryptic soy broth (TSB) or on mannitol soy flour agar plates at 30°C.  Catabolic tests and growth  experiments were performed using mineral salts medium supplemented with 0.5% yeast extract ( M S M Y E : ( N H ) S 0 0.1 g L" , NaCI 0.1 g L ' , M g S 0 - 7 H 0 0.2 g L" , C a C l 1  4  2  1  4  0.01 g L" , yeast extract 0.5 g L" , K H P 0 1  1  4  1  2  1.0 g L" , K H P 0 1  4  2  2  2  0.5 g L" , p H 7.2) and 1  4  aromatic compounds of interest at concentrations of 3.6 m M to 6 m M .  When  appropriate, thiostrepton at 50 (j,g ml" was included for selection and maintenance of 1  plasmid containing strains. For D N A extraction or protoplast preparation, strains were cultivated in Y E M E (liquid medium) supplemented with 0.5% glycine and 5 m M M g C l  2  at 30°C (Hopwood et al, 1985). E. coli DH5oc was grown in Luria-Bertani (LB) medium (supplemented with 100 \xg ampicillin ml" ), when maintaining pUC-based plasmids) at 1  37°C.  Figure  9: Map of expression vector pIJ680 (Hopwood et al, 1985)  30  31  2.3  A  L i b r a r y c o n s t r u c t i o n  a n d g e n e  c l o n i n g  Lambda D A S H II (Stratagene) genomic D N A phage library of chromosomal  Streptomyces sp. D7 fragments was constructed by ligating 9-22 kb Sau3AI partially digested chromosomal D N A fragments into the BamHl site of the phage arms.  Phage  carrying genomic D N A fragments were mixed, in soft nutrient agar, with Escherichia coli X L 1-Blue MRA(P2) cells and plated on N Z Y agar plates. The plates were incubated at 37°C overnight and the resulting plaques (approximately 4000 - between 300 and 400 plaques per plate) were lifted with Hybond-N nylon membranes (Amersham).  The  library was screened by hybridization at 60°C with a y P-ATP-labeled 56-mer 32  oligonucleotide probe 3717C (5'- GC(CG) TAC G A C G A C CT(CG) CG(CG) T A C T T C CT(CG) G A C AC(CG) CT(CG) G A G A A G  G A G GG(CG) C A G CT(CG) C T -3')  derived from protein amino-terminal sequencing data.  O f the approximately 4000  plaques in the library, twelve hybridized strongly to the probe. Lambda phage from one of these plaques were isolated and propagated in X L 1-Blue MRA(P2) E. coli for D N A isolation (see Section 2.3.1). The vdcB, vdcC and vdcD genes were subcloned on a 4.4 kb BamHl fragment into pUC19 in Escherichia coli DH5oc M C R (Gibco BRL).  2.3.1 L a m b d a phage D N A preparation  Lambda D A S H II phage D N A carrying Streptomyces sp. D7 chromosomal D N A BamHl fragments was isolated by the following protocol. Ten milliliters of L B liquid medium was inoculated with one agar plug (from the NZY/soft agar/phage plates (described above) containing a phage plaque), 50 uL of X L 1-Blue MRA(P2) E. coli (in 0.01 M M g S 0 , O.D.6Q0 -0.5), 100 uL of 1 M M g S 0 , and shaken at 37°C overnight. After the 4  4  32  incubation period, 100 uL of chloroform was added and the culture was shaken for an additional 2 minutes at 37°C, then centrifuged at room temperature for 10 minutes at 5000 x g. The aqueous phase was saved, and the following were added: 100 uL 1 M M g S 0 , 10 ml T M buffer (50 m M Tris-HCl pH 7.4, 10 m M M g S 0 ) , 32 iaL 10 mg/ml 4  4  DNAse and 10 mg/ml RNAse. The solution was incubated at room temperature for 15 minutes, then 2 ml of 5 M NaCI and 2.2 g of PEG (m.w. 6000-8000) were added and allowed to dissolve completely. The mixture was the incubated for 15 minutes on ice, then centrifuged for 10 minutes at 4°C at 10000 x g. The supernatant was discarded, leaving the phage pellet, which was resuspended in a minimum of 300 uL of T M buffer. The suspension was transferred to a 1.8 ml Eppendorf tube, 300 uL of chloroform was added, mixed, then the tube was centrifuged for 5 minutes at 12000 x g. The aqueous phase was transferred to a new Eppendorf tube, and the chloroform extraction step was repeated until no interface was observed between the chloroform and aqueous phases. Fifteen microliters of 0.5 M EDTA, 30 uL of 5 M NaCI, and 350 uL of tris-buffered phenol was added, vortexed, then centrifuged for 5 minutes at 12000 x g and the aqueous phase was saved. One final chloroform extraction (350 uL) was performed, then 875 uL of 100% cold ethanol was added and incubated at -20°C overnight to precipitate the D N A . After centrifugation at 12000 x g, the ethanol was removed, the D N A pellet was air dried briefly, and resuspended in 50 to 100 uL of TE buffer.  2.4 D N A s e q u e n c i n g a n d a n a l y s i s .  Automated D N A sequencing was performed using the AmpliTaq PRISM kit (Applied Biosystems) with a standard thermocycling program provided by the manufacturer, with  33  variations in the annealing temperature to match the melting temperature of the sequencing primer being used. Sequencing reactions were carried out by the Nucleic Acid Protein Sequencing (NAPS) Unit at the University of British Columbia and electrophoresed on an A B I Model 377 D N A sequencing apparatus (Applied Biosystems). Nucleic acid sequence was analyzed by the Wisconsin Package Version 10 (Genetics Computer Group) on a Sun Microsystems SparcStation5 (Sun Microsystems).  2.5  S o u t h e r n  b l o t t i n g a n d  h y b r i d i z a t i o n  Genomic D N A preparations from Streptomyces sp. D7 and Streptomyces setonii 75Vi2 were  digested  with the  restriction endonuclease  Sail overnight  at  37°C  and  electrophoresed in a 0.7% agarose gel. The gel was photographed, soaked in 0.15N HC1 for 15 minutes for depurination (to facilitate transfer of high molecular weight fragments) and soaked in alkaline hybridization solution (0.6M NaCl, 0.4M NaOH) for an additional 15 minutes. The treated gel was then Southern blotted to a positively charged nylon membrane (Boehringer Mannheim) using the same alkaline solution as a transfer buffer. After disassembly of the transfer apparatus, the membrane was rinsed briefly in 2 X sodium saline citrate (SSC) and baked at 80°C for one hour. Hybridizations utilized a rotating incubation chamber (Hybaid) and glass hybridization bottles. The membrane was soaked in 2X SSC, rolled, placed in a glass hybridization bottle with hybridization solution (5X Denhardt's Solution, 6X SSPE, 0.1% SDS) and prehybridized at 65°C for several hours. After prehybridization, the  P-labeled probe (a- P or y- P, depending on  the labeling procedure) was denatured at 95 °C for 5 minutes, cooled on ice briefly, centrifuged, then added to the  hybridization bottle  containing the  membrane.  34  Hybridization was carried out overnight at 65°C.  The wash regimen consisted of 2  washes of 2X SSC, 0.1% SDS for 10 minutes at room temperature, I X SSC, 0.1% SDS for 15 minutes at 65°C once, then a brief room temperature rinse in 0.1X SSC, 0.1% SDS. The washed blot was semi-dried, then placed in plastic wrap and exposed to film (Kodak X - A R ) for several hours to overnight. A n identical hybridization procedure was used during the phage library screening process, and also subsequent cloning manipulations.  2.6  C h e m i c a l  a n a l y s e s  Culture supernatants were filtered through 0.45 urn syringe filters and processed through a C-18 hydrophobic interaction column attached to a H P L C system (Hewlett Packard, Model 1050).  Conditions for separation were 30% phosphoric acid/water, 70%  methanol, with a flow rate of 1.0 ml min" . Retention times for vanillic acid and guaiacol 1  under these conditions are 5 minutes and 4 minutes, respectively.  Integrated peak areas  corresponding to compounds in supernatant samples were calibrated against known concentrations of vanillic acid and guaiacol standards. Additional analysis of supernatant samples was performed using a Cary 1 Bio ultraviolet/visible spectrophotometer (Varian). For U V analysis, vanillic acid characteristically displays a primary absorbance at 250 nm and a secondary absorbance at 285 nm, while guaiacol absorbs at 275 nm.  2.7  R N A  isolation  a n d analysis  Total R N A was isolated from cells grown under two different sets of conditions. Primary cultures were grown in 25 ml Y E M E for 48 hours before cells were pelleted by  35  centrifugation, washed twice with sterile water and resuspended in minimal media (MSMYE). For induced cultures, the media was supplemented with 3.6 mM vanillic acid, while no additional substrates were added to the uninduced control. These cultures were then allowed to grow an additional three hours after which cells were pelleted and washed as before. Total RNA was isolated using standard RNA isolation techniques (Hopwood et al, 1985; Kirby et al, 1967). electrophoresis and transfer  For transcript detection, Northern gel  were performed according to the  manufacturer's  recommendations for the NorthernMax kit (Ambion). 15 ug total RNA was loaded per lane in a polyacrylamide gel and 1 ug of an RNA standard ladder (NEB) was included for size comparison. After electrophoresis was complete, the RNA ladder lane was excised and stained with ethidium bromide to allow for visualization and to confirm RNA integrity. Transcript was detected using a probe specific for the vdcC gene. To generate the probe, traditional  double-stranded PCR was performed on pKCEl using  oligonucleotides vdcC.F and vdcC.R (Table 2). Following amplification, excess dNTPs and oligonucleotides were removed using a QIAquick PCR Purification Kit (Qiagen). This product was then used as template for asymmetric PCR with only the vdcC.R primer. This resulted in a single-stranded PCR product that was complementary to the predicted RNA transcript. During chain elongation, P-dCTP was provided in place of j2  dCTP in the dNTP mix to allow for direct incorporation of radiolabel. The extension product was purified and allowed to hybridize with the immobilized RNA at 60°C for 24 hours. Excess probe was removed by washing as directed by the NorthernMax protocol, and the hybridizing transcript was visualized by exposure to autoradiography film (Kodak XAR) for exposure and development.  36  Table 2 (including next page): Polymerase chain reaction (PCR) primers used in this study. Engineered restriction enzyme sites are underlined. Purpose Length Sequence (5' —» 3') Primer (nt) Hybridization probes vdcB.F  26  ACAGGTCAGCGACAGG TTTGAGGTGG  Forward amplification of vdcB gene DNA.  vdcB.R  21  TACGGGGCAGGGGACT TCAGG  Reverse amplification of vdcB gene DNA.  vdcC.F  20  GGCGACGCCGCCTGAA GTCC  Forward amplification of vdcC gene D N A .  vdcC.R  20  GGGTCGGTCGGTGTCA GACG  Reverse amplification of vdcC gene DNA.  vdcD.F  20  CACCGATCCTCACTGA AAGG  Forward amplification of vdcD gene D N A .  vdcD.R  20  CATAGACCGCGTGCCG GTCG  Reverse amplification of vdcD gene DNA.  vdcBCD.FX  26  CGGATCCAGTGACAGG TTTGAGGTGG  Forward amplification of vdcB, vdcBC and vdcBCD genes for expression in pIJ680. Engineered BamHI site.  vdcBCD.RX  28  AGTCTAGACCGGCGTC GGAGGGATGACC  Reverse amplification of vdcD, vdcCD and vdcBCD genes for expression in pIJ680. Engineered Xbal site.  vdcB.RX  21  TTCTAGACAGGGGACT TCAGG  Reverse amplification of vdcB gene for expression in pIJ680. Engineered Xbal site.  vdcC.FX  29  CGGATCCCCCGTAAAG GAATTCACCATGG  Forward amplification for vdcC gene and vdcCD genes for expression in pIJ680. Engineered BamHI site.  pIJ680 expression  37  vdcBC.RX  29  TTCTAGATCAGACGCG GGCCGCGATCAGG  Reverse amplification for vdcB, vdcC and vdcBC genes for expression in pIJ680. Engineered Xbal site.  vdcD.FX  28  CACGGATCCTCACTGA AAGGACAACTCC  Forward amplification of vdcD gene for expression in pIJ680. Engineered BamHl site.  vdcB.pETF  21  TCATATGCGGTTGGTCG TGGG  Forward amplification of vdcB gene for expression in pET22b(+). Engineered Ndel site.  vdcB.pETR  21  TTCTCGAGAGGGGACT TCAGG  Reverse amplification of vdcB gene for expression in pET22b(+). Engineered Xhol site.  vdcC.pETF  23  GGAATTCCATATGGCCT ATGACG  Forward amplification of vdcC gene for expression in pET22b(+). Engineered Ndel site.  vdcC.pETR  18  CGCGGGCTCGAGTCAG GC  Reverse amplification of vdcC gene for expression in pET22b(+). Engineered Xhol site.  pET22b(+) expression  38  2.8 G e n e e x p r e s s i o n  2.8.1 Cloning and expression in pET22b(+) in Escherichia coli BL21 D N A encoding the vdcB and vdcC genes was PCR-amplified, using primers listed in Table 2, to incorporate Ndel and Xhol sites upstream and downstream, respectively of the target gene(s) prior to cloning into the pET22b(+) vector (Novagen). These plasmid constructs were transformed into E. coli BL21(DE3), which were grown to OD 0.4, then induced with IPTG to activate expression by the T7 polymerase system, characteristic of the pET22b(+) vector. Expression was performed for two hours, at which time the cells were pelleted and protein extracted. To optimize protein expression, induction of the T7 system was performed at 37°C, 30°C and 25°C, using 0.1 m M or 1 m M IPTG. Reductions in temperature and IPTG concentration are commonly used methods to increase the chances of obtaining properly folded proteins.  2.8.2 Cloning and expression in pIJ702 in S. lividans 1326 The 4.4 kb BamHI D N A fragment containing the vdcBCD gene cluster was inserted into the Streptomyces cloning vector pIJ702 at the unique Bglil site. Insertion at this site places the cluster downstream of the mel promoter, thereby disrupting transcription of the tyrosinase gene that serves as a color selection marker for transformants. pIJ702 carrying the insert in the same orientation as the mel promoter was designated p K C S l ; conversely, a vector construct with the insert in the opposite orientation to the promoter was designated pKCS2 (Figure 11).  39  Figure 11: pIJ702 and the 4 kb BamHl Streptomyces sp. D7 genomic D N A fragment inserted in both orientations in the tyrosinase gene (mei) promoter.  40 2.8.3 C l o n i n g a n d expression i n pIJ680 i n S. lividans 1326 In order to identify the gene sequences encoding the decarboxylase, D N A regions were PCR amplified using specific primers (Table 2) which included a BamHI site upstream, and a Xbal site downstream, of the gene(s).  The PCR-generated genes were cloned  downstream of the aph promoter in BamHl-Xbal cut pIJ680, replacing most of the aph gene. The following genes were amplified using this BamHI-Xbal P C R cloning strategy for ligation into pIJ680, creating new plasmids designated in brackets: vdcB (pKCS4), vdcC (pKCS5), vdcD (pKCS6), vdcBC (pKCS7), vdcCD (pKCS8), vdcBCD (pKCS3). pIJ680 and the PCR-generated inserts are shown in Figure 12.  A l l plasmids were  transformed individually into 5*. lividans 1326 and the resulting recombinant hosts were screened for the ability to decarboxylate vanillic acid.  fl m CO r - oo r o co co in co co co  o oo oo o  X. X. X. ^ X. a a a aa a  Figure 12: pIJ680 and the PCR-generated gene combinations inserted downstream of the promoter for the aminoglycoside phosphotransferase (aph) gene.  42  2.9  E n z y m e  assays  Late log phase mycelia harvested from Y E M E cultures were washed with phosphate buffer (10 m M Tris pH 8.0, 1 m M EDTA, 1 m M DTT) and resuspended in the same buffer, containing protease inhibitors (100 jag ml" PMSF, 1 ug ml" Pepstatin A ) , at a 1  1  ratio of 0.1. ml buffer per 1 ml of original culture. Samples were sonicated as previously described and centrifuged at 12,000 x g for 15 minutes to remove insoluble cell debris. Soluble cell extracts were tested for decarboxylase activity by adding vanillic acid or comparative substrates to a final concentration of 1 m M . Samples were incubated for 15 minutes at 25°C, at which time they were analyzed by scanning the U V range from 300 nm to 200 nm. Soluble cell extract without substrate was used as a background in the reference cuvette. To test enzyme activity under anaerobic conditions, nitrogen gas was slowly bubbled through assay sample tubes prior to addition of substrate.  2.10  R e c o m b i n a n t  p r o t e i n  p u r i f i c a t i o n  f r o m  E. coli B  L 2 1 ( D E 3 )  As mentioned in Section 2.8.1, vdcB and vdcC were recombinantly expressed using the pET22b(+) plasmid vector in E. coli BL21(DE3). Attempts were made to purify the proteins produced by these strains.  Under all expression conditions, inclusion body  material was obtained, and thus efforts were mainly focused on extracting and refolding the insoluble protein aggregates. However, in the event that minute amounts of soluble, active enzyme were produced, cell lysates were passed through nickel-based affinity columns to purify the histidine-tagged recombinant proteins.  43  2.10.1 L y s a t e p r e p a r a t i o n  Ten milliliters of L B medium containing 100 pg/mL ampicillin were inoculated with material from one colony of the appropriate E. coli BL21(DE3) expression host and grown overnight at 37°C with shaking.  Subsequently, 50 ml of prewarmed media  containing 100 pg ml" ampicillin was inoculated with 2.5 ml of the overnight culture and 1  grown at 37°C, with shaking, until the  OD600  reached approximately 0.6 (about one  hour). IPTG was added to a final concentration of either 1 m M or 0.1 m M , and cultures were grown for an additional 4-5 hours at 37°C, 30°C, or 25°C, depending on the desired expression conditions. As has already been mentioned, reduction of IPTG concentration and growth temperature is believed to reduce the amount of inclusion body formation due a slowing of the expression process. After the expression period, cells were harvested by centrifugation at 4000 x g for 20 minutes. The cell pellet was resuspended in 5 mL of lysis buffer (50 m M N a H P 0 , pH 8.0; 300 m M NaCl; 10 m M imidazole) and subjected 2  4  to a freeze/thaw cycle using dry ice/ethanol and cold water, repeated three times. The sample was then sonicated with a microtip 6 times, on ice, at 10 seconds per burst with 10 second pauses, at 200-300 watts. The lysate was then centrifuged at 10000 x g at 4°C for 30 minutes. At this point, the supernatant contained any soluble recombinant protein, while the pellet contained the large mass of inclusion body material, visible as a white layer on top of the light yellow cell debris.  2.10.2 Inclusion body protein p u r i f i c a t i o n , solubilization a n d r e f o l d i n g  The pellet was resuspended in 0.1 culture volume of inclusion body wash buffer (200 m M Tris-HCI, pH 7.5, 100 m M EDTA, 10% Triton X-100) and recentrifuged. This wash  44 step was repeated, and the resulting purified inclusion bodies (of which the wet weight was noted) were solubilized in inclusion body solubilization buffer (500 m M CAPS, pH 11.0, 0.3% N-laurosarcosine) at 10-20 mg ml" for 15 minutes at room temperature. The 1  (mostly) solubilized solution was centrifuged at 10000 x g for 10 minutes at room temperature, and the supernatant was removed to a clean tube.  The soluble protein  solution was placed in dialysis tubing, and the sample was dialyzed against 20 m M TrisH C l , pH 8.5, 0.1 m M DTT, using at least three buffer changes of greater than 50 times the sample volume. Each dialysis step was performed for at least 3 hours at 4°C. After dialysis, the sample was concentrated using a Centricon-10, 10000 m.w. cut-off ultrafiltration device and analyzed by SDS-PAGE for purity.  2.10.3 Soluble protein purification procedure  The soluble fraction of the recombinant E. coli BL21(DE3) cell lysates were processed through nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography  matrix  columns in an attempt to purify any proteins that were not produced as inclusion bodies. N i - N T A mini spin columns (Qiagen, catalogue #31313) were used for small-scale experiments, while nickel resin (ProBond, Invitrogen, catalogue #R801-01) was packed in a larger column for scaled-up procedures. Column loading, washing and elution were performed according to the manufacturers' instructions.  2.11 C o n s t r u c t i o n o f a k n o c k o u t a l l e l e o f t h e  vdcC g e n e  To elucidate the function of vdcC, the gene encoding the 52 kDa protein, a disrupted allele was created by removing a portion of the 5' region of the gene and replacing it with  45  the thiostrepton resistance gene (tsr). This strategy utilized p S L l 190 (Pharmacia), which contains a superlinker with 78 restriction enzyme sites, allowing for more versatility in the construction process. The allele was designed for gene knockout experiments, which are described in subsequent sections. The allele construction strategy, using pIJ702, p S L l 190 and pUC19, is shown in Figure 13.  Figure 13: Strategy for a knockout of the vdcC gene using the thiostrepton (tsr) resistance gene, (a) Preparation of the thiostrepton resistance marker cassette; (b) Insertion of the thiostrepton resistance cassette in vdcC to create the knockout allele; (c) protoplast transformation and double crossover event.  46  8 i |  (a)  M  Zmtp  Bell  Bell  BamHI, Bglll S  Bcll^  I *  tsr  I S "9  Bell  1  1,1 kb  ligation  Dral Ncol  Ncol, Dral  Ncol  Dral(blunt)  ( s f t  NOTE: tsr " gene orientation could be in either direction. 1  47  KNOCKOUT ALLELE  4«  P L A T E P R O T O P L A S T S O N R E G E N E R A T I V E MEDIA, S E L E C T F O R T H I O S T R E P T O N  RESISTANCE  49  2.12  Streptomyces  sp.  D7  p r o t o p l a s t p r e p a r a t i o n  a n d  t r a n s f o r m a t i o n  Streptomyces sp. D7 protoplasts were prepared and transformed as described for Streptomyces lividans 1326 in Section 2.1.2, with the exception that R6 agar plates were used instead of R5.  The regeneration time for Streptomyces sp. D7 was several days  slower than that observed for S. lividans 1326.  The recipe for R6 solid regeneration agar medium is as follows (grams per liter): mix and autoclave 200 g sucrose, 10 g dextrose, 1 g casamino acids, 0.05 g M g S O ^ H ^ O , 0.1 g K2SO4, 20 g agar. After autoclaving, add 11 g glutamate, 1 mL standard trace elements solution, 7 g CaCl -2H 0 and 100 mL MOPS buffer (0.1 M , pH 7.2). 2  2.13  2  E. coli - Streptomyces  i n t e r s p e c i e s  c o n j u g a t i o n  To obtain a gene knockout mutant, attempts were made to transform Streptomyces sp. D7 mycelia by conjugation with E. coli. As an evaluation of this method, E. coli SI7-1 carrying the vector pPM801 (aph , tra ) was mated with Streptomyces sp. D7 mycelia to +  +  assess whether the vector, which encodes resistance to the antibiotic kanamycin, was transferred between the species.  2.13.1 E. coli S 1 7 - l / p P M 8 0 1 preparation  One colony of E. coli S17-l/pPM801 was diluted in 5 mL of L B broth, supplemented with 10 pg/mL kanamycin, and incubated for 8 hours at 37°C. The grown culture was diluted 1:10 with fresh L B broth with kanamycin.  50 2.13.2 Streptomyces sp. D7 preparation a n d mating  Streptomyces sp. D7 spores were scraped from a lawn of bacteria on a mannitol-soy agar plate and resuspended in 5 mL of TES buffer (0.05 M , p H 7.2). The suspension was heat-shocked at 50°C for 10 minutes, then placed under cold tap water to stop the shock process. The spores were then centrifuged at 3000 x g and resuspended in 1 mL of L B broth. 150 uL of the spore suspension was plated on an AS-1 agar plate (Hopwood et al, 1985) and the spores were allowed to grow for 2 to 4 hours at 30°C. Subsequently, 100 pL of the E. coli S17-l/pPM801 preparation (see above) was plated on top of the Streptomyces sp. D7 spores. After overnight growth of the E. coli layer, the layer was gently scraped off using a sterile glass spreader and 0.05 M TES buffer, p H 7.2. A n antibiotic overlay, consisting of 2.5 mL of 50 ug/mL nalidixic acid and 25 jag/mL kanamycin, was spread over each plate. Plates were allowed to incubate for a further 6 days, at which time they were observed for any ex-conjugants.  2.14 Reagents and enzymes A l l reagents used were of the highest quality, and purchased from Sigma Chemical Company unless noted otherwise.  Restriction endonucleases and other modification  enzymes were obtained from Gibco B R L , New England Biolabs, Boehringer Mannheim or Pharmacia.  51  3. RESULTS 3.1  1 6 s r D N A  s e q u e n c e  identification  Sequence of the 505 bp PCR product amplified from Streptomyces sp. D7 16s r D N A was matched against the GenBank database using the B L A S T - N program (Altschul et al, 1990). The result was an exact match (data not shown) with the 16s rDNA sequence from Streptomyces sp. strain B71277, an isolate from the B B S R C Institute for Food Research in Reading, United Kingdom (Hutson & Collins, 1997).  Studies, i f any,  involving Streptomyces sp. strain B71277 remain unpublished at the time of writing this thesis, and attempts to contact the authors of the GenBank submission failed to generate a response. This identification procedure confirmed Streptomyces sp. D7 as a member of the streptomycetales.  Previously, the microorganism had been referred  to as  Streptomyces violaceusniger, as identified by fatty acid methyl ester analysis (Chow, M.Sc.  thesis, 1996). However, the results of the 16s r D N A analysis, considered a more  powerful tool for taxonomy (J. Davies, personal communication), suggest otherwise.  3.1  C l o n i n g  o fthe  V D C  g e n e  cluster  Edman degradation sequencing of protein 3717, isolated from protein 2D-PAGE gels (previous work described in Section 1.6), yielded the following N-terminal amino acid sequence: A Y D D L R Y F L D T L E K E G Q L L R I T .  This sequence matched well with the  amino-terminal sequence of />-hydroxybenzoate  carboxy-lyase from the anaerobe,  Clostridium hydroxybenzoicum (He and Wiegel, 1995) as shown in Figure 14.  The  deduced degenerate oligonucleotide probe 3717C (for sequence, see Materials and  52  Methods) was synthesized and hybridized against a lambda D A S H II phage library (Stratagene) of Streptomyces D7 genomic D N A . A phage clone, designated 3717C(+), hybridized strongly to the probe (Figure 15). D N A from phage clone 3717C(+) was purified and digested with the restriction enzyme BamHI (Figure 16). This restriction digest revealed that the phage clone carried an approximately 13 kb Streptomyces sp. D7 genomic D N A insert.  Streptomyces genomic D N A BamHI digestion products of  approximately 4.4 kb, 3.0 kb, 2.2 kb, 1.9 kb and <1 kb were subcloned individually into pUC19 for further manipulations and sequencing (Figure 17). The pUC19 subclones were designated p K C E l through pKCE6 (Figure 18). p K C E l contains the 4.4 kb BamHI fragment encoding the vanillate decarboxylase gene cluster, while pKCE2 contains a 527 bp Sail fragment that encodes the amino-terminal region of the vdcC gene.The entire sequence of the 4.4 kb Streptomyces sp. D7 D N A fragment (GenBank accession number AF134589) is shown in Figure 19.  Sequence analysis revealed that the gene encoding protein 3717 was contained on a 4.4 kb BamHI fragment, and was determined to be the second gene in a cluster of at least three genes, designated vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp), as depicted in Figure 20. B L A S T - X sequence analyses (Altschul et al, 1990) revealed that the gene cluster, in whole or in part, is present in a variety of microorganisms.  The vdcB  translation product is highly similar to phenylacrylate decarboxylase (PAD) from Saccharomyces cerevisiae. The yeast P A D contains a putative trans-membrane domain close to the amino-terminus (annotated in GenBank accession number S62017), which is highly  conserved  among  other  hypothetical  P A D homologues  from  various  53  microorganisms, as revealed by genome projects (this region is highlighted in the amino acid sequence alignments shown in Figure 21). The vdcC translation product is also highly similar to hypothetical proteins identified in various microbial genome sequencing projects, in addition to the amino-terminal similarity to 4-hydroxybenzoate carboxy-lyase as revealed from the Edman degradation sequencing of protein 3717. Dendrograms of the vdcB and vdcC translation products in comparison to other microbial homologues are shown in Figure 21. Unlike the first two genes in the cluster, the vdcD translation product shows similarity only to a hypothetical protein from Bacillus subtilis. Although these genes have homologues in a number of microbial genomes, they are not always clustered and only Bacillus subtilis contains all three genes in the same order as Streptomyces sp. D7*. Other microorganisms contain vdcB and vdcC homologues, but at different chromosomal locations. Interestingly, Sphingomonas aromaticivorans strain F199 possesses homologues to vdcB and vdcC and vdcD on its 184 kb catabolic plasmid p N L l (Romine et al, 1999, GenBank accession AF079317). Plasmid p N L l contains a variety of genes encoding enzymes for the degradation of a number of toxic organic chemicals, and among these genes lie (in order; identifications by similarity only): orfl244 (vdcB, phenylacrylate decarboxylase), pchFa (p-cresol methylhydroxylase), vdh (vanillin oxidoreductase), orfl272 (vdcC), orfl280 (vdcD).  * Interestingly, the Bacillus  subtilis  strain did not metabolize vanillic acid.  54  SD7 CHB  2 6  YDDLRYFLDTLEKEGQLL  I III l l  +  I  +  I l  19  +  YRDLREFLEVLXQXGXLI  23  Figure 14: Amino-terminal sequence alignment of Streptomyces sp. D7 protein 3717 (SD7) and Clostridium hydroxybenzoicum /7-hydroxybenzoate carboxy-lyase (CHB) (He and Wiegel, 1995). (|) = identity; (+) = similarity. Note that three amino acid residues of the C hydroxybenzoicum enzyme were not identified (marked as ' X ' ) .  55  Figure 15: Lambda D A S H II phage clone carrying the putative vanillate decarboxylase gene. Phage plaque lift was hybridized to P-labeled oligonucleotide probe 3717C and exposed to X-ray film for several hours with an intensifying screen. A n arrow indicates the position of the hybridizing phage clone. 32  56  Figure 16: D N A purified from phage clone 3717C(+) and digested with BamHI (Lane 1). Molecular size markers (Lane M) are denoted in kilobases. Streptomyces sp. D7 genomic D N A fragments were separately purified and ligated into pUC19 to form the p K C E series of plasmids. The gel consists of 0.7% agarose in T A E buffer, stained with ethidium bromide.  57  M1 M2 A  B  C  D E  Figure 17: p K C E l (Lane A), pKCE3 (Lane B), pKCE4 (Lane C), pKCE5 (Lane D) and pKCE6 (Lane E) purified plasmid D N A , digested with BamHl. A n arrow denotes the position of linear pUC19. M l = X-Hindlll molecular size standards, in kb; M2 = 1 kb molecular size standards, in kb. The gel consists of 0.7% agarose in T A E buffer, stained with ethidium bromide.  58  Lambda phage 3717C(+) BamHI digestion, cloning of fragments into p U C 1 9  pKCEl (7.1 kb)  SD7 Sa/I cut pSUB1 DNA (527 bp)  pKCE3 (5.7 kb)  pKCE4 (4.9 kb)  pKCE5 (4.6 kb)  pKCE6 (3.5 kb)  Sa/I digestion, s u b c l o n i n g of 527 bp DNA fragment  pKCE2 (3.2 kb)  Figure 18: Plasmids carrying subfragments of the BamHI genomic D N A fragment from Streptomyces sp. D7, which was cloned in lambda D A S H II phage clone 3717C(+). Plasmids are designated p K C E l through pKCE6, and are described in the text.  59  1 c c g c g t c c a g a t c g c t c c g t agcgtgaaag c a c a g g t c a g  vdcB-»  cgacaggttt  gaggtggtcc  ccgttcggtg gtcctgtccc gtgtccgccc ggttcgttcc atcaggaccg cgacgcaggc atgctcgaac aacccgcaga gacctgcccg cgatccttcg  tccgtcttct gctgggcgcg tagcggacgt gcaccgacgg gatacgccga tcgtcctcgt tcgcccgcat ccgtcgacga cgcccgccgc gcgacgccgc cgcagcttcc ctgcccgagc cccgccctcc cacggctcct gagcaggtgg gaggaagcac cttcccctct gtctcccgcg atccaggtca catctgcgca gaccccgtga atggcgggag gacgtgccct atagaggggc atccgcgtgg atgccgtgga cagctgcgcg atggtgatca gccatgacga gtcgacccgt gacgacgtcg gccggcatca ggcaacttct cagcgcctga vdcD -> gjj§aaccacc acgtcccccg cgcacgatcg acggcggagg cgttcctggc cccacatggg tcgctcgcac ggaactcatt gggccccatc tctggatgca ggccgtactg caaggacgtc cctgaccgcg tccctccgac  61 121 181 241 301 3 61 421 481 541 601  tf|||cggttg ggagaatctg caccaccatc cacgcaccac catggtgatc agggctcgtc cccgcgcgag gggcgtgcaa catcgtcgat ccggcgctgg  gtcgtgggaa cgccagttgc gagatggaga cccgaggacc gtgccgtgct gcccgggccg acaccgctga ctggtgccgc cacgtggtgg gccgggatgc  tgaccggggc cgggcgtgga cgggcctgtc agggcgccac ccatgaagac ccgacgtggt gcgagatcca ccatgcccgc cccgcatcct gcgccgcccg  gacgggtgcc gacacatctc cgtggccgag catctcctcc cctcgccggg tctcaaggag cctccagaac cttctacaac cgaccagttc cgccgccgcc  661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 18 61 1921 1981 2041  c||||§agtccc tcgacacctt cggatctcgc acttcgacaa gggccaacca aggagttcgc cctggcgtga tccgcctcaa acccggagga tcggcaccaa aggccgagga tggcgatcgt ccctgcgcgg gggggagcga ccttcggcga aacgcgtctc acgagtgcga ccgagttccc tctccacggc cgccgcacgg tcaacctgcc tcgtcatccc gcagcaagat ccactccggc  ctgccccgta ggagaaggag ggcggccgcc cgtcaagggc cgcgctcgcg gcggcgctgg gaacacccag cgacggagac ccgggacgac ccggctcgcc gaagggcgag ggccgggatg cgcgcccgcg ggtcgtcata gttcaccggt gtaccggcac ctacctcgtc cgaggtgcag caagcggtac gctcggctac gcaggtcatg caacctgtcg gatcatcgat caaggacctg  aaggaattca gggcagctgc aacgcgaccg ttcaccgacg ctcgggttgc gacgccttcc gagggcgagg ggtggcttct ttcggcaagc ttccaccctg gacctgccca ccgatggcgt cccatcgcca gagggcgtca cattactcgg gaaccggtct ggacccaaca gccgtcaacg ggcggcttcg gtggcccagg tgggcgatgt gtcctggaac gcgacgacgc cccgagaccg  cc|S||gccta tgcgcatcac gccgcatcgg cccgcatcgc cgaagaacac ctgtcgcccc acgtcgacct a'tctcgacaa agaacgtcgg ccatgcacga tcgccatcac acgaccagag ccgcgccgct tcgagtcccg gggggcgccg tcgagtcgct cgtgcgtgcc ccatgtacac ccaaggccgt tgatcctcgt ccgccaaggt tcgcgcccgc cggtcgcccc cagagtgggc  tgacgacttg cgacgaggtg cgagaacgcc gatgaacgtg gccggtcaag cgagcgccgc gttctcggtg ggccgccgtc cacctaccgc cgtggcccag cctcggcaac cgagtacgag caccggcttc caagcgccga catgcccgtc ctacctcggc gctgctcaag gcacggcctg cggcatgcgc cgacgaggac caacccgaag cgcgcagccc ggacgtccgc cgcccgcctg  2101 2161 2221 2281 2341 2401 24 61 2521 2581 2 641 2701 2761 2821 2881 2941  tcgcggcccg tgcccgttga tcccaggcgt aacccgcacg acatcgagaa acgcccggag agtgagggcg tccgtctcgc gacgcgctac cgctgaccgg tgacaccgct gccgcgctgg tcgtatcgcg aaccccggca gccggtcagg  cgtc|||jlcac atgcccccgc gtgggacgtg ccgcacccgg cgccatcgag agaacgtccg ggtggtgccc gcagtgaacg ctttcgcctg ggccgaaggc ccgttcctcg gcacctctgt gttatcacgt cctgcgtgcg cctcgtgccc  ccaccgatcc tgcgcctttg gtccagtgcg cgtgacgcct gtgcccgcgg ccagaacctg aactttccct tagaaaatat ccccgtcacg gcacagttcc ctgcacgaat cgtggctgcc acgcgtgccc cctcgaccgg g  tcactgaaag aggacatctc gccgctgtct acccggacag tgccgccact aacgggcccg catatacacg tcggtagtag gcaggcacac tgtccaaccc gcgcgggcaa gccgcgtcgc gcaagttggc cacgcggtct  gacaactccc cctgcttgcc ctacacctgg cttcaagctg gctcaagH!§ gcgtcgagct tagttgctac gtagtgcaac caacggtgtt agagactgaa ccaccgttct ccgccgcggc cagtcgtgga atgtcggtca  vdcC->  Figure 19: Nucleotide sequence of the vdc gene cluster, featuring vdcB, vdcC and vdcD. Putative Shine-Delgarno sites have been underlined, and start (ATG, GTG) and stop (TGA) codons are JCAPTAUZLD IN 130LI) A\'p"sTlAI)l;.p. for each open reading frame.  60  RBS  RBS  1  —  RBS  I  vdcB  1  vdcC  vdcD  //  BamHI  BamHI  0.6 kb  1.4 kb  0.2 kb  Figure 20: Schematic diagram o f the 4.4 kb B a m H I genomic D N A fragment from Streptomyces sp. D7, containing the V D C gene cluster. Putative ribosome binding site ( R B S ) locations have been indicated with arrows.  61  Figure 2 1 (following pages): Amino acid sequence alignments between the translation products of vdcB (a) and vdcC (b) and their respective putative homologues from various microorganisms. Dendrograms depicting these alignments are shown for the products of the vdcB gene (c) and the vdcC gene (d). These dendrograms and sequence alignments were produced using the Pileup program, which is part of the Wisconsin Package Version 10 bioinformatics software package (GCG). eco: Escherichia coli; sph: Sphingomonas aromaticivorans; bsu: Bacillus subtilis; str: Streptomyces sp. D7; scv: Saccharomyces cerevesiae; arc: Archaeoglobus fulgidus; meb: Methanobacterium thermoautotrophicum; mec: Methanococcus jannaschii; pyr: Pyrococcus horikoshii; aqx: Aquifex aeolicus; bfi: Bacillus firmis; chl: Chlamydia trachomatis; hpy: Helicobacter pylori; mbr: Methanobrevibacter smithii; rho: Rhodospirillum rubrum; syn: Synechocystis PCC6803; P A D : homologue of phenylacrylate decarboxylase; C: homologue of vdcC The region of the vdcB homologues that is a putative membrane association domain is jholrit-ri anil  62  (a) ecoPAD sphPAD bsuPAD strPAD scvPAD arcPAD mebPAD me c PAD pyrPAD aqxPAD bfiPAD chlPAD hpyPAD  :  -MKAEFKRKGGG  MWGITG •  LWGMTG -L.WGMTG  ;WAITG  MLLFPRRTNIAFFKTTGIFANFPLLGRTITTSPSFLTHKLSKEVTRASTSPPR  n-KWAIiTG -~~MKV :'"|1TIAMTG -, l n v c I T G  '-IIVAITG ; -MSERIEKIMTVGITGj :  10 ecoPAD sphPAD bsuPAD StrPAD s cvPAD arcPAD mebPAD mecPAD pyrPAD aqxPAD bfiPAD chlPAD hpyPAD  "LIVGISq  •  20  30  40  .ywGISG  MKJVLGISG 50  60  ASGAIYGVR  .LQVLRDVTDIETHLVMSQAARQTLSLETDFSLREVQALADV ATGSVYGLR7.LELLRETGGWETHLVMSPAALLNIREELPEGKARLEALADV A T G A I F G V R .LQWLK.AAGVETHLVVSPWANVTIKHETGYTLQEVEQLATY  TH VH TY  ATGAPFGVSI'JLENLRQLPGVETHLVLSRWARTTIELETGLSVAEVSALADV  TH  ATGVALGIR.iLQVLKEL  . SVETHLVISKWGAATMKYETDWEPHDVAALATK A S G Q I L G I B L . J I E K L T E L . GAEVYAVASRAAKITLKAETDYDEGYVREIATK ASGVIYGEEiiLKALRGA.GVRIGLMITDTAREIIRYELGIEPGALEELAD A S G V I Y A K R i L E V L K D R . .AEVNLIISNSAKKIIKEELDIDWKEIKKLAT A S G S I Y G I K .YEILKKL. GHDVILLASKTGIKVAKYETGME IT  TY YY E. .CF D. .YY P. .DF  ASGVIYGIK.iLQVLEEL . DFSVDLVISRNAKWLKEEHSLTFEEVLKGLK  NVRIH  ASGGMYGVR-.TQELLRQ. EYKVHLVLTEAAWQVFKEELLLDTTDRQKVIHELFGDLPGELHTH A S G I V L A V f l .VSELARL . GHHIDVIISPSAQKTLYYEL DTKSFLSTIPQNFHNQIVLH A S G I P L A I g g J F L E K L P K . . EIEVFWASKNAHWALEESNINLKNAMK DLRPSGTFF 70 80 90 100 110 120  ecoPAD DARDIAASISSGSFQT..LGMVILPCSIKTLSGIVHSYTDGLLTRAADWLKERRPLVLCVRE sphPAD NVRNVGASIASGSFVC..EGMAIAPCSMRTLGAVAHALSDNLITRAADVMLKERRRLVMITRE bsuPAD SHKDQAAAISSGSFDT..DGMIVAPCSMKSLASIRTGMADNLLTRAADVMLKERKKLVLLTRE StrPAD HPEDQGATISSGSFRT..DGMVIVPCSMKTLAGIRTGYAEGLVARAADWLKERRRLVLVPRE scvPAD SVRDVSACISSGSFQH..DGMIWPCSMKSLAAIRIGFTEDLITRAADVSIKENRKLLLVTRE arcPAD DEDEIAAPFASGSFRH..DGMAVVPCSIKTASSIAYGIADNLIARAADVTLKEKRRLVLAIRE mebPAD DASDFTTSINSGSS..PFRAMVIAPCTMKTLSAIANGYAENSLTRAADVCLKERRDLVLVPRE mecPAD ENDDFFSPLASGSN..KFDAVWVPCSMKTLSAIANGYSANLIVRVCDIALKERRKLIIMPRE pyrPAD DEDDLFAPIASGSY..PFDAMVIAPCSMKTLGAIANGFSYNLITRAADVTLKERRKLILLIRE aqxPAD EENDFTSPLASGSRLVHYRGVYWPCSTNTLSCIANGINKNLIHRVGEVALKERVPLVLLVRE bfiPAD DLHDYAAPIASGSYRSA..GMVIIPCSMGTLSGMAHGASGNLLERTADVMLKEKRKLVIVPRE ChlPAD HISSIESSVSSGS..NTIDATIIVPCSVATVAAISCGLADNLLRRVADVALKEKRPLILVPRE hpyPAD NEQDIHASIASGSY..GIHKMAIIPASMDMVAKIAHGFGGDLISRSASVMLKEKRPLLIAPRE 130 140 150 160 170 180 ecoPAD TPLHLGHLRLMTQAAEIGAVIMPPVPAFYHRPQSLDDVINQTVNRVLDQFAITLPEDLFARWQ • sphPAD APLNLAHLRNMTACTEMGAVIFPPVPAFYARPTSLADWDHTCMRVLDLFGLHAKSE..KRWQ bsuPAD TPLNQIHLENMLALTKMGTIILPPMPAFYNRPRSLEEMVDHIVFRTLDQFGIRLPE..AKRWN strPAD TPLSEIHLQNMLELARMGVQLVPPMPAFYNNPQTVDDIVDHWARILDQFDLPAPA..ARRWA scvPAD TPLSSIHLENMLSLCRAGVIIFPPVPAFYTRPKSLHDLLEQSVGRILDCFGIHADT..FPRWE arcPAD APLHSGHLKTLARLAEMGAVIFPPVLSFYTRPKSVDDLIEHTVSRIAEQLGVEVDYRRWG mebPAD TPLRSVHLENMLRVSREGGIILPAMPGFYHKPASIEDMADFIAGKVLDVLGI..ENDLFRRWT mecPAD MPFNSIHLENMLKLSNLGAIVMPPIPAFYNKPKNVNDIINFVVGRVLDILGI..DNSLFKRWG pyrPAD TPLNLVHVQNMLKIIQAGGIIMPASPAFYTKPKTIDDMVNFIIGKILDLLGI..THNLYRRWG aqxPAD APYNEIHLENMLKITRMGGVWPASPAFYHKPQSIDDMINFWGKLLDVLRI. . EHNLYKRWR bfiPAD TPLHDIHLENMLKLSKMGATILPAMPGYYHLPKTIDDLINFLVGKALDSLGV..EHTLFTRWG ChlPAD APLSAIHLENLLKLAQNGAVILPPMPIWYFKPQTAEDIANDIVGKILAILQL..DSPLIKRWE hpyPAD MPLSAIMLENLLKLSHSNAIIAPPMMTYYTQSKTLEAMQDFLVGKWFDSLGI..ENDLYPRWG 190 200 210 220 230 240 250  ecoPAD sphPAD bsuPAD StrPAD SCVPAD mebPAD mecPAD pyrPAD aqxPAD bfiPAD chlPAD hpyPAD  GA GLSKEAASLVPGAGQMEGN GIEKQKGGAGMRAARAAARS FGDAA GIKSK ~ GKDI TV MREDD G E NPR MN 260 270  ~ ~ 280  290  300  310  64  ( b )  mebC mbj-C mecC pyrC rhoC bsuC strC sphCaqxC C ecoC synC hpyc chlC a r c  ~  MRNFLDKIGEEALV MDIKDENIIE ~~~ —~ MREIINKLNP. I I MVMKMLREIVES FEDL W ~ ~ ~~~~MERDFSGSPRVIADLGRIIDRLEALGRLVR MAYQDFREFLAALEKEGQLLTVNEEVKPEPDLGASARAASNLGDKSPALLFNNIY MAYDDLRS FLDTLEKEGQLLRITDEVLPEPDLAAAANATGRIGENAPALHFDNVK MTMNDLPNRARSISSLRDFLELLEDAGQAITWSDAVMPEPGVRNIAVAASRDANGAPAIVFDNIT MGYKYRDLHDFIKDLEKEGELVRIKEPLSPILEITEVTDRVCKMPGGGKALLFENP. MAYEDLREFIGRLEDKGELARVKHEVSPILEMSEVADRTVK. . AGGKALLFERP . MDAMKYNDLRDFLTLLEQQGELKRITLPVDPHLEITEIADRTLR..AGGPALLFENP. MARDLRGFIQLLETRGQLRRITAEVDPDLEVAEISNRMLQ. .AGGPGLLFENV. MRDFLKLLKKHDELKIIDTPLEVDLEIAHLAYIEAKKPNGGKALLFTQPI MFSLRSLVDYLRSQHELIDIHVPVDPHLEIAEIHRRVVERE. . GPALLF. . . . 10 20 30 40 50 60 .  -~~ _—„  mebC VEDEVSTSFEAASILREHPRDL..VILKNLKESDIPVISGLCNTREKIALSLNCRVHEITHRIVE mbrC ITTELSSEFEVAKELRKYPKDT..VIIKNVKGYDLPIISGICNTREKIAKSINCEVSEITQKIIE mecC IDKADKK. FGVSRILKKYDGKP. . VYIKDVNGFE . . WGNL . CSRETLSKIFNVKKEDFIFFMLD pyrC IDKPVKKELELTKFLLKYKDKP..VLFKDVEGWE..VAGNLWSSRERIAKFLNTDNKGLLELLYE rhoC VRSEVDPRHDLAGIAARFEGGPQAVLFEKVAGHAYPVFVGLYWSRELLGALFDQPETALPQHVAA bsuC GYHNAR IAMNVIGSWPNHAMMLGMPKDTPVKEQFFEFAKRY StrC GFTDAR IAMNVHGSWANHALALGLPKNTPVKEQVEEFARRW sphC GYPGKR LAVGVHGSWDNIALLLGRPKGTTIRELFFEIAGRWGD aqxC KGYRIPVLTNLYGSEKRIKKALGYEN . . . LEDIGWKLYRILKPEVPKTFLEKIKKLPE arcC KGYDIPVFMNAFGTERRMKLALEVER...LEEIGERLLSALEFR.PSSFMDALKGVGM ecoC KGYSMPVLCNLFGTPKRVAMGMGQEDVSALREVGKLLAFLKEPEPPKGFRDLFDKLPQ synC KGSPFPVAVNLMGTVERICWAMNMDHPLELEDLGKKLALLQQPKPPKKISQAIDFGKV hpyC RKEHDQIKTFGMPVLMNAFGSFKRLDLLL....KTPIESLQQRMQAFLHFNAPKNFTEGLKVLKD ChlC ....HQVKGSPFPVLTNLFGTRRRVDLLFPDLSSDLFEQIIHLLSS PPSFSSLWKHRSL 70 80 90 100 110 120 130 mebC AMENP....T..PISSVGGLDGYRSGRADLSELPILRHYRRDGGPYITAGVIFARDPDT....GV mbrC ASDNP. ... I. .KVDKFTDFSDYNTTEANLDKIPILTHYKRDGGKYITAGWFARDPET. . . .GI ' mecC - AMEKEKEGKL. . KINNKLKEKYIVEIPENIKNWPIPIYYEKDAGAYITSGWWYDKDY . . . . GY pyrC AMEKPKPFSV. .VEKAEFLKN...REKVNLLELPIPKYYPKDGGPYLTSAMVIA..KKE FV rhoC SIKSWQSAPVDPLWADGPVLEVTEAEVDLSTLPIPIHALEDGGPYFDAAWIAKDPET . . . . GV bsuC ..DQFPMPVKREETAPFHEN.EITEDINLFDILPLFRINQGDGGYYLDKACVISRDLEDPDNFGK StrC ..DAFPVAPERREEAPWRENTQEGEDVDLFSVLPLFRLNDGDGGFYLDKAAWSRDPEDRDDFGK sphC ..QEAQISFVPEAQAPVHE.CRIEQDINLYDVLPVYRINEYDGGFYIGKASVASRDPLDPDNFGK aqxC LKKLNDAIPKWKRGKVQEEVIMGD.INLED.LPILKCWPKDGGRYITFGQVITKDPES....GI arcC LKDFMSFIPK. . KTGKAPCKEWAE . . SLDK. FPILKCWPKDAGRFITFPWITKDPET . . . . GE ecoC FKQVLNMPTKRLRGAPCQQKIVSGDDVDLNR.IPIMTCWPEDAAPLITWGLTVTRGPHK....ER synC LFDVLKAKPGRNFFPPCQEWIDGENLDLNQ.IPLIRPYPGDAGKIITLGLVITKDCET....GT hpyC LWDLRHIFPKKTTRPK.DLIIKQDKEVNLLD.LPVLKTWEKDGGAFITMGQVYTQSLDH....QK ChlC FKRGISALGMRKRHLR.PSPFLYQDAPNLSQ.LPMLTSWPEDGGPFLTLPLVYTQSPEN....GV 140 150 160 170 180 190 t  mebC mbrC mecC pyrC rhoC bsuC StrC sphC aqxC arcC ecoC  RNASIHRMMVIGDDRLAVRI.VPRHLYTYLQK..AEERGEDLEIAIAIGMDPATLLATTT...SI QNASIHRMLVLDDKRLVIRI.VPRNLYTYFQK..AQKLGKDLEIAIAIGMDPAILLASTT...SI .NLSIHRILVKDDYLVIRMV.EQRHLHFLYNK..ALKEKGYLDVAIVIGVHPAVLLAGST...SA .NVSFHRMMVLDEERAVIRL.VPRHLYSMWKD..SVEHGEELEVRIVLGNPVHLLLAGAT...SV RNASIQRFQVIGKDRLVINIDAGRHLGLYLDK..AAARGEPLAFTLNVGVGPGVHFAAAAPAEAA QNVGIYRMQVKGKDRLGIQPVPQHDIAIHLRQ..AEERGINLPVTIALGCEPVITTAASTP...L QNVGTYRIQVIGTNRLAFHPA.MHDVAQHLRK..AEEKGEDLPIAITLGNDPVMAIVAGMP...M QNVGIYRLQIQGPDTFTLMTIPSHDMGRQIMA..AEREGVPLKIAVMLGNHPGLAAFAATP...1 RNVGLYRLQVLDKDKLAVHWQIHKDGNHHYWK..AKRLGKKLEVAIAIGGEPPLPYVASAP...L MNAGMYRMQVFDGKTTGMHWQIHKHGAEHFRK.MAEKGGGKIEVAVAIGVDPATLYAATAP...L QNLGIYRQQLIGKNKLIMRWLSHRGGALDYQEWCAAHPGERFPVSVALGADPATILGAVTP...V 200 210 220 230 240 250 260  65 synC hpyC chlC  PNVGVYRLQLQSKTTMTVHWLSVRGGARHLRK..AAEQGKKLEVAIALGVDPLIIMAAATP...1 KNLGMYRLQVYDKNHLGLHWQIHKDSQLFFHEYAKAKV..KMPVSIAIGGDLLYTWCATAP...L PNLGMYRMQRFDKETLGLHFQIQKGGGAHFFE..AEQKKQNLPVTVFLSGNPFLILSAIAP...L 200 210 220 230 240 250 260  mebC PIDADEMEVANTFH....EGELELVRCEGVDMEVPPAEIILEGRILCGVRE.REGPFVDLTDTYD mbrC PIDYNEMDVANAFK....NGELTLIKCG..DLEVPQADIILEGKISVSETS.AEGPFVDLTDTYD mecC DITFDELKFAAAL....LGGEIGVFELDNGLL.VPEAEFIIEGKIL.PEVD.DEGPFVDITGTYD pyrC AYGVSELEIASAISLKAFGRPLEVINLDGIPT.PVDSEFVFKAKIT.DEVA.DEGPFVDITGTYD rhoC PVETDELGIASAFHGAPLELVAGTV...GPVEMVAHAMWALECEIRPGEVH.AEGPFAEVTGYYA bsuC LYDQSEYEMAGAIQGEPYR.IVKSKLSD..LDVPWGAEWLEGEIIAGEREY.EGPFGEFTGHYS strC AYDQSEYEMAGALRGAPAP.IATAPLTG..FDVPWGSEWIEGVIESRKRRI.EGPFGEFTGHYS sphC GYEESEYSYASAMMGAPIR.LTKSG.NG..IDILADSEIVIEAELQPGGREL.EGPFGEFPGSYS aqxC PPEVDEYLFAGIIMERPVE.LVKGLTVD..LEYPANAEIAIEGYVDPEEPLVDEGPFGDHTGFYT arcC PSGISEFMFAGFIRKERLK.VTECETVD..LLVPANAEIILEGYVRVDEMRV.EGPFGDHTGYYT ecoC PDTLSEYAFAGLLRGTKTE.WKCISND..LEVPASAEIVLEGYIEQGET.APEGPYGDHTGYYN synC PVDLSEWLFAGLYGGSGVA.LAKCKTVD..LEVPADSEFVLEGTITPGEM.LPDGPFGDHMGYYG hpyC PYGIYELMLYGFMRGKKAR.VMPCLSNS..LSVPSDCDIVIEGFVDCEKLEL.EGPFGDHTGYYT ChlC PENVPELLFCSFLQNKKLSFVEKHPQSG..HPLLCDSEFILTGEAVAGERR.PEGPFGDHFGYYS 270 280 290 300 310 320 mebC mbrC mecC pyrC rhoC bsuC StrC sphC aqxC arcC ecoC synC hpyC ChlC  VVRDEPVISLERMHIRKD.AMYHAILPAGF.EHRLLQGLPQEPRIYRAVKNTVPTVRNVVLTEGG IIRDQPIINLSKMHIKKDNPHYHGILSAGF.EHKLLQGLPQEPRIFKSVKNAVPTVENWLTEGG IVRKQPIIKIEKLY.RKEKPIFHALLPGGI.EHKTLMGMPQEPRILKGVRNTVPTVKNIVLTEGG IVRKQPIVIFEEMY. HVDDPIFHALLPGGY. EHYMLMGLPKEPQIYASVKKWPKVHGVRLTEGG RVEPRPLVRVKRIH.RRRAPIFHTLL.SGA.EVFNSVGLLGEANVLALLRVQVPGVEDVYFSHGG GGRSMPIIKIKRVYHRNNPIFEHLYLGMPWTECDYMIGINTCVPLYQQLKEAYPNEIVA.VNAMY GGRRMPVIRVERVSYRHEPVFESLYLGMPWNECDYLVGPNTCVPLLKQLRAEFP.EVQA.VNAMY GVRKAPIFKVTAVSHRRDPIFENIYIGRGWTEHDTLIGLHTSAPIYAQLRQSFP.EVTA.VNALY PVDKYPQMHVTAIVMRKDPIYLTTIVGRPPQE.DKYLGWATERIFLPLIKFNLPEWDYHLPAEG PPEPYPVFHITHITHRENPIYHATWGKPPME.DAWLGKATERIFLPILRMMHPEIVDINLPVEG EVDSFPVFTVTHITQREDAIYHSTYTGRPPDE.PAVLGVALNEVFVPILQKQFPE1VDFYLPPEG GVEDSPLVRFQCLTHRKNPVYLTTFSGRPPKE.EAMMAIALNRIYTPILRQQVSEITDFFLPMEA PIEPYPVLEVKTISYKKDSIYLATWGKPPLE.DKYMGYLTERLFLPLLQMNAPNLIEYYMPENG LTHDFPIFKCNCLYHKKDAIYPATWGKPFQE.DFFLGNKLQELLSPLFPLIMPGVQDLKSYGEA 330 340 350 360 370 380 390  mebC mbrC mecC pyrC rhoC bsuC StrC sphC aqxC. arcC ecoC s ynC hpyC ChlC  CCWLHAAVSIKKQTEGDGKNVIMAALAAHPSL. . KHVVWDEDIDVLDPEEIEYAIATRVKGDD CCWLHAAISINKQTEGDGKNAIMAALSAHPSL. , . K H A W V D T D V D V F D P Q D I E Y A I A T R V K G D R CCWLHAWQIEKRTEGDGKNAILAAFASHPSL. . . K H V I W D D D I N I F D I N D V E Y A I A T R V Q G D K CMWLHAWSITKQHEGDGKNAILAAFAGHPSL. ,. K R W W D E D V N I Y D D R E V E W A I A T R F Q P D R CGFYHCWKIAQKRAGWAKQAILATFAAFPPL. , . K M V T W D E D V D I R N G R D V E W A M T T R L D A K T THGLIAIVSTKTRYGGFAKAVGMRALTTPHGLGYCKMVIWDEDVDPFNLPQVMWALSTKMHPKH THGLMVIISTAKRYGGFAKAVGMRAMTTPHGLGYVAQVILVDEDVDPFNLPQVMWAMSAKVNPKD QHGLTGIISVKNRMAGFAKTVALRALSTPHGVMYLKNLIMVDADVDPFDLNQVMWALSTRTR.AD CFHNFCFVSIKKRYPGHAFKVAYALLG.LGLMSLEKHIWFDDWINVQDIGEVLWAWGNNVDPQR AFHNLAIVSIKKRYPGQAKKVMYAIWG. TGMLSLTKIVWVDDDVNVHDMREWWAVTSRFDPAR CSYRLAWTIKKQYAGHAKRVMMGVWSFLRQFMYTKFVIVCDDDVNARDWNDVIWAITTRMDPAR LSYKAAIISIDKAYPGQAKRAALAFWSALPQFTYTKFVIVVDKSINIRDPRQWWAISSKVDPVR VFHNLILAKIHTRYNAHAKQVMHAFWGVGQMSFVKHAIFVNEDAPNLRDTNAIIEYILENF..SK GFHALAAAIVKERYWKEALRSALRILGE.GQLSLTKFLWITDQSVDLENFPSLLECVLERMNFDR 400 410 420 430 440 450  mebC mbrC mecC pyrC rhoC bsuC  DILIVPGARGSSLDPAA.LPDGTTTKVGVDATAPL.ASAEKFQRVSRSE DLMIVPNVRGSSLDPVA.ESDGTTTKIGLDATKSL.KTLDKFERVSFGE DIVIISGAKGSSLDPSSDLKNKLTAKVGVDATMSLIKGREHFERAKIPDK DLVIIPNARGS SLDPSG..KDGLTAKWGIDATKPLDKKKE.FEKASLDF GILVIENAFGHGLNPT..FPNYLGTKVGFDCTRPFPHT.PAFDRAKTKAMTLDGLDIVGAKR DAVIIPDLSVLPLDPGSNPSGITHKMI.LDATTPVAPETRG.HYSQPLDSPLTTKEWEQKLMDLM 460 470 480 490 500 510 520  66 StrC sphC aqxC arcC ecoC synC hpyC chlC  DVWIPNLSVLELAPAAQPAGISSKMI.IDATTPVAPDVRG.NFSTPAKDLPETAEWAARLQRLI DIIVLPNMPAVPIDPSAWPGKGHRLI.IDATSYLPPDPVG.EAHLVTPPTGDEIDALSKRIREM DVLIL.KGPIDVLDHATNEVGFGGK.MIIDATTKWKEEGYTREWPEVIEMSPEVKKRIDEIWDRL DVVILPPSPTDSLDHSAYIPNLAGK.LGIDATKKWRDEGYEREWPDWEMDAETKRKVDAIWNEI DTVLVENTPIDYLDFASPVSGLGSK.MGLDATNKWPGE.TQREWGRPIKKDPDVVAHIDAIWDEL DVFILPETPFDSLDFASEKIGLGGR.MGIDATTKIPPE.TDHEWGEVLESDPAMAEQVSQRWAEY ENALISQGVCDALDHASPEYAMGGK.LGIDATSK SNTPYPTLLNDSALLALLQDKMQNIV DLLILSETANDTLDYTGSGFNKGSKGIFLGVGAPIRSLPRRYRGPSLPGISQIGVFCRGCLVLET 460 470 480 490 500 510 520  bsuC strC sphC aqxC arcC ecoC synC hpyC ChlC  NK : AARV QLGALS GIE RNMVL AIFNNGKSA GLGDINLTEVNPNLFGYDV LLKQYYPHTRNPICVISVEKKDKSVIELAKNLLGFEEHLRIVIFVEHASNDLNNPYMLLWRIVNN SLQQLDIPALLKEPHLADWPLVILVEDLSSALSSTKEFIWRTFTRSSPATDLHIPVSQITNHKVS 530 540 550 560 570 580  hpyC ChlC  IDARRDILTSKHCFFIDATNKGVMDKHFREWPTETNCSMEVIENLKKKGLLKDFETLNQKFHLTH YTPPMILNALMKPPYPKEVEADEATQNLVSSRWHSYFP— 590 600 610 620 630 640 650  hpyC  SFSTHKEDL 660  ~ 670  680  690  700  710  67  Escherichia coli Sphingomonas  aromaticivorans  Bacillus subtilis  Streptomyces sp. D 7  Saccharomyces  cerevisiae  Archaeoglobus  fulgidus  Methanobacterium  Pyrococcus  thermoautotroph  horikoshii  Methanococcus  — Aquifex  jannaschii  aeolicus  Bacillus firmus  Helicobacter Chlamydia  pylori  trachomatis  Methanobacterium thermoautotrophicum  ( d )  Methanobrevibacter Methanococcus Pyrococcus  smithii  jannaschii  horikoshii  Rhode-spirillum rubrum I—  Bacillus subtilis  I  Streptomyces s p . D 7  '  ' I  Synechocystis  PCC6803  Escherichia coli  '  Chlamydia r — — Aquifex I  _  Sphingomonas  Archaeoglobus  trachomatis  aeolicus Helicobacter pylori  fulgidus  aromaticivorans  69 3.2 D e t e c t i o n o f a p u t a t i v e t r a n s c r i p t i o n a l r e g u l a t o r y g e n e  Preliminary D N A sequencing of the region upstream of the V D C gene cluster indicates the presence of a putative divergently transcribed regulatory gene, the position of which is illustrated in Figure 22. B L A S T - X analysis of the translation product of the region matched strongly to a putative positive transcriptional activator from the Streptomyces coelicolor A3(2) genome (Figure 23).  70  BamHI  BamHI  4.4 kb BamHI fragment in p S U B l  Figure 22: Location of the putative divergent regulatory gene in relation to the V D C gene cluster.  71  embICAA19943| (AL031107) p u t a t i v e t r a n s c r i p t i o n a l r e g u l a t o r coelicolor] Length = 301  [Streptomyces  Score = 119 b i t s (295), Expect = 5e-26 I d e n t i t i e s = 86/256 (33%), P o s i t i v e s = 111/256 (42%), Gaps = 4/256 (1%) Frame = -1 Query: 1180 FHQLIGVRGRPLRMWVDFVEHELRPGSWLWIRPGQVQRFGPDLAAADGVIVLFQPGFLPP FH ++ G P+R +DF E+E G LWIRPGQV RF P+ G ++ QPGFLP S b j c t : 54 FHIVMLFTGGPVRHMIDFAEYEASAGDLLWIRPGQVHRFAPE-GEYRGTVLTMQPGFLPR  1001 112  Query: 1000 TTVSLAHMDPPYE-QRPSVLEGSDAE—GVRRALDHLVHEYGAMASLPLQAHTE-XXXXX 833 TV + Y P +L +A G+ AL+ L EY. +LPL HT S b j c t : 113 ATVEATGL YRYDLPPLLHPDEARLAGLTAALEQLRREYEDATTLPLSLHTAVLRHTL 169 Query: 832 S b j c t : 170 Query: 652 S b j c t : 218 Query: 472 S b j c t : 278  XXXXXXXXXXXRGPAPRPTAAFGNVPSLSHXXXXXXXXXXGASTNTRGRSATAPHLTRRV 653 G A P S G +TN V SAFLLRLAHLAAGSARAARQGRAEAPGDSTFVLFRDAVERGFATN HSV 217 DDYAAALGYSSXXXXXXXXXXXXXXANQYVDDRVLLEAKRLLQHSGLTAREVTVRLGFTD 473 YA ALGYS ++D RV+LEAKRLL H+ + V +GF D SAYADALGYSRRTLVRAVRAATGETPKGFIDKRVVLEAKRLLAHTEMPIGRVGAAVGFPD 277 ASDFTKFFRLRTGMTPGAFR 413 A++F+KFF+ T TP AFR AANFSKFFQQHTDQTPAAFR 297  Figure 23: B L A S T - X result indicating that the nucleotide region immediately upstream of vdcB encodes a polypeptide similar to a putative transcriptional regulator from S. coelicolor A3(2). Query = Streptomyces sp. D7 translated nucleotide sequence; Subject = S. coelicolor A3(2) genomic D N A translation product.  72  3.3 M e s s e n g e r R N A  a n a l y s e s  Messenger R N A was isolated from Streptomyces sp. D7 under both uninduced and vanillic acid-induced conditions, blotted to a positively charged nylon membrane, then probed with P-labeled vdcC P C R amplification product. The resulting autoradiogram 32  revealed that a transcript of approximately 2.3 kb was synthesized in vanillic acidinduced cells (Figure 24). A transcript of this size corresponds to the expected length of all three V D C genes and their associated intergenic regions. This result indicates that the gene cluster may be transcribed from a single, inducible promoter.  73  Figure 24: Northern blot hybridization of PCR-amplified, radiolabeled vdcC D N A against m R N A isolated from uninduced Streptomyces sp. D7 (Lane 1) and Streptomyces sp. D7 induced with 3.6 m M vanillic acid (Lane 2). R N A size standards are indicated (Lane M ) .  74 3.4  D e t e c t i o n  o f V D C  c l u s t e r genes i n  Streptomyces setonii 7 5 V i 2  Southern hybridization of radiolabeled vdcB, vdcC, and vdcD P C R products to SaRdigested S. setonii 75Vi2 genomic D N A revealed that all three genes of the cluster appear to be present in the characterized vanillate decarboxylating strain (Figure 25). At the time of the writing of this report, a lambda D A S H II phage clone of S. setonii 75Vi2 genomic D N A , which hybridizes strongly to radiolabeled vdcC probe, is being analyzed and sequenced (M.K. Pope, collaborative work) in order to further characterize the V D C cluster of 5*. setonii 75Vi2. Preliminary sequence information reveals that within the phage clone D N A insert, a cytochrome P-450 gene is located within several kilobases of the putative V D C gene cluster in the genome of S. setonii 75Vi2. In addition, the vdcB homologue in S. setonii 75Vi2 has been sequenced, with further sequencing underway to uncover the remaining genes. As mentioned earlier, cytochrome P-450 enzymes have been implicated in the demethylation of methoxyl groups on guaiacol by S. setonii 75Vi2 (Sutherland, 1986). No cytochrome P-450 gene was observed in the vicinity of the V D C gene cluster in Streptomyces sp. D7, which supports the observation that no transformation of guaiacol could be detected in the organism. These observations will be described in more detail in the Discussion section to follow.  75  Figure 25: Southern blot hybridization of Sa/I-digested chromosomal D N A from Streptomyces sp. D7 (Lane 1) and Streptomyces setonii 75Vi2 (Lane 2) with radiolabeled vdcB, vdcC and vdcD (as indicated) PCR-amplified D N A probes at 65 °C. Approximate molecular sizes (in kilobases) are shown on the left.  76  3.5 G e n e k n o c k o u t s t u d i e s  Gene knockout experiments involve transforming cells with an inactivated form (allele) of the gene being studied, for homologous recombination with the wild type gene on the host's chromosome. Inactivation is usually achieved by replacing a section of the gene with an antibiotic resistance cassette. resistance gene is commonly used.  In streptomycete procedures, the thiostrepton  These experiments are commonly used for gene  identification and/or functional analyses, and disruption and inactivation of the V D C gene cluster would allow determination of the functions of each of the three genes in vivo. In order to do this, Streptomyces sp. D7 had to be transformed with a disrupted allele of each gene, to obtain knockout mutants by double crossover recombination events with the chromosome. Protoplasts of Streptomyces sp. D7 were prepared, which regenerated efficiently on R6 agar plates designed for that purpose. However, in spite of many attempts, these protoplasts were not transformable with pIJ702 as a control plasmid using standard Streptomyces protoplast genetic manipulations. Likewise, attempts to introduce pPM801 into Streptomyces sp. D7 mycelia by interspecies Streptomyces-E. coli conjugation procedures failed to produce ex-conjugants expressing the kanamycin resistance marker gene of the vector. These observations suggest that Streptomyces sp. D7 may possess a potent restriction-modification system, which degrades foreign D N A . Streptomyces is particularly well known for being highly resistant to transformation procedures (Oh & Chater, 1997), which is the major reason why the vast majority of genetic research on these organisms has been conducted with either Streptomyces lividans or Streptomyces coelicolor, organisms for which transformation procedures have been well established. The resistance of Streptomyces sp. D7 to genetic transformation  77 made it necessary to devise alternative methods to characterize the functions of the V D C gene cluster. I decided to express each gene independently to examine their activity in Escherichia coli and/or Streptomyces lividans 1326 as surrogate host systems, which is described in Section 3.6.  3.6 G e n e e x p r e s s i o n  3.6.1 Expression with pET22b(+) in E. coli BL21(DE3) Initially, the goal of the project was to clone individual V D C cluster genes behind the T7 polymerase promoter in the expression vector pET22b(+) (Novagen), to obtain high amounts of active enzymes in the expression strain Escherichia coli BL21(DE3). Studies of the purified, active protein would allow the deduction of the function of each enzyme in the catabolism of vanillic acid. However, although the vdcB and vdcC genes were successfully expressed in E. coli, the proteins were formed as inclusion bodies, no matter how the conditions for expression were varied to achieve proper protein folding. For example, protein expression was carried out at 37°C and 25°C, with both 1 m M and 0.1 m M IPTG used for induction of the T7 R N A polymerase system. Attempts were made to solubilize and refold the inclusion body material, but with no established biochemical assay and only a putative phenotype, it was decided that streptomycete vectors and host systems would be more appropriate for the expression of the V D C genes and the determination of their function(s). A typical example of SDS-PAGE-visualized protein over expressed using pET22b(+) in E. coli BL21(DE3) is shown in Figure 26, in which the vdcC gene product was produced as inclusion bodies at 37°C with 0.1 m M IPTG used  78  to induce protein synthesis. A n example of inclusion body formation at lower induction temperatures is shown in Figure 27.  79  M  1  2  Figure 26: SDS-PAGE (12.5% acrylamide) of cell extracts showing the expression product of the vdcC gene, produced using pET22b(+) in E. coli BL21(DE3) at 37°C with 0.1 m M IPTG for induction. Lane 1: cell lysate from uninduced cells; Lane 2: cell lysate from IPTG-induced cells. The position of the vdcC expression product is denoted by an arrow. Molecular weight markers are shown on the left, in kilodaltons.  80  M  1  3  4  3 2 . 5  Figure 27: SDS-PAGE (12.5% acrylamide) of cell extracts showing the expression product of the vdcC gene, produced using pET22b(+) in E. coli BL21(DE3) at 25°C and 30°C with 0.1 m M IPTG for induction. Lane 1: soluble cell lysate, 25°C induction; Lane 2: insoluble protein, 25°C induction. Lane 3: soluble cell lysate, 30°C induction; Lane 4: insoluble protein, 25°C induction. The position of the vdcC expression product is denoted by an arrow. Molecular weight markers are shown on the left, in kilodaltons.  81  In hindsight, the strategy to express the vdcB and vdcC genes independently most likely would not have been successful, even i f the proteins had been synthesized as soluble, active enzymes. As is detailed in Section 3.6.2 and Section 3.6.3 (below), it appears as though expression of at least the vdcC and vdcD genes, simultaneously, is required to produce vanillate decarboxylase activity.  3.6.2 E x p r e s s i o n w i t h pIJ702 i n S. lividans 1326  Gene expression in Streptomyces is commonly performed using the high copy number plasmid, pIJ702 (Gusek & Kinsella, 1992). The vector, in combination with S. lividans 1326, has been used extensively to study antibiotic biosynthesis gene structure and expression.  S. lividans 1326 carrying p K C S l acquired the ability to efficiently  decarboxylate vanillic acid to guaiacol, while 5*. lividans 1326 carrying pKCS2 produced extremely low amounts of guaiacol (Figure 28). 5*. lividans 1326 wild type did not perform any detectable vanillic acid decarboxylation.  These results suggest that  transcription of the genes required for vanillic acid decarboxylation by S. lividans 1326 carrying p K C S l occurs from the constitutive mei promoter. There is possibly a low level of transcription from a natural promoter, however, as observed for S. lividans 1326 carrying pKCS2 that contains the gene cluster in the opposite orientation from the mei promoter (Figure 28 (b)).  3.6.3 E x p r e s s i o n w i t h pIJ680 i n S. lividans 1326  The results of the aph promoter - vdc gene fusions are shown in Figure 30. S. lividans 1326 carrying pKCS3 (ydcBCD) or pKCS8 (vdcCD) converted vanillic acid to guaiacol  82  at approximately the same rate as the wild type strain, Streptomyces sp. D7. Sonicated cell-free extracts of S. lividans 1326 expressing the VDC  system via pKCS3 exhibited  decarboxylation of vanillic acid under both aerobic and anaerobic conditions (Figure 29). These results confirm the involvement of the cloned gene products in a non-oxidative system.  Transcription of vdcC and vdcD, as demonstrated by pKCS8, results in vanillate decarboxylation by S. lividans 1326. Therefore, it is.the.vdcC gene that possibly encodes the active enzyme, and the 239 bp vdcD gene may encode a protein that is essential for enzyme stability or activation. The vdcD gene product of approximately 9 kDa seems too small to be the decarboxylase itself, as subunits for known aromatic acid decarboxylases range from 28 kDa to 66 kDa (Table 3).  3.7  S u b s t r a t e  specificity  Sonicated cell extracts of S. lividans 1326/pKCS8 were used to test the specificity of the Streptomyces sp. D7 vanillate decarboxylase towards aromatic acids similar to vanillic acid. The following compounds were tested: 4-methoxy-3-hydroxybenzoate (isovanillic acid), 3,4-dimethoxybenzoate (veratrate), 3,4-dihydroxybenzoate (protocatechuate), 4hydroxy-3,5-dimethoxybenzoate  (syringate),  3,4,5-trihydroxybenzoate  (gallate),  3-  phenylpropenoate (frvms-cinnamate). The structures of these compounds are shown in Figure 31. The U V spectrophotometry scans of assay mixtures at the time of substrate addition, fifteen minutes post-addition and several hours post-addition were identical, suggesting that the enzyme did not decarboxylate these compounds.  By contrast,  83  ultraviolet spectrophotometry demonstrated vanillic acid decarboxylation to guaiacol after fifteen minutes in the soluble fractions of sonicated cell extracts, as shown in Figure 29.  84 A.  3.5  Time (h)  Figure 28: Decarboxylation of vanillic acid to guaiacol by recombinant Streptomyces lividans 1326 strains, (a) S. lividans 1326 carrying p K C S l ; (b) S. lividans 1326 carrying pKCS2; (c) S. lividans 1326 wild type. Vanillic acid concentration is represented by squares ( • ) ; guaiacol concentration is represented by circles (•).  85  Figure 29: Results of incubating the insoluble and soluble fractions of sonicated cell extracts of 5". lividans 1326 - pKCS8 with 1 m M vanillic acid for fifteen minutes at 25°C under both aerobic (O2) and anaerobic (N2) conditions. These scans indicate that the decarboxylase activity is in the soluble fraction, and that the enzyme is active under both aerobic and anaerobic conditions. Similar experiments using other aromatic acid substrates did not demonstrate any biotransformation to decarboxylated derivatives. The X for guaiacol (275 nm) is indicated. max  86  plJ680-aph  V D C Activity Paph  pKCS4  dcB  v  Paph  pKCS5  ^  vdcC  ;.:  Paph  pKCS6  ^  vdcD  Paph  pKCS7  ^  _^  v d c e  c C  ^ap/7  pKCS8  ^  '  pKCS3  —  ^cC  vcfcD  aph  \-—  vdcB  vdcC  vdcD  -  Figure 30: Results of placing the vdc genes under control of the aminoglycoside phosphotransferase promoter (P h) in pIJ680. ap  87  Substrate tested  Decarboxylation'  Structure C0 H 2  YES  Vanillic acid ^ ^ O C H OH C0 H  3  2  NO  Isovanillic acid OH CH C0 H  3  2  Veratric acid  NO OCH  3  OCH C0 H  3  2  NO  Syringic acid H CO'^j^ OCH v  3  3  C0 H 2  S7  NO  Gallic acid OH OH C0 H 2  f-cinnamic acid  NO  Figure 31: Chemical structures of vanillic acid and similar aromatic acids used to test the substrate specificity of vanillate decarboxylase.  88  4 . CONCLUSIONS The goal of this study was to determine the genetic basis of the process of non-oxidative decarboxylation of vanillic acid in Streptomyces sp. D7.  It was demonstrated that  Streptomyces sp. D7 performs the reaction by expressing products of a co-transcribed, three-gene cluster comprised of vdcB, vdcC and vdcD. The genes vdcC and vdcD encode proteins that catalyze the decarboxylation; vdcB is possibly involved in upstream reactions that convert more complex substrates to vanillic acid.  Furthermore, the  catabolic gene cluster appears to be controlled by the vdcA gene, which displays sequence similarity to the translated product of a putative positive transcriptional regulatory gene identified in the Streptomyces coelicolor A3 (2) genome sequencing project.  This is, to my knowledge, the first report of the genes associated with the  process of non-oxidative decarboxylation of aromatic acids in a microorganism. Further studies of this system should allow these genes to be incorporated into metabolically engineered microorganisms for the production of industrially useful chemical products such as guaiacol, catechol and adipic acid.  89  5. D I S C U S S I O N 5.1  A r o m a t i c  a c i d  n o n - o x i d a t i v e  d e c a r b o x y l a s e s  a r e m u l t i - s u b u n i t  e n z y m e s  Although there are many reports of microbial non-oxidative decarboxylases active on aromatic acids in the literature, only recently have enzyme purifications been successful, as these proteins appear to be unstable in cell-free extracts. Of the enzymes purified thus far, all share one common feature: they are single polypeptides, which.form multi-subunit enzyme complexes. However, depending on substrate and organism, the molecular mass of the polypeptide, as well as the number of subunits, is variable. Several examples of aromatic acid non-oxidative decarboxylases and their subunit configurations are listed in Table 3.  I have demonstrated that Streptomyces sp. D7 produces a protein of  approximately 52 kDa when grown in the presence of vanillic acid, and suggest that two additional functionally related proteins of 36 kDa and 9 kDa are also synthesized. The exact functions of these proteins remain unknown, although it appears that vdcC encodes the vanillic acid decarboxylase. Experiments in which vdcC and vdcD were expressed under the control of the aph promoter in pIJ680 resulted in vanillate decarboxylase activity, and it is possible that the 52 kDa VdcC is a subunit of a complex similar to nonoxidative decarboxylases described in the literature (Table 3). The amino-terminus of the VdcC protein is highly similar to the limited amino acid sequence obtained from the purified /?-hydroxybenzoate carboxy-lyase of Clostridium hydroxybenzoicum.  The  Clostridium enzyme was purified and characterized, but only limited amino acid sequence was obtained.  The enzyme is responsible for decarboxylation of p-  hydroxybenzoate to phenol, a dead-end metabolite. With the exception of the amino acid sequence similarity between the vanillic acid induced protein of Streptomyces sp. D7 and  90  Enzyme 4-hydroxybenzoate decarboxylase  Organism Clostridium hydroxybenzoicum  Configuration 350 kDa (6 subunits of 57 kDa)  Reference He and Wiegel, 1995  3,4-dihydroxybenzoate decarboxylase  Clostridium hydroxybenzoicum  270 kDa (5 subunits of 57 kDa)  He and Wiegel, 1996  4,5 -dihydroxyphthalate decarboxylase  Pseudomonas testosteroni  150 kDa (4 subunits of 38 kDa)  Nakazawa and Hayashi, 1978  4,5 -dihydroxyphthalate decarboxylase  Pseudomonas fluorescens  420 kDa (6 subunits of 66 kDa)  Pujar and Gibson, 1985  2,3 -dihydroxybenzoate decarboxylase  Aspergillus niger  120 kDa (4 subunits of 28 kDa)  Kamath et al, 1987  2,3 -dihydroxybenzoate decarboxylase  Trichosporon cutaneum  66.1 kDa (2 subunits of 36.5 kDa)  Anderson and Dagley, 1981  3,4,5trihydroxybenzoate decarboxylase  Pantoea agglomerans T71  320 kDa (6 subunits of 57 kDa)  Zeida et al, 1998  Table 3: Variations in subunit size and configuration - characteristics of some microbial aromatic acid non-oxidative decarboxylases.  91  the C. hydroxybenzoicum enzyme, it is difficult to assign the exact function(s) of the vdcC or vdcD gene products in the reaction. Neither polypeptide shows a relationship to any characterized enzymes in the databases, although the amino terminal sequence of the C. hydroxybenzoicum enzyme was noted to have weak similarity to uroporphyrinogen decarboxylase of Synechococcus sp (He & Wiegel, 1995).  However, extending this  comparison to the Streptomyces sp. D7 protein would be speculative, as the Synechococcus sp. uroporphyrinogen decarboxylase amino-terminal sequence bears almost no resemblance to the Streptomyces sp. D7 protein. The product of vdcB has primary amino acid sequence highly similar to phenylacrylate decarboxylase (PAD) from yeast. In light of this functional implication, it may be possible that the vdcB product is involved in transformation of acrylic phenolic compounds to substrates suitable for downstream gene products, possibly VdcC. The putative trans-membrane region noted in the yeast PAD GenBank database entry is highly conserved among the bacterial P A D homologues' amino-terminal regions (Figure 32). product of the vdcB gene is membrane associated.  Therefore, it is possible that the Further biochemical research is  needed to elucidate the true roles of the components of the V D C gene cluster.  5.2  D i s t r i b u t i o n  o ft h e V D C  g e n e cluster  a m o n g  s t r e p t o m y c e t e s  The results of the Southern hybridization experiments demonstrate that the homologues of the V D C cluster genes are also apparently present in the genome of S. setonii 75Vi2. This observation reinforces the notion that the gene cluster described in this thesis is responsible for vanillate decarboxylation (recall that S. setonii 75Vi2 is the streptomycete well characterized in the literature for vanillate decarboxylation).  Of thirteen  92  streptomycetes screened in similar Southern blots (results not shown), only three - S. setonii 75Vi2, Streptomyces sp. D7, and Streptomyces sp. 2065 (another environmental isolate from our collection) appeared to carry these genes. This observation suggests that the ability to decarboxylate vanillic acid to guaiacol is not a widespread trait among streptomycetes.  Degradation via demethylation to protocatechuate, by the enzyme  vanillate demethylase, is likely the more common route for catabolism of vanillic acid in streptomycetes, as well as other prokaryotes. In fact, a cosmid olthe S. coelicolor A3(2) genome sequencing project contains homologues of the vanillate demethylase genes from Pseudomonas sp. It was previously demonstrated, by the colorimetric Rothera assay, that protocatechuate 3,4-dioxygenase is induced in Streptomyces sp. D7 in the presence of vanillic acid, suggesting that the initial attack on vanillic acid in this organism is performed by vanillate demethylase as well as vanillate decarboxylase (Chow, 1996). However, it is apparent from the current study that vanillate decarboxylase is very highly expressed in Streptomyces sp. D7, as evidenced by the almost equimolar conversion of vanillic acid to guaiacol by Streptomyces sp. D7 mycelia. If vanillate demethylase is synthesized in response to vanillate, the levels of that enzyme are likely very low relative to vanillate decarboxylase.  5.3 S u b s t r a t e s p e c i f i c i t y  The apparent high substrate specificity of the V D C system is perhaps not surprising in light of other non-oxidative decarboxylase studies. Most decarboxylases studied thus far have been shown to be very specific for one substrate, although several are active against two related aromatic acids.  For example, gallate decarboxylase from Pantoea  93  agglomerans  T71 is highly substrate specific (Zeida et al,  1998), while p-  hydroxybenzoate carboxy-lyase from Clostridium hydroxybenzoicum is active against both/>-hydroxybenzoate and protocatechuate (He & Wiegel, 1995). Klebsiella aerogenes was biochemically demonstrated to produce a number of non-oxidative decarboxylases, each enzyme specific for a different aromatic acid substrate (Grant & Patel, 1969). It will be interesting in future studies to determine the catalytic sites of these enzymes, in order to elucidate which amino acid residues affect substrate binding.  5.4 Primary structure motifs Microbial non-oxidative decarboxylase systems reported in the literature (Grant & Patel, 1969; Yoshida & Yamada, 1985; Nakajima et al, 1992; Huang et al, 1993; Santha et al, 1995; He & Wiegel, 1995; He & Wiegel, 1996; Zeida et al., 1998) all have minimal or no requirements  of cofactors for activity.  The vanillate decarboxylase system of  Streptomyces sp. D7 appears active in the absence of oxygen. Consistent with this, amino acid sequence analysis of all three V D C gene products failed to reveal the presence of any cofactor binding motifs, including N A D and F A D , which are characteristic of oxidative enzymes.  5.5 Transcriptional activation The proteomics analyses presented previously (Chow, 1996), combined with the genetic analysis provided in this study, together demonstrate that genes encoding proteins linked to vanillic acid decarboxylation are induced, directly or indirectly, by vanillic acid itself. This observation is supported by the northern blot data, indicating that the V D C gene  94  cluster is transcribed in one polycistronic mRNA product upon induction by vanillic acid. These results suggest that transcription of the gene cluster is under tight regulatory control. In fact, preliminary nucleotide sequence analysis upstream of the V D C gene cluster revealed a divergent putative regulatory gene.  Future work will complete the  characterization of the putative regulatory gene and allow elucidation of its relationship to the V D C gene cluster. The mechanism by which vanillic acid enters streptomycete mycelia and activates transcription of the V D C gene cluster should prove to be an interesting model of environmental sensing and response. proportion of two component signal transduction regulators.  Streptomyces has a high Recent data from the S.  coelicolor A3(2) genome project predicts a total of 160 two-component regulators, approximately double the number found in any other genome thus far analyzed (D. Hopwood, personal communication).  5.6 B i o d e g r a d a t i o n — a r e s u l t o f t h e m i c r o b i a l c o m m u n i t y g e n e p o o l  The wide distribution of homologues of the V D C genes throughout the microbial world, mostly encoded by chromosomes, but also plasmid-borne as in the case of p N L l in Sphingomonas aromaticivorans, suggests that these gene products provide useful metabolic abilities for their hosts.  However, non-oxidative decarboxylation of most  aromatic acids yields toxic phenolic compounds and, in many cases cited in the literature, the microorganisms do not possess appropriate mechanisms to further degrade the compounds produced by these dead-end pathways..  For example, in this study,  Streptomyces sp. D7 was able to rapidly convert vanillic acid to guaiacol, but was unable to further degrade the guaiacol. In another example, Clostridium hydroxybenzoicum decarboxylated j?-hydroxybenzoate to phenol, and protocatechuate to catechol without  95  further metabolism. Non-oxidative decarboxylation remains an enigma of microbial metabolism (Frost & Draths, 1995) and in the case of the C. hydroxybenzoicum enzymes, He and Weigel mention that "a direct metabolic function in the bacterium is not known for either of the two enzymes" (He & Weigel, 1996). The functions of these seemingly toxic metabolic reactions of microorganisms are not readily apparent; however, in natural ecosystems, it can be imagined that other organisms in a consortium would mineralize and remove these toxins from the environment. For example, Streptomyces setonii 75Vi2 (Pometto III et al, 1981; Sutherland, 1986) and a Moraxella sp. (Sterjiades, et al, 1982) were demonstrated to demethylate methoxylated aromatic compounds such as guaiacol to catechol, leading to subsequent mineralization. Evidence implicating cytochrome P-450 systems was provided in both cases. The preliminary sequence data we have obtained, indicating that a cytochrome P-450 gene is located within a few kilobases of the V D C gene cluster in 5*. setonii 75Vi2, suggests that vanillate decarboxylation to guaiacol is associated with demethylation of guaiacol to catechol in that organism. S. setonii 75Vi2 was also observed to degrade phenol (Antai and Crawford, 1982), adding to its reputation as a catabolically diverse organism.  Streptomyces sp. D7 does not appear to be as  catabolically diverse as S. setonii 75Vi2, and from additional sequencing studies of the ends of BamHl subclones of the 13 kb genomic library clone, does not appear to possess a cytochrome P-450 gene nearby the V D C gene cluster.  This observation is in  accordance with the fact that the strain does not metabolize guaiacol - that is, guaiacol is a dead end metabolite.  One could speculate that while S. setonii 75Vi2 possesses a  complete complement of lignin-related aromatic acid catabolic genes, Streptomyces sp. D7 acquired (from another streptomycete or microorganism) only a partial set. Indeed,  96  there is a growing focus on biodegradation, not from the standpoint of an individual microorganism, but rather as a coordinated function of the entire gene pool (Wackett, 1999). Efficient biodegradation is therefore likely the result of natural consortiums of microorganisms. The observation that Streptomyces sp. D7 converts vanillic acid to the toxic guaiacol, but is unable to remove the guaiacol from its environment appears to support this view. However, a microorganism producing metabolites toxic to itself defies the principles of natural selection. The observation of the production of vast amounts of guaiacol from vanillic acid by Streptomyces sp. D7 is based on laboratory experiments, in which unnaturally high concentrations of vanillic acid were fed to the microorganism. In contrast, natural settings such as forest soils most likely do not have high amounts of vanillic acid freely available to the microorganisms. The release of such low molecular weight aromatic compounds from lignin is a rate-limited process, determined by the activity of fungi and abiotic processes. In addition, guaiacol produced by organisms such as Streptomyces sp. D7 would most likely be further degraded by other organisms in the environment. Therefore, Streptomyces sp. D7 would not be living with toxic levels of guaiacol, even if its vanillate decarboxylase system were a result of the acquisition or deletion of certain catabolic genes from its genome, leading to incomplete degradation of vanillic acid.  5.7 Sphingomonas aromaticivorans F199 i n  t h em i s s i n g  c a t a b o l i c p l a s m i d  p N L l :  filling  l i n k s  Recent publication of the entire sequence of the 184 kb catabolic plasmid pNLl of Sphingomonas aromaticivorans F199 (Romine et al, 1999) sheds additional light on the role of the VDC gene cluster in biodegradation processes.  pNLl is a conjugative  97 plasmid, which contains 186 open reading frames, 70 of which are likely associated with catabolism or transport of aromatic compounds. On this plasmid are 15 gene clusters encoding biodegradative enzymes.  Among genes encoding biphenyl and p-cresol  degradative enzymes, lie homologues to vdcB, vdcC and vdcD. Amino acid sequence alignments between the Streptomyces sp. D7 vdcB and vdcC genes and the Sphingomonas homologues are shown in Figure 27.  Between the vdcB and vdcC  homologues, there are two ORFs not found in Streptomyces sp. D7: pchFa (p-cresol methylhydroxylase) and vdh (vanillin oxidoreductase).  Figure 32 (following pages): Amino acid sequence comparisons between the Streptomyces sp. D7 vdcB translation product (strPAD) and its Sphingomonas p N L l homologue (sphPAD) (a), and the Streptomyces sp. D7 vdcC translation product (strC) and its Sphingomonas p N L l homologue (sphC) (b).  98  (a) sphPAD strPAD Consensus  50 1 MKRMWGITG ATGSVYGLRL LELLRETGGW ETHLVMSPAA L L N I R E E L P E M.RLVVGMTG ATGAPFGVRL LENLRQLPGV ETHLVLSRWA R T T I E L E T G L G- ETHLV-S--A 1—E G-RL L E - L R M-R-VVG-TG ATG  sphPAD strPAD Consensus  100 51 GKARLEALAD VVHNVRNVGA SIASGSFVCE GMAIAPCSMR TLGAVAHALS SVAEVSALAD VTHHPEDQGA TISSGSFRTD GMVIVPCSMK TLAGIRTGYA GM-I-PCSM- TL —A ALAD V-H GA - I - S G S F  sphPAD strPAD Consensus  150 101 DNLITRAADV MLKERRRLVM ITREAPLNLA HLRNMTACTE MGAVIFPPVP EGLVARAADV VLKERRRLVL V P R E T P L S E I HLQNMLELAR MGVQLVPPMP MG PP-P HL-NM — L — R A A D V -LKERRRLV- — R E - P L  sphPAD strPAD Consensus  200 151 A F Y A R P T S L A DVVDHTCMRV LDLFGLHAKS EKRWQGLSKE AASLVPGAGQ AFYNNPQTVD DIVDHVVARI LDQFDLPAPA ARRWAGMRAA RAAARSFGDA -A AFY — P D-VDH R- L D - F - L - A — —RW-G  sphPAD strPAD Consensus  201 MEGN A  99  b  1  StrC sphC Consensus  50 MAYDDLRSFL DTLEKEGQLL R I T D E V L P E P DLAAAANATG MTMNDLPNRA R S I S S L R D F L ELLEDAGQAI TWSDAVMPEP GVRNIAVAAS LR-FL — L E — G Q — D-V-PEP A-A—  strC sphC Consensus  51 100 RIGENAPALH FDNVKGFTDA RIAMNVHGSW ANHALALGLP KNTPVKEQVE RDANGAPAIV FDNITGYPGK RLAVGVHGSW DNIALLLGRP K G T T I R E L F F R A P A — FDN—G R-A—VHGSW -N-AL-LG-P K-T E  strC sphC Consensus  101 150 EFARRW..DA FPVAPERREE APWRENTQEG EDVDLFSVLP LFRLNDGDGG EIAGRWGDQE AQISFVPEAQ APVHE.CRIE QDINLYDVLP VYRINEYDGG E-A-RW AP—E - D — L — V L P —R-N—DGG  strC sphC Consensus  151 200 FYLDKAAVVS RDPEDRDDFG KQNVGTYRIQ VIGTNRLAFH PA.MHDVAQH FYIGKASVAS RDPLDPDNFG KQNVGIYRLQ IQGPDTFTLM TIPSHDMGRQ F Y — K A - V - S RDP-D-D-FG KQNVG-YR-Q — G HD  strC sphC Consensus  201 250 LRKAEEKGED L P I A I T L G N D PVMAIVAGMP MAYDQSEYEM AGALRGAPAP IMAAEREGVP LKIAVMLGNH PGLAAFAATP IGYEESEYSY ASAMMGAPIR AE — G — L - I A — L G N - P — A — A — P — Y — S E Y — A - A — G A P —  strC sphC Consensus  300 251 IATAPLTGFD VPWGSEVVIE GVIESRKRRI EGPFGEFTGH YSGGRRMPVI LTKSG.NGID I L A D S E I V I E AELQPGGREL EGPFGEFPGS YSGVRKAPIF G-D EGPFGEF-G- Y S G - R — P — SE-VIE  strC sphC Consensus  301 RVERVSYRHE KVTAVSHRRD -V—VS-R—  strC sphC Consensus  400 351 VNAMYTHGLM V I I S T A K R Y G GFAKAVGMRA MTTPHGLGYV AQVILVDEDV VNALYQHGLT GIISVKNRMA GFAKTVALRA LSTPHGVMYL KNLIMVDADV I-VD-DV VNA-Y-HGL- - I I S R — GFAK-V—RA — T P H G — Y -  strC sphC Consensus  450 401 DPFNLPQVMW AMSAKVNPKD DVVVIPNLSV LELAPAAQPA G I S S K M I I D A DPFDLNQVMW ALSTRTR.AD DIIVLPNMPA VPIDPSAVVP GKGHRLIIDA G IIDA DPF-L-QVMW A-S P-A D D—V-PN  strC sphC Consensus  489 451 TTPVAPDVRG NFSTPAKDLP ETAEWAARLQ R L I A A R V — TSYLPPDPVG EAHLVTPPTG DEIDALSKRI REMQLGALS T PD—G  350 PVFESLYLGM PWNECDYLVG PNTCVPLLKQ LRAEFPEVQA P I F E N I Y I G R GWTEHDTLIG LHTSAPIYAQ LRQSFPEVTA Q LR—FPEV-A P - F E — Y - G - -W-E-D-L-G — T — P  100 The pchFa gene was originally isolated from Pseudomonas putida N C I M B 9866, and encodes an enzyme that oxidizes the methyl group of /?-cresol to an aldehyde. The vdh gene was originally isolated from Pseudomonas fluorescens A N 103 (Walton, Genbank, 1997), and encodes an NAD -dependent oxidoreductase, which converts the aldehyde +  vanillin to vanillic acid. These genes are followed by homologues to vdcC and vdcD, for which this thesis gives evidence (pKCS7 in S. lividans 1326) that the gene products in Streptomyces sp. D7 are involved in vanillic acid non-oxidative decarboxylation. The location of the vdc gene homologues in relation to other genes on p N L l is shown in Figure 33. From this one can create a catabolic scenario for these genes on plasmid p N L l , that phenylacrylate derivatives are transformed to aromatic aldehydes, which are oxidized to aromatic acids, then non-oxidatively decarboxylated to catechol derivatives for mineralization via catechol dioxygenase ring cleavage enzymes. In fact, the metacleavage enzyme catechol 2,3-dioxygenase is found on p N L l . It should be noted that the translation products of the pchFa, vdh, vdcB and vdcC homologues on p N L l were approximately 55% identical to their Streptomyces sp. D7 and Pseudomonas spp. counterparts, and therefore may be similar in function but not substrate specificity. For example, the p N L l genes may encode non-oxidative, decarboxylase enzymes that transform  toluene  derivatives  instead  of  lignin-related  aromatic  compounds.  Streptomyces sp. D7 did not contain the aldehyde oxidoreductase gene found on p N L l , and therefore  would not be expected to carry through the bioconversion of  phenylacrylates to aromatic acids. Indeed, & lividans 1326 carrying pKCS8 effectively decarboxylated vanillic acid to guaiacol, no observable biotransformation activity was noted when the organism was exposed to ferulic acid, the acrylate derivative of vanillic  101  acid. The role of the V D C gene cluster for Streptomyces sp. D7 in nature remains to be determined, and one issue that arises from this study is whether or not the organism acquired the cluster through horizontal gene transfer events during evolution. Standard plasmid isolation procedures performed on Streptomyces sp. D7 did not reveal the presence of any plasmids, but this issue should be investigated further in future studies, perhaps using pulsed field gel electrophoresis techniques. Another question is whether the organism originally carried all the genes necessary for vanillic acid mineralization, but lost some downstream catabolic genes (such as those for the degradation of guaiacol) through deletion events.  Finally, it is possible that the V D C gene cluster encodes  enzymes for which nature did not intend to decarboxylate vanillic acid, but rather another substrate not tested in the work presented here. Streptomyces sp. D7 could be able to degrade a yet to be determined complex substrate with a vanillic acid-like degradation intermediate, which is processed by the genes described in this thesis work;  102  B. pNL1 ORF Functional description of closest relative  orf1244 orf1272 orf1280  Phenylacrylic acid decarboxylase homolog conserved hypothetical protein conserved hypothetical protein  % identity to 8. subtilis ORF 50 47 32  F I G U R E 33: p N L l of Sphingomonas aromaticivorans. (a) Physical map of p N L l and the location of the V D C gene cluster homologues; (b) listing of the p N L l open reading frames similar to ORFs from the Bacillus subtilis genome (From Romine, et al., 1999)  103  5.8  M e t a b o l i c  E n g i n e e r i n g  A p p l i c a t i o n s f o r V a n i l l a t e  D e c a r b o x y l a s e  The elaborate metabolic engineering scheme described for E. coli AB2834, which enables the production of adipic acid using D-glucose as a starting material, is an example of what can be achieved by mixing and matching catabolic genes in an appropriate host microorganism (Draths & Frost, 1994).  Similarly, the genes encoding the components of the vanillate decarboxylase system of Streptomyces sp. D7 could be used in engineered systems for the transformation of lignin-rich plant and timber waste to useful commodity or fine chemicals. Engineering such a system would probably not be as complex as the development of E. coli AB2834 for conversion of D-glucose to adipic acid. The by-products of wood pulp and other plant manufacturing processes, such as olive oil production (Ramos, J., personal communication), consist of large amounts of methoxylated aromatic chemicals. In theory, these aromatic chemicals could be separated from the waste stream and refined to industrially useful chemicals such as catechol and cis,cis-muconic acid (for drug syntheses), adipic acid (for nylon), or guaiacols (for medicines and scent compounds). To enable this process, a microorganism would have to be modified with a suite of genes encoding enzymes to produce the desired end products. For the refining of vanillate, a microorganism expressing vanillate decarboxylase would be an efficient producer of guaiacol, while a strain expressing genes for vanillate decarboxylase and guaiacoldemethylating Cytochrome P-450 (cloned and sequenced from Streptomyces setonii 75Vi2 by M . K . Pope and S. Baily in this laboratory) would produce catechol. If this catechol producing strain is supplemented with the gene for catechol 1,2-dioxygenase,  104  adipic acid could be produced. A diagram of this proposed scheme is shown in Figure 34. With the current low prices for petroleum products, commercialization of such biorefining technology is not practical as the desired products can be inexpensively produced from benzene. However, with the continually changing supply and demand of petroleum, and with increasingly stringent environmental regulations being imposed on industry, biorefining technology may become not only practical, but necessary.  105  COOH Lignin solubilization product  VANiLLATE DECARBOXYLASE  U s e d for fragrances and medicines  Used as a drug synthesis precursor  Used as a drug synthesis precursor  HOOC-  F I G U R E 34: Vanillate decarboxylase, Cytochrome P-450 and catechol 1,2-dioxygenase can be used in combinations to produce various industrially useful chemicals from vanillic acid as a starting material.  106  5.9 Future directions  The cloning of the genes required for vanillic acid non-oxidative decarboxylation, as presented in this work, will allow us to expand our knowledge of the reaction. Purification of active recombinant protein products encoded by the gene cluster will enable detailed reaction kinetics to be determined for the decarboxylation process. Future studies will shed light on the structure and catalytic function of vanillic acid decarboxylase from Streptomyces sp. D7. It will be interesting to determine i f the numerous sequence homologues from microbial genome databases are indeed other nonoxidative decarboxylases.  The transcriptional regulation of the V D C gene cluster in  response to environmental stimuli will be another interesting aspect to investigate. Accordingly, one experiment planned for the near future will be to clone the entire V D C gene cluster, including the divergent putative regulatory gene, into a suitable plasmid expression vector for expression in S. lividans 1326. It has already been demonstrated by 2D-PAGE and m R N A analyses that in Streptomyces sp. D7, the vanillate decarboxylase gene cluster is inducible in response to vanillic acid. A recombinant S. lividans 1326 strain carrying the V D C gene cluster and the associated regulatory gene should also demonstrate vanillic acid inducibility, unless the regulatory system requires other components not present in S. lividans 1326. Non-oxidative decarboxylases represent connections between the two major branches of aromatic acid catabolism, characterized by either catechol or protocatechuate central intermediates (Figure 35). By joining these two pathways, this class of enzymes supports the concept of aromatic catabolism as being a web of interconnecting biodegradative processes, and not separate, distinct pathways (Crawford and Olson, 1978). From an applied standpoint, knowledge gained from such  107  Lignin C0 H 2  VDC T  OCH  3  OH guaiacol  OH vanillate  Cytochrome P-  VanA, VanB C0 H 2  PDC , OH OH protocatechuate PcaG, PcaH mineralization  OH catechol CatA mineralization  F I G U R E 35: Non-oxidative decarboxylases represent connections between the two major branches of aromatic acid catabolism, characterized by either catechol or protocatechuate central intermediates. VDC = vanillate decarboxylase; PDC = protocatechuate decarboxylase; VanA, VanB = vanillate demethylase; PcaG, PcaH = protocatechuate 3,4-dioxygenase; CatA = catechol 1,2-dioxygenase  108  endeavors should ultimately allow this class of enzyme to be used for a variety of metabolic engineering applications. Currently, the world's major chemical companies are refocusing their business strategies towards producing many bulk commodity chemicals via microbial fermentations (Alper, 1999). 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