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Characterization of an ethanologenic yeast inhibiting atypical galactose metabolism Keating, Jeffrey Desmond

Abstract

In the near future, biomass-derived energy is predicted to substantially complement that generated from petroleum. However, certain types of biomass employed as substrates in the microorganism-mediated production of renewable fuelethanol contain significant amounts of the recalcitrant hexose sugar galactose. The consumption of galactose in hexose sugar-fermenting yeasts is often delayed with respect to other sugars, such as glucose and mannose, because of an intrinsic preference for carbon sources requiring less energy in the preparatory reactions preceding glycolysis. This work comprised the search for, and characterization of anethanologenic yeast capable of efficiently assimilating galactose. Screening experiments conducted with wild-type Saccharomyces cerevisiae strains identified one isolate (Y-1528) exhibiting exceptionally fast galactose fermentation. The absence of conventional glucose repression, including a preference for galactose as carbon source and notable delays in the utilization of glucose and mannose, was demonstrated in mixed sugar fermentations. Endogenous extracellular glucose was observed during double sugar fermentations of galactose and mannose. This glucose was traced to supplied galactose by radioisotope labeling, suggesting involvement of UDP-galactose 4-epimerase in the responsible reaction mechanism(s).Sub-cellular fractionation was employed in an attempt to ascertain enzyme localization in Y-1528. Fermentations of lignocellulosic substrate mixtures by Y-1528 illustrated better performance than that accomplished by a reference yeast strain, and again showed a preference for galactose. Mixed cultures of Y-1528 and the same reference strain demonstrated accelerated hexose sugar consumption, and no detrimental effects from competition, during synthetic and lignocellulosic substrate fermentations. Glucose repression was absent in mixed culture fermentations. Fermentations of synthetic sugar mixtures augmented with lignocellulosic inhibitory compounds showed Y-1528 to have better performance than a reference yeast strain, despite a global detrimental effect relative to inhibitor-free fermentations. Cell recycle batch fermentations of spent sulfite liquor illustrated the toxic effect of the hardwood variant, as well as a net loss of performance from all strains tested. Y-1528 was taxonomically confirmed as S. cerevisiae. UDP-galactose 4-epimerase chromatographic purification was unsuccessful, but a partial sequence of the enzyme, showing complete identity with type sequence, was obtained by electrophoretic separation, liquid chromatography, and mass spectrometry. A significantly mutated UDP-galactose 4-epimerase gene was successfully sequenced.

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