Elucidating physiological roles of Pichia stipitis alcohol dehydrogenases in xylose fermentation and shuffling promoters for multiple genes in Saccharomyces cerevisiae to improve xylose fermentation

Xylose is the second most abundant sugar in biomass hydrolysate, and the focus of this thesis is to understand and improve xylose fermentation in yeasts Saccharomyces cerevisiae and Pichia stipitis .; To eliminate rate limiting steps in engineered S. cerevisiae for xylose fermentation, we describe here a useful metabolic engineering tool to optimize expression levels for multiple genes, Multiple-Gene Promoter Shuffling (MOPS). This method approaches an optimized levels of gene overexpression by fusing varied strength promoters to genes of interest for a particular pathway. MGPS was implemented in a yeast xylose fermentation by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase (TAL1), transketolase (TKL1 ) and pyruvate kinase (PYK1) in the Saccharomyees cerevisiae strain FPL-YSX3. This host strain has integrated xylose metabolizing genes including xylose reductase, xylitol dehydrogenase and xylulose kinase. We found the optimal combination for ethanol production to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1. The MGPS method could easily be adapted into other eukaryotic and prokaryotic organisms, to optimize expression of genes for industrial fermentations.; The other objective of the present work is to elucidate the physiological roles of alcohol dehydrogenase (Adh) isozymes in yeast P. stipitis. With the recent sequencing of the P. stipitis genome, biochemical properties of Adhs were studied by fusing 6X histidine tag (his6) was fused to carboxyl end of Adh1, Adh2, Adh4, Adh5 and Adh7. The primary Adhs Adh1and Adh2 were strictly NAD(H) linked, secondary Adhs Adh4, Adh5 and Adh7 were NADP(H) linked. The kinetic properties of Adh1 and Adh2 indicated that Adh1 was bifunctional in ethanol metabolism, whereas Adh2 was a major aldehyde dehydrogenase. The deletion of ADH1 impaired the ethanol oxidation capacity in the resulting strain and supported the kinetic data. The same strain accumulated xylitol under microaerobic condition indicated that ethanol synthesis in P. stipitis was a net NAD + gaining step, compared to a NAD+ neutral step in conventional yeasts. Interestingly Adh7 could partially complement growth of a xylitol dehydrogenase null mutant on xylose, probably due to its weak xylitol metabolizing activity and regeneration of NADPH.