Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI from T. thermophilus was produced, and endogenous xylulokinase from S. cerevisiae was overproduced in S. cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene from T. thermophilus on a replicative plasmid generated recombinant xylose utilizing S. cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.

Saccharomyces cerevisiae mutants, in which open reading frames (ORFs) displaying similarity to the aldo-keto reductase GRE3 gene have been deleted, were investigated regarding their ability to utilize xylose and arabinose. Reduced xylitol formation from D-xylose in gre3 mutants of S. cerevisiae suggests that Gre3p is the major Dxylose-reducing enzyme in S. cerevisiae. Cell extracts from the gre3 deletion mutant showed no detectable xylose reductase activity. Decreased arabitol formation from Larabinose indicates that Gre3p, Ypr1p and the protein encoded by YJR096w are the major arabinose reducers in S. cerevisiae. The ypr1 deletion mutant showed the lowest specific L-arabinose reductase activity in cell extracts, 3.5 mU/mg protein compared with 7.4 mU/mg protein for the parental strain with no deletions, and the lowest rate of arabitol formation in vivo. In another set of S. cerevisiae strains, the same ORFs were overexpressed. Increased xylose and arabinose reductase activity was observed in cell extracts for S. cerevisiae overexpressing the GRE3, YPR1 and YJR096w genes. These results, in combination with those obtained with the deletion mutants, suggest that Gre3p, Ypr1p and the protein encoded by YJR096w are capable of xylose and arabinose reduction in S. cerevisiae. Both the D-xylose reductase and the L-arabinose reductase activities exclusively used NADPH as co-factor.