BACKGROUND Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity. RESULTS An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 +/- 0.02 h-1) in both strains. CONCLUSION Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.
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