Monday, May 5, 2008
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Bioethanol production from xylose using recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Akinori Matsushika1, Seiya Watanabe2, Tsutomu Kodaki2, Keisuke Makino2, and Shigeki Sawayama1. (1) Biomass Technology Research Center (BTRC), National Institute of Advanced Industrial Science and Technology, 2-2-2 Hirosuehiro, Kure, Hiroshima, 737-0197, Japan, (2) Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan

 Saccharomyces cerevisiae is used widely for industrial ethanol production. However, S. cerevisiae is naturally unable to metabolize xylose, which is the second major sugar present in hard woods and herbs, so its fermentation is essential for the economic conversion of lignocellulose to ethanol. Accordingly, heterologous expression of genes for xylose reductase (XR) and xylitol dehydrogenase (XDH) cloned from Pichia stipitis (PsXR and PsXDH, respectively) in S. cerevisiae has been studied extensively with regard to ethanol fermentation from xylose. In addition, overexpression of the xylulokinase (XK) gene from S. cerevisiae has been shown to aid xylose utilization. However, it has not yet been applied to the industrial bio-process, mainly due to the unfavorable excretion of xylitol which occurs during xylose fermentation. Intracellular redox imbalance caused by the different coenzyme specificities between PsXR (with NADPH) and PsXDH (with NAD+) has been thought to be one of the main factors that promote xylitol excretion. To reduce xylitol formation, we have already generated several PsXDH mutants (e.g., ARSdR mutant) with complete reversal of coenzyme specificity toward NADP+ by site-directed mutagenesis. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability and measured the efficiency of ethanol fermentation from xylose. The results of such fermentation analyses indicated that improved fermentation performance, as seen by the increased ethanol production, decreased xylitol excretion, and faster xylose consumption, was observed in a strain expressing genes of PsXR and NADP+-dependent PsXDH and endogenous XK as compared with the reference strain expressing the wild-type XDH.

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