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Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis

¹ Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan
² Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
³ CREST, JST (Japan Science and Technology Agency), Gokasho, Uji, Kyoto 611-0011, Japan
⁴ International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan

Correspondence
Seiya Watanabe
irab{at}iae.kyoto-u.ac.jp

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD⁺-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k_cat/K_m with NADH)/(k_cat/K_m with NADPH)] compared with the wild-type (WT), which was due to decrease of k_cat with NADPH in the R276H mutant and increase of K_m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP⁺ and NADH/NAD⁺ ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Two supplementary tables are available with the online version of this paper.

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