title: Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.
abstract: 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.
authors.affiliation: CREST, JST (Japan Science and Technology Agency), Gokasho, Uji, Kyoto 611-0011, Japan.|Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.|Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abu Saleh
authors.affiliation: CREST, JST (Japan Science and Technology Agency), Gokasho, Uji, Kyoto 611-0011, Japan.|Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Pack
authors.affiliation: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Narayana
authors.initials: N
authors.lastname: Annaluru
authors.affiliation: CREST, JST (Japan Science and Technology Agency), Gokasho, Uji, Kyoto 611-0011, Japan.|Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kodaki
authors.affiliation: International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan.|CREST, JST (Japan Science and Technology Agency), Gokasho, Uji, Kyoto 611-0011, Japan.|Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Makino
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D007218:Industrial Microbiology / Q000379:methods; D009243:NAD / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D014994:Xylose / Q000378:metabolism*
chemical_list: D009243:NAD; D000431:Ethanol; D014994:Xylose; D000449:Aldehyde Reductase