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Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae.
Biotechnol J. 2009 May; 4(5):684-94.BJ

Abstract

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD(+)-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP(+), which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP(+) and NAD(+). One of the XDH variants utilized NADP(+) about 4 times more efficiently than NAD(+). This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.

Authors+Show Affiliations

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19452479

Citation

Krahulec, Stefan, et al. "Engineering of a Matched Pair of Xylose Reductase and Xylitol Dehydrogenase for Xylose Fermentation By Saccharomyces Cerevisiae." Biotechnology Journal, vol. 4, no. 5, 2009, pp. 684-94.
Krahulec S, Klimacek M, Nidetzky B. Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae. Biotechnol J. 2009;4(5):684-94.
Krahulec, S., Klimacek, M., & Nidetzky, B. (2009). Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae. Biotechnology Journal, 4(5), 684-94. https://doi.org/10.1002/biot.200800334
Krahulec S, Klimacek M, Nidetzky B. Engineering of a Matched Pair of Xylose Reductase and Xylitol Dehydrogenase for Xylose Fermentation By Saccharomyces Cerevisiae. Biotechnol J. 2009;4(5):684-94. PubMed PMID: 19452479.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae. AU - Krahulec,Stefan, AU - Klimacek,Mario, AU - Nidetzky,Bernd, PY - 2009/5/20/entrez PY - 2009/5/20/pubmed PY - 2009/9/8/medline SP - 684 EP - 94 JF - Biotechnology journal JO - Biotechnol J VL - 4 IS - 5 N2 - Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD(+)-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP(+), which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP(+) and NAD(+). One of the XDH variants utilized NADP(+) about 4 times more efficiently than NAD(+). This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation. SN - 1860-7314 UR - https://www.unboundmedicine.com/medline/citation/19452479/Engineering_of_a_matched_pair_of_xylose_reductase_and_xylitol_dehydrogenase_for_xylose_fermentation_by_Saccharomyces_cerevisiae_ L2 - https://doi.org/10.1002/biot.200800334 DB - PRIME DP - Unbound Medicine ER -