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High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae.
Metab Eng. 2012 Jul; 14(4):336-43.ME

Abstract

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.

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Authors+Show Affiliations

Kim SR
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Ha SJ
No affiliation info available
Kong II
No affiliation info available
Jin YS
No affiliation info available

MeSH

D-Xylulose ReductaseEthanolFermentationGenetic EngineeringHomologous RecombinationMetabolic EngineeringPichiaPlasmidsSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsXylitolXylose

Pub Type(s)

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

Language

eng

PubMed ID

22521925

Citation

Kim, Soo Rin, et al. "High Expression of XYL2 Coding for Xylitol Dehydrogenase Is Necessary for Efficient Xylose Fermentation By Engineered Saccharomyces Cerevisiae." Metabolic Engineering, vol. 14, no. 4, 2012, pp. 336-43.
Kim SR, Ha SJ, Kong II, et al. High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae. Metab Eng. 2012;14(4):336-43.
Kim, S. R., Ha, S. J., Kong, I. I., & Jin, Y. S. (2012). High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae. Metabolic Engineering, 14(4), 336-43. https://doi.org/10.1016/j.ymben.2012.04.001
Kim SR, et al. High Expression of XYL2 Coding for Xylitol Dehydrogenase Is Necessary for Efficient Xylose Fermentation By Engineered Saccharomyces Cerevisiae. Metab Eng. 2012;14(4):336-43. PubMed PMID: 22521925.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae. AU - Kim,Soo Rin, AU - Ha,Suk-Jin, AU - Kong,In Iok, AU - Jin,Yong-Su, Y1 - 2012/04/13/ PY - 2011/12/13/received PY - 2012/03/20/revised PY - 2012/04/05/accepted PY - 2012/4/24/entrez PY - 2012/4/24/pubmed PY - 2012/10/27/medline SP - 336 EP - 43 JF - Metabolic engineering JO - Metab Eng VL - 14 IS - 4 N2 - The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains. SN - 1096-7184 UR - https://www.unboundmedicine.com/medline/citation/22521925/High_expression_of_XYL2_coding_for_xylitol_dehydrogenase_is_necessary_for_efficient_xylose_fermentation_by_engineered_Saccharomyces_cerevisiae_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7176(12)00044-4 DB - PRIME DP - Unbound Medicine ER -