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Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation.
Metab Eng. 2001 Jul; 3(3):226-35.ME

Abstract

To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction.

Authors+Show Affiliations

Department of Applied Microbiology, Lund University, 221 00 Lund, Sweden.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

11461145

Citation

Anderlund, M, et al. "Expression of Bifunctional Enzymes With Xylose Reductase and Xylitol Dehydrogenase Activity in Saccharomyces Cerevisiae Alters Product Formation During Xylose Fermentation." Metabolic Engineering, vol. 3, no. 3, 2001, pp. 226-35.
Anderlund M, Rådström P, Hahn-Hägerdal B. Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation. Metab Eng. 2001;3(3):226-35.
Anderlund, M., Rådström, P., & Hahn-Hägerdal, B. (2001). Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation. Metabolic Engineering, 3(3), 226-35.
Anderlund M, Rådström P, Hahn-Hägerdal B. Expression of Bifunctional Enzymes With Xylose Reductase and Xylitol Dehydrogenase Activity in Saccharomyces Cerevisiae Alters Product Formation During Xylose Fermentation. Metab Eng. 2001;3(3):226-35. PubMed PMID: 11461145.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation. AU - Anderlund,M, AU - Rådström,P, AU - Hahn-Hägerdal,B, PY - 2001/7/20/pubmed PY - 2001/10/26/medline PY - 2001/7/20/entrez SP - 226 EP - 35 JF - Metabolic engineering JO - Metab Eng VL - 3 IS - 3 N2 - To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction. SN - 1096-7176 UR - https://www.unboundmedicine.com/medline/citation/11461145/Expression_of_bifunctional_enzymes_with_xylose_reductase_and_xylitol_dehydrogenase_activity_in_Saccharomyces_cerevisiae_alters_product_formation_during_xylose_fermentation_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7176(01)90190-9 DB - PRIME DP - Unbound Medicine ER -