title: Improved Xylose Metabolism by aCYC8Mutant of Saccharomyces cerevisiae.
abstract: EngineeringSaccharomyces cerevisiaefor the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However,S. cerevisiaelacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means ofin vivoengineering, we have developed a quadruple hexokinase deletion mutant ofS. cerevisiaethat evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressorCYC8, orSSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressedHXT10,HXT13,HXT15, andHXT16Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCEThe yeastSaccharomyces cerevisiaeis used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands.
authors.forename: Jeroen G
authors.initials: JG
authors.lastname: Nijland
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands.
authors.forename: Hyun Yong
authors.initials: HY
authors.lastname: Shin
authors.affiliation: DSM Biotechnology Center, Delft, the Netherlands.
authors.forename: Leonie G M
authors.initials: LGM
authors.lastname: Boender
authors.affiliation: DSM Biotechnology Center, Delft, the Netherlands.
authors.forename: Paul P
authors.initials: PP
authors.lastname: de Waal
authors.affiliation: DSM Biotechnology Center, Delft, the Netherlands.
authors.forename: Paul
authors.initials: P
authors.lastname: Klaassen
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands a.j.m.driessen@rug.nl.
authors.forename: Arnold J M
authors.initials: AJM
authors.lastname: Driessen
mesh_terms: D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
chemical_list: C065455:CYC8 protein, S cerevisiae; D009004:Monosaccharide Transport Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014994:Xylose; D005947:Glucose
keywords: evolutionary engineering; sugar transporter; transcriptome; xylose transport; yeast