title: Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.
abstract: OBJECTIVE
  Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed.
  
  
  RESULTS
  Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose.
  
  
  CONCLUSIONS
  Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption.
  
  
  CONCLUSIONS
  Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Reznicek
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: S J
authors.initials: SJ
authors.lastname: Facey
authors.affiliation: DSM, AX Delft, The Netherlands.
authors.forename: P P
authors.initials: PP
authors.lastname: de Waal
authors.affiliation: DSM, AX Delft, The Netherlands.
authors.forename: A W R H
authors.initials: AW
authors.lastname: Teunissen
authors.affiliation: Jandebontbioconsultancy, Wageningen, The Netherlands.
authors.forename: J A M
authors.initials: JA
authors.lastname: de Bont
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, Groningen, The Netherlands.
authors.forename: J G
authors.initials: JG
authors.lastname: Nijland
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, University of Groningen, Groningen, The Netherlands.
authors.forename: A J M
authors.initials: AJ
authors.lastname: Driessen
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Hauer
mesh_terms: D001692:Biological Transport; D019020:Directed Molecular Evolution; D005947:Glucose / Q000378:metabolism; D057166:High-Throughput Screening Assays; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
chemical_list: C106901:GAL2 protein, S cerevisiae; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014994:Xylose; D005947:Glucose
keywords: Gal2; biofuels; biotechnology; sugar co-consumption; yeasts