Cited by 301Fungal and yeast genetics researchBiofuel production and bioconversionMicrobial Metabolic Engineering and Bioproduction

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

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Farwick, Alexander, Bruder, Stefan, Schadeweg, Virginia, Oreb, Mislav, Boles, Eckhard·2014·Proceedings of the National Academy of Sciences

Abstract

All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.

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Crossref · 10.1073/pnas.1323464111

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  • Adaptive evolutionary engineering based on metabolic engineering can allow microorganisms to quickly obtain excellent phenotypes, but there are problems such as unclear gene targets and negative mutation interference.

    “Adaptive evolutionary engineering based on metabolic engineering can allow microorganisms to quickly obtain excellent phenotypes, but there are problems such as unclear gene targets and negative mutation interference [90,91].”

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Authors
Farwick, A., Bruder, S., Schadeweg, V., Oreb, M., & Boles, E.
Affiliations
Goethe University Frankfurt (DE)
Venue
Proceedings of the National Academy of Sciences
Published
2014-03-24
Volume
111
Issue
14
Pages
5159-5164
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
OpenAlex
W2118069954
Cited by
298
References
54
Citations by year
2026: 20 · 2025: 12 · 2024: 17 · 2023: 20 · 2022: 25 · 2021: 35 · 2020: 28 · 2019: 26 · 2018: 26 · 2017: 35 · 2016: 26 · 2015: 22 · 2014: 6
FWCI
12.16
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
hexose transporters, GAL2, XylE, sugar specificity, competitive inhibition, lignocellulosic biomass, Saccharomyces cerevisiae
MeSH terms
Biological Transport, Glucose, Hydrolysis, Models, Molecular, Monosaccharide Transport Proteins, Saccharomyces cerevisiae, Xylose, Protein Engineering, Mutagenesis, Site-Directed

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