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Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake
Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
Univ Groningen, Groningen Biomol Sci & Biotechnol Inst GBB, Dept Mol Microbiol, Nijenborgh 7, NL-9747 AG Groningen, Netherlands..
Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
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2018 (English)In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 6, article id foy062Article in journal (Refereed) Published
Abstract [en]

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantarum L-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h(-1) on L-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on L-arabinose in the presence of D-glucose and D-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2(N376T) or Gal(2N376I) substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2(T89I) substitution, which was subsequently also detected in the independently evolved strain IMS0010. In C-14-sugar-transport assays, Gal2(N376S), Gal2(N376T) and Gal(2N376I) substitutions showed a much lower glucose sensitivity of L-arabinose transport and a much higher Km for D-glucose transport than wild-type Gal2. Introduction of the Gal2(N376I) substitution in a non-evolved strain enabled growth on L-arabinose in the presence of D-glucose. Gal2(N376T), T89I and Gal2(T89I) variants showed a lower K-m for L-arabinose and a higher K-m for D-glucose than wild-type Gal2, while reverting Gal2(N376T), T89I to Gal2(N376) in an evolved strain negatively affected anaerobic growth on L-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.

Place, publisher, year, edition, pages
Oxford University Press, 2018. Vol. 18, no 6, article id foy062
Keywords [en]
yeast, pentose fermentation, L-arabinose, transporter engineering, laboratory evolution, bioethanol, gene duplication
National Category
Microbiology
Identifiers
URN: urn:nbn:se:kth:diva-239501DOI: 10.1093/femsyr/foy062ISI: 000449353000011PubMedID: 29860442Scopus ID: 2-s2.0-85053495681OAI: oai:DiVA.org:kth-239501DiVA, id: diva2:1266492
Note

QC 20181128

Available from: 2018-11-28 Created: 2018-11-28 Last updated: 2020-03-09Bibliographically approved

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van Maris, Antonius J. A.

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