Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive l-arabinose uptake

Maarten Verhoeven, Jasmine Bracher, Jeroen G. Nijland, Jonna Bouwknegt, Jean Marc Daran, Arnold J.M. Driessen

Research output: Contribution to journalArticleScientificpeer-review

7 Citations (Scopus)
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Abstract

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-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 Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I 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 Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 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.
Original languageEnglish
Pages (from-to)1-15
JournalFEMS Yeast Research
Volume18
Issue number6
DOIs
Publication statusPublished - 2018

Keywords

  • yeast
  • pentose fermentation
  • l-arabinose
  • transporter engineering
  • laboratory evolution
  • bioethanol
  • gene duplication

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