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

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 Gal2(N376T) or Gal2(N376I) 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 (14)C-sugar-transport assays, Gal2(N376S), Gal2(N376T) and Gal2(N376I) substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher K(m) 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.