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 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

Nyckelord

yeast

pentose fermentation

L-arabinose

transporter engineering

laboratory evolution

bioethanol

gene duplication

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