Mutations in the galactose-transporter gene GAL2 enable anaerobic growth of glucose-phosphorylation-negative, arabinose-fermenting yeast strains in the presence of glucose. Saccharomyces cerevisiae CEN.PK113-7D

L-Arabinose is the second most abundant pentose sugar in lignocellulosic plant biomass hydrolysates used for bioethanol production. Engineered S. cerevisiae strains can consume L-arabinose via the Gal2 galactose transporter, but Gal2-mediated L-arabinose uptake is strongly inhibited by D-glucose.An engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantarum L-arabinose pathway and the Penicillium chrysogenum arabinose transporter PcAraT, was constructed by CRISPR-Cas9-supported genome editing., Anaerobic sequential batch cultivation on glucose-xylose-arabinose mixtures yielded evolved strains that grew on L-arabinose in the presence of D-glucose and D-xylose. Whole-genome sequencing of four evolved strains showed that GAL2 had been duplicated in all strains, with both copies encoding the same, strain-dependent amino-acid substitution at position 376 of Gal2. In one strain, one of the two GAL2 alleles additionally encoded a T89I substitution. Introduction of the N376I substitution in a non-evolved strain enabled growth on L-arabinose in the presence of D-glucose. In sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I exhibited a lower glucose sensitivity of L-arabinose uptake than wild-type Gal2 and a decreased Km and Vmax for D-glucose transport. The T89I substitution was also identified in S. cerevisiae IMS0010, a strain previously engineered and evolved for pentose/hexose co-consumption. Gal2N376T, T89I and Gal2T89I showed a lower Km for L-arabinose and a higher Km for D-glucose than wild-type Gal2. Reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on arabinose. Reverse engineering of GAL2 mutations into a non-evolved strain indicated that improved utilization of L-arabinose was primarily due to the Gal2N376T substitution, while the Gal2N376T, T89I substitution and expression of PcAraT contributed to L-arabinose uptake when L-arabinose concentrations were low relative to those of D-glucose.Sequential batch cultures on sugar mixtures of a glucose-phosphorylation-negative yeast strain was successfully used to select for xylose- and glucose-insensitive growth on L-arabinose. Identification of causal mutations in GAL2 provided insight into the evolution of a strain with two new, functionally different L-arabinose transporters. Combination of these two evolved Gal2 variants with a heterologously expressed fungal arabinose transporter enabled the fastest rate of anaerobic L-arabinose fermentation in S. cerevisiae reported to date.