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

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.

Pentose Metabolism in Saccharomyces cerevisiae: The Need to Engineer Global Regulatory Systems

“…More recently, a glucose‐phosphorylation‐negative strain S. cerevisiae , which can activate galactose regulon in the absence of glucose, when engineered to express arabinose metabolic genes displayed a growth rate of 0.26 h −1 , the highest growth rate achieved in pentose sugars, thus far. Although, similar expression of arabinose metabolic genes has been attempted before, even from the same lab, combining that with galactose regulon activation and eliminating glucose‐phosphorylation seemed to have increased growth rate several hundred‐folds without the need for ALE . Growth rates of representative arabinose consuming strains are listed in Table .…”

“…Genome‐wide expression profiles of strains that grew with regulon assistance in both galactose and xylose had several growth‐related genes as well as transcription factors upregulated when compared to strains that constitutively expressed galactose or xylose metabolic genes . Similarly, activation of galactose regulon along with removing HXK2 ‐controlled carbon catabolite repression seemed to have resulted in the fastest arabinose‐growing S. cerevisiae strain, stressing the importance for higher systems‐level regulation for growth on a non‐native sugar …”

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

“…Upon overexpression of NcLAT1 and MtLAT1 in a L-arabinose metabolizing S. cerevisiae strain, L-arabinose utilization, growth and ethanol production was improved. Sequence alignment showed that a conserved asparagine, i.e., N376 of Gal2 (Farwick et al, 2014;Verhoeven et al, 2018), has been replaced into phenylalanine in both NcLAT1 and MtLAT1 (Li et al, 2015). This residue is very critical for the specificity of Hxt transporters for D-glucose, and its substitution in the aforementioned transporters might explain why L-arabinose transport is relatively insensitive to Dglucose inhibition which will be discussed in more detail in the engineering section.…”

“…By means of evolutionary engineering of a D-glucosephosphorylation-negative S. cerevisiae strain on mixed sugar feedstocks (glucose-xylose-arabinose), a strain was evolved that grows on L-arabinose in the presence of D-glucose and Dxylose (Verhoeven et al, 2018). Genome sequencing revealed a mutation of the conserved N376 of Gal2 that corresponds to the key residue in Hxt transporters that determines specificity.…”

“…In Gal2, this residue was mutated into either a serine, threonine or isoleucine. The Gal2 mutants all showed a decreased D-glucose sensitivity of L-arabinose transport which was accompanied by a reduction of the affinity and V max of D-glucose transport (Wisselink et al, 2012;Verhoeven et al, 2018). Reported Larabinose transport affinities range from 57 mM (Subtil and Boles, 2011) and 371 mM (Knoshaug et al, 2015) (Table 2).…”