Abstract
Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.
Highlights
Second-generation bioethanol, produced from lignocellulosic biomass hydrolysates, is a promising alternative transport fuel with multiple major benefits over fossil fuels and first-generation bioethanol
MD4 AST1D405I/AST1D405I/AST1D405I/AST1D405I strains (Fig 5B). These results indicate that the AST1D405I mutation can further enhance the protective effect of AST2N406I against high concentrations of inhibitors, but that by itself it does not have a detectable effect on inhibitor tolerance under the experimental conditions used
In this work we have demonstrated the efficacy of Whole-genome transformation (WGT) for improvement of selectable phenotypes in industrial yeast strains
Summary
Second-generation bioethanol, produced from lignocellulosic biomass hydrolysates, is a promising alternative transport fuel with multiple major benefits over fossil fuels and first-generation bioethanol. A second challenge is the high level of inhibitors present in lignocellulose hydrolysates that severely reduce the yeast fermentation rate and yield, in particular that of xylose [2,3]. The cheaper, harsh methodologies used for pretreatment of the lignocellulosic biomass result in higher levels of inhibitors, while the more gentle methodologies that result in less toxicity compromise the economic viability of the industrial process due to their higher cost [4,5,6]. In addition to the production of bioethanol from lignocellulosic biomass, these challenges apply to the production of other bio-based chemicals with 2G yeast cell factories, in which toxicity of many of these chemicals further aggravates the burden for the yeast
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