A Novel Ast2 Mutation Generated Upon Whole-Genome Transformation Of Saccharomyces Cerevisiae Confers High Tolerance To 5-Hydroxymethylfurfural (Hmf) And Other Inhibitors

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 AST2(N406I) 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. AST1(D405I)) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2(N406I) 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.

Author summaryThe use of lignocellulosic biomass from waste streams or energy crops is highly favored over the use of fossil resources for the production of biofuels or bio-based chemicals in the fight against climate change. However, the pretreatment and hydrolysis of the biomass generates large amounts of inhibitors that compromise the subsequent fermentation of the released sugars. In this work, we have used a rarely applied technology, whole-genome transformation with DNA from an inhibitor tolerant species to obtain cellulosic yeast strains with improved inhibitor tolerance. This resulted in a new highly efficient gene tool, AST2(N406I), for targeted improvement of inhibitor tolerance in different yeast strain backgrounds and active against multiple inhibitors. A highly surprising result from this work is that the origin of the donor DNA from an inhibitor tolerant strain is essential to obtain stable inhibitor-tolerant transformants by whole-genome transformation but that none of the mutations, including the causative mutation, AST2(N406I), was present in the genomic DNA of the donor strain. A tentative explanation is that incoming protective DNA fragments are maintained as extrachromosomal DNA, allowing proliferation of the host strain under the selective condition until it can generate itself a spontaneous mutation in its own DNA that takes over the protective function, after which the heterologous DNA is easily lost.