A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to HMF and other inhibitors

The 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, AST2N406I, 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, AST2N406I, 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.