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Phenolic inhibitors in lignocellulosic hydrolysates interfere with the performance of fermenting microorganisms. Among these, coniferyl aldehyde is one of the most toxic inhibitors. In this study, genetically stable Saccharomyces cerevisiae mutants with high coniferyl aldehyde resistance were successfully obtained for the first time by using an evolutionary engineering strategy, based on the systematic application of increasing coniferyl aldehyde stress in batch cultures. Among the selected coniferyl aldehyde-resistant mutants, the highly resistant strain called BH13 was also cross-resistant to other phenolic inhibitors, vanillin, ferulic acid and 4-hydroxybenzaldehyde. In the presence of 1.2 mM coniferyl aldehyde stress, BH13 had a significantly reduced lag phase, which was less than 3 h and only about 25% of that of the reference strain and converted coniferyl aldehyde faster. Additionally, there was no reduction in its growth rate, either. Comparative transcriptomic analysis of a highly coniferyl aldehyde-resistant mutant revealed upregulation of the genes involved in energy pathways, response to oxidative stress and oxidoreductase activity in the mutant strain BH13, already under non-stress conditions. Transcripts associated with pleiotropic drug resistance were also identified as upregulated. Genome re-sequencing data generally supported transcriptomic results and identified gene targets that may have a potential role in coniferyl aldehyde resistance.

Genomic and transcriptomic analysis of a coniferyl aldehyde-resistant Saccharomyces cerevisiae strain obtained by evolutionary engineering