Metabolic and genomic characterisation of stress-tolerant industrial Saccharomyces cerevisiae strains from TALENs-assisted multiplex editing.

TALENs-assisted multiplex editing (TAME) toolbox was previously established and used to successfully enhance ethanol stress tolerance of Saccharomyces cerevisiae laboratory strain. Here, the TAME toolbox was harnessed to improve and elucidate stress tolerances of S. cerevisiae industrial strain. One osmotolerant strain and one thermotolerant strain were selected from the mutant library generated by TAME at corresponding stress conditions, and exhibited 1.2-fold to 1.3-fold increases of fermentation capacities, respectively. Genome resequencing uncovered genomic alterations in the selected stress-tolerant strains, suggesting that cell wall and membrane-related proteins might be major factors behind improved tolerances of yeast to different stresses. Furthermore, amplified mitochondrial DNA might also have an important impact on increased stress tolerance. Unexpectedly, none of predesigned target potential TALENs modification sites showed any genomic variants in sequenced genomes of the selected strains, implicating that the improved stress tolerances might be due to indirect impacts of genome editing via TALENs rather than introducing genomic variants at potential target sites. Our findings not only confirmed TAME could be a useful tool to accelerate the breeding of industrial strain with multiple stress tolerance, but also supported the previous understandings of the complicated mechanisms of multiple stress tolerance in yeast.