Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.