Abstract

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars that can be fermented to ethanol. However, the inhibitory substances found in this complex feedstock adversely affect yeasts used for the fermentation of the sugars in SSL. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome shuffling based on large-scale population recursive meiotic recombination. Populations of UV-induced yeast mutants more tolerant to hardwood spent sulfite liquor (HWSSL) were 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 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. These results demonstrate 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. The genome of R57, the most inhibitor-tolerant strain generated in this study, was sequenced by massively parallel sequencing and twenty single nucleotide polymorphism mutations were located. Many of these mutations affect genes that correlate with known stress responses to the types of inhibition found in lignocellulosic hydrolysates. Cross-referencing the mutation findings with RNA-seq-derived differential gene expression analysis of R57 yields genes and biological processes that are likely playing determinant roles in the HWSSL tolerance trait of R57. The strongest findings support important roles for the following mutation-bearing proteins and biological processes: stress response transcriptional repressor, Nrg1p; NADPH-dependent glutamate dehydrogenase, Gdh1p, and a modified nitrogen usage and assimilation physiology including alterations to aromatic amino acid synthesis pathways and the associated Aro1p; protein homeostasis machinery including heat-shock 70-family proteins, especially Ssa1p, and Ubp7p and Art5p, which are related to ubiquitin-mediated proteolysis. Redox-associated metabolites NADPH, glutathione, mainly via GSH1 mutation, and iron are also implicated. Overall, these data provide important findings for understanding inhibition by multi-inhibitory lignocellulosic substrates; a meiotic recombination-mediated engineering strategy for generating inhibition-tolerant strains that may be unobtainable through classical evolutionary engineering; novel genetic targets for future rational biocatalyst design and inhibitor-tolerance studies; and a promising work-flow model for generating strains with desirable and complex phenotypic traits along with understanding of the genetic factors and biological processes involved in those traits.