Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials

Background: Production of bioethanol from lignocellulosic biomass requires the development of robust microorganisms that can tolerate the stressful conditions prevailing in lignocellulosic hydrolysates. Several inhibitors are known to affect the redox metabolism of cells. In this study, Saccharomyces cerevisiae was engineered for increased robustness by modulating the redox state through overexpression of GSH1, CYS3 and GLR1, three genes involved in glutathione (GSH) metabolism.Results: Overexpression constructs were stably integrated into the genome of the host strains yielding five strains overexpressing GSH1, GSH1/CYS3, GLR1, GSH1/GLR1 and GSH1/CYS3/GLR1. Overexpression of GSH1 resulted in a 42% increase in the total intracellular glutathione levels compared to the wild type. Overexpression of GSH1/CYS3, GSH1/GLR1 and GSH1/CYS3/GLR1 all resulted in equal or less intracellular glutathione concentrations than overexpression of only GSH1, although higher than the wild type. GLR1 overexpression resulted in similar total glutathione levels as the wild type. Surprisingly, all recombinant strains had a lower [reduced glutathione]:[oxidized glutathione] ratio (ranging from 32-67) than the wild type strain (88), suggesting a more oxidized intracellular environment in the engineered strains. When considering the glutathione half-cell redox potential (Ehc), the difference between the strains was less pronounced. Ehc for the recombinant strains ranged from -225 to -216 mV, whereas for the wild type it was estimated to -225 mV. To test whether the recombinant strains were more robust in industrially relevant conditions, they were evaluated in simultaneous saccharification and fermentation (SSF) of pretreated spruce. All strains carrying the GSH1 overexpression construct performed better than the wild type in terms of ethanol yield and conversion of furfural and HMF. The strain overexpressing GSH1/GLR1 produced 14.0 g L-1 ethanol in 48 hours corresponding to an ethanol yield on hexoses of 0.17 g g-1; while the wild type produced 8.2 g L-1 ethanol in 48 hours resulting in an ethanol yield on hexoses of 0.10 g g-1.Conclusions: In this study, we showed that engineering of the redox state by modulating the levels of intracellular glutathione results in increased robustness of S. cerevisiae in SSF of pretreated spruce.