Transcriptomic Analysis Of Glutathione-overproducing Saccharomyces Cerevisiae Mutant Based On RNA-Seq

Glutathione (GSH), a kind of small bioactive peptide compound, widely exists in mosteukaryotic cells and many bacteria. Due to numerous important physiological functions, such asdetoxification, antioxidant and anti-aging, it has been broadly applied in pharmacy, foodadditives and cosmetic industries.The mutant strain Saccharomyces cerevisiae Y518was screened out from the collectionsof nitrosoguanidine and ultraviolet treated Saccharomyces cerevisiae2-10515. The intracellularGSH content of Y518reached12.02±0.09mg/g, which was about55%higher than that of thewild type2-10515. In this study, the RNA-seq based transcriptomic approach was performedfor exploring the potential mechanisms contributing to GSH overproduction in Y518.A total of1628differentially expressed genes (fold-change>=2.0, FDR<=0.001) wereidentified by RNA-Seq, among of which,1125were up-regulated and503were down-regulated.Among these raised genes, SUL1, SUL2, MET14and MET5were gathered in sulfur absorptionpathway, MET17and CYS4were enriched in cysteine biosynthesis pathway, GSH1wasinvolved in GSH biosynthesis pathway, SKN7, SOD1, CTT1and GRX2were concentrated inoxidative stress response.Physiological and biochemical indicators analysis showed activity of the rate-limitingenzyme γ-glutamylcysteine synthase of GSH biosynthesis and oxidative stress response relatedenzymes (Cu/Zn superoxide dismutase, catalase and glutathione peroxidase) was significantlyhigher in Y518than in2-10515. Intracellular cysteine and reactive oxygen species level ofY518were also observably enhanced. In addition, it was found that2-10515had a normalbreathing ability for its red colonies, while Y518might have some functional defects ofrespiration for its white colonies using triphenyl tetrazolium chloride staining. In the meanwhile, complex III activity of Y518was seriously declined by determination of themitochondrial respiratory chain complexes (I, II, III, IV) activities.There were six mutation sites in the coding region of COR1which caused four mutativeamino acids in Y518by comparison of the subunits coding genes of complex III with2-10515.All these results suggested the endogenous oxidative stress might exist in Y518induced bysome uncertain functional defects of the mitochondrial respiratory chain caused by complex IIImutation, which then caused a series of oxidative stress reactions. To counteract the oxidativestress, the transcription regulator SKN7was enhanced to regulate the improvement of GSH1 expression, which strongly adjusted the increase of intracellular GSH level. Moreover, theoveraccumulation of intracellular GSH in Y518was also connected with enhanced activity ofthe rate-limiting enzyme γ-GCS, up-regulated genes of sulfur assimilation and cysteinebiosynthesis pathway and increased content of the key precursor amino acid cysteine.The mechanism of GSH overaccumulation in the mutant Y518was explained throughRNA-Seq based transcriptomic analysis, combined with physiological and biochemicalindicators analysis, which might provide new strategies for further genetically modified andconstruction of GSH overproducing strains.