Unraveling The Mechanisms Of Oxidative Stress Response In Saccharomyces Cerevisiae Through Inverse Metabolic Engineering

Excessive oxidative stress poses significant damages to yeast cells during fermentation process, resulting in the degeneration, crosslink or breakage of the macro-molecular substances such as lipid, protein and DNA, and finally decreases the fermentation ability of the industrial strain. In this study the inverse metabolic engineering technique based on the theory of "from phenotype to genotype" was employed to identify the key genes related to the oxidative stress response in Saccharomyces cerevisiae BY4741. Firstly, the global transcription machinery engineering (gTME) was employed to elicit S, cerevisiae phenotypes of higher tolerance against oxidative stress caused by H2O2; secondly, the transcriptome sequencing technique (RNA-sequencing, RNA-seq) was used to recognize the differentially expressed genes between the mutant strain and control strain with or without oxidative stress. And finally the Gene Onology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the identified key genes were carried out to further study the response mechanism of the oxidative stress in S. cerevisiae. The main conclusions were as follows:(1) The inducible promoter GAL1in the shuttle vector pYES2was replaced by the constitutive promoter ADH1to reconstruct the new vector pZHW4. The full size of pZHW4was6.14kb, carrying ADH1promoter and CYC1terminator.(2) Error-prone PCR was used to introduce random mutations into the general transcription factor encoding genes SPT15and TAF25in S. cerevisiae. After enzyme digestion and ligation, the recombinant vector pZHW4-sp/15and/or pZHW4-ta/25were constructed successfully and transformed into S. cerevisiae BY4741by electroporation process to generate the mutant libraries, respectively. The libraries capacity containing the mutated transcription factors spt15and/or taf25were about1×105cfu/mL and could be used for large-scale screening of oxidation-stress-tolerant strains.(3) Two strains from two plasmid-based mutagenesis libraries (spt15and taf25), which exhibited significant increases in oxidative stress tolerance, were successfully isolated. At moderate H2O2shock (1.5mM), compared to the control strain, the biomass of the mutant strain spt15-5and taf25-3increased83.3%and50%, respectively, after12h cultivation with1.5mM H2O2treatment.(4) Several mutations were observed in the native transcription factors. For example, five mutation sites, in which serine was substituted for arginine (S136R), and similarly, K138I, R141G, G147R, K167N, respectively, were detected in spt15-5. For taf25-3, seven site mutations resulted in six amino acid replacements, including D106Y, R108Q, Q158R, P160T, K180I, and L188P were also observed.(5) Compared to the control strain, the intracellular reactive oxygen species (ROS) levels in the mutant strains spt15-5and taf25-3were decreased by52.86%and89.36%, respectively. Catalase (CAT) and superoxide dismutase (SOD) activities of the two mutants increased under H2O2stress conditions (for strain spt15-5, CAT and SOD activities increased by54.3%and38.4%, respectively; for strain taf25-3, the two enzyme activities increased by37.9%and67.7%, respectively.).(6) Fermentation experiments revealed that lag phase of the mutant strain taf25-3decreased by12h compared to the control one. Meanwhile, a25.5%increase in ethanol volumetric productivity was also monitored in mutant strain taf25-3.(7) Compared to the control strain, a total of1006genes with significantly differential expression levels were identified in the mutant strain taf25-3upon oxidation stress. Fifteen transcription factor-encoding genes were determined, most of which displayed consistent up-regulated signature expressions in response to the challenge of2mM H2O2. Real-time PCR was employed to validate the expression profiles of the identified key genes. Based on GO and KEGG enrichment analysis, the identified genes were involved in many metabolic pathways including ribosomal synthesis, rRNA metabolism, RNA synthesis, carbon metabolism, fatty acid degradation, peroxisomal, and synthesis of several amino acids, MAP kinase and cAMP-dependent protein kinase A (PKA) signalling pathways.This study enriched the understanding of the molecular mechanisms of oxidative stress response in yeast cells, and provided a theoretical basis for the selection and improvement of S. cerevisiae strains with desired characteristics...