Regulation Of Glucose Metabolism On Oxidative Stress Tolerance In Saccharomyces Cerevisiae

Saccharomyces cerevisiae strain has been widely used in industrial fermentation because of its clear genetic background,short growth cycle and superior growth adaptability.However,during fermentation progress,S.cerevisiae is often subjected to a variety of stresses that lead to oxidative cellular damage,which in turn significantly reduces product yield.As one of the most important carbon metabolic pathways in S.cerevisiae cells,the glycolytic pathway could regulate several processes such as cell growth,development,and production of metabolites.At present,it is not clear whether the strength of glycolytic metabolic activity could regulate the tolerance of S.cerevisiae cell and the specific regulatory mechanism.This work assessed cellular stress tolerance,especially oxidative tolerance,by attenuating the glycolytic metabolic activity of S.cerevisiae,and elucidated the metabolic pathways that might be involved in its regulation.The main findings are as follows.1.Knockdown of HXK1,ENO1,TDH1,PFK1 and FBP1,key genes of the glycolytic metabolic pathway,respectively,using CRISPR/Cas technology,to reduce S.cerevisiae glycolytic metabolic activity,and then to assess the regulatory effects of knockdown on cellular stress tolerance and ROS content.The results indicated that the S.cerevisiae strain with deletion of PFK1 showed the most significant changes,with reduced ROS content and increased oxidative tolerance.Analysis of the expression of genes related to the oxidative metabolic pathway in wild strain and △PFK1 mutant strain using q RT-PCR technology.The results suggested that knocking out PFK1 achieved a reduction in intracellular ROS content mainly by inhibiting ROS production.Meanwhile,PFK1 knockdown promoted SOD enzyme expression,which alleviated the extent of intracellular ROS accumulation to some extent.2.To further understand the overall regulation of PFK1 in S.cerevisiae cells,transcriptomic analysis of wild strain and △ PFK1 mutant strain was performed using a transcriptomic strategy.The results showed that the two strains expressed a total of 40 differentially expressed genes,including 15 upregulated genes and 25 down-regulated genes;they mainly focused on metabolic pathways such as amino acid biosynthesis,cell cycle,and signal transduction processes.Eighteen differentially expressed genes were selected and the accuracy of the transcriptome data was verified using q RT-PCR,and the results showed that the results of the data from the histological analysis were reliable.3.Based on the results of transcriptomic analysis,the VTC1 gene,which is related to vesicle as well as autophagic functions,was selected and the regulation of VTC1 on cellular oxidative tolerance was verified;the results revealed that the deletion of VTC1 gene could reduce the oxidative tolerance of yeast cells.The above results suggested that PFK1 knockdown can upregulate VTC1 gene expression,thus enhancing vesicular H-ATPase activity to provide more energy,as well as enhancing cellular autophagy,and further improving oxidative tolerance in S.cerevisiae.