The Molecular Mechanism Of Response To D-fructose Stress Through Glutathione Metabolism Pathway In Zygosaccharomyces Rouxii

Zygosaccharomyces rouxii is a well-known fermentation yeast.In our previous study,it was interesting that Z.rouxii could produce higher levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone(HDMF)in 120 g/L D-fructose and 180 g/L Na Cl involved YPD medium at 5 d.Under sugar and salt double osmotic pressure,exploring the stress response of Z.rouxii in the early stages of culture and revealing its resistance mechanism indicates important theoretical and applicable research values.To explore the resistance mechanisms of Z.rouxii under D-fructose stress,the physiological and biochemical indexes,the antioxidant enzymes activity,the expression of genes,the glutathione metabolism pathway by multi-omics analysis,and the key enzyme gene bioinformatics of glutathione peroxidase(GSH-Px)were studied.Transcriptomics,proteomics and metabolomics techniques were used to analyze the molecular mechanism of response to D-fructose stress through glutathione metabolism pathway in Z.rouxii.The experimental results are as follows:(1)Results of the physiological and biochemical indexes of Z.rouxii under D-fructose stress: the living cell numbers and cell areas in YPD+Fru groups were significantly lower than in the controls at the initial stage(p<0.01),while those higher than in YPD groups at the late stage(p<0.001).(2)Results of the antioxidant capacity and the expression of antioxidant enzymes genes of Z.rouxii under D-fructose stress: the lipid peroxidation and the relative electrical conductivity of the yeast cell membrane were higher than in YPD groups(p<0.01)during the fermentation periods.And the YPD+Fru group also present lower inhibition superoxide anion ability.The activity of catalase(CAT)in the D-fructose stress group was always higher than that in the control group.Only the activity of GSH-Px was found to be significantly increased at 1 d except for other enzymes activities(p<0.001).GSH-Px gene under D-fructose stress conditions were expressed at higher levels than those of under YPD groups.Combining the levels of transcription and enzymes activity data,those could understand that exogenous D-fructose had a stressing effect on Z.rouxii at the early stage of culture.With the fermentation periods,D-fructose was no longer a stressor substance for the Z.rouxii,and changed to be the nutrient for growth of Z.rouxii in the later stages.During the whole process,GSH-Px was the main defense enzyme andCAT was the sustained defense enzyme.(3)Results of the multi-omics analysis of glutathione metabolism pathway: some gene-encoding enzymes on the glutamate,cysteine and glycine biosynthesis pathways and key genes involved in glutathione synthesis were significantly upregulated(p<0.01).The proteomics expression levels of other genes were consistent with the results of q RT-PCR except Serine hydroxymethyl transferase(SHMT).D-fructose contents gradually decreased with fermentation time and the most important intermediate metabolites,the content of ?-Glutamylcysteine and Glutathione amide,which were always higher than in YPD group.The metabolic flux towards of glutathione synthesis in YPD+Fru groups was increased.(4)Results of GSH-Px bioinformatics analysis: the bioinformatics on the base of the full-length sequence of Z.rouxii GSH-Px gene in NCBI were conducted.The sequence of GSH-Px gene was 624 bp and encoding 207 amino acids.The relative molecular weight of the protein was predicted to be 23120.84 Da.It was a hydrophilic protein,no transmembrane regions,and no signal peptides.In conclusion,the expression of antioxidant enzymes in the oxidative stress systems was enhanced under the D-fructose stress.Through the multi-omics analysis of glutathione metabolism pathway,the key enzyme genes in the pathway are differentially expressed so that increasing glutathione synthesis to resistance oxidative stress.