Analysis Of The Physiological Functions Of Methionine In The H2s Overflow Metabolism Of Saccharomyces Cerevisivae

Hydrogen sulfide?H₂S?imparting an unpleasant odour?rotten eggs?at low olfactory threshold,is an important volatile sulfur compound,and can affect the sensory quality of wine.The formation of H₂S resultes from Saccharomyces cerevisiae,thus the research on the regulation mechanism of sulfur metabolism of S.cerevisiaeis is valuable for H₂S control in wine industry.Previous studies has showed that the addition of methionine could inhibit H₂S release.Therefore,to dissect the role of methionine biosyethesis pathway in the H₂S production,a strain S.cerevisiae 32y12 with high yield of H₂S was used to analyse intracellular amino acid metabolites by overexpressing key genes involved in methionine synthesis pathway.The main results are as follows:?1?Strain S.cerevisiae 32y12 was used for alcoholic fermentation under gradient concentrations of methionine and aspartic acid.The result showed that the growth and fermentation characteristics of the strain did not affect by the exogenous addition of methionine and aspartic acid.However,the production of H₂S greatly reduced from 217 ppm to 2.63 ppm and 0.17 ppm when added 119.7 mg/L and 76.8 mg/L methionine,repectively,which suggested that the exogenous addition of methionine could significantly reduce the H₂S production of S.cerevisiae 32y12.?2?Eight strains with overexpression of key genes?HOM3/2/6,ACS1/2,MET2/17/6?which is involved in the methionine synthesis pathway,were successfully constructed in strain32y12?ura3 by plasmid pY26.The fermentation and H₂S-producing characteristics of recombinant strains were evaluated.The results showed that MET2,MET17 and HOM3overexpressing strains had very low or no H₂S production compared to the control strains?strain with empty pY26 plasmid?.Specifically,the H₂S production of the control strain was217 ppm,while the MET2,MET17 and HOM3 overexpressing strains was 0.87 ppm,29 ppm and 5.26 ppm,respectively.ACS1 overexpressing strains exhibited low H₂S production characteristic with a yield of 141 ppm,however,HOM2 overexpressing strain exhibited a high and unstable H₂S production characteristic with an average yield of 324 ppm.There was no differences in H₂S production between MET6,HOM6,ACS2 overexpressing strains and the control strain,which the yield achieved to 171 ppm,220 ppm and 207 ppm,respectively.Although genes both encode the acetyl-CoA synthetase,ACS1 overexpression strain always produced lower H₂S than the ACS2 overexpression strain.Moreover,the results of physical and chemical indexes of the simulated wine showed that the fermentation characteristics of strain 32y12 were not affected by the overexpression genes in methionine biosynethsis pathway.?3?The amino acid metabolites of the MET2,MET17 and HOM3 overexpression strains with low-yield H₂S were analysed at the initial,exuberant and the late stage of H₂S release process.The results showed that the most sulfur-containing amino acids in S.cerevisiae were accumulated during the process of H₂S production.Moreover,there was a significant difference between the overexpression strains and the control strain in amino acid metabolites by OPLS-DA analysis method,which suggested that the overexpression strains may cause the low H₂S production of yeast by facilating the synthesis of sulfur-containing amino acids in the downstream of sulfur metabolism pathway.