Identification And Functional Analysis Of Genes In Hydrogen Sulfide Metabolisim In Saccharomyces Cerevisiae

The production hydrogen sulfide?H₂S?during fermentation in wine industry deteriorate the quality of wine due to its off-flavors.The characters of H₂ S production show significant differences between individual strains.Therefore,it is necessary to comprehend the mechanism of H₂ S production,in order to deal with danger caused by H₂ S releases in industry.In this study we choose MET17/CYS4/CYS3 and THI5 gene family,had relationship with cofactor VB6,as key gene and analysis their capability in H₂ S over release,according to results of RNA-sequence and resequencing data.The main results were as followed:1.According to previous results of RNA-sequence in different strains,when comparing with high and low H₂ S production,32y12 and 112y4,respectively:?1?The gene family of THI5/11/12/13 showed significantly differences in high H₂ S production strain,illustrating cofactor VB6 as the key factor responsible for high H₂ S production;?2?Though the transcription level of MET17/CYS3/CYS4 did not change significantly,the enzyme encoded by MET17/CYS3/CYS4 were VB6-dependent according to sulfur metabolism pathway,and it remained uncovered relationship between VB6 and its function,also the high H₂ S production phenotype.2.Combined the advantage of low and high-H₂ S production strain and used 112y4 as parent strain,we isolated a haploid strain 112y4-25 of non-H₂ S production,low-yielding of higher alcohols and high yielding of ethyl substances.A reusable deletion method for construction of yeast auxotrophic mutants with non-resistant selective marker was successfully applied to construct single auxotrophic mutants??ura3?DU-3 as well as double auxotrophic mutants??ura3?leu2?DU-3 in 32y12.This result provided a genetic manipulation platform for further analysis of the physiological functions in the H₂ S metabolism of S.cereviviae.3.The results of controlling expression level of MET17/CYS4/CYS3 and VB6 as their coenzyme factor at different concentration revealed that high production of H₂ S barely related to the concentration of VB6,therefore,pointed the difference of H₂ S phenotype probably concerned with VB6 synthesis capacity.4.CRISPR-Cas9 was applied successfully to delete gene family of THI5,the first diploid of THI5 family knockout.