Analysis Of Serine Biosysthesis Pathway In The H₂S Overflow Metabolism Of Saccharomyces Cerevisivae

Hydrogen sulfide（H₂S）as an important volatile sulfur compound,imparting an unpleasant odour（rotten eggs）at low perception threshold,which is the major concern encountered in wine industry.The formation of H₂S resulting from sulfur metabolism depend on the rate of S^2-synthesis and depletion.As a key node in the downstream S^2-metabolism,serine plays an important role in S^2-depletion and H₂S production in S.cerevisivae.Therefore,to dissect the role of serine biosyethesis pathway in the H₂S overflow in S.cerevisivae will provide practical foundation for H₂S control in wine industry.The main results and conclusions are as followed:（1）To investigate the expression level of SER33（encoded phosphoglycerate dehydrogenase,EC 1.1.1.95）on H₂S production,which is a key gene in serine biosynthesis pathway,high H₂S production strain 32y12 was used to construct three strains with gradient expressions of SER33 by plasmid pY16（with promoters of TEF）and pY26（with promoters of TEF,GPD）.As the results demonstrated,the expression level of SER33 in three constructed strains significantly increased to 82,272 and 500 fold,compared with control during the middle of fermentation.Meanwhile,compared with the control（375.00 ppm）,the production of hydrogen sulfide greatly accumulated with SER33 overexpressed,especially increased to 776.19 ppm,631.75 ppm,621.43ppm,respectively.However,the production of hydrogen sulfide showed no significant difference among the strains with SER33 gradient overexpressed.The result provided new direction for deepen analysis of the physiological functions in the hydrogen sulfide metabolism of S.cereviviae.（2）Strains with SER1/SER2/SER33 overexpression and deletion were constructed to study the influence of serine biosynthesis pathway on sulfur metabolism.The results showed that no significant difference of H₂S was shown in strains with elevated SER1/SER2 compared to the control,whereas the significant amount of H₂S increased in strain with elevated SER33.As expected,SER33 deletion significantly decresed H₂S release during fermentation.In addition,we studied the time course expression,at the transcriptomic level,of genes involved in the sulfate assimilation by analyzing the genes related to sulfur metabolism,and correlated transcriptomic differences with metabolites production during fermentation.The main results demonstrated that:1)The overexpression of SER1/SER2/SER33 could significantly increase intracellular amino metabolites（including sulfur metabolites）,especially in the the SER2overexpressed strain.2)MET17(control S^2-to synthesis homecysteine)downregulated in SER33 overexpression strain,which also caused block in homocysteine before the late of fermentation.This may be possibly responsible for high H₂S release in SER33 overexpression strain.（3）To investigate the influence of serine on H₂S production in S.cereviviae during fermentation,high H₂S production strain 32y12 was used to compare their H₂S releases by gradient exogenous serine under 300 N mg/L and 150 N mg/L yeast assimilation nitrogen concentration.As the results demonstrated,compared with the nitrogen concentration of 300N mg/L,exogenous serine addition under nitrogen concentration of 150 N mg/L could significantly increase H₂S production with 5 fold（140.7 mg/L）and 10 fold（281.5 mg/L）serine addition,respectively.Moreover,strains with SER33 overexpression and deletion were used for further investigate the influence of de novo serine biosynthesis on H₂S production in S.cereviviae.The results showed H₂S production in SER33 overexpression strain reduced with exogenous serine addition under the nitrogen concentration of 300 N mg/L,whereas under 150 N mg/L nitrogen concentration,its H₂S production increased about 114%-212%with exogenous serine addition.For SER33 deletion strain,its H₂S production decreased under all nitrogen conditions.In addition,compared with its initial strain S.cereviviae 32y12,exogenous serine addition under 150 N mg/L could not increase H₂S production.The results provided support for research on serine availability in yeast（de novo synthesis and exogenous addition）and its utilization.