Functional Analysis Of A New Gene Of Saccharomyces Cerevisiae Rice Wine Strain And Screening Of Yeast Strains With Low Yields Of Urea

Saccharomyces cerevisiae is the main microorganism of rice wine fermentation and also is the key factor affecting the quality of rice wine.However,compared with strains from Japan sake and European and American wines,few studies have been reported on Saccharomyces cerevisiae from yellow wine,especially on gene function.Yellow rice wine is an unique ancient wine in China,with a long history.Yellow rice wine yeast has undergone continuous evolution and had been adapted to the yellow rice wine fermentation environment,forming its unique fermentation advantages.A haploid rice wine brewing strain D2 with high alcohol tolerance was screened in the laboratory.Bioinformatics analysis was used to compare it with the model experiment strains S288 c at the genome level difference,and found that the protein encoded by g5170 gene contains an amidase,which can catalyzes the hydrolysis of an amide group(CO-NH2)to generate a carboxylic acid,amine or ammonia.These results indicated that the gene may play an important role for the growth and metabolism of yellow wine yeast strains.The new gene has been functionally studied and verified in the laboratory,and it was found that the tolerance to ethanol decreased after D2 knockout of the new gene g5170,so the new gene may be related to the tolerance to ethanol.In this experiment,the new gene g5170 was introduced into model yeast BY4741 for expression through vector construction,and the expression was verified by RT-PCR experiment.In the ethanol tolerance experiment,it was found that the tolerance to ethanol and growth rate of the recombinant yeast strain were improved,which further verified that the new gene g5170 may be related to ethanol tolerance.In order to further develop excellent yeast strains of yellow rice wine and improve the quality of yellow rice wine,this study screened the low-production urea strain of yeast strains containing g5170 gene in the early stage,so as to reduce EC(ethyl carbamate)content in yellow rice wine.In the screening work,arginine,the main substrate produced by urea during the fermentation,was used as the only amino acid in this experiment,and the low urea producing yeast strain was screened through the analysis of growth kinetics,alcohol content,urea content,reducing sugar content,arginine content and the expression of CAR1,a urea gene.The results showed that there was a positive correlation between CAR1 expression and urea content,and the correlation coefficient was 0.874.Finally,two relatively excellent low urea production yeast strains D2 and KN2 were obtained,and the two yeast strains were used for the laboratory simulated fermentation detection,and the urea content was relatively low and there was a significant difference with strain N85(p=0.0078).To sum up,in this paper,the ethanol resistance function of yeast gene g5170 and the selection and breeding of yeast strains with excellent low urea production in rice wine were studied.Finally,D2 and KN2 strains with better performance in ethanol resistance and low urea production were screened out.However,the specific mechanism of its performance still needs further study.