Research On The Construction And Application Of Saccharomyces Cerevisiae Strains For Low-production Of Higher Alcohols

Higher alcohols play an important part in the flavor of yellow rice wine.Higher levels of higher alcohols can lead to unpleasant tastes in wine and strong intoxication.In order to reduce the content of higher alcohols in yellow rice wine,the aldehydes dehydrogenase encoding gene ALD6 was overexpressed by terminators with different intensities,and a series of low-producing higher alcohols strains were constructed and optimized for fermentation.The main research contents and results are as follows:(1)Research the effect of different intensity terminators on gene expression levels.Using the constitutive strong promoter PGK1p and eight different terminators GIC1T,CYC1T,PGK1T,AHC2T,MSH3T,RPL41BT,ATG10T and CPS1? over-express the aldehyde dehydrogenase encoding gene ALD6,screened recombinant strains and detecting them by RT-PCR.At the level of gene transcription,the intensities of these eight terminators were found to be GIC1T<CYC1T<PGK1T<AHC2T<MSH3T<RPL41BT<ATG10T<CPS1T.Eight recombinant strains were fermented with yellow rice wine.The results showed that the increasing trend of acetic acid content and the descending trend of higher alcohols were positively correlated with the terminator strength.Finally,three intensity terminators GIC1T,PGK1T and CPS1T were selected to achieve gradient overexpression of ALD6 gene.(2)Constructed a series of low-production higher alcohols yeast strains.Compared with acetic acid tolerance,lactic acid tolerance,high temperature tolerance,growth performance,and fermentation rate of two Saccharomyces cerevisiae strains HJ-1 and AY12.HJ-1 was eventually selected as the original strain.Separated the haploids strains,and the better haploid strains al and al7 were selected as the original strains of transformants.Overexpressing the ALD6 gene in the haploid al and al7 of Saccharomyces cerevisiae strain HJ-1 used the strong promoter PGK1p and the terminators GIC1T,PGK1T,CPS1T with the scarless knockout system,respectively,meanwhile,deleted the branched-chain amino acid transaminase coding gene BAT2.The a-type recombinant strains alAB-AG,alAB-AP,?l?B-AC and a-type recombinant strains a17?B-AG,a17?B-AP and a17?B-AC were screened.The haplotype recombinant strains carrying the same terminator were fused to diploid recombinant strains HJ?B-AG,HJAB-AP and HJAB-AC.The diploid recombinant strains and the starting strain were simultaneously fermented with yellow rice wine.The fermentation results showed that the acetic acid contents of the recombinant strains HJAB-AG,HJAB-AP and HJAB-AC were 1.26-,1.84-and 2.51-folds compared with the original strain HJ-1,respectively.The lactic acid content in fermentation liquid were 1.23-,1.23-,and 1.26-folds compared with the starting strain HJ-1,respectively.The higher alcohol production was reduced to 39.91%,45.55%and 52.80%,respectively.(3)To improve the alcohol content and decrease the contents of lactic acid and higher alcohols,preliminary optimize the yellow rice wine fermentation process.The best fermentation conditions of yellow rice wine fermentation was that yeast inoculation quantity was 1.4 × 107 cell/g,fermentation time was 6 days,fermentation temperature was 33 ? and the Daqu addition amount was 12 g/100 g raw materials.After yellow rice wine fermentation,the alcohol content was basically the same as the original strain HJ-1 and the higher alcohols content was reduced by 55.87%compared to the original strain HJ-1.The lactic acid content in the fermentation liquid was reduced by 16.64%after optimizing the fermentation conditions.