Mining Of Acid Tolerance Genes And Construction Of High Acid Tolerance Strains Of Schizosaccharomyces Pombe

In the brewing process of Baijiu,the high organic acid content can inhibit the growth of yeast and other microorganisms,resulting in insufficient fermentation power and poor utilization of raw materials.Therefore,the selection and breeding of yeast strains with high acid resistance are of great significance to the production of Baijiu industry.A SP.65（Schizosaccharomyces pombe）with good acid resistance was screened in the previous research,but the molecular mechanism of its response to acid stress is not clear.In this study,the acid tolerant strain SP.65 and model strain ATCC16979 were compared and analyzed under mixed acid stress containing 6 g/L acetic acid and 30 g/L lactic acid by global transcriptome sequencing.Among them,7 differential genes may greatly affect the acid tolerance of yeast;These genes were knocked out and overexpressed by genetic engineering using SP.65 as the starting strain to verify their roles in acid tolerance of S.pombe,and six S.pombe strains with high acid tolerance were constructed.The adaptive response of S.pombe to organic acids was initially explored,which laid the foundation for the subsequent construction of high acid tolerant yeast strains.The main research contents are as follows:（1）Using transcriptome sequencing,95 upregulated genes with significant difference,shared by SP.65 and the model strain ATCC16979 under mixed acid stress,were identified,and further through GO-BP enrichment analysis,the trx1,tpx1,pmp20,gpm1 genes related to redox process were found that may be related to the acid tolerance performance of the strains.Based on the perspective of strain evolution,the differential genes between the model strain ATCC16979 and the acid tolerant strain SP.65 after mixed acid stress treatment were comparatively analyzed,and results showed that 225 genes were upregulated in SP.65,and nucleoside metabolic process related genes pgk1,sam1,SPBC8D2.18C were found by GO-BP enrichment analysis that may be related to the acid tolerance property of the strains.Therefore,7 differential genes were considered as candidate genes for subsequent molecular manipulation.（2）In order to explore the effect of differential genes on the acid tolerance of S.pombe,differential genes single knockout strains were constructed by genetic engineering with SP.65as the starting strain.Through acid tolerance test and growth curve determination,it was found that the final OD₆₀₀ values of recombinant strains with trx1,tpx1,pmp20,gpm1,pgk1,sam1 and SPBC8D2.18C single gene-knockout were decreased by 44.8%,21%,9%,10.2%,12.2%,28.6%and 26.3%,respectively,compared with the starting strain under 16 g/L acetic acid stress.Compared with the starting strain under the condition of 60 g/L lactic acid,the final OD₆₀₀ values of recombinant strains with trx1,tpx1,pgk1 and SPBC8D2.18C single gene-knockout were decreased by 8.2%,12.4%,13.9%and 22.1%,respectively.The above results showed that the differential genes were closely related to the acid tolerance of S.pombe.（3）In order to construct S.pombe with high acid resistance,differential genes overexpression strain were constructed by genetic engineering with SP.65 as the starting strains.Through acid tolerance test and growth curve determination,it was found that gene trx1,tpx1,pmp20,gpm1,pgk1 and SPBC8D2.18C overexpression improved the acetic acid and lactic acid resistance of SP.65 strain,while overexpression of sam1 decreased the acid resistance of the strain.Further,the fermentation performance of the overexpressed recombinant strain was verified by simulated fermentation experiment.The results showed that under the conditions of 35 g/kg lactic acid and 8 g/kg acetic acid,the fermentation performance of SP.65::SPBC8D2.18C was the best.The fermentation basically ended at 148h,and the final ethanol yield was 32.66 g/L,which was 1.59 times higher than that of the starting strain（20.52 g/L）.In conclusion,the over expression of acid resistant genes can promote the fermentation level of Baijiu liquor under acidic conditions.