Breeding Of High-gravity Brewing Yeast And Its Sugar Utilization Mechanism

High-gravity brewing,a common beer production technique that increases yields while decreasing costs,is widely used in lager beer production.However,high-gravity wort would affect the sugar utilization of yeast,causing the slower fermentation rate and the lower fermentation attenuation.Therefore,the breeding of strains fit for high-gravity brewing is critical.In this study,genome shuffling was applied to a lager strain to obtain a mutant strain with improved fermentation rate and attenuation.The related indexes of the original strain and mutant strains at protein,transcriptional,and gene levels were measured during high-gravity brewing to investigate the sugar utilization mechanism of lager yeast.Current study proposed theoretical guidance for screening advantage strains for high-gravity brewing.The main findings are as follows:1.Genome shuffling was applied to a lager yeast G03,and F2-123 with improved fermentation performance in high-gravity brewing was obtained.Strains were fermented in24°P wort at 11°C.The results showed that the alcohol content and real fermentation attenuation of beer fermented with F2-123 were 10.45%(v·v^-1)and 63.00%,which were 8.85%and 6.51%higher than those of beer fermented with G03,respectively.The fermentation cycle of F2-123 beer was 11 days,which was 1 day shorter than that of G03 beer.Meanwhile,the flavor characteristics of F2-123 beer did not change much.Moreover,genetic stability test showed F2-123 kept its performance capability after 45 generations’culture in high-gravity wort.2.Theα-glucoside transport activity andα-glucosidase activity of gene-shuffled strains and G03 were examined during high-gravity brewing.The significantly higher activity ofα-glucoside transporter might be the reason for the improved fermentation performance of gene-shuffled strains.Among them,F2-123 showed the highestα-glucoside transporter activity,which was 34.24%-194.05%higher than that of G03 throughout the fermentation process.The expression levels and sequences of genes encodingα-glucoside transporter（MALT）andα-glucosidase（MALS）were analyzed in these strains.The results showed that there were no mutations occurred in these genes.However,the expression levels of these genes changed during high-gravity brewing.The up-regulation of MALx1,MTY1,and Se AGT1 increased the activity ofα-glucoside transporter.Meanwhile,the MALx1,Se AGT1,and MTY1 were up-regulated sequentially along with the fermentation.3.MALx1,MTY1 and Se AGT1 genes were disrupted and overexpressed in G03,respectively.The maltose and maltotriose utilization-related indexes of the recombinant strains were measured during high-gravity brewing.The results showed that expressions of other MALT and MALS family genes were down-regulation dramatically in gene-disruption strains,resulting in the lower activities ofα-glucoside transporter andα-glucosidase of these strains,and finally caused poor fermentation ability of these strains.Overexpression of MALx1improved the activity ofα-glucoside transporter at the lag phase of beer fermentation.On the contrary,overexpression of MTY1 improved the activity ofα-glucoside transport activity at the late stage of beer fermentation.However,in the gene-overexpression strains,other MALT family genes were down-regulated,therefore,no improvements were found in the fermentation performance of gene-overexpression strains.