Breeding Of Saccharomyces Pastorianus With Weak Flocculation And Gene Difference Analysis Of Flocculation-differentiated Strains

In the beer fermentation process,the flocculation property of Saccharomyces pastorianus is one of the important factors that determine the high quality production of beer.Strong flocculating yeast is beneficial to the separation and reuse of yeast mud.However,the yeast flocculates in advance during the fermentation process,which will cause the slow or stagnation of fermentation,making the tender beer high in sugar content,and further affecting the beer flavor.In addition,too complete flocculation will also reduce the number of yeasts in the tender beer and affect the secondary fermentation and other post-maturation processes.When Saccharomyces pastorianus L-1 is used in industrial production,there are problems such as short yeast suspension stability period,which leads to incomplete fermentation and affects the final beer flavor quality.Although Saccharomyces pastorianus L-2 with reduced flocculation has been selected and bred in the previous studies through domestication,there are problems such as increased mortality rate of recycled yeast mud.Considering that this phenomenon might be caused by the changed genotype of Saccharomyces pastorianus L-2.Therefore,Saccharomyces pastorianus L-1was used as the starting strain in this study,and the atmospheric pressure room temperature plasma mutagenesis technology(ARTP)was used to mutagenize to select the strain with weak flocculation property.In order to look for the information that is closely related to the phenotypic difference between L-1 and L-2.The whole genome sequencing and analysis of Saccharomyces pastorianus L-1 and L-2 were conducted,The main results of this paper are as follows:(1)A series of laboratory scale systems were used to compare the plate growth morphology and fermentation performance of Saccharomyces pastorianus L-1 and L-2,and L-1 was identified as the starting strain for subsequent mutagenesis and breeding.At the same time,a series of laboratory-scale systems were selected to culture L-1 and L-2,and the flocculation capacity was tested and compared with many flocculation detection methods.Finally,the optimized flocculation detection method was selected as the screening method in the mutagenesis process.(2)Saccharomyces pastorianus L-1 was used as the starting strain and mutagenized using the ARTP technology,and nearly 4000 mutagenic strains are obtained;630 primary screening mutagenic strains are obtained through a plate culture method and a graduated cylinder passage culture method;and 82 secondary screening mutagenic strains with weak flocculation are obtained through a first round of secondary screening by a large test tube fermentation system.The second and third rounds of re-screening were conducted using the large test tube fermentation system and the 1 L graduated cylinder fermentation system respectively,and finally three strains with weakened flocculation and good fermentation performance were obtained,named as 179,293 and 361.Compared with L-1,its flocculation ability was reduced by 6.67%,6.67%,and 5.56%,and the mortality rate was reduced by 31.21%,34.57%,and 16.93%,respectively.The acetaldehyde content in the corresponding fermentation broth was reduced by 19.53%,19.17%,and 15.83%,respectively.Meanwhile,the carbon source consumption,α-amino nitrogen consumption,and alcohol production capacity were all improved,with no significant change in the content of major higher alcohols.(3)The nuclear and mitochondrial genomes of Saccharomyces pastorianus L-1 and L-2 were sequenced and the genome assembly at the chromosome level was completed using Illumina and Pac Bio whole genome sequencing technologies.The results showed that the genome sizes of L-1 and L-2 were about 23.80 Mbp and 23.81 Mbp,respectively,and the number of annotated genes was 10377 and 10365,respectively.Saccharomyces pastorianus L-1 was taken as a reference genome to analyze that variation information in L-2: 2839 SNPs exist in L-2,involving 312 genes,651 In Dels exist,involving 38 genes,64 SVs and involving 4 genes;enrichment analysis was performed on the variation genes and enrichment was conducted to carbon source and nitrogen source metabolism,acetaldehyde reduction,life span regulation,and flocculation function direct or indirect and other metabolic pathways;In addition,seven and one specific genes were found between Saccharomyces pastorianus L-1 and L-2,respectively.Among them,two putative proteins in the specific genes compared in L-1 were respectively speculated to have flocculation function and participated in life span regulation.The reasons for the different phenotypes such as flocculation of Saccharomyces pastorianus L-1 and L-2,metabolic ability of carbon and nitrogen source,mortality,and acetaldehyde reduction ability were theoretically explained at the molecular level.