Effects Of Overexpression ? And ? Subunits Coding Genes Of Snf1 Protein Kinase In Baker's Yeast On Its Freezing Tolerance

Frozen dough technology can effectively prolong the shelf life of bread to ensure freshness,which is widely used and gradually replaced the traditional bread production.The activity of baker's yeast after freezing stress is the main factor affecting the quality of frozen dough.Yeast Snf1 protein kinase is an important regulatory protein in the regulation of intracellular energy metabolism and external stress response,but its regulatory effect and pathway under freezing stress have not been reported.In this study,baker's yeast ABY3(Saccharomyces cerevisiae)was used as the research object to explore the effects of overexpression of Snf1 protein kinase ? and ? subunit coding genes SNF1,SIP1,SIP2 and GAL83 on its freezing tolerance for the first time,which provided a new idea for the breeding of industrial baker's yeast.(1)The main results are as follows: the mutant strain(ABY+S)with the overexpression of SNF1 coding gene of Snf1 protein kinase ? subunit was constructed,and the growth performance,freezing tolerance and fermentation performance of the mutant strain(ABY+S1,ABY+S2,ABY+G)with the overexpression of Snf1 protein kinase ?subunit coding genes SIP1,SIP2 and GAL83 were tested.The results showed that the overexpression of Snf1 protein kinase ? subunit coding genes SIP1,SIP2 and GAL83 had negative regulation on the freezing tolerance of baker's yeast,and the survival rate of the mutant strain was much lower than that of its parent strain under freezing stress.The overexpression of Snf1 protein kinase ? subunit coding gene SNF1 had a positive regulatory effect on the freezing tolerance of baker's yeast,and the survival rate of its mutant strain ABY+S under freezing stress was much higher than that of its parent strain43%.In the unsweetened dough fermentation before freezing,the fermentation ability of the mutant strain ABY+S was improved but not significantly,while in the unsweetened dough fermentation after freezing stress,the fermentation power of ABY+S increased8 m L/ h ·g compared with the parent ABY3,and the relative fermentation power increased by 11%.(2)Transcriptome analysis,a total of 1799 genes showed significant differences between ABY+S and the parent ABY3 before freezing,of which 1453 genes were up-regulated and 346 down-regulated.After freezing,a total of 2308 genes were significantly different between ABY+S and its parent ABY3,of which 2035 genes were up-regulated and 273 down-regulated.GO enrichment analysis showed that overexpression of Snf1 protein kinase ? subunit coding gene SNF1 made baker's yeast express a large number of genes related to cell composition to cope with stress environment;at the same time,a large number of gene expressions related to growth and metabolism regulation provided a transcriptional basis for the fermentation ability of mutants superior to their parents.In addition,the expression of genes related to cell stress response was consistent with the strong freezing tolerance phenotype of mutants.KEGG enrichment analysis showed that the overexpression of Snf1 protein kinase ? subunit coding gene SNF1 had the most significant effect on the proteasome of baker's yeast.The mutant strain may enhance the degradation efficiency of misfolded protein under stress by regulating the expression of proteasome-related genes,realize the supervision of cell quality,and ensure that the cells still maintain high growth and metabolic activity after freezing injury.In addition,the regulation of SNF1 gene overexpression on yeast carbon metabolism pathway,amino acid biosynthesis pathway,glycerol metabolism pathway and glycerol phospholipid metabolism pathway is also the reason for the high survival rate and strong fermentation performance of ABY+S.(3)Non-targeted metabolites analysis showed that there were 17 intracellular metabolites significantly different between ABY+S and its parent ABY3 before freezing,of which 10 were up-regulated and 7 down-regulated.After freezing,a total of 21 intracellular metabolites were significantly different between ABY+S and its parent ABY3,of which 11 were up-regulated and 10 down-regulated.Before freezing,consistent with the transcriptome data,the mutant strain with overexpression of SNF1 gene accumulated a large amount of amino acids and glycerol,and the composition of cell membrane changed at the same time.Whether the change of cell membrane composition before freezing can make the cell resist the damage of freezing stress remains to be studied,but the accumulation of high content of amino acids and glycerol in the cell is the main reason for the high survival rate of the mutant strain under freezing stress.After freezing stress,the mutant strain with overexpression of SNF1 gene still maintained higher proline and glycerol content than its parents,so that the cells were better protected under freezing stress.Other differential metabolites were significantly down-regulated,which is the result of the strong carbon metabolism and protein metabolism regulation of mutants;in addition,whether the down-regulation of intracellular hemolytic phospholipids is related to low temperature adaptation needs further study.In this study,the effects of overexpression of Snf1 protein kinase ? and ? subunit coding genes SNF1,SIP1,SIP2 and GAL83 on the freezing tolerance of baker's yeast were analyzed for the first time through the method of system biology,so as to provide theoretical guidance for the breeding of excellent industrial strains and provide new ideas for the development of frozen dough technology.at the same time,it can supplement the basic theory of yeast environmental stress response and improve the global regulation mechanism of Snf1 protein kinase.