Enhancement Of Saccharomyces Cerevisiae Intracellular CAMP Leveland Tolerance Based On Cyclic Adenylate Signaling Pathway

Cyclic adenosine monophosphate(cAMP)plays a very important role in the life activities of living organisms.In this study,the industrial Saccharomyces cerevisiae was selected as the starting strain to investigate the changes of intracellular cAMP level and regulate its tolerance by regulating the activities of adenylate cyclase Cyr1 and phosphodiesterases in cyclic adenylate signaling pathway.The aim of this study was to investigate the effects of genes in the cyclic adenylate signaling pathway in regulating the intracellular cAMP level and tolerance of Saccharomyces cerevisiae.The research contents and results are as follows:(1)With Saccharomyces cerevisiae AY12 a and AY12α as the starting strains,and Kan MX resistance genes as the screening marker genes,the genes PDE1 and PDE2 were knocked out by intracellular homologous recombination method.Four strains were successfully constructed,respectively A1(AY12a-ΔPDE1),A2(AY12a-ΔPDE2),B1(AY12α-ΔPDE1)and B2(AY12α-ΔPDE2).The genes RAS2,GPA2 and CYR1 were overexpressed using the strong promoter PGK1 p and terminator PGK1 t at Gal80 locus of the departure strains,respectively.Six strains were successfully constructed,respectively A3(AY12a-RAS2),A4(AY12a-GPA2),A5(AY12a-CYR1),B3(AY12α-RAS2),B4(AY12α-GPA2)and B5(AY12α-CYR1).The above recombinant strains were hybridized and fused by cell fusion method.Ten strains were constructed,respectively A6(AY12a-ΔPDE1-RAS2),A7(AY12a-ΔPDE2-RAS2),A8(AY12a-ΔPDE1-GPA2),A9(AY12a-ΔPDE2-GPA2),A10(AY12a-ΔPDE1-CYR1),B6(AY12α-ΔPDE1-RAS2),B7(AY12α-ΔPDE2-RAS2),B8(AY12α-ΔPDE1-GPA2),B9(AY12α-ΔPDE2-GPA2)and B10(AY12α-ΔPDE1-CYR1).The 24 hours growth curve of the strains was measured,and no significant difference was found in the growth performance between the modified strains and the starting strains.(2)High performance liquid chromatography was used to measure the intracellular cAMP level of the modified strains.The experimental results showed that the intracellular cAMP level of modified strains A8 and A9 increased by 1.08 times and 0.37 times compared with the starting strain A,respectively.The intracellular cAMP level of modified strains B8 and B9 increased by 0.7 times and 0.3 times compared with the starting strain B,respectively.The results show that the overexpression of the gene GPA2 on the basis of knocking out the gene PDE1 will significantly increase the intracellular cAMP level of Saccharomyces cerevisiae.(3)The growth tolerance of the modified strains was determined by point plate experiment,and the starting strains and the modified strains were placed in the stress environment of 0.4% acetic acid,5% lactic acid,10% ethanol and 8% Na Cl.The experimental results showed: the acetic acid tolerance of strains A8 and A10 were improved compared with the starting strains,but the salt tolerance of strain A10 was decreased.The growth capacity of other modified strains showed an unchanged or decreased trend in different stress environments compared with the starting strains.The results show that overexpression of the gene GPA2 or CYR1 on the basis of knocking out the gene PDE1 will improve the acetic acid tolerance of Saccharomyces cerevisiae AY12a.