Physiological Mechanism Of Fatty Acid Elongase Elo2 In Resistance To Salt Stress In Saccharomyces Cerevisiae

During the industrial fermentation process,industrial strains often encounter various environmental stresses,which could limit performance of srains.To strengthen resistence of industrial strains to salt stress,Saccharomyces cerevisiae BY4741 was chosen as the target strain,adaptive laboratory evolution strategy was applied to improve resistence of Saccharomyces cerevisiae to salt stress,and transcriptomics was used to analyze the potential mechanism of salt stress resistence.The physiological mechanism of Elo2 in resistance to salt stress was studied with the analytical method of liquid chromatography-mass spectrometry and flow cytometry.The main results were described as follows:1.Salt stress resistance strains were selected through adaptive laboratory evolution,and key salt stress resistance pathways were analyzed using transcriptome.The strain XCG001 that can resist to salt stress was obtained by adaptive laboratory evolution.The results showed that the cell concentration of the strain XCG001 increased by 37.3%at 1.5 M NaCl,compared with the wild type strain;the semi-inhibitory concentration of NaCl increased by 55.5%.Further the results of transcriptome data found that glycolysis/gluconeogenesis,pyruvate metabolism,lipid metabolism,signal transduction,fructose and mannose metabolism pathways were up-regulated in strain XCG001,whereas ribosomal and amino acid metabolism pathways were down-regulated;at 1.5 M NaCl,pyrimidine metabolism and lipid metabolism pathways were up-regulated,whereas pyruvate metabolism and transport pathways were down-regulated.2.Overexpression of Elo2 was found to resist salt stress,and its ability to resist salt stress was tested.The most significant up-regulated genes were overexpressed,and it was found that the Elo2 high-expressed strain（XCG010）increased tolerance to salt stress.Elo2 was over-expressed with the two other strong promoters to obtain medium-expressed strain（XCG017）and low-expressed strain（XCG016）.The results showed that the growth of XCG016,XCG017and XCG010 enhanced at 1.0 M NaCl,compared with the control strain XCG002;the semi-inhibitory concentration of NaCl all increased by 33.3%;the cell concentration increased by21.9%,20.8%,and 19.1%,respectively;the survival rate increased by 22.1%,20.8%,and18.0%,respectively.3.The mechanism of Elo2 resisting salt stress was analyzed.The effects of overexpression of Elo2 on cell membrane components and functions,and how Elo2 in response to salt stress were analyzed.Overexpression of Elo2 reconstituted the cell membrane sphingolipid component.At 1.0 M NaCl,compared with the control strain XCG002,the content of C20:0,C22:0 and C24:0 in strain XCG010 increased by 33.1%,106.4%and 31.5%,respectively;the content of PE and PS increased by 18.9%and 15.0%,respectively;the content of IPC（t18:0/26:0）,MIPC（t18:0/22:0（2OH））,MIPC（d18:0/22:0）,MIPC（d20:0/24:0）,M（IP）₂C（d20:0/26:0）,M（IP）₂C（t18:0/26:0（2OH））and M（IP）₂C（d20:0/26:0（2OH））increased by8833.4%,16689.4%,6329.2%,2391.1%,2792.8%,11376.4%and 20806.3%,respectively.Then,it was verified that Elo2 in response to salt stress by enhancing sphingolipid synthesis.It was found that the transcription level of the synthetic sphingolipid in the strain XCG010 was increased by RT-PCR detection.Ceramide synthetase coding gene LAC1 was knocked out in the strain XCG010 to obtain a mutant XCG018,and the growth ability and survival rate were tested.At 1.0 M NaCl,compared with the strain XCG002,the mutant XCG018 growth enhanced;the survival rate increased by 10.2%.Overexpression of Elo2 altered cell membrane function.At 1.0 M NaCl,compared with the control strain XCG002,the cell membrane integrity of the strain XCG010 and mutant XCG018 were increased by 24.4%and 11.2%,respectively.