| Saccharomyces cerevisiae is an important industrial host strain for heterologous gene expression.It has the advantages of complete expression system,simple culture and convenient gene manipulation.?-estradiol inducible promoter subsystem is a completely exogenous regulatory element for Saccharomyces cerevisiae,and its non-target transcription effect is very few.It can control gene expression conveniently and provide great experimental flexibility for relevant research of Saccharomyces cerevisiae.Neuric acid is the core component of nerve cells in brain and nerve tissue.It is effective in treating psychosis,peroxisome diseases,diabetes,AIDS and other diseases.However,limited sources limit the large-scale production and application of neuroacids.Therefore,it is necessary to explore the methods of biotransformation and genetically modified biosynthesis to improve the ability of Saccharomyces cerevisiae to synthesize neuroacids in order to meet the market demand.In this paper,we designed and synthesized a general molecular biology tool,?-estradiol-inducible promoter,which was not inhibited by glucose,as a metabolic regulatory element,using nutritionally deficient Saccharomyces cerevisiae YS58 as the starting strain,and applied it to the regulation of neuroacid synthesis genes.1.The inducible promoter subsystem of beta-estradiol was successfully constructed.The recombinant plasmid vector system(pRS416-PReGall-GFP-TCYc,YEplac112-PTEF1-Aritificial transativator-TCYC)was successfully constructed using pRS416 and YEplac112 as starting plasmids in Saccharomyces cerevisiae YS58.2.The induction conditions of inducible promoter of beta-estradiol were optimized.The detection conditions were as follows:the expression of GFP was detected by 475 nm excitation light and 509 nm emission light in the enzyme labeling apparatus.The induction concentration was 0-0.1?M,and the thermal induction condition was that the thermal induction was carried out at 37? for 15 minutes every 8 hours.Under these conditions,the inducible promoter of beta-estradiol could obtain better regulation ability in Saccharomyces cerevisiae.3.Neuric acid production in Saccharomyces cerevisiae YS58 system using?-estradiol inducible promoter was investigated.Recombinant plasmids pRS416-PReGoll-KCS-Tcyc,pRS416-PReGall-FAE1-TCYC and pRS416-PReGall-ELOVL1-Tcyc were constructed using beta-estradiol inducible promoter,and the pathway of neuroacid synthesis was enhanced in Saccharomyces cerevisiae.The results showed that strains expressing ELOVL1 gene with neuroacid content could reach 0.57%,followed by strains expressing KCS gene with neuroacid content of 0.48%,while strains expressing FAE1 gene could only reach 0.39%.4.The effect of Elo2 knockout on neuroacid synthesis in Saccharomyces cerevisiae was investigated.Firstly,three strains of Saccharomyces cerevisiae,Elo2?-?-Estradiol-ELOVL1,Elo2?-?-Estradiol-KCS and Elo2?-?-Estradiol-FAE1,were constructed.For the above three strains,Elo2?-?-Estradiol-ELOVL1 strain had the highest neuroacid content(0.68%),followed by Elo2A-?-Estradiol-KCS strain(0.60%)and Elo2?-?-Estradiol-FAE1(0.51%).Compared with the strains without Elo2 gene knockout,the neuroacid content of three Saccharomyces cerevisiae strains with Elo2 gene knockout increased by 19.3%,25.0%and 30.7%,respectively. |
PDF Full Text Request