Construction Of Genetically Engineered Saccharomyces Cerevisiae Biosynthesizing Parahydroxybenzoic Acid

Parahydroxybenzoic acid is an important precursor for the synthesis of parabens.It has important application value.The traditional production method is using active ingredients in petroleum for synthesis,but it is environmentally polluted.Therefore,in recent years,More and more researchers are involved in the biosynthesis of p-hydroxybenzoic acid.In this study,Saccharomyces cerevisiae was used as the host to construct the production pathway of p-hydroxybenzoic acid,and the production of p-hydroxybenzoic acid was improved by increasing the flux of related pathways.1.A synthetic route to p-hydroxybenzoic acid was constructed in S.cerevisiae.Introduce ubiC gene which from Escherichia coli and Pseudomonas putida into S.cerevisiae to construct the production route which from chorismate to p-hydroxybenzoic acid.By comparing genes from different sources,it is determined that the ubiC gene derived from E.coli behaves better in S.cerevisiae At the same time,the effect of different promoters on ubiC gene expression was compared.The highest yield reached 11.34±0.13 mg·L-1.2.Reforming shikimic acid pathway in S.cerevisiae.Over-expressing the Aro4 gene,constructing the plasmid 181-TEF2p-Aro4-CYCLt-TEF1p-ubiC-CYCLt,which releasing feedback inhibition,the yield reached 32.09±0.77 mg·L-1.On this basis,the E.coli derived shikimic acid pathway gene was integrated into S.cerevisiae genome,and S.cerevisiae self shikimic acid pathway gene,Arol,was expressed.The overproduction of the chorismate synthase gene,Aro2,resulted in the production of p-hydroxybenzoic acid,it reached 35.22±1.74 mg·L-1.3.The pentose phosphate pathway was modified in S.cerevisiae.The ZWF1 gene promoter was replaced by the strong promoter TEF1p in the genome.At the same time,overexpression of the pentose phosphate pathway genes TKL1 and RKI1 increased the yield by 6.96%.Finally,the knockdown of the pentose phosphate pathway negative regulator RICl gene results in a 5.97%increase in p-hydroxybenzoic acid production.4.The glycolytic pathway was modified.Knocking out the glycolytic pathway by-pass gene PYK2 to improve the accumulation of PEP.Except this,the gene GAPN which from Streptococcus mutans was introduced to increase the glycolysis pathway flux,when it combines with Aro2 gene to express,constructing the plasmid 195-ACT1-Aro2-PGI1t-GPD1p-GAPN-PGI1t the yield reached 38.60±1.96 mg·L-1.