Exploration Of Lipid Metabolism Regulation In Yarrowia Lipolytica

Polyunsaturated fatty acid have important physiological functions on organisms,and its application in the field of medicine,food and health care products are very widely.In recent years, the use of microbial fermentation to produce PUFAs has become a research hot spot.In this study,we use genetic engineering means to build Yarrowa lipolytica, which can produce PUFAs with important physiological functions.First,cloning the ?-9 elongase gene of PUFAs synthetic ways,and connect to the expression vector pINA-1297, yielding the reco mbinant p INA1297-?9E.The use o f restrict io n endo nuclease BglⅡ linear ized recombinant vector pINA1297-?9E,Yarrowa lipolytica po1 f was transformed with pINA1297-?9E by electroporation method and the positive transformants YL?9E were obtained by YNBcasa plates. PCR was used to verify the successful integration into the genome of Yarrowa lipolytica.The growth rate of transgenic strains was much higher than that of wild strain in YPD medium. Analysis of gas chromatography/mass spectrometer(GC-MS) showed that eicosadienoic acid(EDA; C20:2n-6) was detected and their percentage to total fatty acids in transgenic Yarrowa lipolytica was 14.8%, which indicated that Euglena gracilis ?9-elongase gene was expressed in transgenic Yarrowa lipolytica.In order to improve the biomass of engineering yeast YL?9E, the conditions for engineering yeast YL?9E fermentation were optimized by response surface methodology. First of all, the best carbon source and concentration, nitrogen source and concentration, and the optimal initial p H value was determined by the single factor experiment, the response surface optimization analysis of Box-Behnken experimental design. According to response surface analysis,the optimum conditions for engineering yeast YL?9E fermentation were glucose of 5.12%,yeast powder of 2.34%,the initial pH value of 4.83. Under these conditions,the biomass of engineering yeast YL?9E was 29.56 g/L,compared to the previous10.2 g/L increased 2.9 times.?-9 elongase pathway is important for the synthesis pathway of PUFAs,cloning the key enzyme genes in this pathway, including the ?9-elongase gene, ?8-desaturase gene,?5- desaturase gene,?17-desaturase gene.The four genes by means of fusion PCR respectively connected to the respective promoter,terminator,form A,B,C,D four genes expression components,and connect to the expression vector pINA-1297,yielding the recombinant pINA1297-ABCD.Yarrowa lipolytica po1 f was transformed with pINA1297-ABCD by electroporation method and the positive transformants YL-ABCD were obtained by YNBcasa plates.PCR was used to verify the successful integration into the genome of Yarrowa lipolytica.