Cloning Of Kcs Genes From Microalgar And Malania Oleifera, And The Preliminary Exploration Of Producing Nervonic Acid By Yarrowia Lipolytica

Nervonic acid(C24:1) is one the most comman very long chain monunsaturanted fatty acid, and it shows great potentials in pharmaceutical and chemical industries. Now it is mostly producted from no animal. In recent years, engineered for vegetable oil exploitation and utilization have been developed, but there are a few sources, and quality of production is difficult to control. This paper introduces that oleaginous yeast (Yarrowia lipolytica) products nervonic acid through gene transformation and optimization of culture. The exogenous gene,3-ketoacyl-CoA synthase from microalgae (3-ketoacyl-CoAsynthase, KCS), DGA1 gene and ELO2 gene from yeast and FAE1 gene from Arabidopsis thaliana, are used to construct multiple gene expression plasmids:pMT-ZKCS, pMT-ZKCS-ELO2, pMT-DGA1-FAE1 using Gibson Assembly method. Subsequently we transformed them in yeast successfully and efficiently. To some extent, the mutants are valuable:the proportion of all fatty acid in dry yeast mutant increased from 12%to 35%; composition of yeast fatty acid changed, the content of very long chain fatty acids C24:0 improved and the mutant with FAE1 gene can produce the C22:1 that are not the original fatty acid in yeast. The establishment of the molecular biology mechanism of a complete set of effective transformation methods of Yarrowia lipolytica, solid the foundation for the later research.In addition, through the research and analysis of Malania oleifera fruit, we cloned 3-ketoacyl-CoA synthase gene by the methods of high-efficient touch-down PCR and short primers PCR and TAIL-PCR and did the analysis to this key gene of very long fatty acid elongase.