Metabolic Engineering Of Yarrowia Lipolytica For Production Of Nervonic Acid

Nervonic acid is a natural ingredient of neurocyte and tissues of brain.It is also a recognized and important substance which can repair and unclog damaged neural pathways of brain,restore the activity of nerve endings and promote growth and development of neurocyte in the world,so nervonic acid has become the best choice for prevention and treatment of related diseases of brain.In nature,the source of nerve acid is scarce,which can not meet the demand of the market at all.In this paper,metabolic pathways of yarrowia lipolytica was modified by technology of genetic engineering,and engeneering strains which can produce nervonic acid was obtained.The specific results are as follows:(1)Uracil auxotrophy strain URA3? po1 g of yarrowia lipolytica was obtained after URA3 genes were knockout by technology of homologous recombination.(2)Recombinant expression plasmids of pMT-DGA1-SCD_HGR,pMT-BtFAE1-AtFAE1_LEU,pMT-MaKCS_URA,pMT-MoKCS_URA and pMT-CgKCS_URA were obtained by constructing expression plasmids using endogenous genes SCD(stearoyl-CoA desaturase)and DGA1(diacylgycerol acyltransferase 1)and exogenous genes AtFAE1(fatty acid elongase 1 from Arabidopsis thaliana),BtFAE1(fatty acid elongase 1 from Brassica tournefortii),MaKCS(?-ketoacyl-CoA synthase from Microalgae),MoKCS(?-ketoacyl-CoA synthase from Malania oleifera)and CgKCS(?-ketoacyl-CoA synthase from Cardamine graea)by method of Gibson Assembly.(3)Recombinant strain URA3 ? Po1g(pMT-DGA1-SCD/pMT-BtFAE1-AtFAE1)was obtained by introducing into URA3? po1 g using recombinant expression plasmids of pMT-DGA1-SCD_HGR and pMT-BtFAE1-AtFAE1_LEU by method of lithium acetate transformation succesfully.On this basis,recombinant strains URA3? Po1g(pMT-DGA1-SCD/pMT-BtFAE1-AtFAE1/pMT-MaKCS),URA3? Po1g(pMT-DGA1-SCD/p MT-BtFAE1-AtFAE1/pMT-MoKCS)and URA3 ? Po1g(pMT-DGA1-SCD/pMT-BtFAE1-AtFAE1/pMTCgKCS)were obtained succesfully by introducing into URA3? Po1g(pMT-DGA1-SCD/ pMT-BtFAE1-AtFAE1)using pMT-MaKCS_URA,pMT-MoKCS_URA and pMT-CgKCS_URA respectively.(4)Engineering strains of yarrowia lipolytica URA3 ? Po1g(pMT-DGA1-SCD/ pMT-BtFAE1-AtFAE1/pMT-CgKCS)which can produce nervonic acid were obtained succesfully after shaking flask culture,extracting lipids and methyl esterification of fatty acid by using methods of chloroform-methanol and sulfuric acid-methanol respectively and detecting fatty acid methyl ester by GC-MS.Nervonic acid content of URA3? Po1g(pMT-DGA1-SCD/pMT-BtFAE1-AtFAE1/pMT-CgKCS)-7 monoclone is the highest,accounting for 1.5% of its lipids,and proportion of lipids for cell dry weight is 3 times than control strain URA3? Po1 g.(5)A complete and effective molecular biology mechanism for the study of yarrowia lipolytica was established,which provides theoretical and experimental basis for industrialization of production of nervonic acid using yarrowia lipolytica.