The Function Of Thioesterase And Its Effect On Production Of Conjugated Linoleic Acid In Oleaginous Fungus Mortierella Alpina

Conjugated linoleic acid(CLA)refers to a group of positional and geometrical isomers of linoleic acid(LA)with conjugated double bonds,two of which(cis-9,trans-11 and trans-10,cis-12 CLA)are known to possess biological activity.But different isomers of CLA have different physiological functions.In order to meet the requirement of CLA in the field of medicine and nutrition,production of pure isomers is very important.Products from chemical synthesis are a mixture of CLA isomers,and there are some negative effects on the environment by this way.Thus,microbial production may be crucial to address these issues and has become one of the most promising alternative.Mortierella alpina(M.alpina)can accumulate fatty acids(FAs)up to approximately 50% of its dry cell weight.But CLA cannot be synthesed in the wild strain.However,LA accounts for about 10% of the total fatty acids(TFAs)and can be used as a potential substrate of linoleic acid isomerase.In the previous study,the linoleic acid isomerase(PAI)has been expressed in M.alpina to produce trans-10,cis-12-CLA,but the recombinant strains had a low production of CLA,and the low amount of free fatty acids(FFAs)in M.alpina may be the primary limiting factor for CLA production.Studied have showed that thioesterases(TEs)can reinforce the flux from acyl-CoAs to FFAs,improve the production of FFAs.In this study,we explore the role of TEs in mediating fatty acids metabolism in Mortierella alpina;the TE gene and PAI gene were also co-overexpressed to explore TE effect on production of conjugated linoleic acid.The main research results are as follows:1.The expression levels of TEs genes were analysed by RT-qPCR during FA synthesis in M.alpina.The expression levels of ACOT8 I were significantly elevated at the beginning of the FA accumulation period,showing a 7-fold increase.In contrast,ACOT8 II,ACOT9I and ACOT9 II did not change obviously.These results indicated that the expression of ACOT8 I was related to the FA metabolism.2.The role of autologous acyl-CoA thioesterase ACOT8 I in mediating intracellular lipid metabolism in oleaginous fungus M.alpina.The binary expression vector containing ACOT8 I was constructed;the vector was inserted into the genome of M.alpina via Agrobacterium tumefaciens-mediated transformation.The contents of TFAs were the same in the recombinant strains as in the wild-type strain,whilst the production of FFAs was enhanced from about 0.9%(wild-type)to 2.8%(recombinant),a roughly 3-fold increase.LA constituted about 9% of the TFAs in the FFA fraction in the recombinant strains,but only about 1.3% in the control strain.Overexpression of the TE ACOT8 I in the oleaginous fungus M.alpina reinforced the flux from acyl-CoAs to FFAs,improved the production of FFAs.3.The TE gene and PAI gene were also co-overexpressed to explore TE's effect on production of conjugated linoleic acid in oleaginous fungus M.alpina.First the binary expression vector containing ACOT8 I and oPAI was constructed by homologous recombination cloning method,The ACOT8 I gene was inserted into the genome of M.alpina via Agrobacterium tumefaciens-mediated transformation.The FA compositions revealed that TE has no obvious role in promoting the production of CLA.