Production Of Conjugated Linoleic Acid By Heterologous Expression Of Linoleic Acid Isomerase In Oleaginous Fungus Mortierella Alpina

Conjugated linoleic acids(CLAs) are a class of isomers of linoleic acid possessing conjugated double bonds. The two most important CLA isomers that have been well studied are cis-9,trans-11-CLA and trans-10,cis-12-CLA. In recent years, CLAs have been increasingly attracting attention for their biological functions,such as anti-carcinogenic and –atherosclerosis effects,modulation of the immune system and regulation of lipid metabolism. and the latter in particular was also shown to have body fat-lowering and muscle mass-increasing effects. Microbial production of a pure CLA isomer is more reproducible than chemical synthesis methods,and has become one of the most promising alternative to nutritional supplement use. Mortierella alpina,which can accumulate fatty acids up to approximately 50% of its dry cell weight(DCW),the relative abundance and proportion of its PUFAs makes M. alpina a valuable source of some specific nutritional supplements,along with a record of complete safety. As it can accumulate a relatively higher content of linoleic acid(about 10% of TFA),M. alpina appeared to be a promising organism for producing CLA. M. alpina is incapable of synthesis of CLA and the incorporation of trans-10,cis-12-CLA into the PUFAs of M. alpina via PAI conversion will significantly increase the commercial value of M. alpina. In this study,PAI was heterologous expressed in Mortierella alpina to explore the production of trans-10,cis-12-CLA. Through different strategies of the optimization to increase production of conjugated linoleic acid such as codon optimization,addition of exogenous substrates and specific inhibitors. Our research provides a new valuable platform for basic research and industrial production of CLA in oleaginous microorganisms. In this paper,the main research results are as follows:1. The construction of M. alpina PAI-expressing recombinant strains. First,the nature PAI gene(PAI) was amplified by PCR with the plasmid p MD19T-PAI as template. Then the PAI sequence was optimized according to the codon usage bias of M. alpina and acquire the optimized version of PAI(o PAI). The binary vector p BIG2-ura5s-PAI and p BIG2-ura5s-o PAI were structured and electro-transferred into A. tumefaciens C58C1 for Agrobacterium tumefaciens mediated transformation(ATMT). Stable M. alpina transformations were selected and the ATMT conditions were optimized.2. The study of PAI expression level in M. alpina recombinant strains. The transcription level and protein level of PAI and o PAI gene were analyzed. The transcription of all target genes were at the same level in all recombinant strains and not affected by codon optimization. Compared with wild type M. alpina, there were no significant changes in content and composition of the fatty acids in recombinant strains. CLA was detected in one of the recombinant named M. alpina-o PAI-3,which was identified as trans-10,cis-12-CLA by GC-MS. This result indicated that the PAI expressed by M. alpina has catalytic activity.3. The rate limiting factor of CLA production and its optimization strategy. The existence of fatty acids in M. alpina were mainly identified as the form of triglycerides via TLC analysis. The insufficient of available substrate may be the reason of low CLA production. With addition of 0.2%(w/w) free LA and 5 μM ACSL inhibitor, the trans-10,cis-12-CLA reached up to 1.02 %(w/w) and 1.21 %(w/w) of the TFA in M. alpina. The trans-10,cis-12-CLA reached up to 4.05 %(w/w) with addition of both free LA and ACSL inhibitor in the medium. The CLA content increased by nearly 80 times compared with before. The results indicating that M. alpina has a promising potential in biosynthesis of CLA.