Study On The Effect Of Co-expressing Strategy On EPA-producing Mortierella Alpina

Eicosapentaenoic acid(EPA),one of the omega-3 series of long-chain polyunsaturated fatty acids,has many special physiological functions such as anticoagulation,prevention of cardiovascular disease and possenssion of hypolipidemic action.Thus,it has been used in the health care products industry,food industry,and feed industry.The oil-producing microorganism Mortierella alpina(M.alpina)capable of accumulating 40% arachidonic acid(AA)of the total fatty acids has been used for the industrial production of AA.M.alpina possesses EPA synthesis pathway but is only able to produce merely 2% EPA under low temperature conditions.In our previous research,a recombinant M.alpina capable to synhesize significantly increased EPA under normal temperature was successfully constructed through heterologous expression of an omega-3 fatty acid desaturase(oPpFADS17)derived from P.parasitica.Besides,the gucose-6-phosphate dehydrogenase(G6PD2)gene,one of the key gene for NADPH resources of M.alpina,was homologous expressed and a recombinantrole M.alpina with obvious total fatty acids increase were achieved.In order to further improve the total lipids content and EPA content of M.alpina,the oPpFADS17 and G6PD2 are simultaneously expressed by using the double expression cassette strategy and 2A peptide strategy.The main results of this project are as follows:1.In the double expression cassette strategy,recombinants M.alpina with total lipids content increased by about 20%-was achived.The gene transcription level and activity levels of G6PD2 as well as NADPH content of the recombinants was 3-5 times,2 times and 20% higher than the wild strain,indicating that G6PD2 was successfully overexpressed in the recombinant strains,and effectively promoted the total lipid content.While,the EPA content of the recombinant strains does not reach the theoretical content.Transcription level of oPpFADS17 of M.alpina-oPpFADS17 reduced to 20% was observed,which may explain the low EPA content of the recombinant strains.2.In the 2A peptide strategy,a recombinant strains with 30.6% EPA of which 77% AA was canalized was achieved.Transcript level of oPpFADS17 gene was increased by about 20%.While,the transcription level,enzyme activity and NADPH content of G6PD2 gene were similar to the wild strain.Thus,overexpression of oPpFADS17 gene and G6PD2 gene by the 2A peptide strategy can significantly increase EPA content.Total fatty acid of the above recombinant strain does not reach theoretical value may be due to the unchanged NADPH content.