Screening Of PuFAs-producing Soil Fungi And Construction Of Transgenic Strains With Yield Of EPA

Eicosapentaenoic acid（EPA）,which belongs to theω-3 polyunsaturated fatty acids（PuFAs）,is one of the essential fatty acids,it got widespread attention because of its physiologicalfunctions,suchasanticoagulant,loweringbloodlipidlevel,anti-inflammatory,anti-cancer,preventing and treating cardiovascular disease and diabetes,etc.Production of EPA by microbial fermentation is a hotspot in recent years owning to the resource restriction and marine pollution of traditional production by the extraction of deep-sea fish oil.At present,the yield of EPA produced by microbial fermentation is too low to applied to commercialization production,so breeding high yield EPA strains is necessary.In this study,we focus on getting the strain with high EPA production.First,several strains with PuFAs lipids were isolated from the alpine cold environment;next,the key enzyme genes of the lipids biosynthesis,diacylglycerol acyltransferase（DGAT）were cloned;then,the genetic transformation system of the spores of Mortierella alpina by Agrobacetrium tumefaciens LBA4404-mediated transformation was established;finally,the transgenic Mortierella alpina strain with EPA production was constructed.The results were as follows:（1）Several strains with PuFAs were isolated.First,a lot of strains were isolated from the soil of Qinlin mountains and Dabie mountains.Then,the key enzyme in EPA biosythesis,delta-6 desaturase gene was used as molecular marker for targeted screening based on PCR amplification.And the lipid composition of the obtained strains were detected by GC-MS.The results showed that seven strains can accumulate ARA,but no one accumulate EPA.The strains of high ARA yield were classified and identified by the ITS sequence analysis,the results indicates these strains belong to Mortierella fungi.（2）A MaDGAT gene was cloned from Mortierella alpina and expressed in the Saccharomyces cerevisiae H1246.First,the MaDGAT gene was clone of from Mortierella alpina by RT-PCR.Then,a recombinant vector expressing MaDGAT,pYES2-DGAT,was constructed and transformed into strain H1246,a neutral,lipid-deficient quadruple mutant.The thin layer chromatography（TLC）and Nile red fluorescence microscope analysis showed that the recombinant vector restored TAG,the lipids content of the recombinant strain increased by 35.6%.（3）The transgenic Mortierella alpina strain with EPA production was constructed.First,a genetic transformation system of the spores of Mortierella alpina by Agrobacetrium tumefaciens LBA4404-mediated transformation,using the DsRed fluorescent protein as screening marker,was established.The methods are as follows:mix equal volume of 10⁶/mL Mortierella alpina spores with Agrobacterium at a OD₆₀₀00 of 0.8,and then add AS to the final concentration of 150μg/mL.Spread the mix into PDA plate with 72μg/mL clethodim after co-cultivating for 48 h under the temperature of 24℃and rotation of 200 rpm,and the strains were observed for the red fluorescence to isolating positive transformants after grown at 28℃for 4 to 6 days.Then,a vector expressing MaDGAT and delta-17 desaturase genes was constructed and transformed into Mortierella alpina by the methods above.5 transgenic Mortierella alpina strains with EPA production were isolated,the strain with highest EPA yield was fermentated in shaking flasks,the results indicated that the EPA content was 10.5%of oil,and EPA yield was 0.91g/L.These results laid the foundation for construct transgenetic strains of high EPA yield to realize the ommercialization production.