Enhance ω-3PUFAs Production In Oleaginous Fungus Mortierella Alpina By Overexpressing ω3Fatty Acid Desaturase

Research has shown that ω-3polyunsaturated fatty acids (ω-3PUFAs) have variousphysiological functions. The tranditional sources of ω-3PUFAs are limited, so the yields wereunable to meet the increasing market demand. ω3fatty acid desaturase is the key enzymeresponsible for ω-3PUFAs formation. In oleaginous fungus Mortierella alpina, about50%oftotal fatty acids are arachidonic acid (AA), whereas ω-3PUFAs level is very low. In order toenhance the ω-3PUFAs production, ω3fatty acid desaturase gene (FADS15) from M. alpinawas cloned and homologously overexpressed, using the Agrobacterium tumefaciens-mediatedtransformation.Firstly, the FADS15fragment amplified from pET19b-FADS15which was constructedin our laboratory was used as the template. The vector pBIG2-ura5s-ITs which has a ura5scassette as the select marker was used as the backbone. We constructed the T-DNA binaryvector pBIG2-ura5s-FADS15.Secondly, the genetic transformation system of M. alpina via Agrobacterium tumefaciensmediation was investigated by the concentration of acetosyringone, the concentration ofA.tumefaciens cell suspension, the time and temperature of co-culture. The optimumtransformation condition was when the concentration of acetosyringone was200μmol/L, theconcentration of A.tumefaciens cell suspension OD600was1.0, the time and temperature ofco-culture was36h and23℃.Ten positive strains were successfully screened. Compared to the wild-type M. alpina,the FADS15overexpressing strains MA-ω3Des-1, MA-ω3Des-2, MA-ω3Des-3had6.02-,5.62-and5.92-fold increase in EPA yield, respectively. The colony morphology, myceliummicroscopic morphology and spore production ability of recombinant strains were comparedwith that of wild-type M. alpina. There were no differences in physiological characteristics.Recombinant strain was cultured by shaking culture in different medium, culturetemperature and time, linseed oil supplementation (1%, w/v) and soybean oil supplementation(1%, w/v) in order to investigate the influence of these factors in ω-3PUFAs production. TheBroth medium (GB medium) is more advantageous to accumulate ω-3PUFAs forrecombinant strain. The tempreture transformation strategy was beneficial for the productionof ω-3PUFAs. As the extension of cultivation time, the biomass of the recombinant strainsdidn’t increase, while the content of ω-3PUFAs gradually improved to maximum yield195.56mg/L after14days. The total fatty acids content and biomass of the recombinantstrains were both increased by the addition of linseed oil and soybean oil. Linseed oilsupplementation and soybean oil supplementation increased the EPA production ofrecombinant strain1.71-and2.67-fold, respectively.