Genetic Modification Of Schizochytrium Sp.LU310 And The Effect Mechanism Of Malic Acid On DHA Biosynthesis

Traditional methods for increasing the DHA production of Schizochytrium concentrate more on strain screening,medium optimization and control strategy of Fermentation.With the development of genetic engineering technology,regulating metabolic flux of DHA biosynthesis in genetic level has received more and more attention.Compared with the traditional methods,using genetic engineering technology to change DHA biosynthesis metabolic network rationally can increase the DHA production essentially without increasing the additional fermentation cost.However,DHA biosynthesis mechanism is still at the exploratory stage,as a result that it faces some challenges to regulate and control DHA biosynthesis in genetic level.In this research,a high DHA production strain,Schizochytrium sp.LU310,was studied by constructing gene knock-out and gene over-expression system.A target gene was successfully knockout by the gene knock-out system has reminded us that DHA production can be increased by using the system to disrupt the rate-limiting enzyme in some metabolic pathway which are in competition with DHA biosynthesis.On the other hand,MAT gene was over expression by the gene over-expression system with biomass,total lipid,DHA yield and MAT activity increased by 3%,5.5%,6.6%and 10.2%,respectively.The result illustrates that MAT over-expression can promote the DHA biosynthesis.Malic enzyme can catalyze malic acid to pyruvic acid and NADPH,the latter is for the supply of reducing power in DHA biosynthesis.1.5 g/L malic acid was added to fermentation medium for exploring the effect mechanism of malic acid on DHA biosynthesis in Schizochytrium.In comparison to normal condition,after adding 1.5 g/L malic acid,biomass,total lipid,DHA yield,malic enzyme activity and NADPH content was increased by 2.8%,9.4%,16.5%,21.1%,13%,respectively.This result illustrates that malic acid,as the precursors of malic enzyme,can activate the activity of malic enzyme.As a result,there is more NADPH for the supply of reducing power in DHA biosynthesis with achieving of higher yield of DHA.In addition,fermentation performance is more difference between the MAT over-expression recombinant and wild type under the circumstance of adding 1.5 g/L malic acid.