Metabolic Engineering Of Yarrowia Lipolytica For Improving The Production Of Squalene

As a polyunsaturated triterpenoid,squalene has been widely used in the fields of medicine,food,cosmetics,etc.owing to its multifunction such as anti-cancer,anti-tumor and skin-caring.Compared with the traditional extraction methods,the method of producing squalene from microorganisms has the advantages of high efficiency,environmental friendliness and sustainability.Accordingly,it is of great importance to tailor microorganisms by metabolic engineering strategies for efficiently producing squalene.As the rate-limiting enzyme of the terpene pathway for producing terpenes,3-hydroxy-3-methyl-glutaryl-coenzyme A reductase(HMG1)plays a key role in the biosynthesis of squalene.In this research,Yarrowia lipolytica was used as the chassis for the biosynthesis of squalene.By regulating the expression of the endogenous HMG1 gene and optimizing the fermentation condition,the squalene production in Y.lipolytica was improved.The main results are as follows:(1)By constructing the module of 28 S r DNA-TEFp-HMG1-LIP2 t for overexpressing HMG1 gene,and using URA3 as the selection marker,the engineered strain SH-1 was successfully constructed.The squalene production in the engineered strain SH-1 reached117.97 mg/L,which was 187 folds higher than that of the wild strain.(2)The addition of 20 mmol/L sodium acetate can promote the synthesis of squalene in strain SH-1.However,the squalene production of SH-1 at 118.60 mg/L was not significantly improved.The adding of terbinafine can effectively inhibit the consumption of squalene in strain SH-1.Whereas,it was performed that the production of squalene at 237.84 mg/L is reached by adding terbinafine with 10 mg/L in SH-1 strain during fermentation.(3)Response surface methodology was used to optimize the fermentation conditions for improving squalene production in SH-1 strain.Under the optimized condition with fermentation time at 103.20 h,terbinafine concentration at 0.20 mg/L and sodium acetate concentration at 3.90 mmol/L,the squalene titer was reached 300.00 mg/L,which was 2.5folds higher than that of the initial condition(117.97 mg/L).In conclusion,by regulating the metabolic pathway of squalene biosynthesis and optimzaing the fermentation process,the production of squalene was obviously improved in Y.lipolytica.This investigation provides technical and theoretical basis for the biosynthesis and exploitation of squalene.