Metabolic Engineering Of Yarrowia Lipolytica To Produce Triacetic Acid Lactone

Triacetic acid lactone is widely used as a small molecule polyketide platform compound.The chemical synthesis method using acetic acid as raw material and plant extraction method have many problems such as serious environmental pollution,strong dependence on petroleum resources and a long production cycle.The production of triacetic acid lactone from renewable biomass by constructing microbial cell factories has become a new method to solve the above problems.The Yarrowia lipolytica intracellularly has a high flux of metabolic flow of the polyketide precursor acetyl coenzyme A,which is an advantageous chassis for the industrial production of triacetic acid lactone.Glucose and xylose derived from lignocellulosic can be used as cheap raw materials for microbial synthesis of triacetic acid lactone,but the inefficient conversion of xylose is a key problem that remains to be solved.Therefore,in this project,we constructed and optimized the xylose metabolic pathway,and combined with carbon and energy flow regulation,to achieve the efficient conversion of xylose to triacetic acid lactone.The specific research contents include:(1)Using Yarrowia lipolytica as the starting bacterium,the synthesis pathway of triacetic acid lactone by expressing 2-pyrone synthase was successfully constructed,and the TAL yield reached 0.72 g/L with glucose as the raw material.On this basis,the genes of xylose metabolism pathway from Yarrowia lipolytica and Scheffersomyces stipitis were overexpressed respectively to improve the efficiency of xylose utilization by the engineered bacteria,and the TAL yield could reach 1.27 g/L and 1.60 g/L,respectively.(2)The activity of 2-pyrone synthase is dependent on the cofactor NADPH,and the intracellular NADPH content during the synthesis of triacetic acid lactone by the engineered bacteria Yarrowia lipolytica using glucose and xylose was compared in this study,indicating that the total intracellular reducing power of metabolizing xylose was higher than that of glucose.Furthermore,seven key genes of the NADPH recycling system,including mae,mndhl,mndh2,idp2,zwf1,gndl and gnd2,were overexpressed respectively,but the yield of TAL was not significantly increased,indicating that the reducing power generated by xylose metabolism could satisfy the synthesis of TAL.(3)To improve the efficiency of xylose conversion by engineered bacteria,the effect of carbon to nitrogen ratio,pH and by-products on TAL synthesis were further investigated.The experimental conditions were determined that the optimal carbon to nitrogen ratio was 80:1,0.2 M PBS buffer was used to control the pH,and 1.5 mg/L cerulenin was added to inhibit the synthesis of by-product fatty acids.The yield of TAL was increased to 4.99 g/L,which was 2.1 times higher than that without optimization.It has reached the highest level in the shake flask reported currently.In this study,by optimizing and regulating the metabolic pathway of xylose conversion to synthesize triacetic acid lactone,a Yarrowia lipolytica engineered bacteria that can efficiently utilize xylose to synthesize triacetic acid lactone was constructed successfully,which laid a foundation for the study of lignocellulosic conversion to synthesize polyketide compounds.