Study On The Synthesis Of Cordycepin By Metabolic Engineering Of Yarrowia Lipolytica

Cordycepin,as a nucleoside antibiotic,has been widely used in the fields of drug and food.Although the technology of artificial cultivation for Cordyceps militaris has well-established and turned into the main means for cordycepin production,the long fermentation period,complicated cultivation process and complex operation limit its wide application.Therefore,it is necessary to establish the green and efficient production method of cordycepin to meet the huge market demand.In this paper,Yarrowia lipolytica was used as the chassis to construct the microbial cell factory,for realizing the efficient and green biosynthesis of cordycepin.The main findings were as follows:(1)Using Y.lipolytica Po1f(Δku70)as the chassis,the synthesis pathway of cordycepin derived from C.militaris was integrated into the genome by the r DNA site,and the engineered strain YlCor-01 with a cordycepin production of 827.34 mg/L was obtained.Further,optimizing the cordycepin biosynthetic pathway through using promoter engineering and protein engineering strategies,and the engineered strain YlCor-02 performed the highest cordycepin at 949.45 mg/L.(2)The key genes Yl PGK and Cm NK were overexpressed in the engineered strain YlCor-02 by combining ATP engineering and precursor engineering for improving the intracellular ATP content and the ability to synthesize cordycepin.The engineered strain YlCor-18 with a cordycepin yield of 1585 mg/L was obtained through the above metabolic transformation.(3)The fermentation conditions of the final engineered strain YlCor-18 were optimized from shake flask specification,glucose concentration and adenine addition.The conventional shake flask,30 g/L glucose and 1 g/L adenosine dosage were determined as the optimized fermentation conditions.Under this condition,the engineered strain YlCor-18 was capable of producing 3099.25 mg/L cordycepin for 72 h,and the cordycepin yield was reached at4362.54 mg/L at 168 h through fed-batch fermentation at the shaker level,which was the highest productivity reported so far.This study successfully constructed the high-efficiency cell factory to synthesize cordycepin in Y.lipolytica,laying the foundation for the industrial production of cordycepin,and also providing the guidelines for the establishment of cell factories for other nucleoside antibiotics.