Production Of Artemisinin Precursors By Synthetic Biology Methods

OBJECTIVE Amorpha-4,11-diene synthase is the key enzyme of artemisinin biosynthesis,amorpha-4,11-diene is the precursor of artemisinin biosynthesis. Detection of amorpha-4,11-diene synthase activity in E. coli, determined to yeast mating type that improve yield of amorpha-4,11-diene in yeast.METHODS Construct the E. coli expression vectors and the yeast expression vectors by molecular biology methods,these mothods are enzyme digestion, the connection and transformation,and so on. The E. coli expression vectors are transfomed into E. coli by CaCl? method; The yeast expression vectors are transfomed into S. cerevisia of different mating types by LiOAc method, obtained engineering bacterias of different yeast mating types with the same carrier. Using SDS-PAGE and Western blot detection of the results that the E. coli engineered bacteria is induced; Their fermentation products of yeast engineering bacterias were extracted and then detected by GC-MS to determine the yield of a amorpha-4,11-diene,and then to determine the yeast mating type to improve the yield of amorpha-4,11-diene.RESULTS 1,Identify the function of amorpha-4,11-diene synthase gene,the result prove FPP is cyclizated into amorpha-4,11-diene by ADS,this laid a foundation making a study about artemisinin synthetic biology by using ADS in the future.2,ADS is a sesquiterpene synthase, detected 10 kinds of products including amorpha-4,11-dienefrom the yeast engineering stains with ADS, confirmed the production feature of ADS has not high specificity once again.3,There were no significant effect about the yield of amorpha-4,11-diene in the yeast engineering stains of different mating types (P>0.01).It provides a theoretical basis for the research,to make optimization of medium, optimization of metabolic pathways and other factors effect to the yield of engineering stains by using the yeast engineering stains of different mating types. Speed up the study about metabolic engineering of artemisinin precursors.4,Culture conditions affect the yield of Amorpha -4,11-diene of the yeast engineering stains.5,Construction of pYeDP60/G/dbr2 and pYG450-29/CPR3 yeast expression vectors.