The Biosynthesis Of Lupulone By Engineered E.Coli

Hop?Humulus lupulus?is essential to be supplemented as bittering agentin the brewing process of beer.It has also been used as a source of medicinal secondary metabolites.As one of the secondary metabolites,lupulone has antibacterial and anti-inflammatory,sedative and hypnotic,anti-tumor and other pharmaceutical effects.However,the extraction of lupulone from Humulus lupulus requires a large amount of energy and resource,and chemical synthesis of the complex lupulone is not economically feasible.Therefore,this study proposed the biosynthesis of lupulone by microbial fermentation.In this study,from the starting E.coli strain,the genes of key enzymes inthe biosynthesis pathway of lupulone were engineered into starting strain.Eventually,a biosynthetic pathway from glucose and MVA to lupulone was engineered.Through the analysis and identification of the fermentation products,the presence of lupulone was confirmed,indicating the feasibility of the biosynthesis of lupulone.This is the first study that confirmed lupulone can be biosynthesized by engineered E.coli.The key enzymes of cytosolic-CoA ligase?HlCCL2?,valerophenonesynthase?HlVPS?,isoprene pyrophosphate isomerase?IDI?,PT1 and PT2 are engineered in this study.In flask fermentation,the engineered E.coli could produce trace mount of lupulone.However,the cell growth of enginnered strain was inhibited,which limits the higher production of lupulone.This study further optimized the fermentation conditions to increase the cell growth and lupulone production.After broth optimization,the optimized formula was summarized as follows:from M9 medium,7.5 mL/L MVA,5 g/L yeast cream and 1 g/L betaine,and 15 m L/L leucine was added in broth.The results showed that the biomass(OD₆₀₀)was increased from 4.70 to 14.00 after optimization.