Total Biosynthesis Of Natural Product Humulone

With the fast development of synthetic biology,the microbial production of various natural pharmaceutical compounds has become popular.This study mainly studied a total biosynthetic pathway of humulone,a natural bitter acid in hops.From E.coli Trc-low,the whole pathway of humulone was engineered in starting strain,which can produce humulone from glucose and mevalonate.After fermentation,the product was analyzed by HPLC-MS and ~1HNMR,and the results verified that engineered E.coli can synthesize humulone.The genes of prenyl diphosphate isomerase(IDI),carboxy-CoA ligase(CCL2),pentyl-benzophenone synthase(VPS),isoprenyl transferase-1(PT1),isoprene transferase-2(PT2)and humulone synthase(monooxygenase)were expressed in engineered E.coli.These six genes were constructed into two expression vectors and transformed into starting strain for humulone biosynthesis.After completing the construction of the strain,fermentation and product verification,this study further optimized the fermentation conditions of the engineering strain,studied the effect of mevalonate addition,inoculation amount,and the level of inducer addition on the fermentation of humulone.The results indicated that the mevalonate addition method,inoculum amount,yeast extract and inducer had a certain effect on the fermentation results.Among them,fed-feeding mevalonate,adding 1200 μL of inducer and 2 g of yeast extract can increase the yield of humulone.Under the optimized combination of fermentation conditions,the production of humulone could reach 0.0575 g/L,1.70%of dry cell weight.