Biosynthetic Analgesic Paracetamol—Acetaminophen

Acetaminophen is listed as one of the essential drugs by the World Health Organization.It is mainly used to treat pain and fever and has a huge market.As an unnatural chemical,the industrial production of acetaminophen relies on chemical synthesis by using petroleum-derived carbohydrates as raw materials which is considered to be eco-unfriendly and unsustainable.In this study,we report a new artificial pathway for de novo biosynthesis of acetaminophen from glycerol.In this pathway,glycerol as the initial substrate to generate chorismate via the shikimate pathway,and then is catalyzed by p-aminobenzoic acid synthase to generate p-aminobenzoic acid,followed by hydroxylation to generate p-aminophenol,which is further acetylated to acetaminophen.By exploring and expanding the substrate repertoire of natural enzymes,we have identified and characterized a novel p-aminobenzoic acid monooxygenase and an p-aminophenol N-acetyltransferase.The monooxygenase can catalyze 800mg/L p-aminobenzoic acid produces 553.31 mg/L p-aminophenol,and the N-acetyltransferase can catalyze the conversion of p-aminophenol to acetaminophen with a high conversion rate of 75%.In order to provide sufficient p-aminobenzoic acid,we enhanced the flux of the shikimate pathway,overexpressed p-aminobenzoic acid synthase and increased the supply of glutamine,so that the production of p-aminobenzoic acid reached836.43 mg/L.Subsequently,the entire pathway was assembled,and the de novo biosynthesis of acetaminophen was successfully realized,with a yield of 14.32 mg/L.However,because N-acetyltransferase can not only catalyze the acetylation of p-aminophenol,but also the acetylation of p-aminobenzoic acid to produce p-acetaminobenzoic acid,and the enzyme has a higher affinity for p-aminobenzoic acid,resulting in the formation of a large amount of by-product p-acetaminobenzoic acid has greatly reduced the production efficiency of acetaminophen.Subsequently,temperature response control system was employeed to de novo sythesis of acetaminophen to regulate the expression of N-acetyltransferase,which reduced the accumulation of by-products and greatly increased the yield.With this efforts,the prodcution of Acetaminophen reanhed 120.03 mg/L,which is8.38 times than before using this strategy.This work has realized the microbial production of acetaminophen by designing and constructing an artificial biosynthetic pathway for the first time,which provides the possibility for large-scale biological production of acetaminophen from renewable carbon sources.