Design and construction of an artificial pathway for biosynthesis of acetaminophen in Escherichia coli

Acetaminophen (AAP) is one of the most commonly used drug ingredients that possesses antipyretic and analgesic effects. As an unnatural chemical, AAP is commercially produced by chemical processes using petroleum-derived carbohydrates, such as phenol, as raw materials, which is unsustainable and eco-unfriendly. In this study, we report design and construction of an artificial biosynthetic pathway for de novo production of AAP from simple carbon source. By exploring and expanding the substrate repertoire of natural enzymes, we identified and characterized a novel p-aminobenzoic acid (p-ABA) monooxygenase and an p-aminophenol (p-AP) N-acetyltransferase, which enabled the bacterial production of AAP from p-ABA. Then, we constructed an p-ABA over-producer by screening of p-ABA synthases and enhancing glutamine availability, resulting in 836.43 mg/L p-ABA in shake flasks in E. coli. Subsequent assembly of the entire biosynthetic pathway permitted the de novo production of AAP from glycerol for the first time. Finally, pathway engineering by dynamically regulating the expression of pathway genes via a temperature-inducible controller enabled production enhancement of AAP with a titer of 120.03 mg/L. This work not only constructs a microbial platform for AAP production, but also demonstrates design and construction of artificial biosynthetic pathways via discovering novel bioreactions based on existing enzymes.