Establishing de novo microbial biosynthesis of anisole

Anisole is a valuable aromatic ether and promising green solvent whose industrial manufacture relies exclusively on petroleum-derived phenol and hazardous alkali-mediated methylation. Here, we establish the first de novo microbial biosynthesis of anisole from glucose in Pseudomonas putida KT2440 by integrating retrosynthetic pathway design with growth-coupled directed evolution. Using a selection platform in an engineered double auxotroph, in which growth depends on S-adenosylmethionine-dependent methylation, we evolved a promiscuous plant chavicol O-methyltransferase to yield phenol O-methyltransferase 1 (POMT1; bearing a key D264N substitution), which improved anisole production by 2.3-fold over the wild-type enzyme. To overcome product volatility, we integrated an in situ two-phase extraction system using dodecane that achieved near-quantitative product recovery. Then, we compared two phenol routes from the shikimate pathway, selected the 4-hydroxybenzoate route, and raised precursor supply by deleting competing reactions and introducing feedback-resistant and reduced-flux variants in two shikimate pathway enzymes. The final strain produced up to 0.57 mM anisole (62 mg/L) from glucose.