Establishing de novo microbial biosynthesis of anisole

A de novo microbial pathway in Pseudomonas putida KT2440 produces anisole from glucose, reaching 0.57 mM (62 mg/L) after engineering methylation and shikimate-derived precursor supply.

• Why it matters: Industrial anisole production depends on petroleum phenol and hazardous alkali methylation, so a glucose-based microbial route could reduce reliance on fossil feedstocks and harsh chemistry.
• What they did: The team designed a retrosynthetic pathway and used growth-coupled directed evolution in an engineered double auxotroph where growth depends on S-adenosylmethionine-dependent methylation, evolving a chavicol O-methyltransferase variant (POMT1) with D264N that increased anisole 2.3-fold.
• The result: Adding an in situ two-phase extraction with dodecane enabled near-quantitative recovery, and selecting the 4-hydroxybenzoate shikimate route plus shikimate enzyme edits yielded up to 0.57 mM anisole (62 mg/L) from glucose.

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