Establishing de novo microbial biosynthesis of anisole
- 1Wageningen University & Research
Abstract
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.
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BibTeX
@misc{bruinsma2026establishing,
title = {{Establishing de novo microbial biosynthesis of anisole}},
author = {L. Bruinsma and D. Rosas Gomez and C. La Rocca and J. E. Escrig Molina and V. Martins dos Santos and C. Batianis},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.02.763652},
url = {https://www.biorxiv.org/content/10.64898/2026.11.02.763652v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Establishing de novo microbial biosynthesis of anisole
AU - L. Bruinsma
AU - D. Rosas Gomez
AU - C. La Rocca
AU - J. E. Escrig Molina
AU - V. Martins dos Santos
AU - C. Batianis
PY - 2026
DA - 2026/11/02
PB - bioRxiv
DO - 10.64898/2026.11.02.763652
UR - https://www.biorxiv.org/content/10.64898/2026.11.02.763652v1
AB - 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.
ER -