1. Introduction
Benzoic acid and its salts are widely used in food, pharmaceuticals, and cosmetics as preservative, as they inhibit growth of several yeasts and bacteria. Commercially, benzoate is produced by partial oxidation of toluene with oxygen, catalyzed by cobalt or manganese naphthenates [1]. This conversion can also be performed by microbes, e.g., through the upper pathway encoded on the TOL plasmid pWW0 fromPseudomonas putida mt-2 [2]. Besides environmental issues that arise from its petrochemical production process, microbially produced benzoate is considered to be “natural”, which is a major benefit for applications in food and cosmetics. This, however, requires the production to start from a bio-based substrate.
Despite its industrial relevance and simple structure, only minor efforts have been made to develop a microbial host for the bioproduction of benzoate with only one study demonstrating its de novoproduction [3]. About 460 mg L−1 (3.8 mM) of benzoate was produced with Streptomyces maritimus in a fermentation process using a complex medium (5% tryptone, 3% cornstarch). So far this is the only prokaryotic organism that has been described to natively synthesize benzoate from l‑phenylalanine via β-oxidation of trans ‑cinnamoyl-CoA as part of the enterocin biosynthesis pathway [4].
Pseudomonas taiwanensis is a promising microbial cell factory, especially regarding the production of aromatics. This has been recently demonstrated by our group in multiple studies for de novosynthesis of phenol, 4‑hydroxybenzoate, and trans -cinnamate [5-8]. In this study, a previously generated Pseudomonas taiwanensis trans -cinnamate overproducer was further engineered to enable benzoate production from renewable resources in a mineral medium without supplementation of complex substances or antibiotics. To our knowledge, this is the first de novo synthesis of benzoate in a recombinant microbial cell factory. The intrinsic benzoate catabolic pathway of P. taiwanensis was exploited to produce other industrially relevant chemicals, namely catechol andcis,cis -muconate, thereby establishing a novel biosynthesis pathway for these molecules.