Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli

• Novel pathway to p-hydroxybenzoate, protocatechuate, and catechol was engineered.
• Catechol titers reached up to 451 ± 44 mg/L at 14.3% of theoretical max.
• A batch bioreactor lead to improved catechol production.
• The pathway can further serve as a platform for producing cis,cis-muconate.

p-Hydroxybenzoate, protocatechuate, and catechol represent fine and/or commodity chemicals useful as antioxidants and building-block molecules. To date, however, these species have been largely overlooked as focal end-products. An existing route employing protocatechuate and catechol as intermediates suffers from the need for multiple auxotrophies to preserve precursor (3-dehydroshikimate) availability. A novel, modular route from endogenous p-hydroxybenzoate has been engineered in Escherichia coli for the individual biosynthesis of all three products from renewable glucose while minimizing auxotrophy generation. To enhance endogenous biosynthesis of p-hydroxybenzoate, native chorismate pyruvate lyase (ubiC) was over-expressed. p-Hydroxybenzoate was converted to protocatechuate by a hydroxylase (pobA) from Pseudomonas aeruginosa. Catechol was produced by the additional co-expression of protocatechuate decarboxylase from Enterobacter cloacae. Systematic expression of appropriate pathway elements in phenylalanine overproducing E. coli enabled initial titers of 32 ± 4, 110 ± 8, and 81 ± 15 mg/L for p-hydroxybenzoate, protocatechuate, and catechol, respectively. Disruption of chorismate mutase/prephenate dehydratase (pheA) to preserve endogenous chorismate then allowed maximum titers of 277 ± 2, 454 ± 11, and 451 ± 44 mg/L, respectively, at glucose yields of 5.8, 9.7, and 14.3% of their respective theoretical maxima. Catechol titers were further improved to 630 ± 37 mg/L in a batch bioreactor study. The proposed pathway can furthermore serve as a platform for other bioproducts, including the bioplastics precursor cis,cis-muconate.