The engineering of non-natural pathways is a promising approach to renewable alternatives to conventional petrochemicals. Specific examples include the engineering of <i>Escherichia coli</i> to produce phenol, catechol, and muconic acid directly from glucose. Established biosynthetic routes to all three products suffer from inherent limitations. Phenol can be synthesized from endogenous tyrosine via tyrosine phenol lyase (TPL); TPL suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. Muconic acid can be produced via a one-step extension from catechol (catechol 1,2-dioxygenase), which itself is synthesized from endogenous 3-dihydroshikimate (3DHS) via two heterologous steps (3DHS dehydratase and protocatechuate decarboxylase). 3DHS is a key intermediate in the shikimic acid pathway; significant over-production of both catechol and muconic acid requires deletion of shikimate dehydrogenase (<i>aroE</i>), resulting in auxotrophies necessitating expensive medium formulations. To address these limitations, we have explored multiple novel routes to each of the same three products, all of which stem from endogenous chorismate. In contrast to the established routes, chorismate lies at a strongly rigid metabolic node, facilitating endogenous over-production with minimal disruption of native function. Through development of a modular network, we have engineered two novel pathways to phenol and four to both catechol and muconic acid. Each pathway incorporates different heterologous enzymes and chemistries, resulting in greater reductions in net free energy change compared to established routes. In the case of catechol and muconic acid, the novel routes offer a 2-fold greater reduction in the net free energy change of reaction than the established route (-1037.4 vs. -508.5 kJ/mol). Phenol, catechol, and muconic acid titers as high as 315 ± 26, 631 ± 37, and 813 ± 48 mg/L have been achieved thus far, with strategies to increase final product titers underway. These include pathway de-bottlenecking by development of optimal expression strategies and re-engineering of the host metabolism to maximize production and availability of respective precursors. The most recent findings from these alternative pathways will be contrasted with established routes to provide further insight regarding the biosynthesis of phenol, catechol, and muconic acid, as well as best practices for engineering of non-natural pathways.

Engineering a modular network of nonnatural pathways for aromatic chemicals and muconic acid

249th National Meeting (2015)

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