Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids

Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. In this work, we present a new approach to produce alpha,beta-unsaturated carboxylic acids (alpha,beta-UCAs) using an engineered reversal of the beta-oxidation (r-BOX) cycle. To increase the availability of both aryl-CoAs and enoyl-CoAs for alpha,beta-UCA production, we use an engineered Escherichia coli strain devoid of mixed-acid fermentation pathways and known thioesterases. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase were chromosomally overexpressed under the control of a cumate inducible phage promoter. Native E. coli thioesterase Ydil was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding alpha,beta-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. Coupling of r-BOX with Ydil led to crotonic acid production at titers reaching 1.5 g/L in flask cultures and 3.2 g/L in a controlled bioreactor. The engineered r-BOX pathway was also used to achieve for the first time the production of 2-hexenoic acid, 2-octenoic acid, and 2-decenoic acid at a final titer of 0.2 g/L. The superior nature of the engineered pathway was further validated through the use of in silico metabolic flux analysis, which showed the ability of r-BOX to support growth-coupled production of alpha,beta-UCAs with a higher ATP efficiency than the widely used fatty acid biosynthesis pathway. Taken together, our findings suggest that r-BOX could be an ideal platform to implement the biological production of alpha,beta-UCAs. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.