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

α,β-unsaturated carboxylic acids (α,β-UCAs), which have a carbonyl group conjugated with an alkene are very useful building blocks in organic synthesis. Here, we present a new approach to produce α,β-UCAs  using an engineered reversal of the β-oxidation (r-BOX) cycle. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase,  hydroxyacyl-CoA dehydratase and enoyl-CoA reductase were chromosomally integrated under the control of a cumate inducible phage promoter in an E. coli strain engineered to have high levels of acyl-CoAs by deleting mixed-acid fermentation pathways and known thioesterases. Native E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have the ability to convert trans-enoyl-CoAs to the corresponding α,β-UCAs. Coupling of r-BOX with YdiI led to crotonic acid production at titers reaching 1.5 g/L in flask cultures which is nearly 10 times higher than reported with the use of a fatty acid biosynthesis (FAB)-based platform. 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 revealed that differences in ATP availability between r-BOX and FAB platforms led to a significant difference in product synthesis. Taken together, our findings suggest that the r-BOX pathway could be an ideal platform to implement the biological production of α,β-UCAs.