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

Abstract

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 α,β-unsaturated carboxylic acids (α,β-UCAs) using an engineered reversal of the β-oxidation (r-BOX) cycle. To increase the availability of both acyl-CoAs and enoyl-CoAs for α,β-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 YdiI 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 α,β-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 YdiI led to crotonic acid production at titers reaching 1.5g/L in flask cultures and 3.2g/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 0.2g/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 α,β-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 α,β-UCAs.

Escherichia coli as a host for metabolic engineering

“…Utilizing enzymes of β-oxidation, the iterative nature of Claisen condensation of acyl-CoA with acetyl-CoA enabled the synthesis of many higher alcohols such as hexanol and octanol, while butanol was produced with similar titer than the engineered Clostridial pathway (Dellomonaco et al, 2011). Using this pathway, the CoA-hydrolyzed products such as hydroxy acids, α-β unsaturated acids, and saturated carboxylic acids of the intermediates in the pathway were also produced (Clomburg et al, 2012;Kim et al, 2016).…”

Selective production of decanoic acid from iterative reversal of β‐oxidation pathway

“…As a first step to assess the viability of producing decanoic acid (DA) as the primary product from the β‐oxidation reversal (r‐BOX), we used a fermentation‐ and thioesterase‐deficient strain that already generates medium‐chain acyl‐CoAs (strain JST07). While the functional r‐BOX can be constructed solely using endogenous genes of E. coli (Clomburg, Vick, Blankschien, Rodríguez‐moya, & Gonzalez, ; Vick et al, ), the heterogenous enzymes responsible for both the initial carbon‐carbon bond formation and enoyl‐CoA reduction step were used for this study due to their better performance in producing targeted products (Kim et al, ; Kim et al, ) . The following r‐BOX enzymes were integrated into the chromosome of JST07 under cumate inducible promoter (referred to as strain JST10) (genes shown in parenthesis): (i) β‐ketothiolase from Ralstonia eutropha ( bktB ); (ii) 3‐hydroxyacyl‐CoA dehydrogenase/enoyl‐coA hydratase from E. coli ( fadB ); and (iii) trans enoyl‐CoA reductase from Euglena gracilis ( egTER ) (Kim et al, ).…”

“…The identity of DA was verified using GC‐MS (Figure b). As JST10 expresses a medium‐chain thiolase (BktB), which primarily generates acyl‐CoAs in the C4‐C10 range (Clomburg et al, ; Kim et al, ; Kim et al, ), and FadM's chain length specificity dictates a preference for longer‐chain acyl‐CoAs with little activity on C4‐C8 acyl‐CoAs (Nie et al, ), the combination of both creates an overlap between chain elongation (thiolase) and termination (thioesterase) that favors production of DA (Figure c).…”

“…A previously described modified MOPS medium with glycerol as a major carbon source (Kim et al, ) was used for all cultivations. Fermentations for DA production were conducted either in 25 ml flasks or in 500 ml cylindrical fermentor vessels using higher concentration of glycerol, as described below.…”

“…As an alternative to the FAB pathway, a reversal of the β‐oxidation (r‐BOX) pathway has shown some promise as an iterative platform for carbon elongation that could support the production of DA (Kim, Clomburg, & Gonzalez, ). The encouraging results have been attributed to the high ATP efficiency and orthogonal nature of this pathway, which in turn makes it independent from complex cellular regulation like that affecting the FAB pathway (Cheong, Clomburg, & Gonzalez, ; Kim, Cheong, & Gonzalez, ).…”