title: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
authors.forename: Seohyoung
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
authors.forename: Seokjung
authors.initials: S
authors.lastname: Cheong
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA; Department of Bioengineering, Rice University, Houston, TX, USA. Electronic address: Ramon.Gonzalez@rice.edu.
authors.forename: Ramon
authors.initials: R
authors.lastname: Gonzalez
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology*; D002264:Carboxylic Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D008970:Molecular Weight
chemical_list: D002264:Carboxylic Acids; D029968:Escherichia coli Proteins
keywords: 2-butenoic acid; 2-decenoic; 2-hexenoic acid; 2-octenoic acid; Metabolic engineering; Synthetic biology; α,β-unsaturated carboxylic acids; β-oxidation reversal