Rearrangement of Coenzyme A‑Acylated Carbon Chain Enables Synthesis of Isobutanol via a Novel Pathway in Ralstonia eutropha

Coenzyme A (CoA)-dependent pathways have been explored extensively for the biosynthesis of fuels and chemicals. While CoA-dependent mechanisms are widely used to elongate carbon chains in a linear fashion, branch-making chemistry has not been incorporated. In this study, we demonstrated the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a novel CoA-dependent pathway in recombinant Ralstonia eutropha H16. The designed pathway is constituted of three modules: chain elongation, rearrangement, and modification. We first integrated and optimized the chain elongation and modification modules, and we achieved the production of ∼200 mg/L n-butanol from fructose or ∼30 mg/L from formate by engineered R. eutropha. Subsequently, we incorporated the rearrangement module, which features a previously uncharacterized, native isobutyryl-CoA mutase in R. eutropha. The engineered strain produced ∼30 mg/L isobutanol from fructose. The carbon skeleton rearrangement chemistry demonstrated here may be used to expand the range of the chemicals accessible with CoA-dependent pathways.