Synthesis of Isobutanol and n-Butanol Using Engineered Coenzyme a-Dependent Pathways in Ralstonia Eutropha H16

The use of Coenzyme A-dependent pathways has been well established at a primary method for the biosynthesis of varying chain-length alcohols for use in fuels and chemicals. However, only linear chain alcohols have been able to be produced though this pathway. Recently a new enzyme, isobutryl-CoA mutase, has been theorized to convert linear acyl-CoA molecules to branched molecules. In this work, we characterize this enzyme, and demonstrate the ability to generate isobutanol in Ralstonia eutropha via a renewable feedstock. This is the first example of isobutanol production via a renewable feedstock. The Ralstonia eutropha strain was initially engineered via heterologous gene expression, codon optimization, and promoter strength manipulation of the engineered production pathway to achieve a strain capable of producing ~200mg/L n-butanol via fructose or ~30mg/L via formate. After strain optimization, the isobutryl-CoA mutaste was added to generate isobutanol at ~30mg/L via fructose. This work generated a novel pathway for the production of isobutanol. Further exploration of this enzyme class could expand the biological toolkit available for the bioproduction of other previously unattainable chemicals from a renewable source.