A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols

Production of green chemicals and fuels using metabolically engineered organisms has been a promising alternative to petroleum-based production. Higher chain alcohols (C4-C8) are of interest because they can be used as chemical feedstock as well as fuels. Recently, the feasibility of n-hexanol synthesis using Escherichia coli has been demonstrated by extending the modified Clostridium CoA-dependent n-butanol synthesis pathway, thereby elongating carbon chain length via reactions in reversed β-oxidation, (or β-reduction). Here, we developed an anaerobic growth selection platform that allows selection or enrichment of enzymes for increased synthesis of C6 and C8 linear alcohols. Using this selection, we were able to improve the carbon flux towards the synthesis of C6 and C8 acyl-CoA intermediates. Replacement of the original enzyme Clostridium acetobutylicum Hbd with Ralstonia eutropha homologue PaaH1 increased production of n-hexanol by 10-fold. Further directed evolution by random mutagenesis of PaaH1 improved n-hexanol and n-octanol production. This anaerobic growth selection platform may be useful for selecting enzymes for production of long-chain alcohols and acids using this CoA-dependent pathway.