Engineered microorganisms offer new routes for the production of fuels and chemicals. Acetogenic Clostridium species can efficiently convert industrial waste gases (carbon monoxide and carbon dioxide) into fuel grade ethanol. Several companies, including Lanzatech Ltd. (Auckland, New Zealand) are seeking to commercialise this microbial fermentation process for bioethanol production. Further, Lanzatech have recently demonstrated that three Clostridium species, including C. autoethanogenum,can produce small amounts of the commodity chemical 2,3-butanediol. This suggests that C. autoethanogenum may be useful for producing valuable metabolites other than ethanol. We have been exploring the protein engineering and metabolic engineering required to produce a route to isopropanol. Isopropanol is a valuable chemical intermediate and a widely used industrial solvent, with a global market exceeding NZ$3 billion per annum.

A strain that can ferment CO/CO₂ and produce isopropanol requires an alcohol dehydrogenase (ADH) that can reduce acetone to isopropanol, with high activity and specificity. We have expressed and purified the C. autoethanogenum ADH. We have shown that it is NADPH-dependent, and it can catalyze the reduction of a broad range of substrates, including acetone, butanone and acetaldehyde.

Next, we aimed to change the co-factor preference of ADH from NADPH to NADH. Since the predicted intracellular pool of NADH is over 20-fold larger than the pool of NADPH, an NADH-dependent enzyme is likely to increase the yield of isopropanol production. Structural alignment of NADH- and NADPH-dependent ADHs from a variety of bacteria was used to identify key residues that may be involved in determining cofactor preference. Site-directed mutagenesis was used to engineer an ADH variant which utilizes only NADH as its co-factor. Unexpectedly, the mutations also narrowed the substrate specificity – improving the specificity of the acetone to isopropanol reduction. Crystallization trials of the wild-type and mutant ADH are underway.