초록
<P>Acetogenic bacteria use CO and/or CO<SUB>2</SUB> plus H<SUB>2</SUB> as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen <I>Clostridium autoethanogenum</I> is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the <I>C. autoethanogenum</I> enzymes for lactate and 2,3-butanediol biosynthesis. The putative <I>C. autoethanogenum</I> lactate dehydrogenase was active when expressed in <I>Escherichia coli</I>. The 2,3-butanediol pathway was reconstituted in <I>E. coli</I> by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting <I>E. coli</I> strain produced 1.1 ± 0.2 mM 2<I>R</I>,3<I>R</I>-butanediol (23 μM h<SUP>−1</SUP> optical density unit<SUP>−1</SUP>), which is comparable to the level produced by <I>C. autoethanogenum</I> during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that <I>C. autoethanogenum</I> can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from <I>C. autoethanogenum</I>, identify CaADH as a target for further engineering, and demonstrate the potential of <I>C. autoethanogenum</I> as a platform for sustainable chemical production.</P>