With recent advances in synthetic biology, CO₂ could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO₂ fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO₂ to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO₂, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO₂ reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO₂ reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO₂ reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO₂ reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.

IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO₂ fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO₂ reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. ¹³C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO₂ to CO and oxidize CO during growth on glucose.