Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO ₂ and Oxidation of CO by Clostridium acetobutylicum

Abstract

With recent advances in synthetic biology, CO 2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO 2 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 CO 2 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 CO 2 , 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 CO 2 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 CO 2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO 2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO 2 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 CODHexpressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms. IMPORTANCE Functional expression of CO dehydrogenase (CODH) fromClostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO 2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO 2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13 C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO 2 to CO and oxidize CO during growth on glucose.KEYWORDS CO 2 fixation, CO 2 reduction, CO oxidation, Wood-Ljungdahl pathway, acetogenesis, carbon dioxide fixation, carbon dioxide reduction, metabolic engineering, synthetic biology, acetogens

Maturation of the [Ni–4Fe–4S] active site of carbon monoxide dehydrogenases

“…On the contrary, the CO production does not depend on Cc CooC when the medium is supplemented in nickel (50–100 µM). This study supports the idea that CooC facilitates nickel insertion into the active site of CODH when nickel is at trace levels [ 42 ].…”

Functional Expression of the Clostridium ljungdahlii Acetyl-Coenzyme A Synthase in Clostridium acetobutylicum as Demonstrated by a NovelIn VivoCO Exchange Activity En Route to Heterologous Installation of a Functional Wood-Ljungdahl Pathway

“…To determine whether in vivo CO 2 reduction could be observed, we adapted the CODH assay that had previously been demonstrated on the heterologously expressed C. carboxidivorans CODH/ACS complex in C. acetobutylicum, whereby the reduced ferredoxin pool drives the reduction of headspace CO 2 to CO (Fig. 3A) (10). The 824 (WLP) strain was grown in defined clostridial growth medium (CGM) with 40 g/liter sugar.…”

“…As a second negative control, we used C. acetobutylicum strains that had been engineered to include a heterologous CODH but not the heterologous ACS. C. acetobutylicum/p94_CODH from Carlson and Papoutsakis (10) and C. acetobutylicum/ In the presence of acetyl-CoA labeled with 13 C at the carbonyl carbon, unlabeled headspace CO is exchanged with the acetyl-CoA, resulting in labeled headspace CO. p95ACS␤ were grown as described above. The CO produced showed 1.1% 13 C labeling throughout the course of the fermentation when grown with either [ 13 C]acetate or [ 12 C]acetate (data not shown), which proves that the differential CO labeling observed for the 824 (WLP) strain is in fact due to the acetyl-CoA exchange activity from the ACS.…”

“…The resulting purified enzyme showed CO oxidation activity, but ACS exchange activity was not observed until the enzyme was incubated in a NiCl 2 solution that restored nickel to the A clusters in the ACS subunits. More recently, the CODH/ACS from Clostridium carboxidivorans was overexpressed in C. acetobutylicum, and the resulting strain exhibited both CO 2 reduction and CO oxidation capabilities in vivo (10). However, ACS activity was not observed.…”

Production and properties of enzymes that activate and produce carbon monoxide

“…Heterologous expression in Clostridium acetobutylicum of CODHs from Clostridium carboxidivorans and C. ljungdahlii was reported recently (Carlson & Papoutsakis, 2017; Fast & Papoutsakis, 2018). Characterization of these enzymes has not yet been reported.…”