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

Engineering the Wood-Ljungdahl pathway (WLP) in the established industrial organism Clostridium acetobutylicum would allow for the conversion of carbohydrates into butanol, acetone, and other metabolites at higher yields than are currently possible, while minimizing CO 2 and H 2 release. To this effect, we expressed 11 Clostridium ljungdahlii core genes coding for enzymes and accessory proteins of the WLP in Clostridium acetobutylicum The engineered WLP in C. acetobutylicum showed functionality of the eastern branch of the pathway based on the formation of labeled 5,10-methylenetetrahydrofolate from 13 C-labeled formate, as well as functionality of the western branch as evidenced by the formation of CO from CO 2 However, the lack of labeling in acetate and butyrate pools indicated that the connection between the two branches is not functional. The focus of our investigation then centered on the functional expression of the acetyl-coenzyme A (CoA) synthase (ACS), which forms a complex with the CO dehydrogenase (CODH) and serves to link the two branches of the WLP. The CODH/ACS complex catalyzes the reduction of CO 2 to CO and the condensation of CO with a methyl group to form acetyl-CoA, respectively. Here, we show the simultaneous activities of the two recombinant enzymes. We demonstrate in vivo the classical in vitro ACS carbonyl carbon exchange assay, whereby the carbonyl carbon of acetyl-CoA is exchanged with the CO carbon. Our data suggest that the low heterologous expression of ACS may limit the functionality of the heterologous WLP in C. acetobutylicum IMPORTANCE The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from C. ljungdahlii was heterologously expressed in the obligate heterotroph C. acetobutylicum The functional activity of the CODH was confirmed through both the oxidation and reduction of CO, as had previously been shown for the heterologous CODH from Clostridium carboxidivorans Significantly, a novel in vivo assay for ACS exchange activity using 13 C-tracers was developed and used to confirm functional ACS expression.