Functional Expression of theClostridium ljungdahliiAcetyl-Coenzyme A Synthase inClostridium acetobutylicumas Demonstrated by a NovelIn VivoCO Exchange Activity En Route to Heterologous Installation of a Functional Wood-Ljungdahl Pathway
ABSTRACTEngineering the Wood-Ljungdahl pathway (WLP) in the established industrial organismClostridium acetobutylicumwould allow for the conversion of carbohydrates into butanol, acetone, and other metabolites at higher yields than are currently possible, while minimizing CO2and H2release. To this effect, we expressed 11Clostridium ljungdahliicore genes coding for enzymes and accessory proteins of the WLP inClostridium acetobutylicum. The engineered WLP inC. acetobutylicumshowed functionality of the eastern branch of the pathway based on the formation of labeled 5,10-methylenetetrahydrofolate from13C-labeled formate, as well as functionality of the western branch as evidenced by the formation of CO from CO2. 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 CO2to 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 demonstratein vivothe classicalin vitroACS 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 inC. acetobutylicum.IMPORTANCEThe bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) fromC. ljungdahliiwas heterologously expressed in the obligate heterotrophC. 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 fromClostridium carboxidivorans. Significantly, a novelin vivoassay for ACS exchange activity using13C-tracers was developed and used to confirm functional ACS expression.