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

• Published in: Applied and environmental microbiology Vol. 84; no. 7; p. e02307-17
• Main Authors: Fast, Alan G, Papoutsakis, Eleftherios T
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.04.2018
• Subjects: Acetic acid; Acetone; Aldehyde Oxidoreductases - metabolism; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biotechnology; Butanol; Carbohydrates; Carbon dioxide; Carbon monoxide; Carbon Monoxide - metabolism; Carbonyls; Clostridium; Clostridium - genetics; Clostridium - metabolism; Clostridium acetobutylicum - genetics; Clostridium acetobutylicum - metabolism; Coenzyme A; Coenzyme A Ligases - genetics; Coenzyme A Ligases - metabolism; Dehydrogenases; Enzymes; Exchanging; Gene expression; Gene Expression Regulation, Bacterial; Metabolites; Microorganisms, Genetically-Modified - genetics; Microorganisms, Genetically-Modified - metabolism; Multienzyme Complexes - metabolism; Proteins; Spotlight; Wood; Clostridium acetobutylicum; acetogenesis; CODH/ACS; Wood-Ljungdahl pathway; mixotrophy; acetyl-CoA synthase; carbon monoxide dehydrogenase; metabolic engineering; autotrophy; Clostridium ljungdahlii
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.02307-17

Engineering the Wood-Ljungdahl pathway (WLP) in the established industrial organism would allow for the conversion of carbohydrates into butanol, acetone, and other metabolites at higher yields than are currently possible, while minimizing CO and H release. To this effect, we expressed 11 core genes coding for enzymes and accessory proteins of the WLP in The engineered WLP in showed functionality of the eastern branch of the pathway based on the formation of labeled 5,10-methylenetetrahydrofolate from C-labeled formate, as well as functionality of the western branch as evidenced by the formation of CO from CO 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 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 the classical 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 The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from was heterologously expressed in the obligate heterotroph 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 Significantly, a novel assay for ACS exchange activity using C-tracers was developed and used to confirm functional ACS expression.