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
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...
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Published in | Applied and environmental microbiology Vol. 84; no. 7; p. e02307-17 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Microbiology
01.04.2018
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Citation Fast AG, Papoutsakis ET. 2018. 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. Appl Environ Microbiol 84:e02307-17. https://doi.org/10.1128/AEM.02307-17. |
ISSN: | 0099-2240 1098-5336 |
DOI: | 10.1128/AEM.02307-17 |