Dataset Information

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO₂ and Oxidation of CO by Clostridium acetobutylicum.

ABSTRACT: With recent advances in synthetic biology, CO₂ could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO₂ 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 CO₂ 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 CO₂, 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 CO₂ 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 CO₂ reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO₂ reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO₂ 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 CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO₂ fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO₂ reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. ¹³C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO₂ to CO and oxidize CO during growth on glucose.

SUBMITTER: Carlson ED

PROVIDER: S-EPMC5541230 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

Carlson Ellinor D ED Papoutsakis Eleftherios T ET

Applied and environmental microbiology 20170801 16

With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 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 CO2 to CO, effectively enabling a biological water-gas shift reaction at amb ...[more]

PMID: 28625981

Similar Datasets

Project description: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 CO2 and H2 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 13C-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 CO2 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 13C-tracers was developed and used to confirm functional ACS expression.

Project description:The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.

Dataset Information

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO₂ and Oxidation of CO by Clostridium acetobutylicum.

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets