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Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum

Munier, Edouard, Licandro, Hélène, Beuvier, Eric, Cachon, Rémy·2023·International Microbiology
DOI 10.1007/s10123-022-00316-yPMC PMC10397141

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Abstract

Climate change and environmental issues compel us to find alternatives to the production of molecules of interest from petrochemistry. This study aims at understanding the production of butyrate, hydrogen, and CO 2 from the oxidation of lactate with acetate in Clostridium tyrobutyricum and thus proposes an alternative carbon source to glucose. This specie is known to produce more butyrate than the other butyrate-producing clostridia species due to a lack of solvent genesis phase. The recent discoveries on flavin-based electron bifurcation and confurcation mechanism as a mode of energy conservation led us to suggest a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. While searching for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum , we identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii . A phylogenetic approach encompassing other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirmed these results. A metabolic scheme on the production of butyrate, hydrogen, and CO 2 from the lactate-acetate co-metabolism in C. tyrobutyricum was constructed and then confirmed with data of steady-state continuous culture. This in silico metabolic carbon flux analysis model showed the coherence of the scheme from the carbon recovery, the cofactor ratio, and the ATP yield. This study improves our understanding of the lactate oxidation metabolic pathways and the role of acetate and intracellular redox balance, and paves the way for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum .

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Fig. 1 Proposed scheme for the metabolic pathways involving lactate and acetate conversion in C. tyrobutyricum. LctP, lactate permease; LDH, lactate dehydrogenase; PFOR, pyruvate ferredoxine:oxidoreductase; PTA, phosphotransacetylase; ACK, acetate kinase; THL, thiolase; HBD, 3-hydroxybutyryl-CoA dehydrogenase; CRT, crotonase; BCD, butyryl-CoA dehydrogenase; CAT, CoA-transferase; HYD, hydrogenase
Fig. 1 Proposed scheme for the metabolic pathways involving lactate and acetate conversion in C. tyrobutyricum. LctP, lactate permease; LDH, lactate dehydrogenase; PFOR, pyruvate ferredoxine:oxidoreductase; PTA, phosphotransacetylase; ACK, acetate kinase; THL, thiolase; HBD, 3-hydroxybutyryl-CoA dehydrogenase; CRT, crotonase; BCD, butyryl-CoA dehydrogenase; CAT, CoA-transferase; HYD, hydrogenaseCC BY · PubMed Central
Fig. 2 Chromosome fragments (NZ_CP038158.1) of C. tyrobutyricum CIRM BIA 2237 containing EtfAB and FAD-binding oxidoreductase genes. EtfB (1: EZN00_RS10860, 2: EZN00_RS10795, 3: EZN00_RS08615), EtfA (1: EZN00_RS10865, 2: EZN00_RS10800, 3: EZN00_RS08620), FAD-binding oxidoreductase (1:: EZN00_RS10870; 3: EZN00_RS08625)
Fig. 2 Chromosome fragments (NZ_CP038158.1) of C. tyrobutyricum CIRM BIA 2237 containing EtfAB and FAD-binding oxidoreductase genes. EtfB (1: EZN00_RS10860, 2: EZN00_RS10795, 3: EZN00_RS08615), EtfA (1: EZN00_RS10865, 2: EZN00_RS10800, 3: EZN00_RS08620), FAD-binding oxidoreductase (1:: EZN00_RS10870; 3: EZN00_RS08625)CC BY · PubMed Central
Fig. 3 Putative lactate oxidation cluster of C. tyrobutyricum CIRM BIA 2237. Its identity percentage is compared with those from C. ljungdahlii DSM 13,528 et A. woodii DSM 1030. TR, transcriptional regulator (EZN00_RS08600, CLJU_RS10615, AWO_RS0440); L-lactate permease (EZN00_RS08605, CLJU_RS10610, and AWO_RS04425); lactate racemase (EZN00_RS08610, CLJU_RS10605, and AWO_RS04430); EtfB (EZN00_RS08615, CLJU_RS10600, and AWO_RS04410); EtfA (EZN00_RS08620, CLJU_RS10595, and AWO_RS04415); LDH, lactate dehydrogenase (EZN00_RS08625, CLJU_RS10590, and AWO_RS04420)
Fig. 3 Putative lactate oxidation cluster of C. tyrobutyricum CIRM BIA 2237. Its identity percentage is compared with those from C. ljungdahlii DSM 13,528 et A. woodii DSM 1030. TR, transcriptional regulator (EZN00_RS08600, CLJU_RS10615, AWO_RS0440); L-lactate permease (EZN00_RS08605, CLJU_RS10610, and AWO_RS04425); lactate racemase (EZN00_RS08610, CLJU_RS10605, and AWO_RS04430); EtfB (EZN00_RS08615, CLJU_RS10600, and AWO_RS04410); EtfA (EZN00_RS08620, CLJU_RS10595, and AWO_RS04415); LDH, lactate dehydrogenase (EZN00_RS08625, CLJU_RS10590, and AWO_RS04420)CC BY · PubMed Central
Fig. 4 Phylogenetic analysis of Etf complex protein sequences between different species. Orange, EtfAB coupled with acyl-CoA DH; red, EtfAB coupled with Hbd; green, EtfAB coupled FAD-binding oxidoreductase/LDH and lactate permease; blue, EtfAB-coupled FAD-binding oxidoreductase; black, out of any phylogenetic group
Fig. 4 Phylogenetic analysis of Etf complex protein sequences between different species. Orange, EtfAB coupled with acyl-CoA DH; red, EtfAB coupled with Hbd; green, EtfAB coupled FAD-binding oxidoreductase/LDH and lactate permease; blue, EtfAB-coupled FAD-binding oxidoreductase; black, out of any phylogenetic groupCC BY · PubMed Central
Fig. 5 Scheme of the metabolic flux distribution involving lactate and acetate conversion by C. tyrobutyricum. Using an MFA model with 100 mM–C of substrates (83 mM–C from lactate and 17 mM–C from acetate). All values are expressed in % of total carbon. Hatched arrows represent multiple metabolic reactions
Fig. 5 Scheme of the metabolic flux distribution involving lactate and acetate conversion by C. tyrobutyricum. Using an MFA model with 100 mM–C of substrates (83 mM–C from lactate and 17 mM–C from acetate). All values are expressed in % of total carbon. Hatched arrows represent multiple metabolic reactionsCC BY · PubMed Central

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Citations

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Metadata

Authors
Munier, E., Licandro, H., Beuvier, E., & Cachon, R.
Venue
International Microbiology
Publisher
Springer Science and Business Media LLC
Published
2023-01-07
Volume
26
Issue
3
Pages
501-511
ISSN
1618-1905
DOI
10.1007/s10123-022-00316-y
PMC
PMC10397141
Cited by
18
References
1
Language
en
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