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Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

ABSTRACT: Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO₂, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO₂ reductase (HDCR). We observe an increase in ferredoxin-dependent CO₂ reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.

SUBMITTER: Moon J

PROVIDER: S-EPMC11208171 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

Moon Jimyung J Poehlein Anja A Daniel Rolf R Müller Volker V

Nature communications 20240626 1

Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO<sub>2</sub>, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. ...[more]

PMID: 38926344

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Project description:UnlabelledThe methylenetetrahydrofolate reductase (MTHFR) of acetogenic bacteria catalyzes the reduction of methylene-THF, which is highly exergonic with NADH as the reductant. Therefore, the enzyme was suggested to be involved in energy conservation by reducing ferredoxin via electron bifurcation, followed by Na(+) translocation by the Rnf complex. The enzyme was purified from Acetobacterium woodii and shown to have an unprecedented subunit composition containing the three subunits RnfC2, MetF, and MetV. The stable complex contained 2 flavin mononucleotides (FMN), 23.5 ± 1.2 Fe and 24.5 ± 1.5 S, which fits well to the predicted six [4Fe4S] clusters in MetV and RnfC2. The enzyme catalyzed NADH:methylviologen and NADH:ferricyanide oxidoreductase activity but also methylene-tetrahydrofolate (THF) reduction with NADH as the reductant. The NADH:methylene-THF reductase activity was high (248 U/mg) and not stimulated by ferredoxin. Furthermore, reduction of ferredoxin, alone or in the presence of methylene-THF and NADH, was never observed. MetF or MetVF was not able to catalyze the methylene-THF-dependent oxidation of NADH, but MetVF could reduce methylene-THF using methyl viologen as the electron donor. The purified MTHFR complex did not catalyze the reverse reaction, the endergonic oxidation of methyl-THF with NAD(+) as the acceptor, and this reaction could not be driven by reduced ferredoxin. However, addition of protein fractions made the oxidation of methyl-THF to methylene-THF coupled to NAD(+) reduction possible. Our data demonstrate that the MTHFR of A. woodii catalyzes methylene-THF reduction according to the following reaction: NADH + methylene-THF → methyl-THF + NAD(+). The differences in the subunit compositions of MTHFRs of bacteria are discussed in the light of their different functions.ImportanceEnergy conservation in the acetogenic bacterium Acetobacterium woodii involves ferredoxin reduction followed by a chemiosmotic mechanism involving Na(+)-translocating ferredoxin oxidation and a Na(+)-dependent F1Fo ATP synthase. All redox enzymes of the pathway have been characterized except the methylenetetrahydrofolate reductase (MTHFR). Here we report the purification of the MTHFR of A. woodii, which has an unprecedented heterotrimeric structure. The enzyme reduces methylene-THF with NADH. Ferredoxin did not stimulate the reaction; neither was it oxidized or reduced with NADH. Since the last enzyme with a potential role in energy metabolism of A. woodii has now been characterized, we can propose a quantitative bioenergetic scheme for acetogenesis from H2 plus CO2 in the model acetogen A. woodii.

Project description:Acetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5' untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5'-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5'-untranslated-region bioparts.

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Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

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Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

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