Dataset Information

Proteomics analysis of Methanothermobacter species adapted to growth on carbon monoxide

ABSTRACT: Conversion of biomass-derived syngas (gaseous mixture of mainly H2, CO and CO2) to methane might be a sustainable alternative for the biofuel industry. Via the syngas route more methane can be produced from biomass than via conventional anaerobic digestion. Methanogenic archaea are key players in syngas conversion, but only a few are known to utilize CO (or syngas). Methanothermobacter thermoautotrophicus is one of the few hydrogenotrophic methanogens which has been observed to grow on CO. However, carboxydotrophic growth is slow and is reported to be readily inhibited above 50 kPa CO. The aim of this work was to get more insight of the CO metabolism in hydrogenotrophic archaea and to assess the potential toxic effects of CO towards these microorganisms. Archaeal genomic databases were searched for putative homologues of the Methanothermobacter thermoautotrophicus CODH alpha subunit (containing the catalytic site): the highest scores were for the CODH subunits of Methamothermobacter marburgensis (93% identity) and Methanococcus maripaludis (71%). M. thermoautotrophicus and the other two potential carboxydotrophic strains were incubated with CO and CO + H2 as sole substrates. In addition to M. thermoautotrophicus, M. marburgensis was able to grow methanogenically on CO alone and on CO + H2. In contrast to M. thermoautotrophicus, M. marburgensis was not as strongly inhibited when grown in presence of CO alone and was able to adapt its metabolism, shifting its lag phase from ~500 to ~100 hours. It was observed for both strains that presence of hydrogen stimulates the carbon monoxide metabolism. To gain further insight, the proteome of M. marburgensis culture grown on H2 + CO2 and H2 + CO2 + CO were analysed. Cultures grown with H2 + CO showed relative higher abundance of enzymes involved in CODH/ACS associated reactions and reactions involved in redox metabolism. Overall, the data suggests the strong reducing capacity of CO inhibits the hydrogenotrophic methanogen, making growth on CO as a sole substrate difficult for these organisms.

INSTRUMENT(S): maXis

ORGANISM(S): Acyrthosiphon Pisum (pea Aphid)

SUBMITTER: Hans Wessels

LAB HEAD: Prof. Dr. Alain van Gool

PROVIDER: PXD003661 | Pride | 2016-08-03

REPOSITORIES: Pride

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Publications

Proteomic Analysis of the Hydrogen and Carbon Monoxide Metabolism of Methanothermobacter marburgensis.

Diender Martijn M Pereira Ricardo R Wessels Hans J C T HJ Stams Alfons J M AJ Sousa Diana Z DZ

Frontiers in microbiology 20160704

Hydrogenotrophic methanogenic archaea are efficient H2 utilizers, but only a few are known to be able to utilize CO. Methanothermobacter thermoautotrophicus is one of the hydrogenotrophic methanogens able to grow on CO, albeit about 100 times slower than on H2 + CO2. In this study, we show that the hydrogenotrophic methanogen Methanothermobacter marburgensis, is able to perform methanogenic growth on H2/CO2/CO and on CO as a sole substrate. To gain further insight in its carboxydotrophic metabol ...[more]

PMID: 27458443

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Project description:Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model (GEM). CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a four-fold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by five-fold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin-reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

Project description:Cheap and renewable feedstocks such as the one carbon substrate formate are emerging for sustainable production in a growing chemical industry. By quantitatively analyzing physiology, transcriptome, proteome in chemostat cultivations in combination with computational analyses, we investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates. Continuous cultivations with a specific growth rate of 0.05 h-1 on formate showed high specific substrate uptake rates (47 mmol g‑1 h‑1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcription. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol/g/h) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate+H2/CO2 resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate+H2/CO/CO2, 0.75 ATP/acetate for formate+fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

Dataset Information

Proteomics analysis of Methanothermobacter species adapted to growth on carbon monoxide

Publications

Proteomic Analysis of the Hydrogen and Carbon Monoxide Metabolism of Methanothermobacter marburgensis.

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