Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H2:CO feedstock ratios for enhancing carbon capture efficiency

Abstract

Background: Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO₂) gases into bioproducts and fuels via the Wood-Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H₂:CO to maximize CO₂ utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown.

Results: In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO₂, we established controlled chemostats that facilitated a novel and high (11:1) H₂:CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H₂:CO condition where ~ 50% of the carbon in ethanol is derived from CO₂. Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction-oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood-Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H₂:CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels.

Conclusions: This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H₂:CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

Keywords: Clostridium autoethanogenum; Acetogen; Clostridia; Gas fermentation; Lysine-acetylation; Metabolomics; Multi-omics; Post-translational modification; Proteomics; Redox.

Fig. 1

Minimizing CO as a feedstock will increase direct CO₂ consumption by C. autoethanogenum. (a) The Wood–Ljungdahl pathway is the metabolic pathway that C. autoethanogenum uses to convert CO and CO₂ into ethanol and acetate. Truncated locus tags are provided with “CAETHG_” identifier removed. b A chart showing that as the H₂:CO uptake ratio of chemostats increases, direct CO₂ consumption increases with concomitant and inverse decrease of indirect CO₂ consumption. The solid blue line is the percent of carbon that is being consumed directly from CO₂. The dotted green line represents the percent of carbon that is being taken up as CO₂ then converted to CO. c The reaction stoichiometry of ethanol production under various gas feedstocks. The increase in overall carbon conversion, increase in direct CO₂ into product, and decrease in run length are all shown as a result in the stoichiometric ratios increasing H₂:CO uptake ratios. Free energy change of reactions were calculated with eQuilibrator. CODH: CO dehydrogenase; FDH: formate dehydrogenase; FTHFS: formyltetrahydrofolate synthetase; MTHFC: Methenyltetrahydrofolate cyclohydrolase; MTHFD: Methenyltetrahydrofolate dehydrogenase; MTHFR: Methylenetetrahydrofolate reductase; Pta: phosphotransacetylase; Ack: acetatekinase; CODH/ACS acetyl-CoA synthase/carbon monoxide dehydrogenase complex; Adh/AdhE: Alcohol Dehydrogenase; AOR: acetaldehyde:ferredoxin oxidoreductase; LdhA: Lactate Dehydrogenase; AlsS: Acetolactate synthase catabolic; BudA: acetolactate decarboxylase; 2,3-BDH: 2,3-Butanediol Dehydrogenase; MSW: Municipal solid waste; rWGS: reverse water–gas shift

Fig. 2

Change in H₂:CO ratio shifts chemostats gas uptake profiles while maintaining steady end-product formation. Comparison of major metabolites (A), molar gas uptake rates (B), and observed H₂:CO uptake ratios (C) during triplicate fermentations conducted under 5:1 and 11:1 target uptake condition. Vertical lines indicate when -omics samples were obtained for 5:1 target uptake (solid) and 11:1 target uptake (dashed) conditions

Fig. 3

Lysine-acetylation in Clostridium autoethanogenum was observed in unusually high percentages A diagram of the WLP with percent abundance of acetylated peptides comparing the high and low H₂:CO conditions. The low H₂:CO condition is represented with red, and the high H₂:CO condition in blue. Y-axis for each bar graph represents the percentage of acetylated peptide abundance to total proteome abundances.

Fig. 4

Methylation of WLP proteins. A bar chart depicting the percentage of methylated peptides in proportion to total detected peptides for individual WLP proteins. The red bars represent the 5:1 H₂:CO ratio condition and the blue the 11:1 condition