Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth
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ABSTRACT:
ORGANISM(S): Eubacterium limosum
PROVIDER: GSE97613 | GEO | 2018/07/16
REPOSITORIES: GEO
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Project description:The Wood-Ljungdahl pathway in acetogens converts C1 compounds, such as CO2 and CO, into acetyl-CoA. Similarly, the glycine synthase pathway assimilates C1 compounds into glycine. Partial glycine synthase genes are widely conserved in the Wood-Ljungdahl pathway gene cluster but functional relationship between the pathways in autotrophic condition remains unknown. To comprehend, we assembled Clostridium drakei genome (5.7-Mbp) with intact glycine synthase pathway and constructed a genome-scale metabolic model, iSL836, predicting increased metabolic flux rates of the Wood-Ljungdahl pathway and the glycine synthase-reductase associated reactions under autotrophic conditions. Along with the observation of significant transcriptional activation of genes in the pathways, surprisingly, 13C-labeling experiments and enzyme activity assays confirmed the strain synthesizes glycine and converts into acetyl-phosphate. This study suggests the Wood-Ljungdahl and the glycine synthase-reductase pathways convert CO2 into acetyl-CoA and acetyl-phosphate, respectively. In our knowledge, this is the first report on co-utilization of the pathways under autotrophic growth in acetogen.
2019-08-01 | GSE118519 | GEO
Project description:Acetogenic bacteria utilize cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as inorganic materials such as photo-responsive semiconductors. To reveal how acetogenic bacteria utilize electrons generated from external energy sources to reduce CO2, we developed a nanoparticle-microbe hybrid system and generated RNA-seq results.
2020-09-09 | GSE157613 | GEO
Project description:Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions – hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) – directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.
2023-05-10 | PXD040151 | Pride
Project description:Clostridium ljungdahlii not only utilizes CO, but also H2 as energy source during autotrophic growth. And C. ljungdahlii also grows in fructose fermentation. In theory, fructose is a more energetically favourable energy source than syngas in the fermentation of C. ljungdahlii. However, C. ljungdahlii grows insufficiently in fructose and produces less acetate and ethanol, compared to syngas fermentation. In this study, C. ljungdahlii wild type and mutants were fermented on fructose. C. ljungdahlii produced less ethanol than the ΔadhE1 mutant and consumed less fructose. The ΔadhE1+2 mutant cannot grow in the syngas fermentation and produced less ethanol among the three strains. The results showed that aldehyde dehydrogenase inactivation led to efficient metabolism in C. ljungdahlii and the bifunctional aldehyde/alcohol dehydrogenases inactivation led to decrease metabolism. Thus, comparative transcriptomes among cells grown on fructose of three strains were performed to investigate gene expression profiles based on three biological replicates.
2023-06-27 | E-MTAB-13020 | biostudies-arrayexpress
Project description:Acetate is a simple carboxylic acid that is synthesized in various microorganisms. Although acetate toxicity and tolerance have been studied in many microorganisms, little is known about the effects of exogenous acetate on the cell growth of acetogenic bacteria. In this study, we report the phenotypic changes that occurred in the acetogenic bacterium Clostridium sp. AWRP as a result of an adaptive laboratory evolution under acetate challenge. When compared with the wild-type strain, the acetate-adapted strain displayed a tolerance to acetate up to 10 g L-1 and higher biomass yields in batch cultures, although the metabolite profiles greatly varied depending on culture conditions. Interestingly, genome sequencing revealed that the adapted strain harbored three point mutations in the genes encoding an electron-bifurcating hydrogenase, which is crucial to its autotrophic growth on CO2 + H2, in addition to one in the dnaK gene. Transcriptome analysis revealed the global change in the gene expression profile of the acetate-adapted strain. Strikingly, most genes involved in CO2-fixing Wood-Ljungdahl pathway and auxiliary pathways for energy conservation (e.g., Rnf complex, Nfn, etc.) were significantly down-regulated. In addition, we observed that a couple of metabolic pathways associated with dissimilation of nucleosides and carbohydrates were significantly up-regulated in the acetate-adapted strain as well as several amino acid biosynthetic pathways, indicating that the strain might increase its fitness by utilizing organic substrates in response to the down-regulation of carbon fixation. Further investigation into the carbon fixation degeneration of the acetate-adapted strain will provide practical implications in CO2 + H2 fermentation using acetogenic bacteria for long-term continuous fermentation. The transcriptome profiles of the wild-type Clostridium sp. AWRP and its acetate-tolerant derivative 46T-a were compared.
2022-06-26 | GSE206757 | GEO
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.
2018-03-05 | PXD008367 | Pride
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.
2023-03-08 | PXD026569 | Pride
Project description:Clostridium ljungdahlii derives energy by acetogenesis as a lithotrophic pathway and as part of various organotrophic pathways. Its recently sequenced genome has made it possible to discover changes in gene expression that occur in different growth modes, from which strategies for biofuel production may be developed. C. ljungdahlii was grown with fructose as an organoheterotrophic substrate and also lithoautotrophically, either on syngas or with H2 as the electron donor and CO2 as the electron acceptor. RNA extracted from all three conditions was analyzed by hybridization to a microarray, and gene expression was compared quantitatively by RNA-Seq of C. ljungdahlii grown with fructose and with H2 and CO2. Results. Strongly upregulated (> 10-fold, p < 0.05) genes with both syngas and H2/CO2 encode enzymes that degrade aspartate and arginine through the urea cycle to control the release of ammonia from amino acids. Numerous genes for uptake and degradation of peptides and amino acids, response to sulfur starvation, and molybdopterin-dependent pathways were also significantly (> 2-fold, p < 0.05) upregulated, along with three potentially NADPH-producing pathways for which the key metabolites are (S)-malate, ornithine and 6-phospho-D-gluconate. Upregulation of genes implicated in quorum sensing, sporulation and cell wall remodeling suggests a global and multicellular response to lithoautotrophic conditions. With syngas, (R)-lactate dehydrogenase and associated electron transfer flavoproteins were upregulated, representing a route of electron transfer from ferredoxin to NAD that is independent of the proton-translocating Rnf complex. With H2/CO2, a flavodoxin and histidine biosynthesis enzymes were upregulated. Genes for degradation of purine bases entering the cell by facilitated diffusion were upregulated in lithotrophic cells, whereas genes for degradation of adenine or adenosine derivatives entering by active transport were significantly (2-fold, p < 0.05) downregulated. The most downregulated genes, after those specific to fructose metabolism, encode enzymes of pyrimidine and purine biosynthesis, arginine fermentation to ornithine, threonine biosynthesis and phosphate uptake. Genes for biogenesis of an intracytoplasmic microcompartment were downregulated, within which (S)-1,2-propanediol dehydratase and other enzymes may dispose of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several redox-active proteins, both cytoplasmic and membrane-associated, and predicted cell surface proteins were identified as differentially regulated. Conclusion. The transcriptomic profiles of C. ljungdahlii in lithoautotrophic and organoheterotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide the production of biofuels and commodity chemicals with this species.
2017-06-01 | E-MTAB-3806 | biostudies-arrayexpress
Project description:The unique capability of acetogens to ferment a broad range of substrates renders them ideal candidates for the biotechnological production of commodity chemicals. In particular the ability to grow with H2:CO2 or syngas (a mixture of H2/CO/CO2) makes these microorganisms ideal chassis for sustainable bioproduction. However, advanced design strategies for acetogens are currently hampered by incomplete knowledge about their physiology and our inability to accurately predict phenotypes. Here we describe the reconstruction of a novel genome-scale model of metabolism and macromolecular synthesis (ME-model) to gain new insights into the biology of the model acetogen Clostridium ljungdahlii. The model represents the first ME-model of a Gram-positive bacterium and captures all major central metabolic, amino acid, nucleotide, lipid, major cofactors, and vitamin synthesis pathways as well as pathways to synthesis RNA and protein molecules necessary to catalyze these reactions, thus significantly broadens the scope and predictability. Use of the model revealed how protein allocation and media composition influence metabolic pathways and energy conservation in acetogens and accurately predicted secretion of multiple fermentation products. Predicting overflow metabolism is of particular interest since it enables new design strategies, e.g. the formation of glycerol, a novel product for C. ljungdahlii, thus broadening the metabolic capability for this model microbe. Furthermore, prediction and experimental validation of changing secretion rates based on different metal availability opens the window into fermentation optimization and provides new knowledge about the proteome utilization and carbon flux in acetogens.
2024-01-11 | MODEL2204200003 | BioModels
Project description:Acetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood–Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterization of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor (sA) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in Escherichia coli. Lastly, a protein–protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.
2019-10-28 | GSE108700 | GEO