Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Clostridium Autoethanogenum
SUBMITTER: 
James Heffernan
LAB HEAD: Dr Esteban
PROVIDER: PXD048143 | Pride | 2024-05-09
REPOSITORIES: pride
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Project description:Gas fermentation of CO₂ and H₂ is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO-to-ethanol and presents an opportunity for CO₂-to-ethanol processes. As we have previously characterized its CO₂/H₂ chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO₂/H₂. Seven ALE lineages were generated, all with improved specific growth rates. Developed with 2% CO supplementation of CO₂/H₂, Evolved lineage D has the highest ethanol/acetate of ALE lineages when fermenting CO₂/H₂. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Complete multi-omics analyses at steady-state revealed that although Evolved D has widespread proteome changes, intracellular metabolites prevent phenotype shifts. Yet, we observe numerous insights to CO₂/H₂ metabolism via these multi-omics results and link these to mutations, suggesting novel targets for metabolic engineering.
2024-05-09 | PXD048075 | Pride
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:We conducted whole genome sequencing on eight evolved E. coli strains (S1–S8) and the parental wild-type (WT) strain to identify mutations arising from ofloxacin treatments. These strains (S1-S8), generated through fluoroquinolone-mediated adaptive laboratory evolution (ALE), exhibited varying levels of tolerance and resistance. The ALE experiment involved intermittent antibiotic treatments of eight independent cultures over 22 days. The untreated WT strain served as a baseline to pinpoint mutations in the evolved strains.
2024-12-23 | E-MTAB-14682 | biostudies-arrayexpress
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:This experiment tests the gene expression of the RelA mutant (ORF03691/GSU2236) versus wild type G. sulfurreducens during log phase growth. Chemostat culture, Acetate-limiting electron donor, Fumarate electron acceptor.
2007-02-15 | E-TIGR-200 | biostudies-arrayexpress
Project description:Prolonged cultivation of Saccharomyces cerevisiae in aerobic, glucose-limited chemostat cultures (dilution rate, 0·10 h–1) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg l–1 after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an ‘evolved’ strain revealed a decreased Km, while Vmax was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose l–1, the specific growth rate of the evolved strain was lower than that of the parental strain (0·28 and 0·37 h–1, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type S. cerevisiae are exposed to excess glucose, the evolved strain exhibited a delay of 90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary ‘driving force’ for several of the observed changes remains to be elucidated Keywords: evolution
2007-08-31 | GSE8898 | GEO
Project description:Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to understand how stable polymorphism arose in an Escherichia coli population that evolved for 765 generations under continuous glucose limitation. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguish evolved clones from their common ancestor. However, when co-evolved clones are cultured separately their transcriptional profiles differ markedly from that ancestor, and do so in ways that are consistent with our understanding of how E. coli adapts to glucose limitation. All adaptive clones exhibit reduced activity of the stationary-phase sigma factor ÏS and increased expression of glucose transport genes, including the glycoporin LamB and the galactose transporter MglABC. Other expression differences, such as up-regulation of acetyl-CoA synthetase, are clone-specific and confirm previous reports of acetate cross-feeding in this system. When co-evolved clones are cultured together, transcription profiling reveals another class of genes whose expression in the dominant clone differs from that observed when the clone is cultured by itself. Many of these genes are part of the CpxR-mediated stress response. CpxR activation in monoculture likely results from extracellular accumulation of acetate that is removed by acetate-scavenging strains in co-culture. Targeted gene sequencing reveals that global regulatory mutations in ÏS as well as small-scale regulatory mutations in the maltose and acetyl CoA synthetase operons contribute to the evolution of cross-feeding. Finally, we identified two mutations in the founder that likely pre-disposed the experimental population to develop specialists that thrive on overflow metabolites. Subsequent mutations that lead to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Observations that polymorphism readily evolves in an asexual population, that adaptive mutants arise without large-scale change in genome architecture, and that morphs have both common and unique patterns of gene expression influenced by whether they are cultured separately or together, underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes. Four isolates of E. coli evolved under long-term glucose limitation were grown in glucose-limited chemostat culture to steady state. Each isolate was grown separately (i.e in monoculture) in triplicate and three of the four (CV101, CV103, CV115 and CV116) were grown as a consortium in duplicate. Each experiment, or biological replicate, was sampled twice for monoculture experiments and once for consortium experiments. Total RNA from each sample was competitvely hybridized against total RNA from the common ancestor of the isolates grown at the same time in a separate chemostat using the same feed medium. For monoculture experiments, three hybridizations were done for each experiment: two hybridizations comparison for the first sample and a single hybridization for the second (with the exception of sample Jv116-1A which was only hybridized twice due to lack of sufficient RNA). For consortium experiments, two hybridizations were done for each biological replicate.
2010-05-16 | E-GEOD-17276 | biostudies-arrayexpress
Project description:Clostridium ljungdahlii not only utilizes CO, but also H2 as energy source during autotrophic growth. In theory, CO is a more energetically and thermodynamically favourable energy source than H2 in the gas fermentation of C. ljungdahlii. However, how C. ljungdahlii conserves energy for growth and ethanol/acetate formation grown on CO or CO2/H2 is not in great detail. In this study, C. ljungdahlii was fermented on CO and CO2/ H2 at pH 6.0 with 0.1 MPa gas pressure. C. ljungdahlii produced 27 g/L acetate, 9 g/L ethanol, 8 g/L 2,3-butanediol and traces of lactate in the presence of CO as energy source, while it produced 25.8 0.1 g/L acetate, 1.8 0.1 g/L ethanol, 0.7 0.01g/L 2,3-butanediol and trances of lactate in the same fermentation condition using H2 as energy source. Therefore, comparative transcriptomes between cells grown on CO and cells grown on H2/CO2 were performed to investigate gene expression profiles based on three biological replicates.
2020-04-01 | E-MTAB-8260 | biostudies-arrayexpress
Project description:Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C. autoethanogenum shifts from acetate to ethanol production to maintain ATP homeostasis at higher biomass concentrations but reaches a limit at a molar acetate/ethanol ratio of ∼1. This regulatory mechanism eventually leads to depletion of the intracellular acetyl-CoA pool and collapse of metabolism. We accurately predict growth phenotypes using a genome-scale metabolic model. Modeling revealed that the methylene-THF reductase reaction was ferredoxin reducing. This work provides a reference dataset to advance the understanding and engineering of arguably the first carbon fixation pathway on Earth.
2017-05-22 | GSE90792 | GEO
Project description:In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Experiment Overall Design: Cultivation of microorganisms in chemostats offers numerous advantages for studying the structure and regulation of metabolic networks (11). In chemostat cultures, individual culture parameters can be changed, while keeping other relevant physical and chemical culture parameters (composition of synthetic medium, pH, temperature, aeration, etc.) constant. An especially important parameter in this respect is the specific growth rate, which, in a chemostat, is equal to the dilution rate, which can be accurately controlled. This allows the experimenter to investigate the effects of environmental changes or genetic interventions at a fixed specific growth rate, even if these changes result in different specific growth rates in batch cultures. In a chemostat, growth can be limited by a single, selected nutrient. The very low residual concentrations of this growth-limiting nutrient in chemostat cultures alleviate effects of catabolite repression and inactivation. Furthermore, these low residual substrate concentrations prevent substrate toxicity, which, for example, occurs when S. cerevisiae is grown on ethanol or acetate as the carbon source in batch cultures . Experiment Overall Design: The central goal of the present study is to assess to what extent carbon source-dependent regulation of fluxes through central carbon metabolism in S. cerevisiae is regulated at the level of transcription. To this end, we compare the transcriptome of carbon-limited, aerobic chemostat cultures grown on four different carbon sources: glucose, maltose, ethanol, and acetate. Data from the transcriptome analysis are compared with flux distribution profiles calculated with a stoichiometric metabolic network model. Questions that will be addressed are as follows: (i) does glucose-limited aerobic cultivation lead to a complete alleviation of glucose-catabolite repression; (ii) how (in)complete is our understanding of the genes involved in the transcriptional response of S. cerevisiae to four of the most common carbon sources for this yeast; and (iii) to what extent do transcriptome analyses with microarrays provide a reliable indication of flux distribution in metabolic networks?
2008-06-16 | E-GEOD-8895 | biostudies-arrayexpress