Improved growth of Clostridium thermocellum AG553 via medium supplementation with formate and strain evolution
Ontology highlight
ABSTRACT:
ORGANISM(S): Acetivibrio thermocellus DSM 1313
PROVIDER: GSE78287 | GEO | 2017/01/31
SECONDARY ACCESSION(S): PRJNA313094
REPOSITORIES: GEO
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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: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 | PXD048143 | 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:Hydrogenotrophic methanogenic Archaea are defined by a H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. Hydrogenotrophs respond to H2 starvation both phenotypically and at the level of gene expression; however, the responses during growth on formate have not been characterized. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess, suggesting that energy spilling occurs. Using microarray analysis, we show that the expression of genes encoding F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation, but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). The medium contained 380 mM sodium formate in place of NaCl. The agitation rate was 250 revolutions per minute. The gas mixture initially supplied to the chemostat was H2/Ar/CO2/1% H2S (21/125/40/14 mLs min-1) (Proc Natl Acad Sci USA 104: 8930-8934, 2007). After the OD increased above 0.6 (24 h), the medium flow was turned on at 0.083 L/h. Medium was either non-limiting (described above), formate limiting (modified to contain 200 mM sodium formate and 180 mM NaCl), nitrogen limiting (2 mM NH4Cl), or phosphate limiting (0.08 mM K2HPO4). Also at this point, gas flow through the vessel was shut off, a 1 psi check valve was installed on the exhaust line to maintain positive pressure within the vessel while also allowing for exhaust of gaseous metabolic byproducts, and a 1% Na2S9H2O feed was started into the vessel at 0.83 mL/h. 48 hours later, samples were taken for physiological measurements or microarray analysis. Samples were collected for microarray analysis as follows: 1.5 mLs of chemostat culture (OD660 ~0.65) was centrifuged at 13,000 x g for 30 seconds and supernatant was discarded. The pellet was immediately placed in a dry ice/ethanol bath and stored at -80C until RNA extraction and microarrays could be performed. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.
2013-05-03 | E-GEOD-42111 | biostudies-arrayexpress
Project description:Hydrogenotrophic methanogenic Archaea are defined by a H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. Hydrogenotrophs respond to H2 starvation both phenotypically and at the level of gene expression; however, the responses during growth on formate have not been characterized. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess, suggesting that energy spilling occurs. Using microarray analysis, we show that the expression of genes encoding F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation, but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess.
2013-05-03 | GSE42111 | GEO
Project description:In this work, we investigated intracellular pH homeostasis within the thermoacidophilc methanotroph Methylacidiphilum sp. RTK17.1. Our findings show the proton motive force for this species is primarily generated by a pH gradient across the cellular membrane. In batch experiments, the addition of formate resulted in no observable cell growth and, correspondingly, acidification of the cytosol, decreased formate dehydrogenase activity and (presumably) cell-death. Nevertheless, we were able to demonstrable growth on formate as the sole source of metabolizable energy was possible in steady-state (continuous) cultures following the transition from methanol to formate. Under these conditions, biomass productivity yields on formate were 63% less than for growth on methanol. Transcriptome analysis revealed key genes associated with pH homeostasis, methane, methanol and formate metabolism were significantly regulated in response to growth on formate. Collectively, these results suggest environmental formate represents a utilisable source of energy/carbon to the acidophilic methanotrophs during periods of methane starvations and highlights potential short-comings of traditional batch-culture physiological characterisation studies in acidophilic species.
2020-10-31 | GSE145277 | GEO
Project description:The mitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor it is not obvious link between formate and pyrimidine synthesis. Methods Here we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis. We discover that formate induces CAD phosphorylation. Mechanistically formate induces mTORC1 activity as quantified by S6K1 phosphorylation, which is known to phosphorylate CAD and increase its enzymatic activity. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. We conclude that formate activates mTORC1 and induces pyrimidine synthesis via increasing CAD activity.
2020-09-28 | PXD019347 | Pride
Project description:We observed that high extracellular formate concentrations promote the in vitro invasiveness of glioblastoma (GBM) cells. To identify mechanistically, how formates promotes invasion we chose an unbiased approach of RNAseq.
2023-02-06 | GSE196706 | GEO
Project description:The introduction of alternative CO2-fixing pathways such as formate synthesis and assimilation may improve the efficiency of biological carbon fixation that appears to be limited by the enzymatic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we aimed to establish a formate assimilation pathway in the model cyanobacterium Synechocystis sp. PCC 6803. The formate-tetrahydrofolate ligase (FTL) from Methylobacterium extorquens AM1 was expressed in Synechocystis to enable formate assimilation and reduce the loss of fixed carbon in the photorespiratory pathway. Transgenic strains accumulated serine and 3-phosphoglycerate, and consumed more 2-phosphoglycolate and glycine, which seemed to reflect the efficient utilization of formate. However, labelling experiments showed that the serine accumulation was not due to the expected incorporation of formate. DNA-microarray experiments were performed to analyze possible transcriptome changes due to ftl expression. Marked changes in expression of genes encoding proteins associated with serine biosynthesis and enzymes involved in nitrogen and C1 metabolism revealed that ftl expression had a regulatory impact on these metabolic routes. Our results indicate that the expression of new pathways could have a severe impact on the cellular regulatory network, which hampers the establishment of newly designed pathways.
2020-01-17 | GSE143785 | GEO
Project description:Methanol is considered as an interesting carbon source in biobased microbial production processes. As Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies on the response of this organism to methanol. C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be up-regulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The deletion mutants aldadhE and aldmshC were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF recently identified by us. Similar to ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogeneous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.
2013-09-27 | GSE49936 | GEO