Synthetic Methylotrophic E. coli proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Escherichia Coli
SUBMITTER: 
James C. Liao
LAB HEAD: James C. Liao
PROVIDER: PXD051688 | Pride | 2024-04-24
REPOSITORIES: Pride
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Project description:Background: Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. Results: The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by genetic adaption in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. Conclusion: The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity. The gene expression was analyzed in the methanol tolerant strain Tol1 in comparison to the C. glutamicumWT. Direct comparison in LB complex medium and analysis of expression response to methanol addition in mCGXII minimal medium with 100 mM glucose.
2015-08-01 | E-GEOD-71590 | biostudies-arrayexpress
Project description:Background: Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. Results: The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by genetic adaption in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. Conclusion: The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.
2015-08-01 | GSE71590 | GEO
Project description:Methylomicrobium buryatense 5GB1 is an obligate methylotroph, which grows on methane or methanol with similar growth rates. Core metabolic pathways are similar on both substrates, but recent studies of methane metabolism suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach as well as a 13C tracer approach have been taken to understand core carbon metabolism in M. buryatense 5GB1 during methanol growth, to determine whether such differences occur. Targeted metabolomics analyses were performed on both methane and methanol cultures to identify metabolic nodes with altered fluxes. Several key metabolites showed significant differences in pool size. Noticeably, 2-keto-3-deoxy-6-phosphogluconate (KDPG) showed much larger pools under methanol culture, suggesting the Entner-Doudoroff (ED) pathway was more active. Intermediates in other parts of metabolism also showed differences in pool sizes under methanol growth. A systematic shift of active core metabolism is proposed to explain the changes. In order to distinguish flux partition differences at the C3-C4 node, 13C tracer analysis was also applied to methanol-grown cultures. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol. The resulting new insights into core metabolism of this methanotroph provide an improved basis for future strain design.
2019-04-17 | GSE110541 | GEO
Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1R / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed.
2014-12-04 | GSE63822 | GEO
Project description:Enrichment culture DFE is capable of fermenting the toxic pollutant dichloromethane (DCM; CH2Cl2) into environmentally benign acetate. The dominant organism in the enrichment culture is a novel bacterium in the Peptococcaceae family, “Candidatus Formimonas warabiya” strain DCMF. This metaproteomic study analysed culture DFE during growth with four substrates – DCM, glycine betaine, choline, and methanol – all of which are primarily, if not solely, metabolised by strain DCMF.
2023-03-11 | PXD037334 | Pride
Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth M-BM- on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1RM-BM- / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed. Methyloversatilis universalis FAM5 grown on methanol and methylamine with two biologial replicates for each condition. RNA-Seq was used for transcriptomics.
2014-12-04 | E-GEOD-63822 | biostudies-arrayexpress
Project description:Apicomplexans, such as malaria-causing Plasmodium species, Toxoplasma, and Cryptosporidium, are parasitic protists that invade cells of virtually every animal, including humans. Despite the great burden on human healthcare and global economy, and the role in the environment, our understanding of the biology of these microorganisms is still very limited. Apicomplexans have evolved a complex cell architecture featuring a range of subcellular compartments, both common to all eukaryotes and unique to the phylum, that enable invasion into the host cell and exploiting its resources for growth and replication, rendering apicomplexans such remarkably successful parasites. In such an intricate subcellular landscape, protein function and location in the cell are tightly linked, which is why localising proteins in the cell is a major strategy in the apicomplexan research. Yet, the majority of proteins in model apicomplexans is unknown. In this project, a state-of-the-art spatial proteomics technology hyperLOPIT has been successfully adapted and applied to a model apicomplexan Toxoplasma gondii. Three independent hyperLOPIT experiments have been conducted on T. gondii strain RH extracellular tachyzoites, the invasive stage of the parasite's life cycle responsible for acute infection, providing the first cell-wide protein location atlas of any apicomplexan. This provides revolutionary insight into the apicomplexan cell organization and biology, including greatly expanded proteomes of secretory invasion organelles, the range of adaptive and regulatory responses of the organelles, and the subcellular distribution of evolutionary selection pressure, innovation, and conservation.
2020-10-14 | PXD015269 | Pride
Project description:The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol. The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol.
2022-08-05 | PXD034138 | Pride
Project description:Effects of rotenone of Arabidopsis cell culture compared to methanol treatment as a control at 0, 3 and 12 hours post treatment
2008-09-26 | E-MEXP-1797 | biostudies-arrayexpress
Project description:Aneuploidy results in decreased cellular fitness in many different species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth and poor prognosis, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is a tumour suppressor that is frequently lost in cancer cells. Here, we used a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. We find that BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. The improved fitness correlates with a gain of extra copies of chromosome 18. Notably, changes in pathways that are known to promote tolerance to aneuploidy are evident immediately upon loss of BRG1, providing an environment where karyotype changes associated with a fitness advantage can be explored. At least in some genetic backgrounds, therefore, loss of the SWI/SNF complex can contribute to tumourigenesis through tolerance of aneuploidy.
2022-03-23 | PXD014954 | Pride