Converting Escherichia coli to a synthetic methylotroph growing solely on methanol
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE153477 | GEO | 2020/09/01
REPOSITORIES: GEO
Similar Datasets
Project description:Reprogramming a non-methylotrophic industrial host, such as Saccharomyces cerevisiae, to a synthetic methylotroph reprents a huge challenge due to the complex regulation in yeast. Through TMC strategy together with ALE strategy, we completed a strict synthetic methylotrophic yeast that could use methanol as the sole carbon source. However, how cells respond to methanol and remodel cellular metabolic network on methanol were not clear. Therefore, genome-scale transcriptional analysis was performed to unravel the cellular reprograming mechanisms underlying the improved growth phenotype.
2024-05-02 | GSE173802 | GEO
Project description:A total gene expression approach was applied to study the methylotrophic nature of B. methanolicus by comparing the gene expression in bacteria grown methylotropic compared to non-methylotrophic. Genes of interest with different gene expression were quantified in the same RNA samples by real-time PCR, confirming the results found in the microarray experiment. Genes of special interest that are expressed higher when grown methylotrophic, were the RuMP pathway genes located on the pBM19. Bacillus methanolicus was grown in minimal media with either methanol or mannitol as carbon source. The experiment was preformed in triplicate, with bacterial cultures grown on 3 different days.
2012-05-22 | E-GEOD-35298 | biostudies-arrayexpress
Project description:Methylotrophy is the ability of organisms to grow at the expense of reduced one-carbon compounds such as methanol or methane. Here, we used transposon sequencing combining hyper-saturated transposon mutagenesis with high-throughput sequencing to define the essential methylotrophy genome of Methylobacterium extorquens PA1, a model methylotroph. To distinguish genomic regions required for growth only on methanol from general required genes, we contrasted growth on methanol with growth on succinate, a non-methylotrophic reference substrate. About 500'000 insertions were mapped for each condition, resulting in a median insertion distance of 5 base pairs. We identified 147 genes and 76 genes as specific for growth on methanol and succinate, respectively, and a set of 590 genes as required under both growth conditions. For the integration of metabolic functions, we reconstructed a genome-scale metabolic model and performed in silico essentiality analysis. In total, the approach uncovered 95 previously not described genes as crucial for methylotrophy, including genes involved in respiration, carbon metabolism, transport, and regulation. Strikingly, regardless of the absence of the Calvin cycle in the methylotroph, the screen led to the identification of the gene for phosphoribulokinase as essential during growth on methanol but not on succinate. Genetic experiments in addition to metabolomics and proteomics revealed that phosphoribulokinase serves a key regulatory function. Our data support a model according to which ribulose-1,5 bisphosphate is an essential metabolite that induces a transcriptional regulator driving one-carbon assimilation.
2017-07-11 | PXD006834 | 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:Here, we established for the first time a proteomic analysis of B. methanolicus MGA3, and we used this approach to compare cells grown on methanol and mannitol at 50°C as well as cells grown on methanol at 37°C and 50°C. Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD-dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1,200 different detected proteins, approximately 1,000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the expression of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional TCA cycle that is differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. Protein Identification: Raw data of gel slices with equal molecular weight were loaded together into the software package Progenesis LCMS Version. 4.1 (www.nonlinear.com), a software tool developed for label-free quantification of LC–MS data. Data was analyzed according to Progenesis LCMS analysis in Weisser et al., In brief, for data loading, the option "High Mass Accuracy Instrument" was selected. LC–MS data were normalized and aligned according to manufacturer’s specifications. In the aligning step manual seeding of "three to five" vectors along the retention time gradient was performed followed by automatic alignment. For the identification of peptide features in the corresponding mass spectrometry files, Mascot generic files (.mgf file format) are generated with Progenesis LCMS (using up to five tandem mass spectra for each feature with the top 200 fragment ion peaks ,charge deconvolution and de-isotopting activated). Mgf files were searched by Mascot 2.3 search engine against an amino acid database containing 20,399 entries including 3,418 MGA3 annotated proteins (downloaded from Uniprot on August 2013), 6,651 yeast proteins, 261 known mass spectrometry contaminants as forward entries and from all forward entries except the contaminats, a reverse decoy sequence. Parameters for precursor tolerance and fragment ion tolerance were set to ± 10 ppm and ± 0.6 Da, respectively. Mascot results were loaded into Scaffold v4.05, using the options for protein clustering and high mass accuracy PeptideProphet scoring.
2014-01-28 | PXD000638 | Pride
Project description:Here, we established for the first time a proteomic analysis of B. methanolicus MGA3, and we used this approach to compare cells grown on methanol and mannitol at 50°C as well as cells grown on methanol at 37°C and 50°C. Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD-dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1,200 different detected proteins, approximately 1,000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the expression of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional TCA cycle that is differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. Protein Identification: Raw data of gel slices with equal molecular weight were loaded together into the software package Progenesis LCMS Version. 4.1 (www.nonlinear.com), a software tool developed for label-free quantification of LC–MS data. Data was analyzed according to Progenesis LCMS analysis in Weisser et al., In brief, for data loading, the option "High Mass Accuracy Instrument" was selected. LC–MS data were normalized and aligned according to manufacturer’s specifications. In the aligning step manual seeding of "three to five" vectors along the retention time gradient was performed followed by automatic alignment. For the identification of peptide features in the corresponding mass spectrometry files, Mascot generic files (.mgf file format) are generated with Progenesis LCMS (using up to five tandem mass spectra for each feature with the top 200 fragment ion peaks ,charge deconvolution and de-isotopting activated). Mgf files were searched by Mascot 2.3 search engine against an amino acid database containing 20,399 entries including 3,418 MGA3 annotated proteins (downloaded from Uniprot on August 2013), 6,651 yeast proteins, 261 known mass spectrometry contaminants as forward entries and from all forward entries except the contaminats, a reverse decoy sequence. Parameters for precursor tolerance and fragment ion tolerance were set to ± 10 ppm and ± 0.6 Da, respectively. Mascot results were loaded into Scaffold v4.05, using the options for protein clustering and high mass accuracy PeptideProphet scoring.
2014-01-28 | PXD000637 | Pride
Project description:A total gene expression approach was applied to study the methylotrophic nature of B. methanolicus by comparing the gene expression in bacteria grown methylotropic compared to non-methylotrophic. Genes of interest with different gene expression were quantified in the same RNA samples by real-time PCR, confirming the results found in the microarray experiment. Genes of special interest that are expressed higher when grown methylotrophic, were the RuMP pathway genes located on the pBM19.
2012-05-23 | GSE35298 | 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:P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde and formate) oxidation—yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida (both in EM42 and a double mutant of the characterized formate dehydrogenases in this organism.
2023-04-19 | GSE229763 | GEO
Project description:Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation offers a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as sole source of carbon and energy for growth, Methylorubrum extorquens DM4 (formerly named Methyobacterium extorquens) is a longstanding reference Alphaproteobacteria strain. Here, its primary transcriptome was obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM.
2022-06-27 | E-MTAB-11726 | biostudies-arrayexpress