环境胁迫下大肠杆菌中心碳代谢关键酶酶活性的响应Enzyme Activity in E. coli Central Carbon Metabolism in Response to Environmental Stress

Bioprocess ◽  
2013 ◽  
Vol 03 (03) ◽  
pp. 32-37
Author(s):  
申 铁
Keyword(s):  
Enzyme Activity ◽  
Environmental Stress ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
E Coli ◽  
Central Carbon

scholarly journals The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 447
Author(s):  
Klaudyna Krause ◽  
Monika Maciąg-Dorszyńska ◽  
Anna Wosinski ◽  
Lidia Gaffke ◽  
Joanna Morcinek-Orłowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Protein Interactions ◽  
Carbon Metabolism ◽  
Molecular Mechanisms ◽  
Central Carbon Metabolism ◽  
Replication Initiation ◽  
Cell Physiology ◽  
E Coli ◽  
Central Carbon

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

scholarly journals A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

2013 ◽  
Vol 80 (2) ◽  
pp. 564-573 ◽  
Author(s):  
Nobutaka Nakashima ◽  
Satoshi Ohno ◽  
Katsunori Yoshikawa ◽  
Hiroshi Shimizu ◽  
Tomohiro Tamura
Keyword(s):  
Escherichia Coli ◽  
Carbon Metabolism ◽  
Target Genes ◽  
Central Carbon Metabolism ◽  
Accurate Information ◽  
Content Type ◽  
E Coli ◽  
Antisense Rnas ◽  
Central Carbon ◽  
Multiple Gene Silencing

ABSTRACTWe describe here the construction of a series of 71 vectors to silence central carbon metabolism genes inEscherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared toin silicopredictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that anyE. colistrain can be used and multiple gene silencing is easily possible in any combination.

scholarly journals Dissection of Central Carbon Metabolism of Hemoglobin-ExpressingEscherichia coli by 13C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

2001 ◽  
Vol 67 (2) ◽  
pp. 680-687 ◽  
Author(s):  
Alexander D. Frey ◽  
Jocelyne Fiaux ◽  
Thomas Szyperski ◽  
Kurt Wüthrich ◽  
James E. Bailey ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Carbon Metabolism ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Distribution Analysis ◽  
E Coli ◽  
13C Nuclear Magnetic Resonance ◽  
Central Carbon ◽  
Nuclear Magnetic

ABSTRACT Escherichia coli MG1655 cells expressingVitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and theA. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional13C-labeling of the proteinogenic amino acids and two-dimensional [13C, 1H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressingA. eutrophus FHP or the truncated heme domain (FHPg).E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressingA. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and d-lactate. The implications of these observations for biotechnological applications are discussed.

scholarly journals Thermodynamic constraints are sufficient for the emergence of flux sensors in metabolism

2020 ◽  
Author(s):  
Christian Karl Euler ◽  
Radhakrishnan Mahadevan
Keyword(s):  
Carbon Metabolism ◽  
Thermodynamic Data ◽  
Central Carbon Metabolism ◽  
Regulatory Mechanisms ◽  
E Coli ◽  
Regulatory Interactions ◽  
Central Carbon ◽  
Metabolite Binding ◽  
Flux Sensor

AbstractMetabolism is a precisely coordinated phenomenon, the apparent goal of which is to balance fluxes to maintain robust growth. However, coordinating fluxes requires information about rates, which is not obviously reconcilable with known regulatory mechanisms in which concentrations are sensed through metabolite binding. While flux sensor examples have been characterized, the fundamental principles underlying the phenomenon in general are not well understood. Specifically, the questions of which fluxes can be sensed, and the mechanism by which they are remain open. We address this by showing that the concentrations of substrates of thermodynamically constrained reactions reflect upstream flux and therefore carry information about rates which can be propagated through regulatory interactions to control other fluxes in the network. Using fluxomic, metabolomic, and thermodynamic data in E coli, we show that the concentrations of a few metabolites in central carbon metabolism reflect their producing fluxes and demonstrate that they can transmit information about these rates because of their positions in the network and their roles as effectors.

scholarly journals Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

1999 ◽  
Vol 181 (21) ◽  
pp. 6679-6688 ◽  
Author(s):  
Uwe Sauer ◽  
Daniel R. Lasko ◽  
Jocelyne Fiaux ◽  
Michel Hochuli ◽  
Ralf Glaser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Metabolism ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
E Coli ◽  
Chemostat Cultures ◽  
Central Carbon ◽  
Pep Carboxykinase

ABSTRACT The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. colicells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

scholarly journals Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis inEscherichia coli

2017 ◽  
Author(s):  
Nicholas Horvath ◽  
Michael Vilkhovoy ◽  
Joseph A. Wayman ◽  
Kara Calhoun ◽  
James Swartz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Organic Acids ◽  
Carbon Metabolism ◽  
Protein Production ◽  
Central Carbon Metabolism ◽  
Free Protein ◽  
E Coli ◽  
Central Carbon ◽  
Genome Scale ◽  
Cell Free Protein Synthesis

AbstractCell-free protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamicE. colicell-free protein synthesis (CFPS) models. Model parameters were estimated from a training dataset for the cell-free production of a protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluco-neogenesis. Amino acid biosynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-freeE. colisystem, and should provide a foundation for genome scale, dynamic modeling of cell-freeE. coliprotein synthesis.

scholarly journals Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Uri Barenholz ◽  
Dan Davidi ◽  
Ed Reznik ◽  
Yinon Bar-On ◽  
Niv Antonovsky ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Carbon Metabolism ◽  
Quantitative Proteomics ◽  
Central Carbon Metabolism ◽  
Branch Points ◽  
E Coli ◽  
The Core ◽  
Intermediate Metabolites ◽  
Central Carbon ◽  
Substrate Saturation

A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites. As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage.

scholarly journals Characterisation of putative lactate synthetic pathways of Coxiella burnetii

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255925
Author(s):  
Janine Hofmann ◽  
Mebratu A. Bitew ◽  
Miku Kuba ◽  
David P. De Souza ◽  
Hayley J. Newton ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Coxiella Burnetii ◽  
Carbon Metabolism ◽  
Metabolic Pathways ◽  
Malic Enzyme ◽  
Lactate Production ◽  
Central Carbon Metabolism ◽  
Malolactic Enzyme ◽  
Central Carbon

The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.

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