scholarly journals Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

2002 ◽  
Vol 184 (1) ◽  
pp. 152-164 ◽  
Author(s):  
Marcel Emmerling ◽  
Michael Dauner ◽  
Aaron Ponti ◽  
Jocelyne Fiaux ◽  
Michel Hochuli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glucose Uptake ◽  
Dilution Rate ◽  
Pyruvate Kinase ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Analysis ◽  
E Coli ◽  
General Response

ABSTRACT The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

scholarly journals Flexible Metabolism and Suppression of Latent Enzymes Are Important forEscherichia coliAdaptation to Diverse Environments within the Host

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Christopher J. Alteri ◽  
Stephanie D. Himpsl ◽  
Allyson E. Shea ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Pyruvate Kinase ◽  
Tricarboxylic Acid Cycle ◽  
Financial Burden ◽  
Content Type ◽  
Bacterial Physiology ◽  
E Coli ◽  
Growth Requirements ◽  
Tract Infections

ABSTRACTBacterial metabolism is necessary for adaptation to the host microenvironment. Flexible metabolic pathways allow uropathogenicEscherichia coli(UPEC) to harmlessly reside in the human intestinal tract and cause disease upon extraintestinal colonization.E. coliintestinal colonization requires carbohydrates as a carbon source, while UPEC extraintestinal colonization requires gluconeogenesis and the tricarboxylic acid cycle. UPEC containing disruptions in two irreversible glycolytic steps involving 6-carbon (6-phosphofructokinase;pfkA) and 3-carbon (pyruvate kinase;pykA) substrates have no fitness defect during urinary tract infection (UTI); however, both reactions are catalyzed by isozymes: 6-phosphofructokinases Pfk1 and Pfk2, encoded bypfkAandpfkB, and pyruvate kinases Pyk II and Pyk I, encoded bypykAandpykF. UPEC strains lacking one or both phosphofructokinase-encoding genes (pfkBandpfkA pfkB) and strains lacking one or both pyruvate kinase genes (pykFandpykA pykF) were investigated to determine their regulatory roles in carbon flow during glycolysis by examining their fitness during UTI andin vitrogrowth requirements. Loss of a single phosphofructokinase-encoding gene has no effect on fitness, while thepfkA pfkBdouble mutant outcompeted the parental strain in the bladder. A defect in bladder and kidney colonization was observed with loss ofpykF, while loss ofpykAresulted in a fitness advantage. ThepykA pykFmutant was indistinguishable from wild-typein vivo, suggesting that the presence of Pyk II rather than the loss of Pyk I itself is responsible for the fitness defect in thepykFmutant. These findings suggest thatE. colisuppresses latent enzymes to survive in the host urinary tract.IMPORTANCEUrinary tract infections are the most frequently diagnosed urologic disease, with uropathogenicEscherichia coli(UPEC) infections placing a significant financial burden on the health care system by generating more than two billion dollars in annual costs. This, in combination with steadily increasing antibiotic resistances to present day treatments, necessitates the discovery of new antimicrobial agents to combat these infections. By broadening our scope beyond the study of virulence properties and investigating bacterial physiology and metabolism, we gain a better understanding of how pathogens use nutrients and compete within host microenvironments, enabling us to cultivate new therapeutics to exploit and target pathogen growth requirements in a specific host environment.

scholarly journals Functional analysis of deoxyhexose sugar utilization in Escherichia coli reveals fermentative metabolism under aerobic conditions

2021 ◽  
Author(s):  
Pierre Millard ◽  
Julien Pérochon ◽  
Fabien Letisse
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Sugar Metabolism ◽  
Laboratory Strain ◽  
Flux Analysis ◽  
Aerobic Growth ◽  
Aerobic Conditions ◽  
E Coli ◽  
Metabolic Analysis ◽  
K 12

L-rhamnose and L-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol whereas under aerobic condition, it should be oxidised into lactate and then channelled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on L-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on L-rhamnose and L-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol in anoxic conditions allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on L-fucose, 13 C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strain’s prolonged laboratory use. IMPORTANCE E. coli ’s ability to survive, grow and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolysed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include L-fucose and L-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. coli ’s metabolism and of its functioning in the bacterium’s natural environment.

scholarly journals Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

2013 ◽  
Vol 79 (9) ◽  
pp. 3091-3100 ◽  
Author(s):  
Francesco Falcioni ◽  
Lars M. Blank ◽  
Oliver Frick ◽  
Andreas Karau ◽  
Bruno Bühler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Analysis ◽  
Substrate Uptake ◽  
Mrna Levels ◽  
Microbial Physiology ◽  
Whole Cell ◽  
Content Type ◽  
Proline Hydroxylation

ABSTRACTMicrobial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinantEscherichia coliexpressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alterp4hmRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription ofputAandputP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.

scholarly journals Global Transcription and Metabolic Flux Analysis of Escherichia coli in Glucose-Limited Fed-Batch Cultivations

2008 ◽  
Vol 74 (22) ◽  
pp. 7002-7015 ◽  
Author(s):  
K. Lemuth ◽  
T. Hardiman ◽  
S. Winter ◽  
D. Pfeiffer ◽  
M. A. Keller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Genetic Regulation ◽  
Tca Cycle ◽  
Carbon Limitation ◽  
Oxidative Decarboxylation ◽  
Flux Analysis ◽  
Fed Batch ◽  
K 12

ABSTRACT A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic “work mode.” σS-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

scholarly journals Functional analysis of deoxyhexose sugar utilization in Escherichia coli reveals fermentative metabolism under aerobic conditions

2021 ◽  
Author(s):  
Pierre Millard ◽  
Julien Pérochon ◽  
Fabien Letisse
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Sugar Metabolism ◽  
Laboratory Strain ◽  
Flux Analysis ◽  
Aerobic Growth ◽  
Aerobic Conditions ◽  
E Coli ◽  
Metabolic Analysis ◽  
K 12

ABSTRACTL-rhamnose and L-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol whereas under aerobic condition, it should be oxidised into lactate and then channelled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on L-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on L-rhamnose and L-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol in anoxic conditions allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on L-fucose, 13C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strain’s prolonged laboratory use.IMPORTANCEE. coli’s ability to survive, grow and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolysed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include L-fucose and L-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. coli’s metabolism and of its functioning in the bacterium’s natural environment.

scholarly journals Improved Yield of Recombinant Protein via Flagella Regulator Deletion in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Jae-Ho Han ◽  
Sang Taek Jung ◽  
Min-Kyu Oh
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Production ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Glucose Consumption ◽  
High Yield ◽  
Carbon Substrate ◽  
Flux Analysis ◽  
Flagella Assembly

Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella-related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADP+ ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC- and ptsG-deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced green fluorescent protein yield per glucose consumption increased 1.81-fold in the flhC mutant strain. Thus, our study demonstrates that high-yield production of the recombinant protein was achieved with reduced flagella formation.

scholarly journals Responses of theCentral Metabolism in Escherichia coli to PhosphoglucoseIsomerase and Glucose-6-Phosphate DehydrogenaseKnockouts

2003 ◽  
Vol 185 (24) ◽  
pp. 7053-7067 ◽  
Author(s):  
Qiang Hua ◽  
Chen Yang ◽  
Tomoya Baba ◽  
Hirotada Mori ◽  
Kazuyuki Shimizu
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Ratio ◽  
Pentose Phosphate ◽  
Phosphoglucose Isomerase ◽  
Overflow Metabolism ◽  
Flux Analysis ◽  
Glucose Catabolism

ABSTRACT The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-13C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

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