scholarly journals Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli

2006 ◽  
Vol 72 (2) ◽  
pp. 1164-1172 ◽  
Author(s):  
Annik Nanchen ◽  
Alexander Schicker ◽  
Uwe Sauer
Keyword(s):  
Escherichia Coli ◽  
Dilution Rate ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Pentose Phosphate ◽  
Glyoxylate Shunt ◽  
Parallel Operation ◽  
Physiological Variables

ABSTRACT A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic 13C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h−1. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO2. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h−1 was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.

scholarly journals Rewiring of glycerol metabolism in Escherichia coli for effective production of recombinant proteins

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Chung-Jen Chiang ◽  
Yi-Jing Ho ◽  
Mu-Chen Hu ◽  
Yun-Peng Chao
Keyword(s):  
Escherichia Coli ◽  
Crude Glycerol ◽  
Metabolic Flux ◽  
Raw Materials ◽  
Tricarboxylic Acid Cycle ◽  
Cost Effective ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Glycerol Metabolism ◽  
Related Field

Abstract Background The economic viability of a protein-production process relies highly on the production titer and the price of raw materials. Crude glycerol coming from the production of biodiesel is a renewable and cost-effective resource. However, glycerol is inefficiently utilized by Escherichia coli. Results This issue was addressed by rewiring glycerol metabolism for redistribution of the metabolic flux. Key steps in central metabolism involving the glycerol dissimilation pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle were pinpointed and manipulated to provide precursor metabolites and energy. As a result, the engineered E. coli strain displayed a 9- and 30-fold increase in utilization of crude glycerol and production of the target protein, respectively. Conclusions The result indicates that the present method of metabolic engineering is useful and straightforward for efficient adjustment of the flux distribution in glycerol metabolism. The practical application of this methodology in biorefinery and the related field would be acknowledged.

scholarly journals In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Fabien Le Chevalier ◽  
Isabelle Correia ◽  
Lucrèce Matheron ◽  
Morgan Babin ◽  
Mireille Moutiez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activities ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
E Coli ◽  
Chemical Structures ◽  
In Vivo Characterization ◽  
Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

scholarly journals Sigma S-Dependent Antioxidant Defense Protects Stationary-Phase Escherichia coli against the Bactericidal Antibiotic Gentamicin

2014 ◽  
Vol 58 (10) ◽  
pp. 5964-5975 ◽  
Author(s):  
Jing-Hung Wang ◽  
Rachna Singh ◽  
Michael Benoit ◽  
Mimi Keyhan ◽  
Matthew Sylvester ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Antioxidant Defense ◽  
Physiological State ◽  
Pentose Phosphate ◽  
Phase Cell ◽  
Content Type

ABSTRACTStationary-phase bacteria are important in disease. The σs-regulated general stress response helps them become resistant to disinfectants, but the role of σsin bacterial antibiotic resistance has not been elucidated. Loss of σsrendered stationary-phaseEscherichia colimore sensitive to the bactericidal antibiotic gentamicin (Gm), and proteomic analysis suggested involvement of a weakened antioxidant defense. Use of the psfiAgenetic reporter, 3′-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that Gm generated more reactive oxygen species (ROS) in the mutant. HPF measurements can be distorted by cell elongation, but Gm did not affect stationary-phase cell dimensions. Coadministration of the antioxidantN-acetyl cysteine (NAC) decreased drug lethality particularly in the mutant, as did Gm treatment under anaerobic conditions that prevent ROS formation. Greater oxidative stress, due to insufficient quenching of endogenous ROS and/or respiration-linked electron leakage, therefore contributed to the greater sensitivity of the mutant; infection by a uropathogenic strain in mice showed this to be the case alsoin vivo. Disruption of antioxidant defense by eliminating the quencher proteins, SodA/SodB and KatE/SodA, or the pentose phosphate pathway proteins, Zwf/Gnd and TalA, which provide NADPH for ROS decomposition, also generated greater oxidative stress and killing by Gm. Thus, besides its established mode of action, Gm also kills stationary-phase bacteria by generating oxidative stress, and targeting the antioxidant defense ofE. colican enhance its efficacy. Relevant aspects of the current controversy on the role of ROS in killing by bactericidal drugs of exponential-phase bacteria, which represent a different physiological state, are discussed.

scholarly journals Isolation and characterization of a novel temperate Escherichia coli bacteriophage, Kapi1, which modifies the O-antigen and contributes to the competitiveness of its host during colonization of the murine gastrointestinal tract.

2021 ◽  
Author(s):  
Kat Pick ◽  
Tingting Ju ◽  
Benjamin P. Willing ◽  
Tracy Lyn Raivio
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Molecular Mechanisms ◽  
Shigella Flexneri ◽  
Simulated Intestinal Fluid ◽  
O Antigen ◽  
Isolation And Characterization

In this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri, and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways.  Kapi1 displays unstable lysogeny, and we find that lysogeny is favored during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their non-lysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be.

scholarly journals Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in Escherichia coli

2006 ◽  
Vol 189 (3) ◽  
pp. 1176-1178 ◽  
Author(s):  
Tadashi Ogawa ◽  
Keiko Murakami ◽  
Hirotada Mori ◽  
Nobuyoshi Ishii ◽  
Masaru Tomita ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Dehydrogenase ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Glyoxylate Shunt ◽  
Acid Cycle ◽  
Uncompetitive Inhibition

ABSTRACT Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki ′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

scholarly journals Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

2009 ◽  
Vol 76 (4) ◽  
pp. 1298-1300 ◽  
Author(s):  
Min Jiang ◽  
Shu-wen Liu ◽  
Jiang-feng Ma ◽  
Ke-quan Chen ◽  
Li Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Tricarboxylic Acid Cycle ◽  
Redox Balance ◽  
Growth Conditions ◽  
Tricarboxylic Acid ◽  
Feeding Strategies ◽  
Glyoxylate Shunt ◽  
Aerobic Growth ◽  
Succinate Production ◽  
Acid Yield

ABSTRACT Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.

scholarly journals Virulent Bacteriophages Can Target O104:H4 Enteroaggregative Escherichia coli in the Mouse Intestine

2012 ◽  
Vol 56 (12) ◽  
pp. 6235-6242 ◽  
Author(s):  
Damien Maura ◽  
Matthieu Galtier ◽  
Chantal Le Bouguénec ◽  
Laurent Debarbieux
Keyword(s):  
Escherichia Coli ◽  
Dependent Manner ◽  
Content Type ◽  
Intestinal Pathogens ◽  
Colonization Model ◽  
Mouse Intestine ◽  
Further Development ◽  
Dose Dependent

ABSTRACTIn vivobacteriophage targeting of enteroaggregativeEscherichia coli(EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophagesin vivofor the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers.

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