scholarly journals Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

2006 ◽  
Vol 73 (2) ◽  
pp. 456-464 ◽  
Author(s):  
Y. Zhu ◽  
M. A. Eiteman ◽  
K. DeWitt ◽  
E. Altman
Keyword(s):  
Escherichia Coli ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Ph Control ◽  
Defined Medium ◽  
Nuclear Magnetic Resonance Analysis ◽  
Aerobic Growth ◽  
Resonance Analysis ◽  
Phosphoenolpyruvate Synthase ◽  
Anaerobic Production

ABSTRACT We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter · h). Ca(OH)2 was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N2 for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. 13C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter · h during the anaerobic phase.

scholarly journals High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

2008 ◽  
Vol 74 (21) ◽  
pp. 6649-6655 ◽  
Author(s):  
Yihui Zhu ◽  
Mark A. Eiteman ◽  
Ronni Altman ◽  
Elliot Altman
Keyword(s):  
Escherichia Coli ◽  
Nadh Oxidase ◽  
Pyruvate Dehydrogenase Complex ◽  
Formation Rate ◽  
Batch Process ◽  
Defined Medium ◽  
Coli Strain ◽  
Glycolytic Flux ◽  
Exponential Feeding ◽  
Phosphoenolpyruvate Synthase

ABSTRACT We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h−1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g � h), pyruvate formation rate (1.11 g/g � h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g � h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h−1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter � h and yield of 0.68 g/g.

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 Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

2009 ◽  
Vol 191 (17) ◽  
pp. 5538-5548 ◽  
Author(s):  
Pablo I. Nikel ◽  
Jiangfeng Zhu ◽  
Ka-Yiu San ◽  
Beatriz S. Méndez ◽  
George N. Bennett
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Building Blocks ◽  
Flux Analysis ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13C-labeling experiments in chemostat cultures of a wild-type strain, ΔcreB and ΔarcA single mutants, and a ΔcreB ΔarcA double mutant. Continuous cultures were conducted at D = 0.1 h−1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ΔarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ΔarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

scholarly journals Using γ-Ray Polymerization-Induced Assemblies to Synthesize Polydopamine Nanocapsules

Polymers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1754 ◽  
Author(s):  
Wenwen Jiang ◽  
Xinyue Zhang ◽  
Yafei Luan ◽  
Rensheng Wang ◽  
Hanzhou Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Dynamics ◽  
Interfacial Polymerization ◽  
Dynamics Simulation ◽  
Nuclear Magnetic Resonance Analysis ◽  
Resonance Analysis ◽  
Spherical Structure ◽  
1H Nuclear Magnetic Resonance ◽  
Γ Ray ◽  
Model Drug

This work reports a simple and robust strategy for synthesis of polydopamine nanocapsules (PDA NCs). First, polymer assemblies were synthesized by a γ-ray-induced liquid–liquid (H2O–acrylate) interface polymerization strategy, in the absence of any surfactants. 1H nuclear magnetic resonance analysis and molecular dynamics simulation reveal that the generation of polymer assemblies largely depends on the hydrophilicity of acrylate and gravity of the oligomers at the interface. By virtue of the spherical structure and mechanic stability of the polymer assemblies, PDA NCs are next prepared by the interfacial polymerization of dopamine onto the assemblies, followed by the removal of templates by using ethanol. The polydopamine nanocapsules are shown to load and release ciprofloxacin (CIP, a model drug), such that the CIP-loaded PDA NCs are able to inhibit the growth of Escherichia coli.

scholarly journals Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by Escherichia coli

1999 ◽  
Vol 181 (11) ◽  
pp. 3562-3570 ◽  
Author(s):  
Robert E. London ◽  
Devon L. Allen ◽  
Scott A. Gabel ◽  
Eugene F. DeRose
Keyword(s):  
Escherichia Coli ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Tca Cycle ◽  
Nuclear Magnetic Resonance Study ◽  
Nuclear Magnetic Resonance Analysis ◽  
Pyruvate Formate Lyase ◽  
Resonance Analysis ◽  
Direct Formation ◽  
Nuclear Magnetic

ABSTRACT We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.

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