scholarly journals Alterations of Cellular Physiology in Escherichia coli in Response to Oxidative Phosphorylation Impaired by Defective F1-ATPase

2006 ◽  
Vol 188 (19) ◽  
pp. 6869-6876 ◽  
Author(s):  
Sakiko Noda ◽  
Yuji Takezawa ◽  
Tomohiko Mizutani ◽  
Tomoaki Asakura ◽  
Eiichiro Nishiumi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Pyruvate Dehydrogenase Complex ◽  
Growth Yield ◽  
Glucose Consumption ◽  
Tca Cycle ◽  
Total Activity ◽  
Glycolytic Flux ◽  
Flux Analysis ◽  
Physiological Benefits

ABSTRACT The physiological changes in an F1-ATPase-defective mutant of Escherichia coli W1485 growing in a glucose-limited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo 3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.

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 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 Overflow Metabolism in Escherichia coli during Steady-State Growth: Transcriptional Regulation and Effect of the Redox Ratio

2006 ◽  
Vol 72 (5) ◽  
pp. 3653-3661 ◽  
Author(s):  
G. N. Vemuri ◽  
E. Altman ◽  
D. P. Sangurdekar ◽  
A. B. Khodursky ◽  
M. A. Eiteman
Keyword(s):  
Escherichia Coli ◽  
Consumption Rate ◽  
Nadh Oxidase ◽  
Glucose Consumption ◽  
Tca Cycle ◽  
Overflow Metabolism ◽  
Glucose Consumption Rate ◽  
Redox Ratio ◽  
E Coli ◽  
Acetate Formation

ABSTRACT Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.

scholarly journals Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

2010 ◽  
Vol 76 (19) ◽  
pp. 6529-6540 ◽  
Author(s):  
Vasiliy A. Portnoy ◽  
David A. Scott ◽  
Nathan E. Lewis ◽  
Yekaterina Tarasova ◽  
Andrei L. Osterman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Metabolic Flux ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Physiological Characterization ◽  
Metabolic Flux Distribution ◽  
K 12

ABSTRACT The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of d-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.

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 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 Requirement of ArcA for Redox Regulation in Escherichia coli under Microaerobic but Not Anaerobic or Aerobic Conditions

2003 ◽  
Vol 185 (1) ◽  
pp. 204-209 ◽  
Author(s):  
Svetlana Alexeeva ◽  
Klaas J. Hellingwerf ◽  
M. Joost Teixeira de Mattos
Keyword(s):  
Escherichia Coli ◽  
Redox State ◽  
Redox Regulation ◽  
Flux Distribution ◽  
Pyruvate Dehydrogenase Complex ◽  
Oxygen Availability ◽  
Flux Analysis ◽  
Wild Type ◽  
E Coli ◽  
Intracellular Redox

ABSTRACT In Escherichia coli, the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type E. coli to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type E. coli and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator.

PDH activation by dichloroacetate reduces TCA cycle intermediates at rest but not during exercise in humans

1999 ◽  
Vol 277 (1) ◽  
pp. E33-E38 ◽  
Author(s):  
Martin J. Gibala ◽  
Bengt Saltin
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Tca Cycle ◽  
Glycolytic Flux ◽  
Intense Exercise ◽  
Absolute Increase ◽  
Work Transition ◽  
Tca Cycle Intermediates ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Exercise In Humans

We hypothesized that dichloroacetate (DCA), which stimulates the pyruvate dehydrogenase complex (PDH), would attenuate the increase in muscle tricarboxylic acid cycle intermediates (TCAI) during exercise by increasing the oxidative disposal of pyruvate and attenuating the flux through anaplerotic pathways. Six subjects were infused with either saline (Con) or DCA (100 mg/kg body mass) and then performed a moderate leg kicking exercise for 15 min, followed immediately by intense exercise until exhaustion (Exh; ∼4 min). Resting active fraction of PDH (PDHa) was markedly increased ( P ≤ 0.05) after DCA vs. Con (2.65 ± 0.27 vs. 0.64 ± 0.07 mmol ⋅ min−1 ⋅ kg wet wt−1); however, there were no differences between trials after 1 or 15 min of exercise or at Exh. The sum of five measured TCAI (ΣTCAI; ∼90% of total TCAI pool) was lower ( P ≤ 0.05) after DCA vs. Con at rest (0.78 ± 0.11 vs. 1.52 ± 0.23 mmol/kg dry wt, respectively). However, the net increase in muscle TCAI during the first minute of exercise was higher ( P≤ 0.05) in the DCA trial vs. Con (3.05 ± 0.45 vs. 2.44 ± 0.55 mmol ⋅ min−1 ⋅ kg dry wt−1, respectively), and consequently, the ΣTCAI was not different between trials during exercise. We conclude that DCA reduced TCAI pool size at rest by increasing the flux through PDH and diverting pyruvate away from anaplerotic pathways. The reason for the similar absolute increase in TCAI during exercise is not clear but may be related to 1) an initial mismatch between glycolytic flux and PDH flux that provided sufficient pyruvate for anaplerosis in both trials; or 2) a transient inhibition of PDH flux during the DCA trial due to an elevated resting acetyl-CoA-to-CoASH ratio, which augmented the anaplerotic flux of carbon during the rest-to-work transition.

scholarly journals Construction of a fimbriae absent Escherichia coli by deleting 64 genes and its application for efficient production of poly-3-hydroxybutyrate and L-threonine

2021 ◽  
Author(s):  
Jun Qiao ◽  
Xin Tan ◽  
Hongyu Ren ◽  
Zheng Wu ◽  
Xiaoqing Hu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glucose Consumption ◽  
Tca Cycle ◽  
Efficient Production ◽  
Wild Type ◽  
E Coli ◽  
The Tca Cycle ◽  
Key Genes ◽  
Threonine Biosynthesis ◽  
Better Than

Escherichia coli contains 12 chaperone-usher operons for biosynthesis and assembly of various fimbriae. In this study, each of the 12 operons was deleted in E. coli MG1655, and the resulting 12 deletion mutants all grew better than the wild type, especially in the nutrient-deficient M9 medium. When the plasmid pBHR68 containing the key genes for polyhydroxyalkanoate production was introduced into these 12 mutants, each mutant synthesized more polyhydroxyalkanoate than the wild type control. These results indicate that the fimbriae removal in E. coli benefits cell growth and polyhydroxyalkanoate production. Therefore, all the 12 chaperone-usher operons including 64 genes were deleted in MG1655, resulting the fimbriae absent strain WQM026. WQM026 grew better than MG1655, and no fimbriae structures were observed on the surface of WQM026 cells. Transcriptomic analysis showed that in WQM026 cells the genes related to glucose consumption, glycolysis, flagellar synthesis, and biosynthetic pathways of some key amino acids were up-regulated, while the TCA cycle related genes were down-regulated. When pBHR68 was introduced into WQM026, huge amounts of poly-3-hydroxybutyrate were produced; when the plasmid pFW01-thrA*BC-rhtC containing the key genes for L-threonine biosynthesis and transport was transferred into WQM026, more L-threonine was synthesized than the control. These results suggest that this fimbriae absent E. coli WQM026 is a good host for efficient production of polyhydroxyalkanoate and L-threonine, and has the potential to be developed into a valuable chassis microorganism. IMPORTANCE In this study, we investigated the interaction between the biosynthesis and assembly of fimbriae and intracellular metabolic network in E. coli. We found that eliminating fimbriae could effectively improve the production of polyhydroxyalkanoate and L-threonine in E. coli MG1655. These results contribute to understanding the necessity of fimbriae and the advantages of fimbriae removal for industrial microorganisms. The knowledge gathered from this study may be applied to the development of superior chassis microorganisms.

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