Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of the polyhydroxyalkanoate produced from glucose by Rhodococcus ruber was investigated using nuclear magnetic resonance analysis. Spectra obtained for polymer produced from [6-13C]-, [2-13C]-, and [1-13C]glucose were compatible with carboxylation of pyruvate followed by TCA cycle reactions to yield succinate, which is a precursor of propionyl-CoA in the biosynthesis of 3HV in this organism. Carboxylation of pyruvate may occur directly or involve methylmalonyl-CoA transcarboxylase but the latter process is more probable as it provides a route for recycling oxaloacetate, which is essential for the pathway to operate. The observed incorporation of label from NaH13CO3 into C-3 of 3HV monomers cannot occur via forward reactions of the TCA cycle but may be explained by direct carboxylation of pyruvate, followed by reverse reactions of the TCA cycle to yield succinyl-CoA. This minor route accounts for at least 12% of the carbon flux in the synthesis of 3HV monomer units. Inhibitors of key enzymes of possible pathways for polyhydroxyalkanoate biosynthesis in R. ruber were tested. Addition of monofluoroacetate, an inhibitor of aconitase, to cultures decreased production of 3HV, supporting the proposed major route for 3HV synthesis.Key words: Rhodococcus ruber, polyhydroxyalkanoate, PHA, biosynthesis.

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Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

10.1101/2021.01.29.428863 ◽

2021 ◽

Author(s):

Khushboo Borah ◽

Tom A. Mendum ◽

Nathaniel D. Hawkins ◽

Jane L. Ward ◽

Michael H. Beale ◽

...

Keyword(s):

Metabolic Network ◽

Metabolic Flux ◽

Carbon Sources ◽

Tca Cycle ◽

Flux Analysis ◽

Glyoxylate Shunt ◽

Citrate Cycle ◽

Metabolic Fluxes ◽

The Tca Cycle ◽

Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

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Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.9.3171-3179.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3171-3179 ◽

Cited By ~ 194

Author(s):

Annik Perrenoud ◽

Uwe Sauer

Keyword(s):

Escherichia Coli ◽

Transcriptional Regulation ◽

Redox Regulation ◽

Tca Cycle ◽

Batch Cultures ◽

E Coli ◽

Glucose Catabolism ◽

The Tca Cycle

ABSTRACT Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

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The Glyoxylate Shunt, 60 Years On

Annual Review of Microbiology ◽

10.1146/annurev-micro-090817-062257 ◽

2018 ◽

Vol 72 (1) ◽

pp. 309-330 ◽

Cited By ~ 34

Author(s):

Stephen K. Dolan ◽

Martin Welch

Keyword(s):

Citric Acid ◽

Citric Acid Cycle ◽

Tca Cycle ◽

Oxidative Decarboxylation ◽

Glyoxylate Shunt ◽

Cellular Functions ◽

Acid Cycle ◽

E Coli ◽

80Th Anniversary ◽

The Tca Cycle

2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01327-08 ◽

2008 ◽

Vol 74 (22) ◽

pp. 7002-7015 ◽

Cited By ~ 36

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.

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Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01400-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5960-5968 ◽

Cited By ~ 5

Author(s):

Tomohiro Shimada ◽

Kan Tanaka

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Monitoring System ◽

Carbon Sources ◽

Tca Cycle ◽

Bacterial Cells ◽

Bacterial Luciferase ◽

Content Type ◽

Time Dynamics ◽

The Tca Cycle

ABSTRACTRegulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employingVibrio fischeriluciferase to probe the intracellular metabolic condition inEscherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used aVibrio fischeriluciferase monitoring system to probe the intracellular metabolic condition inEscherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.

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Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

International Journal of Molecular Sciences ◽

10.3390/ijms23020773 ◽

2022 ◽

Vol 23 (2) ◽

pp. 773

Author(s):

Kaikai Wang ◽

Xiaolu Wang ◽

Huiying Luo ◽

Yaru Wang ◽

Yuan Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Flux ◽

Glucose Consumption ◽

Amino Sugar ◽

Fermentation Medium ◽

High Yield ◽

Final Strain ◽

Pharmaceutical Industries ◽

Glucose 6 Phosphate Dehydrogenase ◽

Utilization Strategy

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

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