Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

Zhao, Jing; Shimizu, Kazuyuki

doi:10.1016/s0168-1656(02)00316-4

Cited by 142 publications

(140 citation statements)

References 20 publications

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“…It was speculated that the relatively high C. glutamicum TCA cycle fluxes were needed to generate sufficient reductant for respiration to support the high ATP demands of gluconeogenesis. Similar results were observed in E. coli respiring with acetate, with 30% of flux from isocitrate going to the glyoxylate shunt and 70% through the lower TCA cycle (35). A 13 C-labeling study of Geobacter metallireducens showed high TCA cycle flux from acetate while respiring with Fe 3ϩ with zero flux through the glyoxylate shunt (36).…”

Section: Discussionsupporting

confidence: 63%

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

McKinlay

Oda

Rühl

et al. 2014

Journal of Biological Chemistry

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Background:The metabolism of non-growing microbes is poorly understood. Results: In a nitrogen-starved and non-growing photoheterotrophic bacterium, metabolic flow was diverted to mobilize electrons for H 2 production. Conclusion: During starvation bacteria decouple their metabolism from biosynthesis. Significance: An understanding of metabolic activities of non-growing cells can be used to engineer improved biocatalysts.

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Section: Discussionsupporting

confidence: 63%

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

McKinlay

Oda

Rühl

et al. 2014

Journal of Biological Chemistry

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“…However, the model predicted extremely high reversibility (exch ϭ 0.99) in the reaction that converts oxoglutarate to succinate compared to those in other microorganisms (41). This reaction contains two steps and is usually catalyzed by the enzymes oxoglutarate oxidoreductase (oxoglutarate 3 succinyl-CoA; ⌬G ϭ Ϫ33.5 kJ/mol) and succinylCoA synthetase (succinyl-CoA 3 succinate; ⌬G ϭ Ϫ2.9 kJ/ mol) (25).…”

Section: Resultsmentioning

confidence: 99%

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

Tang

Chakraborty

Martín

et al. 2007

Appl Environ Microbiol

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We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13 C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fenitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO 2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.Geobacter species are known to be one of the dominant groups of microorganisms mediating iron reduction in the environment (21). They have been found to be ubiquitous in a myriad of subsurface environments. Detailed studies of their metabolism have revealed them to be capable of bioremediation of several heavy metals, including uranium, plutonium, technetium, and vanadium, as well as biodegradation of several organic contaminants, including monoaromatic hydrocarbons (16,17,26). More recently, Geobacter species have been used to generate electricity from waste organic matter (2, 15, 19). These unique metabolisms make Geobacter species important players in the contaminated subsurface environment (18). Geobacter metallireducens was the first iron-reducing organism isolated that coupled complete oxidation of organic acids with reduction of iron oxides (20,21). It completely oxidizes organic carbons such as fatty acids, alcohols, and monoaromatic compounds via the tricarboxylic acid (TCA) cycle (3, 20) coupled with the reduction of iron. The genomes of several Geobacter species have been sequenced, and proteome data are also available (5, 24). While the genome sequence and proteome are important for understanding Geobacter, they are not necessarily accurate represe...

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“…The unknown metabolic fluxes were calculated to 11 minimize ε. To estimate the confidence interval for calculated fluxes, a Monte Carlo approach 12 was employed (Zhao and Shimizu 2003). In brief, 20 isotopomer concentration data sets were 13 generated by addition of normally distributed measurement noise to actual measurement data.…”

Section: Reversible 13mentioning

confidence: 99%

Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol‐tolerant Geobacillus strain

Tang

Sapra

Joyner

et al. 2008

Biotech & Bioengineering

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1A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius 2 M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to 3 high ethanol concentrations (10% v/v). We have investigated the central metabolism of this 4 bacterium using both in vitro enzyme assays and 13 C-based flux analysis to provide insights into 5 the physiological properties of this extremophile and explore its metabolism for bioethanol or 6 other bioprocess applications. Our findings show that glucose metabolism in G. 7 thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the 8 TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. 9Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase and 10 phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not 11 be accurately determined using amino acid labelling. When growth conditions were switched 12 from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 13 100 units (gm DCW) -1 ·hr -1 ) through the TCA cycle and oxidative pentose phosphate pathway 14 were reduced from 64±3 to 25±2 and from 30±2 to 19±2, respectively. The carbon flux under 15 microaerobic growth was directed to ethanol, L-lactate (>99% optical purity), acetate, and 16formate. Under fully anaerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid 17 fermentation process and exhibited a maximum ethanol yield of 0.38±0.07 mol mol -1 glucose. In 18 silico flux balance modelling demonstrates that lactate and acetate production from G. 19 thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately three 20 folds, thus indicating that both pathways should be modified to maximize ethanol production. 21

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Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

Cited by 142 publications

References 20 publications

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol‐tolerant Geobacillus strain

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