Distinct metabolic flux modes through the tricarboxylic acid cycle in mesophyll and guard cells revealed by GC-MS-based 13C-positional isotopomer analysis

Summary13C-Metabolic flux analysis (13C-MFA) have greatly contributed to revealing the regulation of plant metabolism. However, mass spectrometry (MS) approaches have hitherto been limited in their power to deduce flux information due to lack of positional information.Here we established an MS-based 13C-positional isotopomer labelling approach and performed a multi-species/cell-types analysis based on previous 13C-MFA to compare flux modes through the tricarboxylic acid (TCA) cycle and associated pathways in mesophyll (MCs) and guard cells (GCs).Both cell types showed high 13C-enrichment in pyruvate. However, GCs and sink MCs, but not source MCs showed high 13C-incorporation into Glu/Gln following provision of 13C-sucrose. Only GCs showed higher 13C-enrichment in the carbon 1 atom of Gln, which is derived from PEPc-mediated CO2 fixation. Increased 13C-enrichment in the carbon 1 of Glu was also observed in both trxo1 and ntra ntrb mutants, but not in wild type Arabidopsis plants, following provision of 13C-glucose.Our results suggest that the mitochondrial thioredoxin system restricts the fluxes from PEPc and glycolysis to Glu in illuminated MCs and reveal that fluxes throughout the TCA cycle of GCs resemble those of sink MCs but operate different non-cyclic flux modes to support Gln synthesis in the light.

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Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

10.1101/2021.08.06.455447 ◽

2021 ◽

Author(s):

Joy Omini ◽

Izabela Wojciechowska ◽

Aleksandra Skirycz ◽

Hideaki Moriyama ◽

Toshihiro Obata

Keyword(s):

Malate Dehydrogenase ◽

Metabolic Flux ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Feedback Regulation ◽

Dynamic Structure ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Enzyme Complex ◽

Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

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Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

Journal of Bacteriology ◽

10.1128/jb.00490-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4452-4461 ◽

Cited By ~ 102

Author(s):

Daniel Amador-Noguez ◽

Xiao-Jiang Feng ◽

Jing Fan ◽

Nathaniel Roquet ◽

Herschel Rabitz ◽

...

Keyword(s):

Metabolic Pathways ◽

Genome Annotation ◽

Clostridium Acetobutylicum ◽

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Anaerobic Bacteria ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Fermentation Products

ABSTRACT Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/α-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

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Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.01942-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7866-7869 ◽

Cited By ~ 85

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Hartwig Schröder ◽

Christoph Wittmann

Keyword(s):

Metabolic Engineering ◽

Corynebacterium Glutamicum ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Start Codon ◽

Flux Analysis ◽

Lysine Production ◽

Acid Cycle ◽

The Tca Cycle

ABSTRACT In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.

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Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

Scientific Reports ◽

10.1038/s41598-021-98314-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Joy Omini ◽

Izabela Wojciechowska ◽

Aleksandra Skirycz ◽

Hideaki Moriyama ◽

Toshihiro Obata

Keyword(s):

Malate Dehydrogenase ◽

Metabolic Flux ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Feedback Regulation ◽

Dynamic Structure ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Enzyme Complex ◽

Acetyl Coa

AbstractMitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

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In vivo 13C MRS in the mouse brain at 14.1 Tesla and metabolic flux quantification under infusion of [1,6-13C2]glucose

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17734101 ◽

2017 ◽

Vol 38 (10) ◽

pp. 1701-1714 ◽

Cited By ~ 7

Author(s):

Marta Lai ◽

Bernard Lanz ◽

Carole Poitry-Yamate ◽

Jackeline F Romero ◽

Corina M Berset ◽

...

Keyword(s):

Mouse Brain ◽

Metabolic Flux ◽

Direct Detection ◽

Tricarboxylic Acid Cycle ◽

Quantitative Information ◽

Glutamine Metabolism ◽

Resonance Spectroscopy ◽

Flux Analysis ◽

Carboxylase Activity

In vivo 13C magnetic resonance spectroscopy (MRS) enables the investigation of cerebral metabolic compartmentation while, e.g. infusing 13C-labeled glucose. Metabolic flux analysis of 13C turnover previously yielded quantitative information of glutamate and glutamine metabolism in humans and rats, while the application to in vivo mouse brain remains exceedingly challenging. In the present study, 13C direct detection at 14.1 T provided highly resolved in vivo spectra of the mouse brain while infusing [1,6-13C2]glucose for up to 5 h. 13C incorporation to glutamate and glutamine C4, C3, and C2 and aspartate C3 were detected dynamically and fitted to a two-compartment model: flux estimation of neuron-glial metabolism included tricarboxylic acid cycle (TCA) flux in astrocytes (Vg = 0.16 ± 0.03 µmol/g/min) and neurons (VTCAn = 0.56 ± 0.03 µmol/g/min), pyruvate carboxylase activity (VPC = 0.041 ± 0.003 µmol/g/min) and neurotransmission rate (VNT = 0.084 ± 0.008 µmol/g/min), resulting in a cerebral metabolic rate of glucose (CMRglc) of 0.38 ± 0.02 µmol/g/min, in excellent agreement with that determined with concomitant 18F-fluorodeoxyglucose positron emission tomography (18FDG PET).We conclude that modeling of neuron-glial metabolism in vivo is accessible in the mouse brain from 13C direct detection with an unprecedented spatial resolution under [1,6-13C2]glucose infusion.

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HISTOCHEMICAL INVESTIGATIONS ON THE IN VIVO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES FROM PELARGONIUM ZONALE: PART II

Journal of Histochemistry & Cytochemistry ◽

10.1177/15.4.202 ◽

1967 ◽

Vol 15 (4) ◽

pp. 202-206

Author(s):

C. JAMES LOVELACE ◽

GENE W. MILLER

Keyword(s):

Sodium Fluoride ◽

Reductase Activity ◽

Tricarboxylic Acid Cycle ◽

Leaf Tissue ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Pelargonium Zonale ◽

Acid Cycle ◽

In Vivo Effects

In vivo effects of fluoride on tricarboxylic acid (TCA) cycle dehydrogenase enzymes of Pelargonium zonale were studied using p-nitro blue tetrazoleum chloride. Plants were exposed to 17 ppb HF, and enzyme activities in treated plants were compared to those in controls. Leaves of control plants were incubated in 5 x 10–3 M sodium fluoride. Injuries observed in fumigation and solution experiments were similar. Leaf tissue subjected to HF or sodium fluoride evidenced less succinic p-nitro blue tetrazoleum reductase activity than did control tissue. Other TCA cycle dehydrogenase enzymes were not observably affected by the fluoride concentrations used in these experiments. Excised leaves cultured in 5 x 10–3 M sodium fluoride exhibited less succinic p-nitro blue tetrazoleum reductase activity after 24 hr than did leaves cultured in 5 x 10–3 M sodium chloride.

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Current Advances in the Biodegradation and Bioconversion of Polyethylene Terephthalate

Microorganisms ◽

10.3390/microorganisms10010039 ◽

2021 ◽

Vol 10 (1) ◽

pp. 39

Author(s):

Xinhua Qi ◽

Wenlong Yan ◽

Zhibei Cao ◽

Mingzhu Ding ◽

Yingjin Yuan

Keyword(s):

Polyethylene Terephthalate ◽

Microbial Consortium ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Microbial Consortia ◽

Plastic Pollution ◽

One Step ◽

Complex Polymers ◽

The One

Polyethylene terephthalate (PET) is a widely used plastic that is polymerized by terephthalic acid (TPA) and ethylene glycol (EG). In recent years, PET biodegradation and bioconversion have become important in solving environmental plastic pollution. More and more PET hydrolases have been discovered and modified, which mainly act on and degrade the ester bond of PET. The monomers, TPA and EG, can be further utilized by microorganisms, entering the tricarboxylic acid cycle (TCA cycle) or being converted into high value chemicals, and finally realizing the biodegradation and bioconversion of PET. Based on synthetic biology and metabolic engineering strategies, this review summarizes the current advances in the modified PET hydrolases, engineered microbial chassis in degrading PET, bioconversion pathways of PET monomers, and artificial microbial consortia in PET biodegradation and bioconversion. Artificial microbial consortium provides novel ideas for the biodegradation and bioconversion of PET or other complex polymers. It is helpful to realize the one-step bioconversion of PET into high value chemicals.

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Metabolic control of nitrogen fixation in rhizobium-legume symbioses

Science Advances ◽

10.1126/sciadv.abh2433 ◽

2021 ◽

Vol 7 (31) ◽

pp. eabh2433

Author(s):

Carolin C. M. Schulte ◽

Khushboo Borah ◽

Rachel M. Wheatley ◽

Jason J. Terpolilli ◽

Gerhard Saalbach ◽

...

Keyword(s):

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Oxygen Demand ◽

Metabolic Model ◽

Redox Balance ◽

Ammonia Assimilation ◽

Nodule Formation ◽

Flux Analysis ◽

Carbon And Nitrogen ◽

Genome Scale

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

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Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7277-7287.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7277-7287 ◽

Cited By ~ 71

Author(s):

Christoph Wittmann ◽

Patrick Kiefer ◽

Oskar Zelder

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Metabolic Flux ◽

Tca Cycle ◽

Pentose Phosphate ◽

Flux Analysis ◽

Phosphotransferase System ◽

Lysine Production ◽

Metabolic Fluxes ◽

Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

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Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

Journal of Bacteriology ◽

10.1128/jb.187.9.2967-2973.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 2967-2973 ◽

Cited By ~ 71

Author(s):

Cuong Vuong ◽

Joshua B. Kidder ◽

Erik R. Jacobson ◽

Michael Otto ◽

Richard A. Proctor ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Redox Status ◽

Tricarboxylic Acid ◽

Polysaccharide Intercellular Adhesin ◽

Low Oxygen ◽

Acid Cycle ◽

The Tca Cycle ◽

High Concentrations

ABSTRACT Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production.

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