Engineered citrate synthase improves citramalic acid generation in Escherichia coli

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.     

Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with ¹³C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by ¹³C-NMR spectroscopy. [2-¹³C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-¹³C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate caboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates. Received: November 26, 1997 / Accepted: May 11, 1998     

α-Ketoisocaproate Alters the Production of Both Lactate and Aspartate from [U-13C]Glutamate in Astrocytes: A 13C NMR Study

Abstract: The present study determined the metabolic fate of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-¹³C]glutamate, 64.1% of the ¹³C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [¹³C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [¹³C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [¹³C]glutamate into the 1,2,3-¹³C₃-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [¹³C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4–7 days was observed. Cells adapted to propionate still required 1–2 days before growth commenced. Incorporation of (2-¹³C), (3-¹³C) or (²H₃)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The ¹³C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate. Received: 28 April 1997 / Accepted: 4 July 1997     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

NMR Spectroscopy of Cultured Astrocytes: Effects of Glutamine and the Gliotoxin Fluorocitrate

Abstract: Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with ¹³C and ³¹P NMR spectroscopy. f2–¹³C]Acetate and [1–¹³C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 μM) inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 μM) inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 100 μM fluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis ^; of the ¹³C labeling of the C-2 versus the C-4 positions in glutamine obtained with [2–¹³C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle. Glutamine and citrate were the main TCA cycle intermediates to be released, but a large amount of lactate formed from TCA cycle intermediates was also released, showing that recycling of pyruvate takes place in astrocytes.     

C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevulinic acid destined for chlorophyll formation in dark-grown Scenedesmus obliquus

The ¹³C-nuclear magnetic resonance (NMR) spectra of chlorophyll a formed in dark-grown Scenedesmus obliquus (Turp.) Kützing in the presence of [1-¹³C]glutamate, [2-¹³C]- and [1-¹³C]glycineshowed that the ¹³C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of chlorophyll a. No specific enrichment of these nine carbon atoms was observed in the spectrum of chlorophyll a formed in the presence of [1-¹³C]-glycine. These labeling patterns provide evidence for the operation of the C₅-pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness.     

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

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 the 13C-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.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.     

Reductive Carboxylation of Propionate to Butyrate in Methanogenic Ecosystems

During the batch degradation of sodium propionate by the anaerobic sludge from an industrial digestor, we observed a significant amount of butyrate formation. Varying the initial propionate concentrations did not alter the ratio of maximal butyrate accumulation to initial propionate concentration within a large range. By measuring the decrease in the radioactivity of [1-¹⁴C]butyrate during propionate degradation, we estimated that about 20% of the propionate was converted to butyrate. Labeled butyrate was formed from [1-¹⁴C]propionate with the same specific radioactivity, suggesting a possible direct pathway from propionate to butyrate. We confirmed this hypothesis by nuclear magnetic resonance studies with [¹³C]propionate. The results showed that [1-¹³C]-, [2-¹³C]-, and [3-¹³C]propionate were converted to [2-¹³C]-, [3-¹³C]-, and [4-¹³C]butyrate, respectively, demonstrating the direct carboxylation on the carboxyl group of propionate without randomization of the other two carbons. In addition, we observed an exchange reaction between C-2 and C-3 of the propionate, indicating that acetogensis may proceed through a randomizing pathway. The physiological significance and importance of various metabolic pathways involved in propionate degradation are discussed, and an unusual pathway of butyrate synthesis is proposed.     

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.     

13C-NMR Study of propionate metabolism by sludges from bioreactors treating sulfate and sulfide rich wastewater

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution¹³ C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented.¹³ C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-¹³C]propionate into [2-¹³C]propionate and the formation of acetate equally labeled in the C₁ and C₂ position. In the absence of sulfate, [3-¹³C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-¹³C]propionate partly into the higher fatty acid 2-methyl[2,3-¹³C]butyrate during the simultaneous degradation of [2,3-¹³C]propionate and butyrate. [4,5-¹³C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

β-Poly(l-malate) production by Physarum polycephalum

β-Poly(l-malate) (PMLA) production in Physarum polycephalum has been followed by using d-[1-¹³C]glucose and Ca¹³CO₃. Nuclear magnetic resonance studies of PMLA showed that the ¹³C label from [1-¹³C]glucose was incorporated in the presence of CaCO₃ into positions C-3 (-CH₂-) and C-4 (-CO-) of the l-malate repeating unit of PMLA. The ¹³C label from Ca¹³CO₃ was incorporated into position C-4 and indicated that not only the endogenous CO₂ but also the exogenous CO₂ from CaCO₃ served significantly as a carbon source for PMLA production. In the absence of CaCO₃, the ¹³C labeling pattern of PMLA from d-[1-¹³C]glucose was almost indistinguishable from that for the natural abundance ¹³C-NMR spectrum of the polymer. These results indicated that l-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO₃ or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO₃. Avidin strongly inhibited the formation of l-malate via carboxylation; the ¹³C labeling pattern of PMLA in the presence of CaCO₃ was almost identical with that for the natural abundance spectrum when avidin was added, indicating that l-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle. Received: 16 March 1999 / Received revision: 5 May 1999 / Accepted: 7 May 1999     

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.     

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.     

In Vivo and In Vitro Metabolomic Analysis of Anaerobic Rice Coleoptiles Revealed Unexpected Pathways

The ability of the rice (Oryza sativa L.) seedling to tolerate extended hypoxia during submergence is largely attributed to the biochemical adaptation of its coleoptile. Rice coleoptiles are capable of sustaining ATP production and cytoplasmic pH, unlike flood-sensitive organs, such as maize shoots. Fermentation reactions leading to the production of ethanol, alanine, succinate, and -aminobutyrate (GAB) are active in both types of tissues and thus may not account for the difference in tolerance. We have shown previously that rice coleoptiles undergo nitrate reduction and metabolism, which is efficient in alleviating cytoplasmic acidosis and regenerating NAD. Here, we employed ¹³C-2-acetate tracer methods with in vivo ¹³C NMR measurement, including in vivo isotopomer analysis, to probe the tricarboxylic acid (TCA) cycle and interacting pathways in rice coleoptiles during anaerobiosis. We found that the TCA cycle underwent multiple turns based on the metabolic scrambling of ¹³C label patterns in glutamine and malate. The in vivo kinetics of the ¹³C label incorporation into glutamic acid, glutamine, and GAB supports a separate pool of glutamate that was derived from the glutamate dehydrogenase reaction and subsequently decarboxylated to yield GAB. Both reactions consume additional H⁺ and/or NADH. Moreover, the higher rate of ¹³C enrichment at C-3 than C-2 of malate suggests the contribution of the glyoxylate cycle to malate synthesis, which could replenish the TCA cycle carbons diverted to GAB, glutamate, and glutamine synthesis. All of the above reactions contribute to the maintenance of glycolysis for energy production.