Oxaloacetate is Converted Directly Into Acetyl Coa to Feed the Citric Acid Cycle

Oxaloacetate

Lastly, the oxaloacetate synthesis through pyruvate carboxylating reduction followed by malate oxidation, which is one of the anaplerotic processes:involves coupling of carboxylation through redox energy.

From: Living Systems As Energy Converters , 1977

Metabolic Pathway Synthesis

Gregory N. Stephanopoulos , ... Jens Nielsen , in Metabolic Engineering, 1998

7.3.1. THE ROLE OF OXALOACETATE

It can be observed in the previous pathways that oxaloacetate is a central metabolite in all of them. Although oxaloacetate was partly bypassed in the pathway of Fig. 7.6b, a key question is whether this metabolite can be bypassed altogether and whether pathways can be constructed for the production of lysine from pyruvate or glucose without the involvement of oxaloacetate at any point. Upon examination of all the pathways generated, it turned out that, within the enzymatic database used, such a situation is impossible. Also, no single reaction surrounding oxaloacetate is fixed in the sense that it is present in all pathways. Particular reactions consuming and producing oxaloacetate may vary; however, the intermediate itself is always present. Oxaloacetate thus is a key node in the production of lysine.

In the pathway of Fig. 7.6b, aspartate and lysine are not derived directly from oxaloacetate, because fumarate is converted to aspartate by a single enzyme. In fact, aspartate is converted into oxaloacetate, rather than the reverse. Thus, in this case, the metabolism in the neighborhood of aspartate, fumarate, malate, and oxaloacetate is quite different from what one would typically find. This portion of the metabolism may suggest that it is possible to derive aspartate without the intervention of oxaloacetate. It turns out, however, that the necessary TCA intermediates (malate or succinate) cannot be produced from glucose without the intervention of oxaloacetate. This constraint necessitates the presence of oxaloacetate in any pathway leading from glucose to lysine.

To further illustrate this point, assume that in addition to glucose we could use succinate as an allowed reactant. A priori biosynthetic classifications would still entail oxaloacetate as a required intermediate. Inspection of the pathway of Fig. 7.6b, however, indicates that succinate can be converted to fumarate and from then on produce aspartate by aspartate amino lyase without the intervention of malate or oxaloacetate. Thus, with succinate as an additional allowed substrate, it is entirely possible to synthesize lysine with a pathway that does not entail oxaloacetate.

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Review of Cellular Metabolism

Gregory N. Stephanopoulos , ... Jens Nielsen , in Metabolic Engineering, 1998

2.3.4. ANAPLEROTIC PATHWAYS

Two of the TCA cycle intermediates, α-ketoglutarate and oxaloacetate serve as precursor metabolites for the biosyntheses of amino acids and nucleotides. ² There is no net synthesis of these two organic acids in the TCA cycle, and their removal for other cellular functions must be compensated for by other means. Reaction sequences that fulfill this role are summarily referred to as anaplerotic pathways. The anaplerotic pathways include (see Fig. 2.10) the following: (1) carboxylation of pyruvate by pyruvate carboxylase; (2) carboxylation of phosphoenolpyruvate by PEP carboxylase; (3) oxidation of malate to pyruvate by the malic enzyme; and (4) the glyoxylate cycle.

The most important anaplerotic pathways are carbon dioxide fixation by either pyruvate carboxylase or PEP carboxylase, leading to the formation of oxaloacetate (Fig. 2.10). Pyruvate carboxylase is activated at a high ATP/ADP ratio and by acetyl-CoA and is inhibited by L-aspartate. Thus, the regulation of this enzyme is almost completely the reverse of that of the pyruvate dehydrogenase complex, which is inhibited by high ATP/ADP or NADH/NAD⁺ ratios and high acetyl-CoA concentrations (Zubay, 1988). In S. cerevisiae, pyruvate carboxylase activity is found in both the cytosol and the mitochondria (Haarasilta and Taskinen, 1977). PEP carboxylase is quite active in many prokaryotes, whereas it has not been identified in fungi. It is regulated similar to pyruvate carboxylase, i.e., inhibited by L-aspartate and activated by acetyl-CoA (Jetten et al., 1994).

Acetyl-CoA cannot be transported through the inner mitochondrial membrane into the cytosol where it is needed as a key precursor for amino acid and lipid biosynthesis. In eukaryotes, if there is an imbalance between the cellular needs for energy and carbon skeletons for biosynthesis, acetyl-CoA synthesis in the cytosol must take place. This may occur by two different pathways: (1) The first utilizes the free transport of citrate through the mitochondrial membrane and involves the enzyme citrate lyase (reaction (13) in Fig. 2.10), which cleaves cytoplasmic citrate to oxaloacetate and acetyl-CoA with concurrent hydrolysis of ATP to ADP. Normally, the requirement for acetyl-CoA exceeds that for oxaloacetate, and the excess oxaloacetate generated by this reaction is converted to L-malate by a cytoplasmic malate dehydrogenase. L-Malate may then either re-enter the mitochondria or be oxidatively decarboxylated to pyruvate by the malic enzyme (reaction (15) in Fig. 2.10). In the oxidation of L-malate by the malic enzyme, NADPH is formed, and this reaction may be a major source for NADPH synthesis in the cytosol of eukaryotes. (2) The second pathway for generation of cytoplasmic acetyl-CoA is via acetate. First, pyruvate is oxidatively decarboxylated to yield acetate

(2.11) ${\text{acetate}+\text{CO}}_{\text{2}}+\text{NADH}+\text{H}+-\text{pyruvate}-{\text{NAD}}^{+}=0$

and, thereafter, acetate is converted to acetyl-CoA by acetyl-CoA synthase:

(2.12) $\text{acetyl}-\text{CoA}+{\text{H}}_{\text{2}}\text{O}+\text{AMP}+{\text{PP}}_{\text{i}}-\text{acetate}-\text{CoA}-\text{ATP}=\text{0}$

This pathway is used by S. cerevisiae (Frenkel and Kitchens, 1977), but in A. nidulans acetyl-CoA synthase is induced by acetate and repressed by glucose (Kelly and Hynes, 1982). Reaction (2.12) therefore is mainly active during growth on acetate.

In the glyoxylate cycle (also referred to as glyoxylate shunt), isocitrate is cleaved by isocitrate lyase [reaction 11 in Fig. (2.10)] to form succinate and glyoxylate, which may react with acetyl-CoA by the action of malate synthase [reaction (12)] to form L-malate. L-Malate may then be converted to isocitrate in a sequence of reactions identical to those of the TCA cycle [reactions (9) and (2) in Fig. 2.10]. The net result of the glyoxylate cycle is, therefore, synthesis of the four-carbon succinate from 2 molecules of acetyl-CoA, and this pathway is important during the metabolism of acetate and fatty acids where acetyl-CoA is a common intermediate.

In eukaryotes, the supply of α-ketoglutarate to the cytosol for biosynthesis is important. This precursor metabolite may be transported across the inner mitochondrial membrane by a specific permease, but it may also be synthesized in the cytosol by a cytosolic isocitrate dehydrogenase. Two different kinds of isocitrate dehydrogenases have been identified in fungi: one NAD⁺ dependent and one linked to NADP⁺. The NAD⁺-isocitrate dehydrogenase is always associated with the mitochondria, and in A. nidulans the NADP⁺ dependent enzyme is repressed by glucose and is mainly active during growth on acetate (Kelly and Hynes, 1982). This may offer an explanation for the existence of two isocitrate dehydrogenases: They allow for the generation of NADPH during growth on acetate, where it is energetically expensive to form NADPH via the PP pathway. For other organisms, activity of the NADP⁺-linked enzyme is also found during growth on glucose, and here it may play an important role in the supply of NADPH.

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Miscanthus

Iris Lewandowski , ... Yasir Iqbal , in Perennial Grasses for Bioenergy and Bioproducts, 2018

2.3.1 C₄ Pathway and Resource Use Efficiency

Miscanthus performs photosynthesis by the C₄ pathway. In this pathway, the first compound formed through CO₂ fixation is a 4-carbon organic acid (oxaloacetate) catalyzed by phosphoenolpyruvate carboxylase. The C ₄ pathway directly influences the resource use efficiency of the crop (Sage and Zhu, 2011). For example, it contributes toward high water use efficiency through reduced evapotranspiration by keeping the stomata closed for longer and fixing the available CO₂ more efficiently than in the C₃ pathway (Byrt et al., 2011). Although Miscanthus is undomesticated, it outperforms many other C₄ species under temperate climatic conditions in terms of resource use efficiency and ability to grow under low-temperature conditions. For example, M.  × giganteus is capable of carrying out photosynthetic activity at temperatures as low as 6°C, even lower than the threshold temperature for maize (Wang et al., 2008). Despite being a C₄ plant, some miscanthus genotypes are cold-tolerant and can survive severe winters (Clifton-Brown and Lewandowski, 2000a).

The aboveground water use efficiency of miscanthus varies greatly depending on climatic conditions. For example, biomass accumulation ranges from 9 to 13   g   DM   ha⁻¹ water under temperate conditions (Beale et al., 1999), but from 3 to 5   g   DM   ha⁻¹ water in Mediterranean conditions (Cosentino et al., 2007). Thus the amount of water required for each kg of biomass accumulation is lower for miscanthus than for maize and sugarcane (Van der Weijde et al., 2013).

Miscanthus achieves high nutrient use efficiency in three ways: (1) low input requirements; (2) recycling of nutrients through litter falling; and (3) translocation of nutrients back to rhizomes. The recycling of nutrients is highly dependent on the efficiency of the translocation process, which in turn is mainly defined by the phenological traits of the genotype and the time of harvesting. Early-flowering genotypes complete the translocation of nutrients more efficiently before frost kills the stems. The nutrient input demand for optimal growth is highly dependent on soil conditions. For each kg of DM yield, miscanthus removes 4.90   g   N, 0.45   g   P, and 7.20   g (Cadoux et al., 2012), which is significantly lower than for other C₄ crops such as sorghum, sugarcane, and maize (Van der Weijde et al., 2013).

Radiation use efficiency is shown to vary with temperature and is reduced by water stress. Hastings et al. (2009) used data from Farage et al. (2006) to develop a temperature-related radiation use efficiency model that has a maximum value of 4.8   g biomass dry matter per MJ radiation. This becomes about 2.35   g in temperate climates, as found by Clifton-Brown et al. (2000). Experiments investigating yield response to nitrogen fertilizer application showed that there was little impact, except where either the soil was sand (Clifton-Brown et al., 2001) or where the miscanthus was harvested before senescence (Danalatos et al., 2007). Some miscanthus genotypes have been shown to be conservative users of water, especially under reduced soil water conditions; others such as the current commercial genotype M.  × giganteus less so (Clifton-Brown and Lewandowski, 2000b).

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AMMONIA ASSIMILATION AND AMINO ACID BIOSYNTHESIS

P.J. LEA , in Techniques in Bioproductivity and Photosynthesis (Second Edition), 1985

14.1.6 Aminotransferases

Aminotransferases catalyse the transfer of the amino group of an amino acid to a 2-oxo acid to yield a new amino acid and 2-oxo acid respectively ⁵ e.g. glutamate-oxaloacetate aminotransferase E.C.2.6.1.1 :-

Glutamate + oxaloacetate → 2-oxoglutarate + aspartate

Extraction buffer:

Tris-HCl (pH 7.5, 40 mM), EDTA 0.25 mM, glutathione 2 mM. All aminotransferases are reversible; therefore the direction chosen may depend upon the substrates available. The enzymes are pyridoxal phosphate requiring, although it is now believed that in plants the coenzyme is bound tightly to the enzyme. The requirement for pyridoxal phosphate for the enzyme under test should be checked. Probably the simplest method of testing for aminotransferase is to incubate the enzyme with 5 mM 2-oxo acid and 5 mM amino acid in 50 mM Tris-HCl buffer pH 7.5 for varying times and stopping the reaction with an equal volume of ethanol. After centrifuging the protein, the extract may be chromatographed on paper or TLC plates in a solvent that gives a good separation of the initial end product amino acid (e.g. butanol: acetic acid: water in proportions 90:10:29 by volume gives a good separation of the amino acids aspartate and alanine). The rate of synthesis of the product amino acid may be determined by the method of Atfield and Morris described in the previous section.

A second method is to incubate the amino acid with a very small amount of 2-oxo acid and determine the product 2-oxo acid by formation of a dinitrophenylhydrazone. Alanine aminotransferase may be assayed by incubating the enzyme with 100 mM alanine and 2 mM 2-oxoglutarate in 0.1 M Tris-HCl buffer pH 7.4. The pyruvate formed may be determined by reaction with 2,4-dinitrophenylhydrazine and measuring the colour at 546 nm. A standard curve of varying pyruvate concentrations must, of course, be constructed.

A third more refined but expensive method is to couple the 2-oxo acid produced to NADH oxidation by an added enzyme. A standard reaction mixture may be set up containing 25 μl 10 mM 2-oxoglutarate, 20 μl 10 mM EDTA, 10 μl 10 mM NADH and 40 μl enzyme extract. If aspartate aminotransferase is to be measured the product is oxaloacetate which is rapidly converted to malate by malic dehydrogenase with the subsequent oxidation of NADH, which may be measured on a spectrophotometer at 340 nm in a similar manner to glutamate dehydrogenase. The final reaction medium in the spectrophotometer cell should also include 25 μl 10 mM aspartate, 0.5 ml 0.1 M HEPES buffer pH 8.0, 0.1 ml of 100-fold diluted commercial malate dehydrogenase and 0.28 ml H₂O. In crude extracts of plants there is often sufficient malate dehydrogenase already present to drive the reaction without further addition of the enzyme. If alanine aminotransferase is to be measured, the product is pyruvate, which may be converted to lactate by lactate dehydrogenase. In this case the final reaction medium should also include 0.1 ml 0.1 M alanine, 0.5 ml 0.1 HEPES buffer pH 7.5, 0.1 ml of 100-fold diluted commercially available lactate dehydrogenase and 0.2 ml H₂O. The activity of the enzyme may be calculated in both cases using a blank reaction cuvette containing no 2-oxoglutarate.

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Production of Dicarboxylic Acid Platform Chemicals Using Yeasts

C. Pais , ... B.S. Ferreira , in Biotransformation of Agricultural Waste and By-Products, 2016

Reductive branch of tricarboxylic acid cycle

Under anaerobic conditions, the reductive pathway of TCA cycle is activated, since succinate is the H-acceptor instead of oxygen, and pyruvate, originated from glycolysis, is converted to oxaloacetate, malate, fumarate, and then succinate ( Fig. 9.1B). This pathway, from phosphoenolpyruvate (which precedes pyruvate in glycolysis) to succinate, requires 2   mol of NADH per mole of succinate produced, which represents a maximum theoretical yield of two molecules of succinate for every glucose molecule, since each molecule of glucose can provide only two molecules of NADH through glycolysis. Therefore, the redirection of the carbon flux only to the anaerobic fermentation pathway is energetically unfavorable, has a theoretical yield higher than the oxidative direction or the glyoxylate shunt (1.71   mol per mole of glucose), and results in net CO₂ fixation. The reductive pathway can be divided in the following steps: (1) pyruvate carboxylation, in which pyruvate in converted in oxaloacetate, a reaction performed by the enzyme pyruvate carboxylase, encoded by gene PYC; (2) oxaloacetate reduction to malate, via the action of malate dehydrogenase, encoded by gene MDH; (3) translation of malate to fumarate, under the action of fumarase (encoded by FUMR); and (4) fumarate reduction performed by fumarate reductase, encoded by genes FRDS1 and OSH1. Yan et al. (2014) reviewed the main obstacles of reductive TCA pathway for succinic acid production: (1) yeast fumarase (FUM) only converts fumarate to malate without the possibility to revert the process to fumarate production; (2) fumarate reductase, the key enzyme involved in the reductive production of succinate (Arikawa et al., 1999), coded by genes FRDS1 (cytosol) and OSM1 (mitochondria) (Fig. 9.1B), are only produced under anaerobic conditions; and (3) a high amount of NADH is consumed by this branch (2   mol of NADH per each mole of succinic acid formed).

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CHLOROPLASTS AND PROTOPLASTS

R.C. LEEGOOD , D.A. WALKER , in Techniques in Bioproductivity and Photosynthesis (Second Edition), 1985

9.3.2 C₄ mesophyll protoplasts

Mesophyll protoplasts from C₄ plants have a rather limited applicability for studies of photosynthetic carbon fixation since they do not fix CO₂ and the plasmalemma is impermeable to natural substrates (pyruvate, phosphoenolpyruvate and oxaloacetate) ⁴ , although they are useful for studies of enzyme localisation. Two alternatives are available. The first is to make intact mesophyll cells from plants such as Digitarla sanguinalis ^{7, 9} . Such cells retain permeability to the natural substrates. The second alternative is to prepare a protoplast extract. The protoplasts are passed through a 20 μm nylon mesh but the chloroplasts are assayed in the presence of the rest of cell contents which include the cytosolic PEP carboxylase. By this means, pyruvate supplied to the chloroplasts can be converted to PEP, PEP to oxaloacetate in the medium and oxaloacetate to malate in the chloroplasts ⁸ .

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Enzymes as Diagnostic Tools

Ram Sarup Singh , ... Ashish Kumar Singh , in Advances in Enzyme Technology, 2019

9.2.1.1.2 Aspartate Aminotransferase

The serum level of AST helps people to diagnose damaged body organs, especially the heart and liver. AST (E.C. 2.6.1.1) catalyzes the transamination of l -aspartate and 2-oxoglutarate into oxaloacetate and glutamate, respectively. In a healthy human adult, AST has a concentration of around 5–40  U/L [51]. However, after severe damage, the AST level rises 10–20-times higher than the normal range. AST is also found in the red blood cells, muscle tissue, and other organs, including the kidney and pancreas. It can be used in combination with other enzymes to monitor myocardial, hepatic parenchymal, and muscle diseases in humans and animals. Moreover, to screen the liver fibrosis in chronic hepatitis B, the AST-to-platelet ratio index could be a useful marker, when transient elastography is not available [12].

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Methods for the Experimental Determination of Metabolic Fluxes by Isotope Labeling

Gregory N. Stephanopoulos , ... Jens Nielsen , in Metabolic Engineering, 1998

9.2.1. DISTRIBUTION OF TCA CYCLE METABOLITE ISOTOPOMERS FROM LABELED PYRUVATE

Figure 9.8 depicts the reactions of pyruvate utilization via the tca cycle and gluconeogenic pathways commonly found in eukaryotic systems. We will assume that phosphoenolpyruvate (pep) is not formed directly from pyruvate via PEP synthetase, but rather is formed from oxaloacetate (OAA) via PEP carboxykinase. Pyruvate is converted to OAA by direct carboxylation, or it can be converted to acetyl-CoA, which then condenses with OAA to form citrate that traverses the TCA cycle and eventually regenerates OAA. Finally, glucose can be formed from PEP via the reverse Embden-Meyerhof scheme of glycolysis or gluconeogenic pathway.

FIGURE 9.8. Pyruvate utilization via the citric acid cycle and the gluconeogenic pathway. The abbreviations used in the subscripts of the fluxes are as follows: GP, gluconeogenic pathway; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACON, aconitase; KGDH, α-ketoglutarate dehydrogenase; SUDH, succinate dehydrogenase; FUM, fumarase; MDH, malate dehydrogenase; PPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase. The abbreviations for metabolites are as follows: PEP, phosphoenolpyruvate; P, pyruvate; AcCoA, acetyl-CoA; C, citrate; IC, isocitrate; K, α-ketoglutarate; S, succinate; F, fumarate; M, malate; O, oxaloacetate; CO₂, carbon dioxide; GLU, glucose. The operation of futile cycles such as PEP → P → O → PEP, is assumed inactive. Other reactions such as glutamate-oxaloacetate transaminase and glutamate dehydrogenase for biosynthesis are ignored.

We begin by writing mass balances for the metabolites identified in the network of Fig. 9.8.

(9.10) $\frac{d{c}_0}{dt}=v_{MDH}+v_{PC}-v_{CS}-V_{PPCK}$

(9.11) $r_{C{O}_2}=v_{IDH}+v_{KGDH}-v_{PC}+v_{PPCK}$

(9.13) $\frac{d{c}_P}{dt}=v_{IMPORT}-v_{PC}=v_{PDH}$

At metabolite steady state there is no accumulation of intracellular metabolites and eqs. (9.3)–(9.13) yield:

(9.14) $v_{\text{CS}}=v_{\text{PDH}}=v_{\text{ACON}}=v_{\text{IDH}}=v_{\text{KGDH}}=v_{\text{SUDH}}=v_{\text{FUM}}=v_{\text{MDH}}$

Similar to the total metabolite concentrations, balances can be written for the concentration of each metabolite isotopomer shown in Fig. 9.6 upon administration of 100% [3-¹³C]pyruvate:

$\frac{d{c}_0}{dt}=[\begin{array}{c}{O}_3\\ O_2\\ O_{34}\\ O_{12}\\ O_{13}\\ O_{24}\\ O_{123}\\ O_{234}\\ O_{23}\end{array}]=v_{MDH}[\begin{array}{c}0\\ 0\\ 0\\ 0\\ M_{13}\\ M_{24}\\ M_{123}\\ M_{234}\\ M_{23}\end{array}]+v_{PC}[\begin{array}{c}\left(\frac{1-C{O}_2^{*}}{2}\right)\\ \left(\frac{1-C{O}_2^{*}}{2}\right)\\ \left(\frac{C{O}_2^{*}}{2}\right)\\ \left(\frac{C{O}_2^{*}}{2}\right)\\ 0\\ 0\\ 0\\ 0\\ 0\end{array}]$

(9.19) $-v_{CS}[\begin{array}{c}{O}_3\\ O_2\\ O_{34}\\ O_{12}\\ O_{13}\\ O_{24}\\ O_{123}\\ O_{234}\\ O_{23}\end{array}]-v_{PPCK}[\begin{array}{c}{O}_3\\ O_2\\ O_{34}\\ O_{12}\\ O_{13}\\ O_{24}\\ O_{123}\\ O_{234}\\ O_{23}\end{array}]$

In the preceding equation, each isotopomer species is represented in relative population, i.e., its concentration is normalized by the total concentration of the same metabolite (for example, O₃ = c_{O, 3}/c _O), and CO₂ ^* is the fraction of CO₂ labeled with ¹³C.

Similar to oxaloacetate, isotopomer balances can be written for citrate/isocitrate (indicated by C), α-ketoglutarate, succinate, malate, and the fraction of CO₂ labeled with ¹³C, CO₂ ^* [eqs. (9.20)–(9.24), respectively]:

(9.20) $\frac{d{c}_C}{dt}[\begin{array}{c}{C}_{24}\\ C_{34}\\ C_{124}\\ C_{346}\\ C_{246}\\ C_{134}\\ C_{2346}\\ C_{1234}\\ C_{234}\end{array}]=v_{CS}[\begin{array}{c}{O}_3\\ O_2\\ O_{34}\\ O_{12}\\ O_{13}\\ O_{24}\\ O_{123}\\ O_{234}\\ O_{23}\end{array}]-V_{IDH}[\begin{array}{c}{C}_{24}\\ C_{34}\\ C_{124}\\ C_{346}\\ C_{246}\\ C_{134}\\ C_{2346}\\ C_{1234}\\ C_{234}\end{array}]$

(9.21) $\frac{d{c}_K}{dt}[\begin{array}{c}{K}_{24}\\ K_{34}\\ K_{124}\\ K_{134}\\ K_{234}\\ K_{1234}\end{array}]=v_{IDH}[\begin{array}{c}{C}_{24}+C_{246}\\ C_{34}+C_{346}\\ C_{124}\\ C_{134}\\ C_{2346}+C_{234}\\ C_{1234}\end{array}]-v_{KGDH}[\begin{array}{c}{K}_{24}\\ K_{34}\\ K_{124}\\ K_{134}\\ K_{234}\\ K_{1234}\end{array}]$

(9.22) $\frac{d{c}_S}{dt}[\begin{array}{c}{S}_{23}\\ S_{13}\\ S_{123}\end{array}]=v_{KGDH}[\begin{array}{c}{K}_{134}+K_{34}\\ K_{24}+K_{124}\\ K_{1234}+K_{234}\end{array}]-v_{SUDH}[\begin{array}{c}{S}_{23}\\ S_{13}\\ S_{123}\end{array}]$

(9.23) $\frac{d{c}_M}{dt}[\begin{array}{c}{M}_{23}\\ M_{13}\\ M_{24}\\ M_{123}\\ M_{234}\end{array}]=v_{SUDH}[\begin{array}{c}{S}_{23}\\ \frac{1}{2}{S}_{13}\\ \frac{1}{2}{S}_{13}\\ \frac{1}{2}{S}_{123}\\ \frac{1}{2}{S}_{123}\end{array}]-v_{MDH}[\begin{array}{c}{M}_{23}\\ M_{13}\\ M_{24}\\ M_{123}\\ M_{234}\end{array}]$

(9.24) $\begin{array}{c}{r}_{CO2}C{O}_2^{*}=v_{IDH}\left(C_{346}+C_{246}+C_{2346}\right)+v_{KGDH}\left(K_{124}+K_{134}+K_{1234}\right)\\ -v_{PC}\left(C{O}_2^{*}\right)+v_{PPCK}\left(O_{34}+O_{24}+O_{234}\right)\end{array}$

At metabolite and isotope steady state, using eqs. (9.22), (9.23), and (9.14), we obtain:

(9.25) $[\begin{array}{c}{M}_{23}\\ M_{13}\\ M_{24}\\ M_{123}\\ M_{234}\end{array}]=[\begin{array}{c}{K}_{134}+K_{34}\\ \frac{1}{2}\left(K_{24}+K_{124}\right)\\ \frac{1}{2}\left(K_{24}+K_{124}\right)\\ \frac{1}{2}\left(K_{1234}+K_{234}\right)\\ \frac{1}{2}\left(K_{1234}+K_{234}\right)\end{array}]$

Similarly, from eqs. (9.20), (9.21), and (9.14):

(9.26) $[\begin{array}{c}{K}_{24}\\ K_{34}\\ K_{124}\\ K_{134}\\ K_{234}\\ K_{1234}\end{array}]=[\begin{array}{c}{O}_3+O_{13}\\ O_2+O_{12}\\ O_{34}\\ O_{24}\\ O_{123}+O_{23}\\ O_{234}\end{array}]$

By combining eqs. 9.25 and (9.26) and inserting the resulting expressions for malate isotopomers into the steady state version of eq. (9.19), the isotopomer balance of oxaloacetate at metabolic and isotopic steady state is obtained:

(9.27) $v_{CS}[\begin{array}{c}0\\ 0\\ 0\\ 0\\ \frac{1}{2}\left(O_3+O_{13}+O_{34}\right)\\ \frac{1}{2}\left(O_3+O_{13}+O_{34}\right)\\ \frac{1}{2}\left(O_{234}+O_{123}+O_{23}\right)\\ \frac{1}{2}\left(O_{234}+O_{123}+O_{23}\right)\\ \left(O_{24}+O_2+O_{12}\right)\end{array}]+v_{PC}[\begin{array}{c}\left(\frac{1-C{O}_2^{*}}{2}\right)\\ \left(\frac{1-C{O}_2^{*}}{2}\right)\\ \left(\frac{C{O}_2^{*}}{2}\right)\\ \left(\frac{C{O}_2^{*}}{2}\right)\\ 0\\ 0\\ 0\\ 0\\ 0\end{array}]=\left(v_{CS}+v_{PC}\right)[\begin{array}{c}{O}_3\\ O_2\\ O_{34}\\ O_{12}\\ O_{13}\\ O_{24}\\ O_{123}\\ O_{234}\\ O_{23}\end{array}]$

Equation (9.27) indicates that all oxaloacetate isotopomers can be obtained from the relative enrichment of carbon dioxide and the citrate synthase and pyruvate carboxylase fluxes only, v _cs and v _PC, respectively. The preceding analysis can also be applied in a straightforward manner to all other isotopomers to derive equations for their determination too in terms of v _cs and v _PC, under metabolic and isotopic steady state. Therefore, v _cs and v _PC are the only unknowns from which the relative populations of all isotopomers can be calculated and, through them, the degree of label enrichment of intermediate metabolites.

Solutions can be expressed conveniently in terms of x, defined as the probability that oxaloacetate exits the TCA cycle via the PEP carboxykinase (PPCK) reaction [then (1 – x) will denote the probability that oxaloacetate will re-enter the TCA cycle via citrate synthase (CS)], and y, defined as the probability of bicarbonate fixed into pyruvate to be labeled with ¹³C, *CO₂. In terms of x, one can write:

(9.28) $\frac{x}{1-x}=\frac{v_{PPCK}}{v_{CS}}=\frac{v_{PC}}{v_{CS}}$

Also, if there is no pathway that generates and consumes CO₂ other than the reactions indicated in this model, y can be expressed exclusively in terms of x as well. Table 9.1 summarizes the results for the relative populations of oxaloacetate and glutamate isotopomers in terms of the two probabilities x and y when 100% enriched pyruvate is used as substrate. The legend of Table 9.1 provides expressions for the determination of y in terms of x. Table 9.1 also shows the relative enrichment of glucose and glutamate molecules synthesized from pyruvate labeled at three different carbon atoms, as indicated. The possible isotopomers generated when 100% enriched [2-¹³C]pyruvate and 100% enriched [1-¹³C]pyruvate, respectively, are used as labeling substrates are shown in Fig. 9.9. We note that the results of Table 9.1 allow the determination of any other isotopomer or metabolite enrichment for comparison with experimental data, when available. Also, G and K are used interchangeably as the isotopomer distributions of glutamate and α-ketoglutarate are identical.

TABLE 9.1. Steady State Distribution of Oxaloacetate (O) and Glutamate (G) Isotopomers and Relative Carbon Enrichments in Glucose and Glutamate Following the Utilization of 100% [3−¹³C]Pyruvate, 100% [2−¹³C]Pyruvate, and 100% [1−¹³C]Pyruvate through the Major Pathways Described in Fig. 9.8 ^a

	[3−13C]Pyruvate	[2−13C]Pyruvate	[1−13C]Pyruvate
y	$y=\frac{1-x}{2+x-x^2}$	$y=\frac{1+x-2x^2}{2+x-x^2}$	$y=\frac{x}{2-x}$
Oxaloacetate isotopomer distribution	$\begin{array}{l}{O}_2=O_3=\frac{x\left(1-y\right)}{2}\hfill \\ O_{12}=O_{34}=\frac{xy}{2}\hfill \\ O_{23}=\frac{\left(1-x\right)x}{\left(1+x\right)}\hfill \\ O_{13}=O_{24}=\frac{\left(1-x\right)x}{2\left(1+x\right)}\hfill \\ O_{123}=O_{234}=\frac{{\left(1-x\right)}^2}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{l}{O}_2=O_3=\frac{x\left(1-y\right)}{2}\hfill \\ O_{13}=O_{24}=\frac{x\left(1-x+y+xy\right)}{2\left(1+x\right)}\hfill \\ O_{14}=\frac{x\left(1-x\right)}{\left(1+x\right)}\hfill \\ O_1=O_4=\frac{{\left(1-x\right)}^2}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{l}{O}_1=O_4=\frac{x\left(1-y\right)}{2}\hfill \\ O_{14}=xy\hfill \\ O=1-x\hfill \end{array}$
Glucose enrichment pattern	$\begin{array}{l}C-1=\frac{1}{\left(1+x\right)}\hfill \\ C-2=\frac{1}{\left(1+x\right)}\hfill \\ C-3=\frac{1-x+xy+x^{2}y}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{l}C-1=\frac{x}{\left(1+x\right)}\hfill \\ C-2=\frac{x}{\left(1+x\right)}\hfill \\ C-3=\frac{1-x+2x^2+xy+x^{2}y}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{l}C-1=0\hfill \\ C-2=0\hfill \\ C-3=\frac{x\left(1+y\right)}{2}\hfill \end{array}$
Glutamate isotopomer distribution	$\begin{array}{l}{K}_{24}=\frac{x\left(2-y-xy\right)}{2\left(1+x\right)}\hfill \\ K_{34}=\frac{x}{2}\hfill \\ K_{124}=\frac{xy}{2}\hfill \\ K_{134}=\frac{x\left(1-x\right)}{2\left(1+x\right)}\hfill \\ K_{234}=\frac{1-x}{2}\hfill \\ K_{1234}=\frac{{\left(1-x\right)}^2}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{l}{K}_{35}=\frac{x\left(1-y\right)}{2}\hfill \\ K_{135}=\frac{x\left(1-x+y+xy\right)}{2\left(1+x\right)}\hfill \\ K_{25}=\frac{x}{1+x}\hfill \\ K_{15}=\frac{1-x}{2}\hfill \\ K_5=\frac{{\left(1-x\right)}^2}{2\left(1+x\right)}\hfill \end{array}$	$\begin{array}{c}K=\frac{2-x-xy}{2}\\ K_1=\frac{x\left(1+y\right)}{2}\end{array}$
Glutamate enrichment pattern	$\begin{array}{l}C-1=\frac{1-x+xy+x^{2}y}{2\left(1+x\right)}\hfill \\ C-2=\frac{1}{1+x}\hfill \\ C-3=\frac{1}{1+x}\hfill \\ C-4=1\hfill \\ C-5=0\hfill \end{array}$	$\begin{array}{l}C-1=\frac{1-x+2x^2+xy+x^{2}y}{2\left(1+x\right)}\hfill \\ C-2=\frac{x}{1+x}\hfill \\ C-3=\frac{x}{1+x}\hfill \\ C-4=0\hfill \\ C-5=1\hfill \end{array}$	$\begin{array}{l}C-1=\frac{x\left(1+y\right)}{2}\hfill \\ C-2=0\hfill \\ C-3=0\hfill \\ C-4==0\hfill \\ C-5=0\hfill \end{array}$

a

y can be obtained exclusively as a function of x, as indicated in the first row. Glutamate (G) and α-ketoglutarate (K) are used interchangeably as the labeling patterns of the two are identical. Glucose synthesized via the gluconeogenic pathway with these substrates has the following enrichment pattern: C-4 = C-3; C-5 = C-2; C-6 = C-1.

FIGURE 9.9. Schematic diagrams of the sequential labeling of metabolite intermediates via multiple turns of the TCA cycle using (a) 100% enriched [2-¹³C]pyruvate and (b) 100% enriched [1-¹³C]pyruvate.

We close this section with a summary of the assumptions invoked in derivation of the expressions of Table 9.1:

•

There is no label recycling due to the operation of futile cycles such as pyruvate → OAA → PEP → pyruvate and malate → pyruvate → OAA → malate.

•

There is no compartmentalization of metabolites, and homogeneous pools exist that are common to the TCA cycle and other pathways.

•

There is no net flux to biosynthesis except in the formation of glucose.

•

There is no label scrambling by the operation of the PPP that would redistribute the label of glucose synthesized via the gluconeogenic pathway.

•

Metabolic and isotopic steady states are reached.

•

Input substrates are isotopically pure. They are 100% enriched at the designated position and none at the undesignated positions.

•

Isotopic dilution due to the presence of unlabeled endogenous pools is negligible.

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CO2 capture by bacteria and their enzymes

Alessandro Senatore , ... Angelo Basile , in Advances in Carbon Capture, 2020

18.2.2 The reductive tricarboxylic acid cycle

The reductive tricarboxylic acid cycle (TCA) or reverse citric acid cycle, reported for the first time in 1966 in a green sulfur bacterium, begins with the conversion of citrate into acetyl-CoA and oxaloacetate by the enzyme ATP-citrate lyase. During this step, a molecule of CO ₂ and ATP are used to catalyze the reaction. The cycle proceeds with the subsequent conversion of acetyl-CoA into pyruvate by the enzyme pyruvate: ferrodoxin oxidoreductase (PFOR) which sequesters a CO₂ molecule for the enzymatic reaction. The biochemical reaction can proceed through one or more steps that depend on the species of microorganism and which leads to the formation of oxaloacetate (Fig. 18.2). Malate dehydrogenase enzyme, converts oxaloacetate into malate while fumarate hydratase converts this molecule into fumarate. A series of other reactions summarized in Fig. 18.6, lead to the regeneration of citrate from isocitrate which is the product of isocitrate dehydrogenase (IDH) (another enzyme which sequester CO₂ for its reaction) [27, 29, 30].

Fig. 18.2. Reductive TCA cycle. Some enzymes of the process are shown in red.

From M.C.W. Evans, B.B. Buchanan, D.I. Arnon, A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium, Proc. Natl. Acad. Sci. USA 55 (4) (1966) 928–934.

Fig. 18.6. The dicarboxylate/4-hydroxybutyrate cycle [28].

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Amino-Based Products from Biomass and Microbial Amino Acid Production

K. Madhavan Nampoothiri , ... Kiran S. Dhar , in Bioenergy Research: Advances and Applications, 2014

Cadaverine

Cadaverine can be overproduced by introduction of an overproduced lysine decarboxylase. The corresponding substrate, l-lysine, is synthesized in E. coli and C. glutamicum by similar pathways covering 10 enzymatic steps initiating from the tricarboxylic acid cycle intermediate oxaloacetate. The three initial steps in this pathway lead to aspartic acid semialdehyde, which is the branch point for biosynthesis of the amino acids, l-methionine, l-threonine, l-isoleucine and l-lysine (Figure 19.4). However, there were substantial differences in the enzyme systems possessed by E. coli and C. glutamicum. When it is LysC from C. glutamicum that is additionally feedback inhibited by l-threonine, it was ThrA from E. coli that is subject to feedback inhibition by l-threonine (Park and Lee, 2010). The tolerance of E. coli for cadaverine seems to be lower compared to putrescine. The biomass formed in the presence of 51   g/l cadaverine was reduced by 30% in comparison to the same molar concentration of putrescine (Qian et al., 2011, 2009). Corynebacterium glutamicum was tested for growth on solid medium and grew even at concentrations of up to 31   g/l cadaverine (Mimitsuka et al., 2007).

Escherichia coli strains overexpressing the lysine decarboxylase gene cadA (b4131) in the wild-type genetic background led to accumulation of 0.8   g/l cadaverine by growing cells. To avoid side reactions of enzymes active with putrescine toward cadaverine, a number of genes were deleted: the spermidine synthase gene speE, the spermidine acetyltransferase gene speG, the putrescine importer gene puuP, the putrescine aminotransferase gene puuA and ygjG, which encodes the initial enzyme of the second putrescine degradation pathway and is known to be active in vitro with cadaverine. The resulting strain was able to accumulate 1.2   g/l cadaverine. Production of cadaverine was increased by 10% as a consequence of enhancing the flux of l-aspartic acid toward l-lysine by overexpression of dapA via promoter exchange. In fed-batch cultivation, this strain produced 9.6   g/l cadaverine (Qian et al., 2011).

Cadaverine production in C. glutamicum was also achieved by insertional inactivation of homoserine dehydrogenase gene, hom (cg1337, Figure 19.4, B-1) with cadA from E. coli. The resultant strain secretes 2.6   g/l cadaverine in the supernatant. The expression of cadA was driven by the strong kanamycin resistance gene promoter. But the strain was auxotrophic for l-methionine, l-threonine, and l-isoleucine (Mimitsuka et al., 2007). A different approach with biosynthetic lysine decarboxylase (LdcC) from E. coli led to 30% more cadaverine production than overexpression of cadA (Kind et al., 2010b). Later the C. glutamicum DAP-3c cadaverine-producing strain's substrate spectrum was broadened for hemicellulose utilization by introducing xylA and xylB genes from E. coli (Buschke et al., 2011). Through various studies reasonable titers and productivities were achieved for putrescine and cadaverine (Table 19.1).

TABLE 19.1. Characteristics of Microbial Putrescine and Cadaverine Production

Polyamine	Substrate	Organism	Cultivation Method	C [g/l]	Y(P/S) [g/g]	References
Putrescine	Glucose	E. coli	Fermentor (fed-batch)	5.1	nd	Eppelmann et al. (2006)
Putrescine	Glucose	E. coli	Fermentor (fed-batch)	24.2	nd	Qian et al. (2009)
Putrescine	Glucose	C. glutamicum	Shake flask	6	0.12	Schneider and Wendisch (2010)
Putrescine	Glucose	C. glutamicum	Fermentor (fed-batch)	19	0.16	Schneider et al. (2012)
Cadaverine	Lysine	E. coli	Fermentor (fed-batch)	69	–	Nishi et al. (2007)
Cadaverine	Glucose	E. coli	Fermentor (fed-batch)	9.6	0.12	Qian et al. (2011)
Cadaverine	Glucose	C. glutamicum	Fermentor (fed-batch)	2.6	0.05	Mimitsuka et al. (2007)
Cadaverine	Glucose	C. glutamicum	Shake flask	3.4	nd	Verseck et al. (2008)
Cadaverine	Glucose	C. glutamicum	Fermentor (fed-batch)	5.0	0.09	Tateno et al. (2007)
Cadaverine	Starch	C. glutamicum	Fermentor (fed-batch)	2.4	0.05	Tateno et al. (2007)
Cadaverine	Glucose	C. glutamicum	Shake flask	1.7	0.17	Kind et al. (2010b)
Cadaverine	Glucose	C. glutamicum	Shake flask	1.1	0.11	Kind et al. (2010b)
Cadaverine	Glucose	C. glutamicum	Shake flask	1.3	0.13	Kind et al. (2010a)
Cadaverine	Glucose	C. glutamicum	Fermentor (fed-batch)	72	nd	Völkert et al. (2010)
Cadaverine	Xylose	C. glutamicum	Shake flask	1.4	0.11	Buschke et al. (2011)
Cadaverine	Hemicellulose hydrolysate	C. glutamicum	Shake flask	2	nd	Buschke et al. (2011)

nd - Not determined

Source: Schneider, J. and Wendisch, V.F. (2011); with modification.

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