Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition

Capture and conversion of CO₂ to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry‐relevant chemicals from CO₂. However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO₂ to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP‐driven malonyl‐CoA synthesis pathway and heterologous phosphoketolase (PHK)‐phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl‐CoA directly from CO₂ in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl‐CoA‐derived biochemicals.     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

The SCP2‐thiolase‐like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2‐thiolase‐like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2‐Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5‐fold reduction of de novo sterol biosynthesis from glucose‐ and acetate‐derived acetyl‐CoA. Fluorescence analyses of EGFP‐tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP‐acetoacetyl‐CoA (1.90 Å) and TbSLP‐malonyl‐CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl‐CoA tightly (K_d 90 µM), acetoacetyl‐CoA moderately (K_d 0.9 mM) and acetyl‐CoA and CoA very weakly. TbSLP possesses low malonyl‐CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075–1096. © 2016 Wiley Periodicals, Inc.     

Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942

Genetic manipulation in cyanobacteria enables the direct production of valuable chemicals from carbon dioxide. However, there are still very few reports of the production of highly effective photosynthetic chemicals. Several synthetic metabolic pathways (e.g., isopropanol, acetone, isoprene, and fatty acids) have been constructed by branching from acetyl-CoA and malonyl-CoA, which are key intermediates for photosynthetic chemical production downstream of pyruvate decarboxylation. Recent reports of the absolute determination of cellular metabolites in Synechococcus elongatus PCC 7942 have shown that its acetyl-CoA levels corresponded to about one hundredth of the pyruvate levels. In short, one of the reasons for lower photosynthetic chemical production from acetyl-CoA and malonyl-CoA was the smaller flux to acetyl-CoA. Pyruvate decarboxylation is a primary pathway for acetyl-CoA synthesis from pyruvate and is mainly catalyzed by the pyruvate dehydrogenase complex (PDHc). In this study, we tried to enhance the flux toward acetyl-CoA from pyruvate by overexpressing PDH genes and, thus, catalyzing the conversion of pyruvate to acetyl-CoA via NADH generation. The overexpression of PDH genes cloned from S. elongatus PCC 7942 significantly increased PDHc enzymatic activity and intracellular acetyl-CoA levels in the crude cell extract. Although growth defects were observed in overexpressing strains of PDH genes, the combinational overexpression of PDH genes with the synthetic metabolic pathway for acetate or isopropanol resulted in about 7-fold to 9-fold improvement in its production titer, respectively (9.9 mM, 594.5 mg/L acetate, 4.9 mM, 294.5 mg/L isopropanol). PDH genes overexpression would, therefore, be useful not only for the production of these model chemicals, but also for the production of other chemicals that require acetyl-CoA as a key precursor.     

Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle

In several sulfate-reducing bacteria capable of complete oxidation of acetate (or acetyl CoA), the citric acid cycle is not operative. No 2-oxoglutarate dehydrogenase activity was found in these organisms, and the labelling pattern of oxaloacetate excludes its synthesis via 2-oxo-glutarate. These sulfate-reducers contained, however, high activities of the enzymes carbon monoxide dehydrogenase and formate dehydrogenase and catalyzed an isotope exchange between CO₂ and the carboxyl group of acetate (or acetyl CoA), showing a direct C-C-cleavage of activated acetic acid. These findings suggest that in the investigated sulfate-reducers acetate is oxidized to CO₂ via C₁ intermediates. The proposed pathway provides a possible explanation for the reported different fluoroacetate sensitivity of acetate oxidation by anaerobic bacteria, for mini-methane formation, as well as for the postulated anaerobic methane oxidation by special sulfate-reducers.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.     

Acetoacetate metabolism in AS-30D hepatoma cells

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetly CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells.The major findings were:

Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l^?1 ethanol and 296 mmol l^?1 acetate and started producing 1·8 mol l^?1 acetone in single‐stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.     

Green leaf volatiles and oxygenated metabolite emission bursts from mesquite branches following light–dark transitions

Green leaf volatiles (GLVs) are a diverse group of fatty acid-derived compounds emitted by all plants and are involved in a wide variety of developmental and stress-related biological functions. Recently, GLV emission bursts from leaves were reported following light–dark transitions and hypothesized to be related to the stress response while acetaldehyde bursts were hypothesized to be due to the ‘pyruvate overflow’ mechanism. In this study, branch emissions of GLVs and a group of oxygenated metabolites (acetaldehyde, ethanol, acetic acid, and acetone) derived from the pyruvate dehydrogenase (PDH) bypass pathway were quantified from mesquite plants following light–dark transitions using a coupled GC–MS, PTR-MS, and photosynthesis system. Within the first minute after darkening following a light period, large emission bursts of both C₅ and C₆ GLVs dominated by (Z)-3-hexen-1-yl acetate together with the PDH bypass metabolites are reported for the first time. We found that branches exposed to CO₂-free air lacked significant GLV and PDH bypass bursts while O₂-free atmospheres eliminated the GLV burst but stimulated the PDH bypass burst. A positive relationship was observed between photosynthetic activity prior to darkening and the magnitude of the GLV and PDH bursts. Photosynthesis under ¹³CO₂ resulted in bursts with extensive labeling of acetaldehyde, ethanol, and the acetate but not the C₆-alcohol moiety of (Z)-3-hexen-1-yl acetate. Our observations are consistent with (1) the “pyruvate overflow” mechanism with a fast turnover time (<1 h) as part of the PDH bypass pathway, which may contribute to the acetyl-CoA used for the acetate moiety of (Z)-3-hexen-1-yl acetate, and (2) a pool of fatty acids with a slow turnover time (>3 h) responsible for the C₆ alcohol moiety of (Z)-3-hexen-1-yl acetate via the 13-lipoxygenase pathway. We conclude that our non-invasive method may provide a new valuable in vivo tool for studies of acetyl-CoA and fatty acid metabolism in plants at a variety of spatial scales.     

Phytogenic biosynthesis and emission of methyl acetate

Acetylation of plant metabolites fundamentally changes their volatility, solubility and activity as semiochemicals. Here we present a new technique termed dynamic ¹³C‐pulse chasing to track the fate of C_1–3 carbon atoms of pyruvate into the biosynthesis and emission of methyl acetate (MA) and CO₂. ¹³C‐labelling of MA and CO₂ branch emissions respond within minutes to changes in ¹³C‐positionally labelled pyruvate solutions fed through the transpiration stream. Strong ¹³C‐labelling of MA emissions occurred only under pyruvate‐2‐¹³C and pyruvate‐2,3‐¹³C feeding, but not pyruvate‐1‐¹³C feeding. In contrast, strong ¹³CO₂ emissions were only observed under pyruvate‐1‐¹³C feeding. These results demonstrate that MA (and other volatile and non‐volatile metabolites) derive from the C_2,3 atoms of pyruvate while the C₁ atom undergoes decarboxylation. The latter is a non‐mitochondrial source of CO₂ in the light generally not considered in studies of CO₂ sources and sinks. Within a tropical rainforest mesocosm, we also observed atmospheric concentrations of MA up to 0.6 ppbv that tracked light and temperature conditions. Moreover, signals partially attributed to MA were observed in ambient air within and above a tropical rainforest in the Amazon. Our study highlights the potential importance of acetyl coenzyme A (CoA) biosynthesis as a source of acetate esters and CO₂ to the atmosphere.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Conversion of glycerol to pyruvate by Escherichia coli using acetate- and acetate/glucose-limited fed-batch processes

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h⁻¹ versus 0.44 g/g at 0.25 h⁻¹), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h⁻¹ versus 0.59 g/g h at 0.10 h⁻¹). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h⁻¹). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h⁻¹, the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.     

Differential effects of acetate on palmitate and octanoate oxidation: Segregation of acetyl CoA pools

In isolated hepatic mitochondria, sodium acetate had little effect on the oxidation of octanoate, but conspicuously inhibited the oxidation of palmitate. This differential effect of acetate on long-chain and short-chain fatty acid oxidation is not due to inhibition of the activation or transfer of long-chain fatty acids into the mitochondria. Both palmitate and octanoate reduced CO₂ production from acetate. Palmitate and octanoate mutually inhibited CO₂ production from each other to the same extent. Acetate stimulated ketogenesis from palmitoyl-1-carnitine to the same extent as it inhibited oxygen uptake and CO₂ production from palmitate. This suggests that acetate causes a redistribution of the end products of palmitate oxidation toward ketogenesis rather than toward total oxidation to CO₂ and H₂O. Acetyl CoA derived from acetate or palmitate may share a common pool or pathway, thus each is mutally inhibitory toward the oxidation of the other. Either because of the existence of separate pools, or because octanoate is the preferred substrate, acetate metabolism does not inhibit O₂ uptake or CO₂ production from octanoate, whereas the oxidation of octanoate dilutes the CO₂ produced from labeled acetate. This may be explained by compartmentation or preferred pathways for the disposition of acetyl CoA derived from different sources.     

Acetate assimilation and the synthesis of alanine,aspartate and glutamate inMethanobacterium thermoautotrophicum

Cultures of the autotrophic bacteriumMethanobacterium thermoautotrophicum were shown to assimilate acetate when grown on CO₂ and H₂ in the presence of acetate. At 1 mM acetate 10% of the cell carbon came from acetate, the rest from CO₂. At higher concentrations the percentage increased to reach a maximum of 65%at acetate concentrations higher than 20 mM. The data suggest that acetate may be an important carbon source under physiological conditions.The incorporation of acetate into alanine, aspartate and glutamate was studied in more detail. The cells were grown on CO₂ and H₂ in the presence of 1 mM U-¹⁴C-acetate. The three amino acids were isolated from the labelled cells by a simplified procedure. Alanine, aspartate and glutamate were found to have the same specific radioactivity. Degradation studies showed that C₁ of alanine C₁ and C₄ of aspartate, and C₁ and C₅ of glutamate were exclusively derived from CO₂, whereas C₂ and C₃ alamine and aspartate, and C₃ and C₄ of glutamate were partially derived from acetate. These findings and the presence of pyruvate synthase, phosphoenolpyruvate carboxylase and -ketoglutarate synthase inM. thermoautotrophicum indicate that CO₂ is assimilated into the three amino acids via acetyl CoA carboxylation to pyruvate, phosphoenolpyruvate carboxylation to oxaloacetate, and succinyl CoA carboxylation to -ketoglutarate.     

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells

There are significant differences between acetyl‐CoA and ATP levels, enzymes of acetyl‐CoA metabolism, and toll‐like receptor 4 contents in non‐activated microglial N9 and non‐differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration‐dependent several‐fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α‐ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl‐CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl‐CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy‐acetyl‐CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl‐CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll‐like receptors and degree of disequilibrium between acetyl‐CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells.