Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

Enzymology of the Acetyl-CoA Pathway of CO2 Fixation

Abstract

We know of three routes that organisms have evolved to synthesize complex organic molecules from CO₂: the Calvin cycle. the reverse tricarboxylic acid cycle, and the reductive acetyl-CoA pathway. This review describes the enzymatic steps involved in the acetyl-CoA pathway, also called the Wood pathway, which is the major mechanism of CO₂ fixation under anaerobic conditions. The acetyl-CoA pathway is also able to form acetyl-CoA from carbon monoxide.

There are two parts to the acetyl-CoA pathway: (1) reduction of CO₂ to methyltetrahydrofolate (methyl-H₄folate) and (2) synthesis of acetyl-CoA from methyl-H, folate, a carboxyl donor such as CO or CO₂, and CoA. This pathway is unique in that the major intermediates are enzyme-bound and are often organometallic complexes. Our current understanding of the pathway is based on radioactive and stable isotope tracer studies, purification of the component enzymes (some extremely oxygen sensitive), and identification of the enzyme-bound intcrmediates by chromatographic, spectroscopic. and electrochemical techniques. This review describes the remarkable series of enzymatic steps involved in acetyl-CoA formation by this pathway that is a key component of the global carbon cycle.     

Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Chloroflexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as electron source. The cultures were subjected to long term labelling experments with ¹³C-labelled acetate or alanine in the presence of sodium fluoroacetate. The presence of fluoroacetate caused the cells to accumulate large amounts of polyglucose which was hydrolysed and analysed by NMR. The labelling patterns of glucose were symmetric and in agreement with carbohydrate synthesis from acetate and CO₂ via pyruvate synthase. The content of carbon derived from added acetate was highest in C2 and C5 of glucose, at least 20% higher than in C1 and C6. About one third of the glucose carbon was derived from added acetate, the rest being from CO₂. Contrary to expectations, in glucose formed in the presence of C1-labelled acetate C1 and C6 contained more label than C2 and C5, and with C2-labelled acetate as the tracer glucose was mainly labelled in C2 and C5. Labelled CO₂ was formed from acetate labelled at either position. The labelling data indicate a new metabolic pathway in C. aurantiacus. It is suggested that the cells form C1-labelled acetyl-CoA from C2-labelled acetyl-CoA and vice versa by a cyclic mechanism involving concomitant CO₂ fixation and that this cycle is the part of the autotrophic CO₂ fixation pathways in C. aurantiacus in which acetyl-CoA is formed from CO₂.The polyglucose of C. aurantiacus appears to have predominantly (1–4)-linked structure with about 10% (1–6)-linkages as revealed by ¹³C-NMR.     

Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

The pathway of autotrophic CO₂ fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO₂-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either ¹⁴CO₂ or [¹⁴C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO₂ fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.     

Autotrophic Carbon Dioxide Assimilation in Thermoproteales Revisited

For Crenarchaea, two new autotrophic carbon fixation cycles were recently described. Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme. Ignicoccus hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO₂ fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route. Thermoproteales would be an exception to this unifying concept, since for Thermoproteus neutrophilus, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO₂ fixation. The corresponding genes were identified in the genome. ¹⁴C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous ¹³C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO₂ fixation in the strict anaerobic Thermoproteales as well as in Desulfurococcales.Two new autotrophic carbon fixation cycles have recently been discovered in the Crenarchaea, one of the two subgroups of the Archaea. The 3-hydroxypropionate/4-hydroxybutyrate cycle functions in the aerobic autotrophic Sulfolobales (7) and the dicarboxylate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) in the anaerobic autotrophic Ignicoccus hospitalis, belonging to the Desulfurococcales (27). These pathways have in common the synthesis of succinyl-coenzyme A (CoA) from acetyl-CoA and two inorganic carbons, although this is accomplished in quite different ways and using different carboxylases. In the 3-hydroxypropionate/4-hydroxybutyrate cycle, acetyl-CoA/propionyl-CoA carboxylase fixes two molecules of bicarbonate, and in the dicarboxylate/4-hydroxybutyrate cycle, pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase are the two carboxylating enzymes. Yet, the regenerations of acetyl-CoA, the primary CO₂ acceptor, from succinyl-CoA are similar in the two pathways.Open in a separate windowFIG. 1.Dicarboxylate/4-hydroxybutyrate cycle for autotrophic CO₂ fixation, as proposed for T. neutrophilus. Enzymes: 1, pyruvate synthase (reduced MV); 2, pyruvate-water dikinase; 3, PEP carboxylase; 4, malate dehydrogenase (NADH); 5, fumarate hydratase; 6, fumarate reductase (reduced MV); 7, succinyl-CoA synthetase (ADP forming); 8, succinyl-CoA reductase (NADPH); 9, succinic semialdehyde reductase (NADPH); 10, 4-hydroxybutyrate-CoA ligase (AMP forming); 11, 4-hydroxybutyryl-CoA dehydratase; 12, crotonyl-CoA hydratase; 13, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 14, acetoacetyl-CoA β-ketothiolase. Fd_red, reduced ferredoxin.Acetyl-CoA regeneration is as follows. The CO₂ fixation product succinyl-CoA is reduced to 4-hydroxybutyrate, which is activated to 4-hydroxybutyryl-CoA and then dehydrated to crotonyl-CoA by 4-hydroxybutyryl-CoA dehydratase. This radical [4Fe-4S] and flavin adenine dinucleotide-containing dehydratase (11, 37) is considered a key enzyme of the 4-hydroxybutyrate part of each pathway. Its product, crotonyl-CoA, is further converted to acetoacetyl-CoA and then to two acetyl-CoA molecules, closing the cycle and generating an additional molecule of acetyl-CoA for biosynthesis. Therefore, two different autotrophic pathways in different crenarchaeal orders share many common enzymes and intermediates.In this context, the order Thermoproteales would constitute an exception within the Crenarchaea, since the reductive citric acid cycle was proposed for Thermoproteus neutrophilus (6, 48-50, 55) and Pyrobaculum islandicum (26). T. neutrophilus is a strictly anaerobic hyperthermophilic archaeon growing autotrophically by reducing sulfur with hydrogen at 85°C and neutral pH (19). It can also assimilate organic compounds, such as acetate or succinate, but only in the presence of CO₂ and H₂, i.e., in a mixotrophic way (48).In the reductive citric acid cycle, succinyl-CoA is further transformed with 2 CO₂ to citrate, followed by citrate cleavage to oxaloacetate and acetyl-CoA. This requires two characteristic enzymes, 2-oxoglutarate synthase (2-oxoglutarate-ferredoxin oxidoreductase) and ATP citrate lyase. The proposal of the functioning of the reductive citric acid cycle in T. neutrophilus was based on the results of a ¹³C retrobiosynthetic analysis of the central carbon metabolism, using ¹³C-labeled succinate and acetate as an additional carbon source, following its incorporation into cellular building blocks. The ¹³C enrichment data of, e.g., glutamate, which is directly derived from 2-oxoglutarate, were consistent with the operation of a reductive citric acid cycle only when further assumptions were made (55). The activities of the enzymes of this cycle were demonstrated with extracts of autotrophically grown cells. However, the measured 2-oxoglutarate synthase and ATP-citrate lyase activity levels were very low and could not support the reported growth rate under autotrophic conditions (6, 48).The recent sequencing of the genome of Pyrobaculum aerophilum, belonging to the Thermoproteales (20), revealed a surprising feature, the presence of a 4-hydroxybutyryl-CoA dehydratase gene without the presence of an ATP-citrate lyase gene. Similar gene patterns are found in the genomes of T. neutrophilus as well as Pyrobaculum calidifontis and P. islandicum, sequenced by the DOE Joint Genome Institute (http://www.jgi.doe.gov/). This indicates a possible functioning of the dicarboxylate/4-hydroxybutyrate cycle in Thermoproteales and brings into question the involvement of the reductive citric acid cycle in autotrophic CO₂ fixation. This study has reinvestigated the pathway of autotrophic CO₂ fixation in Thermoproteus neutrophilus. We provide different lines of evidence for the operation of the dicarboxylate/4-hydroxybutyrate cycle.     

Metabolism of hyperthermophiles

Hyperthermophiles are characterized by a temperature optimum for growth between 80 and 110°C. They are considered to represent the most ancient phenotype of living organisms and thus their metabolic design might reflect the situation at an early stage of evolution. Their modes of metabolism are diverse and include chemolithoautotrophic and chemoorganoheterotrophic. No extant phototrophic hyperthermophiles are known. Lithotrophic energy metabolism is mostly anaerobic or microaerophilic and based on the oxidation of H₂ or S coupled to the reduction of S, SO _inf4 ^sup2- , CO₂ and NO _inf3 ^sup- but rarely to O₂. the substrates are derived from volcanic activities in hyperthermophilic habitats. The lithotrophic energy metabolism of hyperthermophiles appears to be similar to that of mesophiles. Autotrophic CO₂ fixation proceeds via the reductive citric acid cycle, considered to be one of the first metabolic cycles, and via the reductive acetyl-CoA/carbon monoxide dehydrogenase pathway. The Calvin cycle has not been found in hyperthermophiles (or any Archaea). Organotrophic metabolism mainly involves peptides and sugars as substrates, which are either oxidized to CO₂ by external electron acceptors or fermented to acetate and other products. Sugar catabolism in hyperthermophiles involves non-phosphorylated versions of the Entner-Doudoroff pathway and modified versions of the Embden-Meyerhof pathway. The classical Embden-Meyerhof pathway is present in hyperthermophilic Bacteria (Thermotoga) but not in Archaea. All hyperthermophiles (and Archaea) tested so far utilize pyruvate:ferredoxin oxidoreductase for acetyl-CoA formation from pyruvate. Acetyl-CoA oxidation in anaerobic sulphur-reducing and aerobic hyperthermophiles proceeds via the citric acid cycle; in the hyperthermophilic sulphate-reducer Archaeoglobus an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway is operative. Acetate formation from acetyl-CoA in Archaea, including hyperthermophiles, is catalysed by acetyl-CoA synthetase (ADP-forming), a novel prokarvotic enzyme involved in energy conservation. In Bacteria, including the hyperthermophile Thermotoga, acetyl-CoA conversion to acetate involves two enzymes, phosphate acetyltransferase and acetate kinase.The authors are with the Institut für Pflanzenphysiologie und Mikrobiologie. Fachbereich Biologie, Freie Universität Berlin, Königin-Luise-Strasse 12–16 a, D-14195 Berlin, Germany     

Occurrence,biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle

The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO₂ fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon. In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO₂ fixation. It has been shown in A. brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA. The subunit structure of the acyl-CoA carboxylase was shown to be ₄₄₄. Gene cloning revealed that the genes encoding the three subunits are adjacent to each other. accC encodes the -subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the -subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the -subunit (62 kDa subunit, carboxyltransferase subunit). Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately. Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented.     

Question 1: The FeS/H2S System as a Possible Primordial Source of Redox Energy

The theory of chemoautotrophy, as developed by Wächtershäuser, has been subject to experimental studies, which show a possible carbon fixation pathway of several consecutive steps from simple CO₂ to amino acids, using the redox system of iron sulphide and hydrogen sulphide. Main findings were a mimicking of the acetyl-CoA enzyme reaction using the mixed sulphide (Fe,Ni)S and the reduction of dinitrogen to ammonia. Present studies aim at a more detailed investigation of the mechanism of the redox system FeS/H₂S and its properties. For these studies a method to produce and immobilise FeS nanoparticles has been developed.     

Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

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.     

Metabolic pathways and energetics of the acetone-oxidizing,sulfate-reducing bacterium,Desulfobacterium cetonicum

Acetone degradation by cell suspensions of Desulfobacterium cetonicum was CO₂-dependent, indicating initiation by a carboxylation reaction. Degradation of butyrate was not CO₂-dependent, and acetate accumulated at a ratio of 1 mol acetate per mol butyrate degraded. In cultures grown on acetone, no CoA transfer apparently occurred, and no acetate accumulated in the medium. No CoA-ligase activities were detected in cell-free crude extracts. This suggested that the carboxylation of acetone to acetoacetate, and its activation to acetoacetyl-CoA may occur without the formation of free acetoacetate. Acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA, which were oxidized to CO₂ via the acetyl-CoA/carbon monoxide dehydrogenase pathway. The measured intracellular acyl-CoA ester concentrations allowed the calculation of the free energy changes involved in the conversion of acetone to acetyl-CoA. At in vivo concentrations of reactants and products, the initial steps (carboxylation and activation) must be energy-driven, either by direct coupling to ATP, or coupling to transmembrane gradients. The G of acetone conversion to two acetyl-CoA at the expense of the energetic equivalent of one ATP was calculated to lie very close to 0kJ (mol acetone)^-1. Assimilatory metabolism was by an incomplete citric acid cycle, lacking an activity oxidatively decarboxylating 2-oxoglutarate. The low specific activities of this cycle suggested its probable function in anabolic metabolism. Succinate and glyoxylate were formed from isocitrate by isocitrate lyase. Glyoxylate thus formed was condensed with acetyl-CoA to form malate, functioning as an anaplerotic sequence. A glyoxylate cycle thus operates in this strictly anaerobic bacterium. Phosphoenolpyruvate (PEP) carboxykinase formed PEP from oxaloacetate. No pyruvate kinase activity was detected. PEP presumably served as a precursor for polyglucose formation and other biosyntheses.Abbreviations MV ²⁺ Oxidized methyl viologen - PEP Phosphoenolpyruvate - PHB Poly--hydroxybutyrate     

Effect of extra- and intracellular sources of CO<Subscript>2</Subscript> on anaerobic utilization of glucose by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains deficient in carboxylation-independent fermentation pathways

The effect of extra- and intracellular CO₂ sources on anaerobic glucose utilization by Escherichia coli strains deficient in the main pathways of mixed acid fermentation and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO₂ generation in the strains was ensured resulting from the oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Endogenous CO₂ formation by pyruvate dehydrogenase stimulated anaerobic glucose consumption by the strains due to the involvement in the fermentation process of condensation reactions between oxaloacetic acid and acetyl-CoA. The availability of an external CO₂ source (dissolved in medium sodium bicarbonate) promoted utilization of carbohydrate substrate by favoring the predominant participation in the fermentation of reactions directly dependent on phosphoenolpyruvate carboxylation. The positive effect of the availability of exogenous СО₂ was sharply decreased in recombinant strains with the impaired functionality of the reductive branch of the tricarboxylic acid cycle. As a result, intracellular СО₂ generation coupled to acetyl-CoA formation promoted anaerobic glucose utilization by cells of the corresponding mutants more markedly than the presence in the medium of dissolved sodium bicarbonate.     

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C₄ carboxylates into two molecules of acetyl-CoA without loss of CO₂. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.     

Effects of HCO3/CO2 on the cytochrome redox potential and metabolic responses of isolated rat cerebral cortex.

The importance of HCO₃^?/CO₂ to the maintenance of stable metabolic conditions in rat cerebral cortex slices has been investigated. Replacement of bicarbonate buffered media with glycylglycine or phosphate buffered salines resulted in an increased lability of the cytochrome redox potential of brain slices as measured by dual wavelength spectroscopy. Depletion of reduced cytochrome in the electron transport chain was associated with changes in the metabolic responses of tissues to electrical stimulation or elevated concentrations of potassium. This appears primarily as a loss of the late reductive responses of the tissue nicotinamide adenine dinucleotides NAD(P)H to these stimuli and decreased lactic acid output of the tissues. The effects could be largely reversed in a combined glycylglycine and HCO₃^?/CO₂ buffered media. It is suggested that although the reduction of NAD(P) in response to stimulation may be substantially located in the cytosol, it is also modulated by the mitochondrial redox potential. It is suggested that the lability of the redox potential in the absence of HCO₃^?/CO₂ may be related to depletion of TCA cycle intermediates normally replaced by CO₂ fixation.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Genomic signatures of fifth autotrophic carbon assimilation pathway in bathypelagic Crenarchaeota

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO₂-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction.     

Autotrophic carbon dioxide fixation inAcidianus brierleyi

The autotrophic CO₂ fixation pathway inAcidianus brierleyi, a facultatively anaerobic thermoacidophilic archaebacterium, was investigated by measuring enzymatic activities from autotrophic, mixotrophic, and heterotrophic cultures. Contrary to the published report that the reductive tricarboxylic acid cycle operates inA. brierleyi, the enzymatic activity of ATP:citrate lyase, the key enzyme of the cycle, was not detected. Instead, activities of acetyl-CoA carboxylase and propionyl-CoA carboxylase, key enzymes of the 3-hydroxypropionate cycle, were detected only whenA. brierleyi was growing autotrophically. We conclude that a modified 3-hydroxypropionate pathway operates inA. brierleyi.Abbreviations TCA tricarboxylic acid - BV Benzyl viologen