Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.     

Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of ∼66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn²⁺ and stabilization of the nucleotide substrate by Mg²⁺. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 μmol min^–1 (mg enzyme)^–1. The apparent K _m values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent K _m values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium. Received: 20 August 1996 / Accepted: 28 December 1996     

Regulation of phospho(enol)-pyruvate-and oxaloacetate-converting enzymes in Corynebacterium glutamicum

The presence and properties of the enzymes involved in the synthesis and conversion of phospho(enol)pyruvate (PEP) and oxaloacetate (OAA), the precursors for aspartate-derived amino acids, were investigated in three different Corynebacterium strains. This study revealed the presence of both PEP carboxykinase 0.29 mol·min^–1·mg^–1 of protein [units (U)·mg^–1] and PEP synthetase (0.13 U·mg^–1) in C. 2 glutamicum as well as pyruvate kinase (1.4 U·mg^–1) and PEP carboxylase (0.16 U·mg^–1). With the exception of PEP carboxykinase these activities were also present in glucose-grown C. flavum and C. lactofermentum. Pyruvate carboxylase activity was not detected in all three species cultivated on glucose or lactate. At least five enzyme activities that utilize OAA as a substrate were detected in crude extracts of C. glutamicum: citrate synthase (2 U·mg^–1), malate dehydrogenase (2.5 U·mg^–1), glutamate: OAA transaminase (1 U·mg^–1), OAA-decarboxylating activity (0.89 U·mg^–1) and the previously mentioned PEP carboxykinase (0.29 U·mg^–1). The partially purified OAA-decarboxylase activity of C. glutamicum was completely dependent on the presence of inosine diphosphate and Mn²⁺, had a Michaelis constant (K _m) of 2.0mm for OAA and was inhibited by ADP and coenzyme A (CoA). Examination of the kinetic properties showed that adenine nucleotides and CoA derivatives have reciprocal but reinforcing effects on the enzymes catalyzing the interconversion of pyruvate, PEP and OAA in C. glutamicum. A model for the regulation of the carbon flow based on these findings is presented.Correspondence to: M. S. M. Jetten     

Oxaloacetate Production via Carboxylations in Crithidia fasciculata Preparations

SYNOPSIS. Fractions containing soluble enzymes from Crithidia fasciculata had an ADP-linked phosphoenolpyruvate (PEP) carboxykinase. The enzyme produced ATP and oxaloacetate (OAA) from PEP, ADP and HCO⁻₃. OAA was determined as the endproduct of reactions by forming the 2,4-dinitrophenylhydrazone derivative; the hydrazone was identified by thin-layer chromatography. Approximate Michaelis constants (PEP, Mg, HCO⁻₃, ADP) were determined spectrophotometrically by linking OAA production to malic dehydrogenase. The PEP carboxykinase did not utilize GDP, UDP or IDP as cofactors; the metal requirement was also satisfied by Mn. The enzyme was inhibited by the biotin antagonists avidin and desthiobiotin.
A pyruvate carboxylase was also present in the preparations, generating OAA from pyruvate and ATP. The role of both enzymes in OAA production and subsequent production of succinate is discussed with regard to C. fasciculata and other trypanosomatids.     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

Comparative Kinetic Effects of Mn (II), Mg (II) and the ATP/ADP Ratio on Phosphoenolpyruvate Carboxykinases from <Emphasis Type="Italic">Anaerobiospirillum succiniciproducens</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The kinetic affinity for CO₂ of phosphoenolpyruvate PEP⁵ carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn²⁺ and Mg²⁺, the affinity for CO₂ increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO₂ fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160–164).     

Cellulase from Ruminococcus albus and Mixed Rumen Microorganisms

Cellulase in the cultural filtrates of Ruminococcus albus and cellulase extracted from mixed rumen microorganisms were investigated with acid-swollen cellulose and carboxymethylcellulose as substrates. Maximal activity occurred at approximately pH 5.8 and 47 C. Apparent Michaelis constants (K_m) varied between 0.53 and 0.02% carboxymethylcellulose, depending on the level of activity and the method of assay. R. albus cellulase has a lower K_m value than the enzyme extracted from mixed rumen microorganisms. Antisera from rabbits immunized with a cellulase preparation from R. albus inhibited the cellulolytic activity of both systems. Based on the relative degree of inhibition, approximately 20% of the cellulase of the mixed rumen microorganisms was immunologically similar to R. albus cellulase. Ratios of activity in different assay techniques showed the two sources of activity to be similar in the mechanisms of degradation. However, glucose is the main product of cellulose degradation by mixed rumen microorganisms, and cellobiose is the product of degradation by R. albus.     

Competition Between Ruminal Cellulolytic Bacteria for Adhesion to Cellulose

Competition for adhesion to cellulose among the three main ruminal cellulolytic bacterial species was studied using differential radiolabeling (¹⁴C/³H) of cells. When added simultaneously to cellulose, Ruminococcus flavefaciens FD1 and Fibrobacter succinogenes S85 showed some competition; however, both species were surpassed competitively by Ruminococcus albus 20. When R. flavefaciens FD1 and F. succinogenes S85 were already adherent, R. albus 20 adhesion occurred without inhibition but involved R. flavefaciens FD1 detachment. Received: 28 October 1996 / Accepted: 28 January 1997     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Cellobiose and Cellodextrin Metabolism by the Ruminal Bacterium Ruminococcus albus

Ruminococcus albus is an important fibrolytic bacterium in the rumen. Cellobiose is metabolized by this organism via hydrolytic and well as phosphorylytic enzymes, but the relative contributions of each pathway were not clear. The cellobiose consumption rate by exponentially growing cells was less than that of crude extracts (75 versus 243 nmol/min/mg protein). Cellobiose phosphorolytic cleavage was much greater than hydrolytic activity (179 versus 19 nmol/min/mg protein) indicating that phosphorylases were key enzymes in the initial metabolism of the soluble products of cellulose degradation. Cellodextrin phosphorylase appeared to be active against substrates as large as cellohexaose. Phosphorylase activities were cytoplasmic, but hydrolytic activities were associated with both the membrane and cytoplasmic fractions. Free glucose was phosphorylated with a GTP-dependent glucokinase, and this enzyme showed 20-fold higher activity with GTP or ITP (>324 nmol/min/mg protein) than with ATP, UTP, CTP, GDP, or PEP. The activity was decreased at least 57% when mannose, 2-deoxyglucose, or fructose was used as substrate compared with glucose. The K _m s for glucose and GTP were 321 and 247 μM, respectively. Since phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, it is likely that such pathways provide for more efficient growth of R. albus in substrate-limiting conditions like those found in the rumen. Received: 24 February 1997 / Accepted: 31 March 1997     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

PEP-dependent⁴ CO₂-fixation by extracts of Ascophyllum nodosum (L.) Le Jol. is reported. The carboxylation of PEP is Mn²⁺ dependent and ATP is shown to be a product. IDP was found to be less efficient as a phosphate acceptor than ADP and 3-mercaptopicolinic acid inhibited the carboxylation reaction. Extracts decarboxylated OAA only in the presence of ATP and had high activities of MDH and GOT. This evidence, together with the probable absence of PEPC, PEPCTrP, and PC in A. nodosum extracts, favors the view that PEPCK is responsible for the light-independent CO₂-fixation observed in this alga.     

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens Fd-1

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H₂ in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO₂ (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H₂ was formed by a hydrogenase rather than by cleavage of formate, and ¹³C-NMR and¹⁴ C-exchange reaction data indicated that formate was produced by CO₂ reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H₂. This shift suggested that reducing equivalents could be balanced through formate or H₂ production without affecting the yields of the major carbon-containing fermentation endproducts.     

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.     

Effects of phosphoenol pyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum

The phosphoenol pyruvate carboxylase gene (ppc) of lysine-producing Corynebacterium glutamicum and C. lactofermentum strains was inactivated by marker exchange mutagenesis. The mutants lacked completely phosphoenol pyruvate carboxylase (PEP carboxylase) activity, but grew in minimal medium containing glucose as the sole carbon source. In addition, the ppc ^– strains produced equivalent titers of lysine in shake flasks and in 10-l fermentation experiments as their parent strains. To address the question of how ppc ^– Corynebacterium strains generate oxaloacetate (OAA) for their own metabolism as well as for high-level lysine production, we measured the activities of enzymes leading to OAA synthesis. Whereas pyruvate carboxylase activity was not detected in any of the strains, phosphoenol pyruvate carboxykinase (PEP carboxykinase) activity was found to be significantly higher in C. glutamicum ppc mutants compared to the parent strains. On the other hand, PEP carboxykinase activity in C. lactofermentum was essentially absent. As glyxylate cycle enzymes are strongly repressed by glucose, they are not likely to compensate for the lack of PEP carboxylase activity. PEP carboxykinase, among several candidates, could play this role. Correspondence to: M. Gubler     

Molybdate and sulfide inhibit H2 and increase formate production from glucose by Ruminococcus albus

H₂ production from glucose by Ruminococcus albus was almost completely inhibited by 10^–5 M molybdate only when sulfide was present in the growth medium. Inhibition was accompanied by a significant increase in the production of formate. Extracts of molybdate-sulfide-grown cells did not contain hydrogenase activity. Active enzyme in extracts of uninhibited cells was not inhibited by the molybdate-sulfide-containing growth medium. The results indicate that a complex formed from molybdate and sulfide prevents the formation of active hydrogenase and electrons otherwise used to form H₂ are used to reduce CO₂ to formate. Growth was significantly inhibited when molybdate was increased to 10^–4 M. Reversal of growth inhibition but not inhibition of H₂ production occurred between 10^–4 and 10^–3 M molybdate. H₂ production by R. bromei but not by R. flavefaciens, Butyrivibrio fibrisolvens, Veillonella alcalescens, Klebsiella pneumoniae and Escherichia coli was inhibited by molybdate and sulfide.     

Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii

Summary The hydrolysis and fermentation of cellulose (Avicel) by continuous cultures of Ruminococcus albus strain 7 and Methanobrevibacter smithii strain PS were studied. Cellulose destruction ranged from ca. 22% to 71% for 0.25 to 2.27 days solids retention time, respectively. The cellulose hydrolysis rate constant (k) was 1.3 days^–1. Concentrations of soluble reducing sugars were low, showing that cellulose hydrolysis was the rate-limiting step of cellulose fermentation. The estimated methane-based molar growth yield for M. smithii was 2.8 g mol^–1. Its maximum specific growth rate was ca. 4 days^–1. The dissolved H₂ half-saturation constant (K _s ) for methanogenesis was ca. 1 M. The final products of the co-culture were primarily acetate, CH₄ and CO₂ and low levels of ethanol and H₂. The co-culture produced more H₂ (used for reduction of CO₂ to CH₄) and acetate than a monoculture of R. albus. These differences coulb be accounted for by the lower production of ethanol, confirming to the theory of interspecies H₂ transfer. Offprint requests to: M. J. Wolin     

Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17

Summary A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.     

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO₂. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn²⁺ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis.MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography.ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn²⁺, reduced k_cat by 14-fold, and significantly increased the K_m values for Mn²⁺ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the K_m value for MgATP and reduced k_cat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the K_m value for OAA and reduced k_cat respectively.ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn²⁺ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK.General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.