CoA Acceptor Research Articles

Metabolic Engineering of Escherichia coli for Production of Butyric Acid

The butyrate production by engineered Escherichia coli is afflicted by both low titer and low selectivity (defined as the butyrate/acetate (B/A) ratio). To address this issue, a strategy for metabolic engineering of E. coli was implemented including (1) elimination of all major NADH-dependent reactions in the fermentation metabolism, (2) reconstruction of a heterologous pathway leading to butyryl-CoA, (3) recruitment of endogenous atoDA for conversion of butyryl-CoA to butyrate with acetate as a CoA acceptor, and (4) removal of the acetate-synthesis pathway. Grown on glucose (20 g/L) plus acetate (8 g/L), the engineered strain consumed almost all glucose and acetate and produced 10 g/L butyrate as a predominant product within 48 h. It leads to high butyrate selectivity with the B/A ratio reaching 143. The result shows that our proposed approach may open a new avenue in biotechnology for production of butyrate in E. coli.

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct(Re)) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct(Re) was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct(Re). In addition to already known CoA acceptors for Pct Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct(Re). Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg⁻¹ min⁻¹). K(m) values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct(Re) in biotechnological polyester production.

C7orf10 encodes succinate‐hydroxymethylglutarate CoA‐transferase, the enzyme that converts glutarate to glutaryl‐CoA

Glutarate, a side-product in the metabolism of tryptophan and lysine, is metabolized by conversion to glutaryl-CoA by a transferase using succinyl-CoA as a coenzyme donor. The enzyme catalyzing this conversion has not been formally identified. However, a benign form of glutaric aciduria (glutaric aciduria type III) is due to mutations in C7orf10, a putative member of the coenzyme A transferase class III family. In the present work, we show that recombinant human C7orf10 catalyzes the succinyl-CoA-dependent conversion of glutarate to glutaryl-CoA. C7orf10 could use many dicarboxylic acids as CoA acceptors, the best ones being glutarate, succinate, adipate, and 3-hydroxymethylglutarate. Confocal microscopy analysis of CHO cells transfected with a C7orf10-GFP fusion protein indicated that C7orf10 is a mitochondrial protein, in agreement with the presence of a predicted mitochondrial propeptide at its N-terminus. The effect of a missense mutation (p.Arg336Trp) found in the homozygous state in several patients with glutaric aciduria type III and present in the general population at a low frequency was also investigated. The p.Arg336Trp mutation led to the production of insoluble and inactive C7orf10 both in Escherichia coli and in HEK293T cells. These findings indicate that C7orf10 is implicated in the metabolism of glutarate, but possibly also of longer dicarboxylic acids. Homologues of this enzyme are found in numerous bacterial operons comprising also a putative glutaryl-CoA dehydrogenase, indicating that an enzyme with similar specificity exists in prokaryotes.

Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii: a Coenzyme A Loop Saves Energy for Caffeate Activation

The anaerobic acetogenic bacterium Acetobacterium woodii couples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product of carA, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function of carA, we overproduced its protein in Escherichia coli and then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparent K(m) values of 75 ± 5 μM for caffeate and 8 ± 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could use p-coumarate and ferulate but not cinnamate, sinapate, or p-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.

A modified pathway for the production of acetone in Escherichia coli

A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium acetobutylicum (thlA coding for thiolase and adc coding for acetoacetate decarboxylase) and one from Bacillus subtilis or Haemophilus influenzae (teIIsrf or ybgC, respectively, for thioesterase). Expression of this operon in Escherichia coli resulted in the production of acetone starting from the common metabolite acetyl-CoA via acetoacetyl-CoA and acetoacetate. The thioesterases do not need a CoA acceptor for acetoacetyl-CoA hydrolysis. Thus, in contrast to the classic acetone pathway of Clostridium acetobutylicum and related microorganisms which employ a CoA transferase, the new pathway is acetate independent. The genetic background of the host strains was crucial. Only E. coli strains HB101 and WL3 were able to produce acetone via the modified plasmid based pathway, up to 64mM and 42mM in 5-ml cultures, respectively. Using glucose fed-batch cultures the concentration could be increased up to 122mM acetone with HB101 carrying the recombinant plasmid pUC19ayt (thioesterase from H. influenzae). The formation of acetone led to a decreased acetate production by E. coli.

Pathway identification combining metabolic flux and functional genomics analyses: Acetate and propionate activation by Corynebacterium glutamicum

Corynebacterium glutamicum can utilize acetic acid and propionic acid for growth and amino acid production. Growth on acetate as sole carbon source requires acetate activation by acetate kinase (AK) and phosphotransacetylase (PTA), encoded in the pta-ack operon. Genetic and enzymatic studies showed that these enzymes also catalyze propionate activation and were required for growth on propionate as sole carbon source. However, when glucose was present as a co-substrate strain lacking the AK-PTA pathway was still able to utilize acetate or propionate for growth indicating that an alternative activation pathway exists. As shown by 13C-labelling experiments, the carbon skeleton of acetate is conserved during activation to acetyl-CoA in this pathway. Metabolic flux analysis during growth on an acetate–glucose mixture revealed that in the absence of the AK-PTA pathway carbon fluxes in glycolysis, the tricarboxylic acid (TCA) cycle and anaplerosis via PEP carboxylase and/or pyruvate carboxylase were increased, while the glyoxylate cycle flux was decreased. DNA microarray experiments identified cg2840 as a constitutively and highly expressed gene putatively encoding a CoA transferase. Purified His-tagged Cg2840 protein was active as CoA transferase interconverting acetyl-, propionyl- and succinyl-moieties as CoA acceptors and donors. Strains lacking both the CoA transferase and the AK-PTA pathway could neither activate acetate nor propionate in the presence or absence of glucose. Thus, when these short-chain fatty acids are co-metabolized with other carbon sources, CoA transferase and the AK-PTA pathway constitute a redundant system for activation of acetate and propionate.

Properties of Succinyl-Coenzyme A: l -Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.

Characterization and transcription of the genes encoding enzymes involved in butyrate production in Butyrivibrio fibrisolvens.

Genes encoding enzymes that catalyze butyryl-CoA formation from acetyl-CoA in a type II strain of Butyrivibrio fibrisolvens were analyzed. The genes encoding thiolase, beta-hydroxybutyryl-CoA dehydrogenase, butyryl-CoA dehydrogenase, and electron transfer flavoproteins were clustered, but the crotonase gene was not present in this region. The deduced amino acid sequences of these enzymes were similar to those of clostridia. The clustered genes were shown to be cotranscribed. The rate of butyrate production increased with an increase in acetate concentration in the medium up to 5 mM, suggesting that the butyryl-CoA/acetate CoA transferase reaction limits butyrate production. Transcription of the clustered genes was not affected by acetate concentration, suggesting that acetate does not affect the synthesis of enzymes involved in butyryl-CoA formation. These results confirm that acetate stimulates butyrate production by acting as a CoA acceptor in the butyryl-CoA/acetate CoA transferase reaction.

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.

Myristoyl CoA:Protein N-Myristoyltransferase : Subcellular Localization, Activation and Kinetic Behavior in the Presence of Organic Solvents

Myristoyl CoA: protein N-myristoyltransferase (NMT) catalyses the addition of myristate to the amino terminal glycine residue of a number of cellular, eukaryotic and viral proteins. The majority of the catalytic activity of spleen NMT was recovered in the soluble cytosolic fraction (98.2%) compared to the particulate fraction (19.3%). Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates,suggesting that the particulate fraction may contain an inhibitory activity towards NMT. The effects of organic solvents (ethanol and acetonitrile) on bovine spleen NMT were investigated. NMT activity was activated several-fold in a time-and concentration-dependent manner in the presence of cAMP-dependent protein-kinase-derived peptide substrate as fatty acyl CoA acceptor, suggesting that the activation by organic solvents is not due to a solvent effect. Similar activation by ethanol and acetonitrile was also observed with pp60src as substrate, suggesting that the effect of solvent is on NMT and not on the substrate. Gel filtration chromatography indicated that the high catalytic activity of NMT was observed only in the presence of organic solvents: removal of organic solvents from the medium drastically reduced the catalytic activity of NMT, suggesting that NMT did not undergo covalent modification. Kinetic data indicated that ethanol enhanced the Vmax without affecting the Km.

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group.

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria.

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.

Modification of mammalian fatty acid synthetase activity by NADP

Experiments are described which show that the mammalian fatty acid synthetase, in the presence of NADP, synthesizes stoichiometric amounts of enzyme-bound acetoacetyl moieties. The acetoacetyl moieties can neither undergo the normal transfer reaction to a CoA acceptor, nor participate in the normal reaction sequence once NADPH is made available. Our results indicate that, since it is the product of the condensation reaction which accumulates on the inhibited enzyme, the previously held view that NADP inhibits the condensation step in fatty acid synthesis is probably incorrect.

The dissimilation of higher dicarboxylic acids by Pseudomonas fluorscens.

This work provides presumptive evidnce that Pseudomonas fluorescens metabolizes the higher homologs (C6 to C10) of the saturated dicarboxylic acid series by β‐oxidation, mediated by a specific series of inducible enzymes. The initial step in the metabolism of four of these acids (pimelic, suberic, azelaic and sebacic acids) is the formation of the corresponding CoA‐ester. This step is mediated by a single, inducible thiokinase. A thiokinaseless mutant can utilize none of these four dicarboxylic acids. Although adipic acid can also apparently be activated to a slight extent by the thiokinase, the unimpaired ability of the thiokinaseless mutant to grow with adipate shows that there is a second mechanism for the activation of this member of the series. Adipic acid is also activated by an inducible adipate: succinyl‐CoA transferase, which cannot use the higher homologs of the dicarboxylic acid series as CoA acceptors (with the exception of pimelic acid, which is weakly activated). This transferase is probably responsible for adipic acid activation in vivo. Inducible dehydrogenases which can oxidise acyl‐CoA and β‐hydroxyacyl‐CoA derivatives of the higher dicarboxylic acids were also demonstrated, but their substrate specificities were not determined. The former enzyme can dehydrogenate both adipyl‐CoA and azelayl‐CoA; and the latter, β‐hydroxyadipyl‐CoA. A mutant unable to form the enzyme with β‐hydroxy‐adipyl‐CoA dehydrogenase activity cannot grow with any dicarboxylic acid of the C6 to C10 series.

Existence and functions of two enzymes with beta-ketoadipate: succinyl-CoA transferase activity in Pseudomonas florescens.

This work shows that Pseudomonas fluorescens can synthesize two different inducible enzymes which possess β‐ketoadipate: succinyl‐CoA transferase activity. Transferase I is operative in the activation of free β‐ketoadipic acid and is specifically induced in cells grown either with this acid, or with an aromatic precursor (benzoate) oxidized through the β‐ketoadipate pathway. Transferase II is specifically induced in cells grown with saturated dicarboxylic acids of chain length C6 through C10, and its physiological function is that of an adipate: succinyl‐CoA transferase; its ability to activate β‐ketoadipic acid reflects non‐specificity with regard to the CoA acceptor.