Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).     

Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.     

Expression of an acetyl-CoA synthase and a CoA-transferase in Escherichia coli to produce modified taxanes in vivo

Previous in vitro studies revealed that the 10-deacetylbaccatin III 10beta-O-acetyltransferase (DBAT) from Taxus can catalyze the transfer of acetyl, propionyl or n-butyryl from CoA to the C10-hydroxyl of 10-deacetylbaccatin III. Accordingly, Escherichia coli JM109 were transformed to recombinantly express dbat, and this enzyme function was coupled to that of acetyl-CoA synthase (acs, EC 6.2.1.1) expressed from and regulated by genes encoded on the bacterial chromosome. Incubation of the bacteria with 10-deacetylbaccatin III and increasing concentrations of acetic acid revealed an in vivo conversion ( approximately 10%) of substrate to natural product baccatin III (C10-acetylated), which was remarkably similar to the relative conversion without acid supplementation. Incubation of the modified E. coli with 5 mM propionic acid, revealed a fivefold increase in the conversion ( approximately 13%) of 10-deacetylbaccatin III to 10-deacetyl-10-propionylbaccatin III, compared to approximately 2% conversion in the absence of exogenous propionate. To produce the butyrylbaccatin III analog in vivo, bacteria were engineered to co-express the dbat and atoAD (EC 2.8.3.8) genes; the latter encodes an acetoacetate: acetyl-CoA CoA-transferase that activates butyrate to butyryl CoA. The bacteria were incubated with 10-deacetylbaccatin III and 25-100 mM butyrate, and a maximum of approximately 2.6% conversion to 10-butyrylbaccatin III was observed compared to approximately 0.6% conversion when no exogenous butyrate was supplied.     

CaiT of Escherichia coli,a new transporter catalyzing L-carnitine/gamma -butyrobetaine exchange

l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for l-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of l-carnitine for gamma-butyrobetaine, the excreted end product of l-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate l-carnitine uptake. Analysis of l-carnitine counterflow yielded an apparent external K(m) of 105 microm and a turnover number of 5.5 s(-1). Contrary to related proteins, CaiT activity was not modulated by osmotic stress. l-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for l-carnitine and gamma-butyrobetaine according to the substrate/product antiport principle.     

Structure and mechanism of HpcG, a hydratase in the homoprotocatechuate degradation pathway of Escherichia coli

HpcG catalyses the hydration of a carbon-carbon double bond without the aid of any cofactor other than a simple divalent metal ion such as Mg(2+). Since the substrate has a nearby carbonyl group, it is believed that it first isomerises to form a pair of conjugated double bonds in the enol tautomer before Michael addition of water. Previous chemical studies of the reaction, and that of the related enzyme MhpD, have failed to provide a clear picture of the mechanism. The substrate itself is unstable, preventing co-crystallisation or soaking of crystals, but oxalate is a strong competitive inhibitor. We have solved the crystal structure of the protein in the apo form, and with magnesium and oxalate bound. Modelling substrate into the active site suggests the attacking water molecule is not part of the metal coordination shell, in contrast to a previous proposal. Our model suggests that geometrically strained cis isomer intermediates do not lie on the reaction pathway, and that separate groups are involved in the isomerisation and hydration steps.     

Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli

The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily. Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E. coli genome sequencing project. The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK). We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase. These reactions are sufficient to form a metabolic cycle by which E. coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown. The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily.     

Epimerization of 3-hydroxy-4-trans-decenoyl coenzyme A by a dehydration/hydration mechanism catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.

The mechanism of 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3), which is associated with the multienzyme complex of fatty acid oxidation from Escherichia coli, was studied with D-3-hydroxy-4-trans-decenoyl-CoA as a substrate. The E. coli complex catalyzes the rapid and direct dehydration of D-3-hydroxy-4-trans-decenoyl-CoA to 2-trans,4-trans-decadienoyl-CoA, which is slowly hydrated to L-3-hydroxy-4-trans-decenoyl-CoA. A kinetic analysis of the epimerase and its partial reactions established that epimerization of 3-hydroxyacyl-CoAs occurs solely by a dehydration/hydration mechanism. The results of a substrate competition study with L-3-hydroxy-4-trans-decenoyl-CoA and its D-isomer, together with the conclusion from a sequence analysis of the large subunit of the E. coli complex (Yang, X.-Y., Schulz, H., Elzinga, M., and Yang, S.-Y. (1991) Biochemistry 30, 6788-6795), prompt the suggestion that a single active site is responsible for the dehydration of the D- and L-isomers of 3-hydroxyacyl-CoAs.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli

We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.     

Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria

The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.     

Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.     

Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli.

Escherichia coli mutants [coaA16(Fr); Fr indicates feedback resistance] were isolated which possessed a pantothenate kinase activity that was refractory to feedback inhibition by coenzyme A (CoA). Strains harboring this mutation had CoA levels that were significantly elevated compared with strains containing the wild-type kinase and also overproduced both intra- and extracellular 4'-phosphopantetheine. The origin of 4'-phosphopantetheine was investigated by using strain SJ135 [panD delta(aroP-aceEF)], in which synthesis of acetyl-CoA was dependent on the addition of an acetate growth supplement. Rapid degradation of CoA to 4'-phosphopantetheine was triggered by the conversion of acetyl-CoA to CoA following the removal of acetate from the media. CoA hydrolysis under these conditions appeared not to involve acyl carrier protein prosthetic group turnover since [acyl carrier protein] phosphodiesterase was inhibited equally well by acetyl-CoA or CoA. These data support the view that the total cellular CoA content is controlled by modulation of biosynthesis at the pantothenate kinase step and by degradation of CoA to 4'-phosphopantetheine.     

Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in Escherichia coli K-12.

Genes encoding the Escherichia coli IH11165 hemagglutinins with specificity for terminal N-acetyl-D-glucosamine and blood group M antigen, respectively, were cloned by a cosmid cloning procedure. A 22-kilobase-pair subclone expressed both hemagglutination specificities in the nonhemagglutinating E. coli HB101 recipient strain. Derivatives obtained by insertion and deletion mutagenesis expressed either one of the two hemagglutination specificities. Both agglutinins were purified; the agglutinin recognizing terminal N-acetyl-D-glucosamine was associated with a new type of fimbria (G fimbria) with an apparent subunit molecular mass of 19.5 kilodaltons, whereas the blood group M agglutinin (M agglutinin) was nonfimbrial and had an apparent subunit mass of 21 kilodaltons.     

Functional expression and characterization of the two cyclic amidohydrolase enzymes, allantoinase and a novel phenylhydantoinase, from Escherichia coli

A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes from Escherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, the allB gene and a putative open reading frame, tentatively designated as a hyuA (for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to allB, high-level expression of hyuA in E. coli of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for D-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5' position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in E. coli.     

Structural basis for the feedback regulation of Escherichia coli pantothenate kinase by coenzyme A

Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate. CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site. The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively. Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface. The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP. Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state. The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters. The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.     

Periplasmic expression of carbonic anhydrase in Escherichia coli: A new biocatalyst for CO2 hydration

Carbonic anhydrase is a valuable and efficient catalyst for CO₂ hydration. Most often the free enzyme is employed which complicates catalyst recycling, and can increase cost due to the need for protein purification. Immobilization of the enzyme may address these shortcomings. Here we report the development of whole‐cell biocatalysts for CO₂ hydration via periplasmic expression of two forms of carbonic anhydrase in Escherichia coli using two different targeting sequences. The enzymatic turnover numbers (k_cat) and catalytic efficiencies (k_cat/K_M) were decreased by an order of magnitude as compared to the free soluble enzyme, indicating the introduction of transport limitations. However, the thermal stabilities were improved for most configurations (>88% activity retention up to 95°C for three of four whole‐cell biocatalysts), operational stabilities were more than satisfactory (100% retention after 24 h of use for all four whole‐cell biocatalysts), and CO₂ hydration was significantly enhanced relative to the uncatalyzed reaction (～50–70% increase in CaCO₃ precipitate formed). A significant advantage of the whole‐cell approach is that protein purification is no longer necessary, and the cells can be easily separated and recycled in future applications including biofuel production, biosensors, and carbon capture and storage. Biotechnol. Bioeng. 2013; 110: 1865–1873. © 2013 Wiley Periodicals, Inc.