2-ketogluconate and gluconic-acid

Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic

Topics: Deuterium; Gluconates; Glucose; Pseudomonas putida; Sugars

The genetic characterization of

Topics: Gluconates; Glucose; Glycolysis; Oxidation-Reduction; Phosphorylation; Pseudomonas; Substrate Specificity

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.

Topics: Bacterial Proteins; Biosynthetic Pathways; Culture Media; Gene Expression Regulation, Bacterial; Gluconates; Hydrogen-Ion Concentration; PQQ Cofactor; Pseudomonas fluorescens; Transcription Factors

While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in the shift between customary C sources (e.g. glucose or succinate) to the polyol. Transcriptomic results were contrasted with key enzymatic activities under the same culture conditions. Cognate expression profiles revealed that genes encoding enzymes of the Entner-Doudoroff route and other catabolic pathways, e.g. the gluconate and 2-ketogluconate loops, were significantly downregulated on glycerol. Yet, the compound simultaneously elicited a gluconeogenic response that indicated an efficient channelling of C skeletons back to biomass build-up through the glyoxylate shunt rather than energization of the cells through downwards pathways, i.e. tricarboxylic acid cycle and oxidative phosphorylation. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favouring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other C sources.

Topics: Bacterial Proteins; Citric Acid Cycle; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gluconates; Glucose; Glycerol; Glyoxylates; Metabolic Networks and Pathways; Pseudomonas putida; Succinic Acid

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.

Topics: Bacterial Proteins; Bioreactors; Biotransformation; Fermentation; Gluconates; Gluconobacter; Glucose; Hydrogen-Ion Concentration; Oxidation-Reduction; Sugar Alcohol Dehydrogenases

The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.. To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.. The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

Topics: Biomass; Caprylates; Citric Acid; Gluconates; Polyhydroxyalkanoates; Pseudomonas putida

We isolated and characterized novel insoluble phosphate (P)-solubilizing bacteria tolerant to environmental factors like high salt, low and high pHs, and low temperature. A bacterium M6 was isolated from a ginseng rhizospheric soil and confirmed to belong to Burkholderia vietnamiensis by BIOLOG system and 16S rRNA gene analysis. The optimal cultural conditions for the solubilization of P were 2.5% (w/v) glucose, 0.015% (w/v) urea, and 0.4% (w/v) MgCl(2).6H(2)O along with initial pH 7.0 at 35 degrees C. High-performance liquid chromatography analysis showed that B. vietnamiensis M6 produced gluconic and 2-ketogluconic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with P solubilization. Insoluble P solubilization in the optimal medium was about 902 mg l(-1), which was approximately 1.6-fold higher than the yield in NBRIP medium (580 mg l(-1)). B. vietnamiensis M6 showed resistance against different environmental stresses like 10-45 degrees C, 1-5% (w/v) salt, and 2-11 pH range. The maximal concentration of soluble P produced by B. vietnamiensis M6 from Ca(3)(PO(4))(2), CaHPO(4), and hydroxyapatite was 1,039, 2,132, and 1,754 mg l(-1), respectively. However, the strain M6 produced soluble P with 20 mg l(-1) from FePO(4) after 2 days and 100 mg l(-1) from AlPO(4) after 6 days, respectively. Our results indicate that B. vietnamiensis M6 could be a potential candidate for the development of biofertilizer applicable to environmentally stressed soil.

Topics: Burkholderia; Culture Media; DNA, Bacterial; DNA, Ribosomal; Gluconates; Hydrogen-Ion Concentration; Molecular Sequence Data; Panax; Phosphates; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Temperature

To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.. We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.. This study indicated that the P solubility was directly correlated with the organic acids produced.. Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.

Topics: Acetic Acid; Culture Media; Formates; Fructose; Gluconates; Glucose; Hydrogen-Ion Concentration; Nitrates; Panax; Phosphates; Plant Growth Regulators; Plant Roots; Pseudomonas fluorescens; Quaternary Ammonium Compounds; Soil Microbiology; Tartrates

The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism.

Topics: Adenosine Triphosphate; Bacterial Proteins; Fermentation; Gene Expression Profiling; Gluconates; Glucose; Membrane Transport Proteins; Metabolic Networks and Pathways; NADP; Phenols; Porins; Pseudomonas putida; Pyruvate Kinase; Systems Biology

Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37 degrees C and 42 degrees C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 42 degrees C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81-176 demonstrated GADH activity, converting d-gluconate to 2-keto-d-gluconate, that was higher at 42 degrees C than at 37 degrees C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d-gluconate or 2-keto-d-gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.

Topics: Animals; Bacterial Proteins; Campylobacter Infections; Campylobacter jejuni; Cecum; Chickens; Colony Count, Microbial; Electrophoresis, Gel, Two-Dimensional; Gene Deletion; Gene Expression Profiling; Gluconates; Mice; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis; Oxidoreductases; Oxygen; Pectobacterium; Proteome; Sequence Homology, Amino Acid; Temperature; Virulence Factors

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-D-gluconic acid (2-KGA) and 5-keto-D-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO2, 0.05% pCO2) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.

Topics: Fermentation; Gluconates; Gluconobacter oxydans; Glucose; Industrial Microbiology; Mutation

For the conversion of glucose to 5-keto-D-gluconate (5-KGA), a precursor of the industrially important L-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP+, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.

Topics: Bacterial Proteins; Biotransformation; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Gluconates; Gluconobacter oxydans; Glucose; NADP; NADP Transhydrogenases; Oxidoreductases; Plasmids; Recombinant Proteins

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5alpha. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50-95% homology to pqq genes of other bacteria. E. coli DH5alpha expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5alpha expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.

Topics: Cloning, Molecular; Culture Media; DNA Probes; DNA, Bacterial; Durapatite; Enterobacter; Escherichia coli; Gene Order; Genes, Bacterial; Genetic Complementation Test; Gluconates; Hydrogen-Ion Concentration; Molecular Sequence Data; Nucleic Acid Hybridization; Phosphates; PQQ Cofactor; Sequence Analysis, DNA; Soil Microbiology; Solubility

Airborne bacteria isolated from a tannery air environment were screened for the property of solubilization of insoluble zinc oxide and zinc phosphate. Out of 10 strains tested, a strain of Pseudomonas aeruginosa (CMG 823) showed the best solubilization and solubilized both zinc oxide and zinc phosphate. Colonies of the bacterium produced clear haloes on solid medium which contained these insoluble metal compounds, but only when glucose was provided as a carbon source. Solubilization of zinc oxide and phosphate was accompanied by an increase in the H+ concentration of the medium, probably a consequence of the production of 2-ketogluconic acid.

Topics: Air Microbiology; Gluconates; Hydrogen-Ion Concentration; Metals; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Solubility; Zinc; Zinc Oxide

The effect of dissolved oxygen concentration on the metabolism of glucose in Pseudomonas aeruginosa was studied with chemostat cultures using both single-step and gradual transitions from either ammonium or glucose limitation to oxygen limitation and studying transient and steady states. The pathway of glucose metabolism was regulated by the availability of oxygen. The organism responded to oxygen limitation by adjusting its metabolism of glucose from the extracellular direct oxidative pathway, which produces gluconate and 2-oxogluconate, to the intracellular phosphorylative route. This change was a consequence of decreased activities of glucose dehydrogenase and gluconate dehydrogenase and of the transport systems for gluconate and 2-oxogluconate, and an increased activity of glucose transport, while relatively high activities of hexokinase and glucose-6-phosphate dehydrogenase were maintained. Citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities responded to changes in dissolved oxygen concentration rather than to changes in the glucose or ammonium concentrations. The effect of oxygen limitation on the oxo-acid dehydrogenases and aconitase was probably due, wholly or in part, to repression by glucose consequent upon the increase in residual glucose concentration. Succinate dehydrogenase was repressed by an increase in ammonium concentration under an oxygen limitation.

Topics: Ammonium Sulfate; Anaerobiosis; Biological Transport; Citrate (si)-Synthase; Citric Acid Cycle; Gluconates; Glucose; Isocitrate Dehydrogenase; Malate Dehydrogenase; Oxygen

Goddard, J. L. (University of Oklahoma School of Medicine, Oklahoma City), and J. R. Sokatch. 2-Ketogluconate fermentation by Streptococcus faecalis. J. Bacteriol. 87:844-851. 1964.-Streptococcus faecalis 10Cl did not grow with 2-ketogluconate alone as an energy source, but did grow when gluconate was added. More growth was obtained than could be accounted for by the gluconate alone. The requirement for gluconate in the stimulation of growth on 2-ketogluconate was found to be stoichiometric, not catalytic. Glucose did not replace gluconate in this phenomenon, apparently owing to the repression of the 2-ketogluconate pathway by glucose. Resting cells grown on a combination of gluconate and 2-ketogluconate did ferment 2-ketogluconate without added gluconate. Fermentation balance studies with resting cells detected the following products in moles (per mole of 2-ketogluconate): carbon dioxide, 0.98; lactic acid, 0.19; formic acid, 1.42; acetic acid, 0.70; and ethanol, 0.42. 2-Ketogluconate-1-C(14) and -2-C(14) were prepared and fermented. The data were interpreted to show that 90% of the substrate was decarboxylated to carbon dioxide and pentose phosphate. Pentose phosphate was then fermented to pyruvate through the sedoheptulose diphosphate variation of the pentose phosphate pathway found in this organism. The other 10% of the substrate was converted to pyruvate by way of the Entner-Doudoroff pathway. Calculations of the energy available by the above combination of pathways indicated that about 2.3 moles of adenosine triphosphate per mole of 2-ketogluconate could be obtained if the energy available in acetate formation is conserved through the acetokinase reaction.

Topics: Acetates; Arsenicals; Bacteriological Techniques; Biochemical Phenomena; Biochemistry; Carbohydrate Metabolism; Carbon Dioxide; Carbon Isotopes; Enterococcus faecalis; Fermentation; Formates; Gluconates; Glucose; Keto Acids; Lactates; Pyruvates; Research

Topics: Carbon; Gluconates; Glucose; Physiological Phenomena; Pseudomonas aeruginosa

Topics: Acetobacter; Carbohydrate Metabolism; Gluconates; Glucose; Oxidation-Reduction