Gluconate Kinase Research Articles

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans.

ABSTRACTCryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans.IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.

Enzymes of an alternative pathway of glucose metabolism in obligate methanotrophs

Aerobic methanotrophic bacteria utilize methane as a growth substrate but are unable to grow on any sugars. In this study we have shown that two obligate methanotrophs, Methylotuvimicrobium alcaliphilum 20Z and Methylobacter luteus IMV-B-3098, possess functional glucose dehydrogenase (GDH) and gluconate kinase (GntK). The recombinant GDHs from both methanotrophs were homotetrameric and strongly specific for glucose preferring NAD+ over NADP+. GDH from Mtm. alcaliphilum was most active at pH 10 (Vmax = 95 U/mg protein) and demonstrated very high Km for glucose (91.8 ± 3.8 mM). GDH from Mb. luteus was most active at pH 8.5 (Vmax = 43 U/mg protein) and had lower Km for glucose (16 ± 0.6 mM). The cells of two Mtm. alcaliphilum double mutants with deletions either of the genes encoding GDH and glucokinase (gdh─/glk─) or of the genes encoding gluconate kinase and glucokinase (gntk─/glk─) had the lower glycogen level and the higher contents of intracellular glucose and trehalose compared to the wild type strain. The gntk─/glk─ knockout mutant additionally accumulated gluconic acid. These data, along with bioinformatics analysis, demonstrate that glycogen derived free glucose can enter the Entner–Doudoroff pathway or the pentose phosphate cycle in methanotrophs, bypassing glycolysis via the gluconate shunt.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone

BackgroundIn 2006, we found healthy subjects carrying ST131 Escherichia coli in their intestinal microbiota consisting of two populations: a subdominant population of fluoroquinolone-resistant E. coli belonging to subclone H30 (H30-R or subclade C1), the current worldwide dominant ST131 subclone, and a dominant E. coli population composed of antibiotic-susceptible E. coli belonging to subclone H22 (clade B), the precursor of subclone H30. We sequenced the whole genome of fecal H22 strain S250, compared it to the genomes of ExPEC ST131 H30-Rx strain JJ1886 and commensal ST131 H41 strain SE15, sought the H22-H30 genomic differences in our fecal strains and assessed their phenotypic consequences.ResultsWe detected 173 genes found in the Virulence Factor Database, of which 148 were shared by the three ST131 genomes, whereas some were genome-specific, notably those allowing determination of virotype (D for S250 and C for JJ1886). We found three sequences of the FimH site involved in adhesion: two in S250 and SE15 close and identical, respectively, to that previously reported to confer strong intestinal adhesion, and one in JJ1886, corresponding to that commonly present in uropathogenic E. coli. Among the genes involved in sugar metabolism, one encoding a gluconate kinase lacked in S250 and JJ1886. Although this gene was also absent in both our fecal H22 and H30-R strains, H22 strains showed a higher capacity to grow in minimal medium with gluconate. Among the genes involved in gluconate metabolism, only the ghrB gene differed between S250/H22 and JJ1886/H30-R strains, resulting in different gluconate reductases. Of the genes involved in biofilm formation, two were absent in the three genomes and one, fimB, in the JJ1886 genome. Our fecal H30-R strains lacking intact fimB displayed delayed biofilm formation relative to our fecal H22 strains. The H22 strains differed by subclade B type and plasmid content, whereas the H30-R strains were identical.ConclusionsPhenotypic analysis of our fecal strains based on observed genomic differences between S250 and JJ1886 strains suggests the presence of traits related to bacterial commensalism in our H22 strains and traits commonly found in uropathogenic E. coli in our H30-R strains.

Transport and Catabolism of Carbohydrates by Neisseria meningitidis

We identified the genes encoding the proteins for the transport of glucose and maltose in Neisseria meningitidis strain 2C4-3. A mutant deleted for NMV_1892(glcP) no longer grew on glucose and deletion of NMV_0424(malY) prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. N. meningitidis catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. N. meningitidis also contains genes encoding proteins which exhibit similarity to the gluconate transporter (NMV_2230) and gluconate kinase (NMV_2231) of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of NMV_2230 because N. meningitidis was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of NMV_2230 stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of N. meningitidis cells from transgenic mice after intraperitoneal challenge.

A doped polyaniline modified electrode amperometric biosensor for gluconic acid determination in grapes.

In winemaking gluconic acid is an important marker for quantitative evaluation of grape infection by Botrytis cinerea. A screen-printed amperometric bienzymatic sensor for the determination of gluconic acid based on gluconate kinase (GK) and 6-phospho-D-gluconate dehydrogenase (6PGDH) coimmobilized onto polyaniline/poly (2-acrylamido-2-methyl-1-propanesulfonic acid; PANI-PAAMPSA) is reported in this study. The conductive polymer electrodeposed on the working electrode surface allowed the detection of NADH at low potential (0.1 V) with a linear range from 4 × 10−3 to 1 mM (R2 = 0.99) and a sensitivity of 419.44 nA·mM−1. The bienzymatic sensor has been optimized with regard to GK/6PGDH enzymatic unit ratio and ATP/NADP+ molar ratio which resulted equal to 0.33 and 1.2, respectively. Under these conditions a sensitivity of 255.2 nA·mM−1, a limit of detection of 5 μM and a Relative Standard Deviation (RSD) of 4.2% (n = 5) have been observed. Finally, the biosensor has been applied for gluconic acid measurements in must grape samples and the matrix effect has been taken into consideration. The results have been compared with those obtained on the same samples with a commercial kit based on a spectrophotometric enzyme assay and were in good agreement, showing the capability of the bienzymatic PANI-PAAMPSA biosensor for gluconic acid measurements and thus for the evaluation of Botrytis cinerea infection in grapes.

Enhancement ofl-ornithine production by disruption of three genes encoding putative oxidoreductases inCorynebacterium glutamicum

Recently, Corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (GDH) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. In this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to GDH of Bacillus subtilis. Chromosomal in-frame deletion of three genes (NCgl0281, NCgl2582, and NCgl2053) encoding putative NADP⁺-dependent oxidoreductases led to the absence of GDH activity and correlated with increased specific glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. This finding suggested that enhanced carbon flux from glucose was directed toward the oxidative pentose phosphate (PP) pathway, when the mutant was cultivated with 6 % glucose. Consequently, the mutant showed 72.4 % increased intracellular NADPH and 66.3 % increased extracellular L-ornithine production. The enhanced activities of the oxidative PP pathway in the mutant explain both the increased intracellular NADPH and the high extracellular concentration of L-ornithine. Thus, the observed metabolic changes in this work corroborate the importance of NADPH in L-ornithine production from C. glutamicum.

Implication of gluconate kinase activity in l-ornithine biosynthesis in Corynebacterium glutamicum

With the purpose of generating a microbial strain for L-ornithine production in Corynebacterium glutamicum, genes involved in the central carbon metabolism were inactivated so as to modulate the intracellular level of NADPH, and to evaluate their effects on L-ornithine production in C. glutamicum. Upon inactivation of the 6-phosphoglucoisomerase gene (pgi) in a C. glutamicum strain, the concomitant increase in intracellular NADPH concentrations from 2.55 to 5.75 mmol g⁻¹ (dry cell weight) was accompanied by reduced growth rate and L-ornithine production, suggesting that L-ornithine production is not solely limited by NADPH availability. In contrast, inactivation of the gluconate kinase gene (gntK) led to a 51.8 % increase in intracellular NADPH concentration, which resulted in a 49.9 % increase in L-ornithine production. These results indicate that excess NADPH is not necessarily rate-limiting, but is required for increased L-ornithine production in C. glutamicum.

Gluconic acid determination in wine by electrochemical biosensing

Abstract The work describes an amperometric bienzymatic biosensor for gluconic acid based on the coimmobilization of gluconate kinase and 6-phospho- d -gluconate dehydrogenase by crosslinking with glutaraldehyde and bovine serum albumin onto screen-printed carbon electrodes. Experimental design methodology has been used to optimize immobilization and operational conditions. This method shows a reproducibility of 2.94% (n = 8) related to the slopes of calibration curves performed in the concentration range from 7.46 to 71.43 μM, as well as an average capability of detection of 7.46 for a probability of false positive and negative of 0.05. Finally, the developed biosensors have been successfully applied to gluconic acid determination in red wines. This approach represents an interesting alternative to be routinely used at a winery.

Structure of a tRNA-dependent kinase essential for selenocysteine decoding

Compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (Sec), the 21(st) natural amino acid (aa). An essential RNA-dependent kinase, O-phosphoseryl-tRNA(Sec) kinase (PSTK), converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec), the immediate precursor of selenocysteinyl-tRNA(Sec). The sequence of Methanocaldococcus jannaschii PSTK (MjPSTK) suggests an N-terminal kinase domain (177 aa) followed by a presumed tRNA binding region (75 aa). The structures of MjPSTK complexed with ADP and AMPPNP revealed that this enzyme belongs to the P-loop kinase class, and that the kinase domain is closely related to gluconate kinase and adenylate kinase. ATP is bound by the P-loop domain (residues 11-18). Formed by antiparallel dimerization of two PSTK monomers, the enzyme structure shows a deep groove with positive electrostatic potential. Located in this groove is the enzyme's active site, which biochemical and genetic data suggest is composed of Asp-41, Arg-44, Glu-55, Tyr-82, Tyr-83, Met-86, and Met-132. Based on structural comparison with Escherichia coli adenylate kinase a docking model was generated that assigns these amino acids to the recognition of the terminal A76-Ser moieties of Ser-tRNA(Sec). The geometry and electrostatic environment of the groove in MjPSTK are perfectly complementary to the unusually long acceptor helix of tRNA(Sec).

Carbohydrate Metabolism Differences between Subgroup A1 and B2 Strains of B acillus anthracis as Assessed by Comparative Genomics and Functional Genetics

In France, Bacillus anthracis subgroup B2 strains do not metabolize starch or glycogen but can use gluconate, whereas subgroup A1 strains show the inverse pattern. Functional genetic analysis revealed that mutations in the amyS and gntK genes encoding an alpha-amylase and a gluconate kinase, respectively, were responsible for these phenotypes.

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

From the Characterization of the Four Serine/Threonine Protein Kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the Role of PknA and PknB in Cell Division

Corynebacterium glutamicum contains four serine/threonine protein kinases (STPKs) named PknA, PknB, PknG, and PknL. Here we present the first biochemical and comparative analysis of all four C. glutamicum STPKs and investigate their potential role in cell shape control and peptidoglycan synthesis during cell division. In vitro assays demonstrated that, except for PknG, all STPKs exhibited autokinase activity. We provide evidence that activation of PknG is part of a phosphorylation cascade mechanism that relies on PknA activity. Following phosphorylation by PknA, PknG could transphosphorylate its specific substrate OdhI in vitro. A mass spectrometry profiling approach was also used to identify the phosphoresidues in all four STPKs. The results indicate that the nature, number, and localization of the phosphoacceptors varies from one kinase to the other. Disruption of either pknL or pknG in C. glutamicum resulted in viable mutants presenting a typical cell morphology and growth rate. In contrast, we failed to obtain null mutants of pknA or pknB, supporting the notion that these genes are essential. Conditional mutants of pknA or pknB were therefore created, leading to partial depletion of PknA or PknB. This resulted in elongated cells, indicative of a cell division defect. Moreover, overexpression of PknA or PknB in C. glutamicum resulted in a lack of apical growth and therefore a coccoid-like morphology. These findings indicate that pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth.

Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2.

Corynebacterium glutamicum is a Gram-positive soil bacterium that prefers the simultaneous catabolism of different carbon sources rather than their sequential utilization. This type of metabolism requires an adaptation of the utilization rates to the overall metabolic capacity. Here we show how two functionally redundant GntR-type transcriptional regulators, designated GntR1 and GntR2, co-ordinately regulate gluconate catabolism and glucose uptake. GntR1 and GntR2 strongly repress the genes encoding gluconate permease (gntP), gluconate kinase (gntK), and 6-phosphogluconate dehydrogenase (gnd) and weakly the pentose phosphate pathway genes organized in the tkt-tal-zwf-opcA-devB cluster. In contrast, ptsG encoding the EIIGlc permease of the glucose phosphotransferase system (PTS) is activated by GntR1 and GntR2. Gluconate and glucono-δ-lactone interfere with binding of GntR1 and GntR2 to their target promoters, leading to a derepression of the genes involved in gluconate catabolism and reduced ptsG expression. To our knowledge, this is the first example for gluconate-dependent transcriptional control of PTS genes. A mutant lacking both gntR1 and gntR2 shows a 60% lower glucose uptake rate and growth rate than the wild type when cultivated on glucose as sole carbon source. This growth defect can be complemented by plasmid-encoded GntR1 or GntR2.

Fluorometric quantification of total d-gluconate by a flow-injection system using an immobilized-enzyme reactor

A method for the quantification of total d-gluconate by flow-injection analysis was developed using an immobilized-enzyme reactor and fluorescence detection. d-Gluconate was quantified using co-immobilized gluconate kinase (GK) and 6-phosphogluconate dehydrogenase (PGDH) reactor. d-Gluconate was phosphorylated to 6-phospho- d-gluconate by GK in the presence of ATP, and then the 6-phospho- d-gluconate produced was oxidized by PGDH with NADP +. The NADPH produced by the GK-PGDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between the responses and concentrations of d-gluconate was obtained in the ranges of 1.0 × 10 −6–1.6 × 10 −4 M. The relative standard deviation for 10 successive injections was 0.57% at the 0.1 m M level. This analytical method was applied to the quantification of d-gluconate in honeys, vinegars and noble rot wines, and the results showed good agreement with those obtained using the conventional F-kit method.

Conformational Changes During the Catalytic Cycle of Gluconate Kinase as Revealed by X-ray Crystallography

The crystal structure of gluconate kinase from Escherichia coli has been determined to 2.0 Å resolution by X-ray crystallography. The three-dimensional structure was solved by multi-wavelength anomalous dispersion, using a crystal of selenomethionine-substituted enzyme. Gluconate kinase is an α/β structure consisting of a twisted parallel β-sheet surrounded by α-helices with overall topology similar to nucleoside monophosphate (NMP) kinases, such as adenylate kinase. In order to identify residues involved in substrate binding and catalysis, structures of binary complexes with ATP, the ATP analogue adenosine 5′-(β,γ-methylene) triphosphate and the product, gluconate-6-phosphate have been determined. Significant conformational changes are induced upon binding of ATP to the enzyme. The largest changes involve a hinge-bending motion of the NMP bind part and a motion of the LID with adjacent helices, which opens the cavity to the second substrate, gluconate. Opening of the active site cleft upon ATP binding is the opposite of what has been observed in the NMP kinase family so far, which usually close their active site to prevent fortuitous hydrolysis of ATP. The conformational change positions the side-chain of Arg120 to stack with the purine ring of ATP and the side-chain of Arg124 is shifted to interact with the α-phosphate in ATP, at the same time protecting ATP from solvent water. The β and γ-phosphate groups of ATP bind in the predicted P-loop. A conserved lysine side-chain interacts with the γ-phosphate group, and might promote phosphoryl transfer. Gluconate-6-phosphate binds with its phosphate group in a similar position as the γ-phosphate of ATP, consistent with inline phosphoryl transfer. The gluconate binding-pocket in GntK is located in a different position than the nucleoside binding-site usually found in NMP kinases.

Novel pathway for alcoholic fermentation of delta-gluconolactone in the yeast Saccharomyces bulderi.

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.

Enzymes of the Entner-Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture.

The osmotolerant Zymomonas mobilis strain suc40, (containing plasmid pDS3154-inaZ), which is capable of producing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod's relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner-Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner-Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrose metabolism of Z. mobilis suc40/pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium.

Crystallization and preliminary X-ray crystallographic studies of recombinant thermoresistant gluconate kinase GntK from Escherichia coli.

The thermoresistant gluconate kinase GntK from Escherichia coli, an essential enzyme in gluconate metabolism, has been expressed, purified and crystallized. For crystallization, the hanging-drop vapour-diffusion method was used with polyethylene glycol (PEG) 6000 and lithium chloride as precipitants. Three crystal forms belonging to the monoclinic space group C2 or the orthorhombic space groups P2(1)2(1)2(1) and P2(1)2(1)2 were obtained. The unit-cell parameters are a = 75.0, b = 79.3, c = 70.2 A, beta = 105.3 degrees (C2), a = 52.0, b = 79.3, c = 89.8 A (P2(1)2(1)2(1)) and a = 70.1, b = 74.1, c = 78.9 A (P2(1)2(1)2). In all three crystal forms, there are two molecules in the asymmetric unit; the different forms occur in the same crystallization drop. The crystals diffract to at least 2.0 A using synchrotron radiation.

Effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014

The effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014 was investigated. Glucose was completely converted to 2-ketogluconatevia extracellular oxdative pathway and then taken up for cell growth under the condition of sufficient dissolved oxygen concentration. On the other hand, oxygen limitation below dissolved oxygen tension (DOT) value of 20% of air saturation caused the shift of glucose metabolism from the extracellular oxidative pathway to the intracellular phosphorylative pathway. Specific activities of hexokinase and gluconate kinase in intracellular phosphorylation pathway decreased as the DOT increased, while 2-ketogluconokinase activity in extracellular oxidative pathway increased under the same condition. This result can be usefully applied to microbial transformation of glucose to 2-ketogluconate, the synthetic precursor for iso-vitamine C, with almost 100% yieldvia extracellular oxidation by simple DOT control.