Gluconate Dehydrogenase Research Articles

6-BA Delays the Senescence of Postharvest Cabbage Leaves by Inhibiting Respiratory Metabolism.

6-BA, a small molecule compound of cytokinins, has been proven to delay leaf senescence in different species, including Chinese flowering cabbage; however, its specific mechanism remains relatively unknown. In this study, the application of external 6-BA delayed leaf senescence in Chinese flowering cabbage, showing that 6-BA effectively prevented the decrease in the maximum quantum yield (Fv/Fm) and overall chlorophyll content and suppressed the expression of the senescence-associated gene BrSAG12 over a 7-day period of storage. Moreover, treatment with 6-BA decreased the respiratory rate, NAD(H) content, the activities of hexose phosphate isomerase (PHI), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO), and ascorbic acid oxidase (AAO) using enzyme-linked immunosorbent assay, and the transcriptional abundance of related genes by real-time quantitative polymerase chain reaction. Furthermore, 6-BA also increased the activity and expression levels of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphate gluconate dehydrogenase (6-PGDH). The group treated with 6-BA retained elevated levels of NADP (H), ATP, total ATPase, and nicotinamide adenine dinucleotide kinase (NADK) activity, as well as the expression of respiratory enzymes. Molecular docking indicated that 6-BA hinders the glycolysis pathway (EMP), tricarboxylic acid cycle (TCA), and cytochrome pathway (CCP), and sustains elevated levels of the pentose phosphate pathway (PPP) through interactions with the PHI, SDH, 6-PGDH, G6PDH, CCO, and AAO proteins, consequently delaying postharvest leaf senescence in Chinese flowering cabbage.

Overexpression of kojR and the entire koj gene cluster affect the kojic acid synthesis in Aspergillus oryzae 3.042

Kojic acid (KA), a fungal secondary metabolite, has various applications in the cosmetics, pharmaceutical, and food industries. Aspergillus oryzae, the primary strain, has been identified as a koj gene cluster positively responsible for KA biosynthesis. In this study, we obtained transformants T58 and T31, which overexpressed either solo kojR or the entire koj gene cluster, respectively. These transformants exhibited peak KA production on the 5th day of shake flask fermentation, with 32.5 g/L and 26.57 g/L that 324.28 % and 246.87 % higher than the control strain with 7.64 g/L, respectively. Morphological analysis revealed that the highly productive KA strains had reduced conidial production but increased antioxidant capacity. The qRT-PCR analysis revealed that relative expression levels of kojR in the transformants were remarkably higher that the primary cause for the increased KA yield. Moreover, the high expression of kojR could also influence the expression of the key enzymes involved in the KA biosynthesis process, such as glucose dehydrogenase and gluconate dehydrogenase. These findings can assist in discovering more about how the koj gene cluster in A. oryzae influences its growth and KA production. And provides valuable insights into facilitating strain improvement and benefits for the future.

Time-resolved, deuterium-based fluxomics uncovers the hierarchy and dynamics of sugar processing by Pseudomonas putida

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 13C- and 2H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate—unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.

Identification of Pathways for Production of D-Glucaric Acid by Pseudogluconobacter saccharoketogenes.

Pseudogluconobacter saccharoketogenes produces glucaric acid from D-glucose via two pathways, i.e., through D-glucuronic acid or D-gluconic acid. These pathways are catalyzed by alcohol dehydrogenase, aldehyde dehydrogenase, and gluconate dehydrogenase. Although D-glucaraldehyde and L-guluronic acid are also theorized to be produced in pathways throsugh D-glucuronic acid and D-gluconic acid, respectively, no direct data to identify these intermediates have been reported. In this study, the intermediates were purified and identified as D-glucaraldehyde and L-guluronic acid. The substrate specificities of the three enzymes on these intermediates and their oxidation products were studied, and the roles of alcohol, aldehyde, and gluconate dehydrogenases in D-glucaric acid-producing pathways were elucidated using the intermediates. Additionally, the substrate specificities of alcohol and aldehyde dehydrogenases on some alcohols, aldehydes, and aldoses were determined. Alcohol dehydrogenase showed wide substrate specificities, whereas the substrates oxidized by aldehyde dehydrogenase were limited. A 30-L scale reaction using the resting cells of Rh47-3 revealed that D-glucaric acid was produced from D-glucose and D-gluconic acid in 60.3mol% (7.0g/L) and 78.6mol% (22.5g/L) yields, respectively.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli

IntroductionGlycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively.ObjectiveTo deeply understand the effect of the blocking of the pentose phosphate pathway (PPP) or the activation of the glyoxylate shunt, metabolite profiles were analyzed on M4-Δgnd, M4-ΔiclR and M4 mutants.MethodsMetabolomics was performed by FT-IR and GC–MS for metabolite fingerprinting and HPLC for quantification of succinate and glycerol.ResultsMost of the 65 identified metabolites showed lower relative levels in the M4-ΔiclR and M4-Δgnd mutants than those of the M4. However, fructose 1,6-biphosphate, trehalose, isovaleric acid and mannitol relative concentrations were increased in M4-ΔiclR and M4-Δgnd mutants. To further improve succinate production, the synthesis of mannitol was suppressed by deletion of mannitol dehydrogenase (mtlD) on M4-ΔgndΔmtlD mutant that increase ~ 20% respect to M4-Δgnd.ConclusionMetabolomics can serve as a holistic tool to identify bottlenecks in metabolic pathways by a non-rational design. Genetic manipulation to release these restrictions could increase the production of succinate.

Characterization of 3 phylogenetically distinct membrane-bound d-gluconate dehydrogenases of Gluconobacter spp. and their biotechnological application for efficient 2-keto-d-gluconate production.

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82±1.15, 22.9±5.0, and 11.3±1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.

Directional enhancement of 2-keto-gluconic acid production from enzymatic hydrolysate by acetic acid-mediated bio-oxidation with Gluconobacter oxydans

An acetic acid-mediated bio-oxidation strategy with Gluconobacter oxydans was developed to produce valuable 2-ketogluconic acid from lignocellulosic biomass. Metabolically, glucose is firstly oxidized to gluconic acid and further oxidized to 2-keto-gluconic acid by Gluconobacter oxydans. As a specific inhibitor for microbial fermentation generated from pretreatment, acetic acid was validated to have a down-regulated effect on bio-oxidizing glucose to gluconic acid. Nevertheless, it significantly facilitated 2-keto-gluconic acid accumulation and improved gluconate dehydrogenase activity. In the presence of 5.0 g/L acetic acid, the yield of 2-keto-gluconic acid increased from 38.0% to 80.5% using pure glucose as feedstock with 1.5 g/L cell loading. Meanwhile, 44.6 g/L 2-keto-gluconic acid with a yield of 83.5% was also achieved from the enzymatic hydrolysate. 2-keto-gluconic acid production, found in this study, laid a theoretical foundation for the industrial production of 2-keto-gluconic acid by Gluconobacter oxydans using lignocellulosic materials.

Exogenous 2,4-Epibrassinolide Treatment Maintains the Quality of Carambola Fruit Associated With Enhanced Antioxidant Capacity and Alternative Respiratory Metabolism.

Brassinosteroids act by delaying fruit ripening. The effects of different concentrations of 2,4-epibrassinolide (eBL) treatments on carambola fruit ripening were investigated. The results show that treatment of 2.8 mg L–1, eBL with 10 min effectively delays ripening and maintains the quality of carambola fruit. This is achieved by retarding color changes and firmness losses while maintaining high level of soluble protein content and vitamin C, and low organic acid content. eBL-delayed senescence may be due to the inhibition of respiration rate and enhanced antioxidant system. It is noteworthy that eBL treatment markedly reduces the content of fructose-6-phosphate (6-P-F) and enhances the activity of cytochrome oxidase (CCO), and the total activity of glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphate gluconate dehydrogenase (6-PGDH). eBL treatment induces the IAA and GA contents but reduces that of ABA. In general, senescence retardation and quality improvement by eBL treatment may be due to the enhanced antioxidant capacity and altered respiratory pathways.

De Novo Engineering of Corynebacterium glutamicum for l-Proline Production.

l-Proline is an important amino acid that has various industrial applications. Industrial l-proline-producing strains are obtained by the mutagenesis of Corynebacterium glutamicum. In this study, the optimized C. glutamicum genome-editing tools were further applied in the de novo construction of a hyper-l-proline-producing strain. Overexpression of a feedback inhibition-resistant γ-glutamic kinase mutant ProBG149K, deletion of a proline dehydrogenase to block l-proline degradation, overexpression of glutamate dehydrogenase to increase glutamate synthesis flux, the mutation of 6-phosphate gluconate dehydrogenase and glucose-6-phosphate-dehydrogenase in the pentose phosphate pathway to enhance NADPH supply, the deletion of pyruvate aminotransferase to decrease the byproduct l-alanine synthesis, and weakening of α-ketoglutarate dehydrogenase to regulate the TCA cycle were combined to obtain ZQJY-9. ZQJY-9 produced 19.68 ± 0.22 g/L of l-proline in flask fermentation and was also demonstrated at the 3 L bioreactor level by fed-batch fermentation producing 120.18 g/L of l-proline at 76 h with the highest productivity of 1.581 g/L/h.

A Membrane-Bound Gluconate Dehydrogenase from 2-Keto-d-Gluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01: Purification, Characterization, and Gene Identification

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-D-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71U/mg at optimum pH and temperature of 6.0 and 35°C. The Km and Vmax values of calcium D-gluconate were 0.631mM and 0.734mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

Gluconic acid production by gad mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1g/g.

Inoculation of genetically modified endophytic Herbaspirillum seropedicae Z67 endowed with gluconic and 2-ketogluconic acid secretion, confers beneficial effects on rice (Oriza sativa) plants

Background and aims Herbaspirillum seropedicae (Hs) Z67 a diazotrophic endophyte was genetically engineered for secretion of 2-keto-D-gluconic acid by heterologous expression of genes for pqq synthesis and gluconate dehydrogenase to study its beneficial effect on plants.

Analysis of the Activity and Regulon of the Two-Component Regulatory System Composed by Cjj81176_1484 and Cjj81176_1483 of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial diarrheal disease and a frequent commensal of the intestinal tract in poultry and other animals. For optimal growth and colonization of hosts, C. jejuni employs two-component regulatory systems (TCSs) to monitor environmental conditions and promote proper expression of specific genes. We analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode proteins of a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Transcriptome analysis revealed that the regulons of the Cjj81176_1484 (Cjj1484) histidine kinase and the Cjj81176_1483 (Cjj1483) response regulator contain many common genes, suggesting that these proteins likely form a cognate TCS. We found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. We observed that the Cjj1483 response regulator binds target promoters in both unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. This work further expands the signaling mechanisms of C. jejuni and provides additional insights regarding the complex and multifactorial regulation of many genes involved in basic metabolism, respiration, and nutrient acquisition that the bacterium requires for optimal growth in different environments. Bacterial two-component regulatory systems (TCSs) link environmental cues to expression of specific genes that enable optimal bacterial growth or colonization of hosts. We found that the Campylobacter jejuni Cjj1484 histidine kinase and Cjj1483 response regulator function as a cognate TCS to largely repress expression of target genes encoding a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract, as well as other genes encoding proteins for heme or iron acquisition, metabolism, and respiration. We also discovered different modes by which Cjj1483 may mediate repression with and without Cjj1484. This work provides insight into the signal transduction mechanisms of a leading cause of bacterial diarrheal disease and emphasizes the multifactorial and complex regulation of specific biological processes in C. jejuni.

Simultaneous amperometric determination of malic and gluconic acids in wine using screen-printed carbon electrodes

The selective and simultaneous amperometric determination of malic and gluconic acids was carried out using a screen-printed design with two working carbon electrodes. One of the two working electrodes was modified with gold nanoparticles, tetrathiafulvalene (TTF) and malate quinone oxido-reductase enzyme for the sensitive detection of malic acid. Analogously, the other working electrode was modified with TTF and gluconate dehydrogenase enzyme for the sensitive detection of gluconic acid. The array of biosensors responds to gluconic and malic acids giving each anodic currents (+100mV and pH 6.5), with detection capabilities of 1.89μM (α=β=0.05) and 0.79μM to malic and gluconic acids, respectively, at room temperature. The method has been applied to the determination of both analytes in wine, obtaining successful results.

Purification and enzymatic characterization of membrane-bound d-gluconate dehydrogenase from Arthrobacter globiformis

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000Da, 44,000Da and 23,000Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40°C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50°C, and the strict substrate specificity for d-gluconate with the Km of 3.15mmolL−1, 1.04mmolL−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

Metagenome-wide association of microbial determinants of host phenotype in Drosophila melanogaster.

ABSTRACTAnimal-associated bacteria (microbiota) affect host behaviors and physiological traits. To identify bacterial genetic determinants of microbiota-responsive host traits, we employed a metagenome-wide association (MGWA) approach in two steps. First, we measured two microbiota-responsive host traits, development time and triglyceride (TAG) content, in Drosophila melanogaster flies monoassociated with each of 41 bacterial strains. The effects of monoassociation on host traits were not confined to particular taxonomic groups. Second, we clustered protein-coding sequences of the bacteria by sequence similarity de novo and statistically associated the magnitude of the host trait with the bacterial gene contents. The animals had been monoassociated with genome-sequenced bacteria, so the metagenome content was unambiguous. This analysis showed significant effects of pyrroloquinoline quinone biosynthesis genes on development time, confirming the results of a published transposon mutagenesis screen, thereby validating the MGWA; it also identified multiple genes predicted to affect host TAG content, including extracellular glucose oxidation pathway components. To test the validity of the statistical associations, we expressed candidate genes in a strain that lacks them. Monoassociation with bacteria that ectopically expressed a predicted oxidoreductase or gluconate dehydrogenase conferred reduced Drosophila TAG contents relative to the TAG contents in empty vector controls. Consistent with the prediction that glucose oxidation pathway gene expression increased bacterial glucose utilization, the glucose content of the host diet was reduced when flies were exposed to these strains. Our findings indicate that microbiota affect host nutritional status through modulation of nutrient acquisition. Together, these findings demonstrate the utility of MGWA for identifying bacterial determinants of host traits and provide mechanistic insight into how gut microbiota modulate the nutritional status of a model host.

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

2-Ketogluconic acid production by Klebsiella pneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism.

GADH screen-printed biosensor for gluconic acid determination in wine samples

Gluconate dehydrogenase (GADH) biosensors have been developed for the satisfactory determination of gluconic acid in wine samples, without any pretreatment. The biosensors have been fabricated by cross-linking immobilization of GADH onto screen-printed carbon electrodes, containing the mediator tetrathiafulvalene (TTF). Chronoamperograms have been registered at +100mV vs. screen-printed Ag/AgCl electrode by successive additions of a gluconic acid solution in the concentration range from 9.0 to 131.4μM. This method shows a reproducibility of 8.1% (n=3) related to the slopes of these calibration curves and a repeatability of 3.2% (n=5). The procedure has shown an average capability of detection of 9.0μM for a probability of false positive and negative of 0.05. Wine samples have been analyzed with these biosensors, obtaining satisfactory results.

2-Ketogluconic Acid Secretion by Incorporation of Pseudomonas putida KT 2440 Gluconate Dehydrogenase (gad) Operon in Enterobacter asburiae PSI3 Improves Mineral Phosphate Solubilization

Enterobacter asburiae PSI3 is known to efficiently solubilize rock phosphate by secretion of approximately50mM gluconic acid in Tris-buffered medium in the presence of 75mM glucose and in a mixture of seven aldosugars each at 15mM concentration, mimicking alkaline vertisol soils. Efficacy of this bacterium in the rhizosphere requires P release in the presence of low amount of sugars. To achieve this, E. asburiae PSI3 has been manipulated to express gluconate dehydrogenase (gad) operon of Pseudomonas putida KT 2440 to produce 2-ketogluconic acid. E. asburiae PSI3 harboring gad operon had 438U of GAD activity, secreted 11.63mM 2-ketogluconic and 21.65mM gluconic acids in Tris-rock phosphate-buffered medium containing 45mM glucose. E. asburiae PSI3 gad transformant solubilized 0.84mM P from rock phosphate in TRP-buffered liquid medium. In the presence of a mixture of seven sugars each at 12mM, the transformant brought about a drop in pH to 4.1 and released 0.53mM P.