5-ketogluconic-acid and gluconic-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

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.

Topics: Amino Acid Sequence; Carbohydrate Dehydrogenases; Cloning, Molecular; Enzyme Stability; Escherichia coli; Flavin-Adenine Dinucleotide; Genes, Bacterial; Gluconates; Gluconobacter; Models, Biological; Molecular Sequence Data; Mutation; Plasmids; Sequence Homology, Amino Acid; Substrate Specificity; Time Factors; Transformation, Genetic

Gluconobacter oxydans DSM 2343 is known to catalyze the oxidation of 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 and soluble dehydrogenases. In G. oxydans MF1, in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated, formation of the undesired 2-KGA was absent. This mutant strain uniquely accumulates high amounts of 5-KGA in the culture medium. To increase the production rate of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, we equipped G. oxydans MF1 with plasmids allowing the overproduction of the soluble and the membrane-bound 5-KGA-forming enzyme. Whereas the overproduction of the soluble gluconate:NADP 5-oxidoreductase resulted in the accumulation of up to 200 mM 5-KGA, the detected 5-KGA accumulation was even higher when the gene coding for the membrane-bound gluconate-5-dehydrogenase was overexpressed (240 to 295 mM 5-KGA). These results provide a basis for designing a biotransformation process for the conversion of glucose to 5-KGA using the membrane-bound as well as the soluble enzyme system.

Topics: Acetates; Bacterial Proteins; Biotechnology; Carbon; Fermentation; Gene Expression Regulation, Bacterial; Genes, Bacterial; Gluconates; Gluconobacter oxydans; Glucose; Hydrogen-Ion Concentration; Oxidoreductases; Oxygen; Plasmids; Tartrates; Time Factors

Gluconobacter oxydans DSM 2343 (ATCC 621H)catalyzes the oxidation of glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of the industrially important L-(+)-tartaric acid. To further increase 5-KGA production in G. oxydans, the mutant strain MF1 was used. In this strain the membrane-bound gluconate-2-dehydrogenase activity, responsible for formation of the undesired by-product 2-keto-D-gluconic acid, is disrupted. Therefore, high amounts of 5-KGA accumulate in the culture medium. G. oxydans MF1 was equipped with plasmids allowing the overexpression of the membrane-bound enzymes involved in 5-KGA formation. Overexpression was confirmed on the transcript and enzymatic level. Furthermore, the resulting strains overproducing the membrane-bound glucose dehydrogenase showed an increased gluconic acid formation, whereas the overproduction of gluconate-5-dehydrogenase resulted in an increase in 5-KGA of up to 230 mM. Therefore, these newly developed recombinant strains provide a basis for further improving the biotransformation process for 5-KGA production.

Topics: Carbohydrate Dehydrogenases; Cell Membrane; Genetic Enhancement; Gluconates; Gluconobacter oxydans; Glucose; Oxidation-Reduction; Protein Engineering

The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of D: -gluconic acid oxidation was 5-keto-D: -gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.

Topics: Alcohol Oxidoreductases; Alcohols; Amino Sugars; Bacterial Proteins; Cloning, Molecular; Escherichia coli; Gluconates; Gluconobacter; Oxidation-Reduction; Recombinant Proteins; Substrate Specificity; Xanthomonas campestris

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 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.

Topics: Bacterial Proteins; Cloning, Molecular; Escherichia coli; Gluconates; Gluconobacter; Kinetics; L-Iditol 2-Dehydrogenase; Molecular Sequence Data; Oxidoreductases; Recombination, Genetic; Sequence Analysis, DNA; Substrate Specificity

Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cell Compartmentation; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Gluconates; Gram-Negative Aerobic Bacteria; Molecular Sequence Data; Molecular Weight; NADP; Oxidoreductases; Protein Conformation; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity