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PubMed2002

Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IF03255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli.

The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E. coli, Gluconobacter oxydans, and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.

Amino Acid Sequence↗
PubMed2002

Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255.

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100. The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30 degrees C. Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.

Acetic Acid↗
PubMed2003

Construction of a vector plasmid for use in Gluconobacter oxydans.

A host vector system in Gluconobacter oxydans was constructed. An Acetobacter-Escherichia coli shuttle vector was introduced with the efficiency of 10(4) transformants/microg of DNA. Next, aiming for a self-cloning vector, we found a cryptic plasmid (which we named pAG5) of 5648 bp in G. oxydans strain IFO 3171, and sequenced the nucleotides. The plasmid seemed to have only one open reading flame (ORF) for a possible replication protein. Shuttle vectors of Gluconobacter-E. coli were constructed with the plasmid pAG5 and an E. coli vector, pUC18.

Escherichia coli↗
PubMed2006

Heterogeneity of strains assigned to Gluconobacter frateurii Mason and Claus 1989 based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions.

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264(T) was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116(T) was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.

DNA Restriction Enzymes↗
PubMed2006

Modification of the membrane-bound glucose oxidation system in Gluconobacter oxydans significantly increases gluconate and 5-keto-D-gluconic acid accumulation.

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.

Carbohydrate Dehydrogenases↗
PubMed2006

A model system for increasing the intensity of whole-cell biocatalysis: investigation of the rate of oxidation of D-sorbitol to L-sorbose by thin bi-layer latex coatings of non-growing Gluconobacter oxydans.

We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.

Bioreactors↗
PubMed2003

Glucose oxidation by Gluconobacter oxydans: characterization in shaking-flasks, scale-up and optimization of the pH profile.

In this study, the advantage of a novel measuring device for the online determination of oxygen and carbon dioxide transfer rates in shaking-flasks is reported for glucose oxidation by Gluconobacter oxydans. In this fermentation process, this device was used for the characterization of the oxidation pattern of different strains. G. oxydans NCIMB 8084 forms 2,5-diketogluconate from d-glucose in a multi-stage process via three different membrane-bound dehydrogenases. This strain was chosen for a scale-up of the process from shaking-flasks to a 2-l stirred vessel. An enhancement of 2,5-diketogluconate production was realized by controlling the pH at different levels during the fermentation.

Biotechnology↗
PubMed2003

Biotransformation of glucose to 5-keto-D-gluconic acid by recombinant Gluconobacter oxydans DSM 2343.

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.

Bacterial Proteins↗
PubMed2004

Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain.

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.

Bacterial Proteins↗
PubMed2004

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-D-gluconic acid.

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.

Fermentation↗
PubMed2004

Batch and fed-batch production of coenzyme Q10 in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans.

Coenzyme Q(10) (CoQ(10)) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ(10) has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ(10) with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ(10) rather than endogenous CoQ(8) was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ(10) to 0.97 mg l(-1) in batch fermentation. A high cell density (103 g l(-1)) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ(10) concentration to 25.5 mg l (-1) and its productivity to 0.67 mg l(-1) h(-1), which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.

Alkyl and Aryl Transferases↗
PubMed2006

Asymmetric oxidation by Gluconobacter oxydans.

Asymmetric oxidation is of great value and a major interest in both research and application. This review focuses on asymmetric oxidation of organic compounds by Gluconobacter oxydans. The microbe can be used for bioproduction of several kinds of important chiral compounds, such as vitamin C, 6-(2-hydroxyethyl)amino-6-deoxy-alpha-L-sorbofuranose, (S)-2-methylbutanoic acid, (R)-2-hydroxy-propionic acid and 5-keto-D-gluconic acid. Characteristics of the bacteria and research progress on the enantioselective biotransformation process are introduced.

Biotechnology↗
PubMed2006

High-yield 5-keto-D-gluconic acid formation is mediated by soluble and membrane-bound gluconate-5-dehydrogenases of Gluconobacter oxydans.

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.

Acetates↗
PubMed2003

Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans.

An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented. Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process. A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells. On the basis of experimentally validated model calculations, a threshold value of about 60 kg x m(-3) DHA was obtained. The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column. In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material. Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg x m(-3) h(-1). However, it was observed that the maximum achievable productivity had not been reached yet.

Bioreactors↗
PubMed2005

Study of the inhibitory effect of the product dihydroxyacetone on Gluconobacter oxydans in a semi-continuous two-stage repeated-fed-batch process.

The influence of the product inhibition by dihydroxyacetone (DHA) on Gluconobacter oxydans for a novel semi-continuous two-stage repeated-fed-batch process was examined quantitatively. It was shown that the culture was able to grow up to a DHA concentration of 80 kg m(-3) without any influence of product inhibition. The regeneration capability of the reversibly product inhibited culture from a laboratory-scale bioreactor system was observed up to a DHA concentration of about 160 kg m(-3). At higher DHA concentrations, the culture was irreversibly product inhibited. However, due to the robust membrane-bound glycerol dehydrogenase of G. oxydans, product formation was still active for a prolonged period of time. The reachable maximum final DHA concentration was as high as 220 kg m(-3). The lag phases for growth increased exponentially with increasing DHA threshold values of the first reactor stage. These results correlated well with fluorescence in situ hybridization (FISH) measurements confirming that the number of active cells decreased exponentially with increasing DHA concentrations.

Bioreactors↗
PubMed2005

Dextran dextrinase and dextran of Gluconobacter oxydans.

Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.2)-a transglucosidase converting maltodextrins into (oligo)dextran. The enzyme catalyses the transfer of an alpha1,4 linked glucosyl unit from a donor to an acceptor molecule, forming an alpha1,6 linkage: consecutive glucosyl transfers result in the formation of high molecular weight dextran from maltodextrins. In the early 1990s, the group of K. Yamamoto in Japan revived research on DDase, focussing on the purification and characterisation of the intracellular DDase produced by G. oxydans ATCC 11894. More recently, this was taken further by Y. Suzuki and coworkers, who investigated the properties and kinetics of the extracellular DDase formed by the same strain. Our group further elaborated on fermentation processes to optimise DDase production and dextran formation, DDase characterisation and its use as a biocatalyst, and the physiological link between intracellular and extracellular DDase. Here, we present a condensed overview of the current scientific status and the application potential of G. oxydans DDase and its products, (oligo)dextrans. The production of DDase as well as of dextran is first described via optimised fermentation processes. Specific assays for measuring DDase activity are also outlined. The general characteristics, substrate specificity, and mode of action of DDase as a transglucosidase are described in detail. Two forms of DDase are produced by G. oxydans depending on nutritional fermentation conditions: an intracellular and an extracellular form. The relationship between the two enzyme forms is also discussed. Furthermore, applications of DDase, e.g. production of (oligo)dextran, transglucosylated products and speciality oligosaccharides, are summarized.

Biotechnology↗
PubMed2005

Gluconobacter oxydans NAD-dependent, D-fructose reducing, polyol dehydrogenases activity: screening, medium optimisation and application for enzymatic polyol production.

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l(-1), 7.2 g peptone l(-1) and 1.8 g yeast extract l(-1). Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5-10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

Biotechnology↗
PubMed2004

Quinate oxidation in Gluconobacter oxydans IFO3244: purification and characterization of quinoprotein quinate dehydrogenase.

Quinoprotein quinate dehydrogenase (QDH) is a membrane-bound enzyme containing pyrroloquinoline quinone (PQQ) as the prosthetic group. QDH in Gluconobacter oxydans IFO3244 was found to be inducible by quinate and it is not constitutively expressed in the absence of quinate. The purification of holo-form of QDH to nearly homogeneity was achieved. The purified QDH appears to have two subunits of approximately 65 and 21 kDa, which could be the result of proteolysis of single polypeptide. Kinetic analysis indicated that the purified enzyme is much more specific to quinate than QDH from Acinetobacter calcoaceticus. The efficiency of the artificial electron acceptor was also determined.

Alcohol Oxidoreductases↗