| 1. |
Sugiyama M,
Suzuki S,
Tonouchi N,
Yokozeki K,
( 2003 ) Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol. PMID : 12723607 : DOI : 10.1271/bbb.67.584 Abstract >>
Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.
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2. |
Tonouchi N,
Sugiyama M,
Yokozeki K,
( 2003 ) Construction of a vector plasmid for use in Gluconobacter oxydans. PMID : 12619700 : Abstract >>
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.
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3. |
Shinjoh M,
Tazoe M,
Hoshino T,
( 2002 ) NADPH-dependent L-sorbose reductase is responsible for L-sorbose assimilation in Gluconobacter suboxydans IFO 3291. PMID : 11790761 : DOI : 10.1128/jb.184.3.861-863.2002 PMC : PMC139518 Abstract >>
The NADPH-dependent L-sorbose reductase (SR) of L-sorbose-producing Gluconobacter suboxydans IFO 3291 contributes to intracellular L-sorbose assimilation. The gene disruptant showed no SR activity and did not assimilate the once-produced L-sorbose, indicating that the SR functions mainly as an L-sorbose-reducing enzyme in vivo and not as a D-sorbitol-oxidizing enzyme.
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4. |
Felder M,
Verma V,
Gupta A,
( 1999 ) A mutant of gluconobacter oxydans deficient in gluconic acid dehydrogenase PMID : 10518757 : DOI : 10.1111/j.1574-6968.1999.tb08769.x Abstract >>
Gluconobacter oxydans ATCC 9937 was subjected to transposon mutagenesis using Tn5. A non-pigmented mutant was shown to be defective in gluconic acid dehydrogenase and to produce gluconic acid from glucose, whereas the parent strain produced 2, 5-diketogluconic acid. Cloning and sequencing of the region containing the Tn5 insertion showed that the insertion point occurred in an open reading frame homologous (42% amino acid identity) to the ribF genes of Pseudomonas fluorescens and Escherichia coli. The resulting lack of a riboflavin cofactor would explain the loss of enzyme activity.
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5. |
Renouf V,
Claisse O,
Miot-Sertier C,
Lonvaud-Funel A,
( 2006 ) Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis. PMID : 16942997 : DOI : 10.1016/j.fm.2005.01.019 Abstract >>
Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.
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6. |
Ehrensberger AH,
Elling RA,
Wilson DK,
( 2006 ) Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity. PMID : 16531240 : DOI : 10.1016/j.str.2005.11.016 Abstract >>
Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.
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7. |
Shibata T,
Ichikawa C,
Matsuura M,
Takata Y,
Noguchi Y,
Saito Y,
Yamashita M,
( 2000 ) Cloning of a gene for D-sorbitol dehydrogenase from Gluconobacter oxydans G624 and expression of the gene in Pseudomonas putida IFO3738. PMID : 16232778 : Abstract >>
We have cloned a novel gene for d-sorbitol dehydrogenase (SLDH), which efficiently converted D-sorbitol to L-sorbose, from Gluconobacter oxydans G624 (FERM BP-4415). A cosmid library of the genomic DNA was screened by assaying SLDH activity. The inserted DNA from a positive clone was downsized by subcloning into charomid and pUCP plasmid, successively. Sequencing analysis of the DNA responsible for SLDH activity revealed an open reading frame of 1455 bp coding for 485 amino acid residues with a calculated molecular mass of 53,642 Da. The amino acid sequence showed 42.2% identity with a NAD+-dependent mannitol dehydrogenase (MDH), which catalyzed conversion of d-sorbitol to d-fructose, from Pseudomonas fluorescens DSM50106. Since the intact SLDH was found to be very unstable during isolation and purification, this SLDH fused to 6 x His-tag was expressed in Pseudomonas putida IFO3738 and purified by immobilized metal affinity chromatography using cobalt-based resins. The 6 x His-tag SLDH catalyzed the oxidation of D-sorbitol to L-sorbose and exhibited 15 times higher activity in the presence of NADP+ than that of NAD+. These results indicate that the SLDH is a novel kind of dehydrogenase distinct from MDH previously reported.
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8. |
Elfari M,
Ha SW,
Bremus C,
Merfort M,
Khodaverdi V,
Herrmann U,
Sahm H,
Görisch H,
( 2005 ) A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-D-gluconic acid. PMID : 15735967 : DOI : 10.1007/s00253-004-1721-4 Abstract >>
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.
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9. |
Cheng H,
Jiang N,
Shen A,
Feng Y,
( 2005 ) Molecular cloning and functional expression of d-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli. PMID : 16165327 : DOI : 10.1016/j.femsle.2005.08.023 Abstract >>
A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.
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10. |
Vangnai AS,
Promden W,
De-Eknamkul W,
Matsushita K,
Toyama H,
( 2010 ) Molecular characterization and heterologous expression of quinate dehydrogenase gene from Gluconobacter oxydans IFO3244. PMID : 20618134 : Abstract >>
The quinate dehydrogenase (QDH) from Gluconobacter oxydans IFO3244 exhibits high affinity for quinate, suggesting its application in shikimate production. Nucleotide sequence analysis of the qdh gene revealed a full-length of 2475-bp encoding an 824-amino acid protein. The qdh gene has the unusual TTG translation initiation codon. Conserved regions and a signature sequence for the quinoprotein family were observed. Phylogenetic analysis demonstrated relatedness of QDH from G. oxydans to other quinate/shikimate dehydrogenases with the highest similarity (56%) with that of Acinetobacter calcoaceticus ADP1 and lower similarity (36%) with a membrane-bound glucose dehydrogenase of Escherichia coli. The function of the gene coding for QDH was confirmed by heterologous gene expression in pyrroloquinoline quinone-synthesizing Pseudomonas putida HK5.
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11. |
Quintero Y,
Poblet M,
Guillamón JM,
Mas A,
( 2009 ) Quantification of the expression of reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth conditions. PMID : 19200331 : DOI : 10.1111/j.1365-2672.2008.04046.x Abstract >>
The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)-alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB). Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adhA was measured in Acetobacter aceti, Acetobacter pasteurianus, Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adhA expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adhA expression. The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter. Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.
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12. |
Adachi O,
Ano Y,
Toyama H,
Matsushita K,
( 2008 ) A novel 3-dehydroquinate dehydratase catalyzing extracellular formation of 3-dehydroshikimate by oxidative fermentation of Gluconobacter oxydans IFO 3244. PMID : 18540103 : DOI : 10.1271/bbb.70778 Abstract >>
In addition to the cytoplasmic soluble form of 3-dehydroquinate dehydratase (sDQD) (EC 4.1.2.10), a novel form of DQD occurring in the periplasmic space was found in Gluconobacter oxydans IFO 3244. The novel DQD, tentatively designated as pDQD, appeared to have a practical function involved in oxidative fermentation extracellularly coupling with membrane-bound quinoprotein quinate dehydrogenase (QDH) yielding 3-dehydroshikimate from quinate via 3-dehydroquinate. pDQD was not detached from the membrane by mechanical disruption or extraction with high salt, but was solubilized only with detergent. pDQD and sDQD were purified to homogeneity and compared as to their enzymatic properties. They showed the same apparent molecular weights and same catalytic properties, but they were distinct each other in subunit molecular mass, 16 kDa for pDQD and 47 kDa for sDQD.
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13. |
Kim TS,
Patel SK,
Selvaraj C,
Jung WS,
Pan CH,
Kang YC,
Lee JK,
( 2016 ) A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization. PMID : 27633501 : DOI : 10.1038/srep33438 PMC : PMC5025769 Abstract >>
A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.
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14. |
Qi XH,
Zhu JF,
Yun JH,
Lin J,
Qi YL,
Guo Q,
Xu H,
( 2016 ) Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell. PMID : 26975753 : DOI : 10.1016/j.jbiosc.2016.02.009 Abstract >>
Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G. oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80 g/L xylitol by mixed culture from 30 g/L d-xylulose in 28 h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10 g/L xylitol was produced by the mixed culture from 30 g/L d-arabitol in 30 h with a yield of 0.837 g/g, and the max volumetric productivity of 0.990 g/L h was obtained at 22 h. These contrast to the fact that wild-type strain G. oxydans only produced 8.10 g/L xylitol in 30 h with a yield of 0.270 g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains.
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15. |
Sadahiro J,
Mori H,
Saburi W,
Okuyama M,
Kimura A,
( 2015 ) Extracellular and cell-associated forms of Gluconobacter oxydans dextran dextrinase change their localization depending on the cell growth. PMID : 25490393 : DOI : 10.1016/j.bbrc.2014.11.115 Abstract >>
Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium.
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16. |
Valera MJ,
Torija MJ,
Mas A,
Mateo E,
( 2015 ) Cellulose production and cellulose synthase gene detection in acetic acid bacteria. PMID : 25381910 : DOI : 10.1007/s00253-014-6198-1 Abstract >>
The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.
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17. |
Spitaels F,
Wieme A,
Balzarini T,
Cleenwerck I,
Van Landschoot A,
De Vuyst L,
Vandamme P,
( 2014 ) Gluconobacter cerevisiae sp. nov., isolated from the brewery environment. PMID : 24368694 : DOI : 10.1099/ijs.0.059311-0 Abstract >>
Three strains, LMG 27748(T), LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748(T) revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA-DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39-65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748(T) and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 �XC. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748(T), LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748(T) (= DSM 27644(T)) as the type strain.
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18. |
Nishikura-Imamura S,
Matsutani M,
Insomphun C,
Vangnai AS,
Toyama H,
Yakushi T,
Abe T,
Adachi O,
Matsushita K,
( 2014 ) Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans. PMID : 24352733 : DOI : 10.1007/s00253-013-5439-z Abstract >>
Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.
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19. |
( 1997 ) Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain. PMID : 9023923 : PMC : PMC168335 Abstract >>
We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.
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20. |
( 1997 ) Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans. PMID : 9055428 : PMC : PMC168403 Abstract >>
A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.
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21. |
McIntire W,
Singer TP,
Ameyama M,
Adachi O,
Matsushita K,
Shinagawa E,
( 1985 ) Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus. PMID : 4074328 : DOI : 10.1042/bj2310651 PMC : PMC1152798 Abstract >>
An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.
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22. |
Yakushi T,
Komatsu K,
Matsutani M,
Kataoka N,
Vangnai AS,
Toyama H,
Adachi O,
Matsushita K,
( 2018 ) Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans. PMID : 29366965 : DOI : 10.1016/j.pep.2018.01.007 Abstract >>
Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452-459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5'-UTR was higher than that using a plasmid with a short 5'-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5'-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.
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23. |
( 1998 ) Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans. PMID : 9692900 : DOI : 10.1046/j.1432-1327.1998.2550052.x Abstract >>
Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33,898-Da polypeptide that displays high similarity (30-50%) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05% Triton X-100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 microM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.
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