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

Elfari, Mustafa; Ha, Seung-Wook; Bremus, Christoph; Merfort, Marcel; Khodaverdi, Viola; Herrmann, U.; Sahm, Hermann; Görisch, Helmut

doi:10.1007/s00253-004-1721-4

Cited by 40 publications

(29 citation statements)

References 23 publications

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“…However, GOX1231 is the sole gene responsible for 2KGA production, which has already been confirmed by Elfari et al (5). On the other hand, in "G. dioxyacetonicus" IFO 3271, a mutation in gndL did not result in the loss of 2KGA production; therefore, another enzyme is responsible for 2KGA production.…”

Section: Discussionmentioning

confidence: 57%

Membrane-Bound, 2-Keto- d -Gluconate-Yielding d -Gluconate Dehydrogenase from “ Gluconobacter dioxyacetonicus ” IFO 3271: Molecular Properties and Gene Disruption

Toyama

Furuya²,

Saichana³

et al. 2007

Appl Environ Microbiol

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Most Gluconobacter species produce and accumulate 2-keto-D-gluconate (2KGA) and 5KGA simultaneously from D-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotidecontaining GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-␦-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, D-arabitol, and D-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.

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Section: Discussionmentioning

confidence: 57%

Membrane-Bound, 2-Keto- d -Gluconate-Yielding d -Gluconate Dehydrogenase from “ Gluconobacter dioxyacetonicus ” IFO 3271: Molecular Properties and Gene Disruption

Toyama

Furuya²,

Saichana³

et al. 2007

Appl Environ Microbiol

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“…Consistently, mutant membranes showed gluconate dehydrogenase activity without prior addition of PQQ. In the G. oxydans 621H membrane, D-gluconate can be oxidized either by the quinoprotein glycerol dehydrogenase or by the flavin-dependent gluconate-2-dehydrogenase (11,38), yielding 5-D-ketogluconate or 2-D-ketogluconate, respectively. Thus, in the mutant, respiratory energy generation could be driven by the flavin-dependent enzyme.…”

Section: Discussionmentioning

confidence: 99%

Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

Hölscher

Görisch

2006

J Bacteriol

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In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membranebound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with D-mannitol, D-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with D-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQdependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.The acetic acid bacterium Gluconobacter oxydans is characterized by its ability to incompletely oxidize various sugars and alcohols by using membrane-associated dehydrogenases that contain pyrroloquinoline quinone (PQQ) as a cofactor (33). Examples are the quinoprotein glucose dehydrogenase (3) and the quinoprotein glycerol dehydrogenase, which also oxidizes D-gluconate, D-mannitol, D-sorbitol, and other polyols (34). The oxidation reactions take place in the periplasmic space and are coupled to the respiratory chain (19,33). Several oxidation reactions carried out by G. oxydans quinoproteins are of industrial importance, e.g., the conversion of D-gluconate to 5-D-ketogluconate, a precursor for the production of L-tartaric acid, and the formation of L-sorbose, an intermediate in the synthesis of vitamin C (reviewed in reference 9). PQQ has invoked considerable attention due to its positive physiological effects in mammals (47). A possible role of PQQ as a vitamin in mammals has been suggested but is controversially debated (13,28,40).Genes involved in PQQ synthesis have been characterized for several bacteria, including Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens AM1, and Pseudomonas sp. (reviewed in reference 19). In Klebsiella pneumoniae, the PQQ biosynthetic genes are clustered in the pqqABCDEF operon (35). In Pseudomonas aeruginosa, the pqqABCDE operon is separated from the pqqF operon (18,48). Methylobacterium extorquens AM1 contains a ...

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“…However, this deficiency was caused by the lack of flavin as cofactor, so that side effects caused by other deficient flavoproteins could not be excluded. Therefore, Elfari et al [2005] generated a mutant of G. oxydans ATCC 621H, in which the gene encoding the membrane-bound GA2DH was inactivated. This mutant grew in a similar manner as the wild type but formation of 2-KGA was not observed, so that it was clearly demonstrated that GA2DH is responsible for 2-KGA formation.…”

Section: Production Of 2-keto-d -Gluconate and 25-di-keto-d -Gluconatementioning

confidence: 99%

Glucose Oxidation and PQQ-Dependent Dehydrogenases in <i>Gluconobacter oxydans</i>

et al. 2008

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Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).

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

Cited by 40 publications

References 23 publications

Membrane-Bound, 2-Keto- d -Gluconate-Yielding d -Gluconate Dehydrogenase from “ Gluconobacter dioxyacetonicus ” IFO 3271: Molecular Properties and Gene Disruption

Membrane-Bound, 2-Keto- d -Gluconate-Yielding d -Gluconate Dehydrogenase from “ Gluconobacter dioxyacetonicus ” IFO 3271: Molecular Properties and Gene Disruption

Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

Glucose Oxidation and PQQ-Dependent Dehydrogenases in <i>Gluconobacter oxydans</i>

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