Applied Microbiology and Biotechnology

, Volume 66, Issue 6, pp 668–674 | Cite as

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

  • Mustafa Elfari
  • Seung-Wook Ha
  • Christoph Bremus
  • Marcel Merfort
  • Viola Khodaverdi
  • Ute Herrmann
  • Hermann Sahm
  • Helmut GörischEmail author
Applied Genetics and Molecular Biotechnology


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.


Gluconic Acid Miglitol Triparental Mating Pyrroloquinoline Quinone Ketogluconic Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The project was carried out within the framework of the Competence Network Göttingen “Genome research on bacteria” (GenoMik) financed by the German Federal Ministry of Education and Research (BMBF).


  1. Beschkov V, Velizarov S, Peeva L (1995) Some kinetic aspects and modelling of bio-transformation of d-glucose to keto-d-gluconates. Bioprocess Eng 13:301–305CrossRefGoogle Scholar
  2. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 14:459–472Google Scholar
  3. Buchert J, Viikari L (1988) Oxidative d-xylose metabolism of G. oxydans. Appl Microbiol Biotechnol 29:375–379Google Scholar
  4. Buse R, Qazi GN, Träger M, Onken U (1990) Influence of dissolved oxygen tension on the production rate of 2,5-Diketogluconic acid by Gluconobacter melanogenum. Biotechnol Lett 12:111–116Google Scholar
  5. Deppenmeier U, Hoffmeister M, Prust C (2002) Biochemistry and biotechnological applications of Gluconobacter strains. Appl Microbiol Biotechnol 3:233–242Google Scholar
  6. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652Google Scholar
  7. Gillis M, de Ley J (1980) Intra- and intergeneric similarities of the ribosomal ribonucleic acid cistrons of Acetobacter and Gluconobacter. Int J Syst Bacteriol 30:7–27Google Scholar
  8. Gupta A, Felder M, Verma V, Cullum J, Qazi GN (1999) A mutant of Gluconobacter oxydans deficient in gluconic acid dehydrogenase. FEMS Microbiol Lett 179:501–506CrossRefPubMedGoogle Scholar
  9. Gupta A, Singh VK, Qazi GN, Kumar A (2001) Gluconobacter oxydans: its biotechnological applications. J Mol Microbiol Biotechnol 3:445–456Google Scholar
  10. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580PubMedGoogle Scholar
  11. Herrmann U, Merfort M, Jeude M, Bringer-Meyer S, Sahm H (2004) Biotransformation of glucose to 5-keto-d-gluconic acid by recombinant Gluconobacter oxydans DSM 2343. Appl Microbiol Biotechnol 64:86–90CrossRefPubMedGoogle Scholar
  12. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) The genus Acetobacter and Gluconobacter. In: Bergey’s manual of determinative bacteriology, vol 1, 9th edn. Williams & Wilkins, Baltimore, pp 268–274Google Scholar
  13. Kheshgi S, Roberts HR, Bucek W (1954) Studies on the production of 5-keto-gluconic acid by Acetobacter suboxydans. Appl Microbiol 2:183–190PubMedGoogle Scholar
  14. Klasen R, Bringer-Meyer S, Sahm H (1995) Biochemical characterization and sequence analysis of the gluconate: NADP 5-oxidoreductase gene from Gluconobacter oxydans. J Bacteriol 177:2637–2643PubMedGoogle Scholar
  15. Kretzschmar U, Schobert M, Görisch H (2001) The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol. Microbiology 147:2671–2677Google Scholar
  16. Macauley S, McNeil B, Harvey LM (2001) The genus Gluconobacter and its applications in biotechnology. Crit Rev Biotechnol 21:1–25PubMedGoogle Scholar
  17. Matsushita K, Ebisuya H, Ameyama M, Adachi O (1992) Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti. J Bacteriol 174:122–129PubMedGoogle Scholar
  18. Matsushita K, Toyama H, Adachi O (1994) Respiratory chains and bioenergetics of acetic acid bacteria. Adv Microb Physiol 36:247–301PubMedGoogle Scholar
  19. Matsushita K, Fujii Y, Ano Y, Toyama H, Shinjoh M, Tomiyama N, Miyazaki T, Sugisawa T, Hoshino T, Adachi O (2003) 5-Keto-d-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in Gluconobacter species. Appl Environ Microbiol 69:1959–1966Google Scholar
  20. Matzerath I, Kläui W, Klasen R, Sahm H (1995) Vanadate catalysed oxidation of 5-keto-d-gluconic acid to tartaric acid: the unexpected effect of phosphate and carbonate on rate and selectivity. Inorg Chim Acta 237:203–205CrossRefGoogle Scholar
  21. Mostafa HE, Heller KJ, Geis A (2002) Cloning of Escherichia coli lacZ and lacY genes and their expression in Gluconobacter oxydans and Acetobacter liquefaciens. Appl Environ Microbiol 68:2619–2623Google Scholar
  22. Porco A, Alonso G, Istúriz T (1998) The gluconate high affinity transport of GntI in Escherichia coli involves a multicomponent complex system. J Basic Microbiol 38:395–404CrossRefPubMedGoogle Scholar
  23. Salusjärvi T, Povelainen M, Hvorslev N, Eneyskaya EV, Kulminskaya AA, Shabalin KA, Neustroev KN, Kalkkinen N, Miasnikov AN (2004) 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. Appl Microbiol Biotechnol (in press)Google Scholar
  24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  25. Shinagawa, E, Matsushita K, Adachi O, Ameyama M (1981) Isolation and purification of 2-ketogluconate dehydrogenase from Gluconobacter melanogenum. Agric Biol Chem 45:1079–1085Google Scholar
  26. Shinagawa E, Matsushita K, Adachi O, Ameyama M (1983) Selective production of 5-keto-d-gluconate by Gluconobacter strains. J Ferment Technol 61:359–363Google Scholar
  27. Shinagawa E, Matsushita K, Toyama H, Adachi O (1999) Production of 5-keto-d-gluconate by acetic acid bacteria is catalyzed by pyrroloquinoline quinone (PQQ)-dependent membrane-bound d-gluconate dehydrogenase. J Mol Catal B 6:341–350CrossRefGoogle Scholar
  28. Silberbach M, Maier B, Zimmermann M, Büchs J (2003) Glucose oxidation by Gluconobacter oxydans in shaking-flasks, scale-up and optimization of the pH profile. Appl Microbiol Biotechnol 62:92–98CrossRefPubMedGoogle Scholar
  29. Sonoyama T, Tani H, Matsuda K, Kageyama B, Tanimoto M, Kobayashi K, Yagi S, Koyotani H, Mitsishima K (1982) Production of 2-keto-l-gulonic acid from d-glucose by two-stage fermentation. Appl Environ Microbiol 43:1064–1069Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Mustafa Elfari
    • 1
  • Seung-Wook Ha
    • 1
  • Christoph Bremus
    • 2
  • Marcel Merfort
    • 2
  • Viola Khodaverdi
    • 1
  • Ute Herrmann
    • 2
  • Hermann Sahm
    • 2
  • Helmut Görisch
    • 1
    Email author
  1. 1.Fachgebiet Technische BiochemieInstitut für Biotechnologie der Technischen Universität BerlinBerlinGermany
  2. 2.Institut für Biotechnologie1Forschungszentrum Jülich GmbHJülichGermany

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