Bioprocess and Biosystems Engineering

, Volume 26, Issue 5, pp 325–330 | Cite as

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

  • Hikmet Geckil
  • Ze’ev Barak
  • David M. Chipman
  • Sebnem O. Erenler
  • Dale A. Webster
  • Benjamin C. Stark
Original Paper


Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.


Acetoin Bacterial hemoglobin Butanediol Enterobacter aerogenes Vitreoscilla hemoglobin 



This work was supported in part by NSF grant number BES-9309759 and a postdoctoral fellowship from the Council for Higher Education of Israel to H.G. at Ben Gurion University.


  1. 1.
    Stark BC, Webster DA, Dikshit KL (1999) Vitreoscilla hemoglobin: molecular biology, biochemistry, and practical applications. Recent Res Dev Biotechnol Bioeng 2:155–174Google Scholar
  2. 2.
    Wakabayashi S, Matsubara H, Webster DA (1986) Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 322:481–483Google Scholar
  3. 3.
    Park KW, Kim KJ, Howard AJ, Stark BC, Webster DA (2002) Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. J Biol Chem 277:3334–3337CrossRefPubMedGoogle Scholar
  4. 4.
    Geckil H, Gencer S, Kahraman H, Erenler SO (2003) Genetic engineering of Enterobacter aerogenes with Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Res Microbiol 154:425–431CrossRefPubMedGoogle Scholar
  5. 5.
    Tsai PS, Rao G, Bailey JE (1995) Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: new insights from NAD(P)H fluorescence and culture redox potential. Biotechnol Bioeng 47:347–354Google Scholar
  6. 6.
    Geckil H, Stark BC, Webster DA (2001) Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene. J Biotechnol 85:57–66CrossRefPubMedGoogle Scholar
  7. 7.
    Pringsheim EG (1951) The Vitreoscillaceae: a family of colourless, gliding, filamentous organisms. J Gen Microbiol 5:124–149PubMedGoogle Scholar
  8. 8.
    Buddenhagen RE, Webster DA, Stark BC (1996) Enhancement by bacterial hemoglobin of amylase production in recombinant E. coli occurs under conditions of low O2. Biotechnol Lett 102:695–700Google Scholar
  9. 9.
    Kallio PT, Bailey JE (1996) Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of α-amylase and neutral protease in Bacillus subtilis. Biotechnol Prog 12:31–39CrossRefPubMedGoogle Scholar
  10. 10.
    Khosravi M, Webster DA, Stark BC (1990) Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinant Escherichia coli strain. Plasmid 24:190–194PubMedGoogle Scholar
  11. 11.
    DeModena JA, Gutierrez S, Velasco J, Fernandez FJ, Fachini RA, Galazzo JL, Hughes DE, Martin JF (1993) The production of cephalosporin C by Acremonium chrysogenum is improved by the intracellular expression of a bacterial hemoglobin. Bio/Technol 11:926–929Google Scholar
  12. 12.
    Magnolo SK, Leenutaphong DL, DeModena JA, Curtis JE, Bailey JE, Galazzo JL, Hughes DE (1991) Bio/Technol 9:473–476Google Scholar
  13. 13.
    Chen W, Hughes DE, Bailey JE (1994) Intracellular expression of Vitreoscilla hemoglobin alters the aerobic metabolism of Saccharomyces cerevisiae. Biotechnol Prog 10:308–313PubMedGoogle Scholar
  14. 14.
    Wei M-L, Webster DA, Stark BC (1998) Metabolic engineering of Serratia marcescens with the bacterial hemoglobin gene: alterations in fermentation pathways. Biotechnol Bioeng 59:640–646CrossRefPubMedGoogle Scholar
  15. 15.
    Geckil H, Gencer S (2004) Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl Microbiol Biotechnol 63:691–697CrossRefPubMedGoogle Scholar
  16. 16.
    Geckil H, Ates B, Gencer S, Uckun M, Yilmaz I (2004) Membrane permeabilization of Gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of L-asparaginase: an enzyme used in cancer therapy. Process Biochem (in press)Google Scholar
  17. 17.
    Chung JW, Webster DA, Pagilla KR, Stark BC (2001) Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability. J Ind Microbiol Biotechnol 27:27–33CrossRefPubMedGoogle Scholar
  18. 18.
    Fish PA, Webster DA, Stark BC (2000) Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo. J Mol Catal B Enzymatic 9:75–82CrossRefGoogle Scholar
  19. 19.
    Liu SC, Webster DA, Wei M-L, Stark BC (1996) Genetic engineering to contain the Vitreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia. Biotechnol Bioeng 49:101–105CrossRefGoogle Scholar
  20. 20.
    Nasr MA, Hwang KW, Akbas M, Webster DA, Stark BC (2001) Effects of culture conditions on enhancement of 2,4-dinitrotoluene degradation by Burkholderia engineered with the Vitreoscilla hemoglobin gene. Biotechnol Prog 17:359–361CrossRefPubMedGoogle Scholar
  21. 21.
    Patel SM, Stark BC, Hwang KW, Dikshit KL, Webster DA (2000) Cloning and expression of the Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation. Biotechnol Prog 16:26–30CrossRefPubMedGoogle Scholar
  22. 22.
    Magee RJ, Kosaric N (1987) The microbial production of 2,3-butanediol. Adv Appl Microbiol 32:89–161Google Scholar
  23. 23.
    Montville TJ, Hsu AH, Meyer ME (1987) High-efficiency conversion of pyruvate to acetoin by Lactobacillus plantarum during pH-controlled and fed-batch fermentations. Appl Environ Microbiol 53:1798–1802Google Scholar
  24. 24.
    Dikshit KL, Webster DA (1988) Cloning, characterization, and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70:377–386CrossRefPubMedGoogle Scholar
  25. 25.
    Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78CrossRefPubMedGoogle Scholar
  26. 26.
    Erenler SO, Gencer S, Geckil H, Stark BC, Webster DA (2004) Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth an oxygen uptake. Appl Biochem Microbiol 40(3):241–248CrossRefGoogle Scholar
  27. 27.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, p 433Google Scholar
  28. 28.
    Westerfeld WW (1945) Colorimetric determination of blood acetoin. J Biol Chem 161:495–502Google Scholar
  29. 29.
    Keen AR, Walker NJ (1973) Separation of diacetyl, acetoin, and 2,3-butylene glycol by ion-exchange chromatography. Anal Biochem 52:475–481PubMedGoogle Scholar
  30. 30.
    Stormer FC (1972) 2,3-Butanediol biosynthetic system in Aerobacter aerogenes. Methods Enzymol 41:518–533Google Scholar
  31. 31.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  32. 32.
    White D (1995) The physiology and biochemistry of prokaryotes. Oxford University Press, Oxford, pp 285–286Google Scholar
  33. 33.
    Bassit N, Boquien C, Picque D, Corrieu G (1993) Effect of initial oxygen concentration on diacetyl and acetoin production by Lactococcus lactis subsp. lactis biovar diacetylactis. Appl Environ Microbiol 59:1893–1897Google Scholar
  34. 34.
    Beronio PB, Tsao GT (1993) Optimization of 2,3-butanediol production by Klebsiella oxytoca through oxygen transfer rate control. Biotechnol Bioeng 42:1263–1269Google Scholar
  35. 35.
    de Mas C, Jansen NB, Tsao GT (1988) Production of optically active 2,3-butanediol by Bacillus polymyxa. Biotechnol Bioeng 31:366–377Google Scholar
  36. 36.
    Jansen NB, Flickenger MC, Tsao GT (1984) Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724. Biotechnol Bioeng 26:362–369Google Scholar
  37. 37.
    Sablayrolles JM, Goma G (1984) Butanediol production by Aerobacter aerogenes NRRL B199: effects of initial substrate concentration and aeration agitation. Biotechnol Bioeng 26:148–155Google Scholar
  38. 38.
    Byun TG, Zeng AP, Deckwer WD (1994) Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate. Bioproc Biosyst Eng 11:167–175CrossRefGoogle Scholar
  39. 39.
    Zeng AP, Biebl H, Deckwer WD (1990) Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture. Appl Microbiol Biotechnol 33:485–489Google Scholar
  40. 40.
    Mallonee DH, Speckman RA (1988) Development of a mutant strain of Bacillus polymyxa showing enhanced production of 2,3-butanediol. Appl Environ Microbiol 54:168–171Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Hikmet Geckil
    • 1
  • Ze’ev Barak
    • 2
  • David M. Chipman
    • 2
  • Sebnem O. Erenler
    • 1
  • Dale A. Webster
    • 3
  • Benjamin C. Stark
    • 3
  1. 1.Department of BiologyInonu UniversityMalatyaTurkey
  2. 2.Department of Life SciencesBen-Gurion University of the NegevBeer-ShevaIsrael
  3. 3.Biology Division, Department of Biological, Chemical, and Physical SciencesIllinois Institute of TechnologyChicagoUSA

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