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Bioprocess and Biosystems Engineering

, Volume 32, Issue 3, pp 313–320 | Cite as

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by Klebsiella pneumoniae under micro-aerobic conditions

  • Li Zhao
  • Yu Zheng
  • Xingyuan MaEmail author
  • Dongzhi WeiEmail author
Original Paper

Abstract

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.

Keywords

1,3-Propanediol 1,3-Propanediol oxidoreductase Glycerol dehydrogenase Klebsiella pneumoniae Micro-aerobic conditions 

References

  1. 1.
    Zeng AP, Biebl H (2002) Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends. Adv Biochem Eng Biotechnol 74:239–259Google Scholar
  2. 2.
    Xiu ZL, Song BH, Wang ZT, Sun LH, Feng EM, Zeng AP (2004) Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in one and two stage anaerobic cultures. Biochem Eng J 19:189–197CrossRefGoogle Scholar
  3. 3.
    Biebl H, Menzel K, Zeng AP, Deckwer WD (1999) Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 52:289–297CrossRefGoogle Scholar
  4. 4.
    Menzel K, Zeng AP, Deckwer WD (1997) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microb Technol 20:82–86CrossRefGoogle Scholar
  5. 5.
    Forage RG, Foster MA (1982) Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases. J Bacteriol 149:413–419Google Scholar
  6. 6.
    Forage RG, Lin ECC (1982) dha System mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB418. J Bacteriol 151:591–599Google Scholar
  7. 7.
    Knietsch A, Bowien S, Whited G, Gottschalk G, Daniel R (2003) Identification and characterization of coenzyme B12-dependent glycerol dehydratase and diol dehydratase encoding genes from metagenomic DNA libraries derived from enrichment cultures. Appl Environ Microbiol 69:3048–3060CrossRefGoogle Scholar
  8. 8.
    Daniel R, Boenigk R, Gottschalk G (1995) Purification of the 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing and over-expression of the corresponding gene in Escherichia coli. J Bacteriol 177:2151–2156Google Scholar
  9. 9.
    Daniel R, Stuertz K, Gottschalk G (1995) Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. J Bacteriol 177:4392–4401Google Scholar
  10. 10.
    Huang H, Gong CS, Tsao GT (2002) Production of 1,3-propanediol by Klebsiella pneumonia. Appl Biochem Biotechnol 98–100:687–698CrossRefGoogle Scholar
  11. 11.
    Chen X, Zhang DJ, Qi WT, Gao SJ, Xiu ZL, Xu P (2003) Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions. Appl Microbiol Biotechnol 63:143–146CrossRefGoogle Scholar
  12. 12.
    Cheng KK, Liu DH, Sun Y, Liu WB (2004) 1,3-Propanediol production by Klebsiella pneumoniae under different aeration strategies. Biotechnol Lett 26:911–915CrossRefGoogle Scholar
  13. 13.
    Liu HJ, Zhang DJ, Xu YH, Sun YQ, Xiu ZL (2007) Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale. Biotechnol Lett 29:1281–1285CrossRefGoogle Scholar
  14. 14.
    Johnson EA, Levine RL, Lin ECC (1985) Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations. J Bacteriol 164:479–483Google Scholar
  15. 15.
    Johnson EA, Lin ECC (1987) Klebsiella pneumoniae 1,3-Propanediol: NAD+Oxidoreductase. J Bacteriol 169:2050–2054Google Scholar
  16. 16.
    Hao J, Wang W, Tian JS, Li JL, Liu DH (2008) Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing dhaT gene in Klebsiella pneumoniae TUAC01. J Ind Microbiol Biotechnol 35(7):735–741CrossRefGoogle Scholar
  17. 17.
    Ahrens K, Menzel K, Zeng AP, Deckwer W (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation. Biotechnol Bioeng 59:544–552CrossRefGoogle Scholar
  18. 18.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  19. 19.
    Cirde SJ, Stone L, Boruff CS (1945) Acrolein determination by means of tryptophane. Ind Eng Chem Anal Ed 17:259–262CrossRefGoogle Scholar
  20. 20.
    Bentley WE, Mirjalili N, Andersen DC, Davis RH, Kompala DS (1990) Plasmid encoded protein: the principal factor in the “metabolic burden” associated with recombinant bacteria. Biotechnol Bioeng 35:668–681CrossRefGoogle Scholar
  21. 21.
    Slininger PJ, Bothast RJ, Smiley KL (1983) Production of 3-hydroxypropionaldehyde from glycerol. Appl Environ Microbiol 46:62–67Google Scholar
  22. 22.
    Barbirato F, Philippe JG, Philippe S, Boriesl A (1996) 3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 62:1448–1451Google Scholar
  23. 23.
    Barbirato F, Philippe S, Boriesl A (1996) Physiologic mechanisms involved in accumulation of 3-hydroxypropionaldehyde during fermentation of glycerol by Enterobacter agglomerans. Appl Environ Microbiol 62:4405–4409Google Scholar
  24. 24.
    Zheng P, Wereath K, Sun JB, van den Heuvel J, Zeng AP (2006) Over-expression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae. Process Biochem 41:2160–2169CrossRefGoogle Scholar
  25. 25.
    Zhang QR, Teng H, Sun YQ, Xiu ZL, Zeng AP (2008) Metabolic flux and robustness analysis of glycerol metabolism in Klebsiella pneumoniae. Bioprocess Biosyst Eng 31(2):127–135CrossRefGoogle Scholar
  26. 26.
    Zhang YP, Li Y, Du CY, Liu M, Cao ZA (2006) Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metab Eng 8:578–586CrossRefGoogle Scholar
  27. 27.
    Du CY, Yan H, Zhang YP, Li Y, Cao ZA (2006) Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae. Appl Microbiol Biotechnol 69:554–563CrossRefGoogle Scholar
  28. 28.
    McGregor WG, Phillips J, Suelter CH (1974) Purification and kinetic characterization of a monovalent cation-activated glycerol dehydrogenase from Aerobacter aerogenes. J Biol Chem 249:3132–3139Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.State Key Laboratory of Bioreactor Engineering, New World Institute of BiotechnologyEast China University of Science and TechnologyShanghaiPeople’s Republic of China

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