Abstract
Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.
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Abbreviations
- Ace:
-
Acetate acid
- Bdo:
-
Butanediol
- Eth:
-
Ethanol
- Gly:
-
Glycerol
- 3-HPA:
-
3-Hydroxypropionaldehyde
- Lac:
-
Lactate acid
- Pdo:
-
1,3-Propanediol
- Suc:
-
Succinic acid
- PDOR:
-
1,3-Propanediol oxidoreductase
References
Ahrens K, Menzel K, Zeng AP, Deckwer WD (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. Biotech Bioeng 59:544–552
Arntz D, Haas T, Müller A, Wiegand N (1991) Kinetische untersuchung zur hydrat isierung von acrolein. Chem Ing Tech 63:733–735
Barbirato F, Grivet JP, Soucaille P, Bories A (1996a) 3-HPA, an Inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 62:1448–1451
Barbirato F, Soucaille P, Bories A (1996b) Physiologic mechanisms involved in accumulation of 3-hydroxypropionaldehyde during fermentation of glycerol by enterobacter agglomerans. Appl Environ Microbiol 62(12):4405–4409
Biebl H (2001) Fermentation of glycerol by Clostridium pasteurianum—batch and continuous culture studies. J Ind Microbiol Biotechnol 27:18–26
Boenigk R, Bowien S, Gottschalk G (1993) Fermentation of glycerol to 1, 3-propanediol in continuous cultures of Citrobacter freundii. Appl Microbiol Biotechnol 38:453–457
Chi NY, Liu CJ, Liu YH, Zhang QF, Zheng XF (2003) Cloning and expressing of 1,3-propanediol oxidoreductase-encoding gene. Acta Microbiol Sin 43:717–721
Cirde SJ, Stone L, Boruff CS (1945) Acrolein deter mination by means of tryptophane. Ind Eng Chem Anal Ed 17:259–262
Daniel R, Boenigk R, Gottschalk G (1995) Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli. J Bacteriol 177(8):2151–2156
Forage RG, Lin ECC (1982) dha System mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 151:591–599
Hao J, Lin RH, Zheng ZM, Liu HJ, Liu DH (2008) Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions. W J Microbiol Biotechnol. doi:10.1007/s11274-008-9665-y
Kleiner D, Paul W, Merrick MJ (1988) Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria J Gen Microbiol 134(77):1779–1784
Knifton JF, James TG, Slaugh LH, Allen KD, Weider PR, Powell JB (2004) One-step production of 1,3-propanediol from ethylene oxide and syngas with a cobalt-iron catalyst. US Patent 6.750.373
Lin RH, Liu HJ, Zhang J, Hao J, Cheng KK, Liu DH (2005) Effect of 3-hydroxypropionaldehyde on 1,3-propanediol fermentation by Klebsiella pneumoniae and regulation for it. Mod Chem Ind 25(9):47–51
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Skraly FA, Lytle BL, Cameron DC (1998) Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol 64(1):98–105
Vollenweider S, Lacroix C (2004) 3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production. Appl Microbiol Biotechnol 64:16–27
Zheng YY, Cao Y, Fang BS (2004) Cloning and sequence analysis of the dhaT gene of the 1,3-propanediol regulon from Klebsiella pneumoniae. Biotechnol Lett 26:251–255
Zeng AP, Bieb H (2002) Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends. Adv Biochem Eng Biotechnol 74:239–259
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Hao, J., Wang, W., Tian, J. et al. Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing dhaT gene in Klebsiella pneumoniae TUAC01. J Ind Microbiol Biotechnol 35, 735–741 (2008). https://doi.org/10.1007/s10295-008-0340-y
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DOI: https://doi.org/10.1007/s10295-008-0340-y