Overexpression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae

Glycerol dehydratase that catalyses the conversion of glycerol to 3-hydroxypropionaldehyde was previously supposed to be a limiting step in the 1,3-propanediol production from glycerol. In this study, glycerol dehydratase was over-expressed separately or coordinately with 1,3-propanediol oxidoreductase in Klebsiella pneumoniae DSM2026 to investigate its effects on the glycerol fermentation. The overexpression of glycerol dehydratase surprisingly led to a serious impairment of growth of K. pneumoniae in both continuous and batch cultures. Instability of the plasmids bearing the genes encoding glycerol dehydratase and/or 1,3-propanediol oxidoreductase was observed in recombinant cells, especially in anaerobic cultures using glycerol as sole carbon source. It is postulated that an imbalanced conversion of glycerol to the intermediate 3-hydroxypropionaldehyde and its further conversion to 1,3-propanediol and the associated accumulation and toxicity of 3-hydroxypropionaldehyde are responsible for the phenomena observed. Furthermore, the putative regulatory gene dhaR of the dha regulon was also overexpressed in K. pneumoniae. The increased expression of 1,3-propanediol oxidoreductase confirmed the role of DhaR as a positive regulator of the dhaT gene.

[1]  A. Zeng,et al.  Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: I. The phenomena and characterization of oscillation and hysteresis , 2000, Biotechnology and bioengineering.

[2]  Douglas C. Cameron,et al.  Construction and Characterization of a 1,3-Propanediol Operon , 1998, Applied and Environmental Microbiology.

[3]  Jibin Sun,et al.  Comparative Genomic Analysis of dha Regulon and Related Genes for Anaerobic Glycerol Metabolism in Bacteria , 2003, Biotechnology progress.

[4]  P. Soucaille,et al.  Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Zeng,et al.  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. , 1998, Biotechnology and bioengineering.

[6]  G. Gottschalk,et al.  Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase. , 1997, FEMS microbiology letters.

[7]  J. Andrade,et al.  Regulation of Carbon and Electron Flow inClostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures , 2001, Journal of bacteriology.

[8]  A. Lustig,et al.  Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR , 2005, The EMBO journal.

[9]  M. A. Foster,et al.  Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases , 1982, Journal of bacteriology.

[10]  G. Gottschalk,et al.  Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli , 1995, Journal of bacteriology.

[11]  T. Tan,et al.  High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli , 2005, Molecular biotechnology.

[12]  Seraphim Papanikolaou,et al.  Modelling aspects of the biotechnological valorization of raw glycerol: production of citric acid by Yarrowia lipolytica and 1,3‐propanediol by Clostridium butyricum , 2003 .

[13]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[14]  D. Sherratt,et al.  Multimerization of high copy number plasmids causes instability: Cole 1 encodes a determinant essential for plasmid monomerization and stability , 1984, Cell.

[15]  G. Gottschalk,et al.  Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum. , 1998, FEMS microbiology letters.

[16]  A. Bories,et al.  Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation , 2000, Applied Microbiology and Biotechnology.

[17]  R. Hayashi,et al.  Cloning, Sequencing, and High Level Expression of the Genes Encoding Adenosylcobalamin-dependent Glycerol Dehydrase of Klebsiella pneumoniae* , 1996, The Journal of Biological Chemistry.

[18]  A. Zeng,et al.  Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. , 1997, Journal of biotechnology.

[19]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[20]  A. Zeng,et al.  Combined use of proteomic analysis and enzyme activity assays for metabolic pathway analysis of glycerol fermentation by Klebsiella pneumoniae , 2003, Biotechnology and bioengineering.

[21]  E. Lin,et al.  Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase , 1987, Journal of bacteriology.

[22]  S. Papanikolaou,et al.  The effect of raw glycerol concentration on the production of 1,3‐propanediol by Clostridium butyricum , 2004 .

[23]  D. C. Cameron,et al.  1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon , 1991, Applied and environmental microbiology.

[24]  G. Gottschalk,et al.  Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii , 1995, Journal of bacteriology.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  A. Zeng,et al.  Study of two-stage processes for the microbial production of 1,3-propanediol from glucose , 2002, Applied Microbiology and Biotechnology.

[27]  A. Bories,et al.  3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species , 1996, Applied and environmental microbiology.

[28]  S. Papanikolaou,et al.  High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. , 2000, Journal of biotechnology.

[29]  M. Rasch.  The influence of temperature, salt and pH on the inhibitory effect of reuterin on Escherichia coli. , 2002, International journal of food microbiology.

[30]  E. Guedon,et al.  Glycerol dehydratase activity: the limiting step for 1,3‐propanediol production by Clostridium butyricum DSM 5431 , 1996 .

[31]  R. Bothast,et al.  Optimizing aerobic conversion of glycerol to 3-hydroxypropionaldehyde , 1985, Applied and Environmental Microbiology.

[32]  E. Petitdemange,et al.  Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum , 1995, Journal of Industrial Microbiology.

[33]  G. Gottschalk,et al.  Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii , 1996, Journal of bacteriology.

[34]  S. Vollenweider,et al.  3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production , 2004, Applied Microbiology and Biotechnology.

[35]  W. Bentley,et al.  Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria , 1990, Biotechnology and bioengineering.

[36]  P. Gérard,et al.  Properties of Allyl Alcohol-Resistant Mutants of Clostridium butyricum Grown on Glycerol , 1996, Applied and environmental microbiology.

[37]  Z. Rao,et al.  [Construction of novel recombinant Escherichia coli capable of producing 1,3-propanediol]. , 2005, Sheng wu gong cheng xue bao = Chinese journal of biotechnology.

[38]  G. Goma,et al.  High production of 1,3-propanediol from glycerol by Clostridium butyricum VPI 3266 in a simply controlled fed-batch system , 1994, Biotechnology Letters.