Biochemical and Genetic Studies on Vitamin B12 Synthesis in Pseudomonas dentrificans

  • J. Crouzet
  • F. Blanche
  • B. Cameron
  • D. Thibaut
  • L. Debussche
Part of the Industry-University Cooperative Chemistry Program Symposia book series (IUCC)


Vitamin B12 is produced industrially by Pseudomonas denitrificans or by bacteria belonging to Propionibacterium species (i. e. Propionibacterium shermanii or Propionibacterium freudenreichii). Industrial strains were obtained after long lasting and intense programs aimed at improving the fermentation caracteristics and the productivity of natural isolates (Florent, 1986). These programs consist of numerous mutagenesis steps, each of them introducing a property resulting in improved characteristics of the strain for industrial purpose (i. e. higher productivity, higher growth rate in a defined medium, growth to a higher biomass, etc). At each mutagenesis step the clone of interest is selected randomly either (i) for its property to produce higher level of vitamin B12 or (ii) for its resistance to an antibiotic for instance or (iii) for a phenotype related to an improved production of vitamin B12 (Florent, 1986). These selection programs have allowed various companies to improve the production of the starting bacteria. For instance the Pseudomonas denitrificans wild type isolate, strain MB580, from which the industrial strain used by Rhône-Poulenc Santé derives produces 2.5 mg/1 of cobalamin. In the same conditions, the industrial strain presently used produces much more (Florent, 1986).


Industrial Strain Cephalosporium Acremonium Propionibacterium Freudenreichii Cobalamin Synthesis Cobyric Acid 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Babior B. M. 1982. Ethanolamine ammonia-lyase. p. 107–144. In D. Dolphin (ed.), B12, voll. John Willey & Sons, Inc., New-York.Google Scholar
  2. Bagdasarian, M., R. Lurz, B. Rückert, F. C. Franklin, M. M. Bagdasarian, J. Frey, and K. Timmis. 1981. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host vector system for gene cloning in Pseudomonas. Gene, 16, 237-247. Blanche, F., D. Thibaut, M. Couder and J. C. Müller. Identification of corrinoids precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography. Submitted to publication.Google Scholar
  3. Blanche, F., L. Debussche, D. Thibaut, J. Crouzet and B. Cameron. 1989. Purification and Characterisation of S-Adenosyl-L-Methionine: Uroporphyrinogen III methyltransferase from Pseudomonas denitrificans. J. Bacteriol., 171, 4222–4231.PubMedGoogle Scholar
  4. Blanche, F., S. Handa, D. Thibaut, C. L. Gibson, F. J. Leeper and A. R. Battersby. 1988. Biosynthesis of vitamin B12: when is the 12β-methyl group of the vitamin generated by acetate decarboxylation? J. Chem. Soc, Chem. Commun., 1988, 1117–1119.CrossRefGoogle Scholar
  5. Brey, R. N., Banner C. D. B., Wolf J. B., 1986. Cloning of Multiple Genes Involved with Cobalamin (Vitamin B12) Biosynthesis in Bacillus megaterium. J. Bacteriol., 167, 623–630.PubMedGoogle Scholar
  6. Cameron, B., K. Briggs, S. Pridmore, G. Brefort and J. Crouzet, 1989. Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans. J. Bacteriol., 171, 547–557.PubMedGoogle Scholar
  7. Crouzet, J., B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut and L. Debussche. Genetic and sequence analysis of an 8.7 kb Pseudomonas denitrificans fragment carrying 8 genes involved in the transformation of precorrin-2 into cobyrinic acid. Submitted to publication.Google Scholar
  8. Crouzet, J., L. Cauchois, F. Blanche, L. Debussche, D. Thibaut, M.-C. Rouyez, S. Rigault, J.-F. Mayaux and B. Cameron. Nucleotide sequence of 5.4 kb DNA fragment from Pseudomonas denitrificans containing five cob genes and identification of structural genes encoding SAM:uroporphyrinogen III methyltransferase and Cobyrinic acid a, c-diamide synthase. Submitted to publication.Google Scholar
  9. De Bruijn, F. J. and J. R. Lupski. 1984. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene, 27, 131–149.PubMedCrossRefGoogle Scholar
  10. Debussche, L., D. Thibaut, B. Cameron, J. Crouzet, F. Blanche. Purification and characterization of cobyrinic acid a, c-diamide synthase from Pseudomonas denitrificans. Submitted to publication.Google Scholar
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R., 1980. Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium melitoti. Proc. Natl. Acad. Sci. U. S. A., 77, 7347–7351.PubMedCrossRefGoogle Scholar
  12. Escalante-Semerena, J. C, S.-J. Suh and J. R. Roth. 1990. cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium. J. Bacteriol., 172, 273–280.PubMedGoogle Scholar
  13. Florent, J. 1986. Vitamins. p115–158. In H.-J. Pehm and G. Reed (ed.), Biotechnology, vol.4, VCH Verlagsgesellschaft mbH, Weinheim.Google Scholar
  14. Grabau, C. and J. Roth. 1989. A vitamin B12 mutant blocked in aminopropanol synthesis. Abstr. Annu. Meet. Soc. Microbiol., p. 180.Google Scholar
  15. Hopp, T. P. and K. R. Woods. 1981. Prediction of protein antigenic determinants from amino acids sequences. Proc. Natl. Acad. Sci. USA, 78, 3824–3828.PubMedCrossRefGoogle Scholar
  16. Jeter, R. M., Olivera B. M., Roth J. R., 1984. Salmonella typhimurium synthetises cobalamin (vitamin B12) de novo under anaerobic growth conditions. J. Bacteriol., 159:206–213.PubMedGoogle Scholar
  17. Jeter, R. M. and J. R. Roth. 1987. Cobalamin (Vitamin B12) Biosynthetic Genes of Salmonella typhimurium. J. Bacteriol. 169, 3189–3198.PubMedGoogle Scholar
  18. Kanehisa, M. 1984. Use of statistical criteria for screening potential homologies in nucleic acids sequences. Nucleic Acids Res., 12, 203–215.PubMedCrossRefGoogle Scholar
  19. Macdonald, H. and J. Cole. 1985. Molecular cloning and fuctional analysis of the cysG and nirB genes of E. coli K12, Two closely-linked genes required for NADH-dependant reductase activity. Mol. en. Genet. 200, 328–334.CrossRefGoogle Scholar
  20. Miwa, K., T. Tsuchida, O. Kurahashi, S. Nakamori and K. Sano. 1983. Construction of L-threonine overproducing strains of Escherichia coli K-12 using recombinant DNA techniques. Agric. Biol. Chem., 47, 2329–2334.CrossRefGoogle Scholar
  21. Normark, S., S. Bergtröm, T. Edlund, T. Grundström, B. Jaurin, F. Lindberg and O. Olsson. 1983. Overlapping genes. Ann. Rev. Genet., 17, 499–525.PubMedCrossRefGoogle Scholar
  22. Ozaki, A., R. Katsumata, T. Oka and A. Furuya. 1985. Cloning of the genes concerned in phenylalanine biosynthesis in Corynebacterium glutamicum and its application to breeding of a phenylalanine-producing strain. Agric. Biol. Chem., 49, 2925–2930.CrossRefGoogle Scholar
  23. Peakman, T., J. Crouzet, J.-F. Mayaux, S. Busby, S. Mohan, R. Nicholson and J. Cole. Nucleotide sequence, organisation and structural analysis of the products of the nirB to cysG region of the E. coli chromosome. Submitted to publication.Google Scholar
  24. Platt T. 1986. Transcription termination and the regulation of gene expression. Ann. Rev. Biochem., 55, 339–372.PubMedCrossRefGoogle Scholar
  25. Sano, K., K. Ito, K. Miwa and S. Nakamori. 1987. Amplification of the phosphoenolpyruvate carboxylase gene of Brevibacterium lactofermentum to improve amino acid production. Agric. Biol. Chem., 51:597–599.CrossRefGoogle Scholar
  26. Skatrud, P. L., A. J. Tietz, T. D. Ingolia, C. A. Cantwell, D. L. Fisher, J. L. Chapman and S. Queener. 1989. Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Technology, 7, 477–485.CrossRefGoogle Scholar
  27. Stachel, S. E., G. An, C. Flores and E. W. Nester. 1985. A Tn 31acZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. Embo J., 4, 891–898.PubMedGoogle Scholar
  28. Staden R. and A. D. McLachlan. 1982. Codon preference and its use in identifying protein coding regions in long DNA sequences. Nucleic Acid Res., 10, 141–156.PubMedCrossRefGoogle Scholar
  29. Staden R. 1984. Measurements of the effects that coding for a protein has on a DNA sequence and their use for finding genes. Nucl. Acid Res., 12, 551–567.CrossRefGoogle Scholar
  30. Thibaut, D., M. Couder, J. Crouzet, L. Debussche, B. Cameron and F. Blanche. Assay and purification of S-adenosyl-L-methionine: precorrin-2 methyltransferase from Pseudomonas denitrificans. Submitted to publication.Google Scholar
  31. Warren, M., C. A. Roessner, P. J. Santander and A. I. Scott. 1990a. The Escherichia coli cysG encodes S-adenosylmethionine-dependant uroporphyrinogen III methylase. Biochem. J., 265, 725–729.PubMedGoogle Scholar
  32. Warren, M., N. J. Stolowich, P. J. Santander C. A. Roessner, B. A. Sowa and A. I. Scott. 1990b. Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and a novel pyrrocorphin by uroporphyrinogen III methylase. FEBS Lett., 261, 76–80.PubMedCrossRefGoogle Scholar
  33. Wolf, J. B., and R. N. Brey. 1986. Isolation and genetic characterisation of Bacillus megaterium Cobalamin biosynthesis-deficient mutants. J. Bacteriol., 166, 51–58.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • J. Crouzet
    • 1
  • F. Blanche
    • 1
  • B. Cameron
    • 1
  • D. Thibaut
    • 1
  • L. Debussche
    • 1
  1. 1.Department of Biotechnology and Department of Analytical ChemistryRhône-Poulenc Santé, Centre de Recherche de Vitry AlfortvilleVitry sur Seine CédexFrance

Personalised recommendations