Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enhancement of Clavulanic Acid by Replicative and Integrative Expression of ccaR and cas2 in Streptomyces clavuligerus NRRL3585

  • Hung, Trinh Viet (Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (IBR), Sun Moon University) ;
  • Malla, Sailesh (Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (IBR), Sun Moon University) ;
  • Park, Byoung-Chul (Korea Research Institute of Bioscience and Biotechnology) ;
  • Liou, Kwang-Kyoung (Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (IBR), Sun Moon University) ;
  • Lee, Hei-Chan (Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (IBR), Sun Moon University) ;
  • Sohng, Jae-Kyung (Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (IBR), Sun Moon University)
  • Published : 2007.09.30
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Abstract

Clavulanic acid (CA) is an inhibitor of ${\beta}$-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHLl, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHLl3, and a 23.8-fold increase with pNQ1. The integrative pNQl strain has been successfully applied to enhance production.

Keywords

References

  1. Alexander, D. C. and S. E. Jensen. 1998. Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein. J. Bacteriol. 180: 4068-4079
  2. Arias, P., M. A. Fernández-Moreno, and F. Malpartida. 1999. Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J. Bacteriol. 181: 6958-6968
  3. Baggaley, K. H., A. G. Brown, and C. J. Schofield. 1997. Chemistry and biosynthesis of clavulanic acid and other clavams. Nat. Prod. Rep. 14: 309-333 https://doi.org/10.1039/np9971400309
  4. Buckland, B. C., D. R. Omstead, and V. Santamaria. 1977. Novel $\beta$-lactam antibiotics. Comprehensive Biotechnol. 3: 49-67
  5. Brown, A. G., D. Butterworth, M. Cole, G. Hanscombe, J. D. Hood, C. Reading, and G. N. Robinson. 1976. Naturally occurring beta-lactamase inhibitors with antibacterial activity. J. Antibiot. 29: 668-669 https://doi.org/10.7164/antibiotics.29.668
  6. Chater, K. F. 1993. Genetics of differentiation in Streptomyces. Annu. Rev. Microbiol. 47: 685-713 https://doi.org/10.1146/annurev.mi.47.100193.003345
  7. Foulstone, M. and C. Reading. 1982. Assay of amoxicillin and clavulanic acid, the components of augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob. Agents Chemother. 22: 753-762 https://doi.org/10.1128/AAC.22.5.753
  8. Garcia-Dominguez, M., J. F. Martin, B. Mahro, A. L. Demain, and P. Liras. 1987. Efficient plasmid transformation of the $\beta$-lactam producer Streptomyces clavuligerus. Appl. Environ. Microbiol. 53: 1376-1381
  9. Gouveia, E. R., A. Baptista-Neto, A. G. Azevedo, A. C. Badino Jr., and C. O. Hokka. 1999. Improvement of clavulanic acid production by Streptomyces clavuligerus in medium containing soybean derivatives. World J. Microbiol. Biotechnol. 15: 623-627 https://doi.org/10.1023/A:1008942405378
  10. Howarth, T. T. and A. Brown. 1976. Clavulanic acid, a novel $\beta$-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis. J. Chem. Soc. Commun. 266-267
  11. Higgens, C. E. and R. E. Kastner. 1971. Streptomyces clavuligerus sp. nov., a $\beta$-lactam antibiotic producer. Int. J. Syst. Bacteriol. 21: 326-331 https://doi.org/10.1099/00207713-21-4-326
  12. Hung, T. V. 2005. Application of genetic engineering to enhance clavulanic acid production from Streptomyces clavuligerus NRRL3585. MSc Thesis, Sun Moon University, Asan, Korea
  13. Ishida, K., T. V. Hung, H. C. Lee, K. Liou, C. H. Shin, Y. J. Yoon, and J. K. Sohng. 2006. Degradation of clavulanic acid during the cultivation of Streptomyces clavuligerus; Instability of clavulanic acid by metabolites and proteins from the strain. J. Microbiol. Biotechnol. 16: 590-596
  14. Jensen, S. E. and A. S. Paradkar. 1999. Biosynthesis and molecular genetics of clavulanic acid. Antonie Van Leeuwenhoek 75: 125-133 https://doi.org/10.1023/A:1001755724055
  15. Jensen, S. E., A. S. Paradkar, R. H. Mosher, C. Anders, P. H. Beatty, M. J. Brumlik, A. Griffin, and B. Barton. 2004. Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 48: 192-202 https://doi.org/10.1128/AAC.48.1.192-202.2004
  16. Kang, S. G., D. H. Lee, A. C. Ward, and K. J. Lee. 1998. Rapid and quantitative analysis of clavulanic acid production by the combination of pyrolysis mass spectrometry and artificial neural network. J. Microbiol. Biotechnol. 8: 523-530
  17. Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. In: Practical Streptomyces Genetics, pp. 230-249. John Innes Foundation, Norwich, United Kingdom
  18. Li, R., N. Khaleeli, and C. A. Townsend. 2002. Expansion of the clavulanic acid gene cluster: Identification and in vivo functional analysis of three new genes required for biosynthesis of clavulanic acid by Streptomyces clavuligerus. J. Bacteriol. 182: 4087-4095 https://doi.org/10.1128/JB.182.14.4087-4095.2000
  19. Madduri, K. and C. R. Hutchinson. 1995. Functional characterization and transcriptional analysis of the dnrR1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius. J. Bacteriol. 177: 1208-1215 https://doi.org/10.1128/jb.177.5.1208-1215.1995
  20. Marsh, E. N., M. D. T. Chang, and C. A. Townsend. 1992. Two isozymes of clavaminate synthase central to clavulanic acid formation: Cloning and sequencing of both genes from Streptomyces clavuligerus. Biochemistry 31: 12648-12657 https://doi.org/10.1021/bi00165a015
  21. Mayer, A. F. and W. D. Deckwer. 1996. Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations. Appl. Microbiol. Biotechnol. 45: 41-46 https://doi.org/10.1007/s002530050646
  22. Mosher, R. H., A. S. Paradkar, C. Anders, B. Barton, and S. E. Jensen. 1999. Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus. Antimicrob. Agents Chemother. 43: 1215-1224
  23. Paget, M. S., L. Chamberlin, A. Atrih, S. J. Foster, and M. J. Buttner. 1999. Evidence that the extracytoplasmic function sigma factor E is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol. 181: 204-211
  24. Paradkar, A. S. and S. E. Jensen. 1995. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. J. Bacteriol. 177: 1307-1314 https://doi.org/10.1128/jb.177.5.1307-1314.1995
  25. Paradkar, A. S., R. H. Mosher, C. Anders, A. Griffin, J. Griffin, C. Hughes, P. Greaves, B. Barton, and S. E. Jensen. 2001. Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production. Appl. Environ. Microbiol. 67: 2292-2297 https://doi.org/10.1128/AEM.67.5.2292-2297.2001
  26. Parajuli, N., T. V. Hung, I. Kenji, T. T. Hang, H. C. Lee, K. K. Liou, and J. K. Sohng. 2005. Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952. Res. Microbiol. 156: 707-712 https://doi.org/10.1016/j.resmic.2005.03.005
  27. Park, H. S., S. H. Kang, H. J. Park, and E. S. Kim. 2005. Doxorubicin productivity improvement by the recombinant Streptomyces peucetius with high-copy regulatory genes cultured in the optimized media composition. J. Microbiol. Biotechnol. 15: 66-71
  28. Park, N. S., J. S. Myeong, H. J. Park, K. Han, S. N. Kim, and E. S. Kim. 2005. Characterization and culture optimization of regiospecific cyclosporine hydroxylation in rare actinomycetes species. J. Microbiol. Biotechnol. 15: 188-191
  29. Perez-Liarena, F. J., P. Liras, A. Rodríguez-García, and J. F. Martín. 1997. A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: Amplification results in overproduction of both $\beta$-lactam compounds. J. Bacteriol. 179: 2053-2059 https://doi.org/10.1128/jb.179.6.2053-2059.1997
  30. Perez-Redondo, R., A. Rodriguez-Garcia, J. F. Martín, and P. Liras. 1998. The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene. Gene 211: 311-321 https://doi.org/10.1016/S0378-1119(98)00106-1
  31. Reading, E. and M. Cole. 1977. Clavulanic acid: A beta lactamase-inhibiting $\beta$-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 11: 852-857 https://doi.org/10.1128/AAC.11.5.852
  32. Ryu, Y. G., W. Jin, J. Y. Kim, S. H. Lee, and K. J. Lee. 2004. Stringent factor regulates antibiotics production and morphological differentiation of Streptomyces clavuligerus. J. Microbiol. Biotechnol. 14: 1170-1175
  33. Salowe, S. P., E. N. Marsh, and C. A. Townsend. 1990. Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: An unusual oxidative enzyme in natural product biosynthesis. Biochemistry 29: 6499-6508 https://doi.org/10.1021/bi00479a023
  34. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  35. Santamarta, I., A. Rodríguez-García, R. Perez-Redondo, J. F. Martín, and P. Liras. 2002. CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus. J. Bacteriol. 184: 3106-3113 https://doi.org/10.1128/JB.184.11.3106-3113.2002
  36. Sthapit, B., T. J. Oh, R. Lamichhane, K. Liou, H. C. Lee, C. G. Kim, and J. K. Sohng. 2004. Neocarzinostatin naphthoate synthase: An unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus. FEBS Lett. 566: 201-206 https://doi.org/10.1016/j.febslet.2004.04.033
  37. Stutzman-Engwall, K. J., S. L. Otten, and C. R. Hutchinson. 1992. Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J. Bacteriol. 174: 144-154 https://doi.org/10.1128/jb.174.1.144-154.1992
  38. Townsend, C. A. 2002. New reactions in clavulanic acid biosynthesis. Curr. Opin. Chem. Biol. 6: 583-589 https://doi.org/10.1016/S1367-5931(02)00392-7
  39. Townsend, C. A. and M. F. Ho. 1985. Biosynthesis of clavulanic acid: Origin of the C3 unit. J. Am. Chem. Soc. 107: 1066-1068 https://doi.org/10.1021/ja00290a057
  40. Zhang, A. Z., J. S. Renb, K. Harlosb, C. H. McKinnona, I. J. Cliftona, and C. J. Schofielda. 2002. Crystal structure of a clavaminate synthase-Fe (II)-2-oxoglutarate-substrate-NO complex: Evidence for metal centered rearrangements. FEBS Lett. 517: 7-12 https://doi.org/10.1016/S0014-5793(02)02520-6
  41. Zhao, X. Q., K. R. Kim, L. W. Sang, S. H. Kang, Y. Y. Yang, and J. W. Suh. 2005. Genetic organization of a 50-kb gene cluster isolated from Streptomyces kanamyceticus for kanamycin biosynthesis and characterization of kanamycin acetyltransferase. J. Microbiol. Biotechnol. 15: 346-353