Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Development of a Recombinant Streptomyces griseus with sprA and sprB Genes for Proteolytic Enzyme Production

Streptomyces griseus IFO13350 유래 sprA 및 sprB 유전자를 이용한 Pretense 생산균주 개발

  • Hwang Ji-Hwan (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Lee Chang-Kwon (Bio Food and Drug Research Center, Konkuk University) ;
  • Lee Kang-Mu (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Jo Byoung-Kee (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Park Hae-Ryong (Division of Food Science and Biotechnology, Kyungnam University) ;
  • Hwang Yong-Il (Division of Food Science and Biotechnology, Kyungnam University)
  • 황지환 (경남대학교 식품생명공학부) ;
  • 이창권 (건국대학교 바이오 식의학 연구센터) ;
  • 이강무 (경남대학교 식품생명공학부) ;
  • 조병기 (경남대학교 식품생명공학부) ;
  • 박해룡 (경남대학교 식품생명공학부) ;
  • 황용일 (경남대학교 식품생명공학부)
  • Published : 2005.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

방선균 Streptomyces griseus에서 상업적 목적으로 생산되는 protease인 protease는 serine protease, alkaline protease, aminopeptidase및 carboxypeptidase로 구성되어 있는 복합체로서 의 약용 소염제로 널리 사용되어지고 있다. 본 연구에서는 기존에 개발되어 있는 방선균용 integration vector인 pSET152로부터 목적산물의 대량발현을 위해 방선균용 promoter ermE가 cloning된 새로운 integration vector인 pHJ101을 개발하였고, pretense의 생산량 증대에 사용하였다. 새로 개발된 integration vector에 S. griseus protease A를 코드하고 있는 유전자, sprA와 S. griseus pretense B유전자, sprB를 각각 cloning하여 plasmid pHJ201과 pHJ202를 구축하였다. 이들 plasmid들을 S. griseus IFO 13350에 형질전환하여 발현용plasmid가 chromosome에 integration된 재조합 균주 S. gliseus HA와 S. griseus HB를 얻었다. 이들 재조합균주로부터 전체 protease의 생산량을 확인한 결과, 모균주보다 각각 S. griseus HA는 약 5.3 배, S. griseus HB는 약 5 배 정도 생산량이 증대되었다. 이들 결과로부터 특정유전자의 고발현용 integration vector의 제작이 확인되었으며, 전체 protease의 생산량 증대의 가능성이 시사되었다.

Keywords

References

  1. Choi, W.J., J.M. Kim, S.S. Choi, D.K. Kang, and S.K. Hong. 2001. Overproduction of Streptomyces griseus protease a and b induces morphological changes in Streptomyces lividans. J. Microbiol. Biotechnol. 11, 1077-1086
  2. Choi, S.S., W.J. Chi, J.H. Lee, S.S. Kang, B.C. Jeong, and S.K. Hong. 2001. Overexpression of the sprD gene encoding streptomyces griseus protease D stimulates actinorhodin production in Streptomyces lividans. J. Microbiol. 39, 304-313
  3. Hagihara, B., H. Matsubara, M. Nakai, and K. Okunuki. 1957. Crystalline bacterial proteinase. 1. Preparation of crystalline proteinase of Bac. subtilis. J. Biochem. 45, 185-194
  4. Henderson, G., P. Krygsman, C.J. Liu, C.C. Davey, and L.T. Malek. 1987. Characterization and structure of genes for proteases A and B from Streptomyces  griseus. J. Bacteriol. 169, 3778-3784 https://doi.org/10.1128/jb.169.8.3778-3784.1987
  5. Johnson, P., and L.B. Smillie. 1974. The amino acid sequence and predicted structure of Streptomyces griseus protease A. FEBS Letters 47, 1-6 https://doi.org/10.1016/0014-5793(74)80412-6
  6.  Jurasek, L., M.R. Carpenter, L.B. Smillie, A. Gertler, S. Levy, and L.H. ericsson. 1974. Amino acid sequence of Streptomyces griseus protease B, a major component of pronase. Biochem. Biophys. Res. Commun. 61, 1095-1100 https://doi.org/10.1016/S0006-291X(74)80396-7
  7.  Kieser, T., M.J. Bibb, M.J. Buttner, K.F. Chater, and D.A. Hopwood. 2000. Practical Streptomyces Genetics, The John Innes Foundation, Norwich
  8. Koo, B.J., J.M. Kim, S.M. Byun, and S.K. Hong. 1999. Optimal production conditions of Streptomyces griseus Trypsin (SGT) in Streptomyces lividans. J. Biochem. Mol. Biol. 32, 86-91
  9. Marcos, A.T., B. Diez, S. Gutierrez, F.J. Fernandez, J.A. Oguiza, and Martin, J. F. 1995. Three genes hrdB, hrdD and hrdT of Streptomyces griseus IMRU3570, encoding sigma factor-like proteins, are differentially expressed under specific nutritional conditions. Gene 153, 41-48 https://doi.org/10.1016/0378-1119(94)00759-L
  10. Narahashi, Y., K. Shibuya, and M. Yanagita. 1968. Studies on proteolytic enzymes(Pronase) of Streptomyces griseus K-1.II. Separation of exo- and endopeptidases of pronase. J. Biochem. 64, 427-437 https://doi.org/10.1093/oxfordjournals.jbchem.a128914
  11. Nomoto, M., and Y. Narahashi. 1959. A Proteolytic enzyme of Streptomyces griseus.I. purification of a protease of Streptomyces griseus. J. Biochem. 46, 653-667 https://doi.org/10.1093/jb/46.5.653
  12. Park, U., J.W. Suh, and S.K. Hong. 2000. Genetics analysis of absR, a new abs locus of Streptomyces coelicolor. J. Microbiol. Biotechnol. 10, 169-175
  13. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning; A Laboratory Manual, 2nd. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  14. Schmitt-John T., and J.W. Engels. 1992. Promoter constructions for efficient secretion expression in Streptomyces lividas. Appl. Microbiol. Biotechnol. 36, 493-498
  15.  Sidhu, S.S., G.B. Kalmar, L.G. Willis, and T.J. Borgford. 1994. Streptomyces griseus protease C-a novel enzyme of the chymotrypsin superfamily. J. Biol. Chem. 269, 20167-20171
  16.  Trop, M., and Y. Birk. 1970. The specificity of proteases from Streptomyces griseus(pronase). J. Biochem. 116, 19-25 https://doi.org/10.1042/bj1160019
  17.  Ueda K., T. Umeyama, T. Beppu, and S. Horinouchi. 1996. The aerial mycelium-defective phenotype of Streptomyces griseusresulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2). J. Mol. Microbiol. 169, 91-95
  18. Vosbeck, K.D., K.F. Chow, and W.M. Awad, Jr. 1973. The proteolytic enzymes of the K-1 strain of Streptomyces griseusobtained from a commercial preparation(pronase) - Purification and characterization of the aminopeptidases. J. Biol. Chem. 248, 6029-6034
  19.  Vosbeck, K.D., B.D. Greenberg, and W.M. Awad, Jr. 1974. The proteolytic enzymes of the K-1 strain of Streptomyces griseusobtained from a commercial preparation(pronase) - specificity and immobilization of aminopeptidase. J. Biol. Chem. 250, 3981-3987
  20.  Vosbeck, K.D., B.D. Greenberg, M.S. Ochoa, P.L. Whitney,   and W. M. Awad, Jr. 1978. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation(pronase) - Effect of pH, metal ions, and amino acids on aminopeptidease.J. Biol. Chem. 253, 257-260
  21.  Wilkinson, C.J., Z.A. Hughes-Thomas, C.J. Martin (nee Rowe), I. Bohm, T. Mironenko, M. Deacon, M. Wheatcroft, G. Wirtz, J. Staunton, and P.J. Leadlay. F. 2002. Increasing the efficiency of heterologous promoters in actinomycetes. J. Mol. Microbiol. Biotechnol. 4, 416-26
  22. Willy J., J. Schwedock, and R. Losick. 1993. Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor. Genes Dev. 7, 895-903 https://doi.org/10.1101/gad.7.5.895