Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Construction and Analysis of a DNA Microarray for the Screening of Biosynthetic Genes of Secondary-Metabolites formation in Streptomyces

방선균 유래 이차대사 생합성 유전자 분석용 DNA Microarray 제작 및 해석

  • Nam Soo Jung (Department of Biological Science, Myongji University) ;
  • Kang Dae-Kyung (Bio-Resources Institute, Easy Bio System Inc.) ;
  • Rhee Ki Hyeong (College of Industrial Science, Kongju National University) ;
  • Kim Jong-Hee (Department of Biological Science, Myongji University) ;
  • Kang Sang Sun (Division of Science Education, Chungbuk National University) ;
  • Chang Yong Keun (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology) ;
  • Hong Soon-Kwang (Department of Biological Science, Myongji University)
  • 남수정 (명지대학교 생명과학과) ;
  • 강대경 (주)이지바이오시스템 중앙연구소) ;
  • 이기형 (공주대학교 산업과학대학) ;
  • 김종희 (명지대학교 생명과학과) ;
  • 강상순 (충북대학교 과학교육과) ;
  • 장용근 (한국과학기술원 생명화학공학과) ;
  • 홍순광 (명지대학교 생명과학과)
  • Published : 2005.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

Streptomyces produces many kinds of secondary-metabolites including antibiotics. Screening of a new compound and elucidation of a biosynthetic pathway for the secondary metabolites are very important fields of biology, however, there is a main problem that most of the identified compounds are already researched compounds. To solve these problems, a microarray system that is based on the data related to the biosynthetic genes for secondary-metabolites was designed. For the main contents of DNA microarray, the important genes for the bio-synthesis of aminoglycosides, polyenes group, enediyne group, alpha-glucosidase inhibitors, glycopeptide group, and orthosomycin group were chosen. A DNA microarray with 69 genes that were involved in the bio-synthesis for the antibiotics mentioned above was prepared. The usability of the DNA microarray was confirmed with the chromosomal DNA and total RNA extracted from S. coelicolor whose genomic sequence had already been reported.

다양한 균주들을 대상으로 무작위로 신물질을 스크리닝하는 방법은 많은 노력과 시간이 소요되는 방법이며, 신물질을 발견하는 비율도 계속 낮아지고 있다. 따라서 기존 균주들을 대상으로 microarray 기술을 이용한 target-directed screening기술의 개발은, 학문적 뿐만 아니라 산업적으로도 중요한 의미를 가진다. 본 연구에서는, 이미 분리된 방선균각각의 유전체를 대상으로 microarray 분석을 통해, 새로운 생리활성 물질 생산균주 및 생합성 유전자를 확보할 수 있는 기법을 개발하기 위한 기초실험을 수행하였다. 즉, 기존에 알려진 생리활성물질 생합성 유전자들을 확보하여 DNA chip을 제조하였으며, 유전체 염기서열이 밝혀진 S. coelicolor 균주를 대상으로 그 효율성을 검증하였다. 전체적으로 유전자 상동성이 높을수록 반응감도도 높은 편이었으나, 이러한 상환관계가 일치하지 않는 유전자들도 있었다. 이와 같은 문제는, probe 유전자의 G+C 비율$(\%)$을 서로 비슷하게 구성하거나, 반응조건을 최적화 시킨다면 DNA chip의 효율성을 더욱 높일 수 있을 것으로 판단된다. DNA microarray를 통한 생리활성물질 발굴 연구는 세계적으로도 보고된 바 없는 새로운 접근방법으로서, 본 연구에서 시도하고 있는 방법은 발굴 target과 대상을 지정하고 시도되기 때물에, 효율면에서 무작위 스크리닝과는 비교되지 않을 정도로 높을것으로 예상된다. 또한 본 연구와 같은 접근방법을 최적화 시킨다면, 방선균뿐만 아니라 다른 미생물부터 생리활성물질 및 생합성유전자 스크리닝에도 효과적으로 응용할 수 있을 것이다.

Keywords

References

  1. Ahlert J., E. Shepard, N. Lomovskaya, E. Zazopoulos, A. Staffa, B.O. Bachmann, K. Huang, L. Fonstein, A. Czisny, R.E. Whitwam, C.M. Farnet, and J.S. Thorson. 2002. The calicheamicin gene cluster and its iterative type I enediyne PKS. Science 297, 1173-1176 https://doi.org/10.1126/science.1072105
  2. August P.R., L. Tang, Y.J. Yoon, S. Ning, R. Muller, T.W. Yu, M. Taylor, D. Hoffmann, C.G. Kim, X. Zhang, C.R. Hutchinson, and H.G. Floss. 1998. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol. 5, 69-79 https://doi.org/10.1016/S1074-5521(98)90141-7
  3.  Barna J.C.J. and D.H. Williams. 1984. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Ann. Rev. Microbiol. 38, 339-357 https://doi.org/10.1146/annurev.mi.38.100184.002011
  4.  Brautaset T., O.N. Sekurova, H. Sletta, T.E. Ellingsen, A.R. Strlm, S. Valla, and S.B. Zotchev. 2000. Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem. Biol. 7. 395-403 https://doi.org/10.1016/S1074-5521(00)00120-4
  5.  Distler J., A. Ebert, K. Mansouri, K. Pissowotzki, M. Stockmann, and W. Piepersberg. 1987. Gene cluster for streptomycin synthesis in Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity. Nucleic Acids Res. 15, 8041-8056 https://doi.org/10.1093/nar/15.19.8041
  6. Grimm A., K. Madduri, A. Ali, and C.R. Hutchinson. 1994. Characterization of the Streptomyces peucetius ATCC 29050 genes encoding doxorubicin polyketide synthase. Gene 151, 1-10 https://doi.org/10.1016/0378-1119(94)90625-4
  7.  Hopwood, D.A., M.J. Bibb, K.F. Chater, T. Kieser, C.J. Bruton, H.M. Kieser, D.J. Lydiate, C.P. Smith, and J.M. Ward. 1985. Genetic manipulation of Streptomyces : a laboratory manual. The John Innes Foundation, Norwich, England
  8.  Huang J,C. J. Lih, K.H. Pan, and S.N. Cohen. 2001. Global analysis of growth phase responsive gene expression and regulation of antibiotic biosynthetic pathways in Streptomyces coelicolor using DNA microarrays. Genes Dev. 15, 3183-3192 https://doi.org/10.1101/gad.943401
  9. Jung Y.G., S.H. Kang, C.G. Hyun, Y.Y. Yang, C.M. Kang, and J. W. Suh. 2003. Isolation and characterization of bluensomycin biosynthetic genes from Streptomyces bluensis. FEMS Microbiol. Lett.219, 285-289 https://doi.org/10.1016/S0378-1097(03)00019-3
  10. Liu W., S. D. Christenson, S. Standage, and B. Shen. 2002. Biosynthesis of the enediyne antitumor antibiotic C-1027. Science 297, 1170-1173 https://doi.org/10.1126/science.1072110
  11. Long B.H., W.C. Rose, D.M. Vyas, J.A. Matson, and S. Forenza. 2002. Discovery of antitumor indolocarbazoles: rebeccamycin, NSC 655649, and fluoroindolocarbazoles. Curr. Med. Chem. Anti-Canc. Agents. 2, 255-266 https://doi.org/10.2174/1568011023354218
  12. Mahmud T., S. Lee, and H.G. Floss. 2001. The biosynthesis of acarbose and validamycin. Chem. Rec. 1, 300-310 https://doi.org/10.1002/tcr.1015
  13. Russell D. W. and J. Sambrook. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U.S.A
  14. Schwecke T, J.F. Aparicio, I. Molnar, A. Konig, L.E. Khaw, S.F. Haydock, M. Oliynyk, P. Caffrey, J. Cortes, and J.B. Lester. 1995. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc. Natl. Acad. Sci. USA. 92, 7839-7843 https://doi.org/10.1073/pnas.92.17.7839
  15. Sekurova O., H. Sletta, T.E. Ellingsen, S. Valla, and S. Zotchev. 1999. Molecular cloning and analysis of a pleiotropic regulatory gene locus from the nystatin producer Streptomyces noursei ATCC11455. FEMS. Microbiol. Lett. 177, 297-304 https://doi.org/10.1111/j.1574-6968.1999.tb13746.x
  16. Steffensky M., A. Muhlenweg, Z.X. Wang, S.M. Li, and L. Heide. 2000. Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob. Agents Chemother. 44, 1214-1222 https://doi.org/10.1128/AAC.44.5.1214-1222.2000
  17. Stratmann A., T. Mahmud, S. Lee, J. Distler, H.G. Floss, and W. Piepersberg. 1999. The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose. J. Biol. Chem. 274, 10889-10896 https://doi.org/10.1074/jbc.274.16.10889
  18. Takano E, H. Kinoshita, V. Mersinias, G. Bucca, G. Hotchkiss, T. Nihira, C.P. Smith, M. Bibb, W. Wohlleben, and K. Chater. 2005. A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor.Mol. Microbiol. 56, 465-479 https://doi.org/10.1111/j.1365-2958.2005.04543.x
  19. van Wageningen A.M., P.N. Kirkpatrick, D.H. Williams, B.R. Harris, J.K. Kershaw, N.J. Lennard, M. Jones, S.J. Jones, and P.J. Solenberg. 1998. Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic. Chem. Biol. 5, 155-162 https://doi.org/10.1016/S1074-5521(98)90060-6
  20. Weitnauer G., A. Muhlenweg, A. Trefzer, D. Hoffmeister, R.D. Sussmuth, G. Jung, K. Welzel, A. Vente, U. Girreser, and A. Bechthold. 2001. Biosynthesis of the orthosomycin antibiotic avilamycinA: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tu57 and production of new antibiotics. Chem. Biol. 8, 569-581 https://doi.org/10.1016/S1074-5521(01)00040-0
  21. Zotchev S., K. Haugan, O. Sekurova, H. Sletta, T.E. Ellingsen, and S. Valla. 2000. Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455. Microbiology 146, 611-619 https://doi.org/10.1099/00221287-146-3-611