Isolation of N-Iauroyl Tyrosine Antibiotic in E. coli Carrying N-acyl Amino Acid Synthase Gene from Environmental DNA in Korean Soils

한국 토양 환경유래의 N-acyl amino acid synthase 유전자에 의한 대장균 내 항생제 N-lauroyl tyrosine 생산

  • Yeo, Yun-Soo (Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Lim, Yoon-Ho (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Jeong-Bong (Molecular Physiology Division, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Yang, Jung-Mo (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Lee, Chang-Muk (Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Kim, Soo-Jin (Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Park, Min-Seon (Department of Biochemistry and Molecular Biology, Ajou University School of Medicine) ;
  • Koo, Bon-Sung (Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Yoon, Sang-Hong (Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration)
  • 여윤수 (농업생명공학연구원 미생물유전과) ;
  • 임융호 (건국대학교 생물과학공학과) ;
  • 김정봉 (농업생명공학연구원 분자생리과) ;
  • 양정모 (건국대학교 생물과학공학과) ;
  • 이창묵 (농업생명공학연구원 미생물유전과) ;
  • 김수진 (농업생명공학연구원 미생물유전과) ;
  • 박민선 (아주대학교 생화학교실) ;
  • 구본성 (농업생명공학연구원 미생물유전과) ;
  • 윤상홍 (농업생명공학연구원 미생물유전과)
  • Published : 2007.12.31

Abstract

To access the natural product antibiotics produced by uncultured microorganisms, six cosmid libraries of DNA extracted directly from soil samples (environmental DNA, eDNA) were constructed and screened for the production of antibacterial active molecules. Of the approximately 60,000 clones screened, one antibacterial clone (YS92B) was detected. Ethyl acetate extracts of clone YS92B showed antibacterial activity against various pathogenic bacteria (Listeria monocytogenes, Bacillus subtilis, Pseudomonas syringae, Xanthomonas campestris pv. oryzae, Staphylococcus epidemis). Active constituents from cultures of YS92B were isolated and purified using a bioassay-guided fractionation against B. subtilis through a series of procedures (ethyl acetate extraction, Sephadex LH20 column chromatography, High Performance Liquid Chromatography). NMR (Nuclear Magnetic Resonance) spectral analysis of a major antibacterial active YS92B-VII indicated that it is a lauric acid linked to tyrosine. This report describes the characterization of antibacterially active long chain N-acyl derivatives of tyrosine that are produced by eDNA clones hosted in Escherichia coli from Korean soils.

References

  1. Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59, 143-169
  2. Hugenholtz, P., Goebel, B. M., and Pace, N. R. (1998) Impact of culture independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765-4774
  3. Gillespie, D. E., Brady, S. F., Bettermann, A. D., Cianciotto, N. P., Liles, M. R., Rondon, M. R., Clardy, J., Goodman, R. M., and Handelsman, J. (2002) Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Appl. Environ. Microbiol. 68, 4301-4306 https://doi.org/10.1128/AEM.68.9.4301-4306.2002
  4. Pace, N. R. (1997) A molecular view of microbial diversity and the biosphere. Science 276, 734-740 https://doi.org/10.1126/science.276.5313.734
  5. Ellis, R. J., Morgan, P., Weightman, A. J., and Fry, J. C. (2003) Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil. Appl. Environ. Microbiol. 69, 3223-3230 https://doi.org/10.1128/AEM.69.6.3223-3230.2003
  6. Wolfgang, R. S., Daniel, R., and Jaeger, K. E. (2004) Prospecting for biocatalysts and drugs in the genomes of noncultured microorganism. Curr Opin Biotechnol. 15, 285-290 https://doi.org/10.1016/j.copbio.2004.05.006
  7. Entcheva, P., Liebl, W., Johann, A., Hartsch, T., and Streit, W. R. (2001) Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl. Environ. Microbiol. 67, 89-99 https://doi.org/10.1128/AEM.67.1.89-99.2001
  8. Torsvik, V., Goksoyr, J., and Daae., F. L. (1990) High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782-787
  9. Schloss, P.D., and Handelsman, J. (2003) Biotechnological prospects from metagenomics. Curr. Opin. Biotechnol. 14, 303-310 https://doi.org/10.1016/S0958-1669(03)00067-3
  10. Rondon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., and Goodman, R. M. (2000) Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541-2547 https://doi.org/10.1128/AEM.66.6.2541-2547.2000
  11. Brady, S. F., Chao, C. J., and Clardy, J. (2004) Long-chain Nacyltyrosine synthases from environmental DNA. Appl. Environ. Microbiol. 70, 6865-6870 https://doi.org/10.1128/AEM.70.11.6865-6870.2004
  12. Yun, S. S., Lee, J. H., Kim, S. J., Kim, S. S., Park, I. C., Lee, M. H., Koo, B. S., Yoon, S. H., and Yeo, Y. S. (2005) Screening and isolation of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from a metagenomic library of soil DNA. J. Korean Soc. Appl. Biol. Chem. 48, 345-351
  13. Brady, S., and Clardy. J. (2000) Long-chain N-acetyl amino acid antibiotics isolated from heterologously expressed environmental DNA. J. Am. Chem. Soc. 122, 12903-12904 https://doi.org/10.1021/ja002990u
  14. Bissett, D. L., and Robinson, L. R. (2005) Regulation of mammalian keratinous tissue using N-acyl amino acid compositions. US Patent No. 0019356