Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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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
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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.

토양에는 생존하지만 현 기술로는 배양이 불가능한 미생물로부터 천연 항생제를 탐색하기 위해 한국 토양 DNA 단편들을 가진 cosmid library을 대장균에서 제작하였고 약6만개의 clone들을 대상으로 항세균 활성을 보여주는 YS92B를 최종 선발하였다. YS92B클론 배양액의 ethyl acetate추출액은 다양한 병원성 세균의 성장을 in vitro에서 강력히 저해하였다(Listeria monocytogenes, Bacillus subtilis, Pseudomonas syringae, Xanthomonas campestris pv. oryzae, Staphylococcus epidemis). 이 항균활성의 주 물질인 YS92B-VII는 ethyl acetate추출, Sephadex LH20 column chromatography와 HPLC(High Performance Liquid Chromatography)에 의해 순차적으로 분리하였으며 이 과정에서 주 활성물질은 각 피크를 항균검정으로 추적하여 최종 정제하였다. 이 물질은 NMR(Nuclear Magnetic Resornance)에 의한 구조 분석 결과에서 탄소 12개의 포화지방산인 lauric acid가 tyrosine에 결합된 N-lauroyl tyrosine임을 최종 확인하였다. 따라서 본 보고는 한국 토양에서 유래한 고유의 N-acyl amino acid synthase(NAS)유전자가 대장균에 발현되어 생산되는 N-acyl amino acid tyrosine의 특성을 밝히는 것이다.

Keywords

References

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