KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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NDP-sugar production and glycosylation of ${\varepsilon}$-rhodomycinone in Streptomyces venezuelae

Streptomyces Peucetius에서의 ${\varepsilon}$-rhodomycinone 추출 및 이종균주에서의 rhodomycin D 생산 연구

  • Park, Sung-Hee (Institute of Molecular Biology and Genetics, Interdisciplinary Program for Bioengineering, Seoul National University) ;
  • Cha, Min-Ho (Institute of Bio Engineering, School of Chemical and Biological Engineering, Seoul National University) ;
  • Kim, Eun-Jung (Institute of Bio Engineering, School of Chemical and Biological Engineering, Seoul National University) ;
  • Yoon, Yeo-Joon (Division of Nano Sciences, Ewha Womans University) ;
  • Sohng, Jae-Kyung (Institute of Biomolecule Reconstruction, SunMoon University) ;
  • Lee, Hee-Chan (Institute of Biomolecule Reconstruction, SunMoon University) ;
  • Liou, Kwang-Kyoung (Institute of Biomolecule Reconstruction, SunMoon University) ;
  • Kim, Byung-Gee (Institute of Molecular Biology and Genetics, Interdisciplinary Program for Bioengineering, Seoul National University)
  • 박성희 (서울대학교 생물공학 협동과정) ;
  • 차민호 (서울대학교 화학생물공학부) ;
  • 김은정 (서울대학교 화학생물공학부) ;
  • 윤여준 (이화여자대학교 나노과학부) ;
  • 송재경 (선문대학교 생체분자재설계연구소) ;
  • 이희찬 (선문대학교 생체분자재설계연구소) ;
  • 류광경 (선문대학교 생체분자재설계연구소) ;
  • 김병기 (서울대학교 생물공학 협동과정)
  • Published : 2008.02.29
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Abstract

Anthracycline antibiotics doxorubicin (DXR) is clinically important cancer therapeutic agent produced by Streptomyces peucetius. DXR result by further metabolism of rhodomycin D (RHOD) and require a deoxy-sugar component for their biological activity. In this study, production of TDP-L-daunosamine and its attachment to ${\varepsilon}$-rhodomycinone (RHO) to generate RHOD has been achieved by bioconversion in Streptomyces venezuelae that bears eleven genes. S. peucetius seven genes (dnmUTJVZQS) were transformed by plasmid and S. venezuelae two genes desIII, IV and two more S. peucetius drrA, B genes were integrated into chromosomal DNA. To generate the feeding substrate RHO, 6L S. peucetius grown on agar plate was harvested, extracted with organic solvent and then purified using preparative HPLC. Recombinant S. venezuelae grown on agar plate containing RHO was harvested and its n-butanol soluble components were extracted. The glycosylated product of aromatic polyketide RHO using heterologous host S. venezuelae presents the minimal information for TDP-L-daunosamine biosynthesis and its attachment onto aglycone. Moreover, the structure of auxiliary protein, DnrQ, was predicted by fold recognition and homology modeling in this study. This is a general approach to further expand of new glycosides of antitumor anthracycline antibiotics.

Streptomyces peucetius가 생산하는 anthracycline 계열의 doxorubicin은 치료목적으로 사용되는 중요한 항암제 중 하나이다. Doxorubicin은 rhodomycin D에서부터 몇 단계의 생합성 과정을 더 거쳐 생산되는데, 생물학적 활성을 갖기 위해서는 deoxy-sugar의 전이가 반드시 일어나야 한다. 본 논문에서는 이종균주인 Streptomyces venezuelae에 11개의 유전자를 형질 전환하여 TDP-L-daunosamine를 생산하고 이것을 ${\varepsilon}$-rhodomycinone에 전이하여 rhodomycin D를 생산하는 연구를 수행하였다. S. peucetius 유래의 7개 유전자 dnmU, T, J, V, Z, Q, S.를 당 합성 및 전이를 위해 plasmid 형태로 전이하였으며, S. venezuelae의 desIII, IV와 doxorubicin 내성 유전자인 drrA, B는 chromosomal DNA에 삽입하였다. Aglycone 기질인 ${\varepsilon}$-rhodomycinone을 확보하기 위하여 6L의 고체 배지에 S. peucetius를 배양하여 유기용매로 추출하고 preparative HPLC로 분리 정제하였다. 결과적으로 이종균주인 S. venezuelae에서 ${\varepsilon}$-rhodomycinone에 당 전이가 일어난 생산물을 확인함으로써 deoxy-sugar의 생합성 및 전이에 필요한 최소한의 유전적 정보를 확인할 수 있었다. 또한, 유사서열 단백질 모델링을 통하여, 최초로 당 전이 반응에 필수적인 도움효소 DnrQ의 구조를 예측하였다.

Keywords

References

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