KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Development of Doxorubicin overproducing Streptomyces Strain using Protoplast Regeneration

방선균 원형질체 재생에 의한 독소루비신 고생산성 균주개발

  • 박희섭 (인하대학교 생명화학공학부) ;
  • 박현주 (인하대학교 생명화학공학부) ;
  • 김용훈 (인하대학교 생명화학공학부) ;
  • 임상민 (인하대학교 생명화학공학부) ;
  • 김동일 (인하대학교 생명화학공학부) ;
  • 류욱상 (보령제약 중앙연구소) ;
  • 김상린 (보령제약 중앙연구소) ;
  • 김응수 (인하대학교 생명화학공학부)
  • Published : 2003.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. In order to increase doxurubicin productivity, a semi-industrial doxorubicin-producing Streptomyces strain named BR-Dox was cultured in a R2YE liquid medium containing CaCO$_3$, and then converted to a cell wall-free protoplast using lysozyme treatment method, followed by PEG-mediated cell wall regeneration. Among several protoplast-regenerated Streptomyces BR-Dox strains, two independent isolates named BR-Dox4 and BR-Dox6 were visually selected using thin layer chromatography (TLC) based on the pigment overproducing phenotype. Comparing with Streptomyces BR-Dox parental strain, two protoplast-regenerated strains, BR-Dox4 and BR-Dox6 exhibited 25.2％ and 12.2％ higher doxorubicin productivity analyzed by high pressure liquid chromatography (HPLC), respectively. This result suggests that a protoplast-regeneration of an antibiotics-producing Streptomyces strain should be a promising strain development approach for antibiotics overproduction in Streptomyces species.

독소루비신 고 생산성 산업균주인 BR-Dox의 충분한 양의 세포를 얻어내기 위해 R2YE 배지를 사용하였으며, 방선균의 포자형성이 잘 일어나는 R2YE 배지의 각 성분별로 첨가한 결과, CaCO$_3$를 첨가하였을 때 인위적인 원형질체 유도에 적합한 양의 세포를 얻어낼 수 있었다. 독소루비신 생합성 능력이 향상된 균주를 선별하기 위해 BR-Dox를 배양하여 세포를 인위적으로 원형질체로 유도하여 세포벽을 재생시킨 결과, 특이적으로 독소루비신 색소로 추정되는 붉은 색을 많이 내는 콜로니를 선별하여 정성 및 정량분석을 수행하였다. 선별된 파생균주 BR-Dox4와 BR-Dox6의 경우 TLC 정성분석 및 HPLC 정량분석 결과, BR-Dox에 비해 각각 25.2%, 12.2%의 생산성이 향상되었다. 본 연구결과는 인위적 원형질체 도입과 세포벽 재생을 통한 새로운 개념의 방선균 균주개량 방법을 제시하고, 이를 이용한 독소루비신 고생산성 균주개발의 가능성을 제시하였다.

Keywords

References

  1. Chin. J. Antibiot. v.7 New ways to obtain antibiotics Berdy,J.
  2. Bacteriol. Rev. v.37 Advances in Streptomyces coelicolor genetics Hopwood,D.A.;K.F.Chater;J.E.Dowding;A.Vvian
  3. Bacteriol. Rev. v.40 Comparative aspects of development and differentiation in actinomycetes Kalakoutskii,L.V.;N.S.Agre
  4. J. Bacteriol. v.137 Simultaneous loss of multiple differentiated functions in aerial mycelium-negative isolates of Streptomyces Peggy,A.R.;P.A.Mcann;M.A.Pentella;B.M.pogell
  5. Appl. Microbiol. Brotechnol. v.52 Screening of spontaneous and induced mutants in Streptomyces avermitils enhances avermectin production Aikawa,M.;S.A.R.Lopes-Shikida;M.F.Lemos;J.G.C.Pradella;G.Padilla https://doi.org/10.1007/s002530051560
  6. FEMS Microbiol Lett. v.9 Use of cerulenin in strain improvement of the daunorubicin fementation McGuire,J.C.;G.Glotfelty;R.J.White https://doi.org/10.1111/j.1574-6968.1980.tb05624.x
  7. Chem. Rev. v.97 Manipulation of modular polyketide synthase Katz,L. https://doi.org/10.1021/cr960025+
  8. Chem. Rev. v.97 Harnessing the biosynthetic potential of modular polyketide synthase Khosla,C. https://doi.org/10.1021/cr960027u
  9. Chem. Biol. v.7 Formation of functional heterologous complexes using subunits from the picromycin, erythormycin and oleandomycin polyketide synthases Tang,L.;H.Fu;R.McDaniel https://doi.org/10.1016/S1074-5521(00)00073-9
  10. Proc. Natl. Acad. Sci. v.96 Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel unnatural natural products McDaniel,R.;A.Thamchaipenet;C.Gustafsson;H.Fu;M.Betlach;G.Ashley https://doi.org/10.1073/pnas.96.5.1846
  11. Proc. American Society for Microbiology 1994 Regulation and integration of antibiotic production and morphological diffrentiation in Streptomyces spp, In regulation of bacterial differentiation Champness,W.C.;K.F.Chater;P.Piggot(ed.);C.P.Moran(ed.);P.Youngman(ed.)
  12. J. Biosci. Bioeng. v.89 Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis Lee,J.Y.;Y.S.Hwang;S.S.Kim;E.S.Kim;C.Y.Choi https://doi.org/10.1016/S1389-1723(00)80065-1
  13. Biotechnol. Lett. v.23 Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis Hwang,Y.S.;J.Y.Lee;E.S.Kim;C.Y.Choi https://doi.org/10.1023/A:1010377219368
  14. Mol. Microbiol. v.27 Genetic instability of the Streptomyces chromosome Volff,J.N.;J.Altenbuchner https://doi.org/10.1046/j.1365-2958.1998.00652.x
  15. Appl. Microbiol. Biotechnol. v.52 Screening of spontaneous and induced mutants in Streptomyces avermitilis enhances avermectin production Aikawa,M.;S.A.R.Lopes-Shikida;M.F.Lemos;J.G.C.Pradella;G.Padilla https://doi.org/10.1007/s002530051560
  16. J. Genet. v.80 Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis Vetrivel,K.S.;K.Dharmalingam https://doi.org/10.1007/BF02811416
  17. J. Pharm. Biomed. Anal. v.10 Solid-phase extraction and optimized separation of doxorubicin, epirubicin and their metabolites using reversed-phase high-performance liquid chromatography Nicholls,G.;B.J.Clark;J.E.Brown https://doi.org/10.1016/0731-7085(91)80104-H
  18. Antibiot. Khimioter v.33 Use of a protoplasting method for altering the qualitative and quantitative composition of the anthracycline complex in Streptomyces peucetius var. caesius Krasil'nikova,O.L;L.N.Anisova;N.B.Romanova;IuE. Bartoshevich
  19. J. Microbiol. Biotechnol. v.9 Isolation and characterization of soil Streptomyces involved in 2,4-dichloro-phenol oxidation Kang,M.J.;J.K.Kang;E.S.Kim
  20. J. Bacteriol. v.178 Isolation and characterization of a gene from Streptomyces sp. strain C5 that confers on Streptomyces lividans TK24 the ability to convert daunomycin to doxorubicin Dickens,M.L.;W.R.Strohl
  21. J. Bacteriol. v.179 In Vivo and In Vitro Bioconversion of e-Rhodomycinone Glycoside to Doxorubicin: Functions of Daup, Dauk, and DoxA Dickens,M.L.;N.D.Priesley;W.R.Strohl
  22. Appl. Microbiol. Biotechnol. v.52 Glucose repression of anthracycline formation in Streptomyces peucetius var. caesius Escalante,L.;I.Ramos;I.Imriskova;E.Langley;S.Sanchez https://doi.org/10.1007/s002530051562