| Title | Optimized Transformation of Streptomyces sp. ATCC 39366 Producing Leptomycin by Electroporation |
|---|---|
| Author | Yong-Qiang Fan1, Hong-Jian Liu1, Li Yan2, Yu-Shi Luan1, Hai-Meng Zhou2, Jun-Mo Yang3, Shang-Jun Yin4*, and Yu-Long Wang2* |
| Address | 1School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, P. R. China, 2Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, P. R. China, 3Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, Republic of Korea, 4College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, P. R. China |
| Bibliography | Journal of Microbiology, 51(3),318-322, 2013, |
| DOI | 10.1007/s12275-013-2428-y |
| Key Words | Streptomyces sp. ATCC 39366, leptomycin, electrotransformation, E. coli |
| Abstract | Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam- ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×102 CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives. |