title: Optimized transformation of Streptomyces sp. ATCC 39366 producing leptomycin by electroporation.
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 8×10(2) 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.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, PR China.
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Fan
authors.forename: Hong-Jian
authors.initials: HJ
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Yan
authors.forename: Yu-Shi
authors.initials: YS
authors.lastname: Luan
authors.forename: Hai-Meng
authors.initials: HM
authors.lastname: Zhou
authors.forename: Jun-Mo
authors.initials: JM
authors.lastname: Yang
authors.forename: Shang-Jun
authors.initials: SJ
authors.lastname: Yin
authors.forename: Yu-Long
authors.initials: YL
authors.lastname: Wang
mesh_terms: D018274:Electroporation / Q000379:methods*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic / Q000235:genetics
chemical_list: D005231:Fatty Acids, Unsaturated