title: An efficient intergeneric conjugation of DNA from Escherichia coli to mycelia of the lincomycin-producer Streptomyces lincolnensis.
abstract: Streptomyces lincolnensis is a producer of lincomycin, which is a lincosamide antibiotic for the treatment of infective diseases caused by Gram-positive bacteria. S. lincolnensis is refractory to introducing plasmid DNA into cells because of resistance of foreign DNAs and poor sporulation. In this study, a simple and efficient method of transferring plasmids into S. lincolnensis through the intergeneric Escherichia coli-mycelia conjugation was established and optimized for the first time. The recipient mycelia of S. lincolnensis were prepared in liquid SM medium containing 10.3% sucrose for three days. The dispersed mycelia were conjugated with competent E. coli donor cells. The exconjugants were regenerated efficiently on solid mannitol soya flour (MS) medium containing 20 mM MgCl(2). The average conjugation frequency was observed at 1.1 × 10(-4) per input donor cell and validated functionally by transferring two types of vectors containing lincomycin resistance genes lmrA, lmrB and lmrC into S. lincolnensis mycelia. The data of fermentation in shaking flasks showed the lincomycin yield of the exconjugants increased by 52.9% for the multiple copy vector and 38.3% for the integrative one, compared with the parental strain. The efficient and convenient method of intergeneric E. coli-mycelia conjugation in this study provides a promising procedure to introduce plasmid DNA into other refractory streptomycetes.
authors.affiliation: Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Du
authors.affiliation: Anhui Wanbei Pharmaceutical Co., Ltd. No.24, Huaihai Road, Suzhou 234000, China.
authors.forename: Rui-Hua
authors.initials: RH
authors.lastname: Liu
authors.affiliation: Anhui Wanbei Pharmaceutical Co., Ltd. No.24, Huaihai Road, Suzhou 234000, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ying
authors.affiliation: Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, China.
authors.forename: Guang-Rong
authors.initials: GR
authors.lastname: Zhao
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D003227:Conjugation, Genetic / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008034:Lincomycin / Q000096:biosynthesis; D025282:Mycelium / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics*
chemical_list: D018528:ATP-Binding Cassette Transporters; D004269:DNA, Bacterial; D008034:Lincomycin