Du Lei, Liu Rui-Hua, Ying Li, Zhao Guang-Rong
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.
Anhui Wanbei Pharmaceutical Co., Ltd. No.24, Huaihai Road, Suzhou 234000, China.
Int J Mol Sci. 2012;13(4):4797-4806. doi: 10.3390/ijms13044797. Epub 2012 Apr 16.
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.
林肯链霉菌是林可霉素的产生菌，林可霉素是一种林可酰胺类抗生素，用于治疗由革兰氏阳性菌引起的感染性疾病。由于对外源DNA的抗性和产孢能力差，林肯链霉菌难以将质粒DNA导入细胞。在本研究中，首次建立并优化了一种通过属间大肠杆菌-菌丝体接合将质粒导入林肯链霉菌的简单高效方法。林肯链霉菌的受体菌丝体在含有10.3%蔗糖的液体SM培养基中制备三天。将分散的菌丝体与感受态大肠杆菌供体细胞进行接合。接合子在含有20 mM MgCl₂的固体甘露醇大豆粉（MS）培养基上高效再生。观察到平均接合频率为每输入一个供体细胞1.1×10⁻⁴，并通过将两种含有林可霉素抗性基因lmrA、lmrB和lmrC的载体导入林肯链霉菌菌丝体进行功能验证。摇瓶发酵数据表明，与亲本菌株相比，多拷贝载体的接合子林可霉素产量提高了52.9%，整合载体的接合子提高了38.3%。本研究中高效便捷的属间大肠杆菌-菌丝体接合方法为将质粒DNA导入其他难转化的链霉菌提供了一种有前景的方法。
