加纳链霉菌接合转移体系的建立与优化

加纳链霉菌(Streptomyces ghanaensis)是动物饲料添加剂黄霉素的主要生产菌。建立高效稳定的遗传操作系统是对该菌进行分子生物学研究和构建高产黄霉素基因工程菌的基础。以含有基因整合型重组质粒pIJ8630-nsdA的Escherichia coli ET12567/pUZ8002为供体、加纳链霉菌ATCC 14672为受体进行接合转移,通过单因素试验与正交试验对接合转移过程中的关键影响因素进行优化。结果表明,WL50培养基为接合转移最适培养基,当受体加纳链霉菌ATCC 14672孢子在50 ℃条件下热激10 min、37 ℃预培养3 h后,与供体按细胞数比例1∶15混合后涂布于接合转移平板上,30 ℃培养14 h后覆盖50 μg·mL^-1安普霉素和500 μg·mL^-1萘啶酸时,接合频率最高,比优化前提高20.3倍。

Establishment and optimization of Streptomyces ghanaensis conjugation system

Streptomyces ghanaensis is the main producer of the animal feed additive flavomycin. Establishment of a highly efficient genetic manipulation system is foundamental for molecular biology study and construction of efficient flavomycin-producing strains by genetic engineering. With the assistance of the helper plasmid pUZ8002, pIJ8630-nsdA was transferred from the donor strain E.coli ET12567 to the recipient S. ghanaensis ATCC 14672 by intergeneric conjugation. The key factors in the process of conjugational transfer were optimized by single factor experiment and orthogonal test. The results showed that WL50 medium was the optimal medium for conjugational transfer. The conjugation frequency reached the highest as follows: The spores of S. ghanaensis ATCC 14672 were heat-shocked at 50 ℃ for 10 min and pre-cultured at 37 ℃ for 3 h first. Secondly, they were mixed well with the donor in a ratio of 1∶15 and spreaded on the conjugation plates, incubated at 30 ℃ for 14 h. Thirdly, they were covered with 50 μg·mL^-1 apramycin and 500 μg·mL^-1 nalidixic acid. The optimized conjugational frequency was 5.35×10^-6, which was increased by 95.3%.