关键词: 黄霉素, 遗传操作, 接合转移

Abstract: 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%.

Key words: flavomycin, genetic manipulation, conjugation