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目的构建具有双拷贝tylF基因的泰乐菌素基因工程菌,以解决泰乐菌素基因生物合成的限速环节,提高泰乐菌素的发酵产量。方法通过SOE-PCR获得PermE-tylF基因,构建随机整合型重组质粒pBH05,通过接合转移将PermE-tylF基因及其载体随机整合到弗氏链霉菌的基因组中,并通过抗性筛选获得重组菌株D-120。将重组菌株D-120进行摇瓶发酵,采用生物效价测定的二剂量法测定泰乐菌素的发酵单位。结果在相同的发酵条件下,重组菌株泰乐菌素的发酵单位较出发菌株提高32.7%。结论该基因工程菌的构建改善了泰乐菌素生物合成的限速环节,大幅度提高了泰乐菌素的发酵效价。
Objective To construct a tylosin genetically engineered bacteria with double copies of tylF gene to solve the restriction of tylosin gene biosynthesis and improve the fermentation yield of tylosin. Methods PermE-tylF gene was obtained by SOE-PCR. The recombinant plasmid pBH05 was constructed by random integration. The PermE-tylF gene and its vector were randomly integrated into the genome of Streptomyces fradia by conjugation and transfer and the recombinant strain D -120. The recombinant strain D-120 was subjected to shake flask fermentation and the tylosin fermentation unit was measured by a two-dose bioassay. Results Under the same fermentation conditions, the fermentation unit of recombinant strain tylosin increased by 32.7% compared with the original strain. Conclusion The construction of the genetically engineered bacteria improved the rate-limiting step of tylosin biosynthesis and greatly enhanced the fermentation titer of tylosin.