Abstract:[Background] Elucidation of antibiotic biosynthetic pathways can help improve the yields of target compounds and create new compounds with more effectiveness. In-frame deletion of genes is a routine method in the study of natural product biosynthesis. By analyzing the intermediate products accumulated by mutant strains, we can deduce the synthetic pathways of natural products and the functions of involving genes. The biosynthetic gene clusters for natural products are generally more than 20 kb in size, which make the in-frame deletion of each gene time-consuming and labor-intensive. Therefore, it is of great significance to optimize the method for in-frame deletion of genes derived from Streptomyces. [Objective] On the basis of the principle of PCR-targeting, we redesigned a method for gene in-frame deletion in cosmids from Streptomyces gene library and established a technical system for rapid and high-precision in-frame deletion of Streptomyces genes in Escherichia coli. [Methods] We used the ampicillin resistance gene bla as a marker for the screening of PCR-targeting DNA fragments and replaced the in vivo Flp/FRT system by the in vitro Pac I digestion and ligation system to mediate the construction of in-frame deletion vectors.[Results] Using this method, we completed the in-frame deletion of 14 genes in the mildiomycin biosynthetic gene cluster within 6 days. [Conclusion] Compared with the traditional PCR-targeting method, the established method demonstrates improved efficiency of constructing in-frame deletion vectors. The rarity of the Pac I recognition sequence in the Streptomyces genome makes this method universal for constructing in-frame deletion vectors of essential genes in antibiotic biosynthesis gene clusters.
