一种适用于链霉菌异源合成基因簇同框缺失的高效方法

An efficient method for in-frame deletion of heterologous synthetic gene clusters in Streptomyces

摘要:

【背景】对抗生素生物合成途径的阐明有助于提高目标化合物的产量并开发具有更高活性的新化合物。基因的同框缺失是天然产物生物合成研究的常规手段，通过分析突变菌株积累的中间产物，可以帮助推导天然产物的合成途径及相关基因的功能。天然产物生物合成基因簇的大小一般在20 kb以上，对每个基因进行同框缺失耗时耗力，因此，优化链霉菌来源的基因同框缺失的方法有重要的意义。【目的】基于PCR-targeting重新设计了一套在链霉菌柯斯文库质粒上进行基因同框缺失的方法，实现链霉菌基因在大肠杆菌中快速、高效的基因同框缺失的技术体系。【方法】使用氨苄青霉素抗性基因bla作为PCR-targeting DNA片段的筛选标记，同时使用体外的Pac I酶切和酶连系统代替体内的Flp/FRT系统来介导同框缺失的构建。【结果】利用这种方法，在6 d内完成了米多霉素生物合成基因簇中14个基因的同框缺失。【结论】此方法与传统的PCR-targeting方法相比，构建同框缺失载体的效率明显提高；Pac I识别序列在链霉菌基因组上的稀有性使得此方法在构建抗生素生物合成基因簇必需基因的同框缺失载体上具有普适性。

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.