State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Sci China Life Sci. 2017 Sep;60(9):968-979. doi: 10.1007/s11427-017-9119-1. Epub 2017 Aug 21.
Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.
大片段 DNA（基因簇）的遗传修饰在合成生物学和组合生物合成中非常重要，因为它可以促进对天然产物进行合理的设计和修饰，以提高其价值和生产力。在这项研究中，我们开发了一种利用 RecET/RED 介导的聚合酶链反应（PCR）靶向结合 Gibson 组装的无痕、精确修饰大片段基因簇的方法。在该策略中，我们用来自多氧霉素生物合成基因簇的碳氨多聚氧乙酸（CPOAA）取代 nikkomycin 生物合成基因簇中肽段（HPHT）的生物合成基因，在大肠杆菌中生成一个约 40kb 的可重复使用的靶向盒的杂种基因簇。将构建好的基因簇导入 Streptomyces lividans TK23 中进行异源表达，得到并验证了预期的杂种抗生素 polynik A。本研究提供了一种用于基因簇重建和修饰的有效策略，可应用于合成生物学和组合生物合成，以合成新型生物活性代谢物或提高抗生素的产量。
