Ecumicins are potent antituberculosis natural compounds produced by the rare actinomycete Nonomuraea sp. MJM5123. Here, we report an efficient genetic manipulation platform of this rare actinomycete. CRISPR/Cas9-based genome editing was achieved based on successful sporulation. Two genes in the ecumicin gene cluster were further investigated, ecuN and ecuE, which potentially encode a pretailoring cytochrome P450 hydroxylase and the core peptide synthase, respectively. Deletion of ecuN led to an enhanced ratio of the ecumicin compound EcuH16 relative to that of EcuH14, indicating that EcuN is indeed a P450 hydroxylase, and there is catalyzed hydroxylation at the C-3 position in unit₁₂ phenylalanine to transform EcuH16 to the compound EcuH14. Furthermore, promoter engineering of ecuE by employing the strong promoter kasO*P was performed and optimized. We found that integrating the endogenous ribosome-binding site (RBS) of ecuE together with the RBS from kasO*P led to improved ecumicin production and resulted in a remarkably high EcuH16/EcuH14 ratio. Importantly, production of the more active component EcuH16 was considerably increased in the double RBSs engineered strain EPR1 compared to that in the wild-type strain, reaching 310 mg/L. At the same time, this production level was 2.3 times higher than that of the control strain EPA1 with only one RBS from kasO*P. To the best of our knowledge, this is the first report of genome editing and promoter engineering on the rare actinomycete Nonomuraea.

中文翻译：

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稀有放线菌 Nonomuraea sp. 增强具有较高抗结核活性的活性丁黄素成分的产量。MJM5123 使用新型启动子工程策略。

Ecumicins 是一种有效的抗结核天然化合物，由罕见的放线菌Nonomuraea sp.产生。MJM5123。在这里，我们报告了这种稀有放线菌的高效遗传操作平台。基于成功的孢子形成实现了基于 CRISPR/Cas9 的基因组编辑。进一步研究了 ecumicin 基因簇中的两个基因，ecuN和ecuE，它们可能分别编码预定制的细胞色素 P450 羟化酶和核心肽合酶。ecuN 的缺失导致 emumicin 化合物 EcuH16 相对于 EcuH14 的比例增加，表明 EcuN 确实是一种 P450 羟化酶，并且在单元₁₂的 C-3 位置有催化的羟基化苯丙氨酸将 EcuH16 转化为化合物 EcuH14。此外，通过使用强启动子kasO*P对ecuE 进行了启动子工程并进行了优化。我们发现，将ecuE的内源性核糖体结合位点 (RBS)与来自kasO*P的 RBS整合在一起，可提高 ecumicin 的产量，并导致 EcuH16/EcuH14 的比率非常高。重要的是，与野生型菌株相比，双 RBS 工程菌株 EPR1 中活性更强的成分 EcuH16 的产量显着增加，达到 310 mg/L。同时，该生产水平是仅含有一种来自kasO*P 的RBS 的对照菌株 EPA1 的 2.3 倍. 据我们所知，这是关于罕见放线菌Nonomuraea基因组编辑和启动子工程的第一份报告。