The platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products continues to inspire the discovery of new chemistry, enzymology, and medicine. Engineered production of this emerging family of natural products however remains laborious due to the lack of practical systems to manipulate their biosynthesis in the native producing Streptomyces platensis species. Here we report solving this technology gap by implementing a CRISPR-Cas9 system in Streptomyces platensis CB00739 to develop an expedient method to manipulate the PTM, PTN, and PTL biosynthetic machinery in vivo. We showcase the utility of this technology by constructing designer recombinant strains S. platensis SB12051, SB12052, and SB12053, which, upon fermentation in the optimized PTM-MS medium, produced PTM, PTN, and PTL with the highest titers at 836 mg L−1, 791 mg L−1, and 40 mg L−1, respectively. Comparative analysis of these resultant recombinant strains also revealed distinct chemistries, catalyzed by PtmT1 and PtmT3, two diterpene synthases that Nature has evolved for PTM, PTN, and PTL biosynthesis. The ΔptmR1/ΔptmT1/ΔptmT3 triple mutant strain S. platensis SB12054 could be envisaged as a platform strain to engineer diterpenoid biosynthesis by introducing varying ent-copalyl diphosphate-acting diterpene synthases, taking advantage of its clean metabolite background, ability to support diterpene biosynthesis in high titers, and the promiscuous tailoring biosynthetic machinery.

中文翻译：

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通过生物合成途径工程和发酵培养基优化开发platensimycin、platencin和platensilin过量生产者

Platensimycin (PTM)、platencin (PTN) 和 Platensilin (PTL) 天然产物家族不断激发新化学、酶学和医学的发现。然而，由于缺乏实用的系统来操纵天然产物在本地生产的钝顶链霉菌中的生物合成，这种新兴天然产物家族的工程化生产仍然很费力。在这里，我们报告通过在钝顶链霉菌 CB00739 中实施 CRISPR-Cas9 系统来解决这一技术差距，开发一种在体内操纵 PTM、PTN 和 PTL 生物合成机制的便捷方法。我们通过构建设计重组菌株 S.platensis SB12051、SB12052 和 SB12053 展示了这项技术的实用性，这些菌株在优化的 PTM-MS 培养基中发酵后，产生了最高滴度为 836 mg L−1 的 PTM、PTN 和 PTL。分别为 1、791 mg L−1 和 40 mg L−1。对这些重组菌株的比较分析还揭示了由 PtmT1 和 PtmT3 催化的不同化学性质，PtmT1 和 PtmT3 是 Nature 为 PTM、PTN 和 PTL 生物合成而进化的两种二萜合酶。ΔptmR1/ΔptmT1/ΔptmT3 三重突变菌株 S.platensis SB12054 可以被设想为一个平台菌株，通过引入不同的对柯巴基二磷酸作用的二萜合酶来工程二萜生物合成，利用其干净的代谢物背景，支持二萜生物合成的能力高滴度，以及混杂的剪裁生物合成机器。