The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.

中文翻译：

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CRISETR：一种用于生物合成基因簇多重重构的高效技术


天然产物生物合成基因簇 （BGC） 的有效重构以激活沉默的 BGC 是发现新的生物活性天然产物的核心挑战。在此，我们开发了一种简单而强大的 CRISETR （CRISPR/Cas9 和 RecET 介导的重构） 技术，结合了簇状调节间隔短回文重复序列 （CRISPR）/Cas9 和 RecET，用于天然产物 BGC 的多重重构。通过这种方法，可以通过 RecET 介导的高效同源重组与 CRISPR/Cas9 系统之间的协同相互作用来重构天然产物 BGC。我们首先对 CRISERTR 的能力进行了概念验证验证，CRISETR 可以在天然产物 BGC 中实现四个启动子位点的同时替换和单个启动子位点的无标记替换。随后，我们将 CRISETR 应用于包含大量直接重复序列的 74 kb 达托霉素 BGC 的启动子工程，以增强达托霉素的异源产生。我们使用组合设计构建了多个重构的达托霉素 BGC，具有转录强度不同的启动子的不同组合，达托霉素的产量在异源宿主天蓝色链霉菌 A3 中提高了 20.4 倍 （2）。一般来说，CRISETR 对基因簇内重复序列的耐受性增强，能够有效重构多样化和复杂的 BGC，这将大大加速微生物中存在的新型生物活性代谢物的发现。