Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach
Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach
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DOI：
10.1021/acssynbio.8b00361
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发表时间：
2019-01-01
影响因子：
4.7
通讯作者：
Brady, Sean F.
中科院分区：
文献类型：
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作者：
Kim, Seong-Hwan;Lu, Wanli;Brady, Sean F.
在细菌基因组和宏基因组测序工作中鉴定的大多数天然产物生物合成基因簇在实验室生长条件下是沉默的。在这里，我们描述了一种可扩展的生物合成基因簇激活方法，其中基因簇在体外使用CRISPR/Cas9在互操作区域被分解，然后在酵母中使用转化辅助重组（TAR）用PCR扩增的携带合成启动子的短DNA重新组装。这种简单，成本效益高，可扩展的方法允许同时生成重构基因簇的组合文库，消除了理解沉默基因的转录层次结构的需要。在两个测试案例中，使用这种体外分解-TAR重组方法来创建启动子替换基因簇的集合，并行测试这些基因簇以鉴定能够产生次级代谢产物的版本。atolypene（ato）基因簇的激活导致了两种前所未有的细菌环状二萜类化合物atolypene A（1）和B（2）的特征，这两种化合物对人类癌细胞系具有中度细胞毒性。这种简化的体外分解-体内重组方法为沉默基因簇重构提供了一种简化的方法，该方法应有助于从从宏基因组或培养的细菌克隆的沉默基因簇中发现天然产物。
Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene (ato) gene cluster led to the characterization of two unprecedented bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly-in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.