Discovery of a Cryptic Antifungal Compound from Streptomyces albus J1074 Using High-Throughput Elicitor Screens.
Discovery of a Cryptic Antifungal Compound from Streptomyces albus J1074 Using High-Throughput Elicitor Screens.
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DOI：
10.1021/jacs.7b02716
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发表时间：
2017-07-12
影响因子：
15
通讯作者：
Seyedsayamdost MR
中科院分区：
文献类型：
--
作者：
Xu F;Nazari B;Moon K;Bushin LB;Seyedsayamdost MR
微生物次生代谢物生产中一个尚未解决的重要问题是在典型的实验室生长条件下没有表达的生物合成基因簇的丰度。这些所谓的沉默或隐蔽的基因簇是新的天然产物的来源，但它们如何被沉默，以及如何被理性地激活是正在进行的研究领域。我们最近设计了一种化学发生高通量筛选方法(HITES)来发现沉默的生物合成基因簇的小分子激发子。这种方法已经成功地应用于革兰氏阴性细菌，但尚未应用于高产抗生素的链霉菌。在这里，我们开发了一种在链霉菌中高通量转录分析的格式。通过利用插入在中性位置和目标生物合成簇内部的绿色荧光蛋白，作为次生代谢物合成的读出。利用这种方法，我们成功地利用Hites激活了白色链霉菌J1074中的一个沉默基因簇。我们的结果显示细胞毒素依托泊苷和伊维菌素是有效的诱导剂，使我们能够分离和表征所选簇的14个新的小分子产物。其中一个分子是一种新的抗真菌分子，其他几个分子则抑制与癌症有关的半胱氨酸蛋白酶。通过研究这两个激发子的诱导机制，鉴定出了一种在标准生长条件下沉默基因簇的途径特异性转录抑制因子。HITES的成功应用将使人们能够进一步研究链霉菌中无数沉默基因簇的生物调节和化学输出。
An important unresolved issue in microbial secondary metabolite production is the abundance of biosynthetic gene clusters that are not expressed under typical laboratory growth conditions. These so-called silent or cryptic gene clusters are sources of new natural products, but how they are silenced, and how they may be rationally activated are areas of ongoing investigation. We recently devised a chemogenetic high-throughput screening approach (“HiTES”) to discover small molecule elicitors of silent biosynthetic gene clusters. This method was successfully applied to a Gram-negative bacterium; it has yet to be implemented in the prolific antibiotic-producing streptomycetes. Herein we have developed a high-throughput transcriptional assay format in Streptomyces spp. by leveraging eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, we successfully used HiTES to activate a silent gene cluster in Streptomyces albus J1074. Our results revealed the cytotoxins etoposide and ivermectin as potent inducers, allowing us to isolate and structurally characterize 14 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under standard growth conditions. The successful application of HiTES will allow future interrogations of the biological regulation and chemical output of the countless silent gene clusters in Streptomyces spp.
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影响因子：
5.7
作者：
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通讯作者：
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