Exploring a New Dimension of Microbial Secondary Metabolism

摘要 细菌天然产物为治疗药物提供了巨大的来源，并推动了创新 在化学生物学和药理学上。在过去的十年里，很明显， 细菌合成天然产物的能力被大大低估了。基因组与转录组研究进展 测序结合生物信息学方法表明，大多数生物合成基因不是 在标准的实验室生长过程中表达，或者充其量是少量表达;因此，它们的产物不 以足够的滴度合成用于检测和结构/功能表征。这些所谓的“沉默” 或“隐藏的”生物合成基因簇的数量超过组成型活性基因簇的5-10倍。所以他们 代表了细菌代谢的一个新维度，解开它将允许获得大量新的 天然产物和微生物生理学和生物化学的更深层次的见解。 我们最近贡献了高通量激发子筛选（HiTES）方法， 神秘代谢物的发现在这种方法中，筛选小分子文库以鉴定诱导剂 沉默基因簇的基因。随着诱导子的确定，隐藏的代谢物和沉默的调节 集群可以研究。HiTES的几个方面仍然未被探索，它们构成了 当前应用：HiTES仅应用于伯克霍尔德氏菌属。和一些放线菌。其他多产 目前尚未有针对性的。此外，HiTES的潜在机制仍有待确定。 低剂量抗生素已多次被确定为最有效的诱导剂，但监管机构 支持这种反应的途径仍有待阐明。在本申请中，我们寻求地址 通过将HiTES扩展到多产和研究不足的细菌属，以发现新的，神秘的 具有所需生物活性的天然产物，并通过阐明低剂量 抗生素引起隐蔽代谢物的生物合成，重点是β-内酰胺抗生素及其刺激性代谢物的生物合成。 对模式生物泰国伯克霍尔德氏菌的影响。总的来说，这些研究将揭示一个 细菌次级代谢的新维度，发掘了驱动表达的新调控回路 沉默的基因簇，并提供新的天然产物作为可能的治疗线索和来源， 生物合成和药理学研究。

ABSTRACT Bacterial natural products have provided an immense source of therapeutic agents and driven innovation in chemistry, biology, and pharmacology. In the past decade, it has become evident that the capacity of bacteria to synthesize natural products was vastly underestimated. Advances in genome and transcriptome sequencing combined with bioinformatic methods have shown that most biosynthetic genes are not expressed or, at best, sparingly expressed during standard laboratory growth; their products are therefore not synthesized at sufficient titers for detection and structural/functional characterization. These so-called ‘silent’ or ‘cryptic’ biosynthetic gene clusters outnumber constitutively active ones by a factor of 5-10. As such, they represent a new dimension of bacterial metabolism, and unlocking it will allow access to a wealth of new natural products and to deeper insights into microbial physiology and biochemistry. We recently contributed the High-Throughput Elicitor Screening (HiTES) methodology toward the discovery of cryptic metabolites. In this approach, small molecule libraries are screened to identify inducers of selected silent gene clusters. With elicitors identified, the cryptic metabolite and the regulation of the silent cluster can be investigated. Several aspects of HiTES remain unexplored and they form the basis for the current application: HiTES has only been applied to Burkholderia spp. and a few actinomycetes. Other prolific genera have not yet been targeted. Moreover, the mechanism underlying HiTES remains to be determined. Low-dose antibiotics have repeatedly been identified as the most effective elicitors, but the regulatory pathways that underpin this response remain to be elucidated. In the current application, we seek address these topics by expanding HiTES to prolific and understudied bacterial genera for the discovery of new, cryptic natural products with desired biological activities, and by elucidating the mechanisms with which low-dose antibiotics elicit cryptic metabolite biosynthesis, focusing on the β-lactam antibiotics and their stimulatory effect on the model organism Burkholderia thailandensis. Collectively, these studies will shed light on an emerging dimension of bacterial secondary metabolism, unearth new regulatory circuits that drive expression of silent gene clusters, and provide novel natural products as possible therapeutic leads and sources of biosynthetic and pharmacological investigations.