MIRA: Enzymology and Self-Resistance of Natural Product Biosynthesis

摘要 微生物基因组测序和合成生物学的最新进展创造了 天然产物发现的复兴。这种及时的组合为找到 具有新结构和生物活性的天然产物。尽管有这些 潜在的，仍然难以i）直接从生物合成基因预测产物结构 集群（BGC）。这是因为我们对参与天然产物的酶的了解 生物合成仍然是有限的，特别是关于高度编程的酶， 真核生物，如丝状真菌; ii）优先考虑可以导致新的 天然化学结构这主要是由于该领域的重点是充分研究的自然 产品家族和核心生物合成酶;以及iii）将生物活性与 BGC在基因组挖掘工作中的作用。这代表了基因组挖掘和传统 基于表型筛选的发现，其中天然产物分离由生物学指导 活动这项MIRA补助金将通过全面的研究计划解决这些限制 重点是真菌天然产物的发现和生物合成研究。 这个MIRA项目的第一个一般领域是获得核心的基本理解 参与真菌天然产物生物合成的酶。特别是要 重点是理解真菌PKS和NRPS的迭代编程规则。其他 核心酶编程规则的方面，包括环化和非规范结构域， 追究我们还将研究真菌中不寻常的剪裁酶活性， 生物合成途径，重点是PLP依赖性和氧化酶。一些 将进行化合物驱动的生物合成研究。第二个方面是 MIRA项目旨在开发和完善基因组挖掘工具。最重要的研究 在这一领域的活动是基于我们最近开发的抗性基因指导的靶基因组 挖掘，其中我们使用了一个共同的集群，天然产品目标的抗性变体， BGC作为发现具有所需生物活性的天然产物的指南。这一战略还可以 用于指定已知天然产物的生物活性。目标有两个- 1）将可通过抗性基因鉴定的靶标列表扩展至酶， 蛋白质的中心法则，蛋白质运输，代谢等，在这里，我们将执行基因组 挖掘和/或天然产物生物活性表征，以将代谢物与靶标联系起来;以及2） 了解抵抗的机制，这将告诉我们自然是如何进化抵抗的 酶，并完善我们对如何克服潜在抗性的理解。

ABSTRACT Recent progresses in microbial genome sequencing and synthetic biology have created a renaissance in natural product discovery. This timely combination offers great promise to find natural products displaying new structures and biological activities. Notwithstanding such potential, it remains difficult to i) predict product structures directly from biosynthetic gene clusters (BGCs). This is because our knowledge of enzymes that are involved in natural product biosynthesis remains limited, especially with regard to the highly programmed enzymes from eukaryotic organisms such as filamentous fungi; ii) prioritize BGCs that can lead to new-to- nature chemical structures. This is primarily due to the focus of the field on well-studied natural product families and core biosynthetic enzymes; and iii) connect the biological activity with BGCs in genome mining efforts. This represents a gap between genome mining and traditional phenotypical screen-based discovery in which natural product isolation is guided by biological activity. This MIRA grant will address these limitations with a comprehensive research program focused on fungal natural product discovery and biosynthetic investigation. The first general area of this MIRA project is to gain fundamental understanding of core enzymes that participate in the biosynthesis of fungal natural products. In particular, we will focus on understanding the iterative programming rules of fungal PKSs and NRPSs. Other aspects of core enzyme programming rules, including cyclization and noncanonical domains will be investigated. We will also investigate the unusual tailoring enzyme activities of fungal biosynthetic pathways, with emphasis on PLP-dependent and oxidative enzymes. A number of compound driven biosynthetic investigations will be conducted. The second general area of this MIRA project is to develop and refine tools for genome mining. The most important research activity in this area is based on our recently developed resistance gene guided target genome mining, in which we use an co-clustered, resistant variant of the natural product target in the BGC as a guide to discover natural product of desired biological activity. This strategy can also be used to assign biological activities to known natural products. The objectives here are two- fold: 1) to expand the list of targets that may be identified via resistance gene, to enzymes and proteins in the central dogma, protein transport, metabolism, etc. Here we will perform genome mining and/or natural product bioactivity characterization to link metabolites to targets; and 2) to understand the mechanism of resistance, which will teach us how Nature evolves resistant enzymes, and refine our understanding of how to overcome potential resistance.