Refactoring Soft Coral Diterpenoid Biosynthesis

项目摘要 软珊瑚（Alcyonaria）是具有药学相关性的天然产物（NP）的显著来源。 生物活性和复杂的支架，这是独特的海洋真核生物，没有陆地来源的 结构相关的化合物。从软珊瑚中分离的次级代谢产物的组成占主导地位 通过一系列不同的生物活性二萜，具有超过50种独特的支架和超过1500种独特的分离二萜类化合物， 迄今许多珊瑚衍生的二萜具有有效的和选择性的生物活性，包括xenicane， acalycixeniflavin F，其显示出对人白血病细胞系K562的细胞毒性（LC 50 = 200 ng/mL），通过 不确定的行动方式。然而，尽管这些化合物显示出作为先导结构的前景， 尽管珊瑚二萜的药用潜力尚未开发， 从水产养殖中的全合成或分离提供了足够的材料， 试验.通过对已发表的软珊瑚基因组和转录组的生物信息学分析，我们的研究小组 最近发现了一系列萜烯脱氢酶，它们可以合成多种珊瑚的碳氢化合物骨架， 特殊的二萜，包括xeniaphyllene，原型珊瑚二萜的前体，xenicanes。 在这里，我建议使用生物合成的xenicanes作为模型的发展，一个可持续的平台 用于生物活性珊瑚二萜的微生物生产。 该提案旨在通过表征和优化xenicane二萜的生产， 参与xenicane支架合成和氧化的酶的定向进化，最终导致 甲醇营养型酵母Pichia pastoris中通过途径工程重建xenicane生物合成。 萜烯脱氢酶由于其催化效率低，常成为萜类化合物生物合成的限速步骤 和不稳定。在目标1中，我将使用高通量比色底物竞争测定来筛选错误- 倾向于PCR产生的xeniaphyllene萜烯合酶变体，用于改善活性和稳定性。访问 在所有xeniaphyllene的核心骨架中，xeniaphyllene中的稠合环丁烷环必须经历氧化 碳-碳键断裂在最近发表的花粉直感虫染色体水平的基因组组装中， 我们发现一组细胞色素P450与xeniaphyllene合酶共定位。在目标2中，我将探索 这些细胞色素P450的氧化化学，以寻找前所未有的碳-碳裂解酶。最后， xenicane生物合成的重建将在目标3中探索，其中我将使用通用环组装 （uLoop）来有效地设计和构建用于发酵生产 巴斯德毕赤酵母中的xenicanes。这项研究将提供宝贵的见解背后的酶学 具有生物活性的山生烷珊瑚二萜的构建。此外，由此开发的方法 这项工作将有助于重建更多的海洋后生动物生物合成途径，解决供应问题， 这些NP的进一步生物学评价的问题。

Project Summary Soft corals (Alcyonaria) are a remarkable source of natural products (NPs) with pharmaceutically relevant biological activity, and complex scaffolds that are unique to marine eukaryotes with no terrestrial sources of structurally related compounds. The composition of secondary metabolites isolated from soft corals is dominated by a diverse suite of bioactive diterpenes with over 50 unique scaffolds and >1500 unique diterpenoids isolated to date. Many coral-derived diterpenes have potent and selective biological activity, including the xenicane, acalycixeniolide F, which displays cytotoxicity against human leukemia cell line K562 (LC50 = 200 ng/mL) via an undetermined mode of action. However, despite the promise these compounds exhibit as lead structures for the development of novel medicines, the pharmaceutical potential of coral diterpenes remains untapped as neither total synthesis nor isolation from aquaculture have provided sufficient material to enable effective in vivo clinical testing. Through bioinformatic analysis of published soft coral genomes and transcriptomes, our group has recently identified a series of terpene synthases that synthesize the hydrocarbon backbones of multiple coral- specific diterpenes, including xeniaphyllene, the precursor to the archetypal coral diterpenes, the xenicanes. Here, I propose to use the biosynthesis of the xenicanes as model for the development of a sustainable platform for the microbial production of bioactive coral diterpenes. This proposal aims to optimize the production of xenicane diterpenes through characterization and directed evolution of enzymes involved in the synthesis and oxidation of the xenicane scaffold, culminating in the reconstitution of xenicane biosynthesis through pathway engineering in methylotrophic yeast, Pichia pastoris. Terpene synthases are often the rate-limiting step in terpenoid biosynthesis due to their low catalytic efficiency and instability. In Aim 1, I will use a high-throughput, colorimetric substrate competition assay to screen error- prone PCR-generated xeniaphyllene terpene synthase variants for improved activity and stability. To access the core scaffold common to all xenicanes, the fused cyclobutane ring in xeniaphyllene must undergo an oxidative carbon-carbon bond cleavage. In the recently published chromosomal level genome assembly of a Xenia sp., we found a suite of cytochromes P450 co-localized with the xeniaphyllene synthase. In Aim 2, I will explore the oxidative chemistry of these cytochromes P450 in search of the unprecedented carbon–carbon lyase. Finally, the reconstitution of xenicane biosynthesis will be explored in Aim 3, wherein I will use Universal Loop Assembly (uLoop) to efficiently design and construct a refactored biosynthetic pathway for fermentative production of xenicanes in P. pastoris. This research will provide invaluable insights into the enzymology behind the construction of the biologically active xenicane coral diterpenes. Furthermore, the methods developed from this work will facilitate the reconstitution of additional marine metazoan biosynthetic pathways, resolving supply issues for further biological evaluation of these NPs.