Refactoring Soft Coral Diterpenoid Biosynthesis

重构软珊瑚二萜生物合成

• 批准号: 10553605
• 负责人: Vikram Vijay Shende
• 金额: $ 7.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10553605
• 关键词: Anabolism; Aquaculture; Architecture; Binding; Biochemistry; Bioinformatics; Biological; Biological Assay; Carbon; Cell Line; Characteristics; Chemicals; Chemistry; Cloning; Common Core; Complex; Cyclobutanes; Cytochrome P450; Cytochromes; Data; Development; Directed Molecular Evolution; Diterpenes; Engineering; Enzymatic Biochemistry; Enzymes; Eukaryota; Evaluation; Event; Exhibits; Family; Future; Gene Cluster; Genes; Genetic Transcription; Genome; Geranylgeranyl-diphosphate geranylgeranyltransferase; Goals; Hand; Human; Hydrocarbons; In Vitro; K-562; Lead; Leukemic Cell; Lyase; Medicine; Membrane; Methods; Mutagenesis; Natural Products; Natural Source; Oxidases; Oxidoreductase; Pathway interactions; Peripheral; Pharmacologic Substance; Pichia; Production; Publications; Publishing; Reaction; Reporting; Research; Series; Source; Structure; Techniques; Terpenes; Terpenoid Biosynthesis Pathway; Therapeutic; Variant; Vertebral column; Work; Yeasts; anti-cancer; combinatorial; coral; cytotoxicity; design and construction; design,build,test; enzyme biosynthesis; improved; in vivo; insight; lycopene; marine; marine natural product; microbial; model development; novel; oxidation; promoter; rapid detection; reconstitution; research clinical testing; scaffold; secondary metabolite; terpene synthase; transcriptome

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.

项目概要 软珊瑚 (Alcyonaria) 是具有药用价值的天然产物 (NP) 的重要来源 生物活性和复杂的支架是海洋真核生物所独有的，没有陆地来源 结构相关的化合物。从软珊瑚中分离出的次生代谢产物的组成占主导地位 由多种生物活性二萜组成，具有 50 多种独特的支架和超过 1500 种分离的独特二萜 迄今为止。许多珊瑚衍生的二萜具有有效和选择性的生物活性，包括 xenicane、 acalycixeniolide F，通过对人白血病细胞系 K562 (LC50 = 200 ng/mL) 表现出细胞毒性 未确定的作用方式。然而，尽管这些化合物表现出作为先导结构的希望 随着新药的开发，珊瑚二萜的药物潜力仍未得到开发，因为两者都没有 全合成或从水产养殖中分离都提供了足够的材料来实现有效的体内临床 测试。通过对已发表的软珊瑚基因组和转录组进行生物信息分析，我们小组 最近发现了一系列萜烯合酶，可以合成多种珊瑚的碳氢化合物骨架 特定的二萜，包括 xeniaphyllene，原型珊瑚二萜（xenicanes）的前体。 在这里，我建议使用 xenicanes 的生物合成作为开发可持续平台的模型 用于微生物生产具有生物活性的珊瑚二萜。 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 通过甲基营养酵母毕赤酵母中的途径工程重建xenicane生物合成。 由于催化效率低，萜烯合酶通常是萜类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 - PCR 生成的 xeniaphyllene 萜烯合酶变体可提高活性和稳定性。要访问 作为所有 xenicanes 共有的核心支架，xeniaphylne 中的稠合环丁烷环必须经历氧化反应 碳-碳键断裂。在最近发表的 Xenia sp. 染色体水平基因组组装中， 我们发现了一套与 xeniaphylne 合酶共定位的细胞色素 P450。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中进行探索，其中我将使用Universal Loop Assembly (uLoop) 有效设计和构建用于发酵生产的重构生物合成途径 巴斯德毕赤酵母中的 xenicanes。这项研究将为了解其背后的酶学提供宝贵的见解。 具有生物活性的xenicane珊瑚二萜的构建。此外，由此开发的方法 工作将促进重建额外的海洋后生动物生物合成途径，解决供应问题 这些纳米粒子的进一步生物学评价的问题。