Refactoring Soft Coral Diterpenoid Biosynthesis

重构软珊瑚二萜生物合成

基本信息

批准号：
10389172
负责人：
Vikram Vijay Shende
金额：
$ 6.76万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
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/10389172
关键词：
Anabolism Aquaculture Architecture 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 Fermentation Future Gene Cluster Genes Genome Geranylgeranyl-diphosphate geranylgeranyltransferase Goals Hand Human Hydrocarbons In Vitro K-562 Lead Leukemic Cell Lyase Medicine Membrane Methods Mutagenesis Natural Products Oxidases Oxides Oxidoreductase Pathway interactions Peripheral Pharmacologic Substance Pichia Production Publications Publishing Reaction Reporting Research Series Source Structure Techniques Terpenes Terpenoid Biosynthesis Pathway Therapeutic Ursidae Family Variant Vertebral column Work Yeasts anti-cancer combinatorial coral cytotoxicity design and construction design-build-test enzyme biosynthesis improved in vivo insight lycopene marine natural product microbial model development novel oxidation promoter rapid detection reconstitution research clinical testing scaffold terpene synthase transcriptome

项目摘要

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，没有陆地来源从软珊瑚中分离出的次生代谢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，许多源自珊瑚的二萜具有有效且选择性的生物活性，包括 xenicane、 acalycixeniolide F，通过对人白血病细胞系 K562 (LC50 = 200 ng/mL) 表现出细胞毒性然而，尽管有希望，这些化合物仍可作为先导结构。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通过对已发表的软珊瑚基因组和转录组的生物信息分析，我们的小组已经进行了测试。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。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在最近发表的 Xenia sp. 的染色体水平基因组组装中，我们发现了一套与 xeniaphyllene 合酶共定位的细胞色素 P450，我将探索它。这些细胞色素 P450 的氧化化学寻找前所未有的碳-碳裂解酶。目标 3 将探讨 xenicane 生物合成的重构，因此我将使用 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这项研究将为了解巴斯德毕赤酵母背后的酶学提供宝贵的见解。此外，还开发了具有生物活性的 xenicane 珊瑚二萜的方法。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。