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.

项目摘要软珊瑚（Alcyonaria）是具有相关性的天然产物（NP）的显着来源生物活性和复杂的脚手架是海洋真核生物所独有的，没有地面底层来源结构相关的化合物。从软珊瑚分离的次级代谢物的组成占主导地位由一套生物活性二萜的潜水员套件，具有超过50个独特的脚手架和> 1500个独特的二萜许多珊瑚衍生的二萜具有潜力和选择性的生物学活性，包括异氨酸， Acalycixeniolide F，通过A A A型针对人白血病细胞系K562（LC50 = 200 ng/ml）显示细胞毒性。不确定的行动方式。但是，尽管有希望，这些化合物作为铅结构新型药物的开发，珊瑚二萜的药物潜力尚未开发为完全合成或与水产养殖的隔离提供了足够的材料以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的生物信息学分析，我们的小组具有最近确定了一系列萜烯合酶，这些合成酶合成多个珊瑚的烃骨架特定的二萜，包括异苯甲酸，是原型珊瑚二萜的前体，Xenicanes。在这里，我建议将Xenicanes的生物合成作为开发可持续平台的模型用于生物活性珊瑚二萜的微生物产生。该建议旨在通过表征和参与异种烷支架合成和氧化的酶的定向演变，最终导致通过甲基营养酵母，Pichia Pastoris的途径工程重构异乙烷生物合成。萜烯合酶通常是由于其低催化效率而导致的萜类生物合成的速率限制步骤和不稳定。在AIM 1中，我将使用高通量，比色的底物竞争测定法进行屏幕错误 - 俯卧的PCR生成的异苯甲酸萜烯合酶变体可改善活性和稳定性。访问所有异种烷共有的核心支架，异脑中融合的环丁烷环必须经过氧化碳碳键裂解。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异苯甲酸合酶共定位的细胞色素P450。在AIM 2中，我将探索这些细胞色素P450的氧化化学因素寻找前所未有的碳 - 碳裂解酶。最后，异乙烷生物合成的重建将在AIM 3中进行探讨，其中我将使用通用循环组装（uloop）有效设计和构建一种重构的生物合成途径，以发酵生产 P. Pastoris中的Xenicanes。这项研究将提供对背后的酶学的宝贵见解生物活性异乙烷珊瑚二萜的结构。此外，从中发展的方法工作将支持重建额外的海洋后生生物合成途径，解决供应进一步对这些NP的生物学评估的问题。