Elucidating the Biosynthesis of a Model Ladder-Frame Polyether Toxin

阐明梯架聚醚毒素模型的生物合成

• 批准号: 10469542
• 负责人: Timothy R Fallon
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10469542
• 关键词: Algal Blooms; Anabolism; Aquaculture; Back; Biochemical; Biochemistry; Biological Models; CRISPR screen; Candidate Disease Gene; Carbon; Cataloging; Catalogs; Cells; Cessation of life; Chemicals; Ciguatoxins; Clone Cells; Complex; Cryopreservation; DNA; Data Set; Development; Diatoms; Dinophyceae; Ecosystem; Environment; Enzymatic Biochemistry; Enzymes; Event; Experimental Genetics; Family; Fishes; Florida; Fractionation; Fresh Water; Gene Proteins; Genes; Genetic; Genome; Genomics; Geographic state; Gymnodinium breve toxin; Haploidy; In Vitro; Knowledge; Link; Logic; Mammals; Methods; Modeling; Monitor; Mutate; Natural Products; Organism; Pathway interactions; Production; Proteins; Public Health; Reporting; Reproducibility; Research; Ribonucleoproteins; Rice; Route; Shellfish; Source; Tandem Repeat Sequences; Testing; Toxin; Transcriptional Regulation; Water Supply; alpha Toxin; base; bioaccumulation; brevetoxin; candidate identification; causal variant; chlorination; contaminated drinking water; glycosylation; glycosyltransferase; harmful algal blooms; in vitro testing; member; metabolomics; mutant; particle; polyketide synthase; prevent; reconstitution; red tide; tool; transcriptomics

Project Summary/Abstract The societal damage from Harmful Algal Blooms, or HABs, continues to increase globally, with resulting impacts such as fish kills in the wild and in aquaculture, death of marine mammals, and even direct public health concerns though contamination of drinking water supplies or bioaccumulation of HAB toxins in otherwise edible shellfish. DNA based monitoring of HAB toxin biosynthetic genes currently provides a reliable and species-agnostic method to predict the development of a toxic HAB versus an innocuous algal bloom, but the biosynthetic genes for a number of highly impactful HAB compounds are unknown, preventing this monitoring approach across all toxin classes. The large ladder-frame polyether toxins represent one such long standing biosynthetic question, where their long recognized biosynthetic source, namely being polyketide derived natural products, has not yet led to the identification of the causal genes responsible for toxin biosynthesis in any eukaryotic algal producer, such as the “red-tide” dinoflagellates that cause annual toxic “red tide” events in Florida. The lack of clarity regarding dinoflagellate ladder-frame polyether biosynthesis is possibly due to their remarkably intractable genetics. Toxic dinoflagellate species have very large genomes in the 100s of gigabases, genes arrayed in tandem repeats, and a lack of transcriptional regulation, making the causal determinations of gene-chemotype links experimentally difficult. Here, I propose that an alternative toxic microalgae, the haptophyte Prymnesium parvum, producer of the ‘prymnesin’ ladder-frame polyether toxins, and an impactful HAB organism in its own right, is an ideal model system to elucidate the full biosynthetic pathway of a ladder-frame polyether. This proposal aims to identify and characterize the genes and enzymes responsible for prymnesin biosynthesis in Prymnesium parvum. I propose to use computational genomic, transcriptomic, and metabolomic approaches combined with experimental genetic and biochemical approaches to elucidate the biosynthetic pathway for the toxic ‘prymnesin’ ladder-frame polyethers from haploid strains of the experimentally well-suited haptophyte Prymnesium parvum. First, a substrain of P. parvum will be cloned and reference datasets produced and candidate biosynthetic genes cataloged. Second, activity guided fractionation will be used to identify those enzymes in prymnesin biosynthesis using substrates that can be tractably isolated from Prymnesium cultures. Lastly, a functional CRISPR/Cas9 screen will be used to establish causal links to prymnesin biosynthesis for those genes intractable to heterologous reconstitution and in vitro biochemistry. The research would result in a biosynthetic model of prymnesin production, which could be used to develop biosynthetic gene-based monitoring approaches for toxic polyethers in marine and freshwater ecosystems.

项目概要/摘要 有害藻华 (HAB) 造成的社会损害在全球范围内持续增加，导致 诸如野生和水产养殖中的鱼类死亡、海洋哺乳动物的死亡，甚至直接公众的影响 饮用水源污染或 HAB 毒素在其他环境中的生物累积引起的健康问题 可食用的贝类。基于 DNA 的 HAB 毒素生物合成基因监测目前提供了可靠且可靠的方法 与物种无关的方法来预测有毒 HAB 与无害藻华的发展，但 许多具有高度影响力的 HAB 化合物的生物合成基因尚不清楚，因此无法进行监测 涵盖所有毒素类别的方法。大梯架聚醚毒素代表了这样一种长期存在的毒素 生物合成问题，其长期以来公认的生物合成来源，即聚酮化合物衍生的 天然产物，尚未鉴定出负责毒素生物合成的致病基因 任何真核藻类生产者，例如引起每年有毒“赤潮”事件的“赤潮”甲藻 佛罗里达。关于甲藻梯架聚醚生物合成缺乏清晰度可能是由于它们 非常棘手的遗传学。有毒的甲藻物种拥有数百个非常大的基因组 千兆碱基、以串联重复排列的基因以及缺乏转录调控，使得因果关系 基因-化学型联系的确定在实验上很困难。在这里，我建议用一种替代的有毒物质 微藻，触生菌 Prymnesium parvum，“prymnesin”梯架聚醚毒素的生产者， 其本身就是一种有影响力的 HAB 生物体，是阐明完整生物合成的理想模型系统 梯架聚醚的路径。 该提案旨在鉴定和表征负责 prymnesin 的基因和酶 Prymnesium parvum 中的生物合成。我建议使用计算基因组学、转录组学和 代谢组学方法结合实验遗传和生化方法来阐明 来自单倍体菌株的有毒“Prymnesin”梯架聚醚的生物合成途径 实验上非常适合的触生菌 Prymnesium parvum。首先，将克隆 P. parvum 亚种并 生成参考数据集并对候选生物合成基因进行编目。二、活动引导分馏 将用于使用可易于分离的底物来鉴定朴门菌素生物合成中的酶 来自Prymnesium 文化。最后，功能性 CRISPR/Cas9 筛选将用于建立因果关系 Prymnesin 生物合成用于那些难以异源重组和体外生物化学的基因。 该研究将建立一个生产普律霉素的生物合成模型，可用于开发 基于生物合成基因的海洋和淡水生态系统中有毒聚醚监测方法。