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.

项目总结/摘要 有害藻华（HAB）造成的社会损害在全球范围内继续增加， 影响，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 尽管饮用水供应受到污染或有害藻华毒素在其他地方的生物积累， 可食用的贝类。基于DNA的赤潮毒素生物合成基因的监测目前提供了一个可靠的， 物种不可知的方法来预测发展的有毒赤潮与无害的藻华，但 一些高影响力的赤潮化合物的生物合成基因是未知的，阻碍了这种监测 所有毒素类别的方法。大的梯形结构聚醚毒素代表了这样一种长期存在的 生物合成的问题，其中它们的长期公认的生物合成来源，即聚酮衍生物 天然产物，还没有导致确定的致病基因负责毒素生物合成， 任何真核藻类生产者，如“赤潮”甲藻，每年造成有毒的“赤潮”事件， 佛罗里达。关于甲藻梯形框架聚醚生物合成缺乏明确性可能是由于它们的 非常棘手的遗传学有毒的甲藻物种有非常大的基因组， 千兆碱基，基因排列在串联重复序列中，缺乏转录调控，使得因果关系 基因-化学型的测定在实验上是困难的。在这里，我建议， 微藻，附着植物Prymnesium parvum，'prymnesin'梯形框架聚醚毒素的生产者， 和一个有影响力的赤潮生物本身，是一个理想的模型系统，以阐明完整的生物合成 梯形框架聚醚的途径。 这项建议旨在确定和表征的基因和酶负责prymnesin 在Prymnesium parvum.我建议使用计算基因组学，转录组学， 代谢组学方法结合实验遗传学和生物化学方法来阐明 有毒的'prymnesin'梯形框架聚醚的生物合成途径， 实验上非常适合的附着植物小扁藻。首先，将克隆P. parvum的亚株， 产生参考数据集并对候选生物合成基因进行编目。第二，活动引导的分馏 将被用于鉴定在prymnesin生物合成中的那些酶， 来自Prymnesium文化。最后，功能性CRISPR/Cas9筛选将用于建立因果关系， prymnesin生物合成的那些基因难以异源重建和体外生物化学。 这项研究将导致prymnesin生产的生物合成模型，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。