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: 10066290
• 负责人: Timothy R Fallon
• 金额: $ 6.49万
• 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 2023-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066290
• 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.

项目摘要/摘要 有害藻类或哈布斯的社会损害在全球范围内继续增加，导致 诸如鱼类在野外和水产养殖中杀死的影响，海洋哺乳动物的死亡，甚至是公众 健康问题尽管饮用水供应的污染或Hab毒素的生物蓄积否则 可食用的贝类。目前，基于DNA的HAB毒素生物合成基因的监测提供了可靠的和 物种敏锐的方法是预测有毒HAB与无害的藻华的发展，但 许多高度影响力的HAB化合物的生物合成基因尚不清楚，以防止这种监测 在所有毒素类中的方法。大梯形框架聚醚毒素代表了一个长期长时间的 生物合成问题，它们长期识别的生物合成来源，即衍生的聚酮化合物 天然产物尚未导致鉴定导致毒素生物合成的因果基因 任何真核藻类生产商，例如在每年导致每年有毒的“红潮”事件的“红潮”鞭毛鞭毛。 佛罗里达。缺乏关于鞭毛晶状体梯形框架聚集生物合成的明确性，这可能是由于它们的 非常棘手的遗传学。有毒的鞭毛藻种在100s中具有非常大的基因组 千兆称，串联重复的基因以及缺乏转录调节，使因果关系 基因化学型的确定在实验上很难。在这里，我提出了一种替代性有毒 微藻，haptophyte prymesium parvum，“ prymnesin”梯子框架聚醚毒素的生产商， 并且本身就是一个有影响力的HAB组织，是阐明完整生物合成的理想模型系统 梯子框架聚醚的途径。 该提案旨在识别和表征负责PRYMNENSIN的基因和酶 parvum的生物合成。我建议使用计算基因组，转录组和 代谢组方法结合了实验性遗传和生化方法，以阐明 来自单倍体菌株的有毒“ prymnesin”梯形框架聚乙烯的生物合成途径 实验合适的haptophyte prymesium parvum。首先，P。parvum的基质将被克隆，并且 产生的参考数据集和候选生物合成基因分类。其次，活动引导分级 将使用可以裂开的底物鉴定prymnesin生物合成中的这些酶 来自prymesium培养物。最后，功能性CRISPR/CAS9屏幕将用于建立因果关系链接 那些针对异源重建和体外生物化学的基因生物合成的生物合成。 这项研究将导致prynesin的生物合成模型，可用于开发 基于生物合成基因的监测方法，用于海洋和淡水生态系统中的有毒聚乙烯。