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: 10254261
• 负责人: Timothy R Fallon
• 金额: $ 6.64万
• 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/10254261
• 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 有害的藻华或称赤潮在全球范围内造成的社会破坏继续增加，其结果是 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 健康问题，尽管饮用水供应受到污染或在其他情况下HAB毒素的生物积累 可食用的贝类。目前，基于DNA的赤潮毒素生物合成基因监测提供了一种可靠和 物种不可知的方法预测有毒的赤潮与无害的藻华的发展，但 许多高度有效的HAB化合物的生物合成基因尚不清楚，阻碍了这种监测 接近所有毒素类别。巨大的梯形结构聚醚毒素代表了这样一个由来已久的 生物合成问题，它们长期以来公认的生物合成来源，即聚酮的来源 天然产物，尚未导致鉴定负责毒素生物合成的致病基因。 任何真核藻类的生产者，例如引起每年一度的有毒“红潮”事件的甲藻 佛罗里达。关于甲藻类梯形聚醚生物合成的不清楚可能是由于它们的 非常难处理的遗传学。有毒的甲藻物种有非常大的基因组在100s 千兆碱基，基因排列成串联重复序列，以及转录调控的缺乏，导致了原因 基因-化学类型链接的确定在实验上是困难的。在这里，我建议一种有毒的替代方案 微藻是一种触生微藻，是一种微小的扁平苔藓，它会产生一种名为Prymnein的梯形聚醚毒素， 和一个有影响力的赤潮生物本身，是解释完整的生物合成的理想模型系统 梯形框架聚醚的路径。 这项建议的目的是鉴定和表征与催眠素有关的基因和酶。 小地早熟禾的生物合成。我建议使用计算基因组学、转录学和 代谢组学方法与实验遗传学和生化方法相结合来阐明 木霉单倍体菌株合成毒性‘Prymnein’梯形聚醚的途径 实验上非常适合的触位体，微小的Prymneum。首先，将克隆一株微小巴氏杆菌的亚株，并 制作了参考数据集并对候选生物合成基因进行了编目。第二，活动导向分级法 将用于使用可跟踪分离的底物来鉴定Prymnein生物合成中的这些酶 从阳春草的培养中分离出来。最后，将使用功能CRISPR/CAS9屏幕来建立与 异源重组和体外生物化学难解基因的Prymnein生物合成。 这项研究将产生一种生产Prymnein的生物合成模型，该模型可用于开发 海洋和淡水生态系统中有毒聚醚的生物合成基因监测方法。