Biosynthesis and Monitoring of the Cyanobacterial Toxin Anatoxin-a(s)

项目摘要/摘要 淡水有害藻类水华的频率和影响正在增加 由于自然和人为的洪灾事件，世界各地都发生了洪灾。在美国，所有50个州现在都经常经历 重大氰化物HAB事件，包括佛罗里达州和俄亥俄州，近年来已宣布进入紧急状态 应对公共水道中不断增加的氰化毒素负荷，威胁到人类健康、环境和经济 威胁。虽然对有害的蓝藻及其毒素进行了积极的监测，但最具神经毒性的 氰基毒素，即有机磷Anatoxin-a(S)，不受监测。这是水溶性的，对紫外线不敏感，而且 两性离子毒素是出了名的活性，这导致了其环境方面的持续挑战 监控。我们最近从浮游生物中发现了编码Anatoxin-a(S)生物合成的基因 球囊蓝藻扭曲菌ITEP-024。通过一系列的基因组、化学和 生化实验，我们几乎重建了从精氨酸到精氨酸的整个生物合成途径 Anatoxin-a(S)与重组酶。此外，我们还鉴定了几乎完整的Anatoxin-a(S)转录本 从俄亥俄州托莱多伊利湖西部盆地的近岸，这表明托莱多的居民以及 美国其他社区可能会在他们不知情的情况下接触Anatoxin-a(S)， 区域和国家监测机构。我们的发现为这项为期2年的R21应用程序奠定了基础 应用我们的生物合成专业知识来填补关于肺炎的流行、意义和影响的知识空白 环境中的这种关键毒素。我们提出四个具体目标来解决我们的广泛目标。首先，我们计划 完成所有Anatoxin-a(S)生物合成酶的功能分配。第二，我们将广泛地 分析淡水供应中Anatoxin-a(S)基因的后基因组和后转录，并鉴定 产生蓝藻。第三，我们的目标是开发一种针对环境Anatoxin的快速、特异的PCR筛查- A(S)基因检测。第四，我们将评估天然磷酸酶ANSH和商业磷酸酶 作为具有潜在生物修复应用的类毒素-a(S)降解酶。

Project Summary / Abstract Freshwater harmful algal blooms of cyanobacteria or cyanoHABs are increasing in frequency and impact worldwide due to natural and anthropogenic flooding events. In the USA, all 50 states now regularly experience major cyanoHAB incidents, including Florida and Ohio that have declared states of emergency in recent years to address increased cyanotoxin loads in public waterways posing human health, environmental, and economic threats. While harmful cyanobacteria and their toxins are actively monitored, one of the most neurotoxic cyanotoxins, the organophosphate anatoxin-a(s), is not monitored. This water-soluble, UV-insensitive, and zwitterionic toxin is notoriously reactive, which has led to the ongoing challenges in its environmental monitoring. We recently discovered the genes encoding anatoxin-a(s) biosynthesis from the planktonic cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through a series of genomic, chemical, and biochemical experiments, we have nearly reconstituted the entire biosynthetic pathway from arginine to anatoxin-a(s) with recombinant enzymes. Moreover, we identified nearly complete anatoxin-a(s) transcripts from the nearshore Western Basin of Lake Erie at Toledo, Ohio, suggesting that residents in Toledo as well as other communities across the USA may experience anatoxin-a(s) exposure without their knowledge and that of regional and national monitoring agencies. Our discovery sets the stage for this 2-year, R21 application to apply our biosynthetic expertise to fill the gap in knowledge about the prevalence, significance, and impact of this critical toxin in the environment. We propose four specific aims to address our broad goals. First, we plan to complete the functional assignment of all anatoxin-a(s) biosynthesis enzymes. Second, we will broadly analyze metagenomic and metatranscriptomic fresh water supplies for anatoxin-a(s) genes and to identify the producing cyanobacteria. Third, we aim to develop a rapid, PCR screen specific for environmental anatoxin- a(s) gene detection. And fourth, we will evaluate the native phosphatase AnsH and commercial phosphatases as anatoxin-a(s) degradation enzymes with potential bioremediation applications.