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

项目总结/摘要 淡水蓝藻水华的发生频率和影响正在增加 由于自然和人为的洪水事件。在美国，所有50个州现在都经常经历 主要的蓝藻赤潮事件，包括近年来宣布进入紧急状态的佛罗里达和俄亥俄州 解决公共水道中增加的蓝藻毒素负荷对人类健康、环境和经济造成的影响， 威胁虽然有害的蓝藻及其毒素受到积极监测， 没有监测蓝藻毒素，即有机磷酸类毒素-a。这种水溶性，紫外线不敏感， 两性离子毒素是众所周知的反应性，这导致了其环境中的持续挑战。 监测.我们最近发现了编码类毒素-a（s）生物合成的基因， 蓝细菌Sphaerospermopsis torques-reginae ITEP-024。通过一系列的基因组，化学， 通过生物化学实验，我们几乎重建了从精氨酸到 类毒素-A与重组酶。此外，我们鉴定了几乎完整的类毒素-A（S）转录本， 来自俄亥俄州托莱多的伊利湖近岸西盆地，这表明托莱多的居民以及 美国其他社区可能在不知情的情况下接触类毒素A， 区域和国家监测机构。我们的发现为这项为期2年的R21应用奠定了基础， 应用我们的生物合成专业知识，填补有关流行性，意义和影响的知识的差距， 这种关键的毒素。我们提出了四个具体目标，以实现我们的广泛目标。首先，我们计划 以完成所有类毒素-A生物合成酶的功能分配。第二，我们将广泛 分析类毒素-a（s）基因的宏基因组和宏转录组淡水供应，并确定 产生蓝细菌。第三，我们的目标是开发一种快速的PCR筛选环境类毒素- a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.