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.