Production of anatoxin-a and a novel biosynthetic precursor by the cyanobacterium Aphanizomenon issatschenkoi

Cyanobacterial blooms in New Zealand surface water resources have been surveyed and, in response to strict new standards for drinking water, more intensive monitoring for cyanotoxins has been initiated. Aphanizomenon issatschenkoi was recently identified in a New Zealand lake and was found to produce the potent neurotoxin anatoxin-a (ATX). A strain of Aph. issatschenkoi (CAWBG02) was cultured for ATX production and a novel derivative of ATX was found to account for a high proportion of the toxin content in the Aph. issatschenkoi cells. Spectroscopic data (LC-UV, liquid chromatography with ultraviolet absorption detection; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-HRMS, liquid chromatography with high resolution mass spectrometry) identified this derivative as 11-carboxyl anatoxin-a. Although precursors with a carboxyl group on C11 have been postulated in the biosynthetic pathway for ATX from amino acids and acetate, this is the first identification of a specific intermediate. The production of ATX and the intermediate by Aph. issatschenkoi was studied under different growth conditions. Concentrations of ATX and the intermediate increased in the aerated culture to 170 mu g/L and 330 mu g/L, respectively, at 21 days (18 x 10(9) cells/L). Cell concentrations did not markedly increase during subsequent growth to 37 days. ATX concentrations decreased, and 11-carboxyl ATX concentrations continued to increase during this period. Toxin production by Aph. issatschenkoi cells was maximal at 6 days of growth (0.08-0.09 pg/cell each; 2.3 x 10(8) cells/L). Other ATX analogues and metabolites were not detected in the cultures. Freeze-thawing of cultures resulted in complete conversion of the intermediate to ATX with a half-life of 5 min, and this conversion was inhibited by acidification, heating of the culture to 100 degrees C, or addition of methanol. The implications of the findings for mechanisms of biosynthesis of anatoxins by cyanobacteria and for monitoring of water bodies for cyanotoxins are discussed.