Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria and .

In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products-namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), -propargyloxy-carbonyl-L-lysine (Prop-Lys), or -propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in and AP biosynthesis in . Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical-analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging.