Aflatoxin biosynthesis: detection of transient, acetate-dependent intermediates in Aspergillus by kinetic pulse-labeling.

A simple technique was developed for the detection of intermediary metabolites of Aspergillus versicolor that are putative precursors of aflatoxin. Minicolony populations were allowed to metabolize [1,2-14C]acetate over various time intervals. The biosynthetic reactions were quenched by quick-freezing the minicolonies, the cells were disrupted, and the metabolites were extracted into acetone. Small silica thin-layer chromatographic plates were then used to separate any radioactive metabolites present. Elution in two or three different directions was often necessary. Radioautography of the thin-layer chromatography plates provided a sensitive assay for the appearance of the various intermediates in a timing pattern which implicated the sequence of formation. Transient intermediates were distinguished from dead-end metabolites by the rapid formation and disappearance of the former. At least five unknown precursors of versicolorin A, a dead-end metabolite, were recognized. The kinetic pulse-labeling technique should be generally applicable to other fungal species whenever the entrapment of intermediary metabolites in the mycelium poses and technical problem.