Utilization of Purified Pertinent Fungal Enzymes for Development of Probes to Identify Genes Responsible for Aflatoxin Biosynthesis

Abstract

The observation that O-methylsterigmatocystin (OMST) is a metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin (ST) and aflatoxin B₁ (AFB₁) led to the discovery of two enzyme activities responsible for the late stages of aflatoxin synthesis: 1) a methyltransferase (MT) catalyzing the conversion of ST to OMST, and 2) an oxidoreductase (OR) which converts OMST to AFB₁. The anionic MT (168 KDa; two subunits, 110 KDa and 58 KDa) has been purified to homogeneity and well characterized. The oxidoreductase has been partially purified. Polyclonal antibodies against MT have been prepared and amino acid composition of MT determined. A cDNA library, constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB₁ biosynthesis, is screened by a standard Hybrid Select Translation procedure to identify the genes responsible for the biosynthetic steps at the terminus of the aflatoxin pathway.