Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus

Abstract

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.