Identification and characterization of mutants affecting the expression of the stc gene cluster in Aspergillus nidulans.

摘要

Fungi in the genus Aspergillus are capable of producing toxic secondary metabolites called mycotoxins. Of particular interest are aflatoxin (AF) which is produced by the agronomically important species Aspergillus flavus and A. parasiticus and the related compound, sterigmatocystin (ST) which is produced by A. nidulans. The biosynthesis of these compounds is conserved in these fungi both at the enzymatic level and the genetic level. The biochemical pathway leading to ST and AF production has been well described and clusters containing the genes necessary for biosynthesis of these compounds have been described in these species of Aspergillus.;The expression of genes in the pathway is regulated by a positively acting transcription factor called AflR. This dissertation describes a mutant screen to identify genes that regulate the expression of aflR, and the genetic characterization of those mutants.;For mutagenesis, a visual assay was necessary to facilitate the rapid screening of large numbers of possible mutants. ST, however, is not visible with the unaided eye. To overcome this problem, I utilized a genetic block in the ST pathway which leads to the accumulation of the first stable intermediate in the biosynthetic pathway (norsolorinic acid, NOR), which is an orange colored compound. Production of NOR requires the activities of the first four genes in the pathway. The mutant screen is based on the loss of NOR accumulation. As I would predict, NOR- mutants were isolated that are linked to the gene cluster and most likely contain mutations in one or more of the genes required for NOR production. A second group of mutants were identified that are genetically unlinked to the gene cluster and represent genes that have a role in regulating the activity of the gene cluster. Here I present the genetic characterization of both linked and unlinked mutants isolated from the screen.