A LACZ REPORTER FUSION METHOD FOR THE GENETIC-ANALYSIS OF REGULATORY MUTATIONS IN PATHWAYS OF FUNGAL SECONDARY METABOLISM AND ITS APPLICATION TO THE ASPERGILLUS-NIDULANS PENICILLIN PATHWAY

Secondary metabolism, usually superfluous under laboratory conditions, is intrinsically elusive to genetic analysis of its regulation. We de scribe here a method of analyzing regulatory mutations affecting expre ssion of secondary metabolic genes, with an Aspergillus nidulans penic illin structural gene (ipnA [encoding isopenicillin N-synthase]) as a model. The method is based on a targeted double integration of a lacZ fusion reporter gene in a chromosome different from that containing th e penicillin gene cluster. The trans-acting regulatory mutations simul taneously affect lacZ expression and penicillin biosynthesis. One of t hese mutations (npeE1) has been analyzed in detail. This mutation is r ecessive, prevents penicillin production and ipnA'::'lacZ expression, and results in very low levels of the ipnA message at certain times of growth. This indicates that npeE positively controls ipnA transcripti on. We also show that this tandem reporter fusion allows genetic analy sis of npeE1 by using the sexual and parasexual cycles and that lacZ e xpression is an easily scorable phenotype. Haploidization analysis est ablished that npeE is located in chromosome IV, but npeE1 does not sho w meiotic linkage to a number of known chromosome IV markers. This met hod might be of general applicability to genetic analysis of regulatio n of other fungal secondary metabolic pathways.