USE OF REPORTER GENES TO IDENTIFY RECESSIVE TRANS-ACTING MUTATIONS SPECIFICALLY INVOLVED IN THE REGULATION OF ASPERGILLUS-NIDULANS PENICILLIN BIOSYNTHESIS GENES

Starting from three amino acid precursors, penicillin biosynthesis is catalyzed by three enzymes which are encoded by the following three ge nes: acvA (pcbAB), ipnA (pcbC), and aat (penDE). To identify trans-act ing mutations which are specifically involved in the regulation of the se secondary metabolism genes, a molecular approach was employed by us ing an Aspergillus nidulans strain (AXTII9) carrying acvA-uidA and ipn A-lacZ gene fusions integrated in double copies at the chromosomal arg B gene. On minimal agar plates supplemented with X-Gal romo-4-chloro-3 -indolyl-beta-D-galactopyranoside), colonies of such a strain stained blue, which is indicative of ipnA-lacg expression. After mutagenesis w ith UV light, colonies were isolated on agar plates with lactose as th e carbon source, which produced only a faint blue color or no color at all. Such mutants (named Prg for penicillin regulation) most likely w ere defective in trans-acting genes. Control experiments revealed that the mutants studied still carried the correct number of gene fusions. In a fermentation run, mutants Prg-1 and Prg-6 exhibited only 20 to 5 0% of the ipnA-lacZ expression of the wild-type strain and produced on ly 20 to 30% of the penicillin produced by the wild-type strain. Weste rn blot (immunoblot) analysis showed that these mutants contained redu ced amounts of ipnA gene product, i.e., isopenicillin N synthase. Both mutant Prg-1 and mutant Prg-6 also differed in acvA-uidA expression l evels from the wild type. Segregation analysis indicated that for both mutants the Prg phenotype resulted from mutation of a single gene. Tw o different complementation groups, which were designated prgA1 and pr gB1, were identified. However, the specific activity of the aat (penDE ) gene product, i.e., acyl coenzyme A:6-aminopenicillanic acid acyltra nsferase, was essentially the same for the mutants as for the wild-typ e strain, implying that the last step of the penicillin biosynthetic p athway is not affected by the trans-acting mutations identified.