ANALYSIS OF THE REGULATION OF PENICILLIN BIOSYNTHESIS IN ASPERGILLUS-NIDULANS BY TARGETED DISRUPTION OF THE ACVA GENE

To analyse the regulation of the biosynthesis of the secondary metabol ite penicillin in Aspergillus nidulans, a strain with an inactivated a cvA gene produced by targeted disruption was used, acvA encodes delta- (L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), which ca talyses the first step in the penicillin biosynthetic pathway. To stud y the effect of the inactivated acvA gene on the expression of ncvA an d the second gene, ipnA, which encodes isopenicillin N synthase (IPNS) , A. nidulans strain XEPD, with the ncvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridiza tion pattern indicating the presence of the disrupted acvA gene, and t he fusion genes integrated in single copy at the chromosomal argB locu s were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both str ains from 24 to 68 h of a fermentation run. In the acvA disrupted stra in, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction me chanism of regulation of the penicillin biosynthesis gene ipnA and the third gene ant.