Penicillin is one of the most important antibiotics and consequently, its biosynthesis is probably the best understood pathway in fungal secondary metabolism. However, there is little knowledge about the environmental signals and their transmission modulating the expression of the structural genes. In this work, the light-dependent regulator velvet A (VeA) and a central protein kinase C (PkcA) were found to be part of the penicillin biosynthesis regulating network in the fungus Aspergillus nidulans. Evolution of the gene cluster that contains genes of apparently both bacterial and eukaryotic origin has not been fully elucidated yet. The final step of penicillin biosynthesis is catalysed by isopenicillin N acyltransferase, which was characterised in this work, and that is encoded by the aatA gene. Because there is no bacterial homolog, its evolutionary origin remained obscure. As shown in this work, disruption of aatA still enabled penicillin production. Genome mining led to the discovery of the aatB gene which has a similar structure and expression pattern as aatA. Disruption of aatB resulted in a reduced penicillin titre. Surface plasmon resonance analysis and Northern blot analysis indicated that the promoters of both aatA and aatB are bound and regulated by the same transcription factors AnCF and AnBH1. In contrast to aatA, aatB does not encode a peroxisomal targeting signal (PTS1). Overexpression of a mutated aatBPTS1 gene in an aatA disruption strain (leading to peroxisomal localisation of AatB) increased the penicillin titre more than overexpression of the wild-type aatB. Homologs of aatA are exclusively part of the penicillin biosynthesis gene cluster, whereas aatB homologs also exist in non-producing fungi. These findings suggest that aatB is a paralog of aatA. They extend the model of evolution of the penicillin biosynthesis gene cluster by recruitment of a biosynthesis gene and its cis-regulatory sites upon gene duplication.