In the last step of the penicillin pathway, the L-α-aminoadipyl side chain of IPN is ARRY-162 manufacturer substituted by aromatic acyl side chains to form hydrophobic penicillins. This reaction is catalysed by the isopenicillin N acyltransferase (IAT), encoded by the penDE gene [2, 3]. Previous activation of the aromatic acid by a Evofosfamide specific aryl-CoA ligase is required [4, 5]. In P.

chrysogenum, the pcbAB, pcbC and penDE genes are clustered with other ORFs forming an amplifiable DNA unit [6–8]. These other ORFs play only a minor role in the penicillin biosynthesis, since complementation of the npe10 strain (Δpen), which lacks the whole amplified region including the penicillin gene cluster [9, 10], with only the pcbAB, pcbC and penDE genes restored CFTRinh-172 full β-lactam synthesis [8, 11]. The evolutionary origin of the penicillin gene cluster is intriguing [12]. The first two

genes pcbAB and pcbC do not contain introns despite the large size of pcbAB (11 kb); they appear to have been transferred from β-lactam producing bacteria [13–15], unlike the IAT-encoding penDE gene, which contains three introns and seems to have been recruited from the fungal genomes. The last enzyme of the penicillin biosynthetic pathway (IAT) is synthesized as a 40-kDa precursor (proacyltransferase, proIAT), which undergoes an autocatalytic self-processing between residues Gly102-Cys103 in P. chrysogenum. The processed protein constitutes an active heterodimer with subunits α (11 kDa, corresponding to the N-terminal fragment) and β (29 kDa, corresponding to the C-terminal Arachidonate 15-lipoxygenase region) [16–20]. The IAT has up to five enzyme activities related to penicillin biosynthesis [21]. The substitution of the side chain either occurs directly through the IPN acyltransferase activity, or as a two-step process through the IPN amidohydrolase activity,

thus forming 6-aminopenicillanic acid (6-APA) as an intermediate [22]. The P. chrysogenum IAT belongs to the N-terminal nucleophile (NTN) family of proteins and it is capable of self-activation (C. García-Estrada and J.F. Martín, unpublished results), as occurs with other NTN amidohydrolases [23]. This enzyme is located inside microbodies (peroxisomes) [24, 25] and its transport inside the peroxisomal matrix is not dependent on the processing state of the protein; the unprocessed proIAT variant IATC103S is correctly targeted to peroxisomes, although it is not active [26]. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene [27]. It was, therefore, of great interest to characterize the ial gene at the molecular level and its relationship with the penDE gene regarding penicillin biosynthesis. Results Characterization of the ial gene in P. chrysogenum, which encodes a protein (IAL) with high similarity to IAT The genome of P.