As reported here, in silico analysis of the P. chrysogenum genome identified a gene (ial) paralogue of the penDE gene [27] that encodes a protein with high similarity to IAT and is present in most of the genomes of ascomycetes. We have shown in this work that the ial gene is expressed very poorly or not expressed at all in several P. chrysogenum strains

and that generation of ial null mutants does not affect penicillin production. In addition, the ial gene in the npe10-AB·C strain has undergone a point mutation at nucleotide 980 (C to T). After cDNA MG-132 solubility dmso sequence analysis, CBL-0137 cost this point mutation introduces a stop codon after residue 286, which gives rise to a shorter protein (286 amino acids instead of 362) in the npe10-AB·C strain. The lack of activity of the IAL present in this strain might be a consequence of the formation of a truncated version derived from the point mutation, but the fact that after overexpression

of the ial gene (without the point mutation), the GSK690693 mw IAL protein still lacks both the IPN amidohydrolase and IPN acyltransferase activities in vivo, excludes this possibility. Due to the high homology existing between the IAT and IAL proteins we wondered about the reason for the lack of activity in the IAL. The first possible cause was the absence of the PTS1 peroxisomal targeting motif and the consequent putative mislocalization of the IAL. However, when the PTS1 was added

to the C’ end of the IAL, this protein was unable to produce 6-APA or benzylpenicillin in vivo. Strikingly, it has been recently reported that expression of the ial gene homologue in A. nidulans (named aatB) is easily detected and the protein encoded by this gene contributes to penicillin biosynthesis [35]. The A. nidulans aatB-encoded IAL homologue also lacks the canonical PTS1 signal at the D-malate dehydrogenase C’ end, although it is active, indicating that either there might be cryptic PTS1 sequences within this protein as it has been reported in literature [36], or the enzyme is active in the cytosol. The latter possibility is more likely, since addition of the PTS1 signal to the aatB-encoded IAL homologue led to an increase in the penicillin titres [35]. The wild-type IAT is only active when it is self-processed into the α (11.5 kDa, pI: 7.24) and β (28.5 kDa, pI: 6.34) subunits [20, 26, 31]. It is well known that the P. chrysogenum and A. nidulans IATs differ in their ability to maintain the 40-kDa α-β heterodimer in an undissociated form [31]. Whereas the P. chrysogenum proIAT undergoes a quick and efficient self-processing, the A. nidulans proIAT remains partially undissociated. This difference in the processing rate of proIAT is responsible, among other reasons, for the low levels of benzylpenicillin production in A.