coli (Fig. 6D). We confirmed the processing through the analysis of the ~28-kDa subunit by peptide mass fingerprinting. This peptide was identified as the C-terminal part of the IAL, evidencing GSK872 molecular weight that the IAL protein, like the IAT, also undergoes a phenomenon of self-processing. Figure 6 Characterization of the recombinant IAL in E. coli. (A) Agarose gel electrophoresis of the cDNA of the ial gene obtained by RT-PCR (RT). The 1-kb Ladder plus molecular marker (Invitrogen) is indicated as M. (B) Schematic representation of plasmid pULCT-ial. (C) SDS-PAGE showing

the overexpression of the ial gene in E. coli at 37°C. M: molecular mass marker; -I: uninduced cells; 37°C: total cell extracts obtained after a 5h-induction with IPTG at 37°C; I.B.: inclussion bodies obtained after a 5h-induction with IPTG at 37°C. (D) SDS-PAGE showing the overexpression of the ial gene in E. coli at 26°C. M: molecular mass marker; -I: uninduced

cells; 26°C: soluble cell extracts obtained after a 5h-induction with IPTG at 26°C. Note the lack of the 40-kDa band and the presence of the 28-kDa band. (E) Biossay carried out to determine the in vitro phenylacetyl-CoA: selleck compound 6-APA acyltransferase activity (see Methods) present in the soluble extracts of E. coli overexpressing either the ial (IAL) or the penDE (IAT) genes. As a negative control, the reaction mixture was used without the addition of soluble extracts from E. coli overexpressing the penDE gene (C-). Once processing was confirmed, in vitro activity of the processed IAL protein was assessed (see Methods) using the soluble extracts of E. coli obtained after the overexpression of the ial gene at 26°C. As positive control, soluble extracts containing the functional processed IAT, obtained from E. MEK inhibitor coli after overepression of the cDNA of

the wild-type penDE gene at 26°C (using plasmid pPBCαβ as indicated in Methods), were used. Benzylpenicillin formation was tested by bioassay as indicated in Methods. As shown in Fig. 6E, benzylpenicillin was only synthesized in the protein extracts containing the processed wild-type IAT, but not in extract of the processed IAL. This confirms that under in vitro conditions, the IAL protein also lacks enzymatic activities related to the biosynthesis of benzylpenicillin, PF-562271 cell line despite the correct self-processing. Discussion The penicillin biosynthetic pathway has been largely elucidated [14, 32]. In addition to the three main enzymes involved in this process (ACVS, IPNS and IAT), other ancillary proteins are also required, such as a phenylacetyl-CoA ligases, which primes (activates) the aromatic side chain [4, 5] and the phosphopantetheinyl transferase (PPTase), which activates the ACVS and is essential for penicillin biosyntheis in P. chrysogenum [33]. The origin of the pen gene cluster is intriguing, as occurs with the clusters of other fungal secondary metabolites [12, 34].