chrysogenum. This gene includes the sequence encoding the PTS1 (peroxisomal targeting sequece) motif “”ARL”" at the 3′ end, which was introduced using the “”QuikChange® Site-Directed Mutagenesis Kit”" (Stratagene La Jolla, CA, U.S.A.) following the manufacturer’s instructions. Plasmid p43gdh-ial was used as template in the PCR reaction performed with HPLC-purified primers ARLF and ARLR (Appendix). Plasmid pJL43b-tTrp, which contains the ble gene (for bleomycin/phleomycin resistance) and the transcriptional terminator

of the A. nidulans trpC gene, was co-transformed with either p43gdh-ial or p43gdh-ial ARL into the Wis54-1255 strain. Plasmid pPBCαβ has been previously described [26, 31] and was used to overexpress the cDNA of the penDE gene in E. coli. Plasmid pULCT-ial is a derivative of plasmid pULCTαβ [31] and was used to overexpress the ial gene in E. coli. It was constructed as follows: The cDNA of the Selleckchem Nutlin 3a ial gene was amplified by RT-PCR using primers cDElikeF and DelikeR (Appendix). The RT-PCR product was digested with those endonucleases and subcloned into plasmid pULCTαβ, which was previously digested Wortmannin supplier with HindIII, blunt-ended and finally digested with NdeI. Transformation of P. chrysogenum protoplasts Protoplasts were selleck chemicals obtained and transformed as previously described [49, 50]. Selection of transformant clones was performed by resistance to phleomycin

(30 μg/ml). Selection of acetamide-consuming transformants was done as described previously [51]. DNA and RNA isolation, Southern and northern blotting DNA and RNA isolation, Southern and northern blotting were carried out as described 5-FU chemical structure before [7]. The ial gene was used as probe. The signal

provided by the Southern blotting was quantified by densitometry using the “”Gel-Pro Analizer”" software (Media Cybernetics). Intron analysis Identification of introns in the ial gene was performed by RT-PCR using the “”OneStep RT-PCR Kit”" (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Total RNA was extracted from mycelia of the npe10-AB·C·ial strain grown for 48 h in CP medium, using the “”RNeasy Mini Kit”" columns (Qiagen) following the manufacturer’s instructions. RNA was treated with RQ1 RNase-free DNase (Promega Corporation) following the manufacturer’s instructions. Oligonucleotides cDElikeF and DElikeR (see the Appendix) were used for this purpose. The presence of introns was confirmed by sequencing. Derivatization of IPN and 6-APA and HPLC analysis Quantification of IPN and 6-APA in P. chrysogenum filtrates was carried out by HPLC as previously described [11]. Extraction and HPLC analyses of penicillin from filtrates Filtrates or cell extracts (3 ml) were acidified until pH 2.0 with 0.1 N HCl. Benzylpenicillin was extracted by adding n-butyl acetate (3 × 1 ml) and re-extracted from the organic phase with 10 mM phosphate buffer pH 7.5 (3 × 1 ml). This procedure was performed at 4°C.