As described for other fungi [29, 30]
deletion of the P. chrysogenum KU70 homologue increases the frequency of homologous recombination significantly (Marco A. van den Berg, unpublished results). Acetamide-consuming transformants were obtained, purified on fresh media and verified for the correct insertion by PCR. Shake flask experiments demonstrated that the ial null Entinostat datasheet mutant had GSK1904529A order no effect on penicillin production in CP medium supplemented with either precursor, adipate or phenylactetate (103 +/- 1% as compared to both DS17690 and DS54465 strains; 100%). Figure 2 Generation of the ial null mutant in P. chrysogenum. The transcription of the ial gene was blocked by insertion (double crossover; dashed lines) of the amdS selection marker in opposite orientation between the ial gene promoter and the ial ORF. Restriction enzymes indicated: Ba, BamHI; Sb, SbfI; Pm, PmeI. Expression of the ial gene in P. chrysogenum and in vivo role of the IAL in the benzylpenicillin biosynthetic pathway To confirm these results, we carried out different experiments with the engineered strain P. chrysogenum
npe10-AB·C. This strain is a transformed derivative of the npe10 PyrG- strain (Δpen) that contains the pcbAB and pcbC genes, but lacks the wild-type penDE gene [11]. Because of these features, this strain is optimal to assess the putative role of the IAL protein in the benzylpenicillin biosynthetic pathway. The integrity of the ial gene in the learn more npe10-AB·C strain was initially tested by PCR (data not shown) and Southern blotting (Fig. 3A). After digestion of the genomic DNA with HindIII, one 11-kbp band was observed in the npe10-AB·C, size that is coincident with that provided by the Wis54-1255 strain digested with the same MycoClean Mycoplasma Removal Kit restriction enzymes (Fig. 3A). However, after sequencing the ial gene from the npe10-AB·C strain, we found a point mutation at nucleotide 980, where C was changed into T (see Discussion). IPN production by the npe10-AB·C strain was confirmed by HPLC (Fig. 3B). Formation of benzylpenicillin (IPN
acyltransferase activity) and 6-APA (IPN amidohydrolase activity) that might be catalyzed by the IAL, were assessed by growing the npe10-AB·C strain in CP medium. Samples were taken at 48 h and 72 h, but neither 6-APA (Fig. 3C) nor benzylpenicillin (Fig. 3D) were detected by HPLC. This indicates that the npe10-AB·C strain, which contains the ial gene, does not produce these compounds formed in the last step of the penicillin biosynthetic pathway. To test whether the lack of activity is due to a low or null expression rate of the ial gene, northern blot experiments were done with samples taken from the npe10-AB·C and the Wis54-1255 strains grown in CP medium. As shown in Fig. 3E no transcript bands were detected at 24 or 48 h, indicating that this gene is very low or not expressed in P. chrysogenum, in agreement with the absence of detectable ial mRNA in P. chrysogenum NRRL 1951, npe10, Wisconsin54-1255 and DS17690 strains (Marco A.
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