Figure 4

Overexpression of the ial gene in the P. chrysogenum npe10- AB · C strain. (A) The npe10-AB·C strain was co-transformed with plasmids p43gdh-ial and the helper pJL43b-tTrp. Different transformants were randomly selected (T1, T7, T20, T39 and T72) and tested by Southern blotting after digestion of the genomic DNA with Hind III and Kpn I. These enzymes release the full Pgdh-ial-Tcyc1 cassette (2.3 kb) and one 11.0-kb band, which includes the internal wild-type ial gene. Bands of different size indicate integration of fragments of the Pgdh-ial-Tcyc1 cassette in these transformants. Genomic DNA from the npe10-AB·C strain [C] was used as positive control. The λ-Hind III molecular weight marker is indicated as M. (B) Northern blot analysis showing the expression of the ial gene in transformant T7 (npe10-AB·C·ial strain). Expression of the β-actin gene was used as control. (C) Representative chromatogram of the HPLC analysis of the production of 6-APA by the npe10-AB·C·ial strain. The npe10-AB·C·DE strain was used as positive control. As internal control, 6-APA was added to the samples obtained from the npe10-AB·C·ial strain. (D) Representative chromatogram showing the lack of benzylpenicillin production by the npe10-AB·C·ial strain. Filtrates obtained from the npe10-AB·C·DE strain and a sample of pure potassium benzylpenicillin were used as positive controls.