Colonies of <i>P. chrysogenum</i> strain DS54555.

<p>A: Colony of the wild type strain showing a wrinkled surface. B: Colony of the <i>ΔhcpA</i> strain with a smooth surface.</p

HPLC-MS elution profiles of the internal standard reserpine and the highest abundant identified peptides.

<p>Total ion chromatogram (TIC, black) and normalized extracted ion chromatograms (EIC, colored) of the protonated molecule ([M+H]⁺ with 5 ppm accuracy) of the used internal standard reserpine (A), the four highest abundant cyclic tetrapeptides 1 (B), 2 (C), 3 (D), 4 (E) and linear tetrapeptides 11–13 (F), 14–16 (G), 17–19 (H), 20–22 (I) present in the culture broth of <i>P. chrysogenum</i>.</p

Southern blot analysis for the <i>hcpA</i> deletion and concentration of cyclic tetrapeptides in culture broth of host strain.

<p>A: Southern blot hybridization validating the complete gene deletion of <i>hcpA.</i> B: Internal standard corrected concentrations of the cyclic tetrapeptides 1–10 present in the culture broth of the host strain of <i>P. chrysogenum</i> grown for 168 hours. Isomers are presented together as no chromatographic separation was obtained during profiling. No cyclic or linear tetrapeptides could be found in the deletion strain.</p

Hypothetical model for the biosynthesis of compound 1 in <i>P. chrysogenum</i> and <i>A. niger</i> by the HcpA NRPS.

<p>A: Binding of the monomers on the carrier protein domains. Both isolated condensation and thiolation domains C₄ and T₄, missing a preceding adenylation domain, are assumed to correspond to the adenylation domain A₄, located upstream. B: Proposed assembly of compound 1 including condensation domains presumed for catalyzing the formation of the peptide bond.</p

Correlation between the expression level of <i>HcpA</i> and metabolite formation during growth.

<p>A: Time dependent expression level of <i>hcpA</i> as determined by quantitative RT-PCR during growth of <i>P. chrysogenum</i> as monitored by biomass formation. B: Internal standard corrected concentration of the cyclic peptide 1 and its degradation products 11–13 present in the growth media. The concentration of peptides was determined by HPLC-UV-MS.</p