A non-canonical NRPS is involved in the synthesis of fungisporin and related hydrophobic cyclic tetrapeptides in Penicillium chrysogenum

Abstract

The filamentous fungus Penicillium chrysogenum harbors an astonishing variety of nonribosomal peptide synthetase genes, which encode proteins known to produce complex bioactive metabolites from simple building blocks. Here we report a novel non-canonical tetra-modular nonribosomal peptide synthetase (NRPS) with microheterogenicity of all involved adenylation domains towards their respective substrates. By deleting the putative gene in combination with comparative metabolite profiling various unique cyclic and derived linear tetrapeptides were identified which were associated with this NRPS, including fungisporin. In combination with substrate predictions for each module, we propose a mechanism for a 'trans-acting' adenylation domain.

Figure 1. Identified secondary metabolites.

Structures of cyclic tetrapeptides identified in P. chrysogenum.

Figure 2. Correlation between the expression level of HcpA and metabolite formation during growth.

A: Time dependent expression level of hcpA as determined by quantitative RT-PCR during growth of P. chrysogenum 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.

Figure 3. Hypothetical model for the biosynthesis of compound 1 in P. chrysogenum and A. niger by the HcpA NRPS.

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.

Figure 4. Colonies of P. chrysogenum strain DS54555.

A: Colony of the wild type strain showing a wrinkled surface. B: Colony of the ΔhcpA strain with a smooth surface.

Figure 5. Southern blot analysis for the hcpA deletion and concentration of cyclic tetrapeptides in culture broth of host strain.

A: Southern blot hybridization validating the complete gene deletion of hcpA. B: Internal standard corrected concentrations of the cyclic tetrapeptides 1–10 present in the culture broth of the host strain of P. chrysogenum 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.

Figure 6. HPLC-MS elution profiles of the internal standard reserpine and the highest abundant identified peptides.

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 P. chrysogenum.