Screening the biosphere: the fungicolous fungus Trichoderma phellinicola, a prolific source of hypophellins, new 17-, 18-, 19-, and 20-residue peptaibiotics

Abstract

To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino acid, α-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high-resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17-, 18-, and 19-residue peptaibiotics, which display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name 'hypophellin(s)', based on the teleomorph name, is introduced.

Fig. 1

a) Structures and configurations of a,a-dialkylamino acids found in peptaibiotics. b) Building scheme of subfamily-1 (SF1) peptaibiotics, produced by Hypocrea phellinicola. Variable positions are underlined. Minor sequence variations are parenthesized. Deletions of certain amino acid positions are highlighted in different shades: C-terminal deletions are highlighted in dark, deletions of Gln in medium, and deletions of [Aib/Ala]⁶ in light gray. a) Deleted in 17-, 18-, and 19-residues hypophellins. b) Deleted in the 17-residue sequence 29. c) Deleted in 18-residue sequences 11, 12, and 28, and in the 17-residue sequence 29. d) Detected with DTU maXis gradient only. e) Detected with JLU micrOTOF-Q II gradient only.

Fig. 2

Base-peak chromatograms (BPCs) of a) the H. phellinicola specimen screened with the micrOTOF-Q II, b) the H. phellinicola ex-type plate culture screened with the micrOTOF-Q II, and c) the H. phellinicola specimen screened with the maXis. †, co-eluting peptaibiotics, not sequenced; ‡, non-peptaibiotic metabolite.

Fig. 2

Base-peak chromatograms (BPCs) of a) the H. phellinicola specimen screened with the micrOTOF-Q II, b) the H. phellinicola ex-type plate culture screened with the micrOTOF-Q II, and c) the H. phellinicola specimen screened with the maXis. †, co-eluting peptaibiotics, not sequenced; ‡, non-peptaibiotic metabolite.

Fig. 3

Sequencing of compounds 13 and 15 ocontaining a new C-terminal residue with a peak at m/z 804.46, tentatively assigned as tyrosinol (Tyrol)