Two Classes of New Peptaibols Are Synthesized by a Single Non-ribosomal Peptide Synthetase of Trichoderma virens

Abstract

Peptaibols are a group of small peptides having a high ␣-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.

Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize

“…The wild-type strain of T. virens (Gv29-8) isolated by our laboratory, has been sequenced (http:// genome.jgi-psf.org/TriviGv29_8_2/TriviGv29_8_2.home.html) and the strain has been deposited at the Fungal Stock Center (Kansas City, MO). The Dtex2 mutant was obtained from our previous study (Mukherjee et al, 2011). All the mutants were cultivated on either potato dextrose agar (PDA, Difco) with or without hygromycin B amendment or on Vogel's minimal medium supplemented with 1.5 % sucrose (VMS).…”

“…Both of these strategies have been successful in previous functional studies of T. virens genes (Mukherjee et al, 2003b;Mukherjee & Kenerley, 2010;Djonović et al, 2007). Disruption of tex2 was by single cross-over with selection for hygromycin B-resistant colonies (Mukherjee et al, 2011). For each gene disrupted by single cross-over homologous recombination, a primer pair was designed to amplify part of the ORF (around the centre) with SalI and KpnI sites integrated in the primer sites.…”

“…The size of the proteins ranged from 547 to 20 891 amino acids. tex1 and tex2 both encode peptaibol synthetases, which have been described previously (Wiest et al, 2002;Mukherjee et al, 2011). An additional gene predicted to be a peptaibol synthetase with seven complete modules arranged in a linear fashion (tex3) was homologous to tex1.…”

Trichoderma Research in the Genome Era

“…Cloning of peptaibol synthase genes and demonstration of one NRPS catalyzing multiple peptaibols (96,144) 2006 Master regulator of cellulases and xylanases identified in T. reesei (XYR1) (130) 2004 Photoreceptors (Brl1 and Brl2) discovered in T. atroviride (15) …”

“…The Trichoderma genomes have now been shown to contain a large repertoire of small cysteine-rich secreted protein-like genes (67), potentially encoding hundreds of possible elicitors. The challenge is to identify which are relevant and then test the proteins for activity (11 Figure 1) (3,91,92,96). Trichoderma secondary metabolites may be grouped into peptaibols, small NRPs (e.g., gliotoxin, siderophores), polyketides, terpenes, or pyrones.…”