The isolation and characterization of a peptide synthetase gene from Trichoderma virens

Abstract

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Includes bibliographical references (leaves 47-53).Issued also on microfiche from Lange Micrographics.The filamentous fungus Trichoderma has a long history of biological control, protecting numerous plant species against a broad spectrum of soilborne pathogens. Additionally, many species are significant sources of industrial enzymes. Trichoderma isolates have been shown to produce a number of antibiotic compounds, including peptaibols, which are deleterious to both fungi and bacteria. Peptaibols are known for their high α-aminoisobutyric acid content and their production as a mixture of isoforms ranging from 7-20 amino acids in length. The antibiotic activity they exhibit is probably due to an ability to form pores in lipid membranes. These pores conduct ions, leading to loss of osmotic balance and cell death. Because of the potential importance of peptaibols in the biological control of plant diseases, we sought to clone the gene(s) responsible for their synthesis. With their unusual amino acid content, we expected peptaibols to be the product of nonribosomal peptide synthetases (NRPSs). NRPSs have a modular structure in which each module is a semiautonomous unit that recognizes, activates, and incorporates a single residue into the final peptide. We have identified novel peptaibols from Trichoderma virens and cloned the NRPS gene, tex1, responsible for their biosynthesis. Disruption of tex1 in T. virens eliminates peptaibol production, as determined by HPLC analysis. This gene is the first complete peptide synthetase gene cloned from the genus Trichoderma. The 62.8 kb continuous open reading frame represents not only the first peptaibol synthetase gene cloned, but also the largest known open reading frame of any gene in any organism. Tex1 is expressed under all examined conditions, including ungerminated conidia. Since modular order is co-linear with that of the product, we were able to assign substrate amino acid(s) to each module. Comparison of protein sequences between modules is consistent with the module/substrate assignments. Here we report the first genetic evidence showing that peptaibols from T. virens are produced by an enormous peptide synthetase. Manipulation of the abundance or sequence of peptaibols or the expression of tex1 in other fungi or plants could lead to increased suppression of plant pathogens through biological control