The production and characterisation of trichotoxin peptaibols, by Trichoderma asperellum.

Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.

Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum.

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.

Growth and pectinase production by Aspergillus Mexican strain protoplast regenerated under acidic stress.

Protoplasts from Aspergillus sp. FP-180 and Aspergillus awamori NRRL- 3112 were released and regenerated at extreme acidic conditions. The best conditions for protoplast release were 0.8 M KCI, pH 5.8, and 3 h of digestion using mycelia from 12- to 16-h cultures from either Aspergillus sp. FP-180 or A. awamori NRRL-3112. The addition of fresh mycelia to an ongoing digestion after 1 h increased protoplast 4.5-5 times. A regeneration efficiency of 90% was attained at pH 6.0, and it was possible to regenerate protoplasts at pH 1.7 with a regeneration efficiency of 0.5% for Aspergillus sp. FP-180. The LpH-10 strain, derived from protoplast from Aspergillus sp FP-180, was able to regenerate at pH 1.7 and grow at pH values as low as 1.5, values at which the original strain is unable to grow. Regeneration at extreme pH improved the performance of LpH-10 strain. It showed a twofold increase in cell growth at pH 2.0 in liquid culture and a higher pectinolytic activity in relation to that produced by the original strain.

Characterisation of the gptA gene, encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase, from the filamentous fungus, Aspergillus niger.

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.