Advances in the discovery and study of Trichoderma natural products for biological control applications

Abstract

Covering: up to 2025Reducing the prevalence of phytopathogens and their impact on crops is essential to reach sustainable agriculture goals. Synthetic pesticides have been commonly used to control crop disease but are now strongly linked to disease resistance, environmental pollution, depletion of soil biodiversity, and bioaccumulation, leading to adverse effects on human health. As a alternative, the prolific Trichoderma genus has been studied for its biocontrol properties, as well as its ability to promote plant growth and increase nutrient uptake. This is done through various mechanisms, one of which is the production of bioactive natural products with high chemical diversity. These include terpenoids, alkaloids, non-ribosomal peptides, polyketides and RiPPs. One of the most studied examples is 6-pentyl-2H-pyran-2-one, a volatile organic polyketide, which induces systemic acquired resistance, morphogenesis, and natural product biosynthesis in plants. Methods for culturing Trichoderma spp., isolating and characterising unique bioactive metabolites are discussed here, with an emphasis on dereplication strategies using metabolomics to optimise discovery. In addition, the role of genome mining for the study of natural product biosynthesis in Trichoderma, and more generally, filamentous fungi is discussed. Examples of bioinformatics tools available to date are listed here with applications in Trichoderma and other ascomycetes. New advances in genome engineering in Trichoderma are also detailed, providing insights into available strategies for the validation of biosynthetic gene clusters identified using genome mining. Finally, the use of a combination of omics approaches, namely metabologenomics, is presented as a growing field for natural product discovery in fungi.

Fig. 1. Snapshot of the chemical diversity of terpenoids and polyketides from Trichoderma. 6-Pentyl-2H-pyran-2-one (1); trichodermin (2); harzianum A (3); 5-hydroxy-2,3-dimethyl-7-methoxychromone (4); microsphaeropsisin B (5); methyllasiodiplodin (6); harziaphilic acid (7); harzianolic acid A (8); harzianone E (9); 3,7,11-trihydroxy-cycloneran (10); 2-((4aR,5R,8aS,E)-3,3,5-trimethyloctahydronaphthalen-1(2H)-ylidene)propan-1-ol (11); trichobrasinelol (12); trichodermol (13).

Fig. 2. Examples of alkaloids and peptides from Trichoderma. Gliotoxin (14); gliovirin (15); alamethicin (16); harzianin A (17); trichokonin V (18); trichogin GA IV (19); pentadecaibin (20).

Fig. 3. Mechanism of type I iterative PKS and NRPS in fungi. (A) A type I iterative PKS is composed of the essential ketosynthase (KS), acyltransferase (AT), and thioesterase (TE) domains. Additional domains include ketoreductase (KR), enoylreductase (ER) and dehydratase (DH) domains. The biosynthesis starts by the transthiolesterification of an acyl-CoA on the active site thiol of the KS. In parallel, a malonyl-CoA unit is also transferred to the thiol of the phosphopantetheine (PP) prosthetic group of the acyl carrier protein (ACP) group. These two reactions can often be catalysed by the AT. The KS catalyses decarboxylative Claisen condensation to form a β-ketoacyl thioester. The KR then reduces the keto group at the β-carbon into a hydroxyl, the DH further catalyses the formation of a double bond by dehydration of the hydroxyl group, and the ER reduces the double bond. The chain can then be loaded back onto the active site thiol of the KS, and the cycle can continue to then finally be released by the TE domain in the case of non-reducing and partially-reducing PKSs, and a trans-acting hydrolase in the case of highly-reducing PKSs. (B) An NRPS is composed of repeating units of adenylation (A), peptidyl carrier protein (PCP), and condensation (C) domains organised in modules. The number of elongation modules can vary and will dictate the size of the final peptide. The biosynthesis starts with the activation of an amino acid catalysed by the A domain. The A domain binding site sequence will dictate the amino acid to be incorporated into the peptide chain. The activated amino acid is then transferred to the thiol unit of the PP group of the PCP. The C domain then catalyses the formation of a peptide bond between this amino acid and the subsequent amino acid attached to the PCP of the next module, and so on until reaching the termination module. The TE domain then catalyses the release of the peptide.

Fig. 4. Examples of polyketides from Trichoderma. sorbicillin (21); emodin (22); trichoderone (23); hypochromin (24); koninginin A (25); chaunopyran A (26); tricholignan A (27).

Fig. 5. Examples of hybrid molecules from Trichoderma. Treconorin (28); trihazone A (29); ilicicolin H (30); pretenellin A (31).

Fig. 6. Strategies for natural product discovery in fungi. The OSMAC (“One Strain Many Compounds”) approach which includes modulating cultivation conditions or using molecular approaches like epigenetic modifications or ribosome engineering. Dereplication is essential to ensure novelty and is exemplified by the discovery of scopularide H from Scopulariopsis sp. CMB-F115 using GNPS. If the product can be detected, a combination of genomics, transcriptomics and metabolomics can be used to isolate the target SM, identify its structure, and link it to a gene cluster using several genome mining tools. This is the concept behind metabologenomics where the use of pattern-based genome mining coupled with molecular networking can provide insight into which clusters to prioritise for BGC-SM discovery. Validation of the link can then be done via a variety of different genetic engineering strategies such as TF or promoter engineering, heterologous expression or CRISPR/Cas technologies. This was exemplified by the discovery of pestalamide B along with its BGC from the native host Aspergillus brasiliensis and heterologously expressed in A. nidulans.

Sophie Jin

Fabrizio Alberti