Phylogenetic analysis and investigation of RiPP cluster in 50 Trichoderma genomes

Many Trichoderma species have applications in biotechnology, agriculture and human healthcare. Often, secondary metabolites play an important role in the biological activity of the fungus. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a class of secondary metabolites which are genetically encoded in biosynthetic gene clusters (BGCs). These cluster contain modifying enzymes, which allow for a wide spectrum of potential compounds and give the possibility to alter the final product for different purposes. Using antiSMASH we mined the genomes of 50 different Trichoderma strains across multiple species, looking especially into potential RiPP cluster. The analysis of the Trichoderma genomes predicted nine RiPP BGCs as well as a polyketide-RiPP hybrid cluster in 10 different species. A subsequent analysis using BiG-SCAPE revealed that 7 of these clusters are closely related to each other and the already welldescribed ustiloxin-B cluster from Aspergillus flavus. These ustiloxin-like clusters are found exclusively in species belonging to the „Harzianum clade“ and show a very conserved cluster structure. The three unique cluster show no resemblance to the ustiloxin-like cluster and might represent a new class of RiPPs. This is further supported by a multiple sequence alignment between homologous genes in all 10 cluster. One representative of the ustiloxin-like cluster, as well as the three unique clusters were chosen for further investigation. Using RT-qPCR we determined which genes are co-transcribed under different conditions. In order to complement the transcript analysis a classic proteomics experiment was conducted to confirm the expression of the linked genes. These results underscore the diverse biotechnological potential of the genus Trichoderma.