## 3 Results
### Characterization of TSs Provides an Overview of the Terpenoid Biosynthetic Potential in Trichoderma
Most of them contain only polyprenyl synthase or TS C domains. Nevertheless, we found one protein in _T. asperellum_ TR456 containing both domains, which is highly similar to ophiobolin F synthase from _Aspergillus clavatus_, suggesting this specie is able to produce sesterterpenes. Assessment of the Genomic Context of tri5 Genes Reveals Its Potential Involvement in Unknown Biosynthetic Pathways in Trichoderma  
The presence of _tri5_ orthologs in _Trichoderma_ spp. that have not been described as trichothecene-producers, such as _T. gamsii_, _T. asperellum_ and _T. guizhouense_, suggests this gene could be involved in the biosynthesis of different trichodiene derivatives. Multi-sequence alignment of TRI5 proteins showed the active center is highly conserved, sharing DDSRE/DDSIE aspartate-rich motif and NDLFSYFXE triad (Figure 2a). Figure 2: Trichodiene synthases in _Trichoderma_ spp. (**a**) Phylogenetic relations among trichodiene synthase (TRI5) proteins in _Trichoderma_ spp. and multiple alignment of the active center of the proteins were obtained using MAFFT v7.450 [52] and FasTree v2.1.11 [81]. Aspartate-rich metal binding motifs are indicated with asterisks. (**b**) Biosynthetic gene cluster containing _tri5_ and putative functions of the enclosing genes found in strains of _T. gamsii_. (**c**) Phylogenetic distribution of _tri5_ and their associated biosynthetic gene clusters in _Trichoderma_ spp. Phylogenetic relations of _Trichoderma_ spp. were obtained using MAFFT v7.450 [52] and FasTree v2.1.11 [81], using concatenated alignment of _actin_, _calmodulin_ and _transcription elongation factor-1_ genes. Phylogenetic clades, according to [PERSON] et al. (2019) [30], are shown in two different grey colors. Genes belonging to variants of the TRI loci reported in species of the clade Brevicompactum and _B. bassiana_, respectively, are shown in light blue. Genes found associated to _tri5_ (in purple) in _T. gamsii_ are shown in red. Pairwise alignments of each TRI5 protein with that of _T. arundinaceum_ and _T. brevicompactum_ showed 77% of amino-acid identity in _T. guizhouense_, 80-82% in _T. asperellum_, respectively, and 87% in _T. gamsii_. Assessment of the genomic context of _tri5_ genes by antiSMASH 5.0 [93] revealed this gene is included in a 21.2 kb cluster in _T. gamsii_, enclosing another six genes that were named as \(A\), \(B\), \(C\), \(D\), \(E\), and \(F\) (Figure 2b). Characterization based on conserved domains and similarity with characterized proteins in other systems enabled the identification of one regulatory protein, Zn2-C6 transcription factor (TF) (_A_); four tailoring enzymes: oxygenase (_B_), alpha-beta hydrolase (_C_), oxygenase (_D_), and carbonic anhydrase (_F_); and one efflux transporter, Major Facilitator Superfamily (MFS) transporter (_E_). Alignment of these proteins with the TRI (trichothecene) proteins functionally associated to _tri5_ in the trichothecene-producer species of the Brevicompactum clade showed no sequence similarity. Furthermore, the genome of _T. gamsii_ lacks the entire set of genes encoding the TRI proteins, with the exception of a distant related homolog of the gene _tri101_, which has already been reported in other _Trichoderma_ species [43]. In order to search for homologous proteins in the _Trichoderma_ genomes here explored, we used the protein sequences encoded in the cluster found in _T. gamsii_ as queries in BLASTp analyses. Genes \(A\), \(B\) and \(C\) were also found in all the other _Trichoderma_ spp. belonging to the Viride clade, with conserved synteny (Figure 2c), and preliminary BLAST analyses suggest that these genes may be originated by horizontal gene transfer (HGT) from a donor belonging to the Eurotiomycetes. In any case, further analyses are needed in order to better understand the evolutionary origins of these genes. Instead, genes \(D\) and \(F\) are present in some of the genomes analyzed in closely-related species; while gene \(E\) seems to be specific to _T. gamsii_.