## 3 Results
### Characterization of TSs Provides an Overview of the Terpenoid Biosynthetic Potential in Trichoderma
synthases, however, they are rich in TSs of \"uncharacterized groups 1 and 2\", which can only be found in few species outside the clade Viride (Table S4). The sister clade of the TRI5-superfamily group (light green color in Figure 1) contains Class I proteins sharing a conserved terpene synthase C domain (Pfam 03936), which can be found in sesquiTS and monoterpene synthases (monoTS). It contains two groups of sesquiTS including 16 presilphiperfolan-8\(\beta\)-ol synthases, 22 pentalene synthases and two groups of proteins that did not cluster with known TSs (uncharacterized groups 3 and 4, respectively) (Table S4). Presilphiperfolan-8\(\beta\)-ol synthases are absent in Viride species, _T. arundinaceum_ and _T. atrobrunneum_, whereas pentalene synthases were found in all the genomes analysed (Table S4). Although most of these share highly conserved metal-binding motifs, some proteins lack the NSD/DTE triad but contain an additional DDxxD motif. This suggests they could actually synthesize sesquiterpenes others than pentalenene. TSs of \"uncharacterized group 4\" are widely distributed across species, but are particularly represented in _T. virens_ and _T. pleuroticola_ (Table S4). Differently, proteins of \"uncharacterized group 3\" seem to be exclusive to species belonging to the Harzianum clade, and their phylogenetic proximity to both groups of sesquiTS suggests this group is also composed by this type of TSs. We found a large group of PTs, identified as squalene synthases (SQSs) (Pfam 00494; PTH1626:SF2; PS01044) (orange color in Figure 1), showing the conserved TM helix region of 23 residues in their C-terminal [88], and enzymes involved in protein prenylation [89] (red color in Figure 1), such as type I geranylgeranyl transferases (GGTases 1) (PTH11774:SF4), type II geranylgeranyl transferases (GGTases 2) (PTH11774:SF11) and farnesyltransferases (FTases) (PTH1774:SF6). They are present in single copy in the genomes of _Trichoderma_, but the additional SQS found in _T. pleuroti_ indicates that at least one SQS is probably pathway-specific (Table S4). Oxidosqualene cyclases (OSCs) (Pfam 13249; Pfam 13243; PTH11764; PS01074) (light-brown color in Figure 1) are present in single copy in the genomes of _Trichoderma_, showing DCTSE or DCISE aspartate-rich motifs, both variants of the classical DCTAE reported in these proteins [75], and five conserved QW motifs responsible of strengthening the structure of the enzyme [90]. TSs from the sister clade of OSCs are in single copy in all the genomes and contain a conserved squalene synthase-phytoene synthase domain (Pfam 00494; PTH21181:SF13) (dark brown color in Figure 1), but they did not cluster with SQSs and neither with reference lycopene-phytoene synthases (uncharacterized group 5). Copalyl-pyrophosphate/Ent-kaurene synthases (CPS/KS) (PTH31739:SF4; PIRSF 026498) (grey color in Figure 1) were found in _T. asperellum_, which known for its ability for gibberellinism biosynthesis [91]. CPS/KS clustered with other Class II bifunctional enzymes of species from clades Longbrachiatum and Brevicompactum (PTH31739:SF4), but the low sequence similarity of these with CPS/KS indicates they are diterpene synthases (diTS) not involved in ent-kaurene biosynthesis. The last cluster (dark blue color in Figure 1) contains proteins sharing a conserved polyprenyl synthase domain (Pfam 00348). Within this group, GGPP synthases (PTHR12001:SF47; PS00723; PS00444) and FPP synthases (PTHR11525:SF0; PS00723; PS00444) were identified, showing the two characteristic DDxxD motifs usually found on these enzymes [51; 92]. Some species belonging to Harzianum and Brevicompactum clades have two to three copies of these PTs class, suggesting that at least some of them could be actually pathway-specific (Table S4). Analysis also revealed a set of highly conserved indole diTS, whose biosynthetic products have not been reported yet in _Trichoderma_. The last group contains Class I TSs clustering with known chimeric TSs from fungi (chimeric-like), which were absent in species of clade Viride.