1. Characterization of TSs provides an overview of the terpenoid
biosynthetic potential in Trichoderma
We used an in-silico approach in order to assign putative
functions to 387 TS-encoded proteins currently found in the genomes ofTrichoderma spp. Initially, we identified PT and TC proteins
according to their conserved domains. Subsequent detection of the
metal-binding motifs enabled protein classification as Class I, Class II
or Bifunctional enzymes. Clustering-based phylogenetic analysis using
biologically characterized fungal TSs also enabled determining
substrate-specificity, as well as assigning putative functions to 15
groups of proteins. TSs sharing conserved domains and metal-binding
motifs clustered in the same phylogenetic group, each one highlighted in
different colour (Fig. 1 ). TSs accession numbers, TS-content
per each species showing specific portions of the TS inventory, and
phylogenetic tree including protein accession numbers are available in
Supporting Information Table S4 and Fig. S1 .
Analysis revealed specific TSs sharing N-terminal HAD-like (Pfam 13419;
PTHR43611:SF3) and C-terminal TS domains (IPR008930) (light-blue colour
in Fig. 1). Although they did not cluster with known TSs, the presence
of both Class I DDxxE and Class II DxDTT motifs indicates they are
bifunctional enzymes. Interestingly, these seems to be exclusive of
species belonging to clade Viride
(Supporting Information Table S4 ).
We found a vast group of sesquiterpene synthases (sesquiTSs) belonging
to the TRI5 superfamily (Pfam 06330) (dark-green colour in Fig. 1),
which was particularly represented in species of Viride clade
(Supporting Information Table S4 ). It contained 7 trichodiene
synthases (TRI5) (PIRSF001388), 15 longiborneol synthases, and two
groups of proteins that did not cluster with known TSs, and were
therefore named as “uncharacterized group 1” and “2”, respectively.
TRI5 was found in species of the Brevicompactum clade, T. gamsii ,T. asperellum and T. guizhouense . This phylogenetic
distribution indicates that TRI5 is not a monophyletic trait inTrichoderma , opening questions about its evolutionary origin.
Species of Viride clade were the only lacking longiborneol synthases,
which were found highly conserved among species from the other clades.
Instead, proteins of “uncharacterized group 1” were only present in
Viride and some species of the Longibrachiatum clade. In species of
Viride, we found two TSs of “uncharacterized group 2”, while T.
arundinaceum and T. virens had only one.
The sister clade of the TRI5-superfamily group (light-green colour in
Fig. 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-8β-ol synthases and 22 pentalenene synthases, along
with two groups of proteins that did not cluster with known proteins
(“uncharacterized 3” and “4”, respectively) (Supporting Information
Table S4 ). Presilphiperfolan-8β-ol synthases are absent in
Viride species, T. arundinaceum and T. atrobrunneum ,
whereas pentalenene synthases were found in all the genomes analysed.
Although most of these lasts share highly conserved metal-binding
motifs, some proteins lack on 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 the species, but are particularly
represented in T. virens and T. pleuroticola . Differently,
proteins of “uncharacterized group 3” seems to be exclusive of 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; PTHR11626:SF2; PS01044) (orange colour in Fig. 1) and
enzymes involved in protein prenylation (Jeong et al., 2018) (red colour
in Fig. 1), such as type I geranylgeranyl transferases (GGTases 1)
(PTHR11774:SF4), type II geranylgeranyl transferases (GGTases 2)
(PTHR11774:SF11) and farnesyl transferases (FTases) (PTHR11774:SF6). In
SQSs, we also identified a C-terminal TM helix region of 23 residues,
which is universally conserved in all eukaryotic SQSs and it is
responsible of binding the protein to the endoplasmic reticulum
(Linscott et al., 2016). Although each genome contains one of these TSs,
an additional SQS is present in T. pleuroti , indicating that at
least one of them is probably pathway-specific (Supporting Information
Table S4 ).
All genomes of Trichoderma here analysed contain one copy of
oxidosqualene cyclase (OSCs) (Pfam 13249; Pfam 13243; PTHR11764;
PS01074) (light-brown colour in Fig. 1) showing DCTSE or DCISE
aspartate-rich motifs, both variants of the classical DCTAE reported in
these enzymes (Abe, Naito, Takagi, & Noguchi, 2001). Furthermore, these
OSCs contain 5 conserved QW motifs, which are thought to be responsible
of strengthening the structure of the enzyme and stabilize the
carbocation intermediates (Kushiro et al., 2000). Interestingly, TSs
from the sister clade of OSCs contain a conserved squalene
synthase-phytoene synthase domain (Pfam 00494; PTHR21181:SF13)
(dark-brown colour in Fig. 1), but they did not cluster with SQSs
neither with reference lycopene-phytoene synthases. They were therefore
named as “uncharacterized group 5”, of which one is present in each
genome.
Copalyl-pyrophosphate/Ent-kaurene synthases (CPS/KS) (PTHR31739:SF4;
PIRSF 026498) were found in T. asperellum , known for its ability
for gibberellin biosynthesis (Zhao & Zhang 2015). In addition, we found
bifunctional enzymes clustering with CPS/KS in T. citrinoviride ,T. parareesei , T. reesei and species of Brevicompactum
clade (PTHR31739:SF4) (grey colour in Fig. 1), but their low sequence
similarity with CPS/KS indicates
these are diterpene synthases (diTS) not involved in ent-kaurene
biosynthesis.
The last cluster (dark-blue colour in Fig. 1) contain 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 (Wendt, &
Schulz, 1998; Gao et al., 2012). Some species belonging to Harzianum and
Brevicompactum clades have 2-3 copies of these PTs class, suggesting
that at least some of them could be actually pathway-specific
(Supporting Information Table S4 ). Analysis also revealed a set
of highly conserved indole diTS, of which some species have more than
one. Considering that production of indole diterpenes has not been
reported in Trichoderma , our results reveal that these species
have at least the potential to produce these compounds. The last group
contains Class I TSs clustering with known chimeric TSs from fungi, and
were therefore named as chimeric-like, which were absent in species of
Viride clade. Most of these proteins contain only polyprenyl synthase or
terpene synthase C domains. Nevertheless, we found one protein inT. asperellum TR456 containing both domains which is highly
similar to ophiobolin F synthase from Aspergillus clavatus ,
suggesting this specie is able to produce sesterterpenes.