2. Assessment of the genomic context of tri5 genes
reveals its potential involvement in unknown biosynthetic pathways inTrichoderma
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 centre is highly conserved, sharing DDSRE/DDSIE
aspartate-rich motif and NDLFSFYKE triad (Fig. 2a ). Pairwise
alignments of each TRI5 protein with that of T. arundinaceum andT. brevicompactum showed 77% of amino-acid identity in T.
guizhouense , 80-82% in T. asperellum , respectively, and 87% inT. gamsii .
Assessment of the genomic context of tri5 genes by antiSMASH 5.0
(Blin et al., 2019) revealed this gene is included in a 21.2 kb cluster
in T. gamsii , enclosing another 6 genes that were named asA , B , C , D , E and F (Fig.2b ). Manual characterization based on conserved domains and
similarity with characterized proteins in other systems enabled the
identification of four tailoring enzymes, one efflux transporter and one
regulatory protein. The three genes located upstream of tri5encode a Zn2-C6 transcription factor (TF) (A ), oxygenase
(B ), and alpha-beta hydrolase (C ), while the three located
downstream were identified as oxygenase (D ), Major Facilitator
Superfamily (MFS) transporter (E ) and carbonic anhydrase
(F ).
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 on the entire set of
genes encoding the TRI proteins, with the exception of a distant related
homolog of the gene tri101 , which has been already reported in
other Trichoderma species (Proctor et al., 2018). We used the
protein sequences encoded in the cluster found in T. gamsii as
queries in BLASTp analyses to search for homologous proteins in theTrichoderma genomes used here. Genes A , B andC were also found in all the other Trichoderma spp.
belonging to the Viride clade, with conserved synteny (Fig.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 analysed in closely related species; while gene E seems
to be specific to T. gamsii .
These findings suggest the origin of a novel tri5 -associated
cluster in T. gamsii , which is likely involved in the
biosynthesis of trichodiene-derivates with unknown functions. According
to this, tri5 could participate in two different sesquiterpene
biosynthetic pathways in Trichoderma .