Background: Cytochrome P450s form an important group of enzymes involved in xenobiotics degradation and metabolism, both primary and secondary. These enzymes are also useful in industry as biotechnological tools for bioconversion and a few are reported to be involved in pathogenicity. Trichoderma spp. are widely used in industry and agriculture and are known for their biosynthetic potential of a large number of secondary metabolites. For realis- ing the full biosynthetic potential of an organism, it is important to do a genome-wide annotation and cataloguing of these enzymes. Results: Here, we have studied the genomes of seven species (T. asperellum, T. atroviride, T. citrinoviride, T. longibra- chiatum, T. reesei , T. harzianum and T. virens) and identified a total of 477 cytochrome P450s. We present here the classification, evolution and structure as well as predicted function of these proteins. This study would pave the way for functional characterization of these groups of enzymes and will also help in realization of their full economic potential. Conclusion: Our CYPome annotation and evolutionary studies of the seven Trichoderma species now provides opportunities for exploration of research-driven strategies to select Trichoderma species for various applications espe- cially in relation to secondary metabolism and degradation of environmental pollutants. ideal candidates for genome-wide studies to further aug- Background ment their biotechnological applications. The first spe - Trichoderma (Hypocreales, Ascomycota, Dikarya) spe- cies to be sequenced is Trichoderma reesei, industrial cies are among the most common fungi frequently source of cellulases and hemicellulases . This was soon isolated as mycotrophs from various fungi and as sap- followed by whole genome sequencing of two strongly rotrophs from free soil, soil litter, dead wood and rhizo- mycoparasitic species, viz. T. atroviride and T. virens sphere, and includes more than 256 accepted species [1, . A comparative analysis of the mycoparasitic species 2]. These fungi are economically important due to their i.e., T. atroviride and T. virens with that of weaker myco- ability to produce enzymes of industrial importance, abil- parasitic species T. reesei yielded novel information on ity to kill/inhibit many plant pathogenic fungi, to boost the genome-scale differences between these species. In plant immunity and promote plant growth, in addition to general, the mycoparasitic species are enriched in genes their ability to produce a plethora of secondary metabo- involved mycoparasitism and secondary metabolism [1, lites [3, 4]. A few species/strains are known to be oppor- 7, 8]. Four more species, i.e., T. asperellum and T. harzi- tunistic human pathogens . Trichoderma spp. are thus anum (biocontrol species) and T. longibrachiatum and T. citrinoviride (opportunistic human pathogens) were sub- *Correspondence: email@example.com sequently sequenced by US Department of Energy Joint Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Genome Initiative (Mycocosm ; http://jgi.doe.gov/ Research Centre, Trombay, Mumbai 400085, India Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 2 of 15 fungi). However, detailed analyses of these four genomes number of sequence-diverse P450s is yet to be discov- are awaited. ered and explored for functions and diverse activities in Cytochrome P450 genes (CYPs) are found in the all kingdoms. One of the most commonly used resources genomes of prokaryotes and lower and higher eukary- includes the Nelson database (http://drnelson.uthsc. otes. CYPs constitute a large superfamily of heme- edu/cytochromeP450.html) . The grouping scheme thiolate proteins involved in the metabolism of a wide for CYPs is based on amino acid sequence similarity variety of both exogenous and endogenous compounds . The original nomenclature for CYPs is based upon . CYPs are heme b containing monooxygenases amino acid identity where Cyp proteins with at least 40% which were recognized and defined as a distinct class identity are placed in the same family [22, 23]. However, of hemoproteins . Cyp proteins catalyze the regio-, due to various evolutionary mechanisms, a straight for- chemo- and stereospecific oxidation of a vast number of ward nomenclature might be difficult, therefore, fam - substrates under mild reaction conditions, thus accom- ily definition is recommended by integrating phylogeny plishing chemical transformations. These functions make and protein evolution . To each family, Cyp number them important players in xenobiotic degradation and in is designated according to their taxonomic groups. Fun- primary and secondary metabolism. A few such enzymes gal Cyp families are numbered as Cyp51-Cyp69, Cyp501- are also reported to be involved in pathogenicity of plant Cyp699 and Cyp5001-Cyp6999. With rapid increase pathogenic fungi [12–16]. Their diverse functional prop - in discoveries of new Cyp proteins through genome erties reflect their biological roles and make them impor - sequencing, Nelson database lacks efficiency to annotate tant candidates for extensive investigation to explore all Cyp proteins. For higher-level grouping of families diverse aspects of P450 functions and regulation as well identified via the sequence similarity-based scheme, CYP as for biotechnological applications [17, 18]. clan system was first developed and then applied to clas - Cytochrome P450s are categorized into two main sify metazoan CYPs . The CYP clan approach places classes, B (initially assigned as Bacterial) and E (ini- all Cyp families with a monophyletic origin into a single tially assigned as Eukaryotic). Bacterial P450s with three clan and has been successfully applied to classify Cyp component systems [an FAD-containing flavoprotein families in fungi . For example, if new Cyps had equal (NADPH or NADH-dependent reductase), an iron sul- identity to two or more Cyp families, they can be tenta- phur protein, and the P450 hemeprotein] and the fungal tively assigned to a clan in which these families belong. P450 nor (nitric oxide reductase). Clan CYP 55 belong A site dedicated to filamentous fungi has been developed to the ‘B’-class . All the other known P450s from dis- that includes comprehensive information on P450 clans tinct systems, including eukaryotic and bacterial P450s, and families (http://p450.riceblast.snu.ac.kr) . In fila - belong to the ‘E’-class. The eukaryotic microsomal P450 mentous fungi, CYPs are involved in various physiologi- system contains two components, the NADPH:P450 oxi- cal processes including fitness, resistance to xenobiotics doreductase (POR), a flavoprotein containing both FAD and biosynthesis of a vast array of secondary metabolites and FMN, and the P450 monooxygenase containing the with applications in biomedical, agricultural and indus- heme domain. The prokaryotic (bacterial) soluble P450 trial fields [28–31]. monooxygenase P450BM3 (Cyp102) exists as a single Keeping in view the wide spectrum of biotechnological protein with both heme and flavin functional domains. applications of Trichoderma species, and the important The complete CYP complement of one organism, called role that CYPs play in the biology of fungi, we decided to CYPome, is a collection of CYP genes in the genome of annotate and make an inventory of the CYPome in the that species . The current state of knowledge on P450 seven species of Trichoderma that have been sequenced evolution in eukaryotes points to CYP51 as the ances- by JGI. Annotation of these genes would help in commer- tral P450, which is believed to have led to the evolution cial exploitation of these proteins. Earlier, the CYPome of of all the present day P450 families . The expansion several fungal species have been analysed in detail, e.g., and diversification of CYPomes may also provide infor - Aspergillus nidulans , Phanerochete chrysosporium mation on fungal evolutionary adaptation to ecologi- , Mycosphaerella graminicola  and Grosmannia cal niches. A key development affecting applied P450 clavigera . However, this subject has not been cov- research is the need to define and annotate ever-expand - ered in earlier analyses of Trichoderma genomes, except ing genomic information. Various web-based resources for the inclusion of T. reesei in a broad analysis of fungal have been developed to probe and assign various orphan CYPomes . Moreover, a detailed phylogenetic analy- CYPs in numerous genomes, owing to the identifica - sis of Trichoderma CYPome could advance our under- tion of conserved motifs responsible for oxygen and standing of the evolutionary processes of cytochrome heme-binding. These databases reveal that enormous P450 proteins in fungi. Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 3 of 15 Abundance and diversity of cytochrome P450 family/clan Results Identified cytochrome P450 proteins were annotated and CYP proteins in Trichoderma classified into 85 families (Fig. 1) and 37 clans (Fig. 2). Trichoderma CYPome embodies a group of cytochrome Trichoderma species showed diversity in the number of P450 diverse proteins which are predicted to partici- annotated Cyp families (Table 1, Figs. 1, 2, 3). The num - pate in a spectrum of functions involved in primary, bers of annotated Cyp families among Trichoderma spe- secondary and xenobiotic metabolism. A total of 595 cies ranged from 36 (T. atroviride) to 67 (T. harzianum). cytochrome P450 proteins have been identified in seven Annotated CYP clans were also found to be diverse in Trichoderma species. These entries were further analysed Trichoderma (Fig. 3). The highest numbers of CYP clans for the presence of full cytochrome P450 domain which were identified in T. harzianum (31) and T. virens (31). led to the selection of a total 477 Cyp proteins (Table 1) T. asperellum and T. atroviride contained 25 and 22 clan for the detailed study. Entries with incomplete sequences types respectively. Clans CYP52 and CYP65 were found and domains are listed in Additional file 1: Table S1. to be most abundant with 55 and 56 protein entries, Analysis showed that T. harzianum genome harbours respectively (Fig. 3). The number of proteins in the most the highest number of Cyps (101), followed by T. virens abundant clans CYP52 and CYP65 ranged from 6 to 12 (90), T. asperellum (62), T. atroviride (57), T. citrinovir- among Trichoderma species. Clan CYP673 was identi- ide (57), T. reesei (57) and T. longibrachiatum (53). The fied only in T. virens and T. harzianum, containing 1 and number of Cyp proteins for families Cyp5080, Cyp52, 2 members respectively, and was found to be absent in Cyp534, Cyp535, Cyp541 and Cyp618 were found con- other five species. Similarly, clan CYP56 proteins were served among seven Trichoderma species. Cytochrome found to be unique to T. asperellum, T. harzianum and P450 families Cyp504, Cyp505, Cyp5080, Cyp51, Cyp52, T. virens with single entries in each species. Clan540 Cyp528, Cyp534, Cyp535, Cyp539, Cyp541, Cyp548, proteins were found absent in T. citrinoviride, T. longi- Cyp570, Cyp58, Cyp584, Cyp61, Cyp618, Cyp620, Cyp65 brachiatum and T. reesei. Clans CYP5042, 642, 659 and and Cyp671 were found ubiquitously present in Tricho- 677 were identified only in T. virens and were absent in all derma suggesting a conserved role of these proteins. In other species. Trichoderma, Cyp families Cyp5039, Cyp5044, Cyp5046, Cyp5049, Cyp5055, Cyp5057, Cyp5060, Cyp5128, Phyletic distribution of CYP families and clans Cyp5129, Cyp5134, Cyp5168, Cyp5181, Cyp5246, in Trichoderma Cyp5262, Cyp5268, Cyp5292, Cyp5296, Cyp5320, The genome-wide comparisons and annotations of P450s Cyp5334, Cyp5390 and Cyp5391 didn’t have any matches have allowed us to further develop the relationships in Fungal Cytochrome P450 Database (FCPD). The among Cyp families in different Trichoderma species. cytochrome P450 families unique to Trichoderma were To demonstrate the divergence of the primary sequences identified as Cyp5039, Cyp5049, Cyp5055, Cyp5057, and evolutionary relationships of cytochrome P450 fami- Cyp5128, Cyp5129, Cyp5134, Cyp5268, Cyp5292, lies in Trichoderma, a detailed phylogenetic analysis was Cyp5296, Cyp5390 and Cyp5391. These families were carried out using 477 aligned Cyp protein sequences. The predicted to be involved in both xenobiotic and second- phylogenetic tree depicting evolutionary relationships ary metabolism (Table 2). among Trichoderma cytochrome P450 proteins are illus- trated in Fig. 4. Further, the distribution of different CYP Table 1 Taxonomic distribution of putative CYPs in seven Trichoderma species Species Genome size No. of predicted Total Cyp pro- Proteins Clan type Family type Families with no (Mb) genes teins with complete FCPD matches sequences T. asperellum 37.46 12,586 73 62 25 40 7 T. atroviride 36.10 11,863 69 57 22 36 5 T. citrinoviride 33.48 9397 75 57 23 41 6 T. longibrachiatum 32.24 10,792 68 53 21 38 4 T. reesei 34.10 9129 70 57 23 42 4 T. harzianum 40.98 14,095 118 101 31 67 12 T. virens 39.00 12,427 122 90 31 59 12 Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 4 of 15 Table 2 Phylogenetic clustering of Trichoderma CYP families and clans Phylogenetic group ID Total entries CYP family CYP Clan Putative functions 1 33 Cyp5044 , Cyp5078, Cyp5080, Cyp5104, CYP528, CYP531, CYP532 Xenobiotic metabolism Cyp528, Cyp531, Cyp532, Cyp5320 , Cyp631 2 19 Cyp535, Cyp570 CYP507 Xenobiotic metabolism 3 3 Cyp673 CYP673 a a a 4 11 Cyp5055, Cyp5057, Cyp5262 , Cyp537, CYP537, CYP62 Xenobiotic metabolism Cyp62, Cyp684 Secondary metabolism a a 5 35 Cyp5039 , Cyp5094, Cyp5128 , CYP58, CYP677 Secondary metabolism a a Cyp5129, Cyp5292 , Cyp551, Cyp552, Xenobiotic metabolism Cyp58, Cyp677, Cyp680, Cyp682 6 10 Cyp5246 , Cyp53 CYP53 Xenobiotic metabolism 7 3 Cyp630 CYP630 Primary metabolism 8 23 Cyp574, Cyp5076, Cyp5168 , Cyp671 CYP574 Secondary metabolism 9 17 Cyp548 CYP548 Xenobiotic metabolism 10 56 Cyp5117, Cyp561, Cyp563, Cyp65 CYP65 Secondary metabolism 11 3 Cyp627 CYP627 a a 12 62 Cyp5049 , Cyp52, Cyp5296 , Cyp538, CYP52, CYP59 Xenobiotic metabolism Cyp539, Cyp584, Cyp587, Cyp655 a a 13 19 Cyp5181, Cyp5334 , Cyp534, Cyp613, CYP534, CYP613 Xenobiotic metabolism Cyp685 a a 14 39 Cyp5134 , Cyp526, C yp5390 , Cyp617, CYP526, CYP547 Secondary metabolism Cyp618 15 36 Cyp505, Cyp5099, Cyp540, Cyp541 CYP505, CYP540, CYP56 Primary metabolism 16 9 Cyp504 CYP504 Xenobiotic metabolism a a 17 51 Cyp5046 , Cyp5068, Cyp5268 , Cyp530, CYP530, CYP533 Xenobiotic metabolism Cyp5391 , Cyp620, Cyp621 18 1 Cyp5042 CYP5042 19 18 Cyp503, Cyp5090, Cyp559, Cyp611, CYP54, CYP550, CYP559, CYP642, Secondary metabolism Cyp635, Cyp636, Cyp641, Cyp642 CYP657, CYP659 20 29 Cyp5060 , Cyp51, Cyp55, Cyp61 CYP51, CYP55, CYP61 Primary metabolism Corresponding clans for these families are absent in FCPD clans and families in 20 phylogenetic groups with their conserved putative role of Cyp535 and Cyp570 in xeno- putative functions are summarized in Table 2. biotics metabolism. Clan CYP673 in group 3 consists of Evolutionary studies differentiated 477 cytochrome only three members-two from T. harzianum and one P450 proteins from 7 Trichoderma species into 20 phy- from T. virens. logenetic groups (Fig. 4). Group 1 consisted of a total Group 4 consists of 11 proteins from 2 clans (CYP537 of 33 Cyp proteins from clans CYP528, CYP531 and and CYP62). In FCPD, clan CYP537 consists of two CYP532. In Trichoderma, clan CYP531 consists of five families: Cyp537 and Cyp577. In Trichoderma, Cyp577 Cyp families including Cyp5078, Cyp5080, Cyp5104, family is absent and Cyp537 proteins are present only Cyp531 and Cyp631. Group 2 consisted of total 19 pro- in T. asperellum, T. atroviride, T. citrinoviride and T. tein members belonging to clan CYP507. Members of harzianum. In group 4, all identified members of clan clan CYP507 have been predicted to be involved in xeno- CYP62 grouped together. Clan CYP62 in FCPD consists biotic metabolism in Pezizomycotina . In FCPD, clan of three CYP families including CYP62, CYP626 and CYP507 consists of four Cyp families including Cyp 507, CYP684. In Trichoderma, one Cyp62 (T. harzianum) Cyp525, Cyp535 and Cyp570. Of these four families, only and three Cyp684 proteins (one each) were identified in Cyp535 and Cyp570 families are present in Trichoderma T. atroviride, T. harzianum and T. virens. Group 4 also species. Group 2 containing clan CYP507 proteins was contained Cyp50555, Cyp5057 and Cyp5262 proteins. further differentiated into two sub-groups containing The corresponding clans for these three families are families Cyp535 (7 proteins) and Cyp570 (12 proteins) absent in FCPD. Protein Cyp5262 was grouped together respectively. In Trichoderma, all 19 proteins belonging with members of clan CYP537, whereas Cyp50555 and to clan 507 are grouped together in group 2 suggesting Cyp5057 proteins formed a separate subgroup in Group Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 5 of 15 Fig. 1 Cytochrome P450 families identified in Trichoderma 4. Group 5 contained total 35 proteins belonging to clans longibrachiatum, T. reesei and T. virens. These proteins CYP58 and CYP677, which includes diverse Cyp fami- are known to be involved in xenobiotic metabolism. The lies Cyp5039, Cyp5094, Cyp5128, Cyp5129, Cyp5292, second largest phylogenetic Group 10 has 56 Cyps from Cyp551, Cyp552, Cyp58, Cyp677, Cyp680 and Cyp682. the clan CYP65 which are involved in secondary metabo- Clans for Cyp families 5039, 5128, 5129 and 5292 are not lism. It comprised of families Cyp5117, Cyp561, Cyp563 available in FCPD. Trichoderma has only one Cyp677 and Cyp65. Group 11 consists of three Cyp627 proteins. protein i.e., in T. virens which was grouped closely with In Trichoderma, group 12 is the largest with 62 Cyp Cyp5292 in phyletic Group 5. A total of 26 proteins proteins. These Cyps from clans CYP52 and CYP59 belonging to clan CYP58 are identified in Trichoderma. were differentiated separately into two sub-groups. Cyp58 family had a single member in all Trichoderma Clans CYP52 and CYP59 involve members of Cyp52, species analysed except in T. virens (2 proteins). All 7 Cyp538, Cyp539, Cyp584, Cyp587 and Cyp655 families. members of CYP53 clan were grouped together in Group Two entries of Cyp587 family belonging to clan CYP59 6. Cyp53 family was found in all Trichoderma species were grouped together with two proteins each from except T. longibrachiatum. These proteins are involved in Cyp5049 and Cyp5296 families. The corresponding clan xenobiotic metabolism. The group 6 also contained three for Cyp5049 and Cyp5296 families were found to be Cyp5246 proteins, clan for this family is absent in FCPD. absent in FCPD. In group 12, Cyp proteins of clan CYP52 Family Cyp5246 is present only in T. harzianum and T. were grouped together in the separate sub-group. Group virens. Members of Cyp53 and Cyp5246 families were dif- 13 contained 19 Cyps belonging to clans CYP534 and ferentiated in two clear sub-groups. Group 7 consists of CYP613. Two Cyp proteins belonging to family Cyp5181 only three proteins belonging to clan CYP630; one each were also present in group 13. Protein members of from T. harzianum, T. longibrachiatum and T. virens. The groups 12 and 13 were predicted to be involved in xeno- group 8 consists of 23 proteins from clan CYP574 includ- biotic metabolism (Table 2). ing families Cyp5076, Cyp574 and Cyp671. Four mem- Group 14 consists of proteins belonging to clans bers of Cyp5168 family were also clustered in group 8. CYP526 and CYP547 which were differentiated sepa - Group 9 consists of all 17 proteins of clan CYP548. In rately into two sub-groups. In Trichoderma, 2 Cyp fami- Trichoderma, Cyp548 family is ubiquitously present in lies of clan CYP547 were identified that includes Cyp617 all seven species, where T. asperellum and T. atrovir- (7) and Cyp618 (7). Cyp5134 proteins were grouped ide contained four and three proteins respectively fol- together in sub-group containing clan CYP526 proteins. lowed by two each in T. citrinoviride, T. harzianum, T. Group 15 consists of 36 proteins involved in primary Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 6 of 15 Fig. 2 Abundance of cytochrome P450 families and clans in Trichoderma. The heatmap displays the abundance of Cyp protein families among Trichoderma species. Blue bar represent the number of Cyp proteins in a clan Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 7 of 15 Fig. 3 Diversity of cytochrome P450 clans among Trichoderma species. Scatter-plot presents number of proteins in CYP clans in T. asperellum (orange), T. atroviride (green), T. citrinoviride (black), T. harzianum (purple), T. longibrachiatum (dark blue), T. reesei (red) and T. virens (light blue) metabolism that includes members of clans CYP505 CYP659. These clans are involved in secondary metabo - (15), CYP540 (11), CYP541 (7) and Cyp5099 (3). All lism. A total of 29 proteins from 7 Trichoderma species three Cyp5099 proteins belonging to clan CYP56 family corresponding to three clans including CYP51, CYP55 were included in this group. These proteins were identi - and CYP61 were clustered together in group 20. These fied only in T. asperellum, T. harzianum and T. virens. are known to be involved in primary metabolism. In this Cyp5099 proteins were found closely related to Cyp540 group, CYP51 and CYP61 families dominate with 9 and proteins and together formed a separate sub-group. 15 members respectively. Further, all proteins belonging Another sub-group contained all proteins belonging to to Cyp51 were grouped together in group 20. This sug - clan CYP505 which includes Cyp505 and Cyp541 fami- gests that Cyp51 protein which is involved in primary lies. All nine protein members of clan CYP504 were clus- metabolism (sterol biosynthesis) is diversified only to a tered together in group 16. These proteins are known to lesser extent in Trichoderma. In comparison to some of be involved in xenobiotic metabolism. Trichoderma spe- the ascomycetous fungi, which carry multiple CYP51 cies contain single copy of Cyp504 protein except T. har- proteins, T. atroviride and T. harzianum contained two zianum which contains three copies of Cyp504 protein copies each, whereas T. asperellum, T. citrinoviride, T. involved in phenylacetate catabolism . longibrachiatum, T. reesei and T. virens contained only Group 17 is the third largest Cyp group consisting of single copy of Cyp51 protein. 51 Cyps from clans CYP530 and CYP533. In this group, CYP533 is the most dominant clan followed by CYP530. Characteristic motifs of the Trichoderma CYP families Clans CYP530 and CYP533 include Cyp families Cyp530 Several signature motifs are conserved in fungal Cyp (8 proteins) and Cyp5068 (1 protein), and Cyp620 (23 proteins as per pervious findings [26, 35, 37, 38]. In proteins) and Cyp621 (4 proteins) respectively. This group Trichoderma, we identified the characteristic signature also contained Cyp5046 (4), Cyp5391 (8) and Cyp5268 motifs of CYP super family AGXDTT, EXXR, PERW (3) proteins. The corresponding clans for these families and FXXGXRXCXG for each phylogenetic group (Fig. 5). are absent in FCPD. Group 18 contains one Cyp5042 pro- These motifs are functionally essential for the Cyp pro - tein of T. virens. Group19 includes 18 proteins belonging teins. Conserved motif FXXGXRXCXG (also known as to clans CYP54, CYP550, CYP559, CYP642, CYP657 and CXG) is designated as a heme-binding domain [26, 29, 39] Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 8 of 15 Fig. 4 Evolutionary relationships of cytochrome P450 proteins among Trichoderma species. Phylogenetic tree was constructed inferred using the minimum evolution method using MEGA5 software. Phylogenetic groups (1–20) and bootstrap frequencies are shown in the tree. Tree includes Cyp proteins from all seven Trichoderma species including T. asperellum, T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum, T. reesei and T. virens. Each phylogenetic group is indicated by a specific color and includes a conserved cysteine residue that binds to previous reports [37, 40]. In phylogenetic groups 13, 16 the Fe of the heme. In Trichoderma, the cysteine residue and 19, Cyp proteins contain glutamate/aspartate, tyros- of the P450 signature CXG motif is invariantly conserved ine and glycine respectively instead of a phenylalanine in all P450s, whereas two glycine and one phenylalanine residue. Another variant of FXXGXRXCXG motif was residues were also found to be conserved among major- found in groups 1, 6 and 20 where first amino acid resi - ity of phylogenetic groups, which are in accordance with due of the motif was either phenylalanine or tryptophan. Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 9 of 15 Fig. 5 Conserved signature motifs of Trichoderma Cyp proteins for 20 phylogenetic groups Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 10 of 15 Further, in groups 5, 8, 12 and 20, FXXGXRXCXG and enormously wide range of substrate specificities and their FXXGXRXCXA variants were identified. Conserved substrate-binding regions. motif EXXR is present in helix K, on the proximal side of heme and probably is involved in the stabilization of Cytochrome P450s associated with secondary metabolism the core structure of Cyp proteins [26, 35, 39]. In Tricho- related gene clusters derma motif EXXR, glutamic acid and arginine residues A survey of the genomes of seven Trichoderma spp. were found to be highly conserved, whereas two mid- revealed that of the 477 cytochrome P450 genes present dle ‘XX’ residues were found to be highly variable. These in the seven genomes, as many as 100 genes are associ- results are in concurrence with previously reported lit- ated with putative secondary metabolism related gene erature for fungal cytochrome P450 proteins [35, 37, clusters namely NRPS, PKS, NRPS-PKS, NRPS-like, and 40, 41]. Another conserved motif of cytochrome P450 terpene cyclase clusters (Additional file 1: Table S2). protein family is PERW (known as PER) which forms E-R-R triad in Cyp proteins . In Trichoderma, we Discussion found PERW as the predominant signature, in accord- Trichoderma species are the champions of opportunis- ance with previous reports in fungi [27, 35]. Motif PERW tic success . They can be found virtually in all ecologi - was found to be relatively conserved in Trichoderma cal niches, both terrestrial and aquatic. These fungi are with few exceptions that mainly includes phylogenetic capable of parasitizing a wide range of fungal and oomy- groups 19 and 20. Group 19 consists of Cyp proteins cetes species. Many species are known to colonize the from clans CYP54, CYP550, CYP559, CYP642, CYP657 rhizosphere and roots, both externally and internally . and CYP659, which have been predicted to play role in Some are reported to be endophytes  while a few are secondary metabolism. High diversity of PER motif of aggressive parasites on cultivated mushrooms . A few this group could be attributed to the evolving functions species are known to be opportunistic human pathogens of Cyp P450 protein members. Phylogenetic group 20 while some strains are nematode-parasite, demonstrat- consisting of clans CYP51, CYP61 and CYP55 includes ing their ability to parasitize members of animal king- Cyp proteins belonging to both class E (CYP51 and dom . Several Trichoderma strains are plant growth CYP61) and B (CYP55). In this group, variant of PERW enhancers and some can colonize composts . A few motif was identified where clan CYP55 proteins (class strains are known to be xenobiotics degraders. Most spe- B) contained amino acid residues K/E/Q between PER cies are prolific producers of a wide range of secondary and W/Y. The absence of an amino acid residue between metabolites, with a total of more than 1000 compounds arginine and tryptophan residues in “PERW” motif in all chemically characterized . Cytochrome P450s are class E Cyp proteins indicate the early functional diver- important for cells to perform a wide variety functions gence of PERW motif in class B and E cytochrome P450 like primary and secondary metabolism, xenobiotic deg- proteins. These results provide an insight on the struc - radation and cellular defence (e.g., in interaction with ture–function relationships in such a diverse and com- other fungi). Recently, a T. virens P450 (TvCyt2; Protein plex Cyp protein families. Further, we also identified Id. 190045) has been shown to be involved in biocontrol conserved motif, AGXDTT in Trichoderma cytochrome and plant growth promotion . Basidiomycetes are P450 proteins. Motif AGXDTT contributes to oxygen capable of metabolizing a wide range of endogenous and binding and activation . The oxygen-binding domain exogenous compounds by using cytochrome P450s . (AGXDTT) was found to be highly variable in Tricho- Great deal of information is available on the role of P450s derma cytochrome P450 proteins. The terminal threo - in degradation of lignins and polyaromatic hydrocarbons nine residue in AGXDTT motif involved in the formation by white rot fungus Phanerochaete chrysosporium and of the enzyme’s critical oxygen-binding pocket was found brown-rot fungus Postia placenta, as well as medicinal to be replaced predominately by valine in phylogenetic mushrooms like Coriolus versicolor and Lentinula edodes group 16. Other amino acid residues that replaced ter- [48–51]. Role of P450s in colonization of living wood by minal threonine in different groups included serine or the plant pathogen Heterobasidion irregulare is also well methionine. For motifs AGXDTT and CXG, Cyp proteins established . in phyletic groups 13, 19 and 20 (Table 2) were relatively In the present study, Trichoderma CYPome from seven less conserved, suggesting divergence of these Cyp pro- Trichoderma species viz. T. asperellum, T. atroviride, T. tein sequences and their functions in Trichoderma. We citrinoviride, T. harzianum, T. longibrachiatum, T. reesei found that the conserved signature motifs and their vari- and T. virens is annotated. Overall, our analysis identified ants identified in Trichoderma showed few exceptions a total of 477 CYPs in these genomes. To provide support to previous reports. These results suggest Cyp signature for the annotation process, the identified CYPs were also motifs have evolved in Trichoderma to accommodate examined for conserved CYP domain. Our analysis of Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 11 of 15 the CYPome has identified 12 families unique to Tricho- All members of clan CYP65 are involved in secondary derma. All the Trichoderma species examined are a rich metabolism. CYP65 is reported to catalyze the epoxida- source of Cyp proteins (55 in T. longibrachiatum to 100 tion reaction during the synthesis of trichothecenes [56, in T. harzianum). 57] and radicicol . Identification of conserved and In Trichoderma, clan CYP52 consisted of families variable CYP motif signatures among and within phy- Cyp52, Cyp538, Cyp539, Cyp584 and Cyp655. Cyp52 logenetic groups in the present study may provide us family is found only amongst Candida-related spe- information on CYP evolution, structure, and function cies of fungi and these proteins catalyze the conversion in Trichoderma and have application in classification of of fatty acids and alkanes to alpha, omega-dicarboxylic proteins in gene expression analysis . acids . The number of Cyp61 proteins was con - Cyp56 clan, found to be unique to T. asperellum, T. served in all Trichoderma species and these proteins harzianum and T. virens (mycoparasites) has been char- were also grouped together in Group 20. Cyp61 proteins acterized earlier in yeast [59, 60]. Members of Cyp56 clan are involved in primary metabolism. In Saccharomyces are involved in meiotic spore wall biogenesis, particularly cerevisiae, CYP61 codes for sterol 22 desaturase , in dityrosine biosynthesis [59–61]. Members of the clan which is involved in later stages of the ergosterol pathway CYP507, CYP530, CYP531, CYP532 and CYP548 are in metabolizing Ergosta-5,7,24(28)-trienol to Ergosta- known to be involved in xenobiotics metabolism . 5,7,22,24(28)-tetraenol by introducing a C-22(23) double Abundance of these proteins in Trichoderma may be bond in the sterol side chain. Since Cyp61 is involved in related to the ability of these fungi to metabolize a wide the later stages of ergosterol pathway, it is considered to range of xenobiotics, including many fungicides. Simi- have evolved as a result of duplication and diversification larly, ability of Trichoderma spp. to produce a plethora of the CYP51 gene. In ascomycetes and basidiomycetes, of secondary metabolites could be linked to the abun- clan CYP51 is involved in sterol biosynthesis and is rec- dance of P450s belonging to the clan CYP574, CYP58 ognized as the housekeeping CYP, and has been a popu- and CYP65 proteins that have been implicated in tri- lar antifungal target for the control of fungal diseases in chothecene biosynthesis . In T. harzianum, three humans and crop plants [29–31, 55]. In comparison to copies of Cyp504 protein are present as compared to some of the ascomycetous fungi, which carry multiple single copy in other Trichoderma species. Expansion of CYP51 genes, T. atroviride and T. harzianum contained Cyp504 proteins in T. harzianum suggest important role two copies whereas T. asperellum, T. citrinoviride, T. lon- of Cyp504 protein in xenobiotic metabolism. Further, gibrachiatum, T. reesei and T. virens contained only single the family members of Cyp504 were also reported to be copy of Cyp51 protein. In addition, all members of both up-regulated during cuticle infection by insect patho- clans CYP51 and CYP61 which are involved in primary genic fungi Metarhizium anisopliae and M. acridum . metabolism (sterol biosynthesis) are grouped together in Cyp505 family was found to be expanded in T. asperel- group 20, suggesting that both Cyp51 and Cyp61 proteins lum, T. harzianum and T. virens where these species are diversified only to a lesser extent in Trichoderma. contained three Cyp505 proteins each. Cyp505 proteins Motif analysis led to the identification of four signa - are membrane-associated fatty acid hydroxylase . ture motifs in phylogenetic groups, which correspond to Cyp528 family has only one protein entry in all Tricho- the conserved tertiary structure and enzyme functions derma species analysed except T. atroviride where family in spite of the wide sequence diversity and functions of Cyp528 consisted of two proteins. Similarly, Cyp58 fam- Cyp proteins. Modifications found in the heme-binding ily has a single protein entry in all Trichoderma species domain FXXGXRXCXG are more frequently found in analysed except in T. virens where family Cyp58 consisted CYPs with catalytic activity, often not requiring oxygen of two proteins. Previous studies also showed expansion . These results indicate Cyp members of groups 5, of clan Cyp58 proteins in fungi . In Trichoderma, the 8, 12, 13, 16, 19 and 20 may have novel catalytic activi- increase in CYPome size of T. harzianum and T. virens ties in Trichoderma. Some P450s showed variations of may be due to the expansion of certain CYP gene families the signature motifs mainly in AGXDTT, EXXR and or the presence of novel genes that are essential for their FXXGXRXCXG motifs. These results are in accordance lifestyle. Previous reports have associated expansions of with previous reports [38, 41] where it was proposed the fungal CYP families with the evolution of various that these P450s variations may be due to misaligned fungal traits including pathogenicity . Our phyloge- sequences or that the P450s are missing the invariant netic analysis showed uneven distribution of CYP group residues at these motifs. In our study, phylogenetic group sizes in Trichoderma species, which are in concordance 10 containing protein families of clan CYP65 showed with extreme expansions and contractions of certain CYP highly conserved motifs, suggesting functional conser- families in the course of evolution. Expansion of CYP vation of CYP65 clan in analysed Trichoderma species. Chadha et al. Fungal Biol Biotechnol (2018) 5:12 Page 12 of 15 clans in different Trichoderma species could aid them in Annotation of CYPs more competent survival in their respective habitats. The annotation pipeline of the CYPome in the Tricho - Trichoderma spp. are prolific producers of secondary derma species was done in a two-step procedure of metabolites, many with antimicrobial, anticancer and identification and annotation. The identification step of plant growth-promoting properties  Cyps are known CYP family was performed by using Conserved Domain to play central role in biosynthesis if many, if not most of Database (CDD); the cut-off of positive hits was set at −2 the secondary metabolites of plant and microbial origin. E-value of 10 . Entries with incomplete sequences and Till date, however, only a handful of Trichoderma Cyps domain were manually removed from the data. Cyp have been investigated for their role in biosynthesis of proteins with complete conserved cytochrome P450 secondary metabolites [46, 66, 67]. Our present findings domains were further subjected to the annotation proce- suggest that more than 20% of the catalogued Cyps from dure using the Nelson’s P450 database against all named Trichoderma are part of putative secondary metabolism- fungal cytochrome P450s (http://blast.uthsc.edu) with −4 related gene clusters. There is a need for systematic stud - the E-value of 10 . For annotation, sequence simi- ies on the functions of these Cyps which would lead to larity cut-off of 40% was used. For few entries, we have the discovery of novel pathways, metabolites and inter- followed criteria of the phenomenon called family creep mediates with greater biotechnological significance. that allows sequences less than 40% to be included in a family. For such entries, we have used sequence similar- Conclusion ity cut-off of 30% and above. These predicted CYPs were Trichoderma CYPome described in our study is by com- then assigned to the corresponding family and clan types bining information generated from existing databases, based on their highest homology according to the Inter- predicting conserved domains and identifying structural national P450 Nomenclature Committee Databases used motifs in each hypothetical protein. By following interna- by Nelson (http://drnelson.uthsc.edu/CytochromeP450. tionally recognized nomenclature system, we have identi- html)  and the fungal cytochrome P450 database fied novel CYP clans and families unique to Trichoderma. (http://p450.riceblast.snu.ac.kr)  respectively. Phylogenetic analysis elucidated distribution of Cyp fam- ilies and clans in different evolutionary groups and their Structural feature analysis of CYP protein sequences probable functions in metabolism or biosynthesis based Presence of cytochrome P450 conserved domain was on the comparisons with CYPomes of other organisms. confirmed using conserved domain database . To The number of these proteins correlates with the genome reveal phylogenetic group-specific conservation pattern size and many are species-specific. Unfortunately, the of cytochrome P450 proteins, structural features were functions of none of these proteins are known. One rea- explored. To identify cyp conserved signature motifs, son being a lack of systematic studies and annotation of multiple protein sequence alignments for each phyloge- these proteins. Our CYPome annotation and evolution- netic group were built by MAFFT program  using ary studies of seven Trichoderma species now provides E-INS-i iterative refinement method. Alignments were opportunities for exploration of research-driven strate- further refined and viewed using AliView . Consen - gies to select Trichoderma species for various applica- sus logos of the alignments were automatically generated tions especially in relation to secondary metabolism and by WebLogo 3 program  and used for visualization degradation of environmental pollutants. Several of these of the conservation of signature motifs for each phylo- proteins could also have biotechnological applications genetic group. The generated logos were used for the like biotransformation and synthesis of pharmaceutically analysis. important drugs. Phylogenetic reconstruction of CYPs Methods After removal of redundant and incomplete sequences, Sequence data the protein sequences were aligned using MUSCLE . Sequences of Cytochrome P450s were retrieved from the The evolutionary history was inferred using the mini - Joint Genome Institute (JGI) fungal genome database mum evolution method . The bootstrap consensus MycoCosm (http://genome.jgi-psf.org/programs/fungi/ tree inferred from 1000 replicates was taken to represent index.jsf ) for all the species of genus Trichoderma. The the evolutionary history of the taxa analysed . The species included were T. asperellum (CBS 433.97) v1.0, evolutionary distances were computed using the Pois- T. atroviride (IMI 206040) v2.0, T. citrinoviride v4.0, T. son correction method  and are in the units of the harzianum (CBS 226.95) v1.0, T. longibrachiatum (ATCC number of amino acid substitutions per site. The rate 18648) v3.0, T. reesei (QM 6a) v2.0 and T. virens (Gv29-8) variation among sites was modelled with a gamma distri- v2.0. bution (shape parameter = 1). The ME tree was searched Chadha et al. 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Fungal Biology and Biotechnology – Springer Journals
Published: Jun 4, 2018
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