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Article

Comparative Genome-Wide Analysis Underscores the Rapid Expansion of Cytochrome P450s for Secondary Metabolism in the Mycoparasite Pezizomycetes

by
Puleng Rosinah Syed
1,
Tiara Padayachee
2,
Philasande Gamede
2,
Bridget Valeria Zinhle Nkosi
2,
David R. Nelson
3,
Rajshekhar Karpoormath
1,* and
Khajamohiddin Syed
2,*
1
Department of Pharmaceutical Chemistry, College of Health Sciences, University of KwaZulu-Natal, Durban 4000, South Africa
2
Department of Biochemistry and Microbiology, Faculty of Science, Agriculture and Engineering, University of Zululand, KwaDlangezwa 3886, South Africa
3
Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(3), 1251-1268; https://doi.org/10.3390/microbiolres15030084
Submission received: 21 June 2024 / Revised: 15 July 2024 / Accepted: 17 July 2024 / Published: 20 July 2024
Browse Figures
Versions Notes

Abstract

:
Mycoparasite secondary metabolites control fungal infections or diseases in agriculture and human health. Among genes involved in synthesizing secondary metabolites, cytochrome P450 monooxygenases (CYPs/P450s) play a key role in synthesizing and attributing diversity to the secondary metabolites. Despite the importance of P450s, a comparative analysis of P450s in mycoparasites has yet to be reported. This study is aimed at addressing this research gap. Genome-wide analysis of P450s in 43 fungi representing six fungal phyla and three distinct lifestyles, such as mycoparasitic (24 species), saprophytic (5 species), and ectomycorrhizal (14 species), revealed the expansion of P450s in Pezizomycete mycoparasites for the synthesis of secondary metabolites. The number of P450s and their families and subfamilies, the number of secondary-metabolite biosynthetic gene clusters (SMBGCs), and the number of P450s that are part of these SMBGCs were found to be highest in Pezizomycete mycoparasites compared to their counterparts of saprophytes and ectomycorrhiza, indicating P450s also play a key role in mycoparasitism. An analysis of P450 location as part of SMBGCs and the available literature on Pezizomycete P450s revealed that P450s play a key role in the synthesis of anti-fungal secondary metabolites such as trichothecene sesquiterpene, harzianum A, heptelidic acid, and gliotoxin. The mycoparasite Trichoderma virens Tv29.8 P450 CYP68Q3 is found to be a bifunctional enzyme with epoxidation and oxidation capability, and CYP5117A3 performs a Baeyer–Villiger oxidation reaction with regioselectivity. This study serves as a reference for future annotation of P450s in mycoparasites.

1. Introduction

Mycoparasitism occurs when one fungus attacks and parasitizes another. This form of lifestyle appears to be ancestral in fungi [1]. During mycoparasitism, parasitic fungi secrete a variety of compounds, including secondary metabolites (low-molecular-weight metabolites that are not required for growth or development but do provide ecological benefits under certain environmental conditions) that aid in the colonization of another fungus [2]. Mycoparasitism research has long been focused on using mycoparasites or their derived compounds, such as secondary metabolites, as biological control agents for plant fungal infections [3].
Mycoparasites can be found across the fungal phyla (Table 1). Among mycoparasites, Trichoderma species are well known for their role in mycoparasitism and as biological control agents of plant fungal pathogens [4]. Trichoderma species exhibit their antagonist activity against a fungus in several ways, such as secreting extracellular hydrolytic enzymes or secondary metabolites with anti-fungal activity or promoting plant growth to fight fungal diseases [4].
Trichoderma species produce a wide range of secondary metabolites that directly or indirectly help them to parasitize fungi [28,29,30]. Trichoderma species secondary metabolite types include non-ribosomal peptides (NRPs), polyketides (PKSs), terpenoids, and pyrones [28,29]. The well-known secondary metabolites produced by these species include gliotoxin and gliovirin, peptaibols, siderophores, trichothecenes (trichodermin and harzianum A), and heptelidic acid (koningic acid) [28,29,30,31,32]. The trichothecenes produced by Trichoderma species are used as a biofungicide to promote plant defence against fungal pathogens [4,30], and heptelidic acid is now being explored as a potential anti-cancer drug [33].
The genes involved in synthesizing some of these metabolites have been identified and found to reside near each other in the DNA, forming a biosynthetic gene cluster (BGC). Three well-known BGCs have been reported in Trichoderma species: the tri BGC involved in the synthesis of trichothecene [14,15,16,17], the vir BGC involved in the synthesis of volatile terpenes and heptelidic acid [12], and the gli BGC involved in the synthesis of gliotoxin [34,35]. Analysis of these three BGCs revealed the presence of cytochrome P450 monooxygenases (CYPs/P450s) as part of the cluster, and these enzymes were found to play a key role in synthesizing respective secondary metabolites [32,34,35,36,37,38]. P450s are heme-containing oxidoreductases ubiquitously present in organisms across the domains of life [39]. P450s perform enzymatic reactions with stereo- and regio-specificity and thus became a key player in biology. P450s follow a typical classification and nomenclature system that assigns names to P450s that reflect their family and subfamily. P450s share more than 40% of the amino acid identity belonging to the same family, and P450s share more than 55% belonging to the same subfamily [40]. The naming system includes assigning the root CYP indicating cytochrome P450, followed by a number indicating its family, then a letter indicating its subfamily, and finally, a number indicating its number within the subfamily [40].
Comparative genome sequence analysis of Trichoderma atroviride and T. virens revealed expansion of P450s in their genomes, and the authors proposed that these P450s, with other genes, were possibly involved in synthesizing secondary metabolites via new secondary-metabolite biosynthetic pathways [19]. Cytochrome P450 TvCyt2 from T. virens is involved in the synthesis of a secondary metabolite that causes antagonistic activity against phytopathogenic fungi and promotes plant growth [41]. Transcriptome analysis of T. atroviride revealed upregulation of three P450s before contact (2-fold), two P450s during contact (2.5- and 1.9-fold), and two P450s after contact (3.3- and 1.8-fold) during mycoparasitism with Rhizoctonia solani [34]. One P450, glic also upregulated before and after contact during T. virens mycoparasitism with R. solani [34]. GliC is involved in synthesizing mycotoxin gliotoxin, which is highly toxic to plant pathogens [42]. It is well established that three P450s (TRI4, TRI22, and TRI23) of T. arundinaceum are involved in trichothecene harzianum A (HA) biosynthesis [15,36,38,43]. Four P450s (Vir1–Vir3 and Vir5) are involved in the synthesis of volatile sesquiterpenes and heptelidic acid [32].
Despite the importance of P450 involvement in the synthesis of biotechnologically valuable compounds in mycoparasites and the availability of many mycoparasite genomes, to date, genome-wide comparative analysis of P450s across the mycoparasites has not been carried out except for analysis of P450s in a few Trichoderma species [36,44]. A total of 91 P450s belonging to 63 P450 families were identified in T. arundinaceum [36]. This study aims to address this research gap by performing genome-wide identification, annotation (assigning the family and subfamily), phylogenetic analysis, and mapping the P450s in secondary-metabolite BGCs in 24 mycoparasites belonging to six different fungal phyla. Furthermore, the study intends to investigate the effect of diverse lifestyles on the P450 content in terms of mycoparasitic, saprophytic, and ectomycorrhizal lifestyles within the class Pezizomycotina.

2. Materials and Methods

2.1. Species and Database

A total of 24 genomes of mycoparasites belonging to six fungal phyla were used in this study (Table 2). Genomes of all the mycoparasites have been published and are available for public use at the Joint Genome Institute (JGI) MycoCosm portal [45]. Fungal species were assigned to different phyla and classes as per the information available at Mycobank [46].

2.2. Genome Mining of Mycoparasites for P450s

For P450 mining in mycoparasites, we followed the well-established procedure previously described by our laboratory [47]. Each mycoparasite genome was searched for P450s using the InterPro code “IPR001128”. The hit protein sequences were downloaded and searched for P450 characteristic motifs, including the EXXR and CXG motifs [48,49]. Hit proteins with all P450 characteristic motifs were considered P450s, and proteins with one of these motifs or short in amino acid length (less than 350 amino acids) were considered P450 fragments. Proteins considered P450s were subjected to P450 family and subfamily analysis.

2.3. Assigning P450 Family and Subfamily

Hit proteins were assigned to different P450 families and subfamilies following the International P450 Nomenclature criteria [40]. Proteins with >40% and >55% amino acid identity were grouped under the same P450 family and subfamily. Proteins with less than 40% identity to the named homologues were assigned to new P450 families. To find the percentage identity with named homologue P450s, hit proteins were subjected to the Basic Local Alignment Search Tool (BLAST) against all named fungal sequences on the Cytochrome P450 Homepage [40]. Based on their percentage identity with the named homologue P450, all the hit proteins were assigned to different P450 families and subfamilies following the International P450 Nomenclature criteria. Supplementary Dataset S1 contains the P450 sequences, given names, P450 fragment sequences, and no-hit proteins.

2.4. Phylogenetic Analysis

Phylogenetic analysis of P450s was carried out following the procedure previously published by our laboratory [47]. The P450 protein sequences were briefly aligned by the MAFFT v6.864 [50] program available at the T-REX web server [51]. The alignments were then automatically subjected to interpret the best tree using the T-REX web server following the maximum likelihood method [51]. Finally, the best-inferred tree was visualized, coloured, and generated by the Interactive Tree Of Life (iTOL) [52].
Table 2. Mycoparasites used in the study, their genome database links, and reference articles are listed in the table. The genomes of mycoparasites were accessed on 24 March 2024.
Table 2. Mycoparasites used in the study, their genome database links, and reference articles are listed in the table. The genomes of mycoparasites were accessed on 24 March 2024.
Species NameGenome VersionPhylumClassSpecies CodeGenome Database LinkReference
Ampelomyces quisqualis HMLAC05119 v1.0AscomycotaPezizomycotinaAmpqui1https://mycocosm.jgi.doe.gov/Ampqui1[5]
Clonostachys rosea IK726 AscomycotaPezizomycotinaCloros1https://mycocosm.jgi.doe.gov/Cloros1[7]
Escovopsis weberi CC031208-10 AscomycotaPezizomycotinaEscweb1https://mycocosm.jgi.doe.gov/Escweb1[12]
Naematelia encephela UCDFST 68-887.2 v1.0AscomycotaPezizomycotinaTreen1https://mycocosm.jgi.doe.gov/Treen1[7]
Trichoderma arundinaceum IBT 40837 AscomycotaPezizomycotinaTriaru1https://mycocosm.jgi.doe.gov/Triaru1[16]
Trichoderma asperelloides T203 v1.0AscomycotaPezizomycotinaTriasper1https://mycocosm.jgi.doe.gov/Triasper1[17]
Trichoderma asperellum CBS 433.97 v1.0AscomycotaPezizomycotinaTrias1https://mycocosm.jgi.doe.gov/Trias1[19]
Trichoderma atroviridev2.0AscomycotaPezizomycotinaTriat2https://mycocosm.jgi.doe.gov/Triat2[17]
Trichoderma citrinoviride TUCIM 6016 v4.0AscomycotaPezizomycotinaTrici4https://mycocosm.jgi.doe.gov/Trici4[23]
Trichoderma gamsii T6085 AscomycotaPezizomycotinaTrigam1https://mycocosm.jgi.doe.gov/Trigam1[17]
Trichoderma guizhouense NJAU 4742 AscomycotaPezizomycotinaTrigui1https://mycocosm.jgi.doe.gov/Trigui1[25]
Trichoderma hamatum GD12 AscomycotaPezizomycotinaTriham1https://mycocosm.jgi.doe.gov/Triham1[17]
Trichoderma harzianum CBS 226.95 v1.0AscomycotaPezizomycotinaTriha1https://mycocosm.jgi.doe.gov/Triha1[17]
Trichoderma longibrachiatum ATCC 18648 v3.0AscomycotaPezizomycotinaTrilo3https://mycocosm.jgi.doe.gov/Trilo3[53]
Trichoderma pleuroti TPhu1 AscomycotaPezizomycotinaTriple1https://mycocosm.jgi.doe.gov/Triple1[19]
Trichoderma virens Gv29-8 v2.0AscomycotaPezizomycotinaTriviGv29_8_2https://mycocosm.jgi.doe.gov/TriviGv29_8_2[19]
Tremella mesenterica Friesv1.0BasidiomycotaAgaricomycotinaTreme1https://mycocosm.jgi.doe.gov/Treme1[15]
Caulochytrium protostelioides ATCC 52028 single-cellv1.0CaulochytriomycotaCaulochytriomycotinaCaupr_SCcombhttps://mycocosm.jgi.doe.gov/Caupr_Sccomb[7]
Caulochytrium protostelioides ATCC 52028 v1.0CaulochytriomycotaCaulochytriomycotinaCaupr1https://mycocosm.jgi.doe.gov/Caupr1[8]
Dimargaris cristalligena RSA 468 single-cell v1.0KickxellomycotaKickxellomycotinaDimcrSC1https://mycocosm.jgi.doe.gov/DimcrSC1[10]
Piptocephalis cylindrospora RSA 2659 single-cell v3.0ZoopagomycotaZoopagomycotinaPipcy3_1https://mycocosm.jgi.doe.gov/Pipcy3_1[54]
Syncephalis pseudoplumigaleata Benny S71-1 single-cell v1.0ZoopagomycotaZoopagomycotinaSynps1https://mycocosm.jgi.doe.gov/Synps1[7]
Thamnocephalis sphaerospora RSA 1356 single-cell v1.0ZoopagomycotaZoopagomycotinaThasp1https://mycocosm.jgi.doe.gov/Thasp1[7]
Rozella allomycis CSF55 Rozellomycota Rozal1_1https://mycocosm.jgi.doe.gov/Rozal1_1[7]
Note: The genome sequence release version is listed for a mycoparasite if available. Mycobank did not give a class for Rozella allomycis CSF55; thus, it was not listed. Species codes are the same as those listed at the Joint Genome Institute (JGI) MycoCosm portal [45].

2.5. Identification of P450s That Are Part of Secondary-Metabolite BGCs (SMBGCs)

P450s that are part of SMBGCs were identified following the method recently described by our laboratory [55]. Each of the SMBGCs listed in the Joint Genome Institute’s MycoCosm site [45] for each mycoparasite was manually searched for gene/protein sequences in that cluster. If a P450 was listed as a part of the cluster, the P450 protein ID and its sequence were noted and matched with its assigned name. The cluster ID, cluster type, scaffold information (genomic location), and P450s that were part of the cluster were presented in a table format as a standard practice. Twenty-one out of twenty-four mycoparasite SMBGCs were analysed in this study, as three mycoparasite SMBGCs are unavailable at JGI Mycocosm [45].

2.6. Saprophytic and Ectomycorrhizal Pezizomycete P450s and SMBGCs

For comparative analysis, information on saprophytic and ectomycorrhizal Pezizomycete P450s and SMGBCs was retrieved from Nsele et al. [47] and used for comparison with mycoparasites.

2.7. Functional Prediction of P450s

This study involved a large number of P450s and consisted of in silico components. Thus, we predicted the mycoparasite P450 functions based on the available literature and the characterized homologue P450s. It should be noted that in this study, we assigned P450 names to the genes/proteins used in the literature to enable researchers to properly cite these P450s in the future.

3. Results and Discussion

3.1. Pezizomycete Mycoparasites Have More P450s in Their Genomes

Genome-wide analysis of P450s in 43 fungi representing six phyla and three distinct lifestyles revealed the presence of 2341 hit proteins (Figure 1 and Table S1). Further analysis of hit proteins for characteristic P450 motifs (as indicated in Section 2.2) showed that not all hit proteins are P450s. Among the hits, 2084 had all of the P450 characteristic motifs and were thus designated P450s; 220 were P450 fragments, 7 were false positives, and 30 were different proteins, so they were marked as no hits (Table S1). The existence of false positives and no hits suggests that automatic P450 allocation is not always accurate, and manual P450 curation is required to determine the correct number of P450s in an organism. Among all fungal species analysed in this study, Clonostachys rosea IK726 has the highest number of P450s (218) followed by T. virens Gv29-8 (118 P450s), T. harzianum CBS 226.95 (117 P450s), T. guizhouense NJAU 4742 (104 P450s), and T. arundinaceum IBT 40837 (101 P450s), and Caulochytrium protostelioides has the lowest number of P450s (one P450) (Figure 1). Apart from Pezizomycotina mycoparasites, Thamnocephalis sphaerospora RSA 1356 single-cell has the highest number of P450s (38 P450s) followed by Dimargaris cristalligena RSA 468 single-cell (12 P450s) (Figure 1). Species from Pezizomycotina include 16 mycoparasites with 1332 P450s, five saprophytes with 204 P450s, and 14 ectomycorrhizal fungi with 464 P450s (Figure 1). The remaining eight mycoparasites are from four fungal phyla with 84 P450s (Figure 1). This indicates that among Pezizomycetes, mycoparasites have the highest number of P450s in their genomes, as the average number of P450s was 83 compared to 41 P450s in saprophytes and 33 P450s in ectomycorrhizal fungi. Furthermore, contrasting P450 patterns can be seen among mycoparasites of different phyla (Figure 1 and Table S1). The basidiomycete Tremella mesenterica Fries has the lowest number of P450s (eight P450s) compared to its counterpart basidiomycetes, such as white-rot or brown-rot fungi, indicating the loss of P450s [14]. In contrast, the mycoparasitic ascomycetes, especially from Pezizomycotina, had the highest number of P450s compared to their saprotrophic or ectomycorrhizal fungi, strongly suggesting the rapid expansion of P450s in Pezizomycete mycoparasites. Notably, such an expansion of P450s in fungi has been attributed to helping the species adapt to their ecological niche [56,57].

3.2. Pezizomycete Mycoparasites Have Highly Diverse P450 Families and Subfamilies

Based on the International P450 Nomenclature Committee rules [40] and phylogenetic analysis (Figure 2), the 1416 P450s from 24 mycoparasites can be grouped into 243 P450 families and 447 P450 subfamilies (Table S2). Phylogenetic analysis revealed that all P450s belonging to the same family aligned together on the tree, suggesting the nomenclature is correct. Among P450 families, CYP65 has the most members (108 P450), followed by CYP620 (51 P450s), CYP539 (41 P450s), CYP548, and CYP505 (39 P450s) (Table S2). Apart from this, only 42 P450 families had more than ten members, and the rest of the 201 P450 families had less than ten members, including 102 P450 families that were represented by a single member (Table S2).
Analysis of P450 families and subfamilies among 43 fungi revealed that mycoparasite C. rosea IK726 has the highest number of P450 families and subfamilies (Figure 1 and Tables S1 and S3). A total of 24 of the 43 fungi are mycoparasites with the highest number of P450 families (Figure 1 and Table S1), indicating that mycoparasite P450s are highly diverse. A detailed analysis of P450 families and subfamilies, with their respective members annotated in 24 mycoparasites, is presented in Table S3. One peculiar characteristic of mycoparasites was the presence of more than one copy of CYP51 and CYP61 P450s (Table 3). Fourteen mycoparasites from Ascomycota had more than one copy of these P450s (Table 3). All the fourteen mycoparasites listed in Table 3 have two copies of CYP51 and CYP61 except C. rosea IK726, T. guizhouense NJAU 4742, and T. harzianum CBS 226.95, which have three copies of CYP61. A point to be noted is that generally, fungal species have one copy of these P450s, and the presence of more than one copy of these genes is also observed for a few fungal species of Ascomycota [58,59].
Having more than one copy of these CYP51 and CYP61 P450s involved in the synthesis of ergosterol [60,61], an essential component of the fungal cell membrane [62], gives mycoparasites an advantage of synthesizing additional ergosterol and thus may overcome fungal cell membrane targeting metabolites [2,63]. However, experimental validation is needed to support the rationale of having more than one copy of these P450s.
A total of 1332 P450s from 16 Pezizomycete mycoparasites can be grouped into 214 P450 families and 397 P450 subfamilies (Table S4). A comparison of P450 families and subfamilies indicated that mycoparasites have more P450 families and subfamilies in their genomes than saprophytic and ectomycorrhizal Pezizomycetes (Figure 3A). Fifteen P450 families were commonly found in all Pezizomycetes analysed in this study, indicating their important role in these species’ physiology (Figure 3A). Pezizomycete mycoparasites shared 7 and 10 P450 families with saprophytes and ectomycorrhiza (Figure 3A). However, Pezizomycete mycoparasites had the highest number of P450 families that are unique (182 P450 families) compared to saprophytes (57 P450 families) and ectomycorrhiza (40 P450 families), indicating that mycoparasites have highest P450 family diversity (Figure 3A). Further comparison of P450 families among all mycoparasites (24 species) revealed that 55 P450 families were shared among all mycoparasites irrespective of their phyla (Figure 3B), suggesting these P450 families might play an important role in mycoparasite physiology, including in mycoparasitism, and thus are conserved in these species.

3.3. Mycoparasites Have a Large and Diverse Number of SMBGCs

A total of 713 SMBGCs were found in 21 mycoparasites, with an average of 34 SMBGCs in their genomes (Table S5). Among SMBGC types, PKS was dominant with 230 BGCs, followed by NRPS with 183 clusters, NRPS-like with 156 BGCs, TC with 58 BGCs, PKS-like with 42, HYRBID with 38 BGCs, and DMAT with 7 BGCs (Table S5). This indicates that mycoparasites produce more polyketide secondary metabolites than other secondary metabolites. Comparative analysis of BGCs among mycoparasites revealed that C. rosea IK726 has the highest number of BGCs (73), followed by T. pleuroti TPhu1 (60 BGCs) and T. virens Gv29-8 (58 BGCs). Among saprophytes, the highest number of 10 BGCs was found in three species: Morchella importuna CCBAS932, M. importuna SCYDJ1-A1, and Pyronema confluens CBS100304. Among ectomycorrhiza, the highest number of 14 BGCs was found in Sphaerosporella brunnea Sb_GMNB300, followed by 10 BGCs in Tuber melanosporum Mel28 (Table S5).
A distinct feature was observed upon genome-wide analysis of SMBGCs among Pezizomycetes (Figure 4). Among Pezizomycetes, mycoparasites have more SMBGCs in their genome than saprophytes and ectomycorrhiza (Figure 4 and Table S5). A total of 676 SMBGCs were found in 16 mycoparasites, with an average of 31 SMBGCs, compared to 45 SMBGCs in five saprophytes (with an average of 9 SMBGCs) and 12 SMBGCs in 14 ectomycorrhiza (with an average of 9 SMBGCs) Pezizomycetes (Figure 4 and Table S5). This indicates an almost four-fold increase in SMBGCs in mycoparasites compared to saprotrophic and ectomycorrhizal Pezizomycetes.
A further difference was observed concerning the dominant SMBGC types among Pezizomycetes (Figure 4 and Table S5). The PKS type with 230 BGCs was dominant in mycoparasites, followed by NRPS with 162 BGCs and NRPS-like with 146 BGCs, whereas NRPS-like, followed by PKS-like, was dominant in both the saprophytes and ectomycorrhiza (Figure 4). This suggests that Pezizomycete mycoparasites produce different types of secondary metabolites from their counterparts, such as saprophytes and ectomycorrhiza. Thus, these metabolites may help mycoparasites to adapt to their ecological niches.

3.4. Mycoparasites Have a Large Number of P450s as Part of Their SMBGCs

Analysis of SMBGCs in mycoparasitic fungi revealed that only 15 mycoparasites have P450s as part of their SMBGCs (Table S6). Among 673 SMBGCs, only 131 SMBGCs have P450s, which indicates that 5% of SMBGCs contain P450s (Table S6). Analysis of SMBGC cluster type with P450s revealed that PKS is the dominant type with 52 clusters, followed by NRPS with 35 clusters, TC with 16 clusters, NRPS-like with 12 clusters, HYBRID with 12 clusters, DMAT with 2 clusters, and PKS-like has a single cluster (Table S6). A comparison of SMBGCs with P450s among 15 species revealed that T. longibrachiatum ATCC 18648 has the highest percentage of SMBGCs with P450s (29%) followed by T. virens Gv29-8 (28% of SMBGCs), T. citrinoviride TUCIM 6016 (27% of SMBGCs), and T. guizhouense NJAU 4742 (24% of SMBGCs) (Table S6). The lowest number of SMBGCs with P450s was found in Ampelomyces quisqualis HMLAC05119 (9% of SMBGCs) and T. hamatum GD12 (7% of SMBGCs) (Table S6).
A total of 191 P450s belonging to 69 P450 families were found to be part of SMBGCs, indicating that 14% of P450s from 15 mycoparasites are possibly involved in secondary-metabolite synthesis (Table S6). Among the P450 families that are part of the SMBGCs, CYP65 was dominant with 16 P450s, followed by CYP584 with 10 P450s, and CYP5392 had 11 P450s (Table S6). The remaining P450 families had a single-digit member part of the SMBGCs, indicating a high diversity of P450s that were part of SMBGCs (Table S6). Mycoparasite SMBGCs have the unique feature of having more than one P450 (Table S6). Two SMBGCs, one from T. virens Gv29-8 and another one from T. asperelloides T203, had four P450s (Table S6). Two SMBGCs had four P450s, 11 SMBGCs had three P450s, 32 SMBGCs had two P450s, and rest of the 86 SMBGCs had a single P450 (Table S6). It is well known that P450s contribute to the diversity of the secondary metabolites [64], and the presence of more than one P450 means they generate diverse secondary metabolites. Furthermore, the expansion of CYP65 members and their dominance as part of the SMBGCs suggests that these family members are expanded (statistically significant as the P-value is 0.01 compared to the second most populated CYP620 family) to create secondary metabolites that help mycoparasites adapt to their ecological niche.
A contrasting feature was observed concerning the number of P450s that were part of SMBGCs among the three Pezizomycetes. Sixteen mycoparasite Pezizomycetes had 200 P450s belonging to 69 P450 families that were part of 136 SMBGCs, indicating 19% of P450s were part of SMBGCs (Table S6). Genome-wide analysis of SMBGCs listed at JGI revealed that none of the secondary-metabolite BGCs of saprotrophic or ectomycorrhizal Pezizomycetes have P450. However, a recent study where a manual search of P450s that are part of SMBGCs revealed that only nine P450s belonging to eight P450 families are found in three species of ectomycorrhizal Pezizomycetes, namely, Sphaerosporella brunnea (six P450s that are part of four BGCs), Trichophaea hybrida (single P450), and Wilcoxina mikolae CBS 423.8 (two P450s that are part of two BGCs) [47]. The two saprophytic and seven ectomycorrhizal Pezizomycetes had no P450 as part of their secondary-metabolite BGCs [47]. A comparison of the P450s that are part of SMBGCs among Pezizomycetes revealed that none of the P450 families are in common between mycoparasites, saprophytes, and ectomycorrhiza.
Based on the above data, one can safely interpret that mycoparasite Pezizomycetes contain many diverse P450s as part of their SMBGCs, enabling them to synthesize many diverse secondary metabolites that will help them successfully prey on other fungi.

3.5. Mycoparasite P450s Are Indeed Involved in the Synthesis of Secondary Metabolites

Functional analysis of P450s from a few mycoparasites, especially Trichoderma, revealed the involvement of P450s in the biosynthesis of secondary metabolites (Figure 5). Three P450s, TRI4, TRI22, and TRI23, named CYP58A4, CYP65T9, and CYP5336B7, respectively, from T. arundinaceum are involved in the synthesis of the trichothecene sesquiterpene harzianum A (HA) [36,43]. CYP58A4 (TRI4), known as trichodiene oxygenase, catalyses the oxygenation of trichodiene at carbon atoms 2, 11, and 13 (C-2, C-11, and C-13) to form isotrichodiol, which can spontaneously cyclize to form EPT (12,13-epoxy-trichothec-9-ene) [38] (Figure 5A). Then, CYP65T9 (TRI22), known as C4-trichothecene monooxygenase, catalyses 4-hydroxylation of EPT to form 4-hydroxy EPT (trichodermol) [38] (Figure 5A). Recent studies indicated that CYP65T9 is highly substrate-specific [43]. CYP5336B7 (TRI23) is involved in the conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioic acid, involving three enzymatic reactions: hydroxylation, alcohol dehydrogenation, and aldehyde dehydrogenation activities (Figure 5A). CYP5336B7 catalyses the hydroxylation of octa-2,4,6-trienoic acid to form the 8-hydroxy derivative of the polyketide (i.e., 8-hydroxy-octa-2,4,6-trienoic acid), and the following two reactions remain to be confirmed [36] (Figure 5A). A point to be noted is that T. arundinaceum trichothecene BGC expands over 40kb, and genes are located in three loci; thus, SMBGC identification programs cannot pick the cluster as observed in this study (Table S6). Interestingly, CYP5336B6 but not CYP5336B7 is found to be part of the PKS cluster in T. arundinaceum (Table S6), indicating genes that are not part of SMBGCs may also be involved in the synthesis of trichothecene in T. arundinaceum [36].
TvCYt2 from T. virens Tv29.8, named CYP620B9 (in this study), is involved in synthesizing secondary metabolites that increase antagonistic activity against fungal pathogens and promote plant health and growth [41]. However, the type of metabolites and the nature of CYP620B9 enzymatic reactions remain to be elucidated.
A gene cluster named “vir” or “hep” involved in the synthesis of heptelidic acid and volatile sesquiterpenes has been characterized from T. virens Tv29.8 and found to contain four P450s, vir1/hepC, vir2/hepD, vir3/hepH, and vir5/hepE, found to be involved in the synthesis of these compounds [32,65] (Figure 5B). As part of this study, these P450s were Vir1/HepC, Vir2/HepD, Vir3/HepH, and Vir5/HepE, named CYP5104A5, CYP5117A3, CYP5094A3, and CYP68Q3, respectively (Table S6). All four P450s were found to be involved in the synthesis of volatile sesquiterpenes [32], but only three P450s, CYP5117A3, CYP5094A3, and CYP68Q3, are involved in the synthesis of heptelidic acid [65] (Figure 5B). The individual function of the CYP5117A3, CYP5094A3, and CYP68Q3 in the biosynthesis of heptelidic acid has been elucidated [65] (Figure 5B). CYP5094A3 converts gamma-cadinene into 8-hydroxy-cadinene [32] (Figure 5B). CYP68Q3 transforms 8-hydroxy-cadinene first into an epoxide and then oxidizes it into a ketone [65] (Figure 5B). CYP5117A3 converts the ketone compound into heptelidic acid [65] (Figure 5B). A point to be noted is that CYP68Q3 acts as a bifunctional P450 performing epoxidation and oxidation reactions, and CYP5117A3 performs a Baeyer–Villiger oxidation reaction with regioselectivity. CYP5117A3 is the first microbial P450 reported to perform a Baeyer–Villiger oxidation reaction [65].
Two P450s, GliC and GliF, as part of the gliotoxin SMBGC found in T. virens Gv29.8 (Table S6), were involved in synthesizing this highly toxic mycotoxin [34,42] (Figure 5C). GliC and GliF of T. virens Gv29.8 were named CYP613Q1 and CYP5085A3 (Figure 5C). Among these two P450s, the CYP613Q1 function has been elucidated. CYP613Q1 is engaged in the second step of the gliotoxin biosynthesis pathway, where it catalyses the hydroxylation of the α-carbon of L-Phe in cyclo-phenylalanyl-serine before bis-glutathionylation (Figure 5C). This suggests that CYP613Q1 performs the hydroxylation of diketopiperazine in the gliotoxin synthesis pathway [42] (Figure 5C).
Apart from the P450s mentioned above, we predict that the P450s listed in Table S6 may be involved in synthesizing different secondary metabolites. However, functional characterization of mycoparasite P450s is needed to confirm their role in synthesizing different secondary metabolites.

4. Conclusions

This study is the best example of observing the phenomenon P450s play a key role in organisms’ adaptation vis a vis lifestyle of organisms impacts P450 content in their genome” in fungi. Mycoparasitism resulted in the highest number of P450s, the highest diversity in the P450 families and subfamilies, a four-fold increase in secondary-metabolite biosynthetic gene clusters, and a greater number of P450s as part of these clusters compared to saprophytic and ectomycorrhizal lifestyle in Pezizomycetes. The study results will help researchers properly identify and cite mycoparasite P450s as per the International P450 Nomenclature System [40]. This also helps to identify homologues or orthologue P450s, thus easily deducing P450s’ functional role in general or in a specific biosynthetic pathway. However, experimental evidence is needed to unravel the role of P450s in synthesizing secondary metabolites and to prove which P450 or P450s are involved in synthesizing specific secondary metabolites in SMBGCs with multiple P450s. Future characterization of mycoparasite P450s will help understand their catalytic versatility and role in synthesizing a wide variety of secondary metabolites and thus explore their biotechnological potential in agriculture and human health.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres15030084/s1, Figure S1: A high-quality picture of phylogenetic analysis of mycoparasite P450s. The CYP65 family that is expanded in mycoparasites is highlighted. Figure S2: Comparative analysis of P450 families among mycoparasitic, saprophytic, and ectomycorrhizal Pezizomycetes. The number in parentheses indicates the number of P450 families. Figure S3: Comparative analysis of P450 families between mycoparasites from different fungal groups. The number in parentheses indicates the number of P450 families. Table S1. Genome-wide analysis and annotation of P450s in the fungal species analysed in the study. Table S2: P450 family and subfamily analysis in the 24 mycoparasitic fungi. Table S3: Comparative analysis of P450 families and subfamilies in individual mycoparasitic fungi. Table S4: Comparative analysis of P450 families and subfamilies in Pezizomycete mycoparasites. Table S5: A comparative analysis of secondary-metabolite biosynthetic gene clusters (SMBGCs) in fungal species. SMBGCs were retrieved from the JGI Mycocosm and presented in the table. The codes for each SMBGC type are the same as those listed at the JGI Mycocosm and are the same type as those in the anti-SMASH database [66]. Table S6: Comparative analysis of secondary-metabolite biosynthetic gene clusters (SMBGCs) and P450s in the SMBGCs in 15 mycoparasites. Standard abbreviations representing the SMBGCs as indicated in anti-SMASH (Antibiotics and Secondary-Metabolite Analysis Shell) [66] were used in the table. Supplementary Dataset S1: P450s, P450 fragments/pseudo, and no-hit proteins were identified in the study. Each protein sequence is represented by its protein ID (from the JGI Mycocosm), assigned a name, species name, and species code.

Author Contributions

Conceptualization, K.S.; methodology, P.R.S., T.P., P.G., B.V.Z.N., R.K., D.R.N. and K.S.; software, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; validation, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; formal analysis, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; investigation, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; resources, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; data curation, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; writing—original draft preparation, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; writing—review and editing, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; visualization, P.R.S., P.G., B.V.Z.N., T.P., R.K., D.R.N. and K.S.; supervision, R.K. and K.S.; project administration, R.K. and K.S.; funding acquisition, R.K. and K.S. All authors have read and agreed to the published version of the manuscript.

Funding

Khajamohiddin Syed expresses sincere gratitude to the University of Zululand (Grant number C686). Postgraduate students Puleng Rosinah Syed, Tiara Padayachee, and Bridget Valeria Zinhle Nkosi thank the National Research Foundation (NRF), South Africa, for postgraduate scholarships (Grant numbers MND210504599108, MND190606443406, and MND200527525406, respectively).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data are presented in the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Comparative analyses of P450s, P450 families, and subfamilies in fungal species analysed in the study. The number next to the bars shows the count for P450s, P450 families, and subfamilies in a fungal species. A detailed analysis is presented in Table S1.
Figure 1. Comparative analyses of P450s, P450 families, and subfamilies in fungal species analysed in the study. The number next to the bars shows the count for P450s, P450 families, and subfamilies in a fungal species. A detailed analysis is presented in Table S1.
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Figure 2. Phylogenetic analysis of mycoparasite P450s. The CYP65 family that is expanded in mycoparasites is highlighted. A high-quality figure is presented in Supplementary Figure S1.
Figure 2. Phylogenetic analysis of mycoparasite P450s. The CYP65 family that is expanded in mycoparasites is highlighted. A high-quality figure is presented in Supplementary Figure S1.
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Figure 3. Comparative analysis of P450 families between mycoparasites and other fungi. (A) Comparative analysis of P450 families among Pezizomycetes with different lifestyles. (B) Comparative analysis of P450 families between mycoparasites from different fungal groups. The numbers indicate the number of P450 families, and the number in parentheses indicates the number of species analysed. Detailed information on P450 families is presented in Figure S2 (for (A)) and Figure S3 (for (B)).
Figure 3. Comparative analysis of P450 families between mycoparasites and other fungi. (A) Comparative analysis of P450 families among Pezizomycetes with different lifestyles. (B) Comparative analysis of P450 families between mycoparasites from different fungal groups. The numbers indicate the number of P450 families, and the number in parentheses indicates the number of species analysed. Detailed information on P450 families is presented in Figure S2 (for (A)) and Figure S3 (for (B)).
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Figure 4. Comparative analysis of secondary-metabolite biosynthetic gene clusters (SMBGCs) in Pezizomycetes. The number in parentheses indicates the number of species analysed for a particular lifestyle, and the number next to the bars shows the number of SMBGCs for a specific type. Detailed information on SMBGCs is presented in Table S5. Abbreviations: PKS, polyketide synthase; NRPS, non-ribosomal peptide synthase; TC, terpene cyclase; DMAT, dimethylallyl-tryptophan.
Figure 4. Comparative analysis of secondary-metabolite biosynthetic gene clusters (SMBGCs) in Pezizomycetes. The number in parentheses indicates the number of species analysed for a particular lifestyle, and the number next to the bars shows the number of SMBGCs for a specific type. Detailed information on SMBGCs is presented in Table S5. Abbreviations: PKS, polyketide synthase; NRPS, non-ribosomal peptide synthase; TC, terpene cyclase; DMAT, dimethylallyl-tryptophan.
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Figure 5. The role of mycoparasite P450s in the biosynthesis of different secondary metabolites. Trichoderma arundinaceum P450 reactions in the biosynthesis of trichothecene sesquiterpene, harzianum A (A), and T. virens Tv29.8 P450 reactions in the biosynthesis of heptelidic acid (B) and gliotoxin (C). For easy identification, for each P450, the generic names used in the literature and the names assigned in this study are listed.
Figure 5. The role of mycoparasite P450s in the biosynthesis of different secondary metabolites. Trichoderma arundinaceum P450 reactions in the biosynthesis of trichothecene sesquiterpene, harzianum A (A), and T. virens Tv29.8 P450 reactions in the biosynthesis of heptelidic acid (B) and gliotoxin (C). For easy identification, for each P450, the generic names used in the literature and the names assigned in this study are listed.
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Table 1. Information on selected mycoparasites.
Table 1. Information on selected mycoparasites.
Species NameInformationReference
Ampelomyces quisqualis HMLAC05119Ampelomyces is an ecologically and economically valuable hyperparasite that can parasitize the hypha, conidiophore, conidium, and perithecium of powdery mildew fungi. Ampelomyces species were among the first fungi used as plant parasitic fungi biocontrol agents. Ampelomyces species produce secondary metabolites such as quinones, phenolic compounds, and lytic enzymes inhibiting fungi or bacteria and have potential antibiotic use against human pathogens. [5,6]
Caulochytrium protostelioides ATCC 52028 single-cell This species is a mycoparasite of Sordaria species and the first example of zoosporic true fungi having aerial sporangia, a unique trait within the Chytridiomycota. The hyphae secrete hydrophobin, a small cysteine-rich protein that develops aerial hyphae. This is the only fungus identified to produce hydrophobins outside the Dikarya (Ascomycota and Basidiomycota).[7]
Clonostachys rosea IK726This species is known for its biocontrol capabilities against a wide variety of plant pathogenic fungi, and various commercial products based on this fungus are available for use in biocontrol applications worldwide. This fungus effectively controls plant diseases caused by fungal and oomycete pathogens such as Alternaria spp., Bipolaris sorokiniana, Botrytis cinerea, Fusarium spp., Helminthosporium solani, Pythium tracheiphilum, and Zymoseptoria tritici.[8]
Dimargaris cristalligena RSA 468 single-cellThis fungus is a mycoparasite from the subphylum Kickxellomycotina (formerly of the Zygomycota). It is primarily a haustorial parasite of Mucorales and is known to produce various secondary metabolites. [7,9]
Escovopsis weberi CC031208-10This fungus is a parasite of Leucoagaricus spp., a garden fungus of leaf-cutting ants. It has adapted to mycoparasitism to the extent that it has lost plant-material-degrading enzymes. This fungus secretes various secondary metabolites that have antifungal and antibacterial activity. [10,11]
Naematelia encephela UCDFST 68-887.2This fugus is a parasite of Stereum sanguinolentum. This fungus has a worldwide presence. [12]
Piptocephalis cylindrospora RSA 2659 single-cellP. cylindrospora is an obligatory parasite of Mucorales that infects the host with specialized haustoria. Haustoria are branched, walled cells that penetrate host cell walls to absorb energy. Strain RSA2659 is grown on the mucoraceous mould Cokeromyces recurvatus. This fungus is distinguished by the presence of relatively large, brownish sporophores with three-dimensional dichotomous branching.[7]
Rozella allomycis CSF55R. allomycis is an obligate parasite of the Blastocladiomycotan fungus Allomyces.[13]
Syncephalis pseudoplumigaleata Benny S71-1 single-cellThis fungus is an obligate parasite of zygomycetes.[7]
Thamnocephalis sphaerospora RSA 1356 single-cellThis fungus is believed to be a weak parasite of other fungi and can be cultivated on Cokeromyces (a Mucoralean). [7]
Tremella mesenterica FriesThis species is a parasite of the Peniophora fungus and has a deceptive appearance as if it is growing on wood. However, it grows on the crust of fungal mycelium.[14]
Trichoderma arundinaceum IBT 40837This species produces the biotechnologically valuable compound harzianum A. Harzianum A is used as a biocontrol agent against plant fungal pathogens and promotes plant defence. [15]
Trichoderma asperelloides T203This fungus was one of the earliest models for understanding Trichoderma mycoparasitism and rhizosphere interactions. It enhanced plant growth potential and tolerance to abiotic stressors and induced systemic resistance to fungal and bacterial disease-causing pathogens. As a result, T. asperelloides strain T203 was the subject of several laboratory, greenhouse, and large-scale field investigations. It was incorporated into commercial agricultural preparations to improve plant development and as a biocontrol agent for plant diseases.[16]
Trichoderma asperellum CBS 433.97T. asperellum is a potent antifungal agent because it can parasitize or hinder the growth and development of other fungi. As a result, this species is used as a biological control agent against a wide range of plant-pathogenic fungi and fungi-like protozoa, including Phytophthora megakarya and nematodes. T. asperellum has also exhibited antibacterial activity via the synthesis of trichotoxin peptaibols and produces various secondary metabolites. [17,18]
Trichoderma atrovirideThis fungus is a biocontrol agent for many economically significant airborne and soilborne plant diseases. Its mycoparasitic activity is due to successful food competition, cell-wall-degrading enzyme synthesis, and antibiosis. This fungus also produces different types of secondary metabolites. [19,20]
Trichoderma citrinoviride TUCIM 6016T. citrinoviride has antifungal activities and nematode resistance, and its use as a biological control of plant fungal pathogens and nematodes has been elucidated.[17,21,22]
Trichoderma gamsii T6085One of the most promising agents for controlling Fusarium head blight (FHB) symptoms and mycotoxin accumulations, combined with mycoparasitic, antagonistic, and competitive activity against Fusarium graminearum, one of the primary causal agents of FHB. [23]
Trichoderma guizhouense NJAU 4742A mycoparasite of F. oxysporum, it has the potential to develop into a novel biofertilizer and biofungicide.[17,24]
Trichoderma hamatum GD12This fungus is unique because it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens, and induce systemic resistance to foliar pathogens.[25]
Trichoderma harzianum CBS 226.95T. harzianum is extensively researched due to its plant protection properties. It produces secondary metabolites with antibiotic characteristics, including peptaibols, harzianic acid, trichoharzianin, and trichodermin. This fungus is used for foliar application, seed, and soil treatments to suppress diseases caused by pathogens such as Botrytis, Fusarium, and Penicillium sp.[17,26]
Trichoderma longibrachiatum ATCC 18648T. longibrachiatum’s parasitic and deadly effects on the cysts of the worm Heterodera avenae have led to its proposed use as a biocontrol agent. Because of its mycoparasitism, it has been investigated for reducing crop fungal diseases.[17,27]
Trichoderma virens Gv29-8This species produces many secondary metabolites with potential biotechnological values, including gliotoxin and gliovirin, which have potential biocontrol activity against Rhizoctonia solani.[19]
Table 3. Analysis of number of copies of CYP51 and CYP61 P450s in mycoparasites.
Table 3. Analysis of number of copies of CYP51 and CYP61 P450s in mycoparasites.
Species NameP450 Family and SubfamilyCount
Clonostachys rosea IK726CYP51F2
CYP61A3
Trichoderma virens Gv29-8 CYP61A2
Thamnocephalis sphaerospora RSA 1356 single-cellCYP51F2
Trichoderma longibrachiatum ATCC 18648CYP61A2
Trichoderma hamatum GD12CYP51F2
CYP61A2
Trichoderma atrovirideCYP51F2
Trichoderma gamsii T6085CYP51F2
CYP61A2
Trichoderma asperellum CBS 433.97CYP61A2
Trichoderma citrinoviride TUCIM 6016CYP61A2
Trichoderma asperelloides T203 CYP61A2
Trichoderma pleuroti TPhu1CYP61A2
Trichoderma arundinaceum IBT 40837CYP51F2
CYP61A2
Trichoderma guizhouense NJAU 4742CYP51F2
CYP61A3
Trichoderma harzianum CBS 226.95CYP51F2
CYP61A3
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Syed, P.R.; Padayachee, T.; Gamede, P.; Nkosi, B.V.Z.; Nelson, D.R.; Karpoormath, R.; Syed, K. Comparative Genome-Wide Analysis Underscores the Rapid Expansion of Cytochrome P450s for Secondary Metabolism in the Mycoparasite Pezizomycetes. Microbiol. Res. 2024, 15, 1251-1268. https://doi.org/10.3390/microbiolres15030084

AMA Style

Syed PR, Padayachee T, Gamede P, Nkosi BVZ, Nelson DR, Karpoormath R, Syed K. Comparative Genome-Wide Analysis Underscores the Rapid Expansion of Cytochrome P450s for Secondary Metabolism in the Mycoparasite Pezizomycetes. Microbiology Research. 2024; 15(3):1251-1268. https://doi.org/10.3390/microbiolres15030084

Chicago/Turabian Style

Syed, Puleng Rosinah, Tiara Padayachee, Philasande Gamede, Bridget Valeria Zinhle Nkosi, David R. Nelson, Rajshekhar Karpoormath, and Khajamohiddin Syed. 2024. "Comparative Genome-Wide Analysis Underscores the Rapid Expansion of Cytochrome P450s for Secondary Metabolism in the Mycoparasite Pezizomycetes" Microbiology Research 15, no. 3: 1251-1268. https://doi.org/10.3390/microbiolres15030084

APA Style

Syed, P. R., Padayachee, T., Gamede, P., Nkosi, B. V. Z., Nelson, D. R., Karpoormath, R., & Syed, K. (2024). Comparative Genome-Wide Analysis Underscores the Rapid Expansion of Cytochrome P450s for Secondary Metabolism in the Mycoparasite Pezizomycetes. Microbiology Research, 15(3), 1251-1268. https://doi.org/10.3390/microbiolres15030084

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