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Article

More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium

by
Fanele Cabangile Mnguni
1,
Tiara Padayachee
1,
Wanping Chen
2,
Dominik Gront
3,
Jae-Hyuk Yu
4,5,
David R. Nelson
6,* and
Khajamohiddin Syed
1,*
1
Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, KwaDlangezwa 3886, South Africa
2
Department of Molecular Microbiology and Genetics, University of Göttingen, 37077 Göttingen, Germany
3
Faculty of Chemistry, Biological and Chemical Research Center, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
4
Department of Bacteriology, University of Wisconsin-Madison, 3155 MSB, 1550 Linden Drive, Madison, WI 53706, USA
5
Department of Systems Biotechnology, Konkuk University, Seoul 05029, Korea
6
Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(13), 4814; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134814
Submission received: 18 January 2020 / Revised: 11 February 2020 / Accepted: 13 February 2020 / Published: 7 July 2020
(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0)
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Abstract

:
Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus, mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s’ association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.

Graphical Abstract

1. Introduction

Cytochrome P450 monooxygenases (CYPs/P450s) are biotechnologically valuable enzymes [1]. P450s have heme (protoporphyrin IX), an iron(III)-containing porphyrin, as a prosthetic group in their structure [2]. Because of the presence of this prosthetic group, these enzymes absorb wavelengths at 450 nm; thus, the name P450s has been assigned to these proteins [3,4,5,6]. Since their identification, a large number of P450s have been identified in almost all living organisms [7] and, surprisingly, in non-living entities as well [8]. The regio- and stereo-specific catalytic nature of these enzymes makes them essential for the survival of some organisms, and these enzymes are thus good drug targets in the case of pathogenic organisms [9,10,11,12,13]. Application of these enzymes in all fields of research continues, and excellent success has been achieved in using them for the production of substances valuable to humans or as drug targets or in drug metabolism, as reported previously [1]. One of the applications of P450s currently being explored is their role in the production of secondary metabolites, compounds with potential biotechnological value, owing to their stereo- and regio-specific enzymatic activity, which contributes to the diversity of secondary metabolites [14,15,16].
Unlike other enzymes, P450 enzymes have a typical nomenclature system established by the International P450 Nomenclature Committee [17,18,19]. According to the committee’s rules, P450s begin with the prefix “CYP” for cytochrome P450 monooxygenase, followed by an Arabic numeral which designates the family, a capital letter designating the subfamily, and an Arabic numeral designating the individual P450 in a family. The annotation of P450s (assigning family and subfamily) follows a rule that all P450s with > 40% identity belong to the same family and all P450s with > 55% identity belong to the same subfamily [17,18,19]. Worldwide, researchers follow this P450 nomenclature system. The nomenclature of P450s is also be verified by phylogenetic analysis to enable their correct annotation, as phylogenetic-based annotation could detect similarity cues beyond a simple percentage identity cutoff, as mentioned elsewhere [20].
The continued genomic rush has resulted in genome sequencing of a large number of species belonging to all biological kingdoms. P450s in the newly sequenced species need to be annotated as per the International P450 Nomenclature Committee rules [17,18,19] to enable researchers to use the same names for functional and evolutionary analysis of P450s. For this reason, large numbers of P450s have recently been annotated in bacterial species belonging to the genera Mycobacterium [21], Bacillus [22], Streptomyces [20], and Cyanobacteria [23]. These studies have revealed numerous P450s involved in the synthesis of different types of secondary metabolites. This type of in silico study is highly important for identifying unique P450s that can be drug-targeted and for P450 evolutionary analysis, as the P450 profiles in species have been found to be characteristic of species’ lifestyle [11,20,22,24,25,26,27].
Among bacterial species, Streptomyces species are well-known for producing over two-thirds of the clinically useful antibiotics in the world [28]. Because of this importance, Streptomyces species have been subjected to exhaustive secondary metabolite production studies [29,30]. Streptomyces P450s play a key role in the production of different secondary metabolites; their contribution to secondary metabolite diversity and applications in drug metabolism have been reviewed extensively [15,16,31,32,33]. In the latest study, comprehensive comparative analysis of P450 and secondary metabolite biosynthetic gene clusters (BGCs) in 48 Streptomyces species was elucidated [20]. The study revealed the presence of novel P450s in Streptomyces species and numerous P450s forming parts of secondary metabolite BGCs [20]. The study results indicated that lifestyle or ecological niches play a key role in the evolution of P450 profiles in species belonging to the genera Streptomyces and Mycobacterium [20].
To date, a large number of Streptomyces species genomes have been sequenced and are available for public use. This provided an opportunity to annotate P450s in these species to analyze and compare their profiles among different bacterial species, including the identification and comparative analysis of P450s involved in the production of secondary metabolites. This study thus aimed to perform genome data mining, annotation, and phylogenetic analysis of P450s in 155 newly available Streptomyces species genomes. It also included the identification and comparative analysis of P450s that are parts of secondary metabolite BGCs among bacterial species belonging to the genera Streptomyces, Bacillus, Mycobacterium, and Cyanobacteria, as the species belonging to these genera are known to have P450s and to produce secondary metabolites.

2. Results and Discussion

2.1. Streptomyces Species Have Large Number of P450s

Genome-wide data mining and annotation of P450s in 203 Streptomyces species (Supplementary Table S1) revealed the presence of 5460 P450s in their genomes (Figure 1, Table 1, and Supplementary Dataset 1). The P450 count in the Streptomyces species ranged from 10 to 69 P450s, with an average of 27 P450s. Apart from the complete P450 sequences, pseudo-P450s (6 hit proteins), P450-fragments (114 hit proteins), P450-derived glycosyltransferase activator proteins (22 hit proteins), and P450 false-positive hits (2 hit proteins) were also found in some Streptomyces species (Supplementary Table S2). The presence of these types of P450 hit proteins in species is common and, because of the nature of these proteins, they were not included in the study for further analysis. Among Streptomyces species, Streptomyces albulus ZPM was found to have the highest number of P450s in its genome (69 P450s) followed by S. clavuligerus (65 P450s); the lowest number of P450s was found in Streptomyces sp. CNT372 and S. somaliensis DSM 40738 (10 P450s each) (Figure 1 and Table 1). Analysis of the most prevalent number of P450s revealed that 19 P450s was the prevalent number in Streptomyces species (Table 1). The average number of P450s in Streptomyces species was found to be higher than in Bacillus species [22] and cyanobacterial species [23], and almost the same as in mycobacterial species [21] (Table 2). A point to be noted is that the number of species greatly influences the average number of P450s and, thus, the higher the number of species in the analysis, the better and more accurate the results, as mentioned elsewhere [20,23]. This is the reason Streptomyces species showed a slightly lower average number of P450s in their genomes compared to mycobacterial species, since only 60 species were employed in the study [21]. Thus, future annotation of P450s in more mycobacterial species will provide accurate insights into this aspect.

2.2. CYP107 Family Was Found to Be Dominant and Conserved in 203 Streptomyces Species

Analysis of P450 families and subfamilies in 203 Streptomyces species revealed that 5460 P450s could be grouped into 253 P450 families and 698 P450 subfamilies (Table 2 and Supplementary Table S3). Among Streptomyces species, S. clavuligerus had the highest number of P450 families (30) and P450 subfamilies (58) in its genome (Table 1). Although S. rimosus rimosus ATCC 10970 had the same number of P450 families as S. clavuligerus, the number of subfamilies was the third highest (52 subfamilies) (Table 1). One interesting observation is that the species with the highest number of P450s did not have the highest number of P450 families, suggesting that some of the P450 families were populated (bloomed). Blooming of P450 families is common across species, and this phenomenon has been observed in different species belonging to different biological kingdoms [24,26,34,35,36]. Phylogenetic analysis revealed that some of the P450 families were scattered across the evolutionary tree (Figure 1). This phenomenon was also observed previously for Streptomyces species P450s, and it has been hypothesized that the phylogenetic-based annotation of P450s could be detecting similarity cues beyond a simple percentage identity cutoff [20]. Analysis of P450 families in the 155 Streptomyces species used in this study revealed the presence of 38 new P450 families, i.e., CYP1200A1, CYP1216A1, CYP1223A1, CYP1228A1, CYP1236A1, CYP1238A1, CYP1265A1, CYP1279A1, CYP1369A1, CYP1432A1, CYP1518A1, CYP1529A1, CYP1543A1, CYP1568A1, CYP159A1, CYP1607A1, CYP1658A1, CYP1759A1, CYP1810A1, CYP1832A1, CYP1866A1, CYP1896A1, CYP1920A1, CYP1929A1, CYP1931A1, CYP1940A1, CYP1941A1, CYP1943A1, CYP1972A1, CYP1984A1, CYP1994A1, CYP2076A1, CYP2080A1, CYP2134A1, CYP2180A1, CYP2349A1, CYP2427A1, and CYP2723A1. A detailed analysis of the number of new P450 families found in different Streptomyces species is presented in Supplementary Table S2.
Among the P450 families, the CYP107 family was found to be dominant, with 1 235 P450s in Streptomyces species, followed by CYP105 with 684 P450s, CYP157 with 525 P450s, and CYP154 with 510 P450s (Figure 2 and Supplementary Table S3), indicating the possible blooming of these families in Streptomyces species, as observed in species belonging to different biological kingdoms [24,26,34,35,36]. It is interesting to note that the CYP107 family was also found to be dominant in the Bacillus species [22], indicating its dominant role in the synthesis of secondary metabolites in both the Streptomyces and Bacillus genera. An interesting pattern was observed when comparing subfamily diversity in the dominant P450 families (Figure 2, Table 3, and Supplementary Table S3). P450 families such as CYP107, CYP105, CYP183, and CYP113 had the highest diversity at the subfamily level, as numerous subfamilies were found in these families (Supplementary Table S3). This phenomenon of the highest diversity in P450 families being found in Streptomyces species is not uncommon, and this proved to be the key contributor in the production of diverse secondary metabolites in Streptomyces species compared to mycobacterial species [20]. Strong support for this argument is the fact that the CYP105 P450 family members in Streptomyces species have been shown to be involved in oxidation of numerous endogenous and exogenous compounds and in the generation of different secondary metabolites [32]. However, in contrast to the diversity at subfamily level for the P450 families CYP107, CYP105, CYP183, and CYP113, the rest of the dominant P450 families had single or double or triple subfamilies, indicating subfamily-level blooming in these P450 families (Table 3).
P450 family conservation analysis revealed that the CYP107 family is conserved in all 203 Streptomyces species (Figure 3 and Supplementary Dataset 3). P450 families such as CYP156, CYP105, CYP154 and CYP157 are also present in the majority of the Streptomyces species (Figure 3 and Supplementary Dataset 3).

2.3. Numerous P450s Involved in Secondary Metabolite Production in Streptomyces Compared to Other Bacterial Species

Analysis of 144 Streptomyces species’ genomes revealed the presence of 4457 BGCs in their genomes (Table 2 and Supplementary Table S4). The number of BGCs found in 144 Streptomyces species was found to be higher than in mycobacterial, Bacillus, and cyanobacterial species (Table 2), indicating the superiority of the Streptomyces species in producing secondary metabolites; two-thirds of the antibiotics used in the world currently come from these species [28]. The average number of BGCs in Streptomyces species was found to be double compared to mycobacterial species and close to four times higher than that in Bacillus and cyanobacterial species (Table 2). Analysis of BGCs revealed that a large proportion of Streptomyces species’ P450s are part of BGCs compared to other bacterial species; 1231 P450s in Streptomyces species compared to 112 in Bacillus species, 204 in mycobacterial species, and 27 in cyanobacterial species (Table 2). A total of 1231 P450s were found to be part of BGCs belonging to 135 P450 families (Figure 4 and Supplementary Table S5). Among 135 P450 families, P450s belonging to the CYP107 family were dominantly present in BGCs, followed by CYP105, CYP157, and CYP154 (Figure 4 and Supplementary Table S5). This clearly suggests that the P450 families that are bloomed in Streptomyces species are actually involved in the production of secondary metabolites. This strongly supports the proposed hypothesis that in Streptomyces species, P450s are evolved to generate secondary metabolites, thus helping these bacteria to thrive in their environment [20]. In order to assess the in silico results generated by this study, in which a large number of Streptomyces species P450s were predicted to be involved in secondary metabolite production, we performed an extensive literature review to identify Streptomyces P450s involved in the production of secondary metabolites. As shown in Table 4, a large number of P450s belonging to different P450 families, as predicted in this study, were found to be involved in the production of different secondary metabolites. This strongly supports the notion that the P450s identified as part of different BGCs in this study produce secondary metabolites.
Analysis of P450 BGCs revealed the presence of 235 types of BGCs, where the BGC type, such as terpene, was dominant, followed by T1PKS, NRPS, and T3PKS (Figure 4 and Supplementary Table S5). A detailed analysis of P450s that are part of BGCs and types of BGCs containing P450s is presented in Supplementary Table S5. Analysis of the linkage between a particular P450 family and BGC revealed that some P450s are linked to a particular BGC (Supplementary Table S4), indicating horizontal transfer of BGCs between Streptomyces species. Streptomyces P450s such as CYP283A are linked to bacteriocin and bottromycin; CYP113K3 is linked to Bacteriocin-Nrps, CYP124G is linked to melanin, and CYP105A is linked to NRPS and butyrolactone. A point to be noted is that horizontal transfer of BGCs among different organisms is well-documented in the literature [37].
Table
Table 4. List of Streptomyces species P450s involved in synthesis of secondary metabolites.
Table 4. List of Streptomyces species P450s involved in synthesis of secondary metabolites.
P450SpeciesFunctionReferences
CYP158A1Streptomyces coelicolor A3(2)Flaviolin biosynthesis[38]
CYP1048A1Streptomyces scabieiThaxtomin (phytotoxin) biosynthesis[39]
CYP105A1Streptomyces griseolusDiterpenoids synthesis[40]
CYP105A3 (P450sca-2)Streptomyces carbophilusPravastatin synthesis[41]
CYP105B28(GfsF) *Streptomyces graminofaciensMacrolide antibiotic synthesis[42,43]
CYP105D6Streptomyces avermitilisFilipin biosynthesis[44]
CYP105D7Streptomyces avermitilisFilipin biosynthesis[45]
CYP105D8Streptomyces tubercidicus strain I-1529Avermectin oxidation[32,46]
CYP105D9Streptomyces sp. JP95Griseorhodin biosynthesis[32,47]
CYP105F2Streptomyces peucetiusOleandomycin biosynthesis[48,49]
CYP105H1Streptomyces noursei ATCC 11455Nystatin biosynthesis[32]
CYP105H3Streptomyces natalensisPimaricin biosynthesis[32,50]
CYP105H4 (AmphN) !Streptomyces nodosusAmphotericin biosynthesis[51,52]
CYP105H5Streptomyces griseusCandidicin biosynthesis[32]
CYP105K1Streptomyces tendae strain Tue901Nikkomycin biosynthesis[32,53]
CYP105K2Streptomyces ansochromogenesNikkomycin biosynthesis[32]
CYP105L1 (TylH1,orf7) !Streptomyces fradiaeTylosin biosynthesis[54,55]
CYP105L4(ChmH1) *Streptomyces bikiniensisChalcomycin biosynthesis[56]
CYP105M1 (orf10) !Streptomyces clavuligerusClavulanic acid antibiotic biosynthesis[57]
CYP105N1Streptomyces coelicolor A3(2)Coelibactin siderophore biosynthesis[58,59]
CYP105P1Streptomyces avermitilisFilipin biosynthesis[44]
CYP105U1Streptomyces hygroscopicusGeldanamycin biosynthesis[60]
CYP105V1Streptomyces sp. HK803Phoslactomycin biosynthesis[32,61]
CYP105AA1Streptomyces tubercidicus strain R922Avermectin oxidation[32,46]
CYP105AA2Streptomyces tubercidicus strain I-1529Avermectin oxidation[32,46]
CYP107A1Streptomyces peucetiusDealkylation of 7-ethoxycoumarin[62]
CYP107A1Saccharopolyspora erythraeaErythromycin biosynthesis[63,64]
CYP107B (HmtN) !Streptomyces himastatinicus ATCC 53653Himastatin biosynthesis[65]
CYP107B (HmtN)Streptomyces himastatinicusHimastatin biosynthesis[66]
CYP107C1Streptomyces thermotoleransCarbomycin biosynthesis[67]
CYP107E40(chmPII) *Streptomyces bikiniensisChalcomycin biosynthesis[56]
CYP107EE2(chmPI) *Streptomyces bikiniensisChalcomycin biosynthesis[56]
CYP107FH5(TamI) *Streptomyces sp. 307-9Tirandamycin biosynthesis[68,69]
CYP107G1Streptomyces rapamycinicusRapamycin biosynthesis[70,71]
CYP107G1 (rapN) !Streptomyces hygroscopicusRapamycin biosynthesis[71,72]
CYP107L1Streptomyces venezuelaeMacrolide antibioitics biosynthesis[73]
CYP107L59(FosK) *Streptomyces pulveraceusFostriecin biosynthesis[74]
CYP107MD3(FosG) *Streptomyces pulveraceusFostriecin biosynthesis[74]
CYP107W1Streptomyces avermitilisOligomycin A biosynthesis[75,76]
CYP112A2Streptomyces rapamycinicusRapamycin biosynthesis[70,71]
CYP113A1Saccharopolyspora erythraeaErythromycin biosynthesis[63,64]
CYP113B1 (TylI) !Streptomyces fradiaeTylosin biosynthesis[54,55]
CYP113D3(HmtT) *Streptomyces himastatinicus ATCC 53653Himastatin biosynthesis[65]
CYP113D3 (HmtT) *Streptomyces himastatinicusHimastatin biosynthesis[66]
CYP113HI (HmtS) *Streptomyces himastatinicusHimastatin biosynthesis[66]
CYP122A2 (rapJ) !Streptomyces hygroscopicusRapamycin biosynthesis[70,71]
CYP122A3Streptomyces hygroscopicusRapamycin biosynthesis[70,71]
CYP122A4 (FkbD) !Streptomyces tsukubaensisFK506 (immunosuppressant) polyketide biosynthesis[77]
CYP129A2Streptomyces peucetiusDoxorubicin biosynthesis[78,79]
CYP129A2 (dox A) !Streptomyces sp. strain C5Doxorubicin biosynthesis[80,81]
CYP131A2 (dnrQ) !Streptomyces sp. strain C5Doxorubicin biosynthesis[80,81]
CYP140M1(TtnI) *Streptomyces griseochromogenesTautomycetin biosynthesis[82]
CYP151A (AurH) !Streptomyces thioluteusAureothin biosynthesis[83]
CYP154A1Streptomyces coelicolor A3(2)Polyketide synthesis and cyclization of a cellular dipentaenone[84,85]
CYP154B1Streptomyces fradiaeTylosin biosynthesis[54,55]
CYP154C1Streptomyces coelicolor A3(2)Macrolide biosynthesis[86]
CYP158A2Streptomyces coelicolor A3(2)Flaviolin biosynthesis[87]
CYP161A2 (PimD) !Streptomyces natalensisPimaricin biosynthesis[88]
CYP161A3 (AmphL) !Streptomyces nodosusAmphotericin biosynthesis[51]
CYP162A1Streptomyces tendaeNikkomycin biosynthesis[53,89]
CYP163A1 (NovI) !Streptomyces spheroidsNovobiocin biosynthesis[90]
CYP163B3 (P450 Sky) !Streptomyces sp. Acta 2897Skyllamycin biosynthesis[91]
CYP170A1Streptomyces coelicolor A3(2)Albaflavenone biosynthesis[92]
CYP170A2Streptomyces avermitilisAlbaflavenone biosynthesis[93]
CYP170B1Streptomyces albusAlbaflavenone biosynthesis[94]
CYP171A1Streptomyces avermitilisAvermectin biosynthesis[95,96]
CYP183A1Streptomyces avermitilisPentalenolactone biosynthesis[96,97]
CYP244A1 (StaN) !Streptomyces sp tp-a0274Rapamycin biosynthesis[70,71]
CYP245A1 (StaP) !Streptomyces sp tp-a0274Rapamycin biosynthesis[70,71]
CYP246A1Streptomyces scabieiThaxtomin (phytotoxin) biosynthesis[98]
CYP248A1Streptomyces thioluteusAureothin biosynthesis[83]
Note: For some P450s, protein notations are given in parentheses. These P450s were annotated in this study (indicated with asterisk superscript) and previously (indicated with exclamation mark) [20] by browsing the individual biosynthetic gene-cluster sequences reported in the literature. To enable readers to match the P450s with the published literature, we have provided protein notations in the parentheses. If known, the name of the secondary metabolite of which P450s are involved in production is indicated in the table.

3. Materials and Methods

3.1. Information on Streptomyces Species and Genome Database

In total, 203 Streptomyces species genomes (permanent and finished draft genomes) available for public use at the Joint Genome Institute Integrated Microbial Genomes and Microbiomes (JGI IMG/M) [99] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [100] were used in this study. The 203 Streptomyces species included 48 Streptomyces species for which P450s and BGCs were annotated previously [20]. For these 48 species, P450 and BGCs data were retrieved from published articles and used in the study [20]. Thus, 155 Streptomyces species were data-mined for P450s and BGCs in this study. Information on the species used in the study is provided in Supplementary Table S1.

3.2. Genome Data Mining and Identification of P450s

Identification and annotation of P450s in Streptomyces species were carried out following a method described elsewhere [20,21,22]. Briefly, each Streptomyces species genome available at JGI IMG/M [99] was searched for P450s using the InterPro code “IPR001128”. The hit protein sequences were then searched for the presence of P450 characteristic motifs such as EXXR and CXG [101]. Proteins having one of these motifs were considered pseudo-P450s, and proteins that were short in amino acid length and lacking both motifs as P450 fragments. Neither the pseudo-P450s nor the P450 fragments were considered for further analysis.

3.3. Allocating Family and Subfamily to P450s

The hit proteins that were collected were subjected to BLAST analysis against bacterial P450s at the website http://www.p450.unizulu.ac.za/. Based on the International P450 Nomenclature Committee rule [17,18,19], proteins with a percentage identity greater than 40% were assigned to the same family as named homolog P450s, and those that had greater than 55% identity were assigned to the same subfamily as named homolog P450s. Proteins that had a percentage identity less than 40% were assigned to a new family.

3.4. Streptomyces P450 Phylogenetic Analysis

Phylogenetic analysis of the Streptomyces P450s was carried out following the method described in the literature [102]. First, the Streptomyces P450 sequences were aligned using the MAFFT v6.864 program with an automatically optimized model option [103], available at the Trex web server [104]. The alignments were then automatically subjected to inference and optimization of the tree by the Trex web server with its embedded weighting procedure, and the best inferred tree was visualized and annotated by iTOL [105].

3.5. Streptomyces P450 Profile Heat-Maps

P450 profile heat-maps were generated following a method published previously [22,27] to check the presence and absence of P450s in Streptomyces species. Briefly, a tab-delimited file was imported into Multi-Experiment Viewer (Mev) [106] and hierarchical clustering using a Euclidean distance metric was used to cluster the data. In total, 203 Streptomyces species formed the vertical axis and P450 family numbers formed the horizontal axis. Data were presented as −3 for family absence (green) and 3 for family presence (red).

3.6. Identification of P450s That Are Part of Secondary Metabolite BGCs

Secondary metabolite BGCs analysis and identification of P450s that are part of these BGCs were carried out following the procedure mentioned previously [102], with slight modification. For each Streptomyces species genome available at JGI IMG/M, the secondary metabolite BGCs were searched for the presence of P450s. The DNA sequence of BGCs with P450s was collected and formatted to fasta format using PSPad editor (http://www.pspad.com/en/). The fasta-formatted files were then used to identify the type of cluster and most similar known clusters using the Antibiotics and Secondary Metabolite Analysis Shell (anti-SMASH) program [107]. The results obtained were recorded on Excel spreadsheets and represented as species-wise BGCs, type and similar known BGCs, percentage similarity to known BGCs, and P450s that are part of specific BGCs. Some Streptomyces species genome IDs did not pass through anti-SMASH analysis, and thus these species were not included in P450s analysis as part of secondary metabolite BGCs. A list of Streptomyces species subjected to anti-SMASH analysis is presented in Supplementary Table S4.

3.7. Data Analysis

All calculations were done following the method described in the literature [23]. The average number of P450s was calculated using the formula: Average number of P450s = Number of P450s/Number of species. The average number of BGCs was calculated using the formula: Average number of BGCs = Total number of BGCs/Number of species. The percentage of P450s that formed part of BGCs was calculated using the formula: Percentage of P450s part of BGCs = 100 × Number of P450s part of BGCs /Total number of P450s present in species. For comparative analysis of P450s and BGCs, information for bacterial species belonging to the genera Bacillus [22], Mycobacterium [21], and Cyanobacteria [23] was resourced from published articles.

4. Conclusions

In the last five decades, research on cytochrome P450 monooxygenases (CYPs/P450s) has mainly focused on their function and structural aspects, with little focus on evolutionary analysis, especially in microbes. The availability of a large number of microbial species genomes gives us an opportunity to focus on exploring the evolutionary aspects of P450s. Because a typical nomenclature system that has been established for P450s, each species genome needs to be data-mined and P450 proteins need to be annotated (assigning family and subfamily). In this way, researchers around the world can make use of uniform P450 names. In this study, we therefore annotated a large number of P450s in 203 Streptomyces species and found 38 new P450 families. Some P450 families were found to be bloomed in Streptomyces species even at the subfamily level. Comparative analysis of key P450 features among different bacterial species revealed that Streptomyces species had a greater number of P450s, more secondary metabolite BGCs, and the highest number of P450s as part of BGCs compared to the bacterial species belonging to the genera Bacillus, Mycobacterium, and Cyanobacteria. This further confirmed that the higher the number of P450s, the higher the secondary metabolite diversity in a species. This was true for Streptomyces species, as large number of P450s were found to be involved in the generation of diverse secondary metabolites. One interesting phenomenon observed was the linkage between a particular P450 family and BGC. This indicates that these BGCs were horizontally transferred among different Streptomyces species. This study is a good addition to the comparative analysis of P450s and BGCs among different bacterial populations. Data presented in the study will serve as a reference for further annotation of P450s in Streptomyces species and other bacterial species. In silico predicted BGCs need to be experimentally validated to assess the secondary metabolites’ biological properties.

Supplementary Materials

Supplementary materials can be found at https://0-www-mdpi-com.brum.beds.ac.uk/1422-0067/21/13/4814/s1.

Author Contributions

Conceptualization, K.S.; data curation, F.C.M., T.P., W.C., D.G., J.-H.Y., D.R.N. and K.S.; formal analysis, F.C.M., T.P., W.C., D.G., J.-H.Y., D.R.N. and K.S.; funding acquisition, K.S.; investigation, F.C.M., T.P., W.C., J.-H.Y., D.R.N. and K.S.; methodology, F.C.M., T.P., W.C., D.G., J.-H.Y., D.R.N. and K.S.; project administration, K.S.; resources, K.S.; supervision, K.S.; validation, F.C.M., T.P., W.C., D.G., J.-H.Y., D.R.N. and K.S.; visualization, F.C.M., T.P., W.C., and K.S.; writing—original draft, F.C.M., T.P., W.C., J.-H.Y., D.R.N. and K.S.; writing—review and editing, F.C.M., T.P., W.C., J.-H.Y., D.R.N. and K.S. All authors have read and agreed to the published version of the manuscript.

Funding

The work presented in this article is part of research funded by a National Research Foundation (NRF), South Africa grant awarded to Khajamohiddin Syed (Grant No. 114159), where all the international authors involved in the study are listed as international collaborators. Fanele Cabangile Mnguni thanks the NRF, South Africa for a DST-NRF Innovation Master’s Scholarship for the year 2019 (Grant No. 117171). Honours student, Tiara Padayachee, thanks the NRF, South Africa for an honours bursary (Grant No. MND190619448759). Dominik Gront was supported by the National Science Centre, Poland (Grant No. 2018/29/B/ST6/01989). Khajamohiddin Syed expresses sincere gratitude to the University of Zululand Research Committee for funding (Grant No. C686) and for the laboratory facilities.

Acknowledgments

The authors want to thank Barbara Bradley, Pretoria, South Africa for English language editing.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Phylogenetic analysis of Streptomyces P450s. In total, 5 460 P450s were used to construct the tree and the dominant P450 families are highlighted in different colors and indicated in the figure. A high-resolution phylogenetic tree is provided in Supplementary Dataset 2.
Figure 1. Phylogenetic analysis of Streptomyces P450s. In total, 5 460 P450s were used to construct the tree and the dominant P450 families are highlighted in different colors and indicated in the figure. A high-resolution phylogenetic tree is provided in Supplementary Dataset 2.
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Figure 2. P450 family and subfamily analysis in 203 Streptomyces species. Only the dominant P450 families with more than 40 P450s are shown in the figure. Detailed data on P450 families and subfamilies are presented in Supplementary Table S3.
Figure 2. P450 family and subfamily analysis in 203 Streptomyces species. Only the dominant P450 families with more than 40 P450s are shown in the figure. Detailed data on P450 families and subfamilies are presented in Supplementary Table S3.
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Figure 3. Heat-map of P450 family conservation analysis in Streptomyces species. In the heat-map, the presence and absence of P450 families are indicated in red and green colors. The horizontal axis represents P450 families and the vertical axis represents Streptomyces species.
Figure 3. Heat-map of P450 family conservation analysis in Streptomyces species. In the heat-map, the presence and absence of P450 families are indicated in red and green colors. The horizontal axis represents P450 families and the vertical axis represents Streptomyces species.
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Figure 4. Analysis of P450s associated with secondary metabolite production in Streptomyces species. (A) Dominant P450 families (families with higher numbers of members) that are part of biosynthetic gene clusters (BGCs) and (B) dominant BGCs (present in higher numbers) containing P450s were presented in the figure. The numbers next to bars indicate the number of P450s in panel A and the number of BGCs in panel B. Detailed information is presented in Supplementary Table S5.
Figure 4. Analysis of P450s associated with secondary metabolite production in Streptomyces species. (A) Dominant P450 families (families with higher numbers of members) that are part of biosynthetic gene clusters (BGCs) and (B) dominant BGCs (present in higher numbers) containing P450s were presented in the figure. The numbers next to bars indicate the number of P450s in panel A and the number of BGCs in panel B. Detailed information is presented in Supplementary Table S5.
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Table
Table 1. Genome-wide data mining and annotation of P450s in 203 Streptomyces species.
Table 1. Genome-wide data mining and annotation of P450s in 203 Streptomyces species.
Species NameP450sNo. FNo. SFSpecies NameP450sNo. FNo. SF
Streptomyces sp. Tu6071221320Streptomyces sp. CNT37210810
Streptomyces purpureus KA281, ATCC 21405221720Streptomyces sp. CNS60616914
Streptomyces sp. W007281224Streptomyces sp. 303MFCol5.2231422
Streptomyces sp. TAA486-18181217Streptomyces acidiscabies 84-104472244
Streptomyces lysosuperificus ATCC 31396251924Streptomyces roseosporus NRRL 11379191016
Streptomyces sp. PVA 94-0720718Streptomyces sp. OspMP-M4519919
Streptomyces sp. SPB78201220Streptomyces sp. AmelKG-A319919
Streptomyces canus 299MFChir4.1281727Streptomyces sp. S419919
Streptomyces sp. FxanaA7301529Streptomyces sp. SM818816
Streptomyces sulphureus DSM 40104261325Streptomyces sp. LaPpAH-199261121
Streptomyces sp. MspMP-M5442041Streptomyces sp. 140Col2.1E22917
Streptomyces coelicoflavus ZG0656171216Streptomyces sp. DvalAA-21241022
Streptomyces pristinaespiralis ATCC 25486181117Streptomyces sp. CNT371171317
Streptomyces sp. LaPpAH-20119819Streptomyces somaliensis DSM 407381089
Streptomyces albulus CCRC 11814642650Streptomyces sp. 351MFTsu5.1221122
Streptomyces viridochromogenes DSM 40736241524Streptomyces sp. DvalAA-83241022
Streptomyces sp. LaPpAH-9524922Streptomyces sp. AmelKG-F2B241723
Streptomyces mirabilis YR139422641Streptomyces sp. CNT302261322
Streptomyces sp. AA1529261524Streptomyces olindensis DAUFPE 5622261422
Streptomyces atratus OK008151014Streptomyces sp. CNY243171416
Streptomyces sp. PsTaAH-130362132Streptomyces sp. AA0539191019
Streptomyces sp. CNT318271524Streptomyces atratus OK807311327
Streptomyces sp. CNH099161216Streptomyces sp. CNS335161317
Streptomyces sp. CNH287161216Streptomyces sp. FxanaC1271524
Streptomyces sp. MnatMP-M77321427Streptomyces sp. WMMB 322191117
Streptomyces zinciresistens K42191118Streptomyces sp. TOR3209201319
Streptomyces sp. So1WspMP-so12th221119Streptomyces sp. AmelKG-E11A241522
Streptomyces sp. GXT613811Streptomyces sp. PP-C4216614
Streptomyces roseosporus NRRL 15998191016Streptomyces sp. DpondAA-E10251022
Streptomyces sp. LaPpAH-108241223Streptomyces sp. HPH0547321832
Streptomyces aurantiacus JA 4570302030Streptomyces sp. DpondAA-A50251022
Streptomyces hygroscopicus ATCC 53653572149Streptomyces sp. TAA040151015
Streptomyces sp. Tu 6176301526Streptomyces sp. PgraA7231020
Streptomyces ghanaensis ATCC 14672352034Streptomyces sp. FxanaD5151115
Streptomyces sp. KhCrAH-337261222Streptomyces sp. LamerLS-316251122
Streptomyces sp. LaPpAH-20219819Streptomyces viridochromogenes Tue57311729
Streptomyces sp. UNC401CLCol151115Streptomyces sp. GBA 94-1020718
Streptomyces sp. SirexAA-H211220Streptomyces sp. CNQ-525181418
Streptomyces turgidiscabies Car8282027Streptomyces sp. SceaMP-e96411836
Streptomyces sp. KhCrAH-40261222Streptomyces mirabilis OK461371631
Streptomyces rimosus rimosus ATCC 10970543052Streptomyces sp. LaPpAH-185442740
Streptomyces gancidicus BKS 13-15181117Streptomyces exfoliatus DSMZ 41693261624
Streptomyces auratus AGR0001351433Streptomyces sp. PsTaAH-137291628
Kitasatospora sp. SolWspMP-SS2h251524Streptomyces sp. Amel2xE9271526
Streptomyces sp. NTK 93717817Streptomyces sp. AmelKG-D3221119
Streptomyces sp. ScaeMP-e48191017Streptomyces prunicolor NBRC 13075441839
Streptomyces sp. HmicA12251424Streptomyces sp. e14281325
Streptomyces griseoaurantiacus M045161116Streptomyces sp. CNX43512912
Streptomyces afghaniensis 772281729Streptomyces sp. HCCB10043171014
Streptomyces sulphureus L180191119Streptomyces sp. JS01241119
Streptomyces sp. KhCrAH-340261222Streptomyces chartreusis NRRL 3882291926
Streptomyces sp. C301727Streptomyces sp. CNY22819919
Streptomyces violaceusniger SPC613812Streptomyces sp. Amel2xB2271325
Streptomyces sp. HGB0020231322Streptomyces sp. LaPpAH-16524922
Streptomyces sp. CNS615271524Streptomyces albulus ZPM682751
Streptomyces tsukubaensis NRRL 18488301830Streptomyces albulus NK660 642750
Streptomyces vitaminophilus DSM 41686181015Streptomyces noursei642652
Streptomyces sp. SA3_actG211220Streptomyces violaceusniger Tu4113501642
Streptomyces bottropensis ATCC 25435 (2517572239)311930Streptomyces bingchenggensis492644
Streptomyces sp. CNQ865161316Streptomyces rapamycinicus632356
Streptomyces sp. CNT360191318Streptomyces sp. 769592449
Streptomyces sp. 142MFCol3.1271424Streptomyces hygroscopicus subsp. jinggangensis 5008381833
Streptomyces sp. ScaeMP-e122251123Streptomyces cattleya NRRL 8058 = DSM 46488 412138
Streptomyces griseoflavus Tu4000201519Streptomyces cattleya NRRL 8057402037
Streptomyces sp. ACT-1 301326Streptomyces hygroscopicus subsp. jinggangensis TL01371833
Streptomyces sp. TAA204181016Streptomyces avermitilis MA-4680522342
Streptomyces sp. SPB74181018Streptomyces collinus341627
Streptomyces sp. CNQ329131013Streptomyces lydicus A02381935
Streptomyces sp. 4F161115Streptomyces lydicus 103321329
Streptomyces sp. KhCrAH-244261222Streptomyces sp. Mg1372136
Streptomyces chartreusis NRRL 12338231523Streptomyces leeuwenhoekii C34(2013)361734
Streptomyces sviceus ATCC 29083191219Streptomyces pratensis/flavogriseus IAF 45291626
Streptomyces sp. CcalMP-8W231220Streptomyces reticuli472643
Streptomyces sp. SS151115Streptomyces griseus281324
Streptomyces sp. CNQ766161316Streptomyces sp. PAMC 26508291626
Streptomyces sp. URHA004116915Streptomyces sp. SirexAA-E241022
Streptomyces sp. CNB091271424Streptomyces davawensis321930
Streptomyces flavidovirens DSM 40150241523Streptomyces cyaneogriseus301428
Streptomyces yeochonensis CN732181118Streptomyces lincolnensis241523
Streptomyces viridosporus T7A, ATCC 39115321931Streptomyces pristinaespiralis HCCB 10218231218
Streptomyces sp. FXJ7.023271223Streptomyces venezuelae231621
Streptomyces mirabilis OV308281427Streptomyces sp. CFMR 7241320
Streptomyces sp. AW19M42271224Streptomyces vietnamensis302029
Streptomyces sp. ATexAB-D23281126Streptomyces xiamenensis 318191219
Streptomyces sp. BoleA517815Streptomyces coelicolor181017
Streptomyces sp. AA4351729Streptomyces albus J107418918
Streptomyces sp. CNS654271022Streptomyces ambofaciens191018
Streptomyces ipomoeae 91-03442643Streptomyces lividans201018
Streptomyces sp. DpondAA-B619919Streptomyces scabiei 87.22301630
Streptomyces sp. PCS3-D2251824Streptomyces glaucescens181117
Streptomyces sp. PRh5572051Streptomyces albus DSM 41398251324
Streptomyces sp. CNR698291726Streptomyces fulvissimus191016
Amycolatopsis sp. 75iv2, ATCC 39116281827Streptomyces sp. CNQ-509161116
Streptomyces cattleya ATCC 35852412138Streptomyces rubrolavendulae201219
Streptomyces sp. WMMB 714211018Streptomyces clavuligerus643058
Streptomyces scabrisporus DSM 41855372736Streptomyces griseochromogenes462440
Streptomyces sp. Ncost-T6T-1251422Streptomyces sp. S10(2016)201520
Streptomyces sp. CNB632161216Streptomyces globisporus231319
Streptomyces mobaraensis NBRC 13819221321Streptomyces sp. CdTB01261725
Streptomyces sp. KhCrAH-43261222Streptomyces parvulus251525
Streptomyces sp. PsTaAH-124321627Streptomyces sp. SAT1251522
Streptomyces sp. Amel2xC10151015
Abbreviations: No. F: number of P450 families; No. SF: number of P450 subfamilies.
Table
Table 2. Comparative analysis of key features of P450s in different bacterial species.
Table 2. Comparative analysis of key features of P450s in different bacterial species.
Streptomyces SpeciesMycobacterial SpeciesBacillus SpeciesCyanobacterial Species
Total No. of species analyzed20360128114
No. of P450s54601784507341
No. of families253771336
No. of subfamilies6981322879
Dominant P450 familyCYP107CYP125CYP107CYP110
Average no. of P450s273043
No. of BGCs *44578981098770
Average no. of BGCs311597
No. of P450s part of BGCs123120411227
Percentage of P450s part of BGCs2211228
ReferenceThis work[20,21][22][23]
Abbreviations: BGC: biosynthetic gene cluster. Symbol: * 103 cyanobacterial species [23] and 144 Streptomyces species were used for BGC analysis.
Table
Table 3. P450 subfamily analysis in the dominant families in 203 Streptomyces species. The number of members in the dominant P450 subfamily is presented. Detailed data on different subfamilies are presented in Supplementary Table S3.
Table 3. P450 subfamily analysis in the dominant families in 203 Streptomyces species. The number of members in the dominant P450 subfamily is presented. Detailed data on different subfamilies are presented in Supplementary Table S3.
P450 FamilyDominant Subfamilies
ABCDEFG
CYP157174 177
CYP154127 16476
CYP156 120
CYP102 78 48
CYP159125
CYP125104
CYP147 73
CYP15891
CYP103579
CYP163 50
CYP18054
CYP17057
CYP124 50
CYP104743
CYP152 42
CYP25123

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Mnguni, F.C.; Padayachee, T.; Chen, W.; Gront, D.; Yu, J.-H.; Nelson, D.R.; Syed, K. More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium. Int. J. Mol. Sci. 2020, 21, 4814. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134814

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Mnguni FC, Padayachee T, Chen W, Gront D, Yu J-H, Nelson DR, Syed K. More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium. International Journal of Molecular Sciences. 2020; 21(13):4814. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134814

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Mnguni, Fanele Cabangile, Tiara Padayachee, Wanping Chen, Dominik Gront, Jae-Hyuk Yu, David R. Nelson, and Khajamohiddin Syed. 2020. "More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium" International Journal of Molecular Sciences 21, no. 13: 4814. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134814

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