**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 [3–6]. 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 [9–13]. 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 [14–16].

Unlike other enzymes, P450 enzymes have a typical nomenclature system established by the International P450 Nomenclature Committee [17–19]. 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 [17–19]. 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 [17–19] 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 [11,20,22,24–27].

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 [15,16,31–33]. 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**
