*Article* **Comparative Analyses of Cytochrome P450s and Those Associated with Secondary Metabolism in** *Bacillus* **Species**

**Bongumusa Comfort Mthethwa 1, Wanping Chen <sup>2</sup> , Mathula Lancelot Ngwenya 1, Abidemi Paul Kappo <sup>1</sup> , Puleng Rosinah Syed 3, Rajshekhar Karpoormath 3, Jae-Hyuk Yu <sup>4</sup> , David R. Nelson 5,\* and Khajamohiddin Syed 1,\***


Received: 25 September 2018; Accepted: 16 October 2018; Published: 16 November 2018

**Abstract:** Cytochrome P450 monooxygenases (CYPs/P450s) are among the most catalytically-diverse enzymes, capable of performing enzymatic reactions with chemo-, regio-, and stereo-selectivity. Our understanding of P450s' role in secondary metabolite biosynthesis is becoming broader. Among bacteria, *Bacillus* species are known to produce secondary metabolites, and recent studies have revealed the presence of secondary metabolite biosynthetic gene clusters (BGCs) in these species. However, a comprehensive comparative analysis of P450s and P450s involved in the synthesis of secondary metabolites in *Bacillus* species has not been reported. This study intends to address these two research gaps. *In silico* analysis of P450s in 128 *Bacillus* species revealed the presence of 507 P450s that can be grouped into 13 P450 families and 28 subfamilies. No P450 family was found to be conserved in *Bacillus* species. *Bacillus* species were found to have lower numbers of P450s, P450 families and subfamilies, and a lower P450 diversity percentage compared to mycobacterial species. This study revealed that a large number of P450s (112 P450s) are part of different secondary metabolite BGCs, and also identified an association between a specific P450 family and secondary metabolite BGCs in *Bacillus* species. This study opened new vistas for further characterization of secondary metabolite BGCs, especially P450s in *Bacillus* species.

**Keywords:** Antibiotics; *Bacillus*; biosynthetic gene clusters; comparative analysis; cytochrome P450 monooxygenase; *Mycobacterium*; P450 diversity percentage; P450 profiling; secondary metabolites

#### **1. Introduction**

Cytochrome P450 monooxygenases, also known as CYPs/P450s, are undoubtedly among the most catalytically-diverse enzymes, performing enzymatic reactions with chemo-, regio- and stereo-selectivity [1–6]. The catalytic diversity combined with chemo-, regio- and stereo-specific oxidation of substrates exerted by P450s are used in diverse biotechnological applications ranging from drug discovery to bioethanol production and synthesis of different secondary metabolites [7–12]. P450s are heme-thiolate proteins ubiquitously found in species belonging to different biological kingdoms, including non-living entities such as viruses [13,14]. In bacteria, P450s have been found to play a key role in enzymatic reactions, leading to the biosynthesis of physiological compounds or the biodegradation of xenobiotics [9,11,15,16].

P450s' role in the synthesis of a diverse array of secondary metabolites has been thoroughly reviewed [8,12]. Secondary metabolites are natural products that are widely-used in human and veterinary medicine, agriculture, and manufacturing, and are known to mediate a variety of microbe-host and microbe-microbe interactions [17]. P450s were found to play a key role in the synthesis of different secondary metabolites, including terpenes, alkaloids, shikimates, polyketides, and peptides [12]. The coding sequences (genes) of enzymes involved in the synthesis of different secondary metabolites, including P450s, were found to be part of gene clusters named biosynthetic gene clusters (BGCs) [17]. Bacterial species have been found to have more than 1000 different types of BGCs involved in the synthesis of known and unknown secondary metabolites [17].

Among bacteria, species belonging to the genus *Bacillus* are ubiquitously present in the biosphere, and are well known for their distinct features with one common characteristic, i.e., making dormant endospores during unfavorable growth conditions [18,19]. Applications of *Bacillus* species across different spectra have been well explored in the industrial, agricultural, and ecological fields, and by academics, against a backdrop of being a well-known human pathogen [18,19]. Comprehensive *in silico* studies detailing *Bacillus* species' ability to produce different secondary metabolites and different types of BGCs have frequently been reported [20,21]. Analysis of 1566 *Bacillus* species' genomes revealed the presence of 20,000 BGCs, most of which were found to produce known secondary metabolites that play a key role in the physiology and development of *Bacillus* species [21]. The study by Grubbs et al. [21] also reported that secondary metabolite alkylpyrones play a key role in inhibiting spore development in *Bacillus* species.

Despite comprehensive analysis of *Bacillus* species' secondary metabolite BGCs, P450s that are part of different BGCs have not been reported. Analysis of P450s in *Bacillus* species date back to 2009, when the authors performed a comparative analysis of P450s in 29 *Bacillus* species, and identified a few P450s belonging to a limited number of P450 families such as CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197 [22]. Among the P450 families identified in *Bacillus* species, the CYP102 P450 family has a special place in P450 research, being one of the most extensively-studied bacterial P450s, for its structural, functional, and biotechnological potential. Even the *Bacillus* species *B. megaterium* has become very famous owing to the identification of CYP102 P450 from this organism [23,24]. *In silico* comparative analysis of P450s in bacterial species is gaining momentum. Recently, a comprehensive comparative analysis of P450s in 60 mycobacterial species has been reported; the authors identified that mycobacteria possess a large number of P450s, and that different mycobacterial categories have characteristic P450 families that can be used as biomarkers to identify different mycobacterial species [25].

The current trend of whole-genome sequencing of organisms resulted in the genome sequencing of a large number of *Bacillus* species genomes. Quite a large number of *Bacillus* species genome sequences are available for public use at Kyoto Encyclopedia of Genes and Genomes—GenomeNet (KEGG) [26]. This gives us the opportunity to perform comprehensive a comparative analysis of P450s in *Bacillus* species as per international P450 nomenclature committee rules [27–29], and to identify P450s involved in the synthesis of different secondary metabolites. Here, we report genome data mining, annotation, phylogenetic and comparative analysis of P450s in 128 *Bacillus* species, including identification of P450s involved in the synthesis of different secondary metabolites. This study also reports comparative analysis of P450s between the genera *Bacillus* and *Mycobacterium*. Last but not least, a previous study reporting BGCs in *Bacillus* species did not clearly indicate BGCs on genomic DNA (gDNA) and plasmid DNA [21]; thus, in this study, gDNA and plasmid DNAs were individually subjected to BGC analysis.
