*2.4. Large Number of P450s Found to Be Part of Secondary Metabolites BGCs in Bacillus Species*

Among 507 P450s identified in 128 *Bacillus* species, 112 P450s (22%) from 50 *Bacillus* species were found to be part of secondary metabolite BGCs (Table 1). Among 13 P450 families, only seven families, namely CYP107, CYP113, CYP134, CYP152, CYP102, CYP109, and CYP1179, were found to be part of different secondary metabolite BGCs (Figure 8). P450 subfamily level analysis revealed that P450s belonging to the subfamilies H and K in the CYP107 family were part of secondary metabolite BGCs, despite subfamily J being dominant in that family (Figure 4). In the CYP152 family, P450s belonging to subfamily A were found to be part of the secondary metabolite BGCs. Analysis of P450s involving secondary metabolite biosynthesis revealed that P450s belonging to the CYP107 family are dominant by 61% (68 P450s) of all P450s (112 P450s) involved in secondary metabolite BGCs, followed by CYP113, CYP152, CYP102, CYP109, and CYP1179 (Figure 8). It is interesting to note that these P450 families are highly-populated in *Bacillus* species (Figures 3 and 5). This further supports the previous hypothesis that species populate specific P450s if they are useful in their adaptation to certain ecological niches or useful in their physiology [31–35]. Considering the large number of P450s, their widespread nature, and part in secondary metabolite BGCs, it can be hypothesized that the P450s belonging to the CYP107, CYP102, CYP109, CYP152, and CYP113 families play a key role in *Bacillus* species' physiology, including synthesis of different secondary metabolites. Despite secondary metabolite BGCs being found on plasmid DNA, no P450 was found to be part of these clusters. Analysis of association between P450 families and secondary metabolite BGCs revealed that CYP107 family P450s were mostly associated with BGCs Nrps-Transatpks-Otherks and Transatpks-Nrps; CYP113 family P450s are associated with Transatpks BGC, and CYP134 family P450s are associated with other, a putative gene cluster (Table 1).



*Bacillus* sp. LM 4-2


**Table 1.** *Cont*.

3 Nrps-Transatpks-Otherks CYP107K1 7 Other CYP102A48 9 Other CYP134A1


**Table 1.** *Cont*.

**Figure 8.** Comparative analysis of P450 families involved in secondary metabolite biosynthesis. The P450 family name, number of P450s and their percentage of the total number of 112 P450s are presented in the figure.

#### *2.5. Bacillus P450s Indeed Involved in the Synthesis of Secondary Metabolites*

Based on *in silico* analysis (in this study), seven P450 families, namely CYP107, CYP113, CYP134, CYP152, CYP102, CYP109, and CYP1179, were identified as part of secondary metabolite BGCs in *Bacillus* species (Figure 8). Functional data available for some P450s confirms that the predicted P450s, in this study, are indeed involved in biosynthesis of different secondary metabolites, and some of the P450 families, such as CYP105, CYP107, and CYP109, have been found to display highly-diverse functions [9,12,36]. CYP102A1 from *B. megaterium* [24,37,38] and CYP152A1 from *B. subtilis* [39,40] were found to be fatty acid hydroxylases. P450s belonging to the CYP106, CYP107, CYP109, and CYP134 families were found to hydroxylate different steroids, albeit with different substrate specificities [22]. CYP134A1 is involved in the synthesis of pulcherriminic acid, a natural product [41], and CYP107H1 (P450 biol) is involved in the synthesis of polyketides [42]. Based on functionally characterized homolog P450s from other organisms, CYP105, CYP107, and CYP109 family P450s have been found to be associated with the degradation and biotransformation of a diverse array of xenobiotics and secondary metabolites [36,43,44]. CYP113 P450s are involved in the biosynthesis of secondary metabolites such as erythromycin [45,46] and tylosin [47,48]. Despite CYP102 and CYP152 P450s being found in secondary metabolite BGCs (in this study), their role in secondary metabolites biosynthesis has not been yet elucidated.
