*2.4. Most CYP139A P450s Are Part of Secondary Metabolite Biosynthetic Gene Clusters*

Analysis of CYP139A P450s as part of secondary metabolite BGCs in mycobacterial species revealed that most of the CYP139A P450s are part of different BGCs (Figure 4A and Table S2). Among 894 CYP139A P450s, 824 CYP139A P450s (92%) were found to be part of secondary metabolic BGCs (Figure 4A). This means 70 CYP139A P450s were not found to be part of any secondary metabolite BGCs. Comparison of CYP139A P450s that are part of BGCs in three categories revealed that most of the CYP139A P450s in MTBC and NTM species were part of BGCs, compared to species of MAC, where fewer than half of CYP139A P450s were part of secondary metabolite BGCs (Figure 4B).

**Figure 4.** CYP139A P450s secondary metabolite BGCs analysis in mycobacterial species. (**A**) Analysis of CYP139A P450s that are part of BGCs. (**B**) Comparative analysis of CYP139A P450s that are part of BGCs and types of BGCs in different mycobacterial categories. Abbreviations: MTBC, *Mycobacterium tuberculosis* complex; MAV, *M. avium* complex; NTM, non-tuberculosis mycobacteria. (**C**) Comparative analysis of CYP139A P450 cluster types. The type of cluster and the number of CYP139A P450s and their percentage in the total number of P450s were presented in the figure. Abbreviation: T1pks, Type 1 polyketide synthase; T2pks, Type 2 polyketide synthase.

Analysis of secondary metabolite BGCs revealed that CYP139A P450s were part of only three different cluster types (Figure 4C and Table S2). Among three different cluster types, CYP139A P450s were found to be present dominantly as part of Type 3-Type 1 polyketide synthase (T3PKS-T1PKS) (97%) compared to T3 PKS (2%) and T1 PKS (1%) (Figure 4C and Table S2). There were 796 CYP139A P450s found to be part of T3PKS-T1PKS, followed by 17 and 11 CYP139 P450s found to be part of T3 PKS and T1 PKS, respectively (Figure 4C and Table S2). Analysis of gene clusters revealed that 824 CYP139A P450s were part of 39 different gene clusters (Figure 4). There were 34 CYP139A P450 gene clusters found in MTBC species, followed by seven gene clusters in NTM species and six gene clusters in MAC (Figure 4B). Among different gene clusters, ML-449 was dominant, with 349 CYP139A P450s followed by methylated alkyl-resorcinol/methylated acyl-phloroglucinol (MAR/MAP) with 104 CYP139A P450s, Nystatin with 74 CYP139A P450s and Jerangolid with 55 CYP139A P450s (Figure 5). Among 39 gene clusters only 11 gene clusters were found to have 10 or more CYP139A P450s (Figure 5). Analysis of DNA sequence percentage identity between CYP139A P450 gene clusters compared to known gene clusters revealed that some of the gene clusters have 100% identity, such as Leucanicidin, MAR/MAP and Micromonolactam (Figure 5), indicating CYP139A P450s are indeed involved in the synthesis of these secondary metabolites.

**Figure 5.** CYP139A P450 gene cluster analysis in mycobacterial species. The 39 gene clusters were presented with their standard abbreviated names as per anti-SMASH. The number next to bars represents the number of CYP139A P450s that is part of that gene cluster. The inset figure shows the percentage identity of CYP139A P450 gene clusters to the known gene clusters available at anti-SMASH. The number next to the bars represents the percentage identity. For some gene clusters the percentage identity is represented with standard deviation (indicated with bars).
