*3.6. Identification of CYP139 P450 Secondary Metabolite BGCs*

BGCs listed on the IMG/M [68] website for each of the mycobacterial species were manually searched for the presence of CYP139 P450s using the protein ID. The BGCs that have CYP139 P450 were selected for further study. The listed BGCs at IMG/M are general [68] and in order to identify the specific type of BGCs, the selected BGCs genome sequences were subjected to secondary metabolite BGCs analysis, as described elsewhere [21]. Briefly, the individual BGC genome sequences downloaded from IMG/M [68] were submitted to anti-SMASH [77]. The type of BGC, percentage similarity to a known cluster and the cluster name were noted. Standard BGC abbreviation terminology developed by anti-SMASH [77] was used in the study.

#### **4. Conclusions**

The advancement of genome sequencing and bioinformatics tools helps significantly in understanding the role of orphan proteins in organisms. This study is an attempt to utilize the availability of quite a large number of mycobacterial species genome sequences and different bioinformatics tools to understand the role of the orphan CYP139 family in mycobacterial species. This study revealed that the CYP139 family indeed plays a role in the synthesis of secondary metabolites in mycobacterial species. Based on the functions of homolog CYP139 P450 gene clusters' secondary metabolites, it can be assumed that these metabolites indeed help mycobacterial species to survive in the host, being part of the cell envelope and inhibiting fibroblast, thus causing tissue weakening and causing ulcers via tissue necrosis. The metabolites that exhibit antibacterial (including antimycobacterial), antifungal and antiviral properties certainly help mycobacterial species to gain the upper hand in the niche area compared to those agents. It would be interesting to determine the roles of CYP139A P450s that are not part of gene clusters. Predictions made in the study are based on the functions of homolog secondary metabolites. However, wet laboratory biosynthesis and functional analysis of secondary metabolites should be carried out to understand the role of these metabolites in mycobacterial physiology. Study results can be used as a reference for future experimental studies.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/11/ 2690/s1.

**Author Contributions:** Conceptualization, R.K. and K.S.; data curation, P.R.S., W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; formal analysis, P.R.S., W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; funding acquisition, R.K., A.P.K. and K.S.; investigation, P.R.S., W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; methodology, P.R.S, W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; project administration, R.K. and K.S.; resources, R.K., A.P.K. and K.S.; supervision, R.K. and K.S.; validation, P.R.S., W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; visualization, P.R.S., R.K. and K.S.; writing—original draft, P.R.S., W.C., D.R.N., A.P.K., J.-H.Y., R.K. and K.S.; writing—review and editing, P.R.S., R.K. and K.S. **Funding:**

Khajamohiddin Syed expresses sincere gratitude to the University of Zululand Research Committee for funding (Grant No. C686) and to the National Research Foundation (NRF), South Africa for a research grant (Grant No. 114159). Puleng Rosinah Syed thanks the NRF, South Africa for a DST-NRF Innovation Master's Scholarship (Grant No. 114575). Rajshekhar Karpoormath (RK) and Puleng Rosinah Syed are grateful to the Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa for providing access to necessary facilities. RK is also thankful to the NRF, South Africa for the research grants (Grant No. 103728 and 112079). Abidemi Paul Kappo is grateful to the South African Medical Research Council (SAMRC) for a research grant (Grant No. PC57009).

**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.

### **References**

1. World Health Organization (WHO). Global Tuberculosis Report 2018. Available online: https://www.who. int/tb/publications/global\_report/en/ (accessed on 22 March 2019).


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