*Article* **Comprehensive Analyses of Cytochrome P450 Monooxygenases and Secondary Metabolite Biosynthetic Gene Clusters in** *Cyanobacteria*

**Makhosazana Jabulile Khumalo 1, Nomfundo Nzuza 1, Tiara Padayachee 1, Wanping Chen 2, Jae-Hyuk Yu 3,4, David R. Nelson 5,\* and Khajamohiddin Syed 1,\***


Received: 22 December 2019; Accepted: 15 January 2020; Published: 19 January 2020

**Abstract:** The prokaryotic phylum *Cyanobacteria* are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria *Bacillus*, *Streptomyces* and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to *Bacillus* and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.

**Keywords:** cytochromes P450 monooxygenases; secondary metabolites; *Cyanobacteria*; biosynthetic gene clusters; gene-cluster diversity percentage; mathematical formula; phylogenetic analysis

#### **1. Introduction**

*Cyanobacteria* are thought to be some of the oldest known photosynthetic organisms that played a major role in the evolution of life by contributing to the oxygenation of the earth's atmosphere [1–5]. These Gram-negative photosynthetic prokaryotes also played a significant role in nitrogen and carbon cycles as they are able to fix atmospheric nitrogen and carbon. It is also well known that cyanobacterial species are responsible for the evolution of plants on earth owing to their endosymbiotic lifestyle,

which led to the development of light-harvesting organelles in plants [6,7]. Cyanobacterial species can be found in diverse terrestrial habitats, ranging from the oceans to fresh-water bodies, soil to desert rocks, and extreme environments such as Antarctic dry valleys, Arctic and thermophilic lakes [8,9].

To survive in a wide range of environments, cyanobacterial species produce diverse natural products comprising both primary and secondary metabolites belonging to the group's non-ribosomal proteins, polyketides, and terpenes and alkaloids. These products have varying activities of anticancer, antiviral, and ultraviolet-protective activities, as well as hepatotoxicity and neurotoxicity [10,11]. Many cyanobacterial species are used as model organisms to understand fundamental processes such as photosynthesis, nitrogen fixation, and circadian rhythm [12–14]. Owing to their amenability to gene manipulation, these organisms have been genetically modified/engineered for the production of valuable human compounds [15,16]. Despite all their highly beneficial characteristics, cyanobacterial species are also known to cause cyanobacterial blooms in water, resulting in the production of toxins that are harmful to humans, wild and domestic animals [17]. An overview of some of the cyanobacterial species' beneficial and harmful characteristics is presented in Table 1.

**Table 1.** Some of the cyanobacterial species well-known characteristics.



**Table 1.** *Cont.*

Currently, world-wide research continues to identify novel secondary metabolites with potential biotechnological value from cyanobacterial species. These secondary metabolites are usually produced by a set of genes that are clustered in an organism, and these clusters are known as secondary metabolite biosynthetic gene clusters (BGCs) [51]. Among different genes involved in the production of secondary metabolites, P450s occupy a special place, as these enzymes contribute to the diversity of secondary metabolites due to regio- and stereo-specific oxidation of substrates [52,53]. Recent studies in different bacterial populations belonging to the genera *Bacillus* [54], *Streptomyces* and *Mycobacterium* [55,56] revealed the presence of a large number of P450s in secondary metabolite BGCs. A paper published in 2010 [11] reported some P450s present in cyanobacterial species where the authors performed blast analysis using P450s from *Nostoc* sp. strain PCC 7120 [57] and *Synechocystis* sp. PCC 6803 [58]. However, to date, a comprehensive comparative analysis of P450s and those associated with secondary metabolism in cyanobacterial species has not been reported. The availability of a large number of cyanobacterial species genomes gives us an opportunity to address these issues. In this study, genome data-mining, annotation and phylogenetic analysis of P450s in 114 cyanobacterial species were performed. The study also reports a comparative analysis of secondary metabolite BGCs in cyanobacterial species and identification of P450s that are part of the different BGCs. Furthermore, a comparative analysis of P450s and secondary metabolite key features of cyanobacterial species with other bacterial species belonging to the genera *Bacillus*, *Streptomyces* and *Mycobacterium* is presented.

#### **2. Results and Discussion**

#### *2.1. Cyanobacterial Species Have Lowest Number of P450s*

Analysis of 41 cyanobacterial genera and species belonging to the unclassified *Cyanobacteria* (Table S1) revealed the presence of P450s in all cyanobacterial genera except in the genera *Thermosynechococcus*, *Atelocyanobacterium* and *Crocosphaera* (Figure 1). This may be due to the lowest number of species genomes being available for analysis in these genera. Furthermore, two species in the unclassified *Cyanobacteria* group did not have P450s (Figure 1). Analysis of P450s at species level revealed that among 114 species, 88 species had P450s and 26 species did not have P450s in their genomes (Figure 1), indicating that most of the species in different genera had P450s.

In total, 341 P450s were found in 88 cyanobacterial species (Figures 2 and 3, Table S2). The analysis also revealed the presence of 13 P450-fragments and 15 P450 false positives in different cyanobacterial species (Table S2). A list of P450s, P450-fragments, and P450 false positives identified in cyanobacterial species, along with their sequences, is presented in Supplementary Dataset 1. The occurrence of P450-fragments and P450 false positives in organisms is quite common and these sequences were not taken for further analysis. Among cyanobacterial species, *Rivularia* sp. PCC 7116 has the highest number of P450s (16 P450s), followed by *Nostocales cyanobacterium* HT-58-2 (13 P450s), and *Nostoc flagelliforme* (12 P450s) (Figure 3 and Table S2). Most of the cyanobacterial species have a single P450 in their genome (Figure 3). Comparative analysis with other bacterial species revealed that cyanobacterial species have the lowest number of P450s compared to *Bacillus* species, *Streptomyces* and mycobacterial species (Table 2). Cyanobacterial species have an average of three P450s in their genomes compared to an average of four P450s in *Bacillus* species [54], 30 P450s in mycobacterial species [59], and 34 P450s in *Streptomyces* species [55] (Table 2).

**Figure 1.** Analysis of P450s in *Cyanobacteria*. P450s were analyzed at both the genus level (**A**) and species level (**B**). The numbers next to the bars indicate the number of species. In Panel A, only species numbers for the species that have P450s are presented. Detailed information is presented in Tables S1 and S2.


**Table 2.** Comparative analysis of key features of P450s in different bacterial species.

Note: \* 103 cyanobacterial species gave results with anti-SMASH (antibiotics & Secondary Metabolite Analysis Shell). Eleven species genomes did not give results. Detailed information on gene clusters is presented in Table S2.

**Figure 3.** Comparative analysis of P450s in cyanobacterial species. The numbers next to bars indicate the number of P450s in each species. The species names with respect to their codes can be found in Table S2.
