Robust Profiling of Cytochrome P450s (P450ome) in Notable Aspergillus spp.

Cytochrome P450s (P450ome) constitute an extended superfamily group of heme-thiolate enzymes identified in all biological domains. P450omes play a critical role in the oxidation of steroids and fatty acids, xenobiotic degradation of hydrophobic compounds, biosynthesis of hormones, and primary and secondary metabolism in organisms. Aspergillus species are among the most economically important fungal organisms in human medicine, industry, and agriculture worldwide. Exploring insight on the genome-wide annotations of cytochrome P450s in Aspergillus species is necessary for their biosynthetic applications. In this present study, we report the identification of 306 cytochrome P450s and their robust profiling in eight notable Aspergillus species (A. carbonarius, A. clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, and A. terreus). Based on the evolutionary relationship, the Aspergillus P450s families clustered into 15 clades, with clades V, I, and XIII recording higher percentages (17.3%, 15.00%, and 14.71%, respectively) of Cyp families. Cyps were classified into 120 families 64 clans, and their putative functions were also elucidated. P450s were predicted to be located in 13 subcellular components, but the endoplasm reticulum was the dominant location across the eight Aspergillus species. Cyps genes of Aspergillus species were associated with seven secondary metabolism-related gene clusters. Elucidating the genome-wide annotations of P450s enzymes in Aspergillus species will form vital potential biotechnological tools that could be harnessed for industrial, pharmaceutical, and agricultural use.


Introduction
Aspergillus is the most extensively studied genus in the fungal kingdom, with 350 identified species so far [1,2]. This holds because of the ubiquitous nature, wide host range, and principally, the economically important roles of Aspergillus species in the medical, industrial, and agricultural sectors globally [3,4]. In medicine, for example, invasive Aspergillosis induced by Aspergillus spp., especially A. fumigatus and A. terreus, are the major causes of death in patients with compromised immunity [5], where rates of mortality in patients with acute leukemia are as high as 50%, thereby making the disease to be considered as the most important human fungal infection [6]. In agriculture, Aspergillus spp. (A. cabonarius, A. flavus, A. niger, A. ochraceus, and A. parasiticus,) infect and produce mycotoxins, such as aflatoxins (aflatoxin B1, B2, G1, and G2), Sterigmatocystin, and ochratoxins, which contaminate wide varieties of agricultural produce/products, such as nuts (walnut, almond, brazil nut, pistachios, groundnuts) dried fruits, cereal/pulse grains (wheat, maize, millet, rice cocoa, and coffee beans), and grapes during pre-harvest or post-harvest periods [7]. Direct consumption of mycotoxin contaminated food produce (primary mycotoxicosis) by humans and animals or feeding on contaminated animal products (secondary mycotoxicosis) by humans have been implicated in posing serious carcinogenic, toxic, or lethal effects worldwide [8,9]. The global industrial application of some Aspergillus spp. has been noteworthy. Aspergillus oryzae, for instance, has been notably certified as a safe fungal species in the synthesis of various industrial enzymes for use in the food-processing industries [10,11]. Several species of Aspergillus have been found to possess the Alfa-glucuronidase enzyme, which is an essential biocatalyst in the biotransformation of lignocellulosic agro-industrial wastes into biofuels [12]. Filamentous fungi species, especially A. niger, among other microorganisms, are known to synthesize phytases enzyme, a form of phosphatases that is used as additives in animal feed and for production of ethanol in food and chemical industry, while the alkaline phosphatases, an enzyme derived from Aspergillus species, such as A. fumigatus, were reported to be an indispensable biotechnological tool for probing of nonisotopic, nucleic acids dephosphorylation, enzyme-linked immunoabsorbent assays (ELISA), and labelling of proteins [13,14]. Cytochrome P450 monooxygenases are distinct multigenic enzymes well known as versatile catalysts of a wide range of biochemical reactions, such as biosynthesis of primary and secondary metabolites in all life systems [15]. The key roles played by CYP protein genes in fungal species, which majorly influence their ubiquitous environmental adaptation and global impact in various economic sectors by enzymatic catalyzing the degradative and oxidative pathways of plethora aliphatic and aromatic hydrocarbon as well as other xenobiotic compounds, have been well documented [15][16][17][18][19]. Given the varying and significant functions regulated by CYP450s in different organisms and availability of their enriched databases, the CYPome of several important fungal species, such as Phanerochete chrysosporium [20], Mycosphaerella graminicola [21], Grosmannia clavigera [22], Trichoderma spp. [23], Cunninghamella elegans [24], and Fusarium species [25], have been comprehensively studied.
However, comparative evolutionary studies among Aspergillus species based on their CYPome are still lacking. Therefore, this present study elucidated and compared the CYP450s evolutionary descents, possible family and clan expansion, putative functions, subcellular localization, and secondary metabolite-related gene clusters among some important Aspergillus species.

Identification of CYP450 Protein Gene Families and Clans in Eight Aspergillus Species
The CYP450 families and clans of each Aspergillus spp. were determined by blasting their respective cyp protein gene sequences in the FCPD (http://p450.riceblast.snu.ac.kr/blast.php, accessed on 3 May 2021) against the cyp sequences of all characterized fungal species available in the database at 1e-5e-value, and the queried sequences were assigned families of their corresponding fungal species at a homology value of ≥40% as a CYP450 family classification criterium approved by the International P450 Nomenclature Committee [23]. The cyp families of each Aspergillus spp. were then used to locate their respective clans in the FCPD (http: //p450.riceblast.snu.ac.kr/clans.php, accessed on 3 May 2021).

Evolutionary Relatedness of Cyp Protein Genes Amongst Eight Aspergillus Species
This analysis was perfumed in the MEGA X software package (http://www.megasoftware. net/, accessed on 3 May 2021), in which the MUSCLE algorithm was employed for the multiple sequence alignment of 306 protein gene sequences. The sequences alignment and phylogenetic tree construction specifications followed as described by Dauda et al. [26].

Prediction of Subcellular Localization of Cyp Protein Genes
Two online servers (http://www.jci-bioinfo.cn/iLoc-Animal, accessed on 3 May 2021) and (http://busca.biocomp.unibo.it/, accessed on 3 May 2021) were explored and harmonized for the prediction of both single and multiple subcellular localization of cyp protein genes in the queried Aspergillus species.

Gene Clusters Associated with Secondary Metabolite Synthesis
Gene clusters responsible for the biosynthesis of secondary metabolites in the eight Aspergillus species were annotated and retrieved from the Joint Genome Institute (https: //jgi.doe.gov/our-science/science-programs/secondary-metabolites/, accessed on 18 April 2021).
Results from Figure 2 and Table 3 have shown that; phyletic group 1 has the highest number of cyp protein entries (46), with all the eight Aspergillus species present and distributed in 15 families and seven clans performing both primary and secondary metabolism functions. Group 1 is observed to be further subdivided into three sub-phyletic clades. A total of 29 cyp protein genes are closely clustered to form subclade 1 with 9 and 5 (CYP65, CYP548, CYP672, CYP566, and CYP5083) cyp families and clans, respectively. In FCPD, 4 out of the 11 Cyp families of Clan CYP65 were in Aspergillus spp. These families include Cyp60, having two cyp genes, one each identified in A. flavus and A. oryzae; Cyp65 family, which contains the highest number (12) of genes in this subclade with four cyp genes identified in A. carbonarius and two genes each in A. clavatus, A. niger, A. oryzae, and A. terreus; Cyp567 has two cyp genes one each identified in A. clavatus and A. nidulans; and Cyp5117, which possesses a cyp gene in A. oryzae. A total of 15 cyp genes from all the eight Aspergillus spp. clustered within a single clan CYP574 to constitute subclade 2. The cyp genes were distributed in five (Cyp671, Cyp628, Cyp670, Cyp669, and Cyp5076) out of seven families of Clan CYP547. Cyp671 contains five cyps (each identified in A. flavus, A. carbonarius, A. nidulans, A. niger, and A. oryzae); four cyp genes in Cyp5076 family were identified each in A. niger, A. flavus, A. fumigatus, and A. terreus. Cyp670 and Cyp669 contain two genes each found in A. niger and A. carbonarius, while Cyp628 family has two Cyps, each located in A. flavus and A. oryzae. Subclade III of group I consists of only two Cyp protein clusters from Cyp578 and Cyp671 identified in A. oryzae and A. niger, respectively, and two clans (CYP578 and CYP574).
Life 2022, 12, 451 6 of 16     The phyletic distribution of the Cyp proteins amongst eight Aspergillus species their annotated families and clans, specific functions and respective number of pr sequences in each clade are presented in Table 3. Phylogenetic tree was inferred using the minimum evolution method with MEGAX software. Each phylogenetic group (I-XV) is indicated by a specific color. Total of five cyp protein genes from four Aspergillus species forms the phyletic group II, which includes two cyp protein genes from A. flavus and one each from A. oryzea, A. niger, and A. fumigatus, clustered to form phyletic group II A total of five cyp protein genes from five clans (CYP5084, CYP58, CYP65, CYP673, and CYP507) clustered to form phyletic group II. CYP55084 clan has two families (Cyp5121 and Cyp5084), but only one cyp gene from Cyp5084 family was identified in A. fumigatus, while a Cyp protein from the only family in CYP673 clan was found in A. oryzea. Of the 14 families in CYP58 clan, only a protein gene from Cyp680 family was located: A. niger. Two cyps were identified in A. flavus from Cyp535 and Cyp567 families in CYP507 and CYP65 clans, respectively.
Clade XV is the phyletic group with the least number (three) of Cyp genes within three Aspergillus species and from three families, namely Cyp687 (A. nidulans), Cyp645 (A. fumigatus), and Cyp682 (A. flavus).

Prediction of Subcellular Localization Analysis of CYP450 Protein Genes of Eight Aspergillus Species
The result of the analysis of subcellular localization of Cyp protein genes in eight Aspergillus spp. is presented in Figure 3. Cyp protein genes of Aspergillus species have been predicted to be localized in 13 subcellular organelles. However, Cyps of the queried Aspergillus species were not distributed across all the 13 organelles. The Cyps protein genes of A. cabonarius and A. terreus were the most widely distributed been localized across all the subcellular components except the cytoskeleton. A total of 11 organelles were predicted to contain at least one Cyp protein gene of A. clavatus, A. flavus, and A. oryzae. All the queried Aspergillus spp. were domiciled in six organelles (cytoplasm, endoplasm reticulum, peroxisome, microsome, cell membrane, and plasma membrane). Amongst the predicted organelles, the endoplasm reticulum dominates, having the highest frequency of cyp protein genes across the Aspergillus species. This is followed by plasma and cell membranes, while the Golgi apparatus had the least dominant subcellular structure.

CYP450s Implicated with Secondary Metabolism-Related Gene Clusters in Aspergillus Species
The results of the analysis in Figure 4 show the involvement of cyp protein genes of Aspergillus species in seven secondary metabolism-related gene clusters, namely DiMethyl Allyl Tryptophan Synthase (DMATS), Hybrid, Non-Ribosomal Peptide Synthetases

CYP450s Implicated with Secondary Metabolism-Related Gene Clusters in Aspergillus Species
The results of the analysis in Figure 4 show the involvement of cyp protein genes of Aspergillus species in seven secondary metabolism-related gene clusters, namely DiMethyl Allyl Tryptophan Synthase (DMATS), Hybrid, Non-Ribosomal Peptide Synthetases (NRPS), Non-Ribosomal Peptide Synthetases-LIKE (NRPS-LIKE), PolyKetide Synthases (PKS), PolyKetide Synthases-LIKE (PKS-LIKE), and Terpene Cyclases (TC). CYP450 gene clusters for secondary metabolism were present in all our queried Aspergillus spp. except for TC in A. fumigatus. PKS was identified as the most dominant secondary metabolism-related gene cluster across the studied Aspergillus species, with an average of 21 cyps. NRPS-LIKE was the next-dominant secondary metabolism-related gene cluster, having an average of 13 cyps, while HYBRID and TC were the least, with three as the average number of cyp proteins. Aspergillus niger was predicted to have the highest number (79) of cyp genes engaged in secondary metabolic activity. This is followed by A. flavus (77), while A. fumigatus had the least (32).

Discussion
P450 genome-wide comparisons and annotations have helped us to establish better the links among Cyp families in various Aspergillus species. A comprehensive phylogenetic analysis was performed to illustrate the divergence of primary sequences and evolutionary connections of cytochrome P450 groups in Aspergillus. The phylogenetic investigations have revealed that the magnitude of the distribution of Cytochrome P450 throughout the fifteen clades differs. In evolutionary terms, the CYPome (the total number of CYPs in a particular species) is very dynamic, and the families, subfamilies, and fraction of CYP genes belonging to each of the eight Aspergillus species vary substantially. These distinctions are reflected in qualitative and quantitative differences in metabolite profiles and physical or developmental traits and adaption to specific ecological niches [27].
According to findings, specific families of P450 found in some species of fungi originated by paralogous evolution of member P450s, allowing the organism to adapt to various ecological niches, including the colonization of plant material [28]. Therefore, P450 family growth is feasible due to P450 member duplication in an organism [29,30]. The existence of several families and clans found in the eight chosen Aspergillus species shows expansions of the fungal CYPs families. We believe this is a result of the evolution of several fungal properties, including pathogenicity. The results also showed unique CYP fam-

Discussion
P450 genome-wide comparisons and annotations have helped us to establish better the links among Cyp families in various Aspergillus species. A comprehensive phylogenetic analysis was performed to illustrate the divergence of primary sequences and evolutionary connections of cytochrome P450 groups in Aspergillus. The phylogenetic investigations have revealed that the magnitude of the distribution of Cytochrome P450 throughout the fifteen clades differs. In evolutionary terms, the CYPome (the total number of CYPs in a particular species) is very dynamic, and the families, subfamilies, and fraction of CYP genes belonging to each of the eight Aspergillus species vary substantially. These distinctions are reflected in qualitative and quantitative differences in metabolite profiles and physical or developmental traits and adaption to specific ecological niches [27].
According to findings, specific families of P450 found in some species of fungi originated by paralogous evolution of member P450s, allowing the organism to adapt to various ecological niches, including the colonization of plant material [28]. Therefore, P450 family growth is feasible due to P450 member duplication in an organism [29,30]. The existence of several families and clans found in the eight chosen Aspergillus species shows expansions of the fungal CYPs families. We believe this is a result of the evolution of several fungal properties, including pathogenicity. The results also showed unique CYP families and clans in some chosen Aspergillus species. We believe that this has a role in the host specificity of the fungus species to plants or animals. According to Rampersad [31], distinct CYPs found in various fungus species may account for the host specificity of each fungus species to a particular plant or animal.
Our study further reveals that irrespective of the Aspergillus spp., the endoplasm reticulum turned out to be the dominant organelle for the localization of their cyp protein genes. Based on how electrons are transferred to their catalytic site from NAD (P) H, cyps have been categorized into four classes, of which fungi and other eukaryotes are most commonly found in class II enzymes, with the endoplasmic reticulum being the dominant subcellular component of their cyps [32]. The cyps protein genes of A. cabonarius and A. terreus were the most distributed across the subcellular components. The localization of P450s in six cell organelles (cytoplasm, endoplasm reticulum, peroxisome, microsome, cell membrane, and plasma membrane) of all the queried Aspergillus spp. indicates the varying significant roles played by these genes in the Aspergillus genus. Fungal species as part of organisms possessing the Class II enzymes are involved in highly diverse functions, including biosynthesis of secondary metabolites (mycotoxins), sterols of membranes, lipid metabolism, and detoxification of xenobiotics and phytoalexins; therefore, their cyps may be localized in more than one subcellular component [32,33].
In response to the surrounding external biotic and abiotic factors, Aspergillus species, like other fungi, are known to bio-synthesize some bioactive molecular compounds referred to as secondary metabolites (SMs), which are maybe either beneficial or harmful to their hosts and environment depending on the species as part of their life cycle [34]. The specific genes responsible for producing these SMs are most often positioned so closely as a cluster [35,36]. Given the significant role played by CYP450 enzymes in the metabolic activity of life systems, the present study predicted the engagement of cyp protein genes of Aspergillus species in the formation of three out of four classes of secondary metabolism-related gene clusters and their hybrids commonly found in fungi as reported by Keller et al. [37]. These biosynthesis gene clusters encode enzymes (polyketide synthases, dimethylallyl tryptophan synthases, non-ribosomal peptide synthetases, and terpene cyclases) characterized by multiple modules and domains, which are vital for the scaffold formation of secondary metabolites [38][39][40][41]. The observed abundance of PKS, NRPS-like, and NRPS in Aspergillus species agrees with the findings of Cox, [42], de Vries et al. [3], and Drott et al. [43], who reported that PKS and NRPS are the most dominant secondary metabolism-related gene clusters in fungal species from which secondary metabolites (SMs) are synthesized. Similarly, the role of P450 genes in the biosynthesis of mycotoxins in fungal species has been well documented [32]. Even though these SMs have not been demonstrated to be essential for the growth and development of Aspergillus spp. [44], they are significant for the colonization of their environment by serving as growth inhibitors of their competitors and chemical communicating signals [40,45]. Several SMs synthesized by Aspergillus species have been identified. For example, lovastatin produced by A. terreus is medically useful for regulating cholesterol in humans [35], while many are detrimental, such as aflatoxins complex from A. niger and A. flavus, gliotoxin by A. fumigatus, and Aspyridones by A. nidulans [35].

Conclusions
Aspergillus species are among the most commercially significant fungal organisms globally, with use in human health, industry, and agriculture. Understanding the genomewide annotations of cytochrome P450s in Aspergillus species is critical for biosynthetic applications. This study revealed a total of 306 cytochrome P450s found in eight Aspergillus species. According to the evolutionary connection, the Aspergillus P450s families are divided into 15 clades, with Clade V containing the most significant Cyp families (17.3%). Cyps were divided into 120 families, 64 clans, and their putative functions. Cytochrome P450 families identified as unique to each of the eight Aspergillus spp. could be a target to be harnessed either for their management or biosynthesis of important secondary metabolites. P450s were anticipated to be found in 13 different subcellular components; however, the endoplasmic reticulum was the most common site in all eight Aspergillus species. The Cyps genes in the Aspergillus species were linked to seven secondary metabolism-related gene clusters. The current study's findings disclosed some pertinent information that may be used for the appropriate management of Aspergillus species, which represent a danger to the sustainable production of various vital crops throughout the world. We conclude that elucidating the genome-wide annotations of P450s enzymes in Aspergillus species would result in critical prospective biotechnological tools that may be used in industrial, medicinal, and agricultural applications.