The presentation describes the preponderance of Cytochrome P450 genes in a chronically polluted soil

1. Elucidation of the CYPome of the Prokaryotic
and Eukaryotic Communities of a Chronically
Polluted Soil
BY
SALAM, L.B. (PhD)
Department of Biological Sciences, Microbiology unit,
Elizade University, Ilara-Mokin, Ondo State, Nigeria

2. INTRODUCTION
Cytochrome P450 is a superfamily of proteins with an iron haem prosthetic
group which are involved in monooxygenase oxidation-reduction reactions
with families cutting across the tree domains of life (Guengerich, 2019).
They play roles particularly in biosynthesis and biotransformation of steroids,
fatty acids, hydrocarbons, xenobiotics, drugs and several toxic compounds
(Zanger and Schwab, 2013; Estevez et al., 2021).
Cytochrome P450s are involved in a plethora of redox reactions including
hydroxylation, epoxidation, demethylation, sulfoxidation, N-oxidation and
decarboxylation (Guengerich and Munro, 2013).
2

3. INTRODUCTION
They are aerotolerant enzymes and apart from their role in oxidative
processes, they also function in reductive dehalogenation and denitration of
diverse persistent organic pollutants under anoxic condition (Behrendorff,
2021).
Their abundance and diversity can be an indicator of stress induced
functional shift in an environment.
Their roles in stress responses in plants including drought, temperature,
salinity, heavy metal herbivore, and myriad biotic stressors was recently
elucidated (Pandian et al. 2020)
3

4. INTRODUCTION
Understanding the potential of pollutants to disrupt community structure and
functions has undergone a shift from culture dependent to culture independent
method, with more emphasis placed on metagenomic approach
The use of metagenomics to gain insight into the genomic potentials
associated with cytochrome P450 in hydrocarbon impacted soils or similar
environmental compartments are relatively few.
In this study, we use metagenomics to elucidate the CYPome of the
prokaryotic and eukaryotic communities of a chronically polluted soil with
phased history of agricultural activities and hydrocarbon contamination.
4

5. MATERIALS & METHODS
5

6. RESULTS
Functional analysis of ORFs of 3S metagenome for cytochrome P450 (CYP)
proteins revealed that 3S CYPome comprises ninety-four (94) cytochrome
P450 proteins cutting across the three domains of life.
The domain Bacteria had 72 CYP families, Eukarya had 21 CYP families,
while the domain Archaea has 1 CYP family.
In prokaryotic community, Bacillus CYPome in 3S metagenome comprises
five CYP families and 13 subfamilies
Streptomyces CYPome in 3S metagenome comprises 16 CYP families and
four standalone CYPs (aziB1, ncsB3, nlmB, scnG) that do not belong to any
of the existing CYP families
6

7. RESULTS
CYP102
31%
CYP106
15%
CYP107
31%
CYP109
15%
CYPX
8%
Bacillus CYPome
CYP102 subfamilies: A1, A2, A3, A5
CYP107 subfamilies: DY1, AA1, H1, J1
CYP106 subfamilies: A1, A2
CYP109 subfamilies: A1, B1
CYP134 (CYPX) subfamily: A1
Functions:
CYP107: Biosynthesis of secondary metabolites
CYP102: Hydroxylation of fatty acids
CYP106 & CYP109: Hydroxylation of steroidal
compounds
CYPX: Pulcherriminic acid biosynthesis
7

8. RESULTS
aziB1
3%
CYP105
24%
CYP107
12%
CYP113
3%
CYP129
3%
CYP147
3%
CYP151
3%
CYP158
6%
CYP161
6%
CYP162
3%
CYP163
6%
CYP170
3%
CYP183
3%
CYP185
3%
CYP244
3%
CYP245
3%
CYP28
3%
ncsB3
3%
NlmB
3%
ScnG
3%
Functions:
CYP105: Secondary metabolite biosynthetic
pathway and xenobiotic catabolism
CYP107: Biosynthesis of antibiotics and
hydroxylation of testosterone and bile acid
Other CYPs: Biosynthesis of diverse secondary
metabolites
Streptomyces CYPome
Major families
CYP105 subfamilies: A1, A3, B1, B2, C1, D1,
H4, L1
CYP107 subfamilies: D1, G1, L1, L14
CYP158 subfamilies: A1, A2
CYP161 subfamilies: A3. C3
CYP163 subfamilies: A1, A3
8

9. RESULTS
Thirty-three CYPs belonging to 24 CYP families were detected as members
of Mycobacterium CYPome in 3S metagenome.
Majority (79%) of the detected CYPs were orphan CYPs that have no known
assigned biological functions.
Seventeen CYP families constitute the Proteobacteria CYPome detected in
3S metagenome. The CYPs were recovered from the genera Pseudomonas,
Bradyrhizobium, Rhizobium, and the myxobacteria species Sorangium
cellulosum
The CYPs are involved in biodegradation of camphor (CYP101A1),
hydroxylation of monoterpene alcohols, α-terpinol and linalool (CYP108A1,
CYP111A2), and biodegradation of thiocarbamate herbicide (CYP116)
9

10. RESULTS
They are also involved in gibberellin biosynthesis (CYP112, CYP114),
detoxification of plant protective agent (CYP103, CYP104), hydroxylation of
fatty acids, conversion of thioridazine to thioridazine-5-sulfoxide, and
biosynthesis of epothilones (CYP109, CYP167, CYP265, CYP266, CYP267)
 Cyanobacteria is represented in 3S metagenome by a Nostoc CYP, CYP110,
which is believed to play a role in fatty acid/alkane hydroxylation.
The archaeal CYP, CYP119A1 Catalyzes in vitro the H2O2-dependent
epoxidation of styrene, cis-β-methylstyrene and cis-stilbene, and hydroxylate
lauric acid
10

11. RESULTS
CYP105
3%
CYP108
3%
CYP123
9%
CYP124
6%
CYP125
6%
CYP126
3%
CYP128
3%
CYP130
3%
CYP135
6%
CYP136
3%
CYP137
3%
CYP138
3%
CYP139
3%
CYP140
9%
CYP141
3%
CYP142
6%
CYP143
6%
CYP144
3%
CYP150
3%
CYP187
3%
CYP226
3%
CYP269
3%
CYP279
3%
CYP51
3%
CYPs with Biological Functions
CYP51, CYP124, CYP142- Sterol biodegradation
CYP128, CYP139, CYP140A7- Biosynthesis of
secondary metabolites, some of which play
prominent role in virulence
Mycobacterium CYPome
 33 CYP families detected
 79% are orphan CYPs with no
assigned biological functions
11

12. RESULTS
The eukaryote CYPome in 3S metagenome comprises 20 CYP families, 92
subfamilies, and ascE, a CYP not assigned to any of the existing CYP families
The animal CYPome in 3S metagenome is predominantly (31.5%)
represented by the members of the CYP4 family.
The detected CYP4 proteins were recovered from Homo sapiens, Sus scrofa,
Rattus norvegicus, Oryctolagus cuniculus, Mus musculus, and Ovis aries,
respectively.
They are primarily involved in metabolism of fatty acids, eicosanoids,
xenobiotics, therapeutic drugs, and also catabolizes leukotrienes and
prostanoids
12

13. RESULTS
The plant CYPome in 3S metagenome comprises 12 CYP families and 48
subfamilies constituting 60% of eukaryote CYP families in the metagenome.
The CYPs that constitute the plant CYPome in the metagenome include
CYP707, CYP709, CYP716, CYP72, CYP720, CYP724, CYP725, CYP734,
CYP735, CYP749, CYP85, and CYP88, respectively
The plant CYPs were recovered from Arabidopsis thaliana, Solanum
lycopersicum, Oryza sativa subsp. Japonica, among others.
 They are involved in oxidative degradation of abscisic acid and salt tolerance
response, triterpenes hydroxylation and carboxylation, secologanin
biosynthesis and plant defense mechanism, gibberellin biosynthesis, etc.
13

14. RESULTS
The fungi CYPome in 3S metagenome comprises three families (CYP505,
CYP51, CYP55), seven subfamilies (CYP505A3, CYP505B1, CYP51A,
CYP51C, CYP55A1, CYP55A2, CYP55A3), and ascE.
The fungi CYPs were annotated for Acremonium egyptiacum, Fusarium
oxysporum, Aspergillus oryzae, Gibberella moniliformis, Aspergillus flavus,
and Fusarium lichenicola, respectively
The CYPs are involved in biosynthesis of ascofuranone and ascochlorin,
hydroxylation of fatty acids, biosynthesis of fumonisin and ergosterol, and
dissimilatory nitrate reduction and denitrification.
14

15. RESULTS
0
5
10
15
20
25
30
35
ascE CYP120 CYP26 CYP2R1 CYP4 CYP505 CYP51 CYP524 CYP55 CYP707 CYP709 CYP716 CYP72 CYP720 CYP724 CYP725 CYP734 CYP735 CYP749 CYP85 CYP88
ABUNDANCE
CYTOCHROME P450
Distribution and Abundance of Eukaryote CYPs in 3S Metagenome
15

16. DISCUSSION
Results of this study revealed relative preponderance of bacterial CYP450
with 72 protein families, 21 in Eukaryotes and 1 in Archaea.
The low number recorded for Archaea may be attributed to the fact that
Archaea are extremophiles adapted to environments with extremes of
environmental conditions.
With a total of 45 out of 72 CYP450 families affiliated to Bacillus,
Streptomyces and Mycobacterium, these genera are the most represented in
the CYPome.
Their dominance have been consistently reported in soils contaminated with
petroleum hydrocarbons, which may be attributed to their resilience to
stressors and extreme conditions, including oligotrophy (Yang et al. 2014;
Salam et al. 2020)
16

17. DISCUSSION
The revelation that more CYP450 families were affiliated to Mycobacterium
than Streptomyces and Bacillus is in consonance with previous studies
(Parvez et al. 2016; Mthethwa et al. 2018).
All the four CYP102 subfamilies identified in this study have been well
studied. Their potential for exploitation in biocatalytic production of drugs,
Short-chain hydrocarbons and xenobiotics has been explored (Kang et al.
2011).
CYP107 have been found to be more highly conserved in Bacillus and
Streptomyces (Mnguni et al. 2020) and many are involved in the synthesis of
macrolide antibiotics (Kim et al. 2020).
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18. DISCUSSION
One of the subfamilies of this group annotated in this study CYP107DY1 is a
recently discovered plasmid encoded protein in Bacillus megaterium which
acts on mevastatin to produce pravastatin, a drug used in the treatment of
hypercholesterolemia (Milhim et al. 2016).
Also, worth mentioning is CYP107J1 (CYPA) detected in this study. The
enzyme encoded by this gene is involved in hydroxylation of polychlorinated
biphenyl (PCB), a notorious environmental pollutant (Sun et al. 2017).
Streptomyces species are well-known producer of chemically diverse
secondary metabolites that are used in human medicine as antibiotics, anti-
infectives, anti-fibrotic, antitumor, and immunosuppressant drugs, and it is
estimated that they produce >50% of commercially available antibiotics (de
Lima Procópio et al. 2012; Senate et al. 2019).
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19. DISCUSSION
It is therefore not surprising that majority (85%; CYP28, CYP105, CYP107,
CYP113, CYP129, CYP151, CYP161, CYP162, CYP163, CYP170, CYP183,
CYP244, CYP245, scnG, ncsB3, aziB1) of the Streptomyces CYPs annotated
in this study are involved in biosynthesis of secondary metabolites (McLean
et al. 2015; Mnguni et al. 2020).
In contrast to Streptomyces species, the genus Mycobacterium comprises of
species that are well-known pathogens of humans and other animals, despite
some saprophytes being present in the genus (Ventura et al. 2007; Parvez et
al. 2016).
The pathogenic nature of mycobacterial species forced them to adapt to
parasitic lifestyles where living in a host, evading the host’s immune system,
and utilizing host carbon sources for survival are the prime tasks (Mnguni et
al. 2020).
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20. DISCUSSION
To sustain this lifestyle, mycobacterial CYPs are carefully acquired to serve
as biocatalyst for the utilization of host lipids or synthesis of novel lipids.
These assertions were strengthened by findings in this study which shows
that though majority of the detected mycobacterial CYPs were orphan CYPs,
the few CYPs with functions were only annotated for sterol biodegradation
(CYP51, CYP124, CYP142), and biosynthesis of secondary metabolites
(CYP128, CYP139, CYP140A7), some of which play prominent role in
Mycobacterium virulence (McLean et al. 2015).
Interest in the M. tuberculosis CYPome is driven mainly by the need to
understand the role the enzymes encoded play in the establishment of
infection as a way of identifying potential drug targets and developing
effective drugs (McLean et al. 2008; Ouellet et al. 2010; Montellano 2018).
20

21. DISCUSSION
Indeed, some of the enzymes in these families, including CYP121, CYP125,
and CYP142 have been found to be promising targets of anti-tuberculosis
therapeutics (Chang et al. 2009; Hudson et al. 2013; Ortiz de Montellano
2018).
Of the seventeen CYP families that constitute the Proteobacteria CYPome
detected in 3S metagenome, quite a number associated with Pseudomonas
species are involved in biodegradation of camphor and hydroxylation of
monoterpene alcohols, α-terpinol, linalool and thiocarbamate herbicide.
Pseudomonas Cytochrome P450 involvement have been documented for
toxic pesticides such as 1,2-dichloromethane and propiconazole (Hage and
Hartman, 1999; Satapute and Kaliwal 2016)
21

22. DISCUSSION
Pseudomonas Cytochrome P450 involvement have been documented for
toxic pesticides such as 1,2-dichloromethane and propiconazole (Hage and
Hartman, 1999; Satapute and Kaliwal 2016)
Recovery of these CYPs from a mechanic workshop where hydrocarbon and
other hydrocarbon derived contaminants were in high concentration could be
a pointer to the role of P450 in the degradation of these pollutants in 3S.
Seven CYPs were annotated for Sorangium cellulosum So ce56, a
myxobacterial soil bacterium reputed as a producer of important secondary
metabolites (Khatri et al. 2010).
Of the seven CYPs, three (CYP167A1, CYP265A1, CYP266A1) were
involved in the biosynthesis and hydroxylation of epothilone D (Kern et al.
2015).
22

23. DISCUSSION
CYP167A1 is responsible for the last step in epothilone biosynthesis in
Sorangium cellulosum So ce56, catalyzing the epoxidation of epothilone D to
epothilone B (Julien et al. 2000) while CYP265A1, CYP266A1 were
involved in hydroxylation of epothilone D to generate hydroxylated
epothilones 14-OH epothilones (Kern et al. 2015).
Epothilones are a family of novel microtubule-stabilizing agents, which
inhibit mitosis and thus prevent cancer cells from dividing via obstruction of
microtubule depolymerization thereby causing G2-M interphase arrest of the
cell cycle with subsequent cytotoxicity and eventual cancer cell apoptosis
(Molner et al. 2000; Goodin et al. 2004).
The detection of these CYPs in 3S soil highlight the importance of the soil
environment as a repository of natural products with extensive functionalities.
23

24. DISCUSSION
The detection of CYP103 and CYP104 involved in detoxification of plant
protective agents at the site of wounding from the phytopathogen Rhizobium
radiobacter and CYP112 and CYP114 involved in the biosynthesis of the
phytohormone gibberellin (Nagel et al. 2018) from Sinorhizobium and
Bradyrhizobium spp. is a confirmation of the historical antecedent of
agricultural activities at the 3S polluted soil.
24

25. DISCUSSION
The recovery of animal CYPs from the 3S soil covering sources such as
Homo sapiens (Humans), Rattus norvegicus (Common rat), Mus musculus
(House mouse), Ovis aris (sheep) and Oryctolagus cuniculus (rabbit) is not
unexpected.
 They represent the common rangers in environments of mechanic workshops
in Nigeria which are usually open and unprotected from domestic animals and
rodents.
CYP4 family, which constituted more than 31 % of the detected eukaryotes
CYPs belong to a group reported to contain more genes than other families of
mammalian CYP found in man and rodents.
25

26. DISCUSSION
The CYP4 are essential in degradation of toxins, xenobiotic and drugs and
the subsequent elimination from the system, thus keeping them at subtoxic
level in the mammalian system (Estevez et al. 2021).
Plant cytochrome are known to play key role in xenobiotic detoxification and
biosynthesis of sterols, pheromones and fatty acid metabolism related growth
and reproduction.
However, stress response involving synthesis of cellular components and
defense compounds including those involved in drought, salt and water
activity related responses have been shown to be cytochrome mediated (Javid
et al. 2011; Jun et al. 2015; Thornton et al. 2020).
26

27. DISCUSSION
This is exemplified in the 3S metagenome plant CYPome by some
subfamilies of families CYP72, CYP707, CYP709, CYP716 which may be
indicative of the fact that if at all, the few plants able to grow in the vicinity of
the petroleum polluted soil recruit stress responses as part of survival strategy.
 Some of the fungi for which CYP genes were annotated in this study such as
Aspergillus flavus, Fusarium oxysporum and Fusarium lichenicola are
common soil flora with well described CYPs.
However, the small number of genes annotated for fungi may not be
unconnected to the limited role they play in the polluted soil, as only those
able to survive the toxicity would persist.
27

28. DISCUSSION
The CYP55 subfamilies identified in this study namely, CYP55A1,
CYP55A2 and CYP55A3 are well documented in the literature as fungal
CYPs functional in denitrification under anoxic condition (Kudo et al. 1996;
Kaya et al. 2004; Shin et al. 2018).
All CYP55 are part of a two-component fungal denitrification system
consisting of NorK and P450nor (Nor) localized in the mitochondrion (Shoun
et al. 2012) and have been identified in Ascomycetous fungi including
Fusarium oxysporum, Fusarium linicola and Aspergillus oryzae (CYP55A5).
28

29. CONCLUSION
Metagenomic assessment of 3S polluted soil revealed diverse CYPomes
spanning prokaryotes and eukaryotes in 94 CYP families.
Although majority of the families (72) were annotated for prokaryotes, these
were mainly in the phyla noted as important hydrocarbon degraders, thus
indicating concurrence in pollution induced reduction in phylotype diversity
and CYPome richness.
Aside the biosynthetic CYPs, detoxification CYPs also made remarkable
appearance, even as there are families especially of animal CYPs whose
presence could be attributed to contamination or deposition from man,
animals and plants.
29

30. CONCLUSION
Poor showing of fungal CYPs in 3S metagenome may be attributed to their
possible limited role in the polluted soil.
Future study will have to be directed to targeting some of the CYP genes
annotated for functions of biotechnological interest with the aim of evaluating
their prospects for useful application.
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31. THANK YOU
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