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The function of Cytochrome (CYP) P450 in plants to enhance detoxification of herbicide metabolism is well-known. However, the knowledge of gene quantification for detecting and detoxifying pollutants and other toxicants by an indigenous plant growing in a contaminated site is limited. The objective of this research is to evaluate the potential of detecting or degrading 2,3,7,8 Tetrachlorodibenzodioxin (TCDD) in soil using a native plant growing in a contaminated site via the gene expression of Cytochrome P450 monooxygenases (P450s) method. The novelty of this research is that P450s in native plants possibly acts as a bioindicator on contaminated land by increasing its gene expression levels induced by the presence of TCDD. In seedling toxicity test and cytochrome enzyme activity test, a significant difference in the root length (range of value: 580.2–799.2 mm) and enzyme activity (range of value: 31.2–82.3 nmolmin−1 g−1 total protein) of such indigenous plant was found in 10 µg/L TCDD treatment when compared to other treatments. 13- and 20-fold levels of gene expression in CYP71C1 and CYP79A61 of the plant growing in a contaminated site were found after 10 µg/L TCDD treatment. The results revealed that such indigenous plant is sensitive to the detection of such persistent organic pollutant in the field site and involves gene expression change facilitated by a plant‒microbe symbiotic association. The current findings can provide an insight to use another option for pollution monitoring using non-standard plant models.Highlights: CYP71C1 and CYP79A61 induction by TCDD. CYP450 is sensitive to detect trace amount of TCDD. Applicable to TCDD detection. [ABSTRACT FROM AUTHOR]

Simple Summary: Enzymes of the detoxification system are known to play a crucial role in insecticide resistance in insects. The role of P450 monooxygenases in providing resistance to chlorfenapyr is ambiguous and of interest due to possible negative cross-resistance to pyrethroids and other insecticides, the resistance to which is ensured by increased activity of these enzymes. This study aimed to perform a biochemical and molecular evaluation of P450 monooxygenases in susceptible and chlorfenapyr-resistant strains of Musca domestica L. depending on sex. The chlorfenapyr-resistant strain of M. domestica L. did not differ from the susceptible strain in terms of the pattern of overall P450-monooxygenase activity: the larvae of both strains had lower enzymatic activity than the adults. However, the development of resistance to chlorfenapyr in house flies was accompanied by an increase in P450-monooxygenase activity without changes in CYP6D1 expression. PCR-RFLP analysis revealed a previously undescribed mutation in the promoter region of the CYP6D1 gene of M. domestica, the effect of which on the gene expression level was not detected. The house fly Musca domestica L. is one of the most common insects of veterinary and medical importance worldwide; its ability to develop resistance to a large number of insecticides is well known. Many studies support the involvement of cytochrome P-450-dependent monooxygenases (P450) in the development of resistance to pyrethroids, neonicotinoids, carbamates, and organophosphates among insects. In this paper, the monooxygenase activity and expression level of CYP6D1 were studied for the first time in a chlorfenapyr-resistant strain of house fly. Our studies demonstrated that P450 activity in adults of the susceptible strain (Lab TY) and chlorfenapyr-resistant strain (ChlA) was 1.56–4.05-fold higher than that in larvae. In females of the Lab TY and ChlA strains, this activity was 1.53- and 1.57-fold higher, respectively (p < 0.05), than that in males, and in contrast, the expression level of CYP6D1 was 21- and 8-fold lower, respectively. The monooxygenase activity did not vary between larvae of the susceptible strain Lab TY and the chlorfenapyr-resistant strain ChlA. Activity in females and males of the ChlA strain exceeded that in the Lab TY strain specimens by 1.54 (p = 0.08) and 1.83 (p < 0.05) times, respectively, with the same level of CYP6D1 expression. PCR-RFLP analysis revealed a previously undescribed mutation in the promoter region of the CYP6D1 gene in adults of the Lab TY and ChlA strains, and it did not affect the gene expression level. The obtained results show that the development of resistance to chlorfenapyr in M. domestica is accompanied by an increase in P450-monooxygenase activity without changes in CYP6D1 expression. [ABSTRACT FROM AUTHOR]

Artemisinin biosynthesis, unique to Artemisia annua, is suggested to have evolved from the ancestral costunolide biosynthetic pathway commonly found in the Asteraceae family. However, the evolutionary landscape of this process is not fully understood. The first oxidase in artemisinin biosynthesis, CYP71AV1, also known as amorpha-4,11-diene oxidase (AMO), has specialized from ancestral germacrene A oxidases (GAOs). Unlike GAO, which exhibits catalytic promiscuity toward amorpha-4,11-diene, the natural substrate of AMO, AMO has lost its ancestral activity on germacrene A. Previous studies have suggested that the loss of the GAO copy in A. annua is responsible for the abolishment of the costunolide pathway. In the genome of A. annua, there are two copies of AMO, each of which has been reported to be responsible for the different product profiles of high-and low-artemisinin production chemotypes. Through analysis of their tissue-specific expression and comparison of their sequences with those of other GAOs, it was discovered that one copy of AMO (AMOHAP) exhibits a different transcript compared to the reported artemisinin biosynthetic genes and shows more sequence similarity to other GAOs in the catalytic regions. Furthermore, in a subsequent in vitro enzymatic assay, the recombinant protein of AMOHAP unequivocally demonstrated GAO activity. This result clearly indicates that AMOHAP is a GAO rather than an AMO and that its promiscuous activity on amorpha-4,11-diene has led to its misidentification as an AMO in previous studies. In addition, the divergent expression pattern of AMOHAP compared to that of the upstream germacrene A synthase may have contributed to the abolishment of costunolide biosynthesis in A. annua. Our findings reveal a complex evolutionary landscape in which the emergence of a new metabolic pathway replaces an ancestral one. [ABSTRACT FROM AUTHOR]

Trichothecenes are a structurally diverse family of toxic secondary metabolites produced by certain species of multiple fungal genera. All trichothecene analogs share a core 12,13-epoxytrichothec-9-ene (EPT) structure but differ in presence, absence and types of substituents attached to various positions of EPT. Formation of some of the structural diversity begins early in the biosynthetic pathway such that some producing species have few trichothecene biosynthetic intermediates in common. Cytochrome P450 monooxygenases (P450s) play critical roles in formation of trichothecene structural diversity. Within some species, relaxed substrate specificities of P450s allow individual orthologs of the enzymes to modify multiple trichothecene biosynthetic intermediates. It is not clear, however, whether the relaxed specificity extends to biosynthetic intermediates that are not produced by the species in which the orthologs originate. To address this knowledge gap, we used a mutant complementation-heterologous expression analysis to assess whether orthologs of three trichothecene biosynthetic P450s (TRI11, TRI13 and TRI22) from Fusarium sporotrichioides, Trichoderma arundinaceum, and Paramyrothecium roridum can modify trichothecene biosynthetic intermediates that they do not encounter in the organism in which they originated. The results indicate that TRI13 and TRI22 could not modify the intermediates that they do not normally encounter, whereas TRI11 could modify an intermediate that it does not normally encounter. These findings indicate that substrate promiscuity varies among trichothecene biosynthetic P450s. One structural feature that likely impacts the ability of the P450s to use biosynthetic intermediates as substrates is the presence and absence of an oxygen atom attached to carbon atom 3 of EPT. Key points: • Trichothecene cytochrome P450 monooxygenases differ in substrate specificity. • Trichothecene 15-hydroxylase (TRI11) has promiscuous substrate specificity. • Fusarium TRI11 modifies evolutionarily divergent biosynthetic intermediates. [ABSTRACT FROM AUTHOR]

The house fly Musca domestica L. is one of the most common insects of veterinary and medical importance worldwide; its ability to develop resistance to a large number of insecticides is well known. Many studies support the involvement of cytochrome P-450-dependent monooxygenases (P450) in the development of resistance to pyrethroids, neonicotinoids, carbamates, and organophosphates among insects. In this paper, the monooxygenase activity and expression level of CYP6D1 were studied for the first time in a chlorfenapyr-resistant strain of house fly. Our studies demonstrated that P450 activity in adults of the susceptible strain (Lab TY) and chlorfenapyr-resistant strain (ChlA) was 1.56–4.05-fold higher than that in larvae. In females of the Lab TY and ChlA strains, this activity was 1.53- and 1.57-fold higher, respectively (p < 0.05), than that in males, and in contrast, the expression level of CYP6D1 was 21- and 8-fold lower, respectively. The monooxygenase activity did not vary between larvae of the susceptible strain Lab TY and the chlorfenapyr-resistant strain ChlA. Activity in females and males of the ChlA strain exceeded that in the Lab TY strain specimens by 1.54 (p = 0.08) and 1.83 (p < 0.05) times, respectively, with the same level of CYP6D1 expression. PCR-RFLP analysis revealed a previously undescribed mutation in the promoter region of the CYP6D1 gene in adults of the Lab TY and ChlA strains, and it did not affect the gene expression level. The obtained results show that the development of resistance to chlorfenapyr in M. domestica is accompanied by an increase in P450-monooxygenase activity without changes in CYP6D1 expression.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme proteins that play a role in organisms' primary and secondary metabolism. P450s play an important role in organism adaptation since lifestyle influences P450 composition in their genome. This phenomenon is well-documented in bacteria but less so in fungi. This study observed this phenomenon where diverse P450 complements were identified in saprophytic and ectomycorrhizal Pezizomycetes. Genome-wide data mining, annotation, and phylogenetic analysis of P450s in 19 Pezizomycetes revealed 668 P450s that can be grouped into 153 P450 families and 245 P450 subfamilies. Only four P450 families, namely, CYP51, CYP61, CYP5093, and CYP6001, are conserved across 19 Pezizomycetes, indicating their important role in these species. A total of 5 saprophyte Pezizomycetes have 103 P450 families, whereas 14 ectomycorrhizal Pezizomycetes have 89 P450 families. Only 39 P450 families were common, and 50 and 64 P450 families, respectively, were unique to ectomycorrhizal and saprophytic Pezizomycetes. These findings suggest that the switch from a saprophytic to an ectomycorrhizal lifestyle led to both the development of diverse P450 families as well as the loss of P450s, which led to the lowest P450 family diversity, despite the emergence of novel P450 families in ectomycorrhizal Pezizomycetes. [ABSTRACT FROM AUTHOR]

The predatory stink bug, Arma custos, is a highly effective beneficial predator of crop pests. The lack of gene information related to xenobiotic detoxification and odorant degrading enzymes in the predator stink bugs to date has limited our ability for more in-depth studies of biological control. Hence, we conducted de novo assembly of the A. custos transcriptome from guts, antennae, and other tiussue samples of 5th instar larvae using Illumina sequencing technology. A total of 91, 50 and 23 genes of cytochrome P450 monooxygenases (CYPs), carboxyl/choline esterases (CCEs) and glutathione S-transferases (GSTs) genes were identified, respectively. Gene expansions of CYP3 and CYP4 clans and the hormone and pheromone processing CCE class were found in A. custos. Analysis of tissue-specific expression patterns showed that 37 CYPs, 14 CCEs and 8 GSTs were enriched in guts, and 6 CYPs, 5 CCEs and 2 GSTs were up-regulated in antennae, suggesting their potential roles on xenobiotics detoxification and ordorant degradation. Gene information data presented here could be useful for a deeper understanding of the ecology, physiology and behavior of this beneficial species and could be helpful to improve their bio-control efficiency.

The deep ocean, one of the largest ecosystems on earth, is dominated by microorganisms that are keystones in the regulation of biogeochemical cycles. However, the evolutionary pathways underlying the specific adaptations required (e.g., high pressure and low temperature) by this unique niche remain understudied. Here, we analyzed the first representatives belonging to the order Acidimicrobiales, a group of marine planktonic Actinobacteriota, that specifically inhabits the aphotic zone of the oceanic water column (>200 m). Compared with their epipelagic counterparts, deep-sea representatives showed the same evolution in genome architecture with higher GC content, longer intergenic spaces as well as higher nitrogen (N-ARSC) and lower carbon (C-ARSC) content in encoded amino acid residue side chains consistent with the higher nitrogen concentration and lower carbon concentration in deep waters compared to the photic zone. Metagenomic recruitment showed distribution patterns that allowed the description of different ecogenomic units within the three deep water-associated genera defined by our phylogenomic analyses (UBA3125, S20-B6 and UBA9410). The entire genus UBA3125 was found exclusively associated with oxygen minimum zones linked to the acquisition of genes involved in denitrification. Genomospecies of genus S20-B6 recruited in samples from both mesopelagic (200-1,000 m) and bathypelagic (1000-4,000 m) zones, including polar regions. Diversity in the genus UBA9410 was higher, with genomospecies widely distributed in temperate zones, others in polar regions, and the only genomospecies associated with abyssal zones (>4,000 m). At the functional level, groups beyond the epipelagic zone have a more complex transcriptional regulation including in their genomes a unique WhiB paralog. In addition, they showed higher metabolic potential for organic carbon and carbohydrate degradation as well as the ability to accumulate glycogen as a source of carbon and energy. This could compensate for energy metabolism in the absence of rhodopsins, which is only present in genomes associated with the photic zone. The abundance in deep samples of cytochrome P450 monooxygenases associated with the genomes of this order suggests an important role in remineralization of recalcitrant compounds throughout the water column. [ABSTRACT FROM AUTHOR]

Substantial human and animal studies support the beneficial effects of ω-3 polyunsaturated fatty acids (PUFAs) on colonic inflammation and colorectal cancer (CRC). However, there are inconsistent results, which have shown that ω-3 PUFAs have no effect or even detrimental effects, making it difficult to effectively implement ω-3 PUFAs for disease prevention. A better understanding of the molecular mechanisms for the anti-inflammatory and anticancer effects of ω-3 PUFAs will help to clarify their potential health-promoting effects, provide a scientific base for cautions for their use, and establish dietary recommendations. In this review, we summarize recent studies of ω-3 PUFAs on colonic inflammation and CRC and discuss the potential roles of ω-3 PUFA-metabolizing enzymes, notably the cytochrome P450 monooxygenases, in mediating the actions of ω-3 PUFAs.

Abstract Background Cytochrome P450 monooxygenases (CYPs) are exciting biocatalysts that catalyzes diverse regio- and stereoselective reactions of a broad range of substrates. The cytochrome P450 genes (CYPomes) of a chronically polluted soil (3S) with history of agricultural activities were deciphered via functional annotation of putative ORFs (open reading frames) using KEGG KofamKOALA, PHMMER, the Cytochrome P450 Engineering Database CYPED v6.0, and the NCBI Batch Web CD-Search tool. Results Annotation revealed the detection of seventy-seven CYP families and eight standalone CYPs cutting across the three domains of life. The prokaryote CYPome has 72 CYP families, 93 subfamilies, and seven standalone CYPs. The phylum Proteobacteria and the genera Streptomyces, Mycobacterium, and Bacillus with 17, 16, 24, and 5 CYP families were predominant, while the domain Archaea was represented by CYP119A1. The phylum Cyanobacteria has two families, while 23 actinobacterial CYPs (other than Streptomyces and Mycobacterium) were also detected. The detected prokaryote CYPs are responsible for biodegradation of camphor, hydroxylation of monoterpene alcohols, biosynthesis of secondary metabolites, and hydroxylation of fatty acids and steroidal compounds. The eukaryote CYPome was represented by seven fungal CYPs (CYP505A3, CYP505B1, CYP51A, CYP51C, CYP55A1, CYP55A2, and CYP55A3) from Acremonium egyptiacum, Fusarium oxysporum, Aspergillus oryzae, Gibberella moniliformis, Aspergillus flavus, and Fusarium lichenicola, respectively, and CYP524A1 from the slime mold, Dictyostelium discoideum. The fungi CYPs were involved in biosynthesis of secondary metabolites, hydroxylation of fatty acids, and nitrate reduction and denitrification. Conclusions This study has established the diverse roles played by CYPs in soil, its implication for soil health and resilience, and its potentials for industrial application.

The cytochrome P450 monooxygenase (CYP) superfamily comprises hemethiolate enzymes that perform remarkable regio- and stereospecific oxidative chemistry. As such, CYPs are key agents for the structural and functional tailoring of triterpenoids, one of the largest classes of plant natural products with widespread applications in pharmaceuticals, food, cosmetics, and agricultural industries. In this review, we provide a full overview of 149 functionally characterised CYPs involved in the biosynthesis of triterpenoids and steroids in primary as well as in specialised metabolism. We describe the phylogenetic distribution of triterpenoid- and steroid-modifying CYPs across the plant CYPome, present a structure-based summary of their reactions, and highlight recent examples of particular interest to the field. Our review therefore provides a comprehensive up-to-date picture of CYPs involved in the biosynthesis of triterpenoids and steroids in plants as a starting point for future research.

Abstract Background Dengue (DENV), chikungunya (CHIKV) and Zika virus (ZIKV), are mosquito-borne viruses of medical importance in most tropical and subtropical regions. Vector control, primarily through insecticides, remains the primary method to prevent their transmission. Here, we evaluated insecticide resistance profiles and identified important underlying resistance mechanisms in populations of Aedes aegypti and Ae. albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations. Methods Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation, G1. Ae. aegypti and Ae. albopictus adults from G1 were tested following World Health Organization (WHO) recommendations and Ae. aegypti G0 adults screened with real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Piperonyl butoxide (PBO) assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance. Statistical analyses were performed using Chi-square test and generalized linear models. Results Loss of susceptibility was observed to all insecticides tested. Mortality rates from tests with 0.25% permethrin varied from 24.27 to 85.89% in Ae. aegypti and from 17.35% to 68.08% in Ae. albopictus. Mortality rates for 0.03% deltamethrin were between 23.30% and 88.20% in Ae. aegypti and between 69.47 and 84.11% in Ae. albopictus. We found a moderate level of resistance against bendiocarb, with mortality rates ranging from 69.31% to 90.26% in Ae. aegypti and from 86.75 to 98.95% in Ae. albopictus. With PBO pre-exposure, we found partial or fully restored susceptibility to pyrethroids and bendiocarb. The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae. aegypti populations from Douala sites resistant to permethrin and deltamethrin. Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae. albopictus samples. F1534C and V1016I mutations were detected in A. aegypti mosquitoes and for the first time V410L was reported in Cameroon. Conclusions This study revealed that Ae. aegypti and Ae. albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated. These findings could guide insecticide use to control arbovirus vectors in Cameroon.

Bemisia tabaci is a threat to agriculture worldwide because of its potential to cause devastating damage to crops. Chlorogenic acid is a bioactive pesticidal phytochemical agent against various insect pests. We here determined the susceptibility of a laboratory strain of B. tabaci to chlorogenic acid and other popular insecticides, and the susceptibility of several field-collected populations to chlorogenic acid. Also, cross-resistance to four common insecticides was measured. Chlorogenic acid had the highest toxicity of all tested insecticides, and all the field-collected populations were susceptible to chlorogenic acid, and little cross-resistance was detected between chlorogenic acid and the other tested insecticides. Furthermore, analysis of enzyme activities and expression of P450 genes in B. tabaci after treatment with LC50 of chlorogenic acid suggested that enhanced P450 activity could be involved in chlorogenic acid detoxification. We subsequently evaluated sublethal effects of chlorogenic acid, and found that treatment with LC25 of chlorogenic acid prolonged duration of two developmental stages, reduced fecundity, and decreased survival rates of treated B. tabaci compared to untreated insects. Overall, these findings demonstrate strong toxicity and significant sublethal effects of chlorogenic acid on B. tabaci, and suggest that overexpression of P450 genes may be associated with chlorogenic acid detoxification. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study's results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s. [ABSTRACT FROM AUTHOR]

Abstract Background Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. Results In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-β) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. Conclusions We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), populations exhibit varying levels of resistance to several insecticide classes, including pyrethroids. Artificial laboratory selection was conducted to investigate mechanisms underlying fenpropathrin resistance. After ten generations of selection, the LC50 for fenpropathrin increased from 0.18 to 11.60 ng/μL, and the associated resistance ratio increased by 96.67-fold. The realized heritability of resistance (h2) to fenpropathrin changed from 0.17 to 0.44 during the first and second round of selection, respectively. We did not find high cross-resistance to imidacloprid (RR = 3.58) or dimethoate (RR = 1.77) in the fenpropathrin-selected strain, but found cross-resistance to bifenthrin (RR = 28.21). RT-qPCR analysis showed that expression of CYP6A2-1 was significantly increased in the selected population relative to the laboratory susceptible population. Our data indicate that D. citri has the capacity to develop a high level of fenpropathrin resistance following persistent selection and that cytochrome P450 (CYP) and glutathione S-transferase (GST) metabolic detoxification may be contributing mechanisms. Rotation programs that include neonicotinoids and organophosphates should manage resistance to fenpropathrin for D. citri. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s/CYPs) are ubiquitous enzymes with unique regio- and stereo-selective oxidation activities. Due to these properties, P450s play a key role in the biosynthesis of natural metabolites. Mycobacterial species are well-known producers of complex metabolites that help them survive in diverse ecological niches, including in the host. In this study, a comprehensive analysis of P450s and their role in natural metabolite synthesis in 2666 mycobacterial species was carried out. The study revealed the presence of 62,815 P450s that can be grouped into 182 P450 families and 345 subfamilies. Blooming (the presence of more than one copy of the same gene) and expansion (presence of the same gene in many species) were observed at the family and subfamily levels. CYP135 was the dominant family in mycobacterial species. The mycobacterial species have distinct P450 profiles, indicating that lifestyle impacts P450 content in their genome vis a vis P450s, playing a key role in organisms' adaptation. Analysis of the P450 profile revealed a gradual loss of P450s from non-pathogenic to pathogenic mycobacteria. Pathogenic mycobacteria have more P450s in biosynthetic gene clusters that produce natural metabolites. This indicates that P450s are recruited for the biosynthesis of unique metabolites, thus helping these pathogens survive in their niches. This study is the first to analyze P450s and their role in natural metabolite synthesis in many mycobacterial species. [ABSTRACT FROM AUTHOR]

Pulses are a group of leguminous crops that are harvested solely for their dry seeds. As the demand for plant-based proteins grows, pulses are becoming important food crops worldwide. In addition to being a rich source of nutrients, pulses also contain saponins that are traditionally considered anti-nutrients, and impart bitterness and astringency. Saponins are plant secondary metabolites with great structural and functional diversity. Given their diverse functional properties and biological activities, both undesirable and beneficial, saponins have received growing attention. It can be expected that redirecting metabolic fluxes to control the saponin levels and produce desired saponins would be an effective approach to improve the nutritional and sensory quality of the pulses. However, little effort has been made toward understanding saponin biosynthesis in pulses, and, thus there exist sizable knowledge gaps regarding its pathway and regulatory network. In this paper, we summarize the research progress made on saponin biosynthesis in pulses. Additionally, phylogenetic relationships of putative biosynthetic enzymes among multiple pulse species provide a glimpse of the evolutionary routes and functional diversification of saponin biosynthetic enzymes. The review will help us to advance our understanding of saponin biosynthesis and aid in the development of molecular and biotechnological tools for the systematic optimization of metabolic fluxes, in order to produce the desired saponins in pulses. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs/P450s) are heme proteins that play a role in organisms’ primary and secondary metabolism. P450s play an important role in organism adaptation since lifestyle influences P450 composition in their genome. This phenomenon is well-documented in bacteria but less so in fungi. This study observed this phenomenon where diverse P450 complements were identified in saprophytic and ectomycorrhizal Pezizomycetes. Genome-wide data mining, annotation, and phylogenetic analysis of P450s in 19 Pezizomycetes revealed 668 P450s that can be grouped into 153 P450 families and 245 P450 subfamilies. Only four P450 families, namely, CYP51, CYP61, CYP5093, and CYP6001, are conserved across 19 Pezizomycetes, indicating their important role in these species. A total of 5 saprophyte Pezizomycetes have 103 P450 families, whereas 14 ectomycorrhizal Pezizomycetes have 89 P450 families. Only 39 P450 families were common, and 50 and 64 P450 families, respectively, were unique to ectomycorrhizal and saprophytic Pezizomycetes. These findings suggest that the switch from a saprophytic to an ectomycorrhizal lifestyle led to both the development of diverse P450 families as well as the loss of P450s, which led to the lowest P450 family diversity, despite the emergence of novel P450 families in ectomycorrhizal Pezizomycetes.

Dendroctonus bark beetles are the most destructive agents in coniferous forests. These beetles come into contact with the toxic compounds of their host's chemical defenses throughout their life cycle, some of which are also used by the insects as kairomones to select their host trees during the colonization process. However, little is known about the molecular mechanisms by which the insects counteract the toxicity of these compounds. Here, two sibling species of bark beetles, D. valens and D. rhizophagus, were stimulated with vapors of a blend of their main kairomones (α-pinene, β-pinene and 3-carene), in order to compare the transcriptional response of their gut. A total of 48 180 unigenes were identified in D. valens and 43 704 in D. rhizophagus, in response to kairomones blend. The analysis of differential gene expression showed a transcriptional response in D. valens (739 unigenes, 0.58–10.36 Log2FC) related to digestive process and in D. rhizophagus (322 unigenes 0.87–13.08 Log2FC) related to xenobiotics metabolism. The expression profiles of detoxification genes mainly evidenced the up-regulation of COEs and GSTs in D. valens, and the up-regulation of P450s in D. rhizophagus. Results suggest that terpenes metabolism comes accompanied by an integral hormetic response, result of compensatory mechanisms, including the activation of other metabolic pathways, to ensure the supply of energy and the survival of organisms which is specific for each species, according to its life history and ecological strategy.

Background: Cytochrome P450 monooxygenases (CYPs) are exciting biocatalysts that catalyzes diverse regio- and stereoselective reactions of a broad range of substrates. The cytochrome P450 genes (CYPomes) of a chronically polluted soil (3S) with history of agricultural activities were deciphered via functional annotation of putative ORFs (open reading frames) using KEGG KofamKOALA, PHMMER, the Cytochrome P450 Engineering Database CYPED v6.0, and the NCBI Batch Web CD-Search tool. Results: Annotation revealed the detection of seventy-seven CYP families and eight standalone CYPs cutting across the three domains of life. The prokaryote CYPome has 72 CYP families, 93 subfamilies, and seven standalone CYPs. The phylum Proteobacteria and the genera Streptomyces, Mycobacterium, and Bacillus with 17, 16, 24, and 5 CYP families were predominant, while the domain Archaea was represented by CYP119A1. The phylum Cyanobacteria has two families, while 23 actinobacterial CYPs (other than Streptomyces and Mycobacterium) were also detected. The detected prokaryote CYPs are responsible for biodegradation of camphor, hydroxylation of monoterpene alcohols, biosynthesis of secondary metabolites, and hydroxylation of fatty acids and steroidal compounds. The eukaryote CYPome was represented by seven fungal CYPs (CYP505A3, CYP505B1, CYP51A, CYP51C, CYP55A1, CYP55A2, and CYP55A3) from Acremonium egyptiacum, Fusarium oxysporum, Aspergillus oryzae, Gibberella moniliformis, Aspergillus flavus, and Fusarium lichenicola, respectively, and CYP524A1 from the slime mold, Dictyostelium discoideum. The fungi CYPs were involved in biosynthesis of secondary metabolites, hydroxylation of fatty acids, and nitrate reduction and denitrification. Conclusions: This study has established the diverse roles played by CYPs in soil, its implication for soil health and resilience, and its potentials for industrial application. [ABSTRACT FROM AUTHOR]

Bemisia tabaci is a threat to agriculture worldwide because of its potential to cause devastating damage to various crops. β-asarone is a bioactive pesticidal chemical originating from Acorus calamus (or "Sweet Flag") plants, and it displays significant lethal effects against insect pests. In this study, we established a baseline of susceptibility to β-asarone from China and patterns of cross-resistance to other popular insecticides. We found that all the 12 field-collected B. tabaci populations exhibited high susceptibility to β-asarone, and there was no cross-resistance detected for other tested insecticides. We subsequently evaluated the sublethal effects of β-asarone on physiology and biochemistry via LC25 treatment (4.7 mg/L). LC25 of β-asarone resulted in prolonged developmental duration and decreased survival rates in B. tabaci nymphs, pseudopupae, and adults. Significant reductions in oviposition duration, fecundity, and hatchability were also observed. Additionally, the metabolic enzyme activity and expression profiles of selected cytochrome P450 monooxygenase (P450) genes following the LC25 treatment of β-asarone suggest that enhanced detoxification via P450s could be involved in the observed sublethal effects. These findings demonstrate the strong toxicity and significant sublethal effects of β-asarone on B. tabaci and suggest that the induced overexpression of P450 genes could be associated with the response to β-asarone. [ABSTRACT FROM AUTHOR]

Background: Dengue (DENV), chikungunya (CHIKV) and Zika virus (ZIKV), are mosquito-borne viruses of medical importance in most tropical and subtropical regions. Vector control, primarily through insecticides, remains the primary method to prevent their transmission. Here, we evaluated insecticide resistance profiles and identified important underlying resistance mechanisms in populations of Aedes aegypti and Ae. albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations. Methods: Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation, G1. Ae. aegypti and Ae. albopictus adults from G1 were tested following World Health Organization (WHO) recommendations and Ae. aegypti G0 adults screened with real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Piperonyl butoxide (PBO) assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance. Statistical analyses were performed using Chi-square test and generalized linear models. Results: Loss of susceptibility was observed to all insecticides tested. Mortality rates from tests with 0.25% permethrin varied from 24.27 to 85.89% in Ae. aegypti and from 17.35% to 68.08% in Ae. albopictus. Mortality rates for 0.03% deltamethrin were between 23.30% and 88.20% in Ae. aegypti and between 69.47 and 84.11% in Ae. albopictus. We found a moderate level of resistance against bendiocarb, with mortality rates ranging from 69.31% to 90.26% in Ae. aegypti and from 86.75 to 98.95% in Ae. albopictus. With PBO pre-exposure, we found partial or fully restored susceptibility to pyrethroids and bendiocarb. The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae. aegypti populations from Douala sites resistant to permethrin and deltamethrin. Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae. albopictus samples. F1534C and V1016I mutations were detected in A. aegypti mosquitoes and for the first time V410L was reported in Cameroon. Conclusions: This study revealed that Ae. aegypti and Ae. albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated. These findings could guide insecticide use to control arbovirus vectors in Cameroon. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg -1 . Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK] n , flexible glycine linkers [GGGGS] n or rigid proline linkers [EPPPP] n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg -1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The commercialization of 2,4-D (2,4-dichlorophenoxyacetic acid) latifolicide in 1945 marked the beginning of the selective herbicide market, with this active ingredient playing a pivotal role among commercial herbicides due to the natural tolerance of monocots compared with dicots. Due to its intricate mode of action, involving interactions within endogenous auxin signaling networks, 2,4-D was initially considered a low-risk herbicide to evolve weed resistance. However, the intensification of 2,4-D use has contributed to the emergence of 2,4-D-resistant broadleaf weeds, challenging earlier beliefs. This review explores 2,4-D tolerance in crops and evolved resistance in weeds, emphasizing an in-depth understanding of 2,4-D metabolic detoxification. Nine confirmed 2,4-D-resistant weed species, driven by rapid metabolism, highlight cytochrome P450 monooxygenases in Phase I and glycosyltransferases in Phase II as key enzymes. Resistance to 2,4-D may also involve impaired translocation associated with mutations in auxin/indole-3-acetic acid (Aux/IAA) co-receptor genes. Moreover, temperature variations affect 2,4-D efficacy, with high temperatures increasing herbicide metabolism rates and reducing weed control, while drought stress did not affect 2,4-D efficacy. Research on 2,4-D resistance has primarily focused on non-target-site resistance (NTSR) mechanisms, including 2,4-D metabolic detoxification, with limited exploration of the inheritance and genetic basis underlying these traits. Resistance to 2,4-D in weeds is typically governed by a single gene, either dominant or incompletely dominant, raising questions about gain-of-function or loss-of-function mutations that confer resistance. Future research should unravel the physiological and molecular-genetic basis of 2,4-D NTSR, exploring potential cross-resistance patterns and assessing fitness costs that may affect future evolution of auxin-resistant weeds. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. Results: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-β) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. Conclusions: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle. [ABSTRACT FROM AUTHOR]

Detoxification of insecticides involving major metabolic/detoxifying enzymes and target-site insensitivity is among the two most important mechanisms of biochemical resistance. Variation in the enzymatic activity highlights upon the prevalence of insecticide resistance pattern in different regions. As such, in order to study the insecticide resistance pattern in the lymphatic filariasis vector, Cx. quinquefasciatus Say (Diptera: Culicidae), field populations were collected from four districts of West Bengal, India, and quantitative enzyme activity of three major detoxifying enzymes was assessed. The enzyme activity of field collected populations was higher than that of the laboratory reared susceptible population and also differed among the study sites. Highest esterases activity was recorded in HCP (Harishchandrapur) of Malda district. DLK (Dalkhola) in Uttar Dinajpur district with a value of 3.016 nM mg protein−1 showed highest monooxygenases activity and MEK (Mekhliganj) showed highest GSTs (glutathione-S-transferases) activity (70.20 × 10–4 mM mg protein−1 min−1). This suggests varying insecticide resistance patterns in most of the studied populations. The finding of this study will be helpful in selecting an effective mosquito control strategy in the endemic districts during outbreaks. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fall armyworm (FAW, Spodoptera frugiperda) is one of the most destructive and invasive pests worldwide and causes significant economic losses. Intensive and frequent use of insecticides has led to the development of resistance in FAW. Adipokinetic hormone (AKH) have been proven to be involved in insecticide resistance in insects. However, the molecular mechanism underlying chlorantraniliprole resistance mediated by AKH signaling in FAW remains unclear. RESULTS: The expression of SpfAKHR was highest in male adults and lowest in 1st instar larvae. SpfAKH was expressed the highest in eggs and the lowest in 6th instar larvae. AKH signaling was involved in the sensitivity of FAW to chlorantraniliprole through a toxicological bioassay, and the combination of chlorantraniliprole and bithionol (an inhibitor of key enzymes in the AKH pathway) significantly increased the mortality of FAW. Chlorantraniliprole significantly induced the expression of ten P450s, SpfAKH and SpfAKHR in FAW. RNA interference against SpfAKHR significantly decreased the P450 content, downregulated the expression of three P450 genes (SpfCYP6B50, SpfCYP321A9 and SpfCYP9A58) and inhibited the resistance of FAW to chlorantraniliprole. The topical application of AKH peptide significantly increased the P450 content, upregulated the expression of five P450 genes (SpfCYP321A9, SpfCY321A8, SpfCYP321A10, SpfCYP321A7 and SpfCYP6AB12), and enhanced the survival of FAW against chlorantraniliprole. CONCLUSIONS: AKH plays an important role in enhancing chlorantraniliprole resistance in FAW by exerting a positive influence on P450 gene expression and P450 content. These results provide valuable insights into insecticide resistance regulation and FAW control strategies. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Descurainia sophia L. (flixweeds) is a noxious broad-leaf weed infesting winter wheat fields in China that has evolved high resistance to tribenuron-methyl. In this work, a brand new gene CYP77B34 was cloned from tribenuron-methyl-resistant (TR) D. sophia and transferred into Arabidopsis thaliana, and the sensitivities of Arabidopsis with or without the CYP77B34 transgene to herbicides with a different mode of actions (MoAs) were tested. Compared to Arabidopsis expressing pCAMBIA1302-GFP (empty plasmid), Arabidopsis transferring pCAMBIA1302-CYP77B34 (recombinant plasmid) became resistant to acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl, protoporphyrinogen oxidase (PPO)-inhibiting herbicides carfentrazone-ethyl and oxyfluorfen. Cytochrome P450 inhibitor malathion could reverse the resistance to tribenuron-methyl, carfentrazone-ethyl and oxyfluorfen in transgenic Arabidopsis plants. In addition, the metabolic rates of tribenuron-methyl in Arabidopsis expressing CYP77B34 were significantly higher than those in Arabidopsis expressing pCAMBIA1302-GFP. Other than that, the transgenic plants showed some tolerance to very-long-chain fatty acid synthesis (VLCFAs)-inhibiting herbicide pretilachlor and photosystem (PS) II-inhibiting herbicide bromoxynil. Subcellular localization revealed that the CYP77B34 protein was located in the endoplasmic reticulum (ER). These results clearly indicated that CYP77B34 mediated D. sophia resistance to tribenuron-methyl and may have been involved in D. sophia cross-resistance to carfentrazone-ethyl, oxyfluorfen, pretilachlor and bromoxynil. [ABSTRACT FROM AUTHOR]

Species belonging to the bacterial phyla Bacteroidetes and Firmicutes represent over 90% of the gastrointestinal microbiota. Changes in the ratio of these two bacterial groups were found to have contrasting health effects, including obesity and inflammatory diseases. Despite the availability of many bacterial genomes, comparative genomic studies on the gene pools of these two bacterial groups concerning cytochrome P450 monooxygenases (P450s), ferredoxins, and secondary metabolite biosynthetic gene clusters (smBGCs) are not reported. This study is aimed to address this research gap. The study revealed the presence of diverse sets of P450s, ferredoxins, and smBGCs in their genomes. Bacteroidetes species have the highest number of P450 families, ferredoxin cluster-types, and smBGCs compared to Firmicutes species. Only four P450 families, three ferredoxin cluster types, and five smBGCs are commonly shared between these two bacterial groups. Considering the above facts, we propose that the contrasting effects of these two bacterial groups on the host are partly due to the distinct nature of secondary metabolites produced by these organisms. Thus, the cause of the contrasting health effects of these two bacterial groups lies in their gene pools. [ABSTRACT FROM AUTHOR]

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha−1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors. [Display omitted] • Cross resistance to four dissimilar ALS inhibitors chemistries reported are in Sinapis alba. • Two mutations Pro197Ser and Trp574Leu (first report) are found. • Ligand docking predicts resistance to tribenuron and imazamox for the Trp574Leu. • Pro197Ser confers resistance only to tribenuron. • Enhanced metabolism can contribute to resistance to tribenuron and imazamox. • Multiple resistance to dicamba could be evolving in S.alba. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study’s results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s.

Cytochrome P450 monooxygenases (P450) participate in the catalytic conversion of biological compounds in a plethora of metabolic pathways, such as the biosynthesis of alkaloids, terpenoids, phenylpropanoids, and hormones in plants. Plants utilize these metabolites for growth and defense against biotic and abiotic stress. In this study, we identified 346 P450 (GmP450) enzymes encoded by 317 genes in soybean where 26 GmP450 genes produced splice variants. The genome-wide comparison of both A-type and non-A-type GmP450s for their motifs composition, gene structure, tissuespecific expression, and their chromosomal distribution were determined. Even though conserved P450 signature motifs were found in all GmP450 families, larger variation within a specific motif was observed in the non-A-type GmP450s as compared with the A-type. Here, we report that the length of variable region between two conserved motifs is exact in the members of the same family in majority of the A-type GmP450. Analyses of the transcriptomic datasets from soybean-Phytophthora sojae interaction studies, quantitative trait loci (QTL) associated with P. sojae resistance, and co-expression analysis identified some GmP450s that may be, in part, play an important role in partial resistance against P. sojae. The findings of our CYPome study provides novel insights into the functions of GmP450s and their involvements in metabolic pathways in soybean. Further experiments will elucidate their roles in general and legume-specific function. [ABSTRACT FROM AUTHOR]

Simple Summary: Eight CYP6 family genes were identified in Grapholita molesta (Busck). The expression of individual gene members differed between the developmental stages and insect tissues. High expression was found in third/fourth-instar larvae and in the midgut and Malpighian tubules. The response patterns of the genes exhibited diverse response patterns to the three representative insecticides were diverse. Cytochrome P450 (CYP) monooxygenases comprise a superfamily of proteins that detoxify xenobiotics and plant secondary metabolites in insects. The CYP6 family is unique to the class Insecta, and its members participate in the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of eight CYP6 family genes from Grapholita molesta (Busck), a global pest of pome fruits. P450 genes with the exception of CYP6AN35, which was most highly expressed in adults, consistently showed high expression in third- or fourth-instar larvae. The analysis of different tissues of adults showed that most of these genes were predominantly expressed in the midgut, Malpighian tubules, and/or fat body. The expression of these eight CYP6 genes was differentially affected by three representative insecticides: malathion (organophosphate), deltamethrin (pyrethroid), and chlorantraniliprole (carbamate). All eight CYP6 genes responded to malathion treatment. Only three CYP6 genes were highly expressed in deltamethrin-treated individuals. Chlorantraniliprole treatment exerted weak effects on gene expression. Interestingly, CYP6AN35 was a highly expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides. This study provides a fundamental understanding of the functions of the CYP6 gene family in insecticide metabolism in G. molesta. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450) participate in the catalytic conversion of biological compounds in a plethora of metabolic pathways, such as the biosynthesis of alkaloids, terpenoids, phenylpropanoids, and hormones in plants. Plants utilize these metabolites for growth and defense against biotic and abiotic stress. In this study, we identified 346 P450 (GmP450) enzymes encoded by 317 genes in soybean where 26 GmP450 genes produced splice variants. The genome-wide comparison of both A-type and non-A-type GmP450s for their motifs composition, gene structure, tissue-specific expression, and their chromosomal distribution were determined. Even though conserved P450 signature motifs were found in all GmP450 families, larger variation within a specific motif was observed in the non-A-type GmP450s as compared with the A-type. Here, we report that the length of variable region between two conserved motifs is exact in the members of the same family in majority of the A-type GmP450. Analyses of the transcriptomic datasets from soybean-Phytophthora sojae interaction studies, quantitative trait loci (QTL) associated with P. sojae resistance, and co-expression analysis identified some GmP450s that may be, in part, play an important role in partial resistance against P. sojae. The findings of our CYPome study provides novel insights into the functions of GmP450s and their involvements in metabolic pathways in soybean. Further experiments will elucidate their roles in general and legume-specific function.

Cytochrome P450 monooxygenase (P450 or CYP) plays an important role in the metabolism of insecticides and plant allelochemicals by insects. CYP321B1 , a novel Spodoptera litura P450 gene, was identified and characterized. CYP321B1 contains a 1488 bp open reading frame (ORF) that encodes a 495 amino acid protein. In fourth instar larvae, the highest CYP321B1 expression levels were found in the midgut and fat body. In the tannin feeding test, tannin can significantly induce the expression of CYP321B1 in the midgut and fat body of 4th instar larvae. To verify the function of CYP321B1, RNA interference and metabolome analysis were performed. The results showed that silencing CYP321B1 significantly reduced the rate of weight gain under tannin induction. Metabolome analysis showed silencing affected 47 different metabolites, mainly involved in secondary metabolite biosynthesis and amino acid metabolism, including amino acids, lipid fatty acids, organic acids and their derivatives. Henoxyacetic acid and cysteamine are the most highly regulated metabolites, respectively. These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism. Functional knowledge about metabolite detoxification genes in this major herbivorous insect pest can provide new insights into this biological process and provide new targets for agricultural pest control. • We identified and characterized CYP321B1 , a novel P450 gene in S. litura. • The expression of CYP321B1 in the midgut and fat body, induced by tannin, increased by 308 times and 166 times, respectively. • Daily weight gain and net weight increase of CYP321B1 silenced larvae were significantly reduced under the tannin induction. • Silencing of CYP321B1 affected metabolites involved in secondary metabolite biosynthesis and amino acid metabolism. • These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism in S. litura larvae. [ABSTRACT FROM AUTHOR]

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura , especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a , is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

Pulses are a group of leguminous crops that are harvested solely for their dry seeds. As the demand for plant-based proteins grows, pulses are becoming important food crops worldwide. In addition to being a rich source of nutrients, pulses also contain saponins that are traditionally considered anti-nutrients, and impart bitterness and astringency. Saponins are plant secondary metabolites with great structural and functional diversity. Given their diverse functional properties and biological activities, both undesirable and beneficial, saponins have received growing attention. It can be expected that redirecting metabolic fluxes to control the saponin levels and produce desired saponins would be an effective approach to improve the nutritional and sensory quality of the pulses. However, little effort has been made toward understanding saponin biosynthesis in pulses, and, thus there exist sizable knowledge gaps regarding its pathway and regulatory network. In this paper, we summarize the research progress made on saponin biosynthesis in pulses. Additionally, phylogenetic relationships of putative biosynthetic enzymes among multiple pulse species provide a glimpse of the evolutionary routes and functional diversification of saponin biosynthetic enzymes. The review will help us to advance our understanding of saponin biosynthesis and aid in the development of molecular and biotechnological tools for the systematic optimization of metabolic fluxes, in order to produce the desired saponins in pulses.

Cytochrome P450 monooxygenases (P450s/CYPs) are ubiquitous enzymes with unique regio- and stereo-selective oxidation activities. Due to these properties, P450s play a key role in the biosynthesis of natural metabolites. Mycobacterial species are well-known producers of complex metabolites that help them survive in diverse ecological niches, including in the host. In this study, a comprehensive analysis of P450s and their role in natural metabolite synthesis in 2666 mycobacterial species was carried out. The study revealed the presence of 62,815 P450s that can be grouped into 182 P450 families and 345 subfamilies. Blooming (the presence of more than one copy of the same gene) and expansion (presence of the same gene in many species) were observed at the family and subfamily levels. CYP135 was the dominant family in mycobacterial species. The mycobacterial species have distinct P450 profiles, indicating that lifestyle impacts P450 content in their genome vis a vis P450s, playing a key role in organisms’ adaptation. Analysis of the P450 profile revealed a gradual loss of P450s from non-pathogenic to pathogenic mycobacteria. Pathogenic mycobacteria have more P450s in biosynthetic gene clusters that produce natural metabolites. This indicates that P450s are recruited for the biosynthesis of unique metabolites, thus helping these pathogens survive in their niches. This study is the first to analyze P450s and their role in natural metabolite synthesis in many mycobacterial species.

BACKGROUND: The control of the housefly, Musca domestica, heavily relies on the application of insecticides. Propoxur, a carbamate, has been widely used for vector control. The housefly populations with high propoxur resistance display point mutations and overexpression of acetylcholinesterase. However, the roles of cytochrome P450 monoxygenases (P450s), as important detoxification enzymes, remain poorly understand in the housefly resistant to propoxur. RESULTS: P450s were implied to contribute to propoxur resistance based on the synergism of piperonyl butoxide (PBO) and the increase of P450 enzyme activity in the near‐isogenic line propoxur resistant strain (N‐PRS). Five P450 genes (CYP6G4, CYP6A25, CYP304A1, CYP6D3, and CYP6A1) by RNA‐sequencing comparison were significantly up‐regulated in the N‐PRS strain with >1035‐fold resistance to propoxur. A total of 13 non‐synonymous mutations of three P450 genes (CYP6G4, CYP6D3, and CYP6D8) were found in the N‐PRS strain. The amino acid substitutions of CYP6D3 and CYP6D8 were probably not resistance‐associated single nucleotide polymorphisms (SNPs) because they were also found in the aabys susceptible strain. However, CYP6G4 variant in the N‐PRS strain was not found in the aabys strain. The conjoint analysis of mutations and a series of genetic crosses exhibited that the housefly propoxur resistance was strongly associated with the mutations of CYP6G4 gene. CONCLUSION: Our results suggested that a combination of up‐regulated transcript levels and mutations of CYP6G4 contributed to propoxur resistance in the housefly. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In light of the widespread use of herbal medicines containing rosmarinic acid (RA) and limited literature available thereon, we investigated the metabolic interactions of RA with human cytochrome P450 monooxygenases (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs). The involvement of selected enzymes (CYP1A2, CYP2C19, CYP2E1, CYP3 A4, UGT1A1, UGT1A6, and UGT2B7) in the metabolism of RA and the inhibitory effect of RA on the enzyme activity were comprehensively evaluated using human recombinant isozyme system. Additionally, concentration-dependent RA metabolism mediated by phase I enzymes (including CYPs) or UGT was investigated in human liver microsome (HLM) system. A significant disappearance of RA was observed in the seven CYP and UGT isoforms studied, indicating their possible involvement in the metabolism of RA. Based on Michaelis-Menten kinetics, the metabolism study using HLM suggests that the UGT system may have a higher capacity and lower affinity for the metabolism of RA than phase I enzyme (including CYP) systems. Moreover, RA weakly inhibited CYP2C9 and 2E1 activities with IC50 values of 39.6 and 61.0 μM, respectively, while moderately inhibiting UGT1A1, 1A6, and 2B7 with IC50 values of 9.24, 19.1, and 23.4 μM, respectively. By constructing Line weaver–Burk plots, the type of inhibition exhibited by RA on CYP and UGT activities was determined as follows: CYP2C19, mixed inhibition; CYP2E1, UGT1A1, UGT1A6, and UGT2B7, competitive inhibition. Based on the comparison of the IC50 and Ki values obtained in the current study with the previously reported plasma concentrations of RA after oral dosing in humans, it is suggested that RA may significantly inhibit the activities of the tested UGTs, rather than CYPs, in clinical settings. Thus, the present study could provide a basis for further studies on clinically significant interactions between UGT substrate drugs and herbal medicines containing RA.

Descurainia sophia L. (flixweeds) is a noxious broad-leaf weed infesting winter wheat fields in China that has evolved high resistance to tribenuron-methyl. In this work, a brand new gene CYP77B34 was cloned from tribenuron-methyl-resistant (TR) D. sophia and transferred into Arabidopsis thaliana, and the sensitivities of Arabidopsis with or without the CYP77B34 transgene to herbicides with a different mode of actions (MoAs) were tested. Compared to Arabidopsis expressing pCAMBIA1302-GFP (empty plasmid), Arabidopsis transferring pCAMBIA1302-CYP77B34 (recombinant plasmid) became resistant to acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl, protoporphyrinogen oxidase (PPO)-inhibiting herbicides carfentrazone-ethyl and oxyfluorfen. Cytochrome P450 inhibitor malathion could reverse the resistance to tribenuron-methyl, carfentrazone-ethyl and oxyfluorfen in transgenic Arabidopsis plants. In addition, the metabolic rates of tribenuron-methyl in Arabidopsis expressing CYP77B34 were significantly higher than those in Arabidopsis expressing pCAMBIA1302-GFP. Other than that, the transgenic plants showed some tolerance to very-long-chain fatty acid synthesis (VLCFAs)-inhibiting herbicide pretilachlor and photosystem (PS) II-inhibiting herbicide bromoxynil. Subcellular localization revealed that the CYP77B34 protein was located in the endoplasmic reticulum (ER). These results clearly indicated that CYP77B34 mediated D. sophia resistance to tribenuron-methyl and may have been involved in D. sophia cross-resistance to carfentrazone-ethyl, oxyfluorfen, pretilachlor and bromoxynil.

Species belonging to the bacterial phyla Bacteroidetes and Firmicutes represent over 90% of the gastrointestinal microbiota. Changes in the ratio of these two bacterial groups were found to have contrasting health effects, including obesity and inflammatory diseases. Despite the availability of many bacterial genomes, comparative genomic studies on the gene pools of these two bacterial groups concerning cytochrome P450 monooxygenases (P450s), ferredoxins, and secondary metabolite biosynthetic gene clusters (smBGCs) are not reported. This study is aimed to address this research gap. The study revealed the presence of diverse sets of P450s, ferredoxins, and smBGCs in their genomes. Bacteroidetes species have the highest number of P450 families, ferredoxin cluster-types, and smBGCs compared to Firmicutes species. Only four P450 families, three ferredoxin cluster types, and five smBGCs are commonly shared between these two bacterial groups. Considering the above facts, we propose that the contrasting effects of these two bacterial groups on the host are partly due to the distinct nature of secondary metabolites produced by these organisms. Thus, the cause of the contrasting health effects of these two bacterial groups lies in their gene pools.

Cytochrome P450 monooxygenases (P450s) play significant roles in protecting organisms from abiotic stress damage. Here, we report the sequence and characterization of a P450s gene (AccCYP4AV1), isolated from Apis cerana cerana Fabricius. The open reading frame of AccCYP4AV1 is 1506 base pairs long and encodes a predicted protein of 501 amino acids and 57.84 kDa, with an isoelectric point of 8.67. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that AccCYP4AV1 is more highly expressed in the midgut than in other tissues. In addition, the highest expression occurs in newly emerged adult workers, followed by the first instar of the larval stage. In addition, the expression of the AccCYP4AV1 was upregulated by low temperature (4 °C), ultraviolet radiation, hydrogen peroxide, paraquat, and dichlorvos treatments. In contrast, AccCYP4AV1 transcription was downregulated by other abiotic stress conditions: exposure to increased temperature (44 °C), deltamethrin, cadmium chloride, and mercury (II) chloride. Moreover, when AccCYP4AV1 was knocked-down by RNA interference, the results suggested that multiple antioxidant genes (AccsHSP22.6, AccSOD2, AccTpx1, and AccTpx4) were downregulated and antioxidant genes AccGSTO1 and AccTrx1 were upregulated. The activity levels of peroxidase and catalase were upregulated in the AccCYP4AV1-knocked-down samples, compared with those in the control groups. These findings suggest that the AccCYP4AV1 protein might be involved in the defense against abiotic stress damage. [ABSTRACT FROM AUTHOR]

Evolution of multiple herbicide resistance in Palmer amaranth across the United States is a serious challenge for its management. Recently, a Palmer amaranth population (KCTR; Kansas Conservation Tillage Resistant) from a long-term conservation tillage research project in Kansas, United States, was found uncontrolled by several commonly used herbicides. Importantly, this field did not have a history of repeated use of some of the herbicides for which the KCTR Palmer amaranth population showed lack of control. The objectives of this study were to confirm the evolution of multiple resistances and determine possible mechanism(s) of resistance in KCTR Palmer amaranth plants. In response to post-emergence application, 28–100% of KCTR Palmer amaranth survived field recommended rates of 2,4-D, ALS-, PS II-, EPSPS-, PPO-, HPPD-inhibitor herbicides, or tank- or pre-mixture of PS II- and HPPD-inhibitor herbicides, confirming evolution of six-way resistance in this Palmer amaranth population. However, this population was found susceptible to the PS I- and glutamine synthetase inhibitor herbicides. Chlorsulfuron-, imazethapyr-, and atrazine-resistant plants did not show any previously reported mutation in ALS and psbA genes, the target sites of these herbicides, respectively. However, the survivors of glyphosate treatment showed amplification of EPSPS gene (up to 88 copies). The KCTR plants pretreated with cytochrome P450 or GST inhibitors along with atrazine, 2,4-D, lactofen, or mesotrione had significantly less biomass accumulation than those treated with herbicides alone. Plants treated with P450 inhibitor followed by imazethapyr showed moderate reduction of biomass in KCTR which was statistically similar to a susceptible Palmer amaranth population treated with imazethapyr. These results suggest predominance of metabolic resistance possibly mediated by cytochrome P450 and GST enzyme activity that may have predisposed the KCTR Palmer amaranth population to evolve resistance to multiple herbicides. This is the first report of evolution of six-way resistance in a single Palmer amaranth population. Appropriate management strategies, including integration of cultural, and mechanical, and herbicide mixtures, are warranted to control such Palmer amaranth populations.

Cytochrome P450 (CYP) monooxygenases comprise a superfamily of proteins that detoxify xenobiotics and plant secondary metabolites in insects. The CYP6 family is unique to the class Insecta, and its members participate in the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of eight CYP6 family genes from Grapholita molesta (Busck), a global pest of pome fruits. P450 genes with the exception of CYP6AN35, which was most highly expressed in adults, consistently showed high expression in third- or fourth-instar larvae. The analysis of different tissues of adults showed that most of these genes were predominantly expressed in the midgut, Malpighian tubules, and/or fat body. The expression of these eight CYP6 genes was differentially affected by three representative insecticides: malathion (organophosphate), deltamethrin (pyrethroid), and chlorantraniliprole (carbamate). All eight CYP6 genes responded to malathion treatment. Only three CYP6 genes were highly expressed in deltamethrin-treated individuals. Chlorantraniliprole treatment exerted weak effects on gene expression. Interestingly, CYP6AN35 was a highly expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides. This study provides a fundamental understanding of the functions of the CYP6 gene family in insecticide metabolism in G. molesta.

Evolution of multiple herbicide resistance in Palmer amaranth across the United States is a serious challenge for its management. Recently, a Palmer amaranth population (KCTR; Kansas Conservation Tillage Resistant) from a long-term conservation tillage research project in Kansas, United States, was found uncontrolled by several commonly used herbicides. Importantly, this field did not have a history of repeated use of some of the herbicides for which the KCTR Palmer amaranth population showed lack of control. The objectives of this study were to confirm the evolution of multiple resistances and determine possible mechanism(s) of resistance in KCTR Palmer amaranth plants. In response to post-emergence application, 28–100% of KCTR Palmer amaranth survived field recommended rates of 2,4-D, ALS-, PS II-, EPSPS-, PPO-, HPPD-inhibitor herbicides, or tank- or pre-mixture of PS II- and HPPD-inhibitor herbicides, confirming evolution of six-way resistance in this Palmer amaranth population. However, this population was found susceptible to the PS I- and glutamine synthetase inhibitor herbicides. Chlorsulfuron-, imazethapyr-, and atrazine-resistant plants did not show any previously reported mutation in ALS and psbA genes, the target sites of these herbicides, respectively. However, the survivors of glyphosate treatment showed amplification of EPSPS gene (up to 88 copies). The KCTR plants pretreated with cytochrome P450 or GST inhibitors along with atrazine, 2,4-D, lactofen, or mesotrione had significantly less biomass accumulation than those treated with herbicides alone. Plants treated with P450 inhibitor followed by imazethapyr showed moderate reduction of biomass in KCTR which was statistically similar to a susceptible Palmer amaranth population treated with imazethapyr. These results suggest predominance of metabolic resistance possibly mediated by cytochrome P450 and GST enzyme activity that may have predisposed the KCTR Palmer amaranth population to evolve resistance to multiple herbicides. This is the first report of evolution of six-way resistance in a single Palmer amaranth population. Appropriate management strategies, including integration of cultural, and mechanical, and herbicide mixtures, are warranted to control such Palmer amaranth populations. [ABSTRACT FROM AUTHOR]