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SUMMARY: Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis in Tabernaemontana litoralis (milky way tree), a prolific producer of rare pseudoaspidosperma‐type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio‐ and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15‐β‐epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15‐α‐epoxytabersonine (lochnericine) found in Catharanthus roseus (Madagascar periwinkle) roots. The pseudovincadifformine 18‐hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10‐hydroxylation of the iboga MIA coronaridine also catalyzes C10‐hydroxylation of voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11‐hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification in T. litoralis, highlighting its potential for further exploration. Significance Statement: New cytochrome P450 monooxygenases (CYP) from Tabernaemontana litoralis can oxidize four different monoterpenoid indole alkaloid (MIA) skeletons, including the rare pseudovincadifformine alkaloid. Our discovery highlights the remarkable versatility of CYP enzymes as catalysts in the biosynthesis of natural products. Such flexibility is a key factor driving the structural diversity of medicinal MIAs, which encompass over 3000 unique structures and hold significant potential for therapeutic development. [ABSTRACT FROM AUTHOR]

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4′-trichlorobiphenyl (PCB28), 2,2′,5,5′-tetrachlorobiphenyl (PCB52), and 2,2′,4,5,5′-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61 µg/L (5,89 nM) for PCB28, 316.98 µg/L (1,03 µM) for PCB52, and 151.1 µg/L (441 nM) for PCB101 from a 20 µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners. [ABSTRACT FROM AUTHOR]

SUMMARY: Triterpenoids (C30‐isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated β‐amyrin‐derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C‐2α and C‐23 hydroxylases/oxidases than C‐28 oxidases. CYP716A233 and CYP716A432 catalyzed β‐amyrin/erythrodiol C‐28 oxidation to produce oleanolic acid. C‐2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C‐23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C‐23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C‐23 hydroxylation. Overall, P450 substrate selectivity suggested that C‐28 oxidation is the first P450‐catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C‐2α/C‐23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds. Significance Statement: Arjuna tree bark contains bioactive triterpenoids that received considerable attention as cardioprotective compounds, but the biosynthetic enzymes remained largely unknown. In this work, transcriptome mining, metabolite analysis, and enzyme biochemical characterization led to identification of six cytochrome P450 monooxygenases (CYP716A233, CYP716A432, CYP716C88, CYP716C89, CYP714E107a, and CYP714E107b) and two cognate P450 reductases (TaCPR1 and TaCPR2) that catalyzed sequential C‐2α, C‐23, and C‐28 oxidations of oleanane scaffolds for the biosynthesis of arjunic acid, arjungenin, and arjunolic acid. [ABSTRACT FROM AUTHOR]

Aedes albopictus is highly prevalent in the northern part of West Bengal and is considered to be responsible for the recent dengue outbreaks in this region. Control of this vector is largely relied on the use of synthetic pyrethroids, which can lead to the development of resistance. In the present study, larvae of three wild Ae. albopictus populations from the dengue‐endemic regions were screened for deltamethrin resistance, and the role of cytochrome P450 monooxygenases (CYPs) was investigated in deltamethrin exposed and unexposed larvae. Two populations were incipient resistant, and one population was completely resistant against deltamethrin. Monooxygenase titration assay revealed the involvement of CYPs in deltamethrin resistance along with an induction effect of deltamethrin exposure. Gene expression studies revealed differential expression of five CYP6 family genes, CYP6A8, CYP6P12, CYP6A14, CYP6N3 and CYP6N6, with high constitutive expression of CYP6A8 and CYP6P12 in all the populations before and after deltamethrin exposure. From these findings, it was evident that CYPs play an important role in the development of deltamethrin resistance in the Ae. albopictus populations in this region. [ABSTRACT FROM AUTHOR]

Abstract Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography–mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.

Abstract Background Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. Results Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield—980.8 mg/L of isatropolone A—achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. Conclusion In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography–mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition. [ABSTRACT FROM AUTHOR]

Selective, one‐step C−H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high‐value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence‐activity landscape of a family of multidomain, self‐sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid‐chain in the lineage leading to the extant, P450‐TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self‐sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation. [ABSTRACT FROM AUTHOR]

Background: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. Results: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield—980.8 mg/L of isatropolone A—achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. Conclusion: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Cell‐free expression systems have emerged as a versatile and powerful platform for metabolic engineering, biosynthesis and synthetic biology studies. Nevertheless, successful examples of the synthesis of complex natural products using this system are still limited. Bicyclomycin, a structurally unique and complex diketopiperazine alkaloid, is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho. Here, we established a modular cell‐free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide. The six cell‐free expressed biosynthetic enzymes, including five iron‐ and α‐ketoglutarate‐dependent dioxygenases and one cytochrome P450 monooxygenase, were active in converting their substrates to the corresponding products. The co‐expressed enzymes in the cell‐free module were able to complete the related partial pathway. In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways (i.e., six enzymes) using the modular cell‐free expression system. This study demonstrates that the modular cell‐free expression system can be used as a robust and promising platform for the biosynthesis of complex antibiotics. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Cell‐free expression systems have emerged as a versatile and powerful platform for metabolic engineering, biosynthesis and synthetic biology studies. Nevertheless, successful examples of the synthesis of complex natural products using this system are still limited. Bicyclomycin, a structurally unique and complex diketopiperazine alkaloid, is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho. Here, we established a modular cell‐free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide. The six cell‐free expressed biosynthetic enzymes, including five iron‐ and α‐ketoglutarate‐dependent dioxygenases and one cytochrome P450 monooxygenase, were active in converting their substrates to the corresponding products. The co‐expressed enzymes in the cell‐free module were able to complete the related partial pathway. In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways (i.e., six enzymes) using the modular cell‐free expression system. This study demonstrates that the modular cell‐free expression system can be used as a robust and promising platform for the biosynthesis of complex antibiotics. [ABSTRACT FROM AUTHOR]

Aldoxime (R1R2C=NOH) and nitrile (R−C≡N) are nitrogen-containing compounds that are found in species representing all kingdoms of life. The enzymes discovered from the microbial "aldoxime-nitrile" pathway (aldoxime dehydratase, nitrile hydratase, amidase, and nitrilase) have been thoroughly studied because of their industrial importance. Although plants utilize cytochrome P450 monooxygenases to produce aldoxime and nitrile, many biosynthetic pathways are yet to be studied. Cyanogenic millipedes accumulate various nitrile compounds, such as mandelonitrile. However, no such aldoxime- and nitrile-metabolizing enzymes have been identified in millipedes. Here, I review the exploration of novel enzymes from plants and millipedes with characteristics distinct from those of microbial enzymes, the catalysis of industrially useful reactions, and applications of these enzymes for nitrile compound production. [ABSTRACT FROM AUTHOR]

Stegomyia aegypti (Linnaeus, 1762) vectors arboviruses of public health concern in urban areas of tropical countries, so it is necessary to reduce its population. Among the control methods used, chemically synthesized molecules have been widely employed, nonetheless, the over usage of the same mechanism of action can result in the resistance selection. Considering the influence of insect resistance with the success of chemical control of vectors, this study aims to assess the susceptibility to organophosphorus of a population of S. aegypti from Paranaguá (Paraná, Brazil), after intense use of malathion during a dengue outbreak. World Health Organization susceptibility tests and expression of Acetylcholinesterase ace-1, cytochrome P450 monooxygenase CYP6N12, and a-esterases CCEae3A genes were evaluated. The mortality rate of wild females (66.5%) indicated their resistance status, furthermore, a new discriminant concentration was detected in this population (3.41%). Exclusively CYP6N12 gene was overexpressed in malathion-resistant females indicating its possible contribution to the transformation of this insecticide. Constant monitoring of insecticide resistance of current and past molecules, mainly in port areas where there is a large flow of species, is crucial for effective use of insecticide in vector control programs. [ABSTRACT FROM AUTHOR]

Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.

Abstract Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism. Graphical Abstract

Cytochrome P450 monooxygenase (CYP) is one of the three detoxification metabolic enzymes in insects, and is involved in the metabolism and transformation of endogenous substances as well as the activation and degradation of exogenous compounds. This study aims to reveal the molecular mechanism of CYP9E2 in Tribolium castaneum in adapting to high-CO2 stress. By predicting the sequence function of CYP9E2, analyzing the temporal and spatial expression profile of TcCYP9E2, and using RNAi to silence TcCYP9E2 combined with a high-CO2 stress treatment, we measured the carbohydrate content, trehalase activity, and gene expression levels in trehalose metabolism of T. castaneum. A bioinformatics analysis showed that the predicted molecular weight of the protein encoded by TcCYP9E2 is 60.15, the theoretical isoelectric point is 8.63, there is no signal peptide, and the protein is hydrophilic. An evolutionary tree analysis showed that TcCYP9E2 belongs to the CYP6 family and belongs to the CYP3 group; and the spatiotemporal expression profile results showed that TcCYP9E2 was highly expressed in the larvae midgut 48 h after injection of dsCYP9E2, with survival rates decreasing with the increase in CO2 concentration. Under the condition of 75% CO2, the contents of glycogen, glucose, ATP, and membrane-bound trehalase decreased significantly after the injection of dsCYP9E2. The expression of TRE-1, TRE-2, and GP in trehalose metabolism and energy pathways was significantly downregulated. [ABSTRACT FROM AUTHOR]

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine. [ABSTRACT FROM AUTHOR]

The application of camptothecin (CPT) and its derivatives to control insect pests has generated significant interest. This study investigated the enzymatic response of cytochrome P450 monooxygenase (CYP) and glutathione S-transferase (GST) genes in the fat body cells of Spodoptera exigua after 10 μM CPT treatment. Additionally, we examined the effects of CPT on the growth and development of S. exigua larvae and detected the relative expression levels of selected CYP and GST genes during the CPT treatment period. Twenty-one CYP and 17 GST genes were identified from the fat body cells of S. exigua by comparative transcriptomic analyses. The relative expression of ten CYP and seven GST genes changed significantly, which suggested that these CPYs and GSTs may be involved in CPT metabolism. During exposure to CPT for 10 days, the development of S. exigua larvae was delayed and was characterized by weight inhibition and a prolonged period of development. The relative expression levels of the selected four CYP genes, CYP9A27, CYP9A186, CYP337B5, CYP321A8, and one GST gene, GSTe7, were significantly changed by CPT treatment compared to the control group. These generated data provide a basis for identifying the CPT metabolism/detoxification genes of S. exigua at the molecular level. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s) are well-known for their crucial roles in the detoxification of xenobiotics. However, whether CYP6CX2 and CYP6CX3, 2 genes from our Bemisia tabaci (B. tabaci) MED/Q genome data were associated with detoxification metabolism and confer resistance to thiamethoxam is unclear. In this study, we investigated the role of CYP6CX2 and CYP6CX3 in mediating whitefly thiamethoxam resistance. Our results showed that mRNA levels of CYP6CX2 and CYP6CX3 were up-regulated after exposure to thiamethoxam. Transcriptional levels of 2 genes were overexpressed in laboratory and field thiamethoxam resistant strains by RT-qPCR. These results indicate that the enhanced expression of CYP6CX2 and CYP6CX3 appears to confer thiamethoxam resistance in B. tabaci. Moreover, linear regression analysis showed that the expression levels of CYP6CX2 and CYP6CX3 were positively correlated with thiamethoxam resistance levels among populations. The susceptibility of whitefly adults was markedly increased after silencing 2 genes by RNA interference (RNAi) which further confirming their major role in thiamethoxam resistance. Our findings provide information to better understand the roles of P450s in resistance to neonicotinoids and suggest that these genes may be applied to develop target genes for sustainable management tactic of agricultural pests such as B. tabaci. [ABSTRACT FROM AUTHOR]

Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism. [ABSTRACT FROM AUTHOR]

Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω‐6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer‐promoting effects. Using LC‐MS/MS‐based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer‐promoting effects of LA, since the LA‐rich diet fails to exacerbate colon cancer in CYP monooxygenase‐deficient mice. Finally, CYP monooxygenase mediates the pro‐cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota‐dependent mechanisms. Overall, these results support that CYP monooxygenase‐mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects. [ABSTRACT FROM AUTHOR]

BackgroundHorehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds.ResultsMining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare.ConclusionsIn a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

The excessive use of chemical herbicides has resulted in evolution of herbicide-resistant weeds. Cytochrome P450 monooxygenases (P450s) are vital detoxification enzymes for herbicide-resistant weeds. Herein, we confirmed a resistant (R) Polypogon fugax population showing resistance to quizalofop-p-ethyl, acetolactate synthase (ALS)-inhibiting herbicide pyroxsulam, and several other ACCase (acetyl-CoA carboxylase)-inhibiting herbicides. Molecular analysis revealed no target-site gene mutations in the R population. Foliar spraying with malathion clearly reversed the quizalofop-p-ethyl phytotoxicity. Higher level of quizalofop-p-ethyl degradation was confirmed in the R population using HPLC analysis. Subsequently, RNA-Seq transcriptome analysis indicated that the overexpression of CYP89A2 gene appeared to be responsible for reducing quizalofop-p-ethyl phytotoxicity. The molecular docking results supported a metabolic effect of CYP89A2 protein on most herbicides tested. Furthermore, we found that low doses of herbicides stimulated the rhizosphere enzyme activities in P. fugax and the increase of rhizosphere dehydrogenase of R population may be related to its resistance mechanism. In summary, our research has shown that metabolic herbicide resistance mediated by CYP89A2, contributes to quizalofop-p-ethyl resistance in P. fugax.

Abstract Background CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. Results Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. Conclusions This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.

Tetranychus gloveri, a recently introduced mite species in Kerala, India has now established in the state as a predominant mite pest on ornamental plants. In this study, the status of acaricide resistance and the activity of detoxification enzymes were investigated in the populations of T. gloveri collected on Adenium plants from six different commercial horticultural nurseries of Thrissur district, Kerala, India. Adulticidal bioassays were performed for fenazaquin and dicofol, while nymphicidal bioassays were conducted for spiromesifen to estimate the LC50. The field populations showed resistance ratios in the range of 1.66–14.38-fold for fenazaquin, 1.06–27.31-fold for spiromesifen and 1.22–3.65-fold for dicofol, compared to the laboratory-maintained reference population. Enzyme assays of detoxifying enzymes revealed enhanced activity of carboxylesterase by 1.03–3.52-fold and cytochrome P450 monooxygenase by 1.01–2.08-fold in the field populations. However, the role of glutathione S-transferase in the development of resistance in different field populations was insignificant. The study recorded the development of resistance in the spider mite, T. gloveri to new generation acaricides spiromesifen and fenazaquin for the first time and also confirmed the significant role of the carboxylesterases and cytochrome P450 monooxygenases in imparting resistance in T. gloveri. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied "self-sufficient" CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters. Key points: • Identified CYP505Es share > 70% amino acid identity. • CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position. • CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter. [ABSTRACT FROM AUTHOR]

Methoxyfenozide is an insecticide with a unique mode of action on the insect ecdysone receptor and has been registered for the control of insect pests all over the world. In the present work, Spodoptera frugiperda was exposed to sublethal and lethal concentrations of methoxyfenozide to determine its impact on specific biological traits, metabolic enzyme activity, and the expression of detoxification enzymes. The result showed that 72-h posttreatment with LC50 and LC70 of methoxyfenozide significantly reduced the fecundity (eggs/female) of the F0 generation compared to those of the control group. However, the duration of the prepupal period was significantly increased. The exposure to LC10, LC30, LC50, and LC70 concentrations of methoxyfenozide significantly extended the developmental duration of larvae in F1 individuals. The fecundity of the F1 generation was significantly decreased, and the population life table parameters of F1 were also significantly affected. The activity of carboxylesterases showed little significant change, whereas the activity of glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) was significantly altered after exposure to LC10, LC30, LC50, and LC70 of methoxyfenozide. In total, 24-, 48-, and 96-h posttreatment with LC10, LC30, LC50, and LC70 of methoxyfenozide could cause upregulation of P450 genes such as CYP6AE44, CYP6B39, CYP9A26, CYP9A58, CYP9A59, and CYP9A60, as well as GST genes including GSTe3, GSTe9, GSTe10, GSTe15, GSTo2, GSTs1, GSTs5, GSTm2, and GSTm3. These findings could be instrumental in elucidating the molecular mechanisms underlying the sublethal and lethal effects of methoxyfenozide to S. frugiperda., (© The Author(s) 2025. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Resistance to multiple herbicides is common in Lolium rigidum. Here, resistance to acetolactate synthase (ALS)- and susceptibility to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides was confirmed in a glyphosate-resistant L. rigidum population (NLR70) from Australia and the mechanisms of pyroxsulam resistance were examined., Results: No ALS target-site mutations nor gene overexpression were detected. Cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) inhibitors (indicators of some certain P450s or GSTs) did not significantly affect the resistance to pyroxsulam. Nevertheless, HPLC analysis showed that plants of the NLR70 population metabolized pyroxsulam faster than plants of the herbicide-susceptible population (SVLR1). RNA sequencing analysis and RT-qPCR validation confirmed that four P450s (CYP709B2, CYP72A14, CYP89A2, CYP94B3), one GT (UGT79), and one ABC transporter (ABCG41) genes were constitutively upregulated in NLR70 plants., Conclusion: This study demonstrates that the glyphosate-resistant L. rigidum population (NLR70) also exhibits resistance to pyroxsulam and identifies six candidate genes associated with non-target site resistance to pyroxsulam. © 2025 Society of Chemical Industry., (© 2025 Society of Chemical Industry.)

Chilo suppressalis is a widely distributed pest occurring in nearly all paddy fields, which has developed high level resistance to different classes of insecticides. Vetiver grass has been identified as a dead-end trap plant for the alternative control of C. suppressalis. In this study, two cytochrome P450 monooxygenase (P450) genes, CsCYP6SN3 and CsCYP306A1 , were identified and characterized, which are expressed at all developmental stages, with the highest expression in the midguts and fat bodies of 3rd instar larvae. Vetiver significantly inhibited the expression levels of CsCYP6SN3 and CsCYP306A1 in 3rd larvae after feeding. RNA interference showed that silencing CsCYP6SN3 and CsCYP306A1 genes dramatically reduced the pupation rate and pupa weight. Feeding on vetiver after silencing CsCYP6SN3 and CsCYP306A1 led to higher mortality compared with feeding on rice. In conclusion, these findings indicated that the expression levels of CsCYP6SN3 and CsCYP306A1 were associated with the lethal effect of vetiver against C. suppressalis larvae and functional knowledge about these two detoxification genes could provide new targets for agricultural pest control. [ABSTRACT FROM AUTHOR]

Pesticide resistance in insects is an example of adaptive evolution occurring in pest species and is driven by the artificial introduction of pesticides. The diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae), has evolved resistance to various insecticides. Understanding the genetic changes underpinning the resistance to pesticides is necessary for the implementation of pest control measures. We sequenced the genome of six resistant and six susceptible DBM individuals separately and inferred the genomic regions of greatest divergence between strains using FST and θπ. Among several genomic regions potentially related to insecticide resistance, CYP6B6-like was observed with significant divergence between the resistant and susceptible strains, with a missense mutation located near the substrate recognition site (SRS) and four SNPs in the promoter. To characterize the relative effects of directional selection via insecticide tolerance ('strain') as compared to acute exposure to insecticide ('treatment'), four pairwise comparisons were carried out between libraries to determine the differentially expressed genes. Most resistance-related differentially expressed genes were identified from the comparison of the strains and enriched in pathways for exogenous detoxification including cytochrome P450 and the ABC transporter. Further confirmation came from the weighted gene co-expression network analysis, which indicated that genes in the significant module associated with chlorantraniliprole resistance were enriched in pathways for exogenous detoxification, and that CYP6B6-like represented a hub gene in the "darkred" module. Furthermore, RNAi knock-down of CYP6B6-like increases P. xylostella sensitivity to chlorantraniliprole. Our study thus provides a genetic foundation underlying selection for pesticide resistance and plausible mechanisms to explain fast evolved adaptation through genomic divergence and altered gene expression in insects. [ABSTRACT FROM AUTHOR]

Osmanthus fragrans flowers have long been used as raw materials in food, tea, beverage, and perfume industries due to their attractive and strong fragrance. The P450 superfamily proteins have been reported to widely participate in the synthesis of plant floral volatile organic compounds (VOCs). To investigate the potential functions of P450 superfamily proteins in the fragrance synthesis of O. fragrans, we investigated the P450 superfamily genome wide. A total of 276 P450 genes were identified belonging to 40 families. The RNA-seq data suggested that many OfCYP genes were preferentially expressed in the flower or other organs, and some were also induced by multiple abiotic stresses. The expression patterns of seven flower-preferentially expressed OfCYPs during the five different flower aroma content stages were further explored using quantitative real-time PCR, showing that the CYP94C subfamily member OfCYP142 had the highest positive correlation with linalool synthesis gene OfTPS2. The transient expression of OfCYP142 in O. fragrans petals suggested that OfCYP142 can increase the content of linalool, an important VOC of the O. fragrans floral aroma, and a similar result was also obtained in flowers of OfCYP142 transgenic tobacco. Combined with RNA-seq data of the transiently transformed O. fragrans petals, we found that the biosynthesis pathway of secondary metabolites was significantly enriched, and many 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes were also upregulated. This evidence indicated that the OfCYP proteins may play critical roles in the flower development and abiotic response of O. fragrans, and that OfCYP142 can participate in linalool synthesis. This study provides valuable information about the functions of P450 genes and a valuable guide for studying further functions of OfCYPs in promoting fragrance biosynthesis of ornamental plants. [ABSTRACT FROM AUTHOR]

Biomacromolecules are known to feature complex three‐dimensional shapes that are essential for their function. Among natural products, ambiguous molecular shapes are a rare phenomenon. The hexapeptide tryptorubin A can adopt one of two unusual atropisomeric configurations. Initially hypothesized to be a non‐ribosomal peptide, we show that tryptorubin A is the first characterized member of a new family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that we named atropopeptides. The sole modifying enzyme encoded in the gene cluster, a cytochrome P450 monooxygenase, is responsible for the atropospecific formation of one carbon‐carbon and two carbon‐nitrogen bonds. The characterization of two additional atropopeptide biosynthetic pathways revealed a two‐step maturation process. Atropopeptides promote pro‐angiogenic cell functions as indicated by an increase in endothelial cell proliferation and undirected migration. Our study expands the biochemical space of RiPP‐modifying enzymes and paves the way towards the chemoenzymatic utilization of atropopeptide‐modifying P450s. [ABSTRACT FROM AUTHOR]

The predatory stink bug P. Lewisi shows potential for Integrated Pest Management programs for controlling Lepidoptera pest insects in crops and forests. The importance of this insect for biological control has stimulated several studies into its biology and ecology. However, P. lewisi has little genetic information available. In the present study, PacBio single-molecule real-time (SMRT) sequencing and Illumina RNA-seq sequencing technologies were used to reveal the full-length transcriptome profiling and tissue-specific expression patterns of P. lewisi. A total of 12,997 high-quality transcripts with an average length of 2,292 bp were obtained from different stages of P. lewisi using SMRT sequencing. Among these, 12,101 were successfully annotated in seven public databases. A total of 67 genes of cytochrome P450 monooxygenases, 43 carboxylesterase genes, and 18 glutathione S-transferase genes were identified, most of which were obtained with full-length ORFs. Then, tissue-specific expression patterns of 5th instar nymphs were analyzed using Illumina sequencing. Several candidate genes related to detoxification of insecticides and other xenobiotics as well as the degradation of odors, were identified in the guts and antennae of P. lewisi. The current study offered in-depth knowledge to understand the biology and ecology of this beneficial predator and related species. [ABSTRACT FROM AUTHOR]

Selective oxyfunctionalization of non-activated C–H bonds remains a major challenge in synthetic chemistry. The biocatalytic hydroxylation of non-activated C–H bonds by cytochrome P450 monooxygenases (CYPs), however, offers catalysis with high regio- and stereoselectivity using molecular oxygen. CYP153s are a class of CYPs known for their selective terminal hydroxylation of n-alkanes and microorganisms, such as the bacterium Alcanivorax dieselolei, have evolved extensive enzymatic pathways for the oxyfunctionalization of various lengths of n-alkanes, including a CYP153 to yield medium-chain 1-alkanols. In this study, we report the characterization of the terminal alkane hydroxylase from A. dieselolei (CYP153A71) for the oxyfunctionalization of medium-chain n-alkanes in comparison to the well-known CYP153A6 and CYP153A13. Although the expected 1-alkanols are produced, CYP153A71 readily converts the 1-alkanols to the corresponding aldehydes, fatty acids, as well as α,ω-diols. CYP153A71 is also shown to readily hydroxylate medium-chain fatty acids. The X-ray crystal structure of CYP153A71 bound to octanoic acid is solved, yielding an insight into not only the regioselectivity, but also the binding orientation of the substrate, which can be used in future studies to evolve CYP153A71 for improved oxidations beyond terminal n-alkane hydroxylation. [ABSTRACT FROM AUTHOR]

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant's kcat/Km values and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols. [ABSTRACT FROM AUTHOR]

The application of camptothecin (CPT) and its derivatives to control insect pests has generated significant interest. This study investigated the enzymatic response of cytochrome P450 monooxygenase (CYP) and glutathione S-transferase (GST) genes in the fat body cells of Spodoptera exigua after 10 μM CPT treatment. Additionally, we examined the effects of CPT on the growth and development of S. exigua larvae and detected the relative expression levels of selected CYP and GST genes during the CPT treatment period. Twenty-one CYP and 17 GST genes were identified from the fat body cells of S. exigua by comparative transcriptomic analyses. The relative expression of ten CYP and seven GST genes changed significantly, which suggested that these CPYs and GSTs may be involved in CPT metabolism. During exposure to CPT for 10 days, the development of S. exigua larvae was delayed and was characterized by weight inhibition and a prolonged period of development. The relative expression levels of the selected four CYP genes, CYP9A27, CYP9A186, CYP337B5, CYP321A8, and one GST gene, GSTe7, were significantly changed by CPT treatment compared to the control group. These generated data provide a basis for identifying the CPT metabolism/detoxification genes of S. exigua at the molecular level.

Strong multi-host adaptability significantly contributes to the rapid dissemination of Hyphantria cunea. The present study explores the involvement of cytochrome P450 monooxygenase (P450) in the multi-host adaptation of H. cunea and aims to develop RNA pesticides targeting essential P450 genes to disrupt this adaptability. The results showed that inhibiting P450 activity notably reduced larval weight and food-intake across seven plants groups. The P450 gene HcCYP6AE178 was highly upregulated in H. cunea larvae from medium- and low-preference host plant groups. Silencing HcCYP6AE178 significantly decreased H. cunea larval body weight, increased larval mortality, inhibited energy metabolism genes expression and interfered with growth regulatory genes expression. Overexpression of HcCYP6AE178 enhanced the tolerance of Drosophila and Sf9 cells to the plant defensive substances cytisine and coumarin. The RNA pesticide CS-ds HcCYP6AE178 constructed using chitosan (CS) exhibited remarkable stability. Treatment with CS-ds HcCYP6AE178 effectively reduced H. cunea larval body weight, heightened larval mortality, and disrupted growth regulatory genes expression in low-preference host plant groups. Combined treatment of CS-ds HcCYP6AE178 and coumarin significantly elevated H. cunea larval mortality compared to coumarin alone, accompanied by the inhibition of growth regulatory genes expression and an abnormal increase in energy metabolism genes expression. Taken together, HcCYP6AE178 is essential for the adaptation of H. cunea to multiple host plants, and RNA pesticides targeting HcCYP6AE178 can effectively impair the performance of H. cunea in different host plants. [Display omitted] • Knocking down HcCYP6AE178 severely reduced multi-host adaptation of H. cunea. • Overexpression of HcCYP6AE178 helps Drosophila to tolerate cytisine and coumarin. • Overexpression of HcCYP6AE178 helps Sf9 cells to tolerate cytisine and coumarin. • The constructed CS-ds HcCYP6AE178 can disrupt the multi-host adaptation of H. cunea. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10 , and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance. [ABSTRACT FROM AUTHOR]

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains. [ABSTRACT FROM AUTHOR]

The wood-boring beetles, including the majority of Cerambycidae, have developed the ability to metabolize a variety of toxic compounds derived from host plants and the surrounding environment. However, detoxification mechanisms underlying the evolutionary adaptation of a cerambycid beetle Pharsalia antennata to hosts and habitats are largely unexplored. Here, we characterized three key gene families in relation to detoxification (cytochrome P450 monooxygenases: P450s, carboxylesterases: COEs and glutathione-Stransferases: GSTs), by combinations of transcriptomics, gene identification, phylogenetics and expression profiles. Illumina sequencing generated 668,701,566 filtered reads in 12 tissues of P. antennata, summing to 100.28 gigabases data. From the transcriptome, 215 genes encoding 106 P450s, 77 COEs and 32 GSTs were identified, of which 107 relatives were differentially expressed genes. Of the identified 215 genes, a number of relatives showed the orthology to those in Anoplophora glabripennis, revealing 1:1 relationships in 94 phylogenetic clades. In the trees, P. antennata detoxification genes mainly clustered into one or two subfamilies, including 64 P450s in the CYP3 clan, 33 COEs in clade A, and 20 GSTs in Delta and Epsilon subclasses. Combining transcriptomic data and PCR approaches, the numbers of detoxification genes expressed in abdomens, antennae and legs were 188, 148 and 141, respectively. Notably, some genes exhibited significantly sexbiased levels in antennae or legs of both sexes. The findings provide valuable reference resources for further exploring xenobiotics metabolism and odorant detection in P. antennata. [ABSTRACT FROM AUTHOR]

Steroids are the second largest class of drugs with a wide range of pharmacological properties. Hydroxylation of steroids seriously affects their biological activities and other properties. However, steroids are mostly sp3 hybridized carbons with numerous C−H bonds far from the functional group that can activate them, and achieving regio‐ and stereo‐selective hydroxylation on steroids is a highly challenging task that is almost impossible to achieve using modern organic synthesis techniques. Interestingly, cytochrome P450 monooxygenases possess the ability to catalyse regio‐ and stereo‐selective oxidations of nonactivated C−H bonds in complex organic molecules under mild conditions. This review summarizes the P450s identified and engineered in recent years that can catalyse steroid nucleus hydroxylation stereo‐ and regio‐selectively. [ABSTRACT FROM AUTHOR]

Acetamiprid is widely used in paddy fields for controlling Nilaparvata lugens (Stål). However, the risk of resistance development, the cross-resistance pattern and the resistance mechanism of acetamiprid in this pest remain unclear. In this study, an acetamiprid-resistant strain (AC-R) was originated from a field strain (UNSEL) through successive selection with acetamiprid for 30 generations, which reached 60.0-fold resistance when compared with a laboratory susceptible strain (AC-S). The AC-R strain (G30) exhibited cross-resistance to thiamethoxam (25.6-fold), nitenpyram (21.4-fold), imidacloprid (14.6-fold), cycloxaprid (11.8-fold), dinotefuran (8.7-fold), sulfoxaflor (7.6-fold) and isoprocarb (8.22-fold), while there was no cross-resistance to etofenprox, buprofezin and chlorpyrifos. Acetamiprid was synergized by the inhibitor piperonyl butoxide (2.2-fold) and the activity of cytochrome P450 monooxygenase was significantly higher in the AC-R strain compared with the AC-S strain, suggesting the critical role of P450. The gene expression results showed that the P450 gene CYP6ER1 was significantly overexpressed in AC-R compared with the AC-S and UNSEL strains. In addition, the RNA interference (RNAi) of CYP6ER1 significantly increased the susceptibility of AC-R to acetamiprid. Molecular docking predicted that acetamiprid and CYP6ER1 had close binding sites, and the nitrogen atoms had hydrogen bond interactions with CYP6ER1. These results demonstrated that the overexpression of CYP6ER1 contributed to acetamiprid resistance in N. lugens. [ABSTRACT FROM AUTHOR]

Abstract Background Tetramycin is a 26-member tetraene antibiotic used in agriculture. It has two components, tetramycin A and tetramycin B. Tetramycin B is obtained by the hydroxylation of tetramycin A on C4. This reaction is catalyzed by the cytochrome P450 monooxygenase TtmD. The two components of tetramycin have different antifungal activities against different pathogenic fungi. Therefore, the respective construction of high-yield strains of tetramycin A and tetramycin B is conducive to more targeted action on pathomycete and has a certain practical value. Results Streptomyces ahygroscopicus S91 was used as the original strain to construct tetramycin A high-yield strains by blocking the precursor competitive biosynthetic gene cluster, disrupting tetramycin B biosynthesis, and overexpressing the tetramycin pathway regulator. Eventually, the yield of tetramycin A in the final strain was up to 1090.49 ± 136.65 mg·L− 1. Subsequently, TtmD, which catalyzes the conversion from tetramycin A to tetramycin B, was overexpressed. Strains with 2, 3, and 4 copies of ttmD were constructed. The three strains had different drops in tetramycin A yield, with increases in tetramycin B. The strain with three copies of ttmD showed the most significant change in the ratio of the two components. Conclusions A tetramycin A single-component producing strain was obtained, and the production of tetramycin A increased 236.84% ± 38.96% compared with the original strain. In addition, the content of tetramycin B in a high-yield strain with three copies of ttmD increased from 26.64% ± 1.97 to 51.63% ± 2.06%.

Triterpenoids constitute a group of specialized plant metabolites with wide structural diversity and high therapeutic value for human health. Cytochrome P450 monooxygenases (CYP) are a family of enzymes important for generating the structural diversity of triterpenoids by catalyzing the site-specific oxidization of the triterpene backbone. The CYP716 enzyme family has been isolated from various plant families as triterpenoid oxidases; however, their experimental crystal structures are not yet available and the detailed catalytic mechanism remains elusive. Here, we address this challenge by integrating bioinformatics approaches with data from other CYP families. Medicago truncatula CYP716A12, the first functionally characterized CYP716A subfamily enzyme, was chosen as the model for this study. We performed homology modeling, structural alignment, in silico site-directed mutagenesis, and molecular docking analysis to search and screen key amino acid residues relevant to the catalytic activity and substrate specificity of the CYP716A subfamily enzyme in triterpenoid biosynthesis. An in vivo functional analysis using engineered yeast that endogenously produced plant-derived triterpenes was performed to elucidate the results. When the amino acids in the signature region and substrate recognition sites (SRSs) were substituted, the product profile of CYP716A12 was modified. We identified amino acid residues that control the substrate contraction of the enzyme (D292) and engineered the enzyme to improve its catalytic activity and substrate specificity (D122, I212, and Q358) for triterpenoid biosynthesis. In addition, we demonstrated the versatility of this strategy by changing the properties of key residues in SRSs to improve the catalytic activity of Arabidopsis thaliana CYP716A1 (S356) and CYP716A2 (M206, F210) at C-28 on the triterpene backbone. This research has the potential to help in the production of desired triterpenoids in engineered yeast by increasing the catalytic activity and substrate specificity of plant CYP716A subfamily enzymes.

BACKGROUND Spirotetramat is a tetramic acid derivative insecticide with novel modes of action for controlling Aphis gossypii Glover in the field. Previous studies have shown that long noncoding RNAs (lncRNAs) and cytochrome P450 monooxygenases (P450s) are involved in the detoxification process. However, the functions of lncRNAs in regulating P450 gene expression in spirotetramat resistance in A. gossypii are unknown. RESULTS: In this study, we found CYP4CJ1, CYP6CY7 and CYP6CY21 expression levels to be significantly upregulated in a spirotetramat‐resistant (SR) strain compared with a susceptible (SS) strain. Furthermore, knockdown of CYP4CJ1, CYP6CY7 and CYP6CY21 increased nymph and adult mortality in the SR strain following exposure to spirotetramat. Drosophila ectopically expressing CYP380C6, CYP4CJ1, CYP6DA2, CYP6CY7 and CYP6CY21 showed significantly decreased mortality after spirotetramat exposure, and CYP380C6, CYP4CJ1 and CYP6CY21 are putative targets of six lncRNAs. Silencing of lncRNAs MSTRG.36649.2/5 and MSTRG.71880.1 changed CYP6CY21 and CYP380C6 expression, altering the sensitivity of the SR strain to spirotetramat. Moreover, MSTRG.36649.2/5 did not compete for microRNA (miRNA) binding to regulate CYP6CY21 expression. CONCLUSION: Our results confirm that CYP380C6, CYP4CJ1, CYP6DA2, CYP6CY7 and CYP6CY21 are potentially involved in the development of spirotetramat resistance in A. gossypii, and MSTRG.36649.2/5 and MSTRG.71880.1 probably regulate CYP6CY21 and CYP380C6 expression other than through the "sponge effect" of competing for miRNA binding. Our results provide a favorable molecular basis for studying cotton aphid P450 genes and lncRNA functions in spirotetramat resistance development. [ABSTRACT FROM AUTHOR]