Genome-wide identification, molecular evolution and gene expression of P450 gene family in Cyrtotrachelus buqueti.

BACKGROUND: Insect Cytochrome P450 monooxygenase (CYPs or P450s) plays an important role in detoxifying insecticides, causing insect populations to develop resistance. However, the molecular functions of P450 gene family in Cyrtotrachelus buqueti genome are still lacking. RESULTS: In this study, 71 CbuP450 genes have been identified. The amino acids length of CbuP450 proteins was between 183 aa ~ 1041 aa. They are proteins with transmembrane domains. The main component of their secondary structure is α-helix and random coils. Phylogenetic analysis showed that C. buqueti and Rhynchophorus ferrugineus were the most closely related. This gene family has 29 high-frequency codons, which tend to use A/T bases and A/T ending codons. Gene expression analysis showed that CbuP450_23 in the female adult may play an important role on high temperature resistance, and CbuP450_17 in the larval may play an important role on low temperature tolerance. CbuP450_10, CbuP450_17, CbuP450_23, CbuP450_10, CbuP450_16, CbuP450_20, CbuP450_23 and CbuP450_ 29 may be related to the regulation of bamboo fiber degradation genes in C. buqueti. Protein interaction analysis indicates that most CbuP450 proteins are mainly divided into three aspects: encoding the biosynthesis of ecdysteroids, participating in the decomposition of synthetic insecticides, metabolizing insect hormones, and participating in the detoxification of compounds. CONCLUSIONS: We systematically analyzed the gene and protein characteristics, gene expression, and protein interactions of CbuP450 gene family, revealing the key genes involved in the stress response of CbuP450 gene family in the resistance of C. buqueti to high or low temperature stress, and identified the key CbuP450 proteins involved in important life activity metabolism. These results provided a reference for further research on the function of P450 gene family in C. buqueti.

St. John's wort extract with a high hyperforin content does not induce P-glycoprotein activity at the human blood-brain barrier.

St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P-gp activity at the human blood-brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)-targeted P-gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P-gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3-6%) for 12-19 days (1800 mg/day), the activity of P-gp at the BBB was assessed by means of PET imaging with the P-gp substrate [11C]metoclopramide and the activity of peripheral P-gp and CYPs was assessed by administering a low-dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P-gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P-gp-mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P-gp induction at the human BBB despite its ability to induce peripheral P-gp and CYPs. Simultaneous intake of SJW with CNS-targeted P-gp substrate drugs is not expected to lead to P-gp-mediated drug interactions at the BBB.

Cytochrome GmGLY1 is Involved in the Biosynthesis of Glycitein in Soybean.

Isoflavones, the major secondary metabolites of interest due to their benefits to both human and plant health, are exclusively produced by legumes. In this study, we profiled the isoflavone content in dry seeds from 211 soybean [Glycine max (L.) Merr.] accessions grown across five environments. Broad and discernible phenotypic variations were observed among accessions, regions, and years of growth. Twenty-six single-nucleotide polymorphisms (SNPs) associated with the sum of glycitein (GLE), glycitin (GL), 6″-O-acetylglycitin (AGL), and 6″-O-malonylglycitin (MGL) contents were detected in multiple environments via a genome-wide association study (GWAS). These SNPs were located on chromosome 11 (8,148,438 bp to 8,296,956 bp, renamed qGly11-01). Glyma.11g108300 (GmGLY1), a gene that encodes a P450 family protein, was identified via sequence variation analysis, functional annotation, weighted gene coexpression network analysis (WGCNA), and expression profile analysis of candidate gene, and hairy roots transformation in soybean. Overexpression of GmGLY1 increased the glycitein content (GLC) in soybean hairy roots and transgenic seeds, while CRISPR/Cas9-generated mutants exhibited decreased GLC and increased daidzein content (DAC). Haplotype analysis revealed that GmGLY1 allelic variations significantly affect the GLC accumulation. These findings enhance our understanding of genes influencing GLC in soybean and may guide breeding for lines with high and stable GLC.

Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

Genome-wide characterization of cytochrome P450 genes reveals the potential roles in fruit ripening and response to cold stress in tomato.

Plant cytochrome P450 (CYP) superfamily, the largest enzyme metabolism family, has been identified in many species and plays a vital role in plant development and stress response via secondary metabolite biosynthesis. A comprehensive identification and functional investigation of CYPs in tomato plants would contribute to deeper understanding of their biological significance. In this study, 268 tomato CYP genes were identified and found to be unevenly located on 12 chromosomes. Based on the phylogenetic analysis, these 268 SlCYPs were classed into two distinct clades (A-type and non-A-type) and nine clans, including 48 families. Moreover, 67 tandem and 22 WGD (whole genome duplication)/segmental duplication events were detected, of which 12 SlCYP genes experienced both WGD/segmental and tandem duplication events, indicating that tandem duplication plays a major role in the expansion of the SlCYP family. Besides, 48 pairs containing 41 SlCYP and 44 AtCYP genes were orthologous, while 216 orthologous pairs were obtained between tomato and potato. The expression level of all SlCYP genes in tomato tissues at different development stages was analyzed, and most expressed SlCYPs showed a tissue-specific pattern. Meanwhile, 143 differentially expressed SlCYPs were identified under cold stress. Furthermore, the RT-qPCR results indicated that SlCYPs may be involved in fruit ripening and cold tolerance in tomato seedlings. These findings provide valuable insights into the evolutionary relationships and functional characteristics of SlCYPs, which can be utilized for further investigation of fruit metabolic pathways and cold tolerance in tomato.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Phytomediated stress modulates antioxidant status, induces overexpression of CYP6M2, Hsp70, α-esterase, and suppresses the ABC transporter in Anopheles gambiae (sensu stricto) exposed to Ocimum tenuiflorum extracts.

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Effects of microplastics on key reproductive and biochemical endpoints of the freshwater microcrustacean Daphnia magna.

Human activities have directly impacted the environment, causing significant ecological imbalances. From the different contaminants resulting from human activities, plastics are of major environmental concern. Due to their high use and consequent discharge, plastics tend to accumulate in aquatic environments. There, plastics can form smaller particles (microplastics, MPs), due to fragmentation and weathering, which are more prone to interact with aquatic organisms and cause deleterious effects, including at the basis of different food webs. This study assessed the effects of two microplastics (polyethylene terephthalate, PET; and polypropylene, PP; both of common domestic use) in the freshwater cladoceran species Daphnia magna. Toxic effects were assessed by measuring reproductive traits (first brood and total number of offspring), and activities of biomarkers involved in xenobiotic metabolism (phase I: cytochrome P-450 isoenzymes CYP1A1, 1A2 and 3A4; phase II/conjugation: glutathione S-transferases; and antioxidant defense (catalase)). Both MPs showed a potential to significantly reduce reproductive parameters in D. magna. Furthermore, PET caused a significant increase in some isoenzymes of CYP450 in acutely exposed organisms, but this effect was not observed in chronically exposed animals. Similarly, the activity of the antioxidant defense (CAT) was significantly increased in acutely exposed animals, but not in chronically exposed organisms. This pattern of effects suggests a possible mechanism of long-term adaptation to the presence of the tested MPs. In conclusion, the herein tested MPs have shown the potential to induce deleterious effects on D. magna mainly observed in terms of the reproductive outcomes. Changes at the biochemical level seems transient and are not likely to occur in long term, environmentally exposed crustaceans.

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Target-site and non-target-site resistance mechanisms confer mesosulfuron-methyl resistance in Alopecurus aequalis.

BACKGROUND: Shortawn foxtail (Alopecurus aequalis Sobol.) is a noxious weed in China. The resistance of A. aequalis developed rapidly due to the long-term application of acetolactate synthase (ALS)-inhibiting herbicides. Here, a suspected mesosulfuron-methyl-resistant A. aequalis population, Aa-R, was collected from a wheat field in China. RESULTS: A dose‒response test showed that the Aa-R population has evolved a high level of resistance to mesosulfuron-methyl, and its growth was suppressed by imazamox, pyroxsulam and bispyribac-sodium. ALS gene sequence analysis revealed that a known resistance-related mutation (Pro-197-Thr) was present in the Aa-R population. Moreover, ALS gene overexpression was detected in the Aa-R population. The mesosulfuron-methyl resistance could be reversed by cytochrome P450 monooxygenase (CYP450) and glutathione S-transferase (GST) inhibitors. In addition, enhanced metabolism of mesosulfuron-methyl was detected in the Aa-R population compared with the susceptible population. NADPH-cytochrome P450 reductase and GST activities were strongly inducible in the Aa-R population. One CYP450 gene, CYP74A2, and one GST gene, GST4, were constitutively upregulated in the Aa-R population. Molecular docking results showed the binding affinity of CYP74A2 and GST4 for the tested ALS-inhibiting herbicides, respectively. CONCLUSION: This study confirmed that target-site resistance and non-target-site resistance involving CYP450 and GST were the main mechanisms involved in resistance in the mesosulfuron-methyl-resistant A. aequalis population.

Effects of cannabidiol and Δ9-tetrahydrocannabinol on cytochrome P450 enzymes: a systematic review.

Due to legal, political, and cultural changes, the use of cannabis has rapidly increased in recent years. Research has demonstrated that the cannabinoids cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) inhibit and induce cytochrome P450 (CYP450) enzymes. The objective of this review is to evaluate the effect of CBD and THC on the activity of CYP450 enzymes and the implications for drug-drug interactions (DDIs) with psychotropic agents that are CYP substrates. A systematic search was conducted using PubMed, Scopus, Scientific Electronic Library Online (SciELO) and PsychINFO. Search terms included 'cannabidiol', 'tetrahydrocannabinol', and 'cytochrome P450'. A total of seven studies evaluating the interaction of THC and CBD with CYP450 enzymes and psychotropic drugs were included. Both preclinical and clinical studies were included. Results from the included studies indicate that both CBD and THC inhibit several CYP450 enzymes including, but not limited to, CYP1A2, CYP3C19, and CYP2B6. While there are a few known CYP450 enzymes that are induced by THC and CBD, the induction of CYP450 enzymes is an understudied area of research and lacks clinical data. The inhibitory effects observed by CBD and THC on CYP450 enzymes vary in magnitude and may decrease the metabolism of psychotropic agents, cause changes in plasma levels of psychotropic medications, and increase adverse effects. Our findings clearly present interactions between THC and CBD and several CYP450 enzymes, providing clinicians evidence of a high risk of DDIs for patients who consume both cannabis and psychotropic medication. However, more clinical research is necessary before results are applied to clinical settings.

Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6ß-OH derivatives, marking the first confirmed C-6ß hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1ß and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 µM.

Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

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.

DeepP450: Predicting Human P450 Activities of Small Molecules by Integrating Pretrained Protein Language Model and Molecular Representation.

Cytochrome P450 enzymes (CYPs) play a crucial role in Phase I drug metabolism in the human body, and CYP activity toward compounds can significantly affect druggability, making early prediction of CYP activity and substrate identification essential for therapeutic development. Here, we established a deep learning model for assessing potential CYP substrates, DeepP450, by fine-tuning protein and molecule pretrained models through feature integration with cross-attention and self-attention layers. This model exhibited high prediction accuracy (0.92) on the test set, with area under the receiver operating characteristic curve (AUROC) values ranging from 0.89 to 0.98 in substrate/nonsubstrate predictions across the nine major human CYPs, surpassing current benchmarks for CYP activity prediction. Notably, DeepP450 uses only one model to predict substrates/nonsubstrates for any of the nine CYPs and exhibits certain generalizability on novel compounds and different categories of human CYPs, which could greatly facilitate early stage drug design by avoiding CYP-reactive compounds.

Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 µg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

Functional Plasticity, Redundancy, and Specificity of Lanosterol 14α-Demethylase in Regulating the Sensitivity to DMIs in Calonectria ilicicola.

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.