Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm （Spodoptera litura） and RNA interference to evaluate its role in commonly used insecticides

Insect cytochrome P450 monooxygenases （CYPs or P450s） play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference （RNAi） and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, β-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal develop- ment indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up- regulated by chlorpyrifos,β-cypermethrin and methomyl with both lethal concentration at 15% （LC15） （50, 100 and 150 μg/mL, respectively） and 50%（LC50） dosages （100,200 and 300μg/mL, respectively）. Addition of piperonyl butoxide （PBO） significantly increased the toxicity ofchlorpyrifos,β-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi- mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin, respectively, at the LCso dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and β-cypermethrin detoxification in S. litura.

Comparative analysis of cytochrome P450-like genes from Locusta migratoria manilensis： expression profiling and response to insecticide exposure

The cytochrome P450 monooxygenase （cytochrome P450） gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and other xenobiotics. To date, very little is known about cytochrome P450 genes in the oriental migratory locust, Locusta migratoria manilensis. In this study, we carried out a genomewide analysis ofcytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures. We identified 15 cytochrome P450-1ike genes from a locust expressed sequence tag database （LocustDB）. Reverse transcription polymerase chain reaction （RT-PCR） analysis showed that most cytochrome P450-1ike genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgnt, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides. Deltamethrin caused significant inductions in 12 h at LD30 （dose to kill 30% of the tested individuals） in the nymphs, whereas malathion and carbaryl did not have significant effect on cytochrome P450 enzyme activity. Further RT-PCR analysis Showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin-treated locusts. Thus, the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450 genes in response to deltamethrin exposure. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels.

Identification and characterization of a cytochrome P450 C YP6CX1 putatively associated with insecticide resistance in Bemisia tabaci

The novel full length of cytochrome P450 gene has been isolated in insecticideresistant （named CYP6CXlvl） and -susceptible （named CYP6CXlv2） Bemisia tabaci, which was identified as B biotype, in Shangjie, Fujian, China （Sj）. CYP6CX1 （1 940 bp contained a 1 557 bp open reading frame） included conserved domains common to CYP6 members, such as heme-binding motif PFGEGPRFCIA, putative ＂meander＂-binding sequence ETLR and PERF in helix-K, oxygen-binding motif AGLDPV and conserved sequence PEKFNP near the carboxyl end. There were four different replacements of amino acid residues between R and S B. tabaci （Thr300 Ala, Thr354Pro, Arg486His and Ile503Thr）, among which the substitution Ile503Thr was located in the substrate recognition sites region. The mRNA transcription level of CYP6CXlvl was 2.38-fold as high as that of CYP6CXlv2. The results indicated that the CFP6CX1 from the B biotype B. tabaci in Sj was one of the CYP6 members, and enhanced CYP6CX1 expression and substitute of amino acid residues might be involved in the resistance mechanisms in field B. tabaci.

Cytochrome P450 monooxygenase specific activity reduction in wheat Triticum aestivum induced by soil roxithromycin stress

Roxithromycin, livestock growth promoter, as widely used medicine and arouses concern because its occurrence and persistence in soil environments. However, effects of roxithromycin in higher plants are still vague. Accordingly, we hypothesized that roxithromycin-con- taminated soil may exhibits ecotoxicological effects in wheat （Triticum aestivum）. In this study, effects induced by a gradient concentration of roxithromycin stress （0.01, 0.1, 1, 10, and 100 mg.kg-1） was investigated in a 7-d soil test in T. aestivum. Results indicated that the specific activity of cytochrome P450 （CYP450） monooxygenase was decreased dramatically with the concentration of roxithro- mycin in soil. The IC50 value was 8.78mg.kg-1 of roxithromycin. On the contrary, the growth related endpoints （i.e., the germination percentage, the biomass and the height）, the content related endpoints （i.e., soluble protein content and CYP450 content）, and the superoxide dismutase （SOD） activity failed to reveal the roxithromy- cin-induced effects. Further analysis revealed that the CYP450 monooxygenase specific activity reduction was enzymatic mechanism mediated, other than oxidative stress induced. We conclude that the soil roxithromycin declined the CYP450 monooxygenase activity in T. aestivum by the inhibition of the enzymatic mechanism. Further efforts can include, but are not limited to, investigation of joint effects induced by combined exposure of roxithromycin and the pesticides and evalua- tion of the similar effects in other higher plants.

HMGR, SQS, β-AS, and Cytochrome P450 Monooxygenase Genes in Glycyrrhiza uralensis

Glycyrrhiza uralensis is frequently used in traditional Chinese medicine. This plant contains a large amount of effective constituents, including triterpenoids and flavonoids. Among them, glycyrrhizin is believed to be the marker compound to evaluate the quality of G. uralensis based on Chinese Pharmacopoeia. Many studies showed that glycyrrhizin possesses various pharmacological activities, such as antibacterial, antiviral, antitumor, anti-inflammatory, and immune-stimulating activities. In this paper, we summarized the cloning, characterization, expression, and polymorphism analysis of several functional genes involved in glycyrrhizin biosynthesis in G. uralensis.

Establishing Modular Cell-Free Expression System for the Biosynthesis of Bicyclomycin from a Chemically Synthesized Cyclodipeptide

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-andα-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 platformforthe biosynthesis of complex antibiotics.

Advances in biosynthesis of triterpenoid saponins in medicinal plants

In recent years, biosynthesis of triterpenoid saponins in medicinal plants has been widely studied because of their active ingredients with diverse pharmacological activities. Various oxidosqualene cyclases, cytochrome P450 monooxygenases, uridine diphosphate glucuronosyltransferases, and transcription factors related to triterpenoid saponins biosynthesis have been explored and identified. In the biosynthesis of triterpenoid saponins, the progress of gene mining by omics-based sequencing, gene screening, gene function verification, catalyzing mechanism of key enzymes and gene regulation are summarized and discussed. By the progress of the biosynthesis pathway of triterpenoid saponins, the large-scale production of some triterpenoid saponins and aglycones has been achieved through plant tissue culture, transgenic plants and engineered yeast cells. However, the complex biosynthetic pathway and structural diversity limit the biosynthesis of triterpenoid saponins in different system. Special focus can further be placed on the systematic botany information of medicinal plants obtained from omics large dataset, and triterpenoid saponins produced by synthetic biology strategies, gene mutations and gene editing technology.

Spatial distribution of insecticide resistant Check update populations of Aedes aegypti and Ae. albopictus and first detection of V410L mutation in Ae. aegypti from Cameroon

Background:Dengue(DENV),chikungunya(CHIKV)and Zika virus(ZIKV),are mosquito-borne viruses of medical importance in most tropical and subtropical regions.Vector control,primarily through insecticides,remains the primary method to prevent their transmission.Here,we evaluated insecticide resistance profles and identifed important underlying resistance mechanisms in populations of Aedes aegypti and Ae.albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations.Methods:Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation,G1.Ae.aegypti and Ae.albopictus adults from G1 were tested following World Health Organization(WHO)recommendations and Ae.aegypti GO adults screened with real time melting curve qPCR analyses to genotype the F1534C,V1016l and V410L Aedes kdr mutations.Piperonyl butoxide(PBO)assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance.Statistical analyses were performed using Chi-square test and generalized linear models.Results:Loss of susceptibility was observed to all insecticides tested.Mortality rates from tests with 0.25%permethrin varied from 24.27%to 85.89%in Ae.aegypti and from 17.35%to 68.08%in Ae.albopictus.Mortality rates for 0.03%deltamethrin were between 23.30%and 88.20%in Ae.aegypti and between 69.47%and 84.11%in Ae.albopictus.We found a moderate level of resistance against bendiocarb,with mortality rates ranging from 69.31%to 90.26%in Ae.aegypti and from 86.75%to 98.95%in Ae.albopictus.With PBO pre-exposure,we found partial or fully restored suscepti bility to pyrethroids and bendiocarb.The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae.aegypti populations from Douala sites resistant to permethrin and deltamethrin.Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae.albopictus samples.F1534C and V1016l mutations were detected in A.aegypti mosquitoes and for the first time V410L was reported in Cameroon.Conclusions:This study revealed that Ae.aegypti and Ae albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated.These findings could guide insecticide use to control arbovirus vectors in Cameroon.

Aflatoxin B1： Toxicity, bioactivation and detoxification in the polyphagous caterpillar Trichoplusia ni

Trichoplusia ni caterpillars are polyphagous foliage-feeders and rarely likely to encounter aflatoxin B1 （AFB1）, a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB 1, we evaluated the toxicity ofAFB 1 to T. ni caterpillars at different developmental stages and found that AFB 1 tolerance significantly increases with larval development. Diet incorporation of AFB 1 at 1μg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3μg/g AFB 1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases （P450s）, reduced the toxicity of AFB 1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as fiavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFBI toxicity, suggesting that P450s are also involved in AFB 1 detoxification in T. ni.

Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana

The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A(CsA)primarily at the 4th N-methyl leucine(MeLeu4),giving rise toγ-hydroxy-N-methyl-L-Leu4-CsA(CsA-4-OH).This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect,thus demonstrating great application potential in both pharmaceutical and cosmetic industries.However,this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine(MeLeu9),indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator.Herein,we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85Å.Together with sequence and 3D structure comparisons,Autodock-based substrate docking,molecular dynamics(MD)simulation,and site-directed mutagenesis,we identified a number of key residues including R294,E264,and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA,setting the stage for better enzymatic preparation of CsA-4-OH.This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.