A Novel Approach to Integrate Human Biomonitoring Data with Model Predicted Dietary Exposures: A Crop Protection Chemical Case Study Using Lambda-Cyhalothrin.

The appropriate use of human biomonitoring data to model population chemical exposures is challenging, especially for rapidly metabolized chemicals, such as agricultural chemicals. The objective of this study is to demonstrate a novel approach integrating model predicted dietary exposures and biomonitoring data to potentially inform regulatory risk assessments. We use lambda-cyhalothrin as a case study, and for the same representative U.S. population in the National Health and Nutrition Examination Survey (NHANES), an integrated exposure and pharmacokinetic model predicted exposures are calibrated to measurements of the urinary metabolite 3-phenoxybenzoic acid (3PBA), using an approximate Bayesian computing (ABC) methodology. We demonstrate that the correlation between modeled urinary 3PBA and the NHANES 3PBA measurements more than doubled as ABC thresholding narrowed the acceptable tolerance range for predicted versus observed urinary measurements. The median predicted urinary concentrations were closer to the median measured value using ABC than using current regulatory Monte Carlo methods.

Isolation and characterization of novel 3,3'-iminodipropionitrile biodegrading Paracoccus communis, from an industrial wastewater treatment bioreactor.

Until now, bacteria able to degrade, 3,3'-iminodipropionitrile (IDPN), a neurotoxin that destroys vestibular hair cells, causing ototoxicity, culminating in irreversible movement disorders, had never been isolated. The aim of this study was to isolate a novel IDPN-biodegrading microorganism and characterize its metabolic pathway. Enrichment was performed by inoculating activated sludge from a wastewater treatment bioreactor that treated IDPN-contaminated wastewater in M9 salt medium, with IDPN as the sole carbon source. A bacterial strain with a spherical morphology that could grow at high concentrations was isolated on a solid medium. Growth of the isolated strain followed the Monod kinetic model. Based on the 16S rRNA gene, the isolate was Paracoccus communis. Whole-genome sequencing revealed that the isolated P. communis possessed the expected full metabolic pathway for IDPN biodegradation. Transcriptome analyses confirmed the overexpression of the gene encoding hydantoinase/oxoprolinase during the exponential growth phase under IDPN-fed conditions, suggesting that the enzyme involved in cleaving the imine bond of IDPN may promote IDPN biodegradation. Additionally, the newly discovered P. communis isolate seems to metabolize IDPN through cleavage of the imine bond in IDPN via nitrilase, nitrile hydratase, and amidase reactions. Overall, this study lays the foundation for the application of IDPN-metabolizing bacteria in the remediation of IDPN-contaminated environments.

Plant glucosinolate biosynthesis and breakdown pathways shape the rhizosphere bacterial/archaeal community.

Rhizosphere microbial community assembly results from microbe-microbe-plant interactions mediated by small molecules of plant and microbial origin. Studies with Arabidopsis thaliana have indicated a critical role of glucosinolates in shaping the root and/or rhizosphere microbial community, likely through breakdown products produced by plant or microbial myrosinases inside or outside of the root. Plant nitrile-specifier proteins (NSPs) promote the formation of nitriles at the expense of isothiocyanates upon glucosinolate hydrolysis with unknown consequences for microbial colonisation of roots and rhizosphere. Here, we generated the A. thaliana triple mutant nsp134 devoid of nitrile formation in root homogenates. Using this line and mutants lacking aliphatic or indole glucosinolate biosynthesis pathways or both, we found bacterial/archaeal alpha-diversity of the rhizosphere to be affected only by the ability to produce aliphatic glucosinolates. In contrast, bacterial/archaeal community composition depended on functional root NSPs as well as on pathways of aliphatic and indole glucosinolate biosynthesis. Effects of NSP deficiency were strikingly distinct from those of impaired glucosinolate biosynthesis. Our results demonstrate that rhizosphere microbial community assembly depends on functional pathways of both glucosinolate biosynthesis and breakdown in support of the hypothesis that glucosinolate hydrolysis by myrosinases and NSPs happens before secretion of products to the rhizosphere.

Immobilization of aldoxime dehydratases on metal affinity resins and use of the immobilized catalysts for the synthesis of nitriles important in fragrance industry.

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Nitrile biosynthesis in nature: how and why?

Covering: up to the end of 2023Natural nitriles comprise a small set of secondary metabolites which however show intriguing chemical and functional diversity. Various patterns of nitrile biosynthesis can be seen in animals, plants, and microorganisms with the characteristics of both evolutionary divergence and convergence. These specialized compounds play important roles in nitrogen metabolism, chemical defense against herbivores, predators and pathogens, and inter- and/or intraspecies communications. Here we review the naturally occurring nitrile-forming pathways from a biochemical perspective and discuss the biological and ecological functions conferred by diversified nitrile biosyntheses in different organisms. Elucidation of the mechanisms and evolutionary trajectories of nitrile biosynthesis underpins better understandings of nitrile-related biology, chemistry, and ecology and will ultimately benefit the development of desirable nitrile-forming biocatalysts for practical applications.

Exploration and utilization of novel aldoxime, nitrile, and nitro compounds metabolizing enzymes from plants and arthropods.

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.

Transcription factor B-H2 regulates CYP9J34 expression conveying deltamethrin resistance in Culex pipiens pallens.

BACKGROUND: Mosquitoes are vectors of various diseases, posing significant health threats worldwide. Chemical pesticides, particularly pyrethroids like deltamethrin, are commonly used for mosquito control, but the emergence of resistant mosquito populations has become a concern. In the deltamethrin-resistant (DR) strain of Culex pipiens pallens, the highly expressed cytochrome P450 9 J34 (CYP9J34) gene is believed to play a role in resistance, yet the underlying mechanism remains unclear. RESULTS: Quantitative polymerase chain reaction with reverse transcription (qRT-PCR) analysis revealed that the expression of CYP9J34 was 14.6-fold higher in DR strains than in deltamethrin-susceptible (DS) strains. The recombinant production of CYP9J34 protein of Cx. pipiens pallens showed that the protein could directly metabolize deltamethrin, yielding the major metabolite 4'-OH deltamethrin. Through dual luciferase reporter assays and RNA interference, the transcription factor homeobox protein B-H2-like (B-H2) was identified to modulate the expression of the CYP9J34 gene, contributing to mosquito resistance to deltamethrin. CONCLUSIONS: Our findings demonstrate that the CYP9J34 protein could directly degrade deltamethrin, and the transcription factor B-H2 could regulate CYP9J34 expression, influencing the resistance of mosquitoes to deltamethrin. © 2023 Society of Chemical Industry.

JH degradation pathway participates in hormonal regulation of larval development of Bombyx mori following λ-cyhalothrin exposure.

λ-Cyhalothrin (λ-cyh), a widely utilized pyrethroid insecticide, poses serious threats to non-target organisms due to its persistence nature in the environment. Exposure to low concentrations of λ-cyh has been observed to result in prolonged larval development in Bombyx mori, leading to substantial financial losses in sericulture. The present study was undertaken to elucidate the underlying mechanisms for prolonged development caused by λ-cyh (LC10) exposure. The results showed that the JH Ⅲ titer was significantly increased at 24 h of λ-cyh exposure, and the JH interacting genes Methoprene-tolerant 2, Steroid Receptor Co-activator, Krüppel-homolog 1, and JH binding proteins were also up-regulated. Although the target of rapamycin (Tor) genes were induced by λ-cyh, the biosynthesis of JH in the corpora allata was not promoted. Notably, 13 JH degradation genes were found to be significantly down-regulated in the midgut of B. mori. The mRNA levels and enzyme activity assays indicated that λ-cyh had inhibitory effects on JH esterase, JH epoxide hydrolase, and JH diol kinase (JHDK). Furthermore, the suppression of JHDK (KWMTBOMO01580) was further confirmed by both western blot and immunohistochemistry. This study has offered a comprehensive perspective on the mechanisms underlying the prolonged development caused by insecticides, and our results also hold significant implications for the safe production of sericulture.

Identification of G Protein-Coupled Receptors (GPCRs) Associated with Lambda-Cyhalothrin Detoxification in Cydia pomonella.

While previous studies have reported G protein-coupled receptor (GPCR)-mediated insecticide resistance in various arthropods, the understanding of GPCR-associated resistance mechanisms in Cydia pomonella remains limited. In this study, a total of 95 CpGPCR genes categorized into four families were identified in C. pomonella. Results revealed high expression levels of the majority of the CpGPCRs during the first larval stage and in the head of C. pomonella. Exposure to lambda-cyhalothrin significantly increased the expression of 15 CpGPCRs, including CpGPCR70, which is highly expressed in all larval stages and shows the highest expression in the midgut. RNA interference (RNAi) demonstrated that downregulation of CpGPCR70 leads to reduced expression of key resistance-related genes and a decreased tolerance of larvae to lambda-cyhalothrin. These findings indicate that CpGPCR70 plays a crucial role in regulating the expression of detoxifying genes involved in lambda-cyhalothrin resistance, offering valuable insights for the development of more effective pest control strategies.

Ioxynil and diethylstilbestrol impair cardiac performance and shell growth in the mussel Mytilus coruscus.

The herbicide ioxynil (IOX) and the synthetic estrogen diethylstilbestrol (DES) are environmentally relevant contaminants that act as endocrine disruptors (EDCs) and have recently been shown to be cardiovascular disruptors in vertebrates. Mussels, Mytilus coruscus, were exposed to low doses of IOX (0.37, 0.037 and 0.0037 mg/L) and DES (0.27, 0.027 and 0.0027 mg/L) via the water and the effect monitored by generating whole animal transcriptomes and measuring cardiac performance and shell growth. One day after IOX (0.37 and 0.037 mg/L) and DES (0.27 and 0.027 mg/L) exposure heart rate frequency was decreased in both groups and 0.27 mg/L DES significantly reduced heart rate frequency with increasing time of exposure (P < 0.05) and no acclimatization occurred. The functional effects were coupled to significant differential expression of genes of the serotonergic synapse pathway and cardiac-related genes at 0.027 mg/L DES, which suggests that impaired heart function may be due to interference with neuroendocrine regulation and direct cardiac effect genes. Multiple genes related to detoxifying xenobiotic substances were up regulated and genes related to immune function were down regulated in the DES group (vs. control), indicating that detoxification processes were enhanced, and the immune response was depressed. In contrast, IOX had a minor disrupting effect at a molecular level. Of note was a significant suppression (P < 0.05) by DES of shell growth in juveniles and lower doses (< 0.0027 mg/L) had a more severe effect. The shell growth depression in 0.0027 mg/L DES-treated juveniles was not accompanied by abundant differential gene expression, suggesting that the effect of 0.0027 mg/L DES on shell growth may be direct. The results obtained in the present study reveal for the first time that IOX and DES may act as neuroendocrine disrupters with a broad spectrum of effects on cardiac performance and shell growth, and that DES exposure had a much more pronounced effect than IOX in a marine bivalve.

Enzymatic properties of a non-classical aldoxime dehydratase capable of producing alkyl and arylalkyl nitriles.

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.

Light induces an increasing release of benzyl nitrile against diurnal herbivore Ectropis grisescens Warren attack in tea (Camellia sinensis) plants.

Herbivore-induced plant volatiles (HIPVs) are critical compounds that directly or indirectly regulate the tritrophic interactions among herbivores, natural enemies and plants. The synthesis and release of HIPVs are regulated by many biotic and abiotic factors. However, the mechanism by which multiple factors synergistically affect HIPVs release remains unclear. Tea plant (Camellia sinensis) is the object of this study because of its rich and varied volatile metabolites. In this study, benzyl nitrile was released from herbivore-attacked tea plants more in the daytime than at night, which was consistent with the feeding behaviour of tea geometrid (Ectropis grisescens Warren) larvae. The Y-tube olfactometer assay and insect resistance analysis revealed that benzyl nitrile can repel tea geometrid larvae and inhibit their growth. On the basis of enzyme activities in transiently transformed Nicotiana benthamiana plants, CsCYP79 was identified as a crucial regulator in the benzyl nitrile biosynthetic pathway. Light signalling-related transcription factor CsPIF1-like and the jasmonic acid (JA) signalling-related transcription factor CsMYC2 serve as the activator of CsCYP79 under light and damage conditions. Our study revealed that light (abiotic factor) and herbivore-induced damage (biotic stress) synergistically regulate the synthesis and release of benzyl nitrile to protect plants from diurnal herbivorous tea geometrid larvae.

Interaction of chlorothalonil and Varroa destructor on immature honey bees rearing in vitro.

Under realistic environmental conditions, bees are often exposed to multiple stressors, especially Varroa destructor and pesticides. In this study, the effects of exposure to NOAEC of chlorothalonil during the larval stage, in the presence or absence of V. destructor, was examined in terms of survival, morphological and transcriptional changes. The interaction between chlorothalonil and V. destructor on the survival of honey bee was additive. V. destructor are the dominant factor in the interaction for survival and transcriptome alternation. The downregulation of the genes related to tissue growth and caste differentiation may directly link to the mortality of honey bees. Either chlorothalonil or V. destructor induces the irregular morphology of trophocytes and oenocytes in the fat body. In addition to irregular shapes, oenocytes in V. destructor alone and double-stressor treatment group showed altered nuclei and vacuoles in the cytoplasm. The interaction of V. destructor and chlorothalonil at the larval stage have potential adverse effects on the subsequent adult bees, with up-regulation of genes involved in lipid metabolism and detoxification/defense in fat body tissue. Our findings provide a comprehensive understanding of combinatorial effects between biotic and abiotic stressors on one of the most important pollinators, honey bees.

Biosynthesis, herbivore induction, and defensive role of phenylacetaldoxime glucoside.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii.

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages; the first barriers to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed ABCH2 to be mainly localized in the legs and head appendages, and more specifically, the apical part of the epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed no affect by ABCH2 silencing, indicating this protein is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket in the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed an ABCH2 substrate. Overall, our findings pinpoint ABCH2 participating in deltamethrin toxicity regulation.

Identification and characterization of cytochrome P450 CYP77A59 of loquat (Rhaphiolepis bibas) responsible for biosynthesis of phenylacetonitrile, a floral nitrile compound.

MAIN CONCLUSION: Cytochrome P450 CYP77A59 is responsible for the biosynthesis of phenylacetonitrile in loquat flowers. Flowers of some plants emit volatile nitrile compounds, but the biosynthesis of these compounds is unclear. Loquat (Rhaphiolepis bibas) flowers emit characteristic N-containing volatiles, such as phenylacetonitrile (PAN), (E/Z)-phenylacetaldoxime (PAOx), and (2-nitroethyl)benzene (NEB). These volatiles likely play a defense role against pathogens and insects. PAN and NEB are commonly biosynthesized from L-phenylalanine via (E/Z)-PAOx. Two cytochrome P450s-CYP79D80 and "promiscuous fatty acid ω-hydroxylase" CYP94A90, which catalyze the formation of (E/Z)-PAOx from L-phenylalanine and NEB from (E/Z)-PAOx, respectively-are involved in NEB biosynthesis. However, the enzymes catalyzing the formation of PAN from (E/Z)-PAOx in loquat have not been identified. In this study, we aimed to identify candidate cytochrome P450s catalyzing PAN formation in loquat flowers. Yeast whole-cell biocatalyst assays showed that among nine candidate cytochrome P450s, CYP77A58 and CYP77A59 produced PAN from (E/Z)-PAOx. CYP77As catalyzed the dehydration of aldoximes, which is atypical of cytochrome P450; the reaction was NADPH-dependent, with an optimum temperature and pH of 40 °C and 8.0, respectively. CYP77As acted on (E/Z)-PAOx, (E/Z)-4-hydroxyphenylacetaldoxime, and (E/Z)-indole-3-acetaldoxime. Previously characterized CYP77As are known to hydroxylate fatty acids; loquat CYP77As did not act on tested fatty acids. We observed higher expression of CYP77A59 in flowers than in buds; expression of CYP77A58 was remarkably reduced in the flowers. Because the flowers, but not buds, emit PAN, CYP77A59 is likely responsible for the biosynthesis of PAN in loquat flowers. This study will help us understand the biosynthesis of floral nitrile compounds.

[A case of poisoning caused by intramuscular injection of esfenvalerate].

Esfenvalerate is a kind of commonly used highly effective pyrethroid insecticide. It is common for people who are poisoned by contact or misuse, but rarely reported for people who are poisoned by intramuscular injection. This paper reports a case of intramuscular injection of esfenvalerate in the Department of Infection, West China Hospital of Sichuan University in November 2021. The patient was intramuscularly injected with about 20 ml of esfenvalerate, inducing the sense of swelling and tingling, degeneration and necrosis of striated muscle tissue at the injection site, also liver function damage and other manifestations. The patient was discharged from hospital after rehydration, accelerating poison metabolism, anti-infection, liver protection and local puncture.

Metabolic Exploration for Cyhalofop-Butyl Antagonism in Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] Following Pretreatment of Malathion.

The present study investigated the effects of broad-spectrum metabolic inhibitors malathion (cytochrome P450 inhibitor) and/or 4-chloro-7-nitrobenzofurazan (NBD-Cl; glutathione S-transferase inhibitor) on the metabolism of cyhalofop-butyl (CyB) in barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes confirmed previously with multiple resistance to two herbicides CyB and florpyrauxifen-benzyl. The metabolic inhibitors were not effective at recovering the sensitivity of resistant barnyardgrass biotypes to CyB treated at the labeled rate (313 g ai ha-1). Rather, treatment with malathion followed by CyB caused antagonism, reducing the efficacy of CyB and promoting the growth of resistant biotypes. Pretreatment with malathion did not influence absorption/translocation of the applied form CyB and its conversion to the active herbicide form cyhalofop-acid (CyA), in both susceptible and resistant biotypes. In contrast, metabolism of the applied form (CyB) decreased 1.5 to 10.5 times by the malathion pretreatment. Taken together, the maintained CyA production against the reduced CyB metabolism could be the mechanism to account for the cause of CyB antagonism observed in barnyardgrass following malathion pretreatment. Additionally, the evolution of CyB resistance in barnyardgrass might be associated with reduced production of CyA in resistant biotypes, independent of activities of cytochrome P450 or GST enzymes.

Engineered aldoxime dehydratase to enable the chemoenzymatic conversion of benzyl amines to aromatic nitriles.

A chemoenzymatic strategy has been implemented to synthesize nitriles from benzyl amines under mild conditions. Aldoxime dehydratase (Oxd) plays a decisive role to convert aldoximes into corresponding nitriles. However, natural Oxds commonly exhibit extremely low catalytic capacity toward benzaldehyde oximes. Here, we engineered the OxdF1 from Pseudomonas putida F1 to enhance its catalytic efficiency toward benzaldehyde oximes by a semi-rational design strategy. The protein structure-based CAVER analysis indicates that M29, A147, F306, and L318 are located adjacent to the substrate tunnel entrance of OxdF1, which were responsible for the transportation of substrate into the active site. After two rounds of mutagenesis, the maximum activities of the mutants L318F and L318F/F306Y were 2.6 and 2.8 U/mg respectively, which were significantly higher than the wild OxdF1 of 0.7 U/mg. Meanwhile, the lipase type B from Candida antarctica was functionally expressed in Escherichia coli cells to selectively oxidize benzyl amines to aldoximes using urea-hydrogen peroxide adduct (UHP) as an oxidant in ethyl acetate. To merge the oxidation and dehydration reactions, a reductive extraction solution was added to remove the residue UHP, which is critical to eliminate its inhibition on the Oxd activity. Consequently, nine benzyl amines were efficiently converted into corresponding nitriles by the chemoenzymatic sequence.