Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts.Here a green and efficient asymmetric hydroxylation of primary and secondary C-H bon...Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts.Here a green and efficient asymmetric hydroxylation of primary and secondary C-H bonds at propargylic positions has been established.A series of optically active propargylic alcohols were prepared with high regio-and enantioselectivity(up to 99%ee)under mild reaction conditions by using P450tol,while the C≡C bonds in the molecule remained unreacted.This protocol provides a green and practical method for constructing enantiomerically chiral propargylic alcohols.In addition,we also demonstrated that the biohydroxylation strategy was able to scaled up to 2.25 mmol scale with the production of chiral propargyl alcohol 2a at a yield of 196 mg with 96%ee,which’s an important synthetic intermediate of antifungal drug Ravuconazole.展开更多
Rice sheath blight(RSB)is a major destructive disease impeding rice production.Identifying key germplasm resources with increased resistance remains a challenge.However,the mechanisms underlying disease resistance are...Rice sheath blight(RSB)is a major destructive disease impeding rice production.Identifying key germplasm resources with increased resistance remains a challenge.However,the mechanisms underlying disease resistance are not yet fully understood.Cytochrome P450 monooxygenases(CYP450s)serve biosynthesis and metabolic detoxification functions in plants,but there is limited information about their role in the response induced by RSB.This study demonstrated that CYT02 belongs to the CYP73A100 subfamily and is a typical member of the CYP450s.Overexpression(OE)in rice of the cytochrome P450 monooxygenase cyt02 conferred increased resistance to RSB and increased vegetative tillering.Cyt02 may increase RSB resistance by regulating plant hormone synthesis,regulate reactive oxygen species(ROS)by coordinating the activity of antioxidant enzymes,and initiate phytoalexin synthesis in response to fungal infection.These research findings have laid a foundation for a deeper understanding of the function of cyt02 and offered a potential target gene for breeding rice varieties resistant to sheath blight.展开更多
Pyrrolobenzoxazines are a rare terpene-amino acid family of natural products with potent biological activities.Here,we reported the full biosynthetic pathway of paeciloxazine(1),a typical pyrrolobenzoxazine,with signi...Pyrrolobenzoxazines are a rare terpene-amino acid family of natural products with potent biological activities.Here,we reported the full biosynthetic pathway of paeciloxazine(1),a typical pyrrolobenzoxazine,with significant insecticidal activity.Base on heterologous expression,chemical complement experiment,and in vitro biochemical assays,we demonstrated the sesquiterpene portion of 1 derived from discontinuously oxidations of amorphdiene,in which P450 monooxygenase PaxH catalyzed a cascade of hydroxylation and epoxidation,while two flavin dependent monooxygenases are involved in the transformation of the esterified tryptophan into a pyrrolobenzoxazine core.Furthermore,a total of 15 compounds were generated through heterologous expression,of which 13,17 and 20 showed potential antiepileptic activity.This study fully elucidated the biosynthetic pathway of paeciloxazine(1)and showed the diversity and complexity of constructing natural products by organisms.展开更多
Methane(CH_(4))has a higher heat capacity(104.9 kcal/mol)than carbon dioxide(CO_(2)),and this has inspired research aimed at reducing methane levels to retard global warming.Hydroxylation under ambient conditions thro...Methane(CH_(4))has a higher heat capacity(104.9 kcal/mol)than carbon dioxide(CO_(2)),and this has inspired research aimed at reducing methane levels to retard global warming.Hydroxylation under ambient conditions through methanotrophs can provide crucial information for understanding the harsh C-H activation of methane.Soluble methane monooxygenase(sMMO)belongs to the bacterial multi-component monooxygenase superfamily and requires hydroxylase(MMOH),regulatory(MMOB),and reductase(MMOR)components.Recent structural and biophysical studies have demonstrated that these components accelerate and retard methane hydroxylation in MMOH through protein-protein interactions.Complex structures of sMMO,including MMOH-MMOB and MMOH-MMOD,illustrate how these regulatory and inhibitory components orchestrate the di-iron active sites located within the four-helix bundles of MMOH,specifically at the docking surface known as the canyon region.In addition,recent biophysical studies have demonstrated the role of MmoR,aσ54-dependent transcriptional regulator,in regulating sMMO expression.This perspective article introduces remarkable discoveries in recent reports on sMMO components that are crucial for understanding sMMO expression and activities.Our findings provide insight into how sMMO components interact with MMOH to control methane hydroxylation,shedding light on the mechanisms governing sMMO expression and the interactions between activating enzymes and promoters.展开更多
The tobacco whitefly,Bemisia tabaci,is a notorious pest affecting various crops globally,and it exhibits high levels of resistance to various insecticides.Afidopyropen is a recently commercialized pyropene insecticide...The tobacco whitefly,Bemisia tabaci,is a notorious pest affecting various crops globally,and it exhibits high levels of resistance to various insecticides.Afidopyropen is a recently commercialized pyropene insecticide for B.tabaci control with high selectivity and a novel mode of action.We previously identified a high level of afidopyropen resistance in a field-collected population after selection in the lab,and named it the HD-Afi strain.In the present study,minimal cross-resistance in the HD-Afi strain was found between afidopyropen and other common chemical agents.However,the P450 enzyme activity in HD-Afi was 2.18 times the level in susceptible strain HD-S.Expression analysis revealed that two of 12 candidate P450 genes,namely CYP6DW3 and CYP4C64,were significantly up-regulated in HD-Afi.Silencing CYP6DW3 and CYP4C64 by RNA interference(RNAi)substantially increased the susceptibility of whitefly adults,confirming their involvement in afidopyropen resistance.Homology modeling and molecular docking analyses demonstrated stable binding of afidopyropen to CYP6DW3 and CYP4C64,with binding free energies of–6.87 and–6.11 kcal mol^(-1),respectively.The findings of this study suggest that the induction of CYP6DW3 and CYP4C64 facilitates afidopyropen detoxification,contributing to the development of resistance in B.tabaci.展开更多
Cytochrome P450 monooxygenases(P450s)play crucial roles in the oxyfunctionalization of non-activated hydrocarbons,thus bridging the gap between simple molecules and high value-added fine chemicals.The introduction of ...Cytochrome P450 monooxygenases(P450s)play crucial roles in the oxyfunctionalization of non-activated hydrocarbons,thus bridging the gap between simple molecules and high value-added fine chemicals.The introduction of P450s into artificially designed cascade reactions provides an exciting opportunity to accomplish challenging reactions and access organic compounds that cannot be achieved by traditional chemical catalysts or by natural metabolic pathways.The main objective of this review is to provide an overview of different types of artificially designed multi-step cascades in which P450s are involved as key catalysts in the biosynthesis of various organic molecules.The different efforts include in vitro multi-enzymatic biocatalytic cascades,in vivo biocatalytic cascades as well as chemo-enzymatic hybrid cascades.Overall,this work provides an overview of cascade reactions involving P450s with various potential applications for the industrial production of food,cosmetics,polymers and pharmaceuticals.展开更多
Microbial enhanced oil recovery(MEOR)is a cost effective and efficient method for recovering residual oil.However,the presence of wax(paraffin)in residual oil can substantially reduce the efficiency of MEOR.Therefore,...Microbial enhanced oil recovery(MEOR)is a cost effective and efficient method for recovering residual oil.However,the presence of wax(paraffin)in residual oil can substantially reduce the efficiency of MEOR.Therefore,microbial dewaxing is a critical process in MEOR.In this study,a bacterial dewaxing agent of three spore-forming bacteria was developed.Among these bacteria,Bacillus subtilis GZ6 produced the biosurfactant surfactin.Replacing the promoter of the surfactin synthase gene cluster(srfA),increased the titer of surfactin in this strain from 0.33 g/L to 2.32 g/L.The genetically modified strain produced oil spreading rings with diameters increasing from 3.5±0.1 to 4.1±0.2 cm.The LadA F10L/N133R mutant was created by engineering an alkane monooxygenase(LadA)using site-directed mutagenesis in the Escherichia coli host.Compared to the wild-type enzyme,the resulting mutant exhibited an 11.7-fold increase in catalytic efficiency toward the substrate octadecane.When the mutant(pIMPpladA2mu)was expressed in Geobacillus stearothermophilus GZ178 cells,it exhibited a 2.0-fold increase in octadecane-degrading activity.Cultures of the two modified strains(B.subtilis GZ6(pg3srfA)and G.stearothermophilus GZ178(pIMPpladA2mu))were mixed with the culture of Geobacillus thermodenitrificans GZ156 at a ratio of 5:80:15.The resulting composition increased the rate of wax removal by 35%compared to the composition composed of three native strains.This study successfully developed a multi-strain bacterial agent with enhanced oil wax removal capabilities by genetically engineering two bacterial strains.展开更多
Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is...Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.展开更多
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, cytochrom...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.展开更多
The 5＇-flanking proximal region of stress-induced gene encoding choline monooxygenase （CMO） was isolated by Adaptor-PCR and TAIL-PCR from halophyte Suaeda liaotungensis K. A total of 2,204 bp DNA sequence was obtai...The 5＇-flanking proximal region of stress-induced gene encoding choline monooxygenase （CMO） was isolated by Adaptor-PCR and TAIL-PCR from halophyte Suaeda liaotungensis K. A total of 2,204 bp DNA sequence was obtained. The transcription start site, which is located at 128 bp upstream to the start ATG, was predicted by the TSSP-TCM program. The functional elements were analysed by PLACE program. The obtained SICMO gene promoter contains the basic elements： TATA-box, CAAT-box, and stress-induced elements, for example, salt responsive element （GAAAAA）, cold responsive elements （CANNTG）, ABA （Abscisic Acid） responsive elements （NAACAA）, water stress element （CGGTTG）, and WUN responsive elements （GTTAGGTTC）. Isolation and analysis of the promoter of the CMO gene from S. liaotungensis lays a foundation for characterising the stress-induced promoter elements, studying the relationship between the structure and function of the promoter, and investigating the molecular mechanism of CMO gene regulation.展开更多
The objectives of this study were as follows: 1) to establish a baseline ethoxyresorufin-O-deethylase (EROD) activity level in channel catfish (Ictalurus punctatus), 2) to assess changes in induction of cytochrome P45...The objectives of this study were as follows: 1) to establish a baseline ethoxyresorufin-O-deethylase (EROD) activity level in channel catfish (Ictalurus punctatus), 2) to assess changes in induction of cytochrome P450 enzyme in channel catfish following exposure to creek water at the discharge point from the Troy (Alabama) Wastewater Treatment Plant (TWWTP) compared to upstream samples from Walnut Creek, 3) to compare EROD activity in populations maintained in laboratory and field settings, and 4) to quantify cytochrome P450 gene expression. Enzyme activity was measured fluorometrically and CYP1 gene expression was analyzed by quantitative real-time reverse transcription polymerase chain reaction. A mean EROD baseline was established at 0.03 nmol/min/μg of protein. The overall mean field effluent (TF) EROD had a significant 5-fold increase over field upstream (UF) exposed catfish;and overall mean laboratory effluent (TL) exposed catfish EROD had a significant 1.8-fold increase over laboratory upstream (UL) exposed catfish. Field exposures generally showed more robust enzyme induction over laboratory exposures on all sampling days. Ex- pression of the CYP1B gene following TF exposure was 6-fold over UF. Results suggested that in situ exposure to wastewater pollutants using caged test organisms provided a much more sensitive local monitor of pollutant exposure and biological impact than ex situ toxicological studies.展开更多
Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine be- taine[1]. Two enzymes are involved in the pathway choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BA...Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine be- taine[1]. Two enzymes are involved in the pathway choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spina- cia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42℃), as determined by immunoblot analysis, but did not respond to cold stress (4℃), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.展开更多
Heavy metal pollution affects soil ecological function.Biochar and compost can effectively remediate heavy metals and increase soil nutrients.The effects and mechanisms of biochar and compost amendments on soil nitrog...Heavy metal pollution affects soil ecological function.Biochar and compost can effectively remediate heavy metals and increase soil nutrients.The effects and mechanisms of biochar and compost amendments on soil nitrogen cycle function in heavy-metal contaminated soils are not fully understood.This study examined how biochar,compost,and their integrated use affected ammonia-oxidizing microorganisms in heavy metal polluted soil.Quantitative PCR was used to determine the abundance of ammonia-oxidizing archaea(AOA)and bacteria(AOB).Ammonia monooxygenase(AMO)activity was evaluated by the enzymelinked immunosorbent assay.Results showed that compost rather than biochar improved nitrogen conversion in soil.Biochar,compost,or their integrated application significantly reduced the effective Zn and Cd speciation.Adding compost obviously increased As and Cu effective speciation,bacterial 16 S rRNA abundance,and AMO activity.AOB,stimulated by compost addition,was significantly more abundant than AOA throughout remediation.Correlation analysis showed that AOB abundance positively correlated with NO_(3)^(-)-N(r=0.830,P<0.01),and that AMO activity had significant correlation with EC(r=-0.908,P<0.01)and water-soluble carbon(r=-0.868,P<0.01).Those seem to be the most vital factors affecting AOB community and their function in heavy metal-polluted soil remediated by biochar and compost.展开更多
The coupled effects of nitrogen source and methane monooxygenase（MMO） on the growth and poly-β-hydroxybutyrate（PHB） accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressi...The coupled effects of nitrogen source and methane monooxygenase（MMO） on the growth and poly-β-hydroxybutyrate（PHB） accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO（s MMO） grew at the highest rate, while N2-fixing bacteria expressing particulate MMO（p MMO） grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content（51.0%） was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB（45.2%） with the expression of p MMO, while N2-fixing bacteria containing p MMO only showed low PHB production capacity（32.1%）. The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of p MMO were greatly improved in the cyclic NO3-N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.展开更多
Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate（PHB）accumulation capacity from methane.Methane was used as...Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate（PHB）accumulation capacity from methane.Methane was used as the carbon source,N2as sole nitrogen source,and oxygen and Cu content were varied.Copper proved essential for PHB synthesis.All cultures enriched with Cu could accumulate high content of PHB（43.2%–45.9%）,while only small amounts of PHB were accumulated by cultures enriched without Cu（11.9%–17.5%）.Batch assays revealed that communities grown with Cu and a higher O2content synthesized more PHB,which had a wider optimal CH4：O2range and produced a high PHB content（48.7%）even though in the presence of N2.In all methanotrophic–heterotrophic communities,both methanotrophic and heterotrophic populations showed the ability to accumulate PHB.Although methane was added as the sole carbon source,heterotrophs dominated with abundances between 77.2%and 85.6%.All methanotrophs detected belonged to type II genera,which formed stable communities with heterotrophs of different PHB production capacities.展开更多
Nitrogen(N) application may lead to niche segregation of soil ammonia-oxidizing archaea(AOA) and bacteria(AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availa...Nitrogen(N) application may lead to niche segregation of soil ammonia-oxidizing archaea(AOA) and bacteria(AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availability. However, the adaptive mechanisms of AOA and AOB under N enrichment remain poorly understood. Stable isotope probing(SIP) microcosm incubation was employed to reveal community changes of active AOA and AOB in a loess soil from a field experiment growing potatoes that received no N(control, CK), low N(LN, 75 kg N ha^(-1)), and high N(HN, 375 kg N ha^(-1)). The results showed that the soil potential nitrification rate(PNR) was measured by culturing of the soil samples from the field experiment. Soil PNR was significantly increased in HN by87.5% and 67.5% compared with CK and LN, respectively. Compared with CK, the^(13)C-amoA genes of soil AOA and AOB in HN had 2.58 × 10~4 and 1.55 × 10~6 copies, representing 1.6-and 16.2-fold increase respectively. It was indicated that AOB dominated soil ammonia oxidation. A phylogenetic analysis of the^(13)C-amoA gene showed that N application significantly increased the proportion of54 d9-like AOA up to 90% in HN, while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared. Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like to Nitrosospira sp. TCH711-like. Our study provides compelling evidence for the emergence and maintenance of active nitrifying communities under the intensified N input to an agricultural ecosystem.展开更多
[ Objective] This study aimed to investigate the function of aliD gene in the inactivation of AHLs. [ Method ] A bacterial isolate, Acinetobacter sp. CA2 from soil, is capable of inactivation of AHLs. A gene designed ...[ Objective] This study aimed to investigate the function of aliD gene in the inactivation of AHLs. [ Method ] A bacterial isolate, Acinetobacter sp. CA2 from soil, is capable of inactivation of AHLs. A gene designed as aliD, which is responsible for AHL-quenching activity and exhibits high similarity with Mo- nooxygenase genes, was cloned from the genomic library of Acinetobacter sp. CA2. [ Result ] The aliD gene in-frame deletion mutant, CA2 AliD, impaired its AHLs inactivating function when mixed with N-（3-oxooctanoyl） -L-homosefine lactone （30C8-HSL）. Expression of AliD in plant pathogenic bacterium Pectobacterium ca- rotovorum subsp, carotovorum Z3-3 significantly reduced the AHLs production and the extracellular pectolytic enzyme activities, and attenuated soft rot disease symptoms on the plants tested, including potato, Chinese cabbage, radish and cabbage. [ Conclusion ] Our study suggests that the aliD gene complemented strain CA2-AliD showes a similar AHLs inactivating function.展开更多
ebisu dwarf (d2) is a mutant caused by mutation in a rice brassinosteroid biosynthetic enzyme gene, CYP90D2/D2, thereby conferring a brassinosteroid-deficient dwarf phenotype. Three newly isolated d2 alleles derived f...ebisu dwarf (d2) is a mutant caused by mutation in a rice brassinosteroid biosynthetic enzyme gene, CYP90D2/D2, thereby conferring a brassinosteroid-deficient dwarf phenotype. Three newly isolated d2 alleles derived from a Nippon- bare mutant library (d2-3, d2-4, and d2-6) produced more severe dwarf phenotypes than the previously characterized null allele from a Taichung 65 mutant library, d2-1. Linkage analysis and a complementation test clearly indicated that the mutant phenotypes in d2-6 were caused by defects in CYP90D2/D2, and exogenous treatment with brassinolide, a bioactive brassinosteroid, rescued the dwarf phenotype of three Nipponbare-derived d2 mutants. However, the content of endogenous bioactive brassinosteroid, castasterone, and the expression of brassinosteroid-response genes indicated that partial suppression of the brassinosteroid response in addition to a brassinosteroid deficiency has occurred in the Nipponbare-derived d2 mutants. Based on these results, we discuss the possibility that wild-type Nipponbare has some defects in an unknown factor or factors related to the brassinosteroid response in rice.展开更多
Serotonin deficiency in major depressive disorder(MDD)has formed the basis of antidepressant drug development and was originally attributed to induction of the major tryptophan(Trp)-degrading enzyme,liver Trp 2,3-diox...Serotonin deficiency in major depressive disorder(MDD)has formed the basis of antidepressant drug development and was originally attributed to induction of the major tryptophan(Trp)-degrading enzyme,liver Trp 2,3-dioxygenase(TDO),by cortisol,leading to decreased Trp availability to the brain for serotonin synthesis.Subsequently,the serotonin deficiency was proposed to involve induction of the extrahepatic Trp-degrading enzyme indoleamine 2,3-dioxygenase(IDO)by proinflammatory cytokines,with inflammation being the underlying cause.Recent evidence,however,challenges this latter concept,as not all MDD patients are immune-activated and,when present,inflammation is mild and/or transient.A wide range of antidepressant drugs inhibit the activity of liver TDO and bind specifically to the enzyme,but not to IDO.IDO induction is not a major event in MDD,but,when it occurs,its metabolic consequences may be masked and overridden by upregulation of kynurenine monooxygenase(KMO),the gateway to production of modulators of immune and neuronal functions.KMO appears to be activated in MDD by certain proinflammatory cytokines and antidepressants with anti-inflammatory properties may block this activation.We demonstrate the ability of the antidepressant ketamine to dock(bind)to KMO.The pathophysiology of MDD may be underpinned by both the serotonin deficiency and glutamatergic activation mediated respectively by TDO induction and N-methyl-D-aspartate receptor activation.Inhibition of TDO and KMO should be the focus of MDD pharmacotherapy.展开更多
基金financial support from the National Natural Science Foundation of China(No.32271537 and 22061049)the Science and Technology Department of Guizhou province(QKHJCZK2021-036 and QKHRCPTGCC-2023-003)+1 种基金the Science and Technology Department of Zunyi(ZSKRPT-2020-5,ZSKH-2018-3,ZSKRPT-2021-5)Zunyi Medical University(QKH-2018-5772-014).
文摘Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts.Here a green and efficient asymmetric hydroxylation of primary and secondary C-H bonds at propargylic positions has been established.A series of optically active propargylic alcohols were prepared with high regio-and enantioselectivity(up to 99%ee)under mild reaction conditions by using P450tol,while the C≡C bonds in the molecule remained unreacted.This protocol provides a green and practical method for constructing enantiomerically chiral propargylic alcohols.In addition,we also demonstrated that the biohydroxylation strategy was able to scaled up to 2.25 mmol scale with the production of chiral propargyl alcohol 2a at a yield of 196 mg with 96%ee,which’s an important synthetic intermediate of antifungal drug Ravuconazole.
基金supported by the Sichuan Province International Science and Technology Innovation Cooperation(2024YFHZ0299)the Project of Science and Technology Department of Sichuan Province(2022YFH0031)Chengdu Science and Technology Bureau(2024-YF05-02168-SN).
文摘Rice sheath blight(RSB)is a major destructive disease impeding rice production.Identifying key germplasm resources with increased resistance remains a challenge.However,the mechanisms underlying disease resistance are not yet fully understood.Cytochrome P450 monooxygenases(CYP450s)serve biosynthesis and metabolic detoxification functions in plants,but there is limited information about their role in the response induced by RSB.This study demonstrated that CYT02 belongs to the CYP73A100 subfamily and is a typical member of the CYP450s.Overexpression(OE)in rice of the cytochrome P450 monooxygenase cyt02 conferred increased resistance to RSB and increased vegetative tillering.Cyt02 may increase RSB resistance by regulating plant hormone synthesis,regulate reactive oxygen species(ROS)by coordinating the activity of antioxidant enzymes,and initiate phytoalexin synthesis in response to fungal infection.These research findings have laid a foundation for a deeper understanding of the function of cyt02 and offered a potential target gene for breeding rice varieties resistant to sheath blight.
基金supported financially by the National Natural Science Foundation of China(Nos.22107122 and 82225042)the CAMS Innovation Fund for Medical Sciences(CIFMS,No.2021I2M-1-029)。
文摘Pyrrolobenzoxazines are a rare terpene-amino acid family of natural products with potent biological activities.Here,we reported the full biosynthetic pathway of paeciloxazine(1),a typical pyrrolobenzoxazine,with significant insecticidal activity.Base on heterologous expression,chemical complement experiment,and in vitro biochemical assays,we demonstrated the sesquiterpene portion of 1 derived from discontinuously oxidations of amorphdiene,in which P450 monooxygenase PaxH catalyzed a cascade of hydroxylation and epoxidation,while two flavin dependent monooxygenases are involved in the transformation of the esterified tryptophan into a pyrrolobenzoxazine core.Furthermore,a total of 15 compounds were generated through heterologous expression,of which 13,17 and 20 showed potential antiepileptic activity.This study fully elucidated the biosynthetic pathway of paeciloxazine(1)and showed the diversity and complexity of constructing natural products by organisms.
基金This research was supported by"Regional Innovation Strategy"(2023RIS-008)and"C1 Gas Refinery Program"(NRF-2015M3D3D3A1A01064876)through the National Research Foundation of Koreafunded by the Ministry of Education(NRF-2017R1A6A1A03015876).
文摘Methane(CH_(4))has a higher heat capacity(104.9 kcal/mol)than carbon dioxide(CO_(2)),and this has inspired research aimed at reducing methane levels to retard global warming.Hydroxylation under ambient conditions through methanotrophs can provide crucial information for understanding the harsh C-H activation of methane.Soluble methane monooxygenase(sMMO)belongs to the bacterial multi-component monooxygenase superfamily and requires hydroxylase(MMOH),regulatory(MMOB),and reductase(MMOR)components.Recent structural and biophysical studies have demonstrated that these components accelerate and retard methane hydroxylation in MMOH through protein-protein interactions.Complex structures of sMMO,including MMOH-MMOB and MMOH-MMOD,illustrate how these regulatory and inhibitory components orchestrate the di-iron active sites located within the four-helix bundles of MMOH,specifically at the docking surface known as the canyon region.In addition,recent biophysical studies have demonstrated the role of MmoR,aσ54-dependent transcriptional regulator,in regulating sMMO expression.This perspective article introduces remarkable discoveries in recent reports on sMMO components that are crucial for understanding sMMO expression and activities.Our findings provide insight into how sMMO components interact with MMOH to control methane hydroxylation,shedding light on the mechanisms governing sMMO expression and the interactions between activating enzymes and promoters.
基金partly supported by research grants from the Outstanding Youth Foundation of the Beijing Academy of Agriculture and Forestry Sciences,China(YXQN202301)the National Natural Science Foundation of China(32272522)the Beijing Natural Science Foundation,China(6232005)。
文摘The tobacco whitefly,Bemisia tabaci,is a notorious pest affecting various crops globally,and it exhibits high levels of resistance to various insecticides.Afidopyropen is a recently commercialized pyropene insecticide for B.tabaci control with high selectivity and a novel mode of action.We previously identified a high level of afidopyropen resistance in a field-collected population after selection in the lab,and named it the HD-Afi strain.In the present study,minimal cross-resistance in the HD-Afi strain was found between afidopyropen and other common chemical agents.However,the P450 enzyme activity in HD-Afi was 2.18 times the level in susceptible strain HD-S.Expression analysis revealed that two of 12 candidate P450 genes,namely CYP6DW3 and CYP4C64,were significantly up-regulated in HD-Afi.Silencing CYP6DW3 and CYP4C64 by RNA interference(RNAi)substantially increased the susceptibility of whitefly adults,confirming their involvement in afidopyropen resistance.Homology modeling and molecular docking analyses demonstrated stable binding of afidopyropen to CYP6DW3 and CYP4C64,with binding free energies of–6.87 and–6.11 kcal mol^(-1),respectively.The findings of this study suggest that the induction of CYP6DW3 and CYP4C64 facilitates afidopyropen detoxification,contributing to the development of resistance in B.tabaci.
基金This study was supported by the National Key Research and Development Program of China(No.2019YFA09005000)the National Natural Science Foundation of China(Nos.21977026&21702052)Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Cytochrome P450 monooxygenases(P450s)play crucial roles in the oxyfunctionalization of non-activated hydrocarbons,thus bridging the gap between simple molecules and high value-added fine chemicals.The introduction of P450s into artificially designed cascade reactions provides an exciting opportunity to accomplish challenging reactions and access organic compounds that cannot be achieved by traditional chemical catalysts or by natural metabolic pathways.The main objective of this review is to provide an overview of different types of artificially designed multi-step cascades in which P450s are involved as key catalysts in the biosynthesis of various organic molecules.The different efforts include in vitro multi-enzymatic biocatalytic cascades,in vivo biocatalytic cascades as well as chemo-enzymatic hybrid cascades.Overall,this work provides an overview of cascade reactions involving P450s with various potential applications for the industrial production of food,cosmetics,polymers and pharmaceuticals.
基金the financial support from Scientific Research Program of Beijing Municipal Commission of Education(KM202110017009,granted to Xiaoyan Guo)Undergraduates Research Training Program of Beijing Institute of Petrochemical Technology(2022J00178,granted to Lizhu Li)。
文摘Microbial enhanced oil recovery(MEOR)is a cost effective and efficient method for recovering residual oil.However,the presence of wax(paraffin)in residual oil can substantially reduce the efficiency of MEOR.Therefore,microbial dewaxing is a critical process in MEOR.In this study,a bacterial dewaxing agent of three spore-forming bacteria was developed.Among these bacteria,Bacillus subtilis GZ6 produced the biosurfactant surfactin.Replacing the promoter of the surfactin synthase gene cluster(srfA),increased the titer of surfactin in this strain from 0.33 g/L to 2.32 g/L.The genetically modified strain produced oil spreading rings with diameters increasing from 3.5±0.1 to 4.1±0.2 cm.The LadA F10L/N133R mutant was created by engineering an alkane monooxygenase(LadA)using site-directed mutagenesis in the Escherichia coli host.Compared to the wild-type enzyme,the resulting mutant exhibited an 11.7-fold increase in catalytic efficiency toward the substrate octadecane.When the mutant(pIMPpladA2mu)was expressed in Geobacillus stearothermophilus GZ178 cells,it exhibited a 2.0-fold increase in octadecane-degrading activity.Cultures of the two modified strains(B.subtilis GZ6(pg3srfA)and G.stearothermophilus GZ178(pIMPpladA2mu))were mixed with the culture of Geobacillus thermodenitrificans GZ156 at a ratio of 5:80:15.The resulting composition increased the rate of wax removal by 35%compared to the composition composed of three native strains.This study successfully developed a multi-strain bacterial agent with enhanced oil wax removal capabilities by genetically engineering two bacterial strains.
文摘Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.
基金the Central Significant Increase or Decrease Program of China(No.2060302-1604-05)NSFC(No.81703639)。
文摘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.
基金This work was supported by the National Natural Sciences Foundation of China (No. 30370806).
文摘The 5＇-flanking proximal region of stress-induced gene encoding choline monooxygenase （CMO） was isolated by Adaptor-PCR and TAIL-PCR from halophyte Suaeda liaotungensis K. A total of 2,204 bp DNA sequence was obtained. The transcription start site, which is located at 128 bp upstream to the start ATG, was predicted by the TSSP-TCM program. The functional elements were analysed by PLACE program. The obtained SICMO gene promoter contains the basic elements： TATA-box, CAAT-box, and stress-induced elements, for example, salt responsive element （GAAAAA）, cold responsive elements （CANNTG）, ABA （Abscisic Acid） responsive elements （NAACAA）, water stress element （CGGTTG）, and WUN responsive elements （GTTAGGTTC）. Isolation and analysis of the promoter of the CMO gene from S. liaotungensis lays a foundation for characterising the stress-induced promoter elements, studying the relationship between the structure and function of the promoter, and investigating the molecular mechanism of CMO gene regulation.
文摘The objectives of this study were as follows: 1) to establish a baseline ethoxyresorufin-O-deethylase (EROD) activity level in channel catfish (Ictalurus punctatus), 2) to assess changes in induction of cytochrome P450 enzyme in channel catfish following exposure to creek water at the discharge point from the Troy (Alabama) Wastewater Treatment Plant (TWWTP) compared to upstream samples from Walnut Creek, 3) to compare EROD activity in populations maintained in laboratory and field settings, and 4) to quantify cytochrome P450 gene expression. Enzyme activity was measured fluorometrically and CYP1 gene expression was analyzed by quantitative real-time reverse transcription polymerase chain reaction. A mean EROD baseline was established at 0.03 nmol/min/μg of protein. The overall mean field effluent (TF) EROD had a significant 5-fold increase over field upstream (UF) exposed catfish;and overall mean laboratory effluent (TL) exposed catfish EROD had a significant 1.8-fold increase over laboratory upstream (UL) exposed catfish. Field exposures generally showed more robust enzyme induction over laboratory exposures on all sampling days. Ex- pression of the CYP1B gene following TF exposure was 6-fold over UF. Results suggested that in situ exposure to wastewater pollutants using caged test organisms provided a much more sensitive local monitor of pollutant exposure and biological impact than ex situ toxicological studies.
文摘Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine be- taine[1]. Two enzymes are involved in the pathway choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spina- cia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42℃), as determined by immunoblot analysis, but did not respond to cold stress (4℃), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.
基金supported by the Hunan Provincial Key Research and Development Project(Nos.2019WK2031 and 2017SK2351)the National Natural Science Foundation of China(No.51408219)+1 种基金the Natural Science Foundation of Hu-nan Province(No.2020JJ5259)the Outstanding Youth Fund Project of the Hunan Education Department(No.18B094)。
文摘Heavy metal pollution affects soil ecological function.Biochar and compost can effectively remediate heavy metals and increase soil nutrients.The effects and mechanisms of biochar and compost amendments on soil nitrogen cycle function in heavy-metal contaminated soils are not fully understood.This study examined how biochar,compost,and their integrated use affected ammonia-oxidizing microorganisms in heavy metal polluted soil.Quantitative PCR was used to determine the abundance of ammonia-oxidizing archaea(AOA)and bacteria(AOB).Ammonia monooxygenase(AMO)activity was evaluated by the enzymelinked immunosorbent assay.Results showed that compost rather than biochar improved nitrogen conversion in soil.Biochar,compost,or their integrated application significantly reduced the effective Zn and Cd speciation.Adding compost obviously increased As and Cu effective speciation,bacterial 16 S rRNA abundance,and AMO activity.AOB,stimulated by compost addition,was significantly more abundant than AOA throughout remediation.Correlation analysis showed that AOB abundance positively correlated with NO_(3)^(-)-N(r=0.830,P<0.01),and that AMO activity had significant correlation with EC(r=-0.908,P<0.01)and water-soluble carbon(r=-0.868,P<0.01).Those seem to be the most vital factors affecting AOB community and their function in heavy metal-polluted soil remediated by biochar and compost.
基金supported by the National Key Scientific and Technology Project for Water Pollution Treatment of China(No.2012ZX07202006)the National Natural Science Foundation of China(No.21477014)
文摘The coupled effects of nitrogen source and methane monooxygenase（MMO） on the growth and poly-β-hydroxybutyrate（PHB） accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO（s MMO） grew at the highest rate, while N2-fixing bacteria expressing particulate MMO（p MMO） grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content（51.0%） was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB（45.2%） with the expression of p MMO, while N2-fixing bacteria containing p MMO only showed low PHB production capacity（32.1%）. The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of p MMO were greatly improved in the cyclic NO3-N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.
基金supported by the National Key Scientific and Technology Project for Water Pollution Treatment of China (No.2012ZX07202006)
文摘Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate（PHB）accumulation capacity from methane.Methane was used as the carbon source,N2as sole nitrogen source,and oxygen and Cu content were varied.Copper proved essential for PHB synthesis.All cultures enriched with Cu could accumulate high content of PHB（43.2%–45.9%）,while only small amounts of PHB were accumulated by cultures enriched without Cu（11.9%–17.5%）.Batch assays revealed that communities grown with Cu and a higher O2content synthesized more PHB,which had a wider optimal CH4：O2range and produced a high PHB content（48.7%）even though in the presence of N2.In all methanotrophic–heterotrophic communities,both methanotrophic and heterotrophic populations showed the ability to accumulate PHB.Although methane was added as the sole carbon source,heterotrophs dominated with abundances between 77.2%and 85.6%.All methanotrophs detected belonged to type II genera,which formed stable communities with heterotrophs of different PHB production capacities.
基金supported by the National Key Basic Research Program of China (No. 2015CB150501)the National Natural Science Foundation of China (No. 41530857)
文摘Nitrogen(N) application may lead to niche segregation of soil ammonia-oxidizing archaea(AOA) and bacteria(AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availability. However, the adaptive mechanisms of AOA and AOB under N enrichment remain poorly understood. Stable isotope probing(SIP) microcosm incubation was employed to reveal community changes of active AOA and AOB in a loess soil from a field experiment growing potatoes that received no N(control, CK), low N(LN, 75 kg N ha^(-1)), and high N(HN, 375 kg N ha^(-1)). The results showed that the soil potential nitrification rate(PNR) was measured by culturing of the soil samples from the field experiment. Soil PNR was significantly increased in HN by87.5% and 67.5% compared with CK and LN, respectively. Compared with CK, the^(13)C-amoA genes of soil AOA and AOB in HN had 2.58 × 10~4 and 1.55 × 10~6 copies, representing 1.6-and 16.2-fold increase respectively. It was indicated that AOB dominated soil ammonia oxidation. A phylogenetic analysis of the^(13)C-amoA gene showed that N application significantly increased the proportion of54 d9-like AOA up to 90% in HN, while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared. Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like to Nitrosospira sp. TCH711-like. Our study provides compelling evidence for the emergence and maintenance of active nitrifying communities under the intensified N input to an agricultural ecosystem.
基金Supported by the Natural Science Foundation of China(30871666,31071725)Special Fund for Agro-scientific Research in the Public Interest(201003066)the National High Technology Research and Development Program of China("863" Program)(2011AA10A205)
文摘[ Objective] This study aimed to investigate the function of aliD gene in the inactivation of AHLs. [ Method ] A bacterial isolate, Acinetobacter sp. CA2 from soil, is capable of inactivation of AHLs. A gene designed as aliD, which is responsible for AHL-quenching activity and exhibits high similarity with Mo- nooxygenase genes, was cloned from the genomic library of Acinetobacter sp. CA2. [ Result ] The aliD gene in-frame deletion mutant, CA2 AliD, impaired its AHLs inactivating function when mixed with N-（3-oxooctanoyl） -L-homosefine lactone （30C8-HSL）. Expression of AliD in plant pathogenic bacterium Pectobacterium ca- rotovorum subsp, carotovorum Z3-3 significantly reduced the AHLs production and the extracellular pectolytic enzyme activities, and attenuated soft rot disease symptoms on the plants tested, including potato, Chinese cabbage, radish and cabbage. [ Conclusion ] Our study suggests that the aliD gene complemented strain CA2-AliD showes a similar AHLs inactivating function.
文摘ebisu dwarf (d2) is a mutant caused by mutation in a rice brassinosteroid biosynthetic enzyme gene, CYP90D2/D2, thereby conferring a brassinosteroid-deficient dwarf phenotype. Three newly isolated d2 alleles derived from a Nippon- bare mutant library (d2-3, d2-4, and d2-6) produced more severe dwarf phenotypes than the previously characterized null allele from a Taichung 65 mutant library, d2-1. Linkage analysis and a complementation test clearly indicated that the mutant phenotypes in d2-6 were caused by defects in CYP90D2/D2, and exogenous treatment with brassinolide, a bioactive brassinosteroid, rescued the dwarf phenotype of three Nipponbare-derived d2 mutants. However, the content of endogenous bioactive brassinosteroid, castasterone, and the expression of brassinosteroid-response genes indicated that partial suppression of the brassinosteroid response in addition to a brassinosteroid deficiency has occurred in the Nipponbare-derived d2 mutants. Based on these results, we discuss the possibility that wild-type Nipponbare has some defects in an unknown factor or factors related to the brassinosteroid response in rice.
文摘Serotonin deficiency in major depressive disorder(MDD)has formed the basis of antidepressant drug development and was originally attributed to induction of the major tryptophan(Trp)-degrading enzyme,liver Trp 2,3-dioxygenase(TDO),by cortisol,leading to decreased Trp availability to the brain for serotonin synthesis.Subsequently,the serotonin deficiency was proposed to involve induction of the extrahepatic Trp-degrading enzyme indoleamine 2,3-dioxygenase(IDO)by proinflammatory cytokines,with inflammation being the underlying cause.Recent evidence,however,challenges this latter concept,as not all MDD patients are immune-activated and,when present,inflammation is mild and/or transient.A wide range of antidepressant drugs inhibit the activity of liver TDO and bind specifically to the enzyme,but not to IDO.IDO induction is not a major event in MDD,but,when it occurs,its metabolic consequences may be masked and overridden by upregulation of kynurenine monooxygenase(KMO),the gateway to production of modulators of immune and neuronal functions.KMO appears to be activated in MDD by certain proinflammatory cytokines and antidepressants with anti-inflammatory properties may block this activation.We demonstrate the ability of the antidepressant ketamine to dock(bind)to KMO.The pathophysiology of MDD may be underpinned by both the serotonin deficiency and glutamatergic activation mediated respectively by TDO induction and N-methyl-D-aspartate receptor activation.Inhibition of TDO and KMO should be the focus of MDD pharmacotherapy.
