scholarly journals Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes

Plants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 577
Author(s):  
Mariko Baba ◽  
Ken-ichi Yamada ◽  
Michiho Ito
Keyword(s):  
Cytochrome P450 ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Volatile Component ◽  
Cloning And Expression ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Components ◽  
Cytochrome P450 Enzyme ◽  
Reaction Products ◽  
High Sequence Similarity

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.

scholarly journals The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

2005 ◽  
Vol 187 (23) ◽  
pp. 7970-7976 ◽  
Author(s):  
Mohini S. Ghatge ◽  
Kevin A. Reynolds
Keyword(s):  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Biosynthetic Gene Cluster ◽  
Conjugative Plasmid ◽  
Cytochrome P450 Monooxygenases ◽  
Phosphate Ester ◽  
High Sequence Similarity ◽  
P450 Monooxygenase ◽  
Complementation Experiment

ABSTRACT Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS 2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS 2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and k cat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.

scholarly journals A Bifunctional Cytochrome P450 Enzyme Involved in the O-Dealkylation or N-Dealkoxymethylation of Chloroacetanilide Herbicides in Rhodococcus sp. B2

2021 ◽  
Author(s):  
hongming liu ◽  
Meng Yuan ◽  
Aimin Liu ◽  
Lei Ren ◽  
Guo-ping Zhu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Draft Genome ◽  
Maximal Activity ◽  
Gas Chromatography Mass Spectrometry ◽  
Genome Comparison ◽  
Cytochrome P450 Enzyme ◽  
P450 Monooxygenase ◽  
Emerging Pollutant ◽  
P450 Enzyme

Abstract Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1°C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

scholarly journals A Bifunctional Cytochrome P450 Enzyme Involved in the O-Dealkylation and N-Dealkoxymethylation toward Chloroacetanilide Herbicides in Rhodococcus sp. B2

2021 ◽  
Author(s):  
hongming liu ◽  
Meng Yuan ◽  
Aimin Liu ◽  
Guo-ping Zhu ◽  
Li-na Sun
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Draft Genome ◽  
Maximal Activity ◽  
Gas Chromatography Mass Spectrometry ◽  
Genome Comparison ◽  
Cytochrome P450 Enzyme ◽  
P450 Monooxygenase ◽  
Emerging Pollutant ◽  
P450 Enzyme

Abstract Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1°C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

scholarly journals GC-MS analysis of volatiles in cinnamon essential oil extracted by different methods

2020 ◽  
Vol 71 (3) ◽  
pp. 372
Author(s):  
T. Yu ◽  
H. Yao ◽  
S. Qi ◽  
J. Wang
Keyword(s):  
Essential Oil ◽  
Steam Distillation ◽  
Volatile Component ◽  
Principal Component ◽  
Industrial Applications ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Components ◽  
Cinnamic Aldehyde ◽  
Microwave Assisted ◽  
Cinnamon Essential Oil

Cinnamon essential oil (CEO) was extracted by three different methods: steam distillation (SD), ultrasound-assisted steam distillation (UASD) and microwave-assisted steam distillation (MASD). The volatiles in CEO were separated and identified by gas chromatography–mass spectrometry (GC-MS), and the differences in volatiles among the three different methods were further analyzed through principal component analysis. The results showed that 36 individual volatile components were present in the CEO from the three different methods. In general, the numbers of aldehydes, esters, alcohols, terpenes, aromatics and ketones were 6, 3, 7, 17, 2, and 1, respectively. The most abundant volatile component was determined to be cinnamic aldehyde. The content of total cinnamic aldehydes, which determines the price of CEO, was the highest among the three methods in the UASD sample (85.633%). Moreover, the highest yield (8.33‰) of essential oil was extracted by the UASD method. Therefore, UASD was the best way for CEO extraction in this research and was recommended for future industrial applications.

Fatty Liver and Impaired Hepatic Metabolism Alters the Congener-Specific Distribution of Polychlorinated Biphenyls (PCBs) in Mice with a Liver-Specific Deletion of Cytochrome P450 Reductase

2020 ◽  
Author(s):  
Xueshu Li ◽  
Chun-Yun Zhang ◽  
Hans-Joachim Lehmler
Keyword(s):  
Cytochrome P450 ◽  
Polychlorinated Biphenyls ◽  
Fatty Liver ◽  
Partition Coefficients ◽  
Hepatic Metabolism ◽  
Gas Chromatography Mass Spectrometry ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
Specific Distribution ◽  
Specific Deletion

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that are linked to adverse health outcomes. PCB tissue levels are determinants of PCB toxicity; however, it is unclear how factors, such as an altered metabolism and/or a fatty liver, affect PCB distribution in vivo. We determined the congener-specific disposition of PCBs in mice with a liver specific deletion of cytochrome P450 reductase (KO), a model of fatty liver with impaired hepatic metabolism, and wildtype (WT) mice. Male and female KO and WT mice were exposed orally to Aroclor 1254, a technical PCB mixture. PCBs were quantified in adipose, blood, brain and liver tissues by gas chromatography-mass spectrometry. PCB profiles and levels in tissues were genotype and sex dependent. PCB levels were higher in the liver from KO compared to WT mice. PCB profiles showed clear differences between tissues from the same exposure group. While experimental tissue : blood partition coefficients in KO and WT mice did not follow the trends predicted using a composition-based model, the agreement between experimental and calculated partition coefficients was still reasonable. Thus, a fatty liver and/or an impaired hepatic metabolism alter the distribution of PCBs in mice and the magnitude of the partitioning of PCBs from blood into tissues can be approximated using composition-based models.<br>

scholarly journals Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Mohamed Ramadan ◽  
Muna Alariqi ◽  
Yizan Ma ◽  
Yanlong Li ◽  
Zhenping Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Sequence Similarity ◽  
High Specificity ◽  
Transformation Method ◽  
High Sequence Similarity ◽  
Single Experiment ◽  
Next Generation Sequencing Technology ◽  
Cotton Transformation ◽  
Assembly Method ◽  
Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

scholarly journals Rapid and sensitive analysis of benzyl isothiocyanate in peel, pulp, and seeds of Carica papaya Linn. by headspace gas chromatography-mass spectrometry

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Yinzheng Ma ◽  
Yingying Wen ◽  
Jinping Chen ◽  
Yunxia Zhang ◽  
Haiying Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Carica Papaya ◽  
Orthogonal Design ◽  
Gas Chromatography Mass Spectrometry ◽  
Volatile Components ◽  
Headspace Gas Chromatography ◽  
Benzyl Isothiocyanate ◽  
Chromatography Mass Spectrometry ◽  
Headspace Gas

AbstractA rapid and sensitive headspace gas chromatography-mass spectrometry (HS-GC–MS) method was established for the determination of benzyl isothiocyanate (BITC) in the peel, pulp, and seeds of Carica papaya Linn. Tween 80 solution with a concentration of 0.002% (w/v) was chosen as a headspace medium for solving the poor solubility of BITC in water without using organic solvents and ensuring high headspace efficiencies. Extraction parameters had been evaluated and optimized by using an orthogonal design with an OA9(34) table. Optimal headspace conditions were obtained when vials were equilibrated at 80 °C for 20 min at a stirring speed of 375 rpm. The calibration curve obtained by using GC–MS was linear in a concentration range of 10–320 ng/mL. The recoveries of peel, pulp, and seeds ranged from 97.3 to 100.6% with RSDs less than 3.0%. The method is simple, rapid, sensitive, and environmentally friendly. It is suitable for analyzing BITC in papaya fruit and is expected to have important application potential in the extraction of water-insoluble volatile components in foods, plants, medicines, and other samples.

The role of cytochrome P450 enzyme genetic variants in cannabis hyperemesis syndrome—A case report

2021 ◽  
Author(s):  
Maxim Kuzin ◽  
Franziskos Xepapadakos ◽  
Isabel Scharrer ◽  
Marc Augsburger ◽  
Chin‐Bin Eap ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Case Report ◽  
Genetic Variants ◽  
Cytochrome P450 Enzyme ◽  
P450 Enzyme ◽  
Cannabis Hyperemesis Syndrome

scholarly journals Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State, Brazil

2021 ◽  
Vol 22 (2) ◽  
pp. 890
Author(s):  
Luccas M. Barata ◽  
Eloísa H. Andrade ◽  
Alessandra R. Ramos ◽  
Oriel F. de Lemos ◽  
William N. Setzer ◽  
...  
Keyword(s):  
Black Pepper ◽  
Plant Diseases ◽  
Gas Chromatography Mass Spectrometry ◽  
Piper Nigrum ◽  
Volatile Components ◽  
Chemical Compositions ◽  
Multivariate Statistical ◽  
Protein Targets ◽  
Fruit Samples ◽  
Sesquiterpene Hydrocarbons

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene (“Equador”/“Guajarina”, I); δ-elemene (“Iaçará”/“Kottanadan”/“Cingapura”, II); elemol (“Clonada”/“Uthirankota”, III) and α-muurolol, bicyclogermacrene, and cubebol (“Bragantina”, IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g−1 FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19–57.22 µU·mL−1), and carotenoids (0.21–2.31 µg·mL−1) displayed significant variations. Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

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