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Peter J Facchini - One of the best experts on this subject based on the ideXlab platform.

  • partial genome assembly of the medicinal plant Ephedra sinica
    bioRxiv, 2021
    Co-Authors: Jeremy S. Morris, Peter J Facchini, Sam Yeaman
    Abstract:

    Ephedra sinica is a high-value medicinal plant that produces important phenylpropylamino alkaloids pseudoephedrine and ephedrine. Few genomics resources exist for E. sinica, which has been characterized as a tetraploid with a monoploid genome size of 8.56 Gb. Here we reported a partial genome assembly of E. sinica (12.8 Gb) based on Illumina short-read sequencing technology at low coverage.

  • an n methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production
    Journal of Biological Chemistry, 2018
    Co-Authors: Jeremy S. Morris, Jillian M Hagel, Ryan A. Groves, Peter J Facchini
    Abstract:

    : Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, β-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.

  • RESEARCH ARTICLE Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis
    2016
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Jillian M Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the bio-chemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-min-ing frameworks for Ephedra sinica and khat plants. Sequence libraries representing a com-bined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidate

  • Characterization of aromatic aminotransferases from Ephedra sinica Stapf
    Amino Acids, 2016
    Co-Authors: Korey Kilpatrick, Efraim Lewinsohn, Jillian M Hagel, Peter J Facchini, Agnieszka Pajak, Mark W. Sumarah, Frédéric Marsolais
    Abstract:

    Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l -glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S -phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

  • transcriptome profiling of khat catha edulis and Ephedra sinica reveals gene candidates potentially involved in amphetamine type alkaloid biosynthesis
    PLOS ONE, 2015
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Jillian M Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.

Efraim Lewinsohn - One of the best experts on this subject based on the ideXlab platform.

  • RESEARCH ARTICLE Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis
    2016
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Jillian M Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the bio-chemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-min-ing frameworks for Ephedra sinica and khat plants. Sequence libraries representing a com-bined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidate

  • Characterization of aromatic aminotransferases from Ephedra sinica Stapf
    Amino Acids, 2016
    Co-Authors: Korey Kilpatrick, Efraim Lewinsohn, Jillian M Hagel, Peter J Facchini, Agnieszka Pajak, Mark W. Sumarah, Frédéric Marsolais
    Abstract:

    Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l -glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S -phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

  • transcriptome profiling of khat catha edulis and Ephedra sinica reveals gene candidates potentially involved in amphetamine type alkaloid biosynthesis
    PLOS ONE, 2015
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Jillian M Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.

  • Ephedra sinica unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.
    2015
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Each unigene is assigned an identifier, which corresponds to a database ID in the ESI-Velvet library. Percent amino acid identity between unigenes and queries is provided. Abbreviations: AAO4, aromatic aldehyde oxidase 4; Ab, Atropa belladonna; Am, Antirrhinum majus; AHAS, acetohydroxyacid synthase; ArAT, aromatic amino acid transaminase; At, Arabidopsis thaliana; BALDH, benzaldehyde dehydrogenase; BDH, benzaldehyde dehydrogenase; BL; benzoate CoA ligase; BZO, benzoyloxyglucosinolate; Ca, Caffea arabica; Ce, Catha edulis; CHD, cinnamoyl-CoA hydratase-dehydrogenase; 4CL, 4-coumaroyl-CoA ligase; Cm, Cucumis melo; CS, caffeine synthase; COR, codeinone reductase; Ds, Datura stramonium; Ec, Eschscholzia californica; Es, Ephedra sinica; KAT, 3-ketoacyl-CoA thiolasae; NMT, N-methyltransferase; PEANMT, phosphoethanolamine N-methyltransferase; PMT, putrescine N-methyltransferase; Ps, Papaver somniferum; PAL, L-phenylalanine ammonia lyase; PDC, pyruvate decarboxylase; Ph, Petunia x hybrida; PPA-AT, prephenate aminotransferase; PRMT, protein arginine N-methyltransferase; Pt, Pinus taeda; RED, reductase; SanR, sanguinarine reductase; Sl, Solanum lycopersicon; SUVH, histone lysine N-methyltransferase, H3L9-specific; TA, transaminase; ThDPC, thiamin diphosphate-dependent carboligase; TNMT, (S)-tetrahydroprotoberberine N-methyltransferase; TR, tropinone reducase.Ephedra sinica unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.

  • Summary of the construction and assembly for three Illumina NGS libraries.
    2015
    Co-Authors: Ryan A. Groves, Efraim Lewinsohn, Jillian M Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Christoph W. Sensen, Peter J Facchini
    Abstract:

    Abbreviations: CED, Catha edulis; ESI, Ephedra sinica; SRA, short-read archive; CDS, coding sequence.Summary of the construction and assembly for three Illumina NGS libraries.

Peiling Wang - One of the best experts on this subject based on the ideXlab platform.

  • ephedrine type alkaloid content of nutritional supplements containing Ephedra sinica ma huang as determined by high performance liquid chromatography
    Journal of Pharmaceutical Sciences, 1998
    Co-Authors: Bill J. Gurley, Peiling Wang, Stephanie F. Gardner
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma-huang), a botanical source of ephedrine-type alkaloids, have been linked to numerous episodes of ephedrine (EPH) toxicity. With passage of the 1994 Dietary Supplement Health and Education Act, nutritional supplements are no longer subject to the same FDA preapproval requirements as food additives, prescription, or nonpre-scription medications. As a consequence, EPH content is not a label requirement for Ephedra-containing supplements. Less stringent labeling requirements, therefore, may contribute to toxicity associated with these products. A validated HPLC method for the determination of ephedrine-type alkaloids, commonly found in Ephedra supplements, is presented. Nine commercially available supplements exhibited considerable variability in alkaloid content (EPH range: 1.08-13.54 mg). Only three products listed EPH content on the label while one exhibited lot to lot variations in EPH of 137%.

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    SummaryNutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed at characterizing the pharmacokinetics of ephedrine after the ingestion of three commercially available ma huang products compared with a 25-mg ephedrine capsule. Pharmacokinetic parameters for botanical ephedrine were similar to those for synthetic ephedrine hydrochloride. Gender-based comparisons of Vss/F and CL/F revealed higher values for women than for men (Vss/F, 3.49 +/- 1.04 vs 2.98 +/- 0.73 l/kg; CL/F, 0.48 +/- 0.11 vs 0.37 +/- 0.11 l/hour x kg). The current study suggests that the increased incidence of ma huang toxicity does not stem from differences in the absorption of botanical ephedrine compared with synthetic ephedrine; rather, it results from accidental overdose often prompted by exaggerated off-label claims and a belief that "natural" medicinal agents are inherently safe.

Stephanie F. Gardner - One of the best experts on this subject based on the ideXlab platform.

  • ephedrine type alkaloid content of nutritional supplements containing Ephedra sinica ma huang as determined by high performance liquid chromatography
    Journal of Pharmaceutical Sciences, 1998
    Co-Authors: Bill J. Gurley, Peiling Wang, Stephanie F. Gardner
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma-huang), a botanical source of ephedrine-type alkaloids, have been linked to numerous episodes of ephedrine (EPH) toxicity. With passage of the 1994 Dietary Supplement Health and Education Act, nutritional supplements are no longer subject to the same FDA preapproval requirements as food additives, prescription, or nonpre-scription medications. As a consequence, EPH content is not a label requirement for Ephedra-containing supplements. Less stringent labeling requirements, therefore, may contribute to toxicity associated with these products. A validated HPLC method for the determination of ephedrine-type alkaloids, commonly found in Ephedra supplements, is presented. Nine commercially available supplements exhibited considerable variability in alkaloid content (EPH range: 1.08-13.54 mg). Only three products listed EPH content on the label while one exhibited lot to lot variations in EPH of 137%.

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    SummaryNutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed at characterizing the pharmacokinetics of ephedrine after the ingestion of three commercially available ma huang products compared with a 25-mg ephedrine capsule. Pharmacokinetic parameters for botanical ephedrine were similar to those for synthetic ephedrine hydrochloride. Gender-based comparisons of Vss/F and CL/F revealed higher values for women than for men (Vss/F, 3.49 +/- 1.04 vs 2.98 +/- 0.73 l/kg; CL/F, 0.48 +/- 0.11 vs 0.37 +/- 0.11 l/hour x kg). The current study suggests that the increased incidence of ma huang toxicity does not stem from differences in the absorption of botanical ephedrine compared with synthetic ephedrine; rather, it results from accidental overdose often prompted by exaggerated off-label claims and a belief that "natural" medicinal agents are inherently safe.

Bill J. Gurley - One of the best experts on this subject based on the ideXlab platform.

  • ephedrine type alkaloid content of nutritional supplements containing Ephedra sinica ma huang as determined by high performance liquid chromatography
    Journal of Pharmaceutical Sciences, 1998
    Co-Authors: Bill J. Gurley, Peiling Wang, Stephanie F. Gardner
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma-huang), a botanical source of ephedrine-type alkaloids, have been linked to numerous episodes of ephedrine (EPH) toxicity. With passage of the 1994 Dietary Supplement Health and Education Act, nutritional supplements are no longer subject to the same FDA preapproval requirements as food additives, prescription, or nonpre-scription medications. As a consequence, EPH content is not a label requirement for Ephedra-containing supplements. Less stringent labeling requirements, therefore, may contribute to toxicity associated with these products. A validated HPLC method for the determination of ephedrine-type alkaloids, commonly found in Ephedra supplements, is presented. Nine commercially available supplements exhibited considerable variability in alkaloid content (EPH range: 1.08-13.54 mg). Only three products listed EPH content on the label while one exhibited lot to lot variations in EPH of 137%.

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    SummaryNutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed

  • ephedrine pharmacokinetics after the ingestion of nutritional supplements containing Ephedra sinica ma huang
    Therapeutic Drug Monitoring, 1998
    Co-Authors: Bill J. Gurley, Stephanie F. Gardner, Laura M White, Peiling Wang
    Abstract:

    Nutritional supplements containing Ephedra sinica (ma huang), a botanical source of ephedrine alkaloids, have been linked to several episodes of ephedrine toxicity and at least 17 deaths, yet these products remain unregulated. Ten subjects were enrolled in a randomized, crossover study aimed at characterizing the pharmacokinetics of ephedrine after the ingestion of three commercially available ma huang products compared with a 25-mg ephedrine capsule. Pharmacokinetic parameters for botanical ephedrine were similar to those for synthetic ephedrine hydrochloride. Gender-based comparisons of Vss/F and CL/F revealed higher values for women than for men (Vss/F, 3.49 +/- 1.04 vs 2.98 +/- 0.73 l/kg; CL/F, 0.48 +/- 0.11 vs 0.37 +/- 0.11 l/hour x kg). The current study suggests that the increased incidence of ma huang toxicity does not stem from differences in the absorption of botanical ephedrine compared with synthetic ephedrine; rather, it results from accidental overdose often prompted by exaggerated off-label claims and a belief that "natural" medicinal agents are inherently safe.