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Peter J. Facchini - One of the best experts on this subject based on the ideXlab platform.
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Evidence for the monophyletic evolution of Benzylisoquinoline Alkaloid biosynthesis in angiosperms.
Phytochemistry, 2020Co-Authors: David K Liscombe, Benjamin P Macleod, Natalia Loukanina, Owi I Nandi, Peter J. FacchiniAbstract:Benzylisoquinoline Alkaloids (BIAs) consist of more than 2500 diverse structures largely restricted to the order Ranunculales and the eumagnoliids. However, BIAs also occur in the Rutaceae, Lauraceae, Cornaceae and Nelumbonaceae, and sporadically throughout the order Piperales. Several of these Alkaloids function in the defense of plants against herbivores and pathogens--thus the capacity for BIA biosynthesis is expected to play an important role in the reproductive fitness of certain plants. Biochemical and molecular phylogenetic approaches were used to investigate the evolution of BIA biosynthesis in basal angiosperms. The occurrence of (S)-norcoclaurine synthase (NCS; EC 4.2.1.78) activity in 90 diverse plant species was compared to the distribution of BIAs superimposed onto a molecular phylogeny. These results support the monophyletic origin of BIA biosynthesis prior to the emergence of the eudicots. Phylogenetic analysis of NCS, berberine bridge enzyme and several O-methyltransferases suggest a latent molecular fingerprint for BIA biosynthesis in angiosperms not known to accumulate such Alkaloids. The limited occurrence of BIAs outside the Ranunculales and eumagnoliids suggests the requirement for a highly specialized, yet evolutionarily unstable cellular platform to accommodate or reactivate the pathway in divergent taxa. The molecular cloning and functional characterization of NCS from opium poppy (Papaver somniferum L.) is also reported. Pathogenesis--related (PR)10 and Bet v 1 major allergen proteins share homology with NCS, but recombinant polypeptides were devoid of NCS activity.
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isolation and characterization of two o methyltransferases involved in Benzylisoquinoline Alkaloid biosynthesis in sacred lotus nelumbo nucifera
Journal of Biological Chemistry, 2020Co-Authors: Ivette M Menendezperdomo, Peter J. FacchiniAbstract:: Benzylisoquinoline Alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value owing to antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-Benzylisoquinoline, aporphine, and bisBenzylisoquinoline structural subclasses and displaying a predominant R enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing > 40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-Benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R and S substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-Benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 μM and 13 μM for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of Alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.
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a single residue determines substrate preference in Benzylisoquinoline Alkaloid n methyltransferases
Phytochemistry, 2020Co-Authors: Jeremy S. Morris, Lisa Yu, Peter J. FacchiniAbstract:Abstract N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including Alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of Benzylisoquinoline Alkaloids (BIAs) involves conversion of the secondary amine (S)-coclaurine into the tertiary amine (S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT). Subsequent enzymatic steps yield the core intermediate (S)-reticuline, from which various branch pathways for the biosynthesis of major BIAs such as morphine, noscapine and sanguinarine diverge. An additional N-methylation yielding quaternary BIAs is catalyzed by reticuline N-methyltransferase (RNMT), such as in the branch pathway leading to the taxonomically widespread and ecologically significant Alkaloid magnoflorine. Despite their functional differences, analysis of primary sequence information has been unable to accurately distinguish between CNMT-like and RNMT-like enzymes, necessitating laborious in vitro screening. Furthermore, despite a recent emphasis on structural characterization of BIA NMTs, the features and mechanisms underlying the CNMT-RNMT functional dichotomy were unknown. We report the identification of structural variants tightly correlated with function in known BIA NMTs and show through reciprocal mutagenesis that a single residue acts as a switch between CNMT- and RNMT-like functions. We use yeast in vivo screening to show that this discovery allows for accurate prediction of activity strictly from primary sequence information and, on this basis, improve the annotation of previously reported putative BIA NMTs. Our results highlight the unusually short mutational distance separating ancestral CNMT-like enzymes from more evolutionarily advanced RNMT-like enzymes, and thus help explain the widespread yet sporadic occurrence of quaternary BIAs in plants. While this is the first report of structural variants controlling mono-versus di-methylation activity among plant NMT enzymes, comparison with bacterial MT enzymes also suggests possible convergent evolution.
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Benzylisoquinoline Alkaloid biosynthesis in opium poppy: an update
Phytochemistry Reviews, 2019Co-Authors: Aparna Singh, Ivette M. Menéndez-perdomo, Peter J. FacchiniAbstract:For nearly eight millennia, opium poppy ( Papaver somniferum ) has been bred and cultivated for therapeutic purposes. The medicinal properties of the plant are conferred by specialized metabolites known as Benzylisoquinoline Alkaloids (BIAs), comprising the narcotic analgesics morphine and codeine, the antimicrobial agent sanguinarine, and the potential anticancer drug noscapine. In addition, naturally occurring thebaine is used for the semi-synthesis of widely prescribed pain-relievers (e.g., oxycodone and hydrocodone), valuable drugs used in the treatment of opioid addiction (i.e., naltrexone), or antidotes for opioid overdose (i.e., naloxone). The complex stereochemistry of many opiates hinders their chemical synthesis and opium poppy remains the sole commercial source of these important pharmaceuticals. For decades, opium poppy has served as a model plant for research aimed at a comprehensive understanding of BIA metabolism. Recent progress in functional genomics has enabled the discovery of a nearly complete collection of BIA biosynthetic genes, many of which are clustered in the opium poppy genome. Advances in synthetic biology have facilitated the successful reconstitution of several BIA biosynthetic pathways in heterologous hosts such as Saccharomyces cerevisiae and Escherichia coli , although the initially low production levels suggest that commercial scale-up will present additional challenges. This review provides an update of key molecular and biochemical aspects of BIA metabolism in opium poppy, including recent biosynthetic gene discoveries, genomic organization, novel BIA transporters, metabolic regulation, and major efforts in the engineering of pathways in plants and microbes.
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purine permease type Benzylisoquinoline Alkaloid transporters in opium poppy
Plant Physiology, 2019Co-Authors: Mehran Dastmalchi, Jillian M Hagel, Limei Chang, Rongji Chen, Lisa Yu, Xue Chen, Peter J. FacchiniAbstract:Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, Benzylisoquinoline Alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in Alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate Alkaloid biosynthesis in opium poppy.
Fumihiko Sato - One of the best experts on this subject based on the ideXlab platform.
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modulation of Benzylisoquinoline Alkaloid biosynthesis by heterologous expression of cjwrky1 in eschscholzia californica cells
PLOS ONE, 2017Co-Authors: Yasuyuki Yamada, Tomoe Shimada, Yukiya Motomura, Fumihiko SatoAbstract:: Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole Alkaloids (MIAs), nicotine Alkaloids, and Benzylisoquinoline Alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in Alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.
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unraveling additional o methylation steps in Benzylisoquinoline Alkaloid biosynthesis in california poppy eschscholzia californica
Plant and Cell Physiology, 2017Co-Authors: Ratmoyo Purwanto, Yasuyuki Yamada, Kentaro Hori, Fumihiko SatoAbstract:: California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active Benzylisoquinoline Alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple Benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.
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tyrosine phosphorylation and protein degradation control the transcriptional activity of wrky involved in Benzylisoquinoline Alkaloid biosynthesis
Scientific Reports, 2016Co-Authors: Yasuyuki Yamada, Fumihiko SatoAbstract:Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis
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Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis
Scientific Reports, 2016Co-Authors: Yasuyuki Yamada, Fumihiko SatoAbstract:Benzylisoquinoline Alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated Alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs.
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pathway engineering of Benzylisoquinoline Alkaloid biosynthesis in transgenic california poppy cells with ectopic expression of tetrahydroberberine oxidase from coptis japonica
Plant Biotechnology, 2012Co-Authors: Yasutaka Matsushima, Hiromichi Minami, Kentaro Hori, Fumihiko SatoAbstract:S)-Tetrahydroberberine oxidase is the enzyme in the last step of berberine biosynthesis. While a previous report described the isolation of cDNA of tetrahydroberberine oxidase (THBO) from cultured Coptis japonica cells, we later found that purified THBO was heavily contaminated by triosephosphate isomerase. Here, we report the re-isolation of THBO cDNA from cultured C. japonica cells and its functional characterization in transgenic California poppy cells. A cDNA clone for (S)-tetrahydroberberine oxidase was isolated from an EST library prepared from high berberineproducing cultured C. japonica cells based on the partial amino acid sequence of the purified enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts of 1728 base pairs revealed an open reading frame that encoded a 540-amino acid polypeptide with putative 28-amino acid signal peptides and a mature polypeptide with a molecular mass of 57,748. A protein blast search also shows that CjTHBO belongs to the FAD-containing berberine bridge enzyme oxidoreductase family. Since all attempts to produce active recombinant CjTHBO in Escherichia coli and Saccharomyces cerevisiae cells failed, we tried to express CjTHBO in California poppy (Eschscholzia californica) cells. When transgenic California poppy cells that ectopically expressed CjTHBO under the control of Cauliflower mosaic virus 35S promoter were established, the transgenic cells showed small but evident new Alkaloid peaks, which were scarcely detected in control cells that did not express CjTHBO. LC-MS analyses showed that these peaks were coptisine and dehydrocheilanthifoline, which were expected to be generated by the reaction of CjTHBO from pathway intermediates, i.e., cheilanthifoline and stylopine. The usefulness of CjTHBO for metabolic engineering in transgenic California poppy cells is discussed.
Yasuyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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modulation of Benzylisoquinoline Alkaloid biosynthesis by heterologous expression of cjwrky1 in eschscholzia californica cells
PLOS ONE, 2017Co-Authors: Yasuyuki Yamada, Tomoe Shimada, Yukiya Motomura, Fumihiko SatoAbstract:: Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole Alkaloids (MIAs), nicotine Alkaloids, and Benzylisoquinoline Alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in Alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.
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unraveling additional o methylation steps in Benzylisoquinoline Alkaloid biosynthesis in california poppy eschscholzia californica
Plant and Cell Physiology, 2017Co-Authors: Ratmoyo Purwanto, Yasuyuki Yamada, Kentaro Hori, Fumihiko SatoAbstract:: California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active Benzylisoquinoline Alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple Benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.
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tyrosine phosphorylation and protein degradation control the transcriptional activity of wrky involved in Benzylisoquinoline Alkaloid biosynthesis
Scientific Reports, 2016Co-Authors: Yasuyuki Yamada, Fumihiko SatoAbstract:Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis
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Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis
Scientific Reports, 2016Co-Authors: Yasuyuki Yamada, Fumihiko SatoAbstract:Benzylisoquinoline Alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated Alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs.
Christina D. Smolke - One of the best experts on this subject based on the ideXlab platform.
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de novo production of the key branch point Benzylisoquinoline Alkaloid reticuline in yeast
Metabolic Engineering, 2015Co-Authors: Isis J. Trenchard, Kate Thodey, Michael S Siddiqui, Christina D. SmolkeAbstract:Abstract Microbial biosynthesis for plant-based natural products, such as the Benzylisoquinoline Alkaloids (BIAs), has the potential to address limitations in plant-based supply of established drugs and make new molecules available for drug discovery. While yeast strains have been engineered to produce a variety of downstream BIAs including the opioids, these strains have relied on feeding an early BIA substrate. We describe the de novo synthesis of the major BIA branch point intermediate reticuline via norcoclaurine in Saccharomyces cerevisiae. Modifications were introduced into yeast central metabolism to increase supply of the BIA precursor tyrosine, allowing us to achieve a 60-fold increase in production of the early Benzylisoquinoline scaffold from fed dopamine with no supply of exogenous tyrosine. Yeast strains further engineered to express a mammalian tyrosine hydroxylase, four mammalian tetrahydrobiopterin biosynthesis and recycling enzymes, and a bacterial DOPA decarboxylase produced norcoclaurine de novo. We further increased production of early Benzylisoquinoline scaffolds by 160-fold through introducing mutant tyrosine hydroxylase enzymes, an optimized plant norcoclaurine synthase variant, and optimizing culture conditions. Finally, we incorporated five additional plant enzymes – three methyltransferases, a cytochrome P450, and its reductase partner – to achieve de novo production of the key branch point molecule reticuline with a titer of 19.2 μg/L. These strains and reconstructed pathways will serve as a platform for the biosynthesis of diverse natural and novel BIAs.
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Engineering strategies for the fermentative production of plant Alkaloids in yeast
Metabolic Engineering, 2015Co-Authors: Isis J. Trenchard, Christina D. SmolkeAbstract:Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the Benzylisoquinoline Alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine Alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676. μg/L stylopine, 548. μg/L cis- N-methylstylopine, 252. μg/L protopine, and 80. μg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization.
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A microbial biomanufacturing platform for natural and semisynthetic opioids
Nature Chemical Biology, 2014Co-Authors: Kate Thodey, Stephanie Galanie, Christina D. SmolkeAbstract:Metabolic engineering of yeast to incorporate plant and bacterial enzymes that construct and decorate morphine, along with spatial engineering to enable a spontaneous chemical reaction, provides strains capable of producing up to 130 mg/l of opioids. Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable Benzylisoquinoline Alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.
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A microbial biomanufacturing platform for natural and semisynthetic opioids
Nature Chemical Biology, 2014Co-Authors: Kate Thodey, Stephanie Galanie, Christina D. SmolkeAbstract:Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable Benzylisoquinoline Alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.
Jillian M Hagel - One of the best experts on this subject based on the ideXlab platform.
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purine permease type Benzylisoquinoline Alkaloid transporters in opium poppy
Plant Physiology, 2019Co-Authors: Mehran Dastmalchi, Jillian M Hagel, Limei Chang, Rongji Chen, Lisa Yu, Xue Chen, Peter J. FacchiniAbstract:Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, Benzylisoquinoline Alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in Alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate Alkaloid biosynthesis in opium poppy.
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plug and play Benzylisoquinoline Alkaloid biosynthetic gene discovery in engineered yeast
Methods in Enzymology, 2016Co-Authors: Jeremy S. Morris, Mehran Dastmalchi, Jillian M Hagel, Limei Chang, Xue Chen, J Li, Peter J. FacchiniAbstract:Benzylisoquinoline Alkaloid (BIA) metabolism has been the focus of a considerable research effort over the past half-century, primarily because of the pharmaceutical importance of several compounds produced by opium poppy (Papaver somniferum). Advancements in genomics technologies have substantially accelerated the rate of gene discovery over the past decade, such that most biosynthetic enzymes involved in the formation of the major Alkaloids of opium poppy have now been isolated and partially characterized. Not unexpectedly, the availability of all perceived biosynthetic genes has facilitated the reconstitution of several BIA pathways in microbial hosts, including yeast (Saccharomyces cerevisiae). Product yields are currently insufficient to consider the commercial production of high-value BIAs, such as morphine. However, the rudimentary success demonstrated by the uncomplicated and routine assembly of a multitude of characterized BIA biosynthetic genes provides a valuable gene discovery tool for the rapid functional identification of the plethora of gene candidates available through increasingly accessible genomic, transcriptomic, and proteomic databases. BIA biosynthetic gene discovery represents a substantial research opportunity largely owing to the wealth of existing enzyme data mostly obtained from a single plant species. Functionally novel enzymes and variants with potential metabolic engineering applications can be considered the primary targets. Selection of candidates from sequence repositories is facilitated by the monophyletic relationship among biosynthetic genes belonging to a wide range of enzyme families, such as the numerous cytochromes P450 and AdoMet-dependent O- and N-methyltransferases that operate in BIA metabolism. We describe methods for the rapid functional screening of uncharacterized gene candidates encoding potential BIA biosynthetic enzymes using yeast strains engineered to perform selected metabolic conversions. As an initial screening tool, the approach is superior to the in vitro characterization of recombinant enzyme candidates, and provides a standardized functional genomics opportunity for otherwise recalcitrant exotic plant species.
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isolation and characterization of o methyltransferases involved in the biosynthesis of glaucine in glaucium flavum
Plant Physiology, 2015Co-Authors: Limei Chang, Jillian M Hagel, Peter J. FacchiniAbstract:Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in Benzylisoquinoline Alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature Alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4′-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable Alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine Alkaloid glaucine, which displays C8-C6′ coupling and four O-methyl groups at C6, C7, C3′, and C4′ as numbered on the 1-Benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-Benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3′ and 4′ hydroxyl groups of certain Alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4′OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4′-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3′-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3′- and 7′-O-methylations of several 1-Benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of Alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.
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transcriptome analysis of 20 taxonomically related Benzylisoquinoline Alkaloid producing plants
BMC Plant Biology, 2015Co-Authors: Jillian M Hagel, Jeremy S. Morris, Limei Chang, Xue Chen, Isabel Desgagnepenix, Crystal D Bross, Scott C Farrow, Ye Zhang, Christoph Wilhelm SensenAbstract:Background Benzylisoquinoline Alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts.
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metabolome analysis of 20 taxonomically related Benzylisoquinoline Alkaloid producing plants
BMC Plant Biology, 2015Co-Authors: Jillian M Hagel, Rupasri Mandal, Donald R Dinsmore, Christoph H Borchers, David S Wishart, Peter J. FacchiniAbstract:Background Recent progress toward the elucidation of Benzylisoquinoline Alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties.
