WO2007011887A2 - Production d'alcaloides de morphinane et de derives de ceux-ci dans des plantes - Google Patents

Production d'alcaloides de morphinane et de derives de ceux-ci dans des plantes Download PDF

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WO2007011887A2
WO2007011887A2 PCT/US2006/027731 US2006027731W WO2007011887A2 WO 2007011887 A2 WO2007011887 A2 WO 2007011887A2 US 2006027731 W US2006027731 W US 2006027731W WO 2007011887 A2 WO2007011887 A2 WO 2007011887A2
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plant
seq
nucleic acid
alkaloid
cell
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WO2007011887A3 (fr
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Nestor Apuya
Steven Craig Bobzin
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Ceres, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/22—Bridged ring systems
    • C07D221/28—Morphinans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • This document relates to methods and materials involved in producing alkaloid compounds, e.g., morphinan alkaloids and derivatives thereof, in plants.
  • this document provides plants and plant cells expressing polypeptides involved in the modification of one or more morphinan alkaloid compounds in plants, e.g., plants of the Papaveraceae family. Methods of making such plants and plant cells, and methods of extracting alkaloid compounds from such plants and plant cells are also provided.
  • morphinan alkaloids in opium poppy are morphine, codeine, and thebaine.
  • Various morphinan alkaloids and derivatives e.g., morphine-6-glucuronide, hydrocodone, and hydromorphone, are produced semisynthetically from naturally occurring alkaloids.
  • Morphinan alkaloids are known for their effects on the mammalian central nervous system, including pain sensation and euphoria. Given the variety of uses and effects of morphinan alkaloids, it would be useful to have novel means of producing such compounds that are more cost-effective and/or efficient.
  • the present invention relates to transgenic plants or plant cells containing recombinant nucleic acids encoding exogenous polypeptides useful in alkaloid compound production.
  • the present invention also relates to methods for producing one or more morphinan alkaloids and/or derivatives in a plant that contains such a recombinant nucleic acid construct.
  • the transgenic plants and other compositions described herein can be used to synthesize morphinan alkaloids and derivatives such as morphine-6-glucuronide, codeinone, morphinone, hydrocodone, and hydromorphone in an efficient and cost-effective manner compared to known semisynthetic production systems.
  • transgenic plants and other compositions described herein can be useful to eliminate the chemical waste associated with known morphinan alkaloid production systems.
  • Production of morphinan alkaloids and derivatives such as morphine-6-glucuronide, codeinone, morphinone, hydrocodone, and hydromorphone inplanta can also benefit farmers by increasing the value of alkaloid producing species as a production crop.
  • new chemical entities may be produced by these transgenic plant cells.
  • a transgenic plant comprises a recombinant nucleic acid construct comprising a nucleic acid encoding a UGT2B, morphine dehydrogenase, or morphinone reductase polypeptide.
  • the nucleic acid can be operably linked to a regulatory region that modulates transcription in the plant.
  • the plant can comprise first and second recombinant nucleic acid constructs, where the first construct comprises a nucleic acid encoding a morphine dehydrogenase polypeptide and the second construct comprises a nucleic acid encoding a morphinone reductase polypeptide.
  • the first construct can comprise a nucleic acid encoding a polypeptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-36, SEQ ID NOs:63-69, and the consensus sequence set forth in Figure 2
  • the second construct can comprise a nucleic acid encoding a polypeptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:39-56, SEQ ID NOs:70-79, and the consensus sequence set forth in Figure 3.
  • the first construct can comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-36, SEQ ID NOs:63-69, and the consensus sequence set forth in Figure 2
  • the second construct can comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:39-56, SEQ ID NOs:70-79, and the consensus sequence set forth in Figure 3.
  • a transgenic plant comprises a recombinant nucleic acid construct comprising a nucleic acid encoding a polypeptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:3-15, SEQ ID NOs: 18-36, SEQ ID NOs:39-80, and the consensus sequences set forth in Figures 1-3.
  • the nucleic acid can be operably linked to a regulatory region that modulates transcription in the plant.
  • the sequence identity can be at least 95%.
  • the nucleic acid can encode a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:3-15, SEQ ID NOs: 18-36, SEQ ID NOs:39-80, and the consensus sequences set forth in Figures 1-3.
  • the plant can be a member of the Papaveraceae, Berberidaceae, Leguminosae, Boraginaceae, Apocynaceae, Asclepiadaceae, Liliaceae, Gnetaceae, Erythroxylaceae, Convolvulaceae, Ranunculaeceae, Rubiaceae, Solanaceae, or Rutaceae family.
  • the plant can be a Papaver somniferum plant.
  • the plant can produce a morphinan alkaloid glycoside.
  • the morphinan alkaloid glycoside can be morphine-6-glucuronide.
  • the plant can produce codeinone or morphinone.
  • the plant can produce hydrocodone or hydromorphone.
  • a plant cell from a plant capable of producing an alkaloid is provided.
  • the plant cell can comprise a recombinant nucleic acid construct comprising a nucleic acid encoding a UGT2B, morphine dehydrogenase, or morphinone reductase polypeptide.
  • the nucleic acid can be operably linked to a regulatory region that modulates transcription in the plant.
  • the plant cell can produce a morphinan alkaloid glycoside.
  • the morphinan alkaloid glycoside can be morphine-6-glucuronide.
  • the plant cell can produce codeinone or morphinone.
  • the plant cell can produce hydrocodone or hydromorphone.
  • the plant cell can be from a member of the Papaveraceae, Berberidaceae, Leguminosae, Boraginaceae, Apocynaceae, Asclepiadaceae, Liliaceae, Gnetaceae, Erythroxylaceae, Convolvulaceae, Ranunculaeceae, Rubiaceae, Solanaceae, or Rutaceae family.
  • the plant cell can be a Papaver somniferum cell.
  • a regulatory region can be a promoter.
  • a promoter can be a tissue- preferential promoter, where the tissue is stem, poppy capsule, vascular, seed pod, or parenchymal tissue.
  • a promoter can be a cell type-preferential promoter, where the cell is a laticifer, sieve element, or companion cell.
  • a method of making a transgenic plant comprises transforming a plant capable of producing an alkaloid with a recombinant nucleic acid construct comprising a nucleic acid encoding a UGT2B, morphine dehydrogenase, or morphinone reductase polypeptide.
  • the nucleic acid can be operably linked to a regulatory region that modulates transcription in the plant.
  • a method of making a transgenic plant comprises transforming a plant capable of producing an alkaloid with a recombinant nucleic acid construct comprising a nucleic acid encoding a polypeptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:3-15, SEQ ID NOs:18-36, SEQ ID NOs:39-80, and the consensus sequences set forth in Figures 1-3.
  • the nucleic acid can be operably linked to a regulatory region that modulates transcription in the plant.
  • a method of producing an alkaloid compound comprises: a) growing a plant capable of producing an alkaloid, where the plant is transformed with a recombinant nucleic acid construct comprising a nucleic acid encoding a UGT2B, morphine dehydrogenase, or morphinone reductase polypeptide, and where the nucleic acid is operably linked to a regulatory region that modulates transcription in the plant; and b) extracting the alkaloid compound from the plant.
  • a method of producing an alkaloid compound comprises: a) growing a plant capable of producing an alkaloid, where the plant is transformed with a recombinant nucleic acid construct comprising a nucleic acid encoding a polypeptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:3-15, SEQ ID NOs: 18-36, SEQ ID NOs:39-80, and the consensus sequences set forth in Figures 1-3, and where the nucleic acid is operably linked to a regulatory region that modulates transcription in the plant; and b) extracting the alkaloid compound from the plant.
  • the growing step of any method described above, of producing an alkaloid compound can comprise growing a plurality of the plants. Any method described above, of producing an alkaloid compound, can further comprise producing straw from the plants after the growing step. The alkaloid compound can be extracted from the straw. The extracting step of any method described above, of producing an alkaloid compound, can comprise extracting the alkaloid compound to a purity of 80% or greater by weight. The method can further comprise the step of formulating a composition suitable for administration to human beings or animals, where the composition comprises the alkaloid compound.
  • the alkaloid compound can be a morphinan alkaloid derivative.
  • the morphinan alkaloid derivative can be a morphinan alkaloid glycoside.
  • the morphinan alkaloid glycoside can be morphine-6-glucuronide.
  • the alkaloid compound can be a morphinan alkaloid.
  • the morphinan alkaloid can be codeinone, morphinone, hydrocodone, or hydromorphone.
  • Figure 1 is an alignment of the amino acid sequence of UGT2B7 (SEQ ID NO: 3) with homologous and/or orthologous amino acid sequences gi
  • the consensus sequence determined by the alignment is set forth.
  • Figure 2 is an alignment of the amino acid sequence of morA (SEQ ID NO: 3) with homologous and/or orthologous amino acid sequences gi
  • Figure 3 is an alignment of the amino acid sequence of morB (SEQ ID NO:39) with homologous and/or orthologous amino acid sequences gi
  • the consensus sequence determined by the alignment is set
  • the present invention is based in part on transgenic plant cells that express a morphinan alkaloid modifying enzyme.
  • en2ymes include uridine diphosphate-glucuronosyltransferases, morphine dehydrogenases and morphinone reductases.
  • Plant cells transformed with nucleic acids encoding such polypeptides are contemplated to synthesize useful morphinan alkaloids or derivatives thereof, e.g., morphine-6-glucuronide, codeinone, morphinone, hydrocodone and hydromorphone, in an efficient and cost-effective manner. These alkaloids are not normally end products in corresponding non-transgenic plants.
  • Transgenic plant cells expressing morphinan alkaloid modifying enzymes are also contemplated to produce novel morphinan alkaloids.
  • polypeptide refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation.
  • the subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including D/L optical isomers. Full- length proteins, analogs, mutants, and fragments thereof are encompassed by this definition.
  • Polypeptides described herein include morphinan alkaloid modifying enzymes.
  • a morphinan alkaloid modifying enzyme can be a uridine diphosphate-glucuronosyltransferase (UGT2B) polypeptide.
  • UGT2B polypeptides form a family of enzymes that catalyze the glucuronidation of endogenous and xenobiotic chemicals. Mammalian UGT2B polypeptides play a key role in several important metabolic functions. Plant cells transformed with nucleic acids encoding UGT2B polypeptides, such as UGT2B7 polypeptides, may produce morphine-6-glucuronide.
  • SEQ ID NO: 3 sets forth the amino acid sequence of a human UGT2B polypeptide suitable for expression in plants, which is designated as UGT2B7 (Ritter et al. (1990) J Biol Chem, 265:7900-7906).
  • a morphinan alkaloid modifying enzyme can comprise the amino acid sequence set forth in SEQ ID NO: 3.
  • a morphinan alkaloid modifying enzyme can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.
  • a morphinan alkaloid modifying enzyme can comprise an amino acid sequence with at least 40% sequence identity, e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:3.
  • Figure 1 Amino acid sequences of homologs and/or orthologs of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 are provided in Figure 1.
  • Figure 1 also includes a consensus amino acid sequence determined by aligning homologous and/or orthologous amino acid sequences with the amino acid sequence set forth in SEQ ID NO:3.
  • the alignment in Figure 1 provides the amino acid sequences of UGT2B7 (SEQ ID NO:3), gi
  • SEQ ID NO:3 Other homologs and/or orthologs of SEQ ID NO:3 include gi
  • a morphinan alkaloid modifying enzyme can comprise a polypeptide having at least 80% sequence identity, e.g., 82%, 85%, 90%, 94%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to gi
  • a morphinan alkaloid modifying enzyme can be a morphine dehydrogenase polypeptide. Morphine dehydrogenase catalyzes the oxidation of morphine and codeine to morphinone and codeinone, respectively. Transgenic plant cells expressing a morphine dehydrogenase may produce the morphinan alkaloids morphinone and codeinone.
  • SEQ ID NO: 18 sets forth the amino acid sequence of a morphine dehydrogenase polypeptide designated morA (Willey et a (1993) Biochem J, 290 (Pt 2):539-544).
  • a morphinan alkaloid modifying enzyme can comprise the amino acid sequence set forth in SEQ ID NO: 18.
  • a morphinan alkaloid modifying enzyme can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 18.
  • a morphinan alkaloid modifying enzyme can comprise an amino acid sequence with at least 40% sequence identity, e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 18.
  • Figure 2 also includes a consensus amino acid sequence determined by aligning homologous and/or orthologous amino acid sequences with the amino acid sequence set forth in SEQ ID NO: 18.
  • the alignment in Figure 2 provides the amino acid sequences of morA (SEQ ID NO: 18), gi
  • SEQ ID NO: 18 Other homologs and/or orthologs of SEQ ID NO: 18 include gi
  • a morphinan alkaloid modifying enzyme can comprise a polypeptide having at least 80% sequence identity, e.g., 82%, 85%, 90%, 94%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to gi
  • a morphinan alkaloid modifying enzyme can be a morphinone reductase polypeptide. Morphinone reductase reduces the 7,8-unsaturated bond of morphinone and codeinone to yield hydromorphone and hydrocodone, respectively. Transgenic plant cells expressing a morphinone reductase polypeptide may produce the morphinan alkaloids hydromorphone and hydrocodone.
  • SEQ ID NO:39 sets forth the amino acid sequence of a morphinone reductase polypeptide designated morB (French and Bruce (1995) Biocheni J, 312 (Pt 3):671-678).
  • a morphinan alkaloid modifying enzyme can comprise the amino acid sequence set forth in SEQ ID NO:39.
  • a morphinan alkaloid modifying enzyme can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:39.
  • a morphinan alkaloid modifying enzyme can comprise an amino acid sequence with at least 40% sequence identity, e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:39.
  • Figure 3 also includes a consensus amino acid sequence determined by aligning homologous and/or orthologous amino acid sequences with the amino acid sequence set forth in SEQ ID NO:39.
  • the alignment in Figure 3 provides the amino acid sequences of morB (SEQ ID NO:39), gi
  • SEQ ID NO:39 Other homologs and/or orthologs of SEQ ID NO:39 include gi
  • a morphinan alkaloid modifying enzyme can comprise a polypeptide having at least 80% sequence identity, e.g., 82%, 85%, 90%, 94%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to gi
  • a morphinan alkaloid modifying enzyme encoded by a recombinant nucleic acid can be a native morphinan alkaloid modifying enzyme, i.e., one or more additional copies of the coding sequence for a morphinan alkaloid modifying enzyme that is naturally present in the cell.
  • a morphinan alkaloid modifying enzyme can be heterologous to the cell, e.g., a transgenic Papaveraceae plant can contain the coding sequence for a morphinan alkaloid modifying enzyme from a Pseudomonas putida bacterium.
  • a morphinan alkaloid modifying enzyme can include additional amino acids that are not involved in modulating morphinan alkaloid levels, and thus can be longer than would otherwise be the case.
  • a morphinan alkaloid modifying enzyme can include an amino acid sequence that functions as a reporter.
  • Such a morphinan alkaloid modifying enzyme can be a fusion protein in which a green fluorescent protein (GFP) polypeptide is fused to, e.g., SEQ ID NO: 3, or in which a yellow fluorescent protein (YFP) polypeptide is fused to, e.g., SEQ ID NO: 18.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • a morphinan alkaloid modifying enzyme includes a purification tag, a chloroplast transit peptide, a mitochondrial transit peptide, or a leader sequence added to the amino or carboxy terminus.
  • Morphinan alkaloid modifying enzyme candidates suitable for use in the invention can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs and/or orthologs of morphinan alkaloid modifying enzymes. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using known morphinan alkaloid modifying enzyme amino acid sequences. Those polypeptides in the database that have greater than 40% sequence identity can be identified as candidates for further evaluation for suitability as morphinan alkaloid modifying enzymes.
  • Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains suspected of being present in morphinan alkaloid modifying enzymes, e.g., conserved functional domains.
  • conserved regions in a template or subject polypeptide can facilitate production of variants of morphinan alkaloid modifying enzymes.
  • conserved regions can be identified by locating a region within the primary amino acid sequence of a template polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains at sanger.ac.uk/Pfam and genome.wustl.edu/Pfam. A description of the information included at the Pfam database is described in Sonnhammer et al, Nucl. Acids Res., 26:320-322
  • conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate. For example, sequences from Homo sapiens and Macaca fascicularis can be used to identify one or more conserved regions. Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. conserveed regions of related polypeptides can exhibit at least 45% amino acid sequence identity, e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity.
  • a conserved region of target and template polypeptides exhibit at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • Amino acid sequence identity can be deduced from amino acid or nucleotide sequences.
  • highly conserved domains have been identified within morphinan alkaloid modifying en2ymes. These conserved regions can be useful in identifying functionally similar (orthologous) morphinan alkaloid modifying enzymes.
  • suitable morphinan alkaloid modifying enzymes can be synthesized on the basis of consensus functional domains and/or conserved regions in polypeptides that are homologous morphinan alkaloid modifying enzymes.
  • Domains are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint” or "signature” that can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three- dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities.
  • a domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
  • FIG. 1-3 Representative homologs and/or orthologs of morphinan alkaloid modifying enzymes are shown in Figures 1-3.
  • Each Figure represents an alignment of the amino acid sequence of a morphinan alkaloid modifying enzyme with the amino acid sequences of corresponding homologs and/or orthologs.
  • Amino acid sequences of morphinan alkaloid modifying enzymes and their corresponding homologs and/or orthologs have been aligned to identify conserved amino acids and to determine consensus sequences that contain frequently occurring amino acid residues at particular positions in the aligned sequences, as shown in Figures 1-3.
  • a dash in an aligned sequence represents a gap, i.e., a lack of an amino acid at that position.
  • Identical amino acids or conserved amino acid substitutions among aligned sequences are identified by boxes.
  • Each consensus sequence is comprised of conserved regions. Each conserved region contains a sequence of contiguous amino acid residues. A dash in a consensus sequence indicates that the consensus sequence either lacks an amino acid at that position or includes an amino acid at that position. If an amino acid is present, the residue at that position corresponds to one found in any aligned sequence at that position.
  • Useful polypeptides can be constructed based on the consensus sequence in Figure 1, Figure 2, or Figure 3.
  • Such a polypeptide includes the conserved regions in the selected consensus sequence, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end.
  • Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes. When no amino acids are present at positions marked by dashes, the length of such a polypeptide is the sum of the amino acid residues in all conserved regions. When amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
  • a morphinan alkaloid modifying enzyme can also be a fragment of a naturally occurring morphinan alkaloid modifying enzyme.
  • the suitability of polypeptides for use as morphinan alkaloid modifying en2ymes can be evaluated by functional complementation studies.
  • a nucleic acid can comprise a coding sequence that encodes any of the morphinan alkaloid modifying enzymes described herein, such as those set forth in SEQ ID NOs:3-15, SEQ ID NOs:18-36, SEQ ID NOs:39-80, and the consensus sequences set forth in Figures 1-3.
  • a recombinant nucleic acid construct can include a nucleic acid comprising less than the full- length coding sequence of a morphinan alkaloid modifying enzyme.
  • a recombinant nucleic acid construct can include a nucleic acid comprising a coding sequence, a gene, or a fragment of a coding sequence or gene in an antisense orientation so that the antisense strand of RNA is transcribed.
  • nucleic acids can encode a polypeptide having a particular amino acid sequence.
  • the degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given morphinan alkaloid modifying enzyme can be modified such that optimal expression in a particular plant species is obtained, using appropriate codon bias tables for that species.
  • nucleic acid and “polynucleotide” are used interchangeably herein, and refer both to RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.
  • Polynucleotides can have any three-dimensional structure.
  • a nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand).
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA siRNA
  • micro-RNA micro-RNA
  • ribozymes cDNA
  • recombinant polynucleotides branched polynucleot
  • An isolated nucleic acid can be, for example, a naturally-occurring DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment).
  • An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid.
  • PCR polymerase chain reaction
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
  • percent sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
  • a subject sequence typically has a length that is more than 80 percent, e.g., more than 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120 percent, of the length of the query sequence.
  • a query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). Chenna et ah, Nucleic Acids Res., 31(13):3497-500 (2003).
  • ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • word size 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3.
  • weight matrix blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: GIy, Pro, Ser, Asn, Asp, GIn, GIu, Arg, and Lys; residue-specific gap penalties: on.
  • the output is a sequence alignment that reflects the relationship between sequences.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
  • ClustalW divides the number of identities in the best alignment by the number of residues compared (gap positions are excluded), and multiplies the result by 100.
  • the output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • exogenous nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism.
  • exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration.
  • a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.
  • a morphinan alkaloid modifying enzyme can be endogenous or exogenous to a particular plant or plant cell.
  • Exogenous polypeptides can include polypeptides that are native to a plant or plant cell, but that are expressed in a plant cell via a recombinant nucleic acid construct, e.g., a California poppy plant transformed with a recombinant nucleic acid construct encoding a California poppy enzyme.
  • expression refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes.
  • a regulatory region also can be exogenous or endogenous to a plant or plant cell.
  • An exogenous regulatory region is a regulatory region that is part of a recombinant nucleic acid construct, or is not in its natural environment.
  • a Nicotiana promoter present on a recombinant nucleic acid construct is an exogenous regulatory region when a Nicotiana plant cell is transformed with the construct.
  • Vectors containing nucleic acids such as those described herein also are provided.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An “expression vector” is a vector that includes a regulatory region.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., chlorosulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • hemagglutinin or FlagTM tag (Kodak, New Haven, CT) sequences
  • FlagTM tag Kodak, New Haven, CT sequences
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • operably linked refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically comprises at least a core (basal) promoter.
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a suitable enhancer is a cis-regulatory element (-212 to - 154) from the upstream region of the octopine synthase (ocs) gene. Fromm e ⁇ al, The Plant Cell, 1:977-984 (1989).
  • the choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue- preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
  • a promoter that is active predominantly in a reproductive tissue e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, 2ygote, endosperm, integument, or seed coat
  • a reproductive tissue e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, 2ygote, endosperm, integument, or seed coat
  • a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well.
  • Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano et al., Plant Cell, 1:855- 866 (1989); Bustos et al, Plant Cell, 1 :839-854 (1989); Green et al, EMBO J., 7:4035-4044 (1988); Meier et al, Plant Cell, 3:309-316 (1991); and Zhang et al, Plant Physiology, 110:1069-1079 (1996).
  • Nucleotide sequences of promoters are set forth in SEQ ID NOs:81-174 and SEQ ID NOs:185-198. It will be appreciated that a promoter may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species.
  • a promoter can be said to be "broadly expressing" when it promotes transcription in many, but not necessarily all, plant tissues.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds.
  • Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO: 156), YP0144 (SEQ ID NO.135), YP0190 (SEQ ID NO:139), pl3879 (SEQ ID NO:155), YP0050 (SEQ ID NO:115), p32449 (SEQ ID NO:157), 21876 (SEQ ID NO:81), YP0158 (SEQ ID NO: 137), YP0214 (SEQ ID NO: 141), YP0380 (SEQ ID NO:150), PT0848 (SEQ ID NO: 106), and PT0633 (SEQ ID NO:87) promoters.
  • CaMV 35S promoter the cauliflower mosaic virus (CaMV) 35S promoter
  • MAS mannopine synthase
  • 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens the figwort mosaic virus 34S promoter
  • actin promoters such as the rice actin promoter
  • ubiquitin promoters such as the maize ubiquitin-1 promoter.
  • the CaMV 35S promoter is excluded from the category of broadly expressing promoters. Root Promoters
  • Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues.
  • root-active promoters are root-preferential promoters, i.e., confer transcription only or predominantly in root tissue.
  • Root-preferential promoters include the YP0128 (SEQ ID NO:132), YP0275 (SEQ ID NO: 143), PT0625 (SEQ ID NO:86), PT0660 (SEQ ID NO:89), PT0683 (SEQ ID NO:94), and PT0758 (SEQ ID NO: 102) promoters.
  • root-preferential promoters include the PT0613 (SEQ ID NO:85), PT0672 (SEQ ID NO:91), PT0688 (SEQ ID NO:95), and PT0837 (SEQ ID NO: 104) promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds.
  • Other examples of root- preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al, Proc. Natl. Acad. Sd. USA, 86:7890-7894 (1989)), root cell specific promoters reported by Conkling et al, Plant Physiol., 93: 1203-1211 (1990), and the tobacco RD2 promoter. Maturing Endosperm Promoters
  • promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used.
  • Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustos et al., Plant Cell, l(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs et al, Plant Cell, l(6):609-621 (1989)), the ACP promoter (Baerson et al, Plant MoI Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase promoter (Slocombe et al, Plant Physiol, 104(4): 167- 176 (1994)), the soybean ⁇ ' subunit of ⁇ -conglycinin promoter (Chen et al, Proc.
  • the napin promoter the Arcelin-5 promoter
  • the phaseolin promoter Bustos et al., Plant Cell, l(9):839-8
  • zein promoters such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter.
  • Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al, MoI. Cell Biol, 13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordein promoter.
  • Other maturing endosperm promoters include the YP0092 (SEQ ID NO:118), PT0676 (SEQ ID NO:92), and PT0708 (SEQ ID NO:97) promoters.
  • Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, the melon actin promoter, YP0396 (SEQ ID NO:196), and PT0623 (SEQ ID NO: 195).
  • promoters that are active primarily in ovules include YP0007 (SEQ ID NO: 110), YPOl 11 (SEQ ID NO: 126), YP0092 (SEQ ID NO: 118), YP0103 (SEQ ID NO:123), YP0028 (SEQ ID NO:113), YP0121 (SEQ ID NO:131), YP0008 (SEQ ID NO:111), YP0039 (SEQ ID NO:114), YPOl 15 (SEQ ID NO:127), YPOl 19 (SEQ ID NO: 129), YP0120 (SEQ ID NO:130), and YP0374 (SEQ ID NO:148).
  • Embryo Sac/Early Endosperm Promoters SEQ ID NO: 110
  • YPOl 11 SEQ ID NO: 126
  • YP0092 SEQ ID NO: 118
  • YP0103 SEQ ID NO:123
  • YP0028 SEQ ID NO:113
  • regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell.
  • a pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
  • Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBankNo. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoI. Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBankNo. AF096096); and FIE 1.1 (U.S. Patent 6,906,244).
  • Arabidopsis viviparous-1 see, GenBankNo. U93215
  • Arabidopsis atmycl see, Urao (1996) Plant MoI. Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505
  • Arabidopsis FIE GeneBank No. AF129516
  • Arabidopsis MEA Arabidopsis FIS2
  • promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBankNo. L05934; Abler (1993) Plant MoI. Biol, 22:10131-1038).
  • promoters include the following Arabidopsis promoters: YP0039 (SEQ ID NO: 114), YPOlOl (SEQ ID NO: 121), YP0102 (SEQ ID NO:122), YPOI lO (SEQ ID NO:125), YPOl 17 (SEQ ID NO:128), YPOl 19 (SEQ ID NO:129), YP0137 (SEQ ID NO:133), DME, YP0285 (SEQ ID NO: 144), and YP0212 (SEQ ID NO: 140).
  • Other promoters that may be useful include the following rice promoters: p530cl0 (SEQ ID NO: 197) and pOsFIE2- 2 (SEQ ID NO: 198).
  • Regulatory regions that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable.
  • Embryo-preferential promoters include the barley lipid transfer protein (Ltpl) promoter ⁇ Plant Cell Rep (2001) 20:647-654), YP0097 (SEQ ID NO:120), YP0107 (SEQ ID NO: 124), YP0088 (SEQ ID NO:117), YP0143 (SEQ ID NO: 134), YP0156 (SEQ ID NO: 136), PT0650 (SEQ ID NO:88), PT0695 (SEQ ID NO:96), PT0723 (SEQ ID NO:99), PT0838 (SEQ ID NO: 105), PT0879 (SEQ ID NO: 108), and PT0740 (SEQ ID NO: 100).
  • Ltpl barley lipid transfer protein
  • Photosvnthetic Tissue Promoters Active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricind), the pine cab6 promoter (Yamamoto et al, Plant Cell Physiol, 35:773-778 (1994)), the Cab-1 promoter from wheat (Fejes et ah, Plant MoI.
  • RbcS ribulose-l,5-bisphosphate carboxylase
  • vascular Tissue Promoters examples include YP0087 (SEQ ID NO: 187), YP0093 (SEQ ID NO: 188),
  • vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell, 3(10): 1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al, Plant Cell, 4(2): 185-192 (1992)), and the rice tungro bacilliform virus (RTBV) promoter (Dai et al, Proc. Natl. Acad. Sci. USA, 101(2):687-692 (2004)).
  • GRP 1.8 promoter Keller and Baumgartner, Plant Cell, 3(10): 1051-1061 (1991)
  • CoYMV Commelina yellow mottle virus
  • RTBV rice tungro bacilliform virus
  • Poppy Capsule Promoters examples include PT0565 (SEQ ID NO:185) and YP0015 (SEQ ID NO:186). Alkaloid Biosynthesis Promoters
  • Inducible promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli.
  • inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought.
  • drought- inducible promoters include YP0380 (SEQ ID NO: 150), PT0848 (SEQ ID NO:106), YP0381 (SEQ ID NO:151), YP0337 (SEQ ID NO:146), PT0633 (SEQ ID NO:87), YP0374 (SEQ ID NO:148), PT0710 (SEQ ID NO:98), YP0356 (SEQ ID NO:147), YP0385 (SEQ ID NO:153), YP0396 (SEQ ID NO:154), YP0388 (SEQ ID NO:193), YP0384 (SEQ ID NO:152), PT0688 (SEQ ID NO:95), YP0286 (SEQ ID NO:145), YP0377 (
  • Nitrogen-inducible promoters include PT0863 (SEQ ID NO: 107), PT0829 (SEQ ID NO: 103), PT0665 (SEQ ID NO:90), and PT0886 (SEQ ID NO: 109).
  • shade-inducible promoters include PR0924 (SEQ ID NO: 192) and PT0678 (SEQ ID NO:93).
  • Basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation.
  • Basal promoters frequently include a "TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation.
  • Basal promoters also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
  • CCAAT box typically the sequence CCAAT
  • promoters include, but are not limited to, leaf- preferential, stem/shoot-preferential, callus-preferential, guard cell-preferential such as PT0678 (SEQ ID NO:93), and senescence-preferential promoters.
  • a 5' untranslated region can be included in nucleic acid constructs described herein.
  • a 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide.
  • a 3 ' UTR can be positioned between the translation termination codon and the end of the transcript.
  • UTRs can have particular functions such as increasing niRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
  • more than one regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • more than one regulatory region can be operably linked to the sequence of a polynucleotide encoding an oil-modulating polypeptide.
  • Regulatory regions such as promoters for endogenous genes, can be obtained by chemical synthesis or by subcloning from a genomic DNA that includes such a regulatory region.
  • a nucleic acid comprising such a regulatory region can also include flanking sequences that contain restriction enzyme sites that facilitate subsequent manipulation.
  • the polynucleotides and recombinant vectors described herein can be used to express or inhibit expression of a gene, such as an endogenous gene involved in alkaloid biosynthesis, e.g., to alter alkaloid biosynthetic pathways in a plant species of interest.
  • a gene such as an endogenous gene involved in alkaloid biosynthesis, e.g., to alter alkaloid biosynthetic pathways in a plant species of interest.
  • expression refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an en2yme, RNA polymerase, and into polypeptide, through translation of mRNA on ribosomes.
  • Up-regulation or “activation” refers to regulation that increases the production of expression products (mRNA, polypeptide, or both) relative to basal or native states
  • down-regulation or “repression” refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states.
  • Modulated level of gene expression refers to a comparison of the level of expression of a transcript of a gene or the amount of its corresponding polypeptide in the presence and absence of a morphinan alkaloid modifying enzyme described herein, and refers to a measurable or observable change in the level of expression of a transcript of a gene or the amount of its corresponding polypeptide relative to a control plant or plant cell under the same conditions (e.g., as measured through a suitable assay such as quantitative RT-PCR, a "Northern blot,” a “Western blot” or through an observable change in phenotype, chemical profile, or metabolic profile).
  • a modulated level of gene expression can include up-regulated or down-regulated expression of a transcript of a gene or polypeptide relative to a control plant or plant cell under the same conditions. Modulated expression levels can occur under different environmental or developmental conditions or in different locations than those exhibited by a plant or plant cell in its native state.
  • RNA interference RNA interference
  • Antisense technology is one well-known method. In this method, a nucleic acid segment from a gene to be repressed is cloned and operably linked to a promoter so that the antisense strand of RNA is transcribed. The recombinant vector is then transformed into plants, as described above, and the antisense strand of RNA is produced.
  • the nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed.
  • a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
  • Constructs containing operably linked nucleic acid molecules in the sense orientation can also be used to inhibit the expression of a gene.
  • the transcription product can be similar or identical to the sense coding sequence of a polypeptide of interest.
  • the transcription product can also be unpolyadenylated, lack a 5' cap structure, or contain an unsplicable intron. Methods of co-suppression using a full-length cDNA as well as a partial cDNA sequence are known in the art. See, e.g., U.S. Patent No. 5,231,020.
  • a nucleic acid in another method, can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
  • Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
  • Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • RNA contains a 5'-UG-3' nucleotide sequence.
  • the construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein.
  • Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
  • tRNA transfer RNA
  • RNA endoribonucleases which have been described, such as the one that occurs naturally in Tetrahymena thermophila, can be useful. See, for example, U.S. Patent No. 4,987,071 and 6,423,885.
  • RNAi can also be used to inhibit the expression of a gene.
  • a construct can be prepared that includes a sequence that is transcribed into an interfering RNA.
  • Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure.
  • One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of the polypeptide of interest, and that is from about 10 nucleotides to about 2,500 nucleotides in length.
  • the length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides.
  • the other strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the antisense strand of the coding sequence of the polypeptide of interest, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence.
  • the loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides.
  • the loop portion of the RNA can include an intron.
  • a construct including a sequence that is transcribed into an interfering RNA is transformed into plants as described above. Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S.
  • nucleic-acid based methods for inhibition of gene expression in plants can be a nucleic acid analog.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, Antisense Nucleic Acid Drug Dev., 7:187-195 (1997) and Hyrup et ah, Bioorgan. Med. Chem., 4:5-23 (1996).
  • the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • transgenic plant cells and plants comprising at least one recombinant nucleic acid construct or exogenous nucleic acid.
  • a recombinant nucleic acid construct or exogenous nucleic acid can include a nucleic acid encoding a morphinan alkaloid modifying enzyme as described herein.
  • a plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division.
  • a plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome.
  • Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
  • transgenic plant cells used in methods described herein constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. Progeny includes descendants of a particular plant or plant line.
  • Progeny of an instant plant include seeds formed on F 1 , F 2 , F 3 , F 4 , F 5 , F 6 and subsequent generation plants, or seeds formed on BCi, BC 2 , BC 3 , and subsequent generation plants, or seeds formed on FiBCi, FiBC 2 , F 1 BC 3 , and subsequent generation plants. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.
  • Transgenic plant cells growing in suspension culture, or tissue or organ culture can be useful for extraction of alkaloid compounds.
  • solid and/or liquid tissue culture techniques can be used.
  • transgenic plant cells can be placed directly onto the medium or can be placed onto a filter film that is then placed in contact with the medium.
  • transgenic plant cells can be placed onto a floatation device, e.g., a porous membrane that contacts the liquid medium.
  • Solid medium typically is made from liquid medium by adding agar.
  • a solid medium can be Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin.
  • an auxin e.g., 2,4-dichlorophenoxyacetic acid (2,4-D)
  • a cytokinin e.g., kinetin.
  • a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation.
  • a suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days.
  • the use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous morphinan alkaloid modifying enzyme whose expression has not previously been confirmed in particular recipient cells.
  • nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrob ⁇ cterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880, 5,204,253, 6,329,571 and 6,013,863.
  • a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art. See, e.g., Allen et ⁇ l, Nature Biotechnology, 22(12):1559-1566 (2004); Chitty et al, Fund. Plant Biol, 30:1045-1058 (2003); and Park et al, J. Exp. Botany, 51(347): 1005-1016 (2000).
  • Suitable plants with which to practice the invention include plants that are capable of producing one or more alkaloids.
  • a plant that is capable of producing one or more alkaloids is capable of producing one or more alkaloids even when it lacks recombinant nucleic acid constructs.
  • a plant from the Solanaceae or Papaveraceae families is capable of producing one or more alkaloids when it lacks a nucleic acid encoding a morphinan alkaloid biosynthesis enzyme.
  • a plant or plant cell may be transgenic for sequences other than the morphinan alkaloid biosynthesis enzymes described herein and still be characterized as capable of producing one or more alkaloids.
  • Useful plant families that are capable of producing one or more alkaloids include the Papaveraceae, Berberidaceae, Leguminosae, Boraginaceae, Apocynaceae, Erythroxylaceae, Convolvulaceae, Asclepiadaceae, Liliaceae, Gnetaceae, Ranunculaeceae, Rubiaceae, Solanaceae, and Rutaceae families.
  • useful genera include the Papaver (e.g., Papaver bracteatum, Papaver orientale, Papaver setigerum, and Papaver somniferuni), Sanguinaria, Dendromecon, Glaucium, Meconopsis, Chelidonium, Eschscholzioideae (e.g., Eschscholzia, Eschscholzia California), and Argemone (e.g., Argemone hispida, Argemone mexicana, and Argemone munit ⁇ ) genera.
  • Papaver e.g., Papaver bracteatum, Papaver orientale, Papaver setigerum, and Papaver somniferuni
  • Sanguinaria Dendromecon
  • Glaucium Glaucium
  • Meconopsis Chelidonium
  • Eschscholzioideae e.g., Eschscholzia, Eschscholzia California
  • Argemone e.g., Argemone hispida, Argemone mexi
  • morphinan producing species include Croton salutaris, Croton balsamifera, Glaucium spp., Papaver spp., Sinomenium acutum, Stephania cepharantha, Litsea sebiferea, Alseodaphne perakensis, Cocculus laurifolius, Duguetia obovata, Rhizocarya racemifera, Beilschmiedia oreophila, Stephania zippeliana, Roemeria refracta, and Papaver nudicale.
  • plant cells are transformed with more than one construct, such that any combination of two gene products capable of using morphinans as substrates are present in the cells.
  • genes encode morphinan alkaloid modifying enzyme polypeptides.
  • plant cells can be transformed with two constructs, the first construct comprising a nucleic acid encoding a UGT polypeptide and the second construct comprising a nucleic acid encoding a morphine dehydrogenase and/or a morphinone reductase polypeptide.
  • plant cells can be transformed with constructs encoding morphine dehydrogenase (e.g., morA) and morphinone reductase (e.g., morB).
  • plant cells can be transformed with three constructs, the first comprising a nucleic acid encoding a UGT polypeptide, the second construct comprising a nucleic acid encoding a morphine dehydrogenase polypeptide, and the third construct comprising a nucleic acid encoding a morphinone reductase polypeptide.
  • each coding sequence is operably linked to a regulatory region.
  • a nucleic acid construct may contain two morphinan alkaloid biosynthesis enzyme coding sequences, each operably linked to a regulatory region. The two regulatory regions in such a construct can have the same nucleotide sequence, or can have different nucleotide sequences.
  • transgenic plant cells described herein in addition to having a nucleic acid encoding a morphinan alkaloid modifying enzyme described herein, also have a construct comprising a nucleic acid encoding one or more regulatory proteins, operably linked to a regulatory region.
  • a nucleic acid encoding a regulatory protein that transactivates a pathway resulting in enhanced accumulation of morphine can be present in a plant along with one or more nucleic acids encoding one or more UGT polypeptides, morphine dehydrogenase polypeptides or morphinone reductase polypeptides, resulting in the accumulation of greater amounts of morphinan alkaloids in planta compared to a control plant that lacks the regulatory protein nucleic acid.
  • Suitable regulatory proteins can be found, e.g., in U.S. Patent Application Ser. Nos. 11/360,459 and 11/360,039.
  • transgenic plant cells described herein in addition to having a nucleic acid encoding a morphinan alkaloid modifying enzyme described herein, also have a construct comprising a nucleic acid that results in inhibition of a competing pathway or that enhances activation of silent genes, operably linked to a regulatory region.
  • a nucleic acid can be, for example, a nucleic acid that is transcribed into an interfering RNA against a berberine bridge enzyme gene.
  • Such a nucleic acid can inhibit the production of benzophenanthridine intermediates, thereby enhancing synthesis of morphinans in planta.
  • nucleic acid encoding a berberine bridge enzyme RNAi When a nucleic acid encoding a berberine bridge enzyme RNAi is present in a plant along with nucleic acids encoding morphine dehydrogenase and morphinone reductase polypeptides, greater amounts of morphinan alkaloids can accumulate in planta compared to a control plant that lacks the berberine bridge enzyme RNAi.
  • a nucleic acid that is transcribed into an interfering RNA against a cytosine DNA methyltransferase is present in a plant, along with a nucleic acid encoding a UGT polypeptide, resulting in greater accumulation of morphine-6-glucuronide inplanta compared to a control plant that lacks the cytosine DNA methyltransferase RNAi.
  • a population of transgenic plants can be screened and/or selected for those members of the population that have a desired trait or phenotype conferred by expression of the transgene. Selection and/or screening can be carried out over one or more generations, which can be useful to identify those plants that have a desired trait, such as an increased level of one or more morphinan alkaloids or morphinan alkaloid derivatives. Selection and/or screening can also be carried out in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be carried out during a particular developmental stage in which the phenotype is exhibited by the plant.
  • the invention also features methods in which one or more alkaloid compounds, e.g., morphinan alkaloids or derivatives thereof, are produced from a plant or cell.
  • the method includes growing a plant or plant cell that has one or more of the recombinant nucleic acid constructs described herein.
  • the alkaloid compound that is produced can be a morphinan alkaloid such as codeinone, morphinone, hydrocodone, hydromorphone, thebaine, codeine, morphine, or a glucuronide derivative of a morphinan alkaloid.
  • the amount of one or more alkaloid compounds is modulated, e.g. increased or decreased, relative to a plant cell not transformed with the recombinant nucleic acid construct.
  • An amount of any one or more of the alkaloids can be increased or decreased, as discussed below.
  • the alkaloid compound that is modulated can be, for example, morphine-6-glucuronide, codeinone, morphinone, hydrocodone, hydromorphone, salutaridine, salutaridinol, salutaridinol acetate, isothebaine, thebaine, neopinone, codeine, morphine, papaverine, narcotine, narceine, hydrastine, glucoronimides, thebaine, or oripavine.
  • more than one alkaloid e.g., two, three, four, five, six, seven, eight, nine, ten, or even more alkaloids, can have their amounts modulated relative to a control plant or cell.
  • at least one alkaloid will be detectable by the analytical technique used, whereas the alkaloid will not be detectable in a corresponding non-transgenic control using the same analytical technique.
  • the detectable alkaloid is a novel compound.
  • the novel compound may be new to the plant species or a new chemical entity.
  • the amount of one or more alkaloid compounds can be increased or decreased in transgenic cells containing a recombinant nucleic acid construct as described herein.
  • the increase is from about 1.5-fold to about 500-fold, or about 2-fold to about 22-fold, or about 25-fold to about 50- fold, or about 75-fold to about 130-fold, or about 5-fold to about 50-fold, or about 5-fold to about 10-fold, or about 10-fold to about 20-fold, or about 10-fold to about 25-fold, or about 20-fold to about 75-fold, or about 10-fold to about 100-fold, or about 40-fold to about 100-fold, or about 30-fold to about 50-fold, or about 100-fold to about 200-fold, or about 150-fold to about 250-fold, or about 200-fold to about 300-fold, or about 300-fold to about 400-fold, or about 350-fold to about 500-fold higher on a fresh or freeze dried weight basis than the amount in a corresponding control cell that lacks the construct.
  • the amounts of two or more alkaloid compounds are increased and/or decreased, e.g., the amounts of two, three, four, five, six, seven, eight, nine, ten (or more) alkaloid compounds are independently increased and/or decreased.
  • the alkaloid compound that is increased in transgenic cells described herein is either not produced or is not detectable in a corresponding control cell that lacks the recombinant nucleic acid construct.
  • the increase in such an alkaloid compound is infinitely high relative to a corresponding control cell that lacks the construct.
  • the increase in amount of one or more alkaloids can be restricted in some embodiments to particular tissues and/or organs, relative to other tissues and/or organs.
  • a transgenic plant can have an increased amount of an alkaloid in leaf tissue relative to root or floral tissue.
  • the amounts of one or more alkaloids are decreased in transgenic cells.
  • a decrease ratio can be expressed as the ratio of the alkaloid in such a transgenic cell on a weight basis (e.g., fresh or freeze dried weight basis) as compared to the alkaloid in a corresponding control cell.
  • the decrease ratio can be from about 0.05 to about 0.90.
  • the ratio can be from about 0.2 to about 0.6, or from about 0.4 to about 0.6, or from about 0.3 to about 0.5, or from about 0.2 to about 0.4.
  • the alkaloid compound that is decreased in transgenic cells is decreased to an undetectable level as compared to the level in a corresponding control cell.
  • the decrease ratio in such an alkaloid compound is zero.
  • the decrease in amount of one or more alkaloids can be restricted in some embodiments to particular tissues and/or organs, relative to other tissues and/or organs.
  • a transgenic plant can have a decreased amount of an alkaloid in leaf tissue relative to root or floral tissue.
  • the amount of an alkaloid compound can be determined by known techniques, e.g., by extraction of alkaloid compounds followed by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). If desired, the structure of the alkaloid compound can be confirmed by GC-MS, LC-MS, nuclear magnetic resonance and/or other known techniques.
  • GC-MS gas chromatography-mass spectrometry
  • LC-MS liquid chromatography-mass spectrometry
  • plant cells are subjected to environmental conditions that facilitate the synthesis of increased amounts of morphinan alkaloids or derivatives thereof.
  • Environmental conditions under which a plant, or a plant or cell culture, is grown can be altered, e.g., by increasing the temperature, increasing the watering rate, or decreasing the watering rate, relative to a control temperature or watering rate.
  • Other environmental conditions that can be altered in order to increase the amount or synthesis rate of morphinan alkaloids or morphinan alkaloid derivatives include the concentration of salt, minerals, hormones, nitrogen, carbon, osmoticum, or known elicitors such as yeast extract, salicylic acid, and methyl jasmonate.
  • an increase or decrease in the amount of an alkaloid in cells of a transgenic plant or cell relative to a control plant or cell is considered statistically significant atp ⁇ 0.05 with an appropriate parametric or non- parametric statistic, e.g., Chi-square test, Student's t-test, Mann- Whitney test, or F-test.
  • a difference in the amount of an alkaloid is statistically significant at p ⁇ 0.01, p ⁇ 0.005, or pO.OOl .
  • a statistically significant difference in, for example, the amount of an alkaloid in cells of a transgenic plant compared to the amount in cells of a control plant indicates that (1) the recombinant nucleic acid present in the transgenic plant alters the amount of the alkaloid in cells and/or (2) the recombinant nucleic acid warrants further study as a candidate for altering the amount of the alkaloid in a plant.
  • the invention also features methods in which one or more secondary metabolites, e.g., alkaloids, are extracted from plant cells containing a recombinant nucleic acid construct described herein.
  • Suitable tissues or organs from which secondary metabolites can be extracted include leaves, roots, stems, bark, flowers, seeds, immature flower pods, seed capsules, embryos, endosperm, cotyledons, trichomes, meristematic tissue, embryogenic cultures, organogenic cultures, cambial cells, or liquid suspension cultures.
  • plant cells in which a morphinan alkaloid or a derivative of a morphinan alkaloid is known or suspected of being present can be separated from cells in which the morphinan alkaloid or derivative is not suspected of being present.
  • Such a separation can enrich for cells or cell types that contain such an alkaloid.
  • a number of methods for separating particular cell types or cell layers are known to those having ordinary skill in the art. For example, cell types may be dissected using laser capture microdissection, or can be captured using a cell sorter by virtue of an epitope tag in the reporter or receptor.
  • Fractionation can be carried out by techniques known in the art. For example, plant tissue can be extracted with 100% methanol to give a crude oil which is partitioned between several solvents in a conventional manner. As an alternative, fractionation can be carried out on gel columns using methylene chloride and ethyl acetate/hexane solvents.
  • a fractionated or unfractionated plant tissue or organ extract is subjected to mass spectrometry in order to identify and characterize one or more morphinan alkaloids or derivatives. See, e.g., WO 02/37111.
  • Mass spectrometry analysis is often suitable for characterizing and identifying particular compounds.
  • electrospray ionization (ESI) mass spectrometry can be used.
  • atmospheric pressure chemical ionization (APCI) mass spectrometry is used. If it is desired to identify higher molecular weight molecules in an extract, matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry can be useful.
  • MALDI-TOF matrix- assisted laser desorption/ionization time-of-flight
  • Such extracts can be crude extracts (for example, less than about 50%, e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%, by weight of the alkaloid compound), partially purified extracts (for example, greater than about 50% and less than about 80%, e.g., 55%, 60%, 65%, 70%, or 75%, by weight of the alkaloid compound), or extensively purified extracts (for example, greater than about 80%, e.g., greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by weight of the alkaloid compound).
  • crude extracts for example, less than about 50%, e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%, by weight of the alkaloid compound
  • partially purified extracts for example, greater than about 50% and less than about 80%, e.g., 55%, 60%, 65%, 70%, or
  • Such extracts can be aqueous extracts or non-aqueous extracts.
  • An extract can also be in a solid form, e.g., a powder.
  • the alkaloid After extraction of a morphinan alkaloid or a morphinan alkaloid derivative from a plant as described herein, the alkaloid can be chemically modified, e.g., by addition, elimination, or substitution of functional groups.
  • compositions suitable for administration to human beings and animals can be formulated into compositions suitable for administration to human beings and animals.
  • Compositions can be formulated for any route of administration, e.g., oral, intravenous, subcutaneous, intramuscular, rectal, transdermal, or topical administration, and can include one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • solid dosage forms for oral administration such as gel capsules, tablets, pills, and powders, can include one or more therapeutic agents with at least one excipient or carrier, such as a buffering agent, an absorption accelerator, a coating, or a disintegrating agent.
  • Gel capsules can contain solid, liquid, and/or semi-solid formulations.
  • a composition in the form of a syrup or elixir can contain a sweetener, an antiseptic agent, a flavoring agent, and/or a colorant.
  • Water- dispersible powders or granules can contain one or more morphinan alkaloids and/or morphinan alkaloid derivatives as a mixture with dispersants, wetting agents, or suspending agents, as well as with sweeteners.
  • suppositories can be prepared with binders, such as cocoa butter, that melt at the rectal temperature.
  • Aqueous suspensions, isotonic saline solutions or sterile, injectable solutions containing pharmacologically compatible dispersants and/or solubilizing agents can be used for parenteral, intranasal, or intraocular administration.
  • compositions can be formulated for delayed release, controlled release, sustained release, or extended release.
  • Compositions including more than one, e.g., two, active ingredients can be formulated such that the release profile of each active ingredient differs.
  • compositions described herein can be used to treat human beings and animals having a condition for which treatment with an alkaloid compound, such as morphine, codeine, or thebaine, is useful.
  • alkaloid compound such as morphine, codeine, or thebaine
  • Examples of such conditions include, without limitation, treatment of moderate to severe, acute and chronic nociceptive pain, such as post-operative pain (van Dorp et al., Anesth Analg, 102(6): 1789-97 (2006)), pain associated with malignant diseases, and neuropathic pain.
  • a therapeutically effective amount of a morphinan alkaloid will vary with the route of administration and with factors such as the age, sex, weight, and condition of the subject being administered and the condition being treated.
  • a suitable dose is typically in the range of 1-1000 mg/70 kg, e.g., 200 mg/70 kg or 5-75 mg/70 kg.
  • the dosage for routes of administration where bioavailability is high e.g., intravenous, subcutaneous, or intranasal, will be lower than for routes with low bio-availability, e.g., oral.
  • the invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
  • UGT2B7 A synthetic version of the human UDP glycosyltransferase 2, polypeptide 7 coding sequence (UGT2B7; SEQ ID NO:1; NCBI accession number NMJ)01074; Ritter et ah, 1990, J Biol Chem 265: 7900-7906) was generated by GeneArt AG (BioPark Regensburg, Germany) and codon-optimized for expression in plants.
  • the synthetic UGT2B7 DNA sequence is set forth in SEQ ID NO:2.
  • a PCR-Script ® plasmid (Stratagene, La Jolla, CA) containing the synthetic UGT2B7 DNA was digested by restriction enzymes Apal and HindIIL The UGT2B7 fragment was ligated into an NB42-35S T-DNA vector that was previously digested with the same set of restriction enzymes. The resulting construct, designated Bin4-35S-UGT2B7, was introduced into Agrobacterium competent cells to generate an Agrobacterium clone for Opium poppy transformation.
  • Synthetic versions of the morphine dehydrogenase ⁇ MorA) and morphinone reductase (MorB) coding sequences from Pseudomonas putida were generated with optimized codons in a manner similar to that described above for the UGT2B7 coding sequence.
  • the synthetic sequence for MorA was based on the sequence corresponding to NCBI accession number M94775 (SEQ ID NO: 16; Willey et ah, 1993, Biochem J, 290:539-544).
  • the synthetic MorA sequence, which was contained in a PCR-Script ® plasmid, is set forth in SEQ ID NO: 17.
  • the synthetic sequence for MorB was based on the sequence corresponding to NCBI accession number U37350 (SEQ ID NO:37; French and Bruce, 1995, Biochem J, 312 (Pt3): 671-678).
  • the synthetic MorB sequence which was contained in a PCR-Script ® plasmid, is set forth in SEQ ID NO:38.
  • the synthetic MorA and MorB sequences were flanked by DNA recombination sequences (attB) for Gateway ® cloning.
  • Plasmid pDONRTM 221 (Invitrogen, Carlsbad, CA) was reacted separately with the PCR-Script ® plasmid containing the synthetic MorA sequence and the PCR-Script ® plasmid containing the synthetic MorB sequence to generate the respective pENTRY plasmids, pENTRY-MorA and pENTRY- MorB.
  • Each pENTRY plasmid was used in an integration reaction with a 35S- promoter-containing T-DNA vector (designated CW_DESTR1-R2_35S) or a 326-promoter-containing T-DNA vector (designated CW_DESTR1 -R2_326) to generate T-DNA plasmids (designated Bin6-35S-MorA, Bin6-35S-MorB, Bin6- 326-MorA, and Bin6-326-MorB).
  • Each T-DNA plasmid was introduced into Agrobacterium for Opium poppy transformation.
  • Opium poppy plants containing the Bin4-35S-UGT2B7, Bin6-35S- MorA, Bin6-35S-MorB, Bin6-326-MorA, or Bin6-326-MorB construct described in Example 1 were generated as follows.
  • CIM Callus Induction Medium
  • EmC a compact light yellow to white spherical embryogenic callus (EmC) usually emerged from the surface of a translucent friable non-embryogenic callus (NEC).
  • NEC translucent friable non-embryogenic callus
  • Agrobacterium clones each containing a T-DNA construct described in Example ⁇ , were inoculated separately into two mL aliquots of YEB liquid medium with antibiotics and incubated overnight at 28°C with shaking. Agrobacterium cells were spun down at 10,000 rpm in a 1.5 mL Eppendorf tube at room temperature (RT) using a micro-centrifuge.
  • the cells were resuspended in 6 mL of liquid Co-Cultivation Medium (CCM: CM with 100 uM acetosyringone, where CM contains MS basal medium, B 5 vitamins, 1 g/L Casamnino acid, 2 mg/L 2,4 D, 0.5 mg/L BA and 6.5 g/L Phytagar) in a 50 mL conical tube to a final OD 60O of 0.06-0.08.
  • CCM liquid Co-Cultivation Medium
  • EmC was infected with Agrobacterium suspension for five minutes with gentle agitation.
  • the transfected EmC was blotted dry with sterile Kimwipe ® paper in a Petri plate before being transferred onto sterile Whatman filter paper contained in CCM.
  • the transfected EmC was incubated at 22°C under low light in a Percival growth chamber for three days for co-cultivation.
  • the transfected EmC was washed three times with 20-30 mL of sterile MiIIiQ-H 2 O with moderate shaking. The last wash was done in the presence of 500 mg/L Carbenicillin. The washed EmC was briefly dried in sterile Kimwipe ® paper and transferred to Recovery Medium (RRM: CIM with 500mg/L carbenicillin). The transfected EmC was incubated at 25 0 C under low light in a Percival growth chamber for 7-9 days. After the recovery period, all calli were transferred to Callus Selection
  • CSM CM with 500mg/L carbenicillin and 5 mg/L bialaphos
  • CSM CM with 500mg/L carbenicillin and 5 mg/L bialaphos
  • the transfected EmC was subcultured every 10 to 12 days. After the second subculture, only bialaphos resistant calli were transferred to fresh CSM.
  • the resistant embryogenic calli typically had a light yellow color.
  • Non-resistant calli typically were light to dark brown in color and were dead or dying.
  • bialaphos resistant calli were transferred to Regeneration Medium 1 (RMl : CM with 250 mg/L carbenicillin, 2 mg/L Zeatin, 0.05 mg/L IBA, 100 mg/L L-Glutamine and 200 mg/L L-Cysteine) and incubated at 25 0 C under high light in a Percival growth chamber with a 16 hour photo period.
  • RMl Regeneration Medium 1
  • bialaphos resistant calli were transferred to Regeneration Medium 2 (RM2: CM with 250 mg/L carbenicillin, 0.5 mg/L Zeatin, 0.05 mg/L IBA, 100 mg/L L-Glutamine and 200 mg/L L-Cysteine).
  • RM2 Regeneration Medium 2
  • Bialaphos resistant EmC continued to grow and differentiate into embryos. These embryos developed into plantlets after 15-20 days.
  • Rooting Medium CM with 250 mg/L carbenicillin, 0.2 mg/L IBA, 50 mg/L L-Glutamine and 4 5 g/L Phytagar
  • Fully regenerated plants were transferred to soil at the appropriate time.
  • Leaf tissues were collected from transgenic plants of independent transformation events and used for qRT-PCR analysis. To serve as a control, 10 similar tissue was also collected from regenerated wild-type lines or lines transformed with a YP188::GFP construct.
  • the Opium poppy EFl b ⁇ elongation factor- Ib gene was used to normalize the expression of the transgenes and the endogenous codeinone reductase (PsCR) gene measured in the samples. Transcription of these genes was monitored for each of the transgenic events using the corresponding set of 20 primers shown below.
  • the Ct value was arbitrarily set at 35 (representing a conservative background level) for the wild-type and YP188::GFP lines in order to have a defined ratio.
  • Example 4 Alkaloid analysis of Transgenic Opium poppy lines
  • tissue samples were collected depending on availability: (1) young rosette leaves from regenerated plants approximately two months after transplanting to soil from regeneration medium, (2) latex from the main leaf vein after young rosette leaves were cut, and (3) latex from capsules/pods approximately seven days after flower opening.
  • Leaf samples were immediately frozen in liquid nitrogen and lyophilized prior to analysis.
  • Latex samples, either from the leaves or capsules, were collected in 200 ⁇ L buffer (100 mM Potassium phosphate buffer pH 7, 500 mM Mannitol, 20 mM ascorbic acid) and immediately frozen. Latex samples from different capsules originating from the same plant but collected at different time points were pooled together prior to analysis.
  • Latex samples were extracted by sonication using methanol as a solvent. An internal standard (oxycodone) was included in the solvent. The sonicated suspension was centrifuged and the resulting supernatant was applied to a spin column. An aliquot of the flow-through was analyzed by LC-MS according to the method described below.
  • Ratios were generated by dividing the normalized value of the transgenic line by the average normalized value of the control. Summary of alkaloid analysis The level of a compound with an ion mass/charge ratio (m/z) of 526 was observed to be increased substantially, from 4X in transformant #4-05 to 45X in transformant #12-04, in Opium poppy lines transformed with the 35S-UGT2B7 construct (Table 5). Based on the molecular weight, this compound appears to be an alkaloid.
  • a subject sequence was considered a functional homolog or ortholog of a query sequence if the subject and query sequences encoded proteins having a similar function and/or activity.
  • a process known as Reciprocal BLAST (Rivera et al, Proc. Natl Acad. ScL USA, 95:6239-6244 (1998)) was used to identify potential functional homolog and/or ortholog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
  • a specific query polypeptide was searched against all peptides from its source species using BLAST in order to identify polypeptides having BLAST sequence identity of 80% or greater to the query polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment.
  • the query polypeptide and any of the aforementioned identified polypeptides were designated as a cluster.
  • the BLASTP version 2.0 program from Washington University at Saint Louis, Missouri, USA was used to determine BLAST sequence identity and E- value.
  • the BLASTP version 2.0 program includes the following parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3) the -postsw option.
  • the BLAST sequence identity was calculated based on the alignment of the first BLAST HSP (High-scoring Segment Pairs) of the identified potential functional homolog and/or ortholog sequence with a specific query polypeptide. The number of identically matched residues in the BLAST HSP alignment was divided by the HSP length, and then multiplied by 100 to get the BLAST sequence identity. The HSP length typically included gaps in the alignment, but in some cases gaps were excluded.
  • the main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search.
  • a query polypeptide sequence "polypeptide A”
  • SA was BLASTed against all protein sequences from a species of interest.
  • Top hits were determined using an E-value cutoff of 10 "5 and a sequence identity cutoff of 35%. Among the top hits, the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
  • top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA.
  • a top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog or ortholog.
  • Functional homologs and/or orthologs were identified by manual inspection of potential functional homolog and/or ortholog sequences.

Abstract

L'invention concerne des compositions et des procédés destinés à produire des composés alcaloïdes, par exemple, des alcaloïdes de morphinane et des dérivés de ceux-ci, dans des plantes. Elle concerne par exemple, une cellule végétale exprimant une enzyme modifiant un alcaloïde de morphinane qui catalyse la synthèse d'un ou plusieurs alcaloïdes de morphinane ou dérivés de ceux-ci.
PCT/US2006/027731 2005-07-18 2006-07-18 Production d'alcaloides de morphinane et de derives de ceux-ci dans des plantes WO2007011887A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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US7598367B2 (en) 2005-06-30 2009-10-06 Ceres, Inc. Early light-induced protein promoters
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CN103270947A (zh) * 2013-03-08 2013-09-04 浙江省农业科学院 一种司牛角组织培养的方法
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US7790874B2 (en) 2006-03-15 2010-09-07 Pioneer Hi-Bred International, Inc. Gene expression modulating element
US7825234B2 (en) 2006-03-15 2010-11-02 Pioneer Hi Bred International Inc Gene expression modulating element
CN103270947A (zh) * 2013-03-08 2013-09-04 浙江省农业科学院 一种司牛角组织培养的方法
CN103270947B (zh) * 2013-03-08 2014-09-03 浙江省农业科学院 一种司牛角组织培养的方法
CN107124887A (zh) * 2014-08-06 2017-09-01 庄信万丰股份有限公司 催化剂及其用途
WO2016020695A1 (fr) * 2014-08-06 2016-02-11 Johnson Matthey Public Limited Company Catalyseur et son utilisation
EP3489356A1 (fr) * 2014-08-06 2019-05-29 Johnson Matthey Public Limited Company Catalyseur et utilisation associée
US10829743B2 (en) 2014-08-06 2020-11-10 Johnson Matthey Public Limited Company Catalyst and use thereof
US10927352B2 (en) 2014-08-06 2021-02-23 Johnson Matthey Public Limited Company Catalyst and use thereof
US10927353B2 (en) 2014-08-06 2021-02-23 Johnson Matthey Public Limited Company Catalyst and use thereof
WO2018015066A1 (fr) 2016-07-18 2018-01-25 Saint-Gobain Glass France Vitrage coupe-feu transparent ayant des propriétés de résistance à l'effraction et anti-panique
WO2018024387A1 (fr) 2016-08-03 2018-02-08 Saint-Gobain Glass France Vitrage transparent, sans éclats, anti-bombardement, doté de propriétés de protection contre l'incendie

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