WO2019113710A1 - Benzylisoquinoline uptake permeases and methods of using - Google Patents

Benzylisoquinoline uptake permeases and methods of using Download PDF

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Publication number
WO2019113710A1
WO2019113710A1 PCT/CA2018/051604 CA2018051604W WO2019113710A1 WO 2019113710 A1 WO2019113710 A1 WO 2019113710A1 CA 2018051604 W CA2018051604 W CA 2018051604W WO 2019113710 A1 WO2019113710 A1 WO 2019113710A1
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seq
benzylisoquinoline
nucleic acid
substrate
compound
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PCT/CA2018/051604
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French (fr)
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WO2019113710A9 (en
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Peter J. Facchini
Jillian M. HAGEL
Limei CHANG
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Serturner Corp.
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin

Definitions

  • the methods and systems disclosed herein relate to a class of chemical compounds known as alkaloids and methods of making alkaloids.
  • the methods and systems disclosed herein relate to a class of alkaloids known as benzylisoquinolines, and to methods and systems involving the use of benzylisoquinoline uptake permeases to produce benzylisoquinolines.
  • Alkaloids are a class of nitrogen containing organic chemical compounds that are naturally produced by opium poppy ⁇ Papaver somniferum), and a range of other plant species belonging to the Papaveraceae family of plants, as well as other plant families including, for example the Lauraceae, Annonaceae, Euphorbiaceae and the Moraceae.
  • the interest of the art in alkaloid compounds is well established and can be explained by the pharmacological properties of these compounds, as well as their utility as feedstock materials in the manufacture of pharmaceutical compounds.
  • the manufacture of alkaloid compounds can involve the conversion of precursor alkaloid compounds into one or more intermediary alkaloid compounds to yield a desired alkaloid compound, for example a desired benzylisoquinoline.
  • enzymes can catalyze the conversion reaction of precursor alkaloid compounds into intermediate alkaloid compounds, or into a desired product alkaloid.
  • alkaloid compounds are not efficiently converted into the desired products, for example, due to substrate inhibition, or they can be converted into products other than the desired alkaloids products, each of which results into low alkaloid product yields.
  • the yields of biosynthetic production systems are frequently lower than desired, while costs are higher than desired.
  • the present disclosure relates to alkaloid compounds, notably a class of alkaloid compounds known as benzylisoquinolines.
  • the present disclosure relates to biosynthetic systems for making benzylisoquinoline compounds.
  • the present disclosure provides, in at least one embodiment, a method of producing a product benzylisoquinoline compound in a host cell, the method comprising:
  • a chimeric nucleic acid comprising as operably linked components (i) a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii) a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell; and
  • the product benzylisoquinoline compound can be (5]-norcoclaurine, (5]-norlaudanosoline, (S)-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S)-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£ ⁇ - reticuline, (7?)-reticuline, salutaridine, salutaridinol, or thebaine.
  • the substrate can be a substrate benzylisoquinoline compound.
  • the substrate benzylisoquinoline compound can be (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S]-coclaurine, (5]-/V-methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (£ ⁇ - reticuline, (/? ⁇ -reticuline, salutaridine, or salutaridinol.
  • the substrate can be a substrate benzylisoquinoline precursor compound.
  • the substrate benzylisoquinoline precursor compound can be L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydroxy- phenylacetaldehyde (4-HPAA]
  • the enzyme complement can comprise one or more benzylisoquinoline biosynthetic enzymes, and the enzymes can be norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0-acetyltransferase (SalAT], or thebaine synthase (TS]
  • NCS norcoclaurine synthase
  • 60MT 6-0-methyltransferase
  • CNMT coclaurine-/V- methyltransferase
  • NMCH coclaurine-/V-methyltransferase
  • NMCH coclaurine
  • the host cell further can have a benzylisoquinoline precursor enzyme complement.
  • the benzylisoquinoline precursor enzyme complement can comprise one or more benzylisoquinoline precursor biosynthetic enzymes, and the enzymes can be tyrosine hydroxylase (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • TRR tyrosine hydroxylase
  • DODC dihydroxyphenyl alanine decarboxylase
  • TYDC tyrosine decarboxylase
  • MAO monoamide oxidase
  • the substrate can be converted into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
  • the substrate can be converted into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
  • the product benzylisoquinoline can be thebaine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS]
  • NCS norcoclaurine synthase
  • 60MT 6-O-methyltransferase
  • the product benzylisoquinoline compound can be thebaine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS] and the cell can further have a benzylisoquinoline precursor enzyme complement
  • the product benzylisoquinoline compound can be salutaridinol
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridinol
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O- acetyltransferase (SalAT]
  • NCS norcoclaurine synthase
  • 60MT 6-O-methyltransferase
  • CNMT coclaur
  • the product benzylisoquinoline compound can be salutaridinol
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridinol
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O-acetyltransferase (SalAT], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of
  • the product benzylisoquinoline compound can be salutaridine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn]
  • the product benzylisoquinoline compound can be salutaridine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and
  • the product benzylisoquinoline compound can be (R] -reticuline
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (R] -reticuline
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’ -0-methyl transferase (4 ⁇ MT], or reticuline epimerase (REPI]
  • the product benzylisoquinoline compound can be (R] -reticuline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (R] -reticuline
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’ -O-methyl transferase (4 ⁇ MT], or reticuline epimerase (REPI]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide
  • the product benzylisoquinoline compound can be (5] -reticuline
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (5] -reticuline
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], or 4’-0-methyltransferase (4 ⁇ MT]
  • the product benzylisoquinoline compound can be (5] -reticuline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5] -reticuline
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase or (4 ⁇ MT]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the product benzylisoquinoline compound can be (S]-3'-hydroxy-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-3’-hydroxy-/V-methylcoclaurine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase, or (NMCH]
  • the product benzylisoquinoline compound can be (5]-3’-hydroxy-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquino
  • the product benzylisoquinoline compound can be (S]-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-/V-methylcoclaurine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], or coclaurine-/V-methyltransferase (CNMT]
  • the product benzylisoquinoline compound can be (S]-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- /V-methylcoclaurine
  • the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], or coclaurine-iV-methyltransferase (CNMT]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the product benzylisoquinoline compound can be (5]-coclaurine
  • the substrate can be a benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-coclaurine, wherein the enzymes are selected from norcoclaurine synthase (NCS], and 6-O-methyltransferase (60MT]
  • the product benzylisoquinoline compound can be (S]-coclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- coclaurine
  • the enzymes can be selected from norcoclaurine synthase (NCS] or 6- O-methyltransferase (60MT] and the cell can further have a benzylisoquinoline precursor enzyme complement
  • the product benzylisoquinoline compound can be (5] -norcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norcoclaurine
  • the enzyme can be norcoclaurine synthase (NCS]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the product benzylisoquinoline compound can be (5]-norlaudanosoline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norlaudanosoline
  • the enzyme can be norcoclaurine synthase (NCS]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the product benzylisoquinoline compound can be (S]-6-0-methyl-norlaudanosoline
  • the substrate can be a benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound (5]-6-0-methyl- norlaudanosoline, wherein the enzymes are selected from norcoclaurine synthase (NCS] and 60MT.
  • the product benzylisoquinoline compound can be (S]-6-0-methyl-norlaudanosoline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-6-0-methyl-norlaudanosoline
  • the enzymes can be selected from 60MT or norcoclaurine synthase (NCS]
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the host cell can further include an electron transfer facilitating protein.
  • the electron transfer facilitating protein can be a cytochrome P450 reductase (CPR]
  • the substrate can be produced in the medium by a cell for co-culturing with the host cell, wherein the co-cultured cell is capable of secreting the substrate into the medium.
  • the cell for co-culturing can be a cell that does not comprise the benzylisoquinoline biosynthetic enzyme complement to produce the product benzylisoquinoline compound.
  • the host cell can be a cell that does not produce the substrate compound.
  • the benzylisoquinoline uptake permease can be a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of
  • SEQ.ID NO: 1 SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
  • nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
  • nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • (B) a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • SEQ.ID NO: 23 SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37; SEQ.ID NO: 39; and SEQ.ID NO: 55
  • nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a]; (e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56; and
  • (B) a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • the product benzylisoquinoline compound can be further converted in the host cell to form a derivative benzylisoquinoline.
  • the derivative benzylisoquinoline can be neopinone, codeine, codeinone or morphine.
  • the conversion to form a derivative benzylisoquinoline can involve the performance of an enzyme-catalyzed reaction by one or more of the enzymes T6-0-demethylase (T60DM], neopinone isomerase (NISO], codeinone reductase (COR], and codeinone-O-demethylase (CODM]
  • T6-0-demethylase T60DM
  • NISO neopinone isomerase
  • COR codeinone reductase
  • CODM codeinone-O-demethylase
  • the method can further include a step comprising recovering the product benzylisoquinoline compound or the derivative benzylisoquinoline.
  • the host cell can be a microbial cell.
  • the host cell can be a bacterial cell.
  • the host cell can be a yeast cell.
  • the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
  • the host cell can be an algal cell.
  • the host cell can be a plant cell.
  • the host cell and cell for co-culturing can be cells belonging to the same biological species.
  • the host cell and cell for co-culturing can be microbial cells.
  • the host cell and cell for co-culturing can be bacterial cells.
  • the host cell and cell for co-culturing can be yeast cells.
  • the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
  • the host cell and cell for co-culturing can be algal cells.
  • the host cell and cell for co-culturing can be plant cells.
  • the cell for co-culturing can comprise a benzylisoquinoline uptake permease.
  • the present disclosure provides, in at least one aspect, a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds, the host cell comprising a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell, and the host cell capable of producing the benzylisoquinoline uptake permease and a product benzylisoquinoline compound when provided with a substrate compound.
  • the product benzylisoquinoline compound can be (5]-norcoclaurine, (5]-norlaudanosoline, (S]-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£ ⁇ - reticuline, (7?] -reticuline, salutaridine, salutaridinol, or thebaine.
  • the substrate can be a substrate benzylisoquinoline compound.
  • the substrate benzylisoquinoline compound can be (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S]-coclaurine, (5]-/V-methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (£ ⁇ - reticuline, (7?] -reticuline, salutaridine, or salutaridinol.
  • the substrate can be a substrate benzylisoquinoline precursor compound.
  • the substrate benzylisoquinoline precursor compound can be L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydrox- phenylacetaldehyde (4-HPAA]
  • the enzyme complement can comprise one or more benzylisoquinoline biosynthetic enzymes, wherein the enzymes can be norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0-acetyltransferase (SalAT], or thebaine synthase (TS]
  • NCS norcoclaurine synthase
  • 60MT 6-0-methyltransferase
  • CNMT coclaurine-/V- methyltransferase
  • NMCH coclaurine-/V-methyltransferase
  • NMCH coclaur
  • the host cell further can have a benzylisoquinoline precursor enzyme complement.
  • the benzylisoquinoline precursor enzyme complement can comprise one or more benzylisoquinoline precursor biosynthetic enzymes, wherein the enzymes can be (TYR], dihydroxyphenyl alanine decarboxylase DODC, tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • the substrate can be converted by the host cell into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
  • the substrate can be converted by the host cell into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
  • the benzylisoquinoline uptake permease can be a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • (B) a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • SEQ.ID NO: 23 SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37, SEQ.ID NO: 39, and SEQ.ID NO: 55;
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and
  • (B) a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • the host cell can be a microbial cell.
  • the host cell can be a bacterial cell.
  • the host cell can be a yeast cell.
  • the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
  • the host cell can be an algal cell.
  • the host cell can be a plant cell.
  • the present disclosure provides, in at least one embodiment, a mixture of cells comprising host cells recombinantly expressing benzylisoquinoline uptake permease and having a benzylisoquinoline enzyme complement to biosynthetically produce a product benzylisoquinoline compound, and cells for co-culturing with the host cells, wherein the cells for co-culturing are capable of secreting a substrate that can be converted by the host cells when co cultured to form the product benzylisoquinoline compound.
  • the cells for co-culturing can be cells that do not include the benzylisoquinoline enzyme complement to produce the benzylisoquinoline product.
  • the host cells do not produce the substrate.
  • the host cells and cells for co-culturing can be cells belonging to the same biological species.
  • the host cells or cells for co-culturing can be microbial cells.
  • the host cells or cells for co-culturing can be bacterial cells.
  • the host cells or cells for co-culturing can be yeast cells.
  • the yeast cells can be Saccharomyces cerevisiae cell or Yarrowia lipolytica cells.
  • the host cells or cells for co-culturing can be algal cells.
  • the host cells or cells for co-culturing can be plant cells.
  • the cells for co-culturing can comprise a benzylisoquinoline uptake permease.
  • the present disclosure provides, in at least one embodiment, a use of a cell according to the present disclosure to convert a substrate and form a product benzylisoquinoline compound.
  • the present disclosure provides, in at least one embodiment, a use of a mixture of cells comprising host cells and cells for co-culturing with the host cells according to the present disclosure to convert a substrate and form a product benzylisoquinoline compound.
  • the present disclosure provides, in at least one embodiment, a product benzylisoquinoline compound produced in accordance with any one of the methods of the present disclosure.
  • FIGS. 1A, IB and 2C show prototype structures of benzylisoquinoline compounds.
  • FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H depict the chemical structures of some benzylisoquinoline compounds, notably (5]-norcoclaurine (FIG. 2 A], (5]- coclaurine (FIG. 2B], (5]-/V-methylcoclaurine (FIG. 2C], (S]-3’-hydroxy-/V- methylcoclaurine (FIG. 2D], (5]-reticuline (FIG. 2E], (7?]-reticuline (FIG. 2F], (5]- norlaudanosoline (FIG. 2G], and (S]-6-0-methyl-norlaudanosoline (FIG. 2H]
  • FIGS. 3A, 3B, 2C, 3D, 3E, 3F and 3G depict the chemical structures of some other benzylisoquinoline compounds, notably salutaridine (FIG. 3A], salutaridinol (FIG. 3B], thebaine (FIG. 3C], codeinone (FIG. 3D], codeine (FIG. 3E], morphine (FIG. 3F] and neopinone (FIG. 3G]
  • FIG. 4A, 4B, 4C, 4D, 4E and 4F depict the chemical structures of some benzylisoquinoline precursor compounds, notably L-tyrosine (FIG. 4A], L- dihydroxyphenyl alanine (L-DOPA] (FIG. 4B], dopamine (FIG. 4C], tyramine (FIG. 4D], 4-hydroxyphenylacetaldehyde (4-HPAA] (FIG. 4E] and 3,4- hydroxyphenylacetaldehyde (3, 4-HPAA]
  • FIG. 5 depicts some chemical reactions involving the conversion of benzylisoquinoline compounds.
  • FIG. 6 depicts some chemical reactions involving the conversion of benzylisoquinoline precursor compounds.
  • FIG. 7 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising L-DOPA as a substrate.
  • FIG. 8 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising dopamine as a substrate.
  • FIG. 9 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising norlaudanosoline as a substrate.
  • FIG. 10 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising L-DOPA as a substrate.
  • FIG. 11 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising dopamine as a substrate.
  • FIG. 12 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising norlaudanosoline as a substrate.
  • FIG. 13A-F depicts certain experimental results obtained, notably uptake of alkaloid compounds (y-axes] by Saccharomyces cerevisiae transformed with one of six plasmids, allowing expression of BUP1 (13A], BUP2 (13B], BUP3 (13C], BUP4 (13D], BUP5 (13E], BUP6 (13F], respectively.
  • Substrates provided exogenously in the media and measured post-incubation in cellular extract are indicated on the x-axes. Black bars and grey-shaded bars represent empty vector and BUP-expressing strains, respectively. Standard error was calculated using four biological replicates representing four individual transformants.
  • FIG. 14A-H depict certain experimental results obtained, notably production of alkaloid compounds by three different Saccharomyces cerevisiae strains (strain 1 (14A-C], strain 2 (14D-F], strain 3 (14G-H]] transformed with one of nine plasmids, allowing expression of BUP1, BUP2, BUP3, BUP4, BUP5, BUP6, BUP7, BUP8, and BUP 9, respectively (x-axes].
  • Exogenously fed substrate compounds are: DOPA (FIG. 14A], dopamine (FIG. 14B], norlaudanosoline (FIG. 14C], (5]- reticuline (FIG. 14D], (7?]-reticuline (FIG. 14E], salutaridine (FIG.
  • FIG. 15 depicts certain experimental results obtained, notably production of alkaloid compounds by 2 different combinations of co-cultured Saccharomyces cerevisiae strains: strain 1 and strain 2 (FIG. 15A and FIG. 15C] and strain 1, strain 2 and strain 3 (FIG. 15B and FIG. 15D).
  • Alkaloids produced are reticuline, salutaridine, thebaine and codeine (FIG. 15A and FIG. 15B] and salutaridine, thebaine and codeine (FIG. 15C and FIG. 15D] (x-axes).
  • Substrates used are L-DOPA (FIG. 15A and FIG. 15B] or S-reticuline (FIG. 15C and FIG. 15D). Strains were transformed with BUP 1 (right hand side of each of FIGS. 15A-15D] or with empty vector (left hand side of each of FIGS. 15A-15D], as indicated.
  • compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below.
  • the claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter.
  • compositions, system or process described below may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
  • compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below.
  • the claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter.
  • compositions, system or process described below may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
  • any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed (e.g . a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5]
  • other terms of degree such as “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.
  • the terms“benzylisoquinoline”, or“benzylisoquinoline compound”, as may be used interchangeably herein are chemical compounds having the prototype structure set forth in FIGS. 1A, IB or 1C. It is noted that certain ring closure reactions of compounds having the prototype structure shown in FIG. 1A can lead to the formation of compounds having the prototype structure shown in FIG. IB or FIG. 1C.
  • Benzylisoquinoline compounds are further intended to include, without limitation, (S]-norcoclaurine, (S]-coclaurine, (5]-/V-methylcoclaurine, (S]-3’-hydroxy-/V- methylcoclaurine, (S]-reticuline, (7?]-reticuline, salutaridine, salutaridinol, thebaine, codeinone, codeine, and morphine.
  • benzylisoquinoline precursor compound refers to a chemical compound that can be converted into a benzylisoquinoline compound and includes, L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4- hydroxy-phenylacetaldehyde (3,4-HPAA] and 4-hydrox-phenylacetaldehyde (4- HPAA]
  • Benzylisoquinoline precursor compounds include compounds that can be directly converted into benzylisoquinoline compound, or compounds that can be converted into benzylisoquinoline compound via another precursor benzylisoquinoline compound.
  • (S]-norcoclaurine refers to a chemical compound having the chemical structure set forth in FIG. 2A.
  • (S]-coclaurine refers to a chemical compound having the chemical structure set forth in FIG. 2B.
  • (S]-/V-methylcoclaurine refers to a chemical compound having the chemical structure set forth in FIG. 2C.
  • (S]-3’-hydroxy-/V-methylcoclaurine refers to a chemical compound having the chemical structure set forth in FIG. 2D.
  • (S]-reticuline refers to a chemical compound having the chemical structure set forth in FIG. 2E.
  • (7?]-reticuline refers to a chemical compound having the chemical structure set forth in FIG. 2F.
  • (S]-norlaudanosoline refers to a chemical compound having the chemical structure set forth in FIG. 2G.
  • (5]-6-0-methyl-norlaudanosoline refers to a chemical compound having the chemical structure set forth in FIG. 2H.
  • salutaridine refers to a chemical compound having the chemical structure set forth in FIG. 3A.
  • salutaridinol refers to a chemical compound having the chemical structure set forth in FIG. 3B.
  • the term "thebaine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3C.
  • codeinone refers to a chemical compound having the chemical structure set forth in FIG. 3D.
  • codeine refers to a chemical compound having the chemical structure set forth in FIG. 3E.
  • morphine refers to a chemical compound having the chemical structure set forth in FIG. 3F.
  • neopinone refers to a chemical compound having the chemical structure set forth in FIG. 3G.
  • L-tyrosine refers to a chemical compound having the chemical structure set forth in (FIG. 4A]
  • L-dihydroxyphenyl alanine or "L-DOPA”, as may be used interchangeable herein, refers to a chemical compound having the chemical structure set forth in (FIG. 4B],
  • dopamine refers to a chemical compound having the chemical structure set forth in FIG. 4C.
  • tyramine refers to a chemical compound having the chemical structure set forth in FIG. 4D.
  • benzylisoquinoline biosynthetic enzyme refers to a polypeptide capable of facilitating the chemical conversion of a first benzylisoquinoline compound into a second benzylisoquinoline compound, or the chemical conversion of a benzylisoquinoline precursor compound into a benzylisoquinoline compound in a single chemical step.
  • Benzylisoquinoline biosynthetic enzymes include norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’- hydroxylase (NMCH], 4’-0-methyltransferase (4 ⁇ MT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0- acetyltransferase (SalAT], and thebaine synthase (TS]
  • benzylisoquinoline precursor enzyme complement refers to one or more benzylisoquinoline precursor biosynthetic enzymes, which together when provided with a substrate molecule, can produce a compound that can act as a substrate compound for a benzylisoquinoline biosynthetic enzyme.
  • Benzylisoquinoline precursor biosynthetic enzymes that can be included in a benzylisoquinoline precursor enzyme complement include tyrosine hydroxylase (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
  • 6-0-methyltransferase or "60MT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any 6-0-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 24, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any 6-0-methyltransferase polypeptide set forth herein, but for the use of synonymous codons.
  • coclaurine-/V-methyltransferase or "CNMT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any coclaurine-/V-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 26, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any coclaurine-methyltransferase polypeptide set forth herein, but for the use of synonymous codons.
  • (S]-/V-methylcoclaurine 3’-hydroxylase or "NMCH”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any (5]-/V-methylcoclaurine 3’-hydroxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 28, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide set forth herein, but for the use of synonymous codons.
  • 4’-0-methyltransferase or “4 ⁇ MT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any 4’-0-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 30, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any 4’-0-methyltransferase set forth herein, but for the use of synonymous codons.
  • reticuline epimerase or “REPI”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any reticuline epimerase polypeptide set forth herein, including, for example, SEQ.ID NO: 32, or (if) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any reticuline epimerase polypeptide set forth herein, but for the use of synonymous codons.
  • salutaridine synthase or “SalSyn”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridine synthase polypeptide set forth herein, including, for example, SEQ.ID NO: 34, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutaridine synthase polypeptide set forth herein, but for the use of synonymous codons.
  • salutaridine reductase or “SalR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridine reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 36, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutaridine reductase polypeptide set forth herein, but for the use of synonymous codons.
  • salutaridinol-7-0-acetyltransferase or “SalAT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridinol-7-0-acetyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 38, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutardinol-7-O-acetyl transferase polypeptide set forth herein, but for the use of synonymous codons.
  • the terms "thebaine synthase” or "TS”, as maybe used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any thebaine synthase polypeptide set forth herein, including, for example, SEQ.ID NO: 40, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any thebaine synthase polypeptide set forth herein, but for the use of synonymous codons.
  • neopinone isomerase or "NISO”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any neopinone isomerase polypeptide set forth herein, including, for example, SEQ.ID NO: 42, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any neopinone isomerase polypeptide set forth herein, but for the use of synonymous codons.
  • codeinone reductase or “COR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeinone reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 44, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeinone reductase polypeptide set forth herein, but for the use of synonymous codons.
  • codeine-O-demethylase or “CODM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeine-O-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 46, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeine-O-demethylase polypeptide set forth herein, but for the use of synonymous codons.
  • T6-0-demethylase or "T60DM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any T6-0-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 58, or (if) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any T6-0-demethylase polypeptide set forth herein, but for the use of synonymous codons.
  • cytochrome P450 reductase or “CPR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any set cytochrome P450 reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 60, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any cytochrome P450 reductase polypeptide set forth herein, but for the use of synonymous codons.
  • benzylisoquinoline uptake permease BUP
  • purine permease or “PUP” refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any benzylisoquinoline uptake permease polypeptide set forth herein, including, for example. SEQ.ID NO: 2, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any benzylisoquinoline uptake permease polypeptide set forth herein, but for the use of synonymous codons.
  • tyrosine hydroxylase refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any tyrosine hydroxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 48, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tyrosine hydroxylase polypeptide set forth herein, but for the use of synonymous codons.
  • tyrosine decarboxylase or "TYDC”, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any tyrosine decarboxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 50, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tyrosine decarboxylase polypeptide set forth herein, but for the use of synonymous codons.
  • dihydroxyphenyl alanine decarboxylase refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any dihydroxyphenyl alanine decarboxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 52, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any dihydroxyphenyl alanine decarboxylase set forth herein, but for the use of synonymous codons.
  • monoamide oxidase or “MAO”, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any monoamide oxidase polypeptide set forth herein, including, for example, SEQ.ID NO: 54, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any monoamide oxidase polypeptide set forth herein, but for the use of synonymous codons.
  • NCS noncoclaurine synthase
  • NCS noncoclaurine synthase
  • SEQ.ID NO: 56 or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any norcoclaurine polypeptide set forth herein, but for the use of synonymous codons.
  • nucleic acid sequence encoding 6 -0-methyl transferase refers to any and all nucleic acid sequences encoding a 6-0-methyltransferase polypeptide, including, for example, SEQ.ID NO: 23.
  • Nucleic acid sequences encoding a 6-0-methyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the 6-0-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any 6-0-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding coclaurine-iV- methyltransferase refers to any and all nucleic acid sequences encoding a coclaurine-iV-methyltransferase polypeptide, including, for example, SEQ.ID NO: 25.
  • Nucleic acid sequences encoding a coclaurine-/V-methyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the coclaurine-/V-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any coclaurine-iV-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding (S]-/V-methylcoclaurine 3’- hydroxylase refers to any and all nucleic acid sequences encoding a (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide, including, for example, SEQ.ID NO: 27.
  • Nucleic acid sequences encoding a (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the (5]-/V-methylcoclaurine 3’-hydroxylase polypeptide sequences set forth herein; or (ii] hybridize to any (S]-/V-methylcoclaurine 3’-hydroxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding 4’ -O-methyl transferase refers to any and all nucleic acid sequences encoding a 4’-0-methyltransferase polypeptide, including, for example, SEQ.ID NO: 29.
  • Nucleic acid sequences encoding a 4’ -O-methyl transferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the 4’-0-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any 4’-0-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding reticuline epimerase refers to any and all nucleic acid sequences encoding a reticuline epimerase polypeptide, including, for example, SEQ.ID NO: 31.
  • Nucleic acid sequences encoding a reticuline epimerase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the reticuline epimerase polypeptide sequences set forth herein; or (ii] hybridize to any reticuline epimerase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding salutaridine synthase refers to any and all nucleic acid sequences encoding a salutaridine synthase polypeptide, including, for example, SEQ.ID NO: 33.
  • Nucleic acid sequences encoding a salutaridine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridine synthase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding salutaridine reductase refers to any and all nucleic acid sequences encoding a salutaridine reductase polypeptide, including, for example, SEQ.ID NO: 35.
  • Nucleic acid sequences encoding a salutaridine reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridine reductase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridine reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding salutaridinol-7-0- acetyltransferase refers to any and all nucleic acid sequences encoding a salutaridinol-7-0-acetyltransferase polypeptide, including, for example, SEQ.ID NO: 37.
  • Nucleic acid sequences encoding a salutaridinol-7-0-acetyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridinol-7-0-acetyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridinol-7-0-acetyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding thebaine synthase refers to any and all nucleic acid sequences encoding a thebaine synthase polypeptide, including, for example, SEQ.ID NO: 39.
  • Nucleic acid sequences encoding a thebaine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the thebaine synthase polypeptide sequences set forth herein; or (ii] hybridize to any thebaine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding neopinone isomerase refers to any and all nucleic acid sequences encoding a neopinone isomerase polypeptide, including, for example, SEQ.ID NO: 41.
  • Nucleic acid sequences encoding a neopinone isomerase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the neopinone isomerase polypeptide sequences set forth herein; or (ii] hybridize to any neopinone isomerase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding codeinone reductase refers to any and all nucleic acid sequences encoding a codeinone reductase polypeptide, including, for example, SEQ.ID NO: 43.
  • Nucleic acid sequences encoding a codeinone reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeinone reductase polypeptide sequences set forth herein; or (ii] hybridize to any codeinone reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding codeine-O-demethylase refers to any and all nucleic acid sequences encoding a codeine-O-demethylase polypeptide, including, for example, SEQ.ID NO: 45.
  • Nucleic acid sequences encoding a codeine-O-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeine-O-demethylase polypeptide sequences set forth herein; or (ii] hybridize to any codeine-O-demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding T6-0-demethylase refers to any and all nucleic acid sequences encoding a codeine-O-demethylase polypeptide, including, for example, SEQ.ID NO: 57.
  • Nucleic acid sequences encoding a T6-0-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the T6-0-demethylase polypeptide sequences set forth herein; or (ii] hybridize to any T6-0-demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding cytochrome P450 reductase refers to any and all nucleic acid sequences encoding a cytochrome P450 reductase polypeptide, including, for example, SEQ.ID NO: 59.
  • Nucleic acid sequences encoding a cytochrome P450 reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the cytochrome P450 reductase polypeptide sequences set forth herein; or (ii] hybridize to any cytochrome P450 reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding a benzylisoquinoline uptake permease refers to any and all nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide, including, for example, SEQ.ID NO: 1.
  • Nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the benzylisoquinoline uptake permease polypeptide sequences set forth herein; or (ii] hybridize to any benzylisoquinoline uptake permease nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding tyrosine hydroxylase refers to any and all nucleic acid sequences encoding a tyrosine hydroxylase polypeptide, including, for example, SEQ.ID NO: 47.
  • Nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the tyrosine hydroxylase polypeptide sequences set forth herein; or (ii] hybridize to any tyrosine hydroxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding tyrosine decarboxylase refers to any and all nucleic acid sequences encoding a tyrosine decarboxylase polypeptide, including, for example, SEQ.ID NO: 49.
  • Nucleic acid sequences encoding a tyrosine decarboxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the tyrosine decarboxylase polypeptide sequences set forth herein; or (ii] hybridize to any tyrosine decarboxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding dihydroxyphenyl alanine decarboxylase refers to any and all nucleic acid sequences encoding a dihydroxyphenyl alanine decarboxylase polypeptide, including, for example, SEQ.ID NO: 51.
  • Nucleic acid sequences encoding a dihydroxyphenyl alanine decarboxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the dihydroxyphenyl alanine decarboxylase polypeptide sequences set forth herein; or (ii] hybridize to any dihydroxyphenyl alanine decarboxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding monoamine oxidase refers to any and all nucleic acid sequences encoding a monoamine oxidase polypeptide, including, for example, SEQ.ID NO: 53.
  • Nucleic acid sequences encoding a monoamine oxidase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the monoamine oxidase polypeptide sequences set forth herein; or (ii] hybridize to any monoamine oxidase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid sequence encoding norcoclaurine synthase refers to any and all nucleic acid sequences encoding a norcoclaurine synthase polypeptide, including, for example, SEQ.ID NO: 55.
  • Nucleic acid sequences encoding a norcoclaurine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the norcoclaurine synthase polypeptide sequences set forth herein; or (ii] hybridize to any norcoclaurine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • nucleic acid refers to a sequence of nucleoside or nucleotide monomers, consisting of naturally occurring bases, sugars and intersugar (backbone] linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof.
  • the nucleic acids of the present disclosure may be deoxyribonucleic nucleic acids (DNA] or ribonucleic acids (RNA] and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The nucleic acids may also contain modified bases.
  • modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine.
  • a sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, nucleic acid sequence, a nucleotide sequence or a nucleoside sequence.
  • polypeptide refers to any and all polypeptides comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequence constituting the polypeptide having such reference SEQ.ID NO, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the polypeptide having such reference SEQ.ID NO, but for the use of synonymous codons.
  • a sequence of amino acid residues may be referred to as an amino acid sequence, or polypeptide sequence.
  • nucleic acid sequence encoding a polypeptide refers to any and all nucleic acid sequences encoding a polypeptide having such reference SEQ.ID NO.
  • Nucleic acid sequences encoding a polypeptide, in conjunction with a reference SEQ.ID NO further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the polypeptide having such reference SEQ.ID NO; or (ii) hybridize to any nucleic acid sequences encoding polypeptides having such reference SEQ.ID NO under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
  • substantially identical it is meant that two amino acid sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical.
  • amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences.
  • a particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22]: 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
  • the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature.
  • a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C.
  • stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate
  • a functional variant refers to polynucleotides or polypeptides capable of performing the same function as a noted reference polynucleotide or polypeptide.
  • a functional variant of the polypeptide set forth in SEQ.ID NO: 2 refers to a polypeptide capable of performing the same function as the polypeptide set forth in SEQ.ID NO: 2.
  • Functional variants include modified a polypeptide wherein, relative to a noted reference polypeptide, the modification includes a substitution, deletion or addition of one or more amino acids. In some embodiments, substitutions are those that result in a replacement of one amino acid with an amino acid having similar characteristics.
  • substitutions include, without limitation (i] glutamic acid and aspartic acid; (i] alanine, serine, and threonine; (iii] isoleucine, leucine and valine, (iv] asparagine and glutamine, and (v] tryptophan, tyrosine and phenylalanine.
  • Functional variants further include polypeptides having retained or exhibiting an enhanced benzylisoquinoline biosynthetic bioactivity.
  • Chimeric nucleic acids refers to at least two linked nucleic acids which are not naturally linked.
  • Chimeric nucleic acids include linked nucleic acids of different natural origins.
  • a nucleic acid constituting a microbial promoter linked to a nucleic acid encoding a plant polypeptide is considered chimeric.
  • Chimeric nucleic acids also may comprise nucleic acids of the same natural origin, provided they are not naturally linked.
  • a nucleic acid constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid encoding a polypeptide obtained from that same cell- type, but not normally linked to the nucleic acid constituting the promoter.
  • Chimeric nucleic acids also include nucleic acids comprising any naturally occurring nucleic acids linked to any non-naturally occurring nucleic acids.
  • substantially pure and isolated describe a compound, e.g., a benzylisoquinoline, polynucleotide or a polypeptide, which has been separated from components that naturally accompany it.
  • a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction] in a sample is the compound of interest.
  • Purity can be measured by any appropriate method, e.g., in the case of polypeptides, by chromatography, gel electrophoresis or HPLC analysis.
  • recovered refers to a more or less pure form of the enzyme, protein alkaloid or benzylisoquinoline.
  • the present disclosure relates to alkaloids.
  • the current disclosure further relates to certain nucleic acids and polypeptides that can be used to make alkaloids.
  • the herein provided methods and compositions are useful in that they facilitate a novel and efficient means of making certain alkaloid compounds, notably benzylisoquinolines.
  • the methods and compositions can yield substantial quantities of product benzylisoquinoline compounds, including codeinone, codeine, morphine, and other benzylisoquinoline compounds.
  • the current disclosure involves the use of benzylisoquinoline uptake permeases to facilitate the production of benzylisoquinoline compounds.
  • the present disclosure provides a method of making benzylisoquinoline compounds in a host cell comprising a benzylisoquinoline uptake permease.
  • the benzylisoquinoline uptake permease can be recombinantly expressed in the host cell.
  • the product benzylisoquinoline compound can be produced from a substrate benzylisoquinoline compound or a precursor thereof.
  • the substrate benzylisoquinoline compound or the precursor of the substrate benzylisoquinoline compound can be included in the growth medium for the host cell.
  • the disclosure provides methods of making benzylisoquinoline compounds involving co-culturing of a host cell comprising a benzylisoquinoline uptake permease, and a cell for co-culturing.
  • the cells for co-culturing comprise a biosynthetic capacity to produce substrate benzylisoquinoline compound, or optionally substrate benzylisoquinoline precursor compounds, which can be used as a substrate by the host cell. It has been found by the inventors that benzylisoquinoline compounds can be unexpectedly efficiently produced in cells comprising benzylisoquinoline uptake permeases.
  • the cells may be used as a source whence the benzylisoquinoline compounds can economically be extracted.
  • the benzylisoquinoline compounds produced in accordance with the present disclosure are useful inter alia in the manufacture of pharmaceutical compositions.
  • the present disclosure provides, in at least one aspect, and in at least one embodiment, a method of producing a product benzylisoquinoline compound in a host cell, the method comprising:
  • a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell; and
  • a variety of compounds can be used as substrates to provide to the host cell for the biosynthetic production of a product benzylisoquinoline compound by the conversion of the substrate into a variety of product benzylisoquinoline compounds.
  • the substrate can be an alkaloid compound.
  • the substrate can be a substrate benzylisoquinoline compound.
  • the substrate can be a substrate precursor benzylisoquinoline compound.
  • (S]-norcoclaurine can be a substrate benzylisoquinoline compound that can be converted into the product benzylisoquinoline compound (5]- coclaurine; or the substrate benzylisoquinoline compound (S]-coclaurine can be converted into the product benzylisoquinoline compound (S]-/V-methylcoclaurine.
  • a substrate benzylisoquinoline compound can any benzylisoquinoline compound that can be converted into another benzylisoquinoline compound, namely the product benzylisoquinoline compound, in a single chemical step, i.e. a step forming no or no substantial amounts of free stable intermediate compounds.
  • a substrate benzylisoquinoline compound can any benzylisoquinoline compound that can be converted into another benzylisoquinoline compound, namely the product benzylisoquinoline compound, in two or more chemical steps, for example, 3, 4, 5, 6, or 7 steps, and each step forming a stable intermediate compound, notably a stable intermediate benzylisoquinoline compound.
  • a variety of substrate benzylisoquinoline precursor compounds can be used to provide to the host cell for the biosynthetic production of a product benzylisoquinoline compound, by the initial conversion of the substrate benzylisoquinoline precursor compound to a substrate benzylisoquinoline compound, and the subsequent conversion of the substrate benzylisoquinoline compound into a product benzylisoquinoline compounds.
  • FIG. 6 shown therein is an example biosynthetic pathway showing the conversion of certain alkaloid compounds, notably substrate benzylisoquinoline precursor compounds into other substrate benzylisoquinoline precursor compounds, in a specified order.
  • the substrate benzylisoquinoline precursor compound L-tyrosine can be converted into another substrate benzylisoquinoline precursor compound, L-DOPA; or the substrate benzylisoquinoline precursor compound L-DOPA can be converted into another substrate benzylisoquinoline precursor compound, dopamine.
  • a substrate benzylisoquinoline precursor compound can be any compound, that can be converted into another substrate benzylisoquinoline precursor compound, in a single chemical step, i.e. a step forming no or no substantial amounts of free stable intermediate compounds.
  • Those of skill in the art will be familiar with other substrate benzylisoquinoline precursor compounds and benzylisoquinoline biosynthetic pathways and thus will be able to identify other substrate benzylisoquinoline precursor compounds.
  • the conversion from a substrate benzylisoquinoline compound into a product benzylisoquinoline compound can be catalyzed, in different embodiments, by benzylisoquinoline biosynthetic enzymes, including norcoclaurine synthase (NCS), 6-O-methyltransferase (60MT), coclaurine-/V-methyltransferase (CNMT), (S)- /V-methylcoclaurine 3’-hydroxylase (NMCH), 4’-0-methyltransferase (4 ⁇ MT), reticuline epimerase (REPI), salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridinol-7-O-acetyltransferase (SalAT), or by a thebaine synthase (TS).
  • NCS norcoclaurine synthase
  • 60MT 6-O-methyltransferase
  • CNMT coclaurine-/V-methyl
  • the conversion of a substrate benzylisoquinoline precursor compound into another substrate benzylisoquinoline precursor compound can be catalyzed, in different embodiments, by a benzylisoquinoline precursor enzyme complement.
  • the benzylisoquinoline precursor enzyme complement can include one or more of the following benzylisoquinoline precursor enzymes: tyrosine hydroxylase (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC), and monoamide oxidase (MAO]
  • benzylisoquinoline compounds can be made including, for example, (5) -norcoclaurine, (5]- norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S)-coclaurine, ( S]-N - methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (S]-reticuline, (/? ⁇ -reticuline, salutaridine, salutaridinol and thebaine.
  • a variety of substrates can be selected and combined with a cell which has a compatible biosynthetic enzyme capacity to convert the substrate into the product benzylisoquinoline compound, in a similar manner as various substrates can be selected and combined with a cell with a compatible biosynthetic enzyme capacity to convert these substrates into salutaridine, as hereinafter described.
  • salutaridine can be made using (7?]-reticuline as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzyme SalSyn.
  • salutaridine can be made using (S]-reticuline as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes REPI and SalSyn.
  • salutaridine can be made using (5]-3’-hydroxy-/V-methylcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using (S]-/V-methylcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using (S]-coclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using (5]-norcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using dopamine and 4-HPAA as a substrate benzylisoquinoline precursor compounds, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using dopamine and 3, 4-HPAA as a substrate benzylisoquinoline precursor compounds, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using L-DOPA and dopamine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme MAO.
  • salutaridine can be made using L-DOPA and tyramine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme MAO.
  • salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR, DODC and MAO.
  • a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR, DODC and MAO.
  • salutaridine can be made using L-DOPA as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes DODC and MAO.
  • a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes DODC and MAO.
  • salutaridine can be made using dopamine and L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR and MAO.
  • a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR and MAO.
  • salutaridine can be made using tyramine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme TYDC.
  • salutaridine can be made using dopamine and L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme TYDC.
  • a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme TYDC.
  • salutaridine can be made using tyramine and dopamine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn.
  • salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, DODC, and MAO.
  • a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, DODC, and MAO.
  • salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, and DODC.
  • a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI and SalSyn
  • the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, and DODC.
  • the product benzylisoquinoline salutaridine can be made in accordance with the methods of present disclosure using a variety of substrate benzylisoquinoline compounds or substrate precursor benzylisoquinoline compounds in combination with a cell including a compatible benzylisoquinoline enzyme complement and, optionally, a benzylisoquinoline precursor enzyme complement.
  • benzylisoquinoline compounds that can be made in accordance herewith are (5)- norcoclaurine, (S)-norlaudanosoline, (S)-6-0-methyl-norlaudanosoline, (5)- coclaurine, (S)-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (Sj- reticuline, (R)-reticuline, salutaridine, salutaridinol and thebaine
  • the present disclosure further includes methods to make these benzylisoquinoline compounds.
  • the product benzylisoquinoline can be thebaine
  • the substrate can be a benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the thebaine precursor compound into thebaine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, SalSyn, SalR, SalAT and TS.
  • the product benzylisoquinoline compound can be thebaine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, SalSyn, SalR, SalAT and TS and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be salutaridinol
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridinol, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, SalSyn, and SalR.
  • the product benzylisoquinoline compound can be salutaridinol
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridinol, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, SalSyn, and SalR
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be salutaridine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, and SalSyn.
  • the product benzylisoquinoline compound can be salutaridine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT, REPI, and SalSyn
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (R)-reticuline
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (R)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT and REPI.
  • the product benzylisoquinoline compound can be (R)-reticuline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (R)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4 ⁇ MT and REPI
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (5]-reticuline
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (S)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, and 4 ⁇ MT.
  • the product benzylisoquinoline compound can be (5]-reticuline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, and 4 ⁇ MT
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (S)-3’-hydroxy-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (5]-3’-hydroxy-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, CNMT, and NMCH.
  • the product benzylisoquinoline compound can be (S)-3’-hydroxy-/V- methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-3’- hydroxy-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, CNMT, and NMCH
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (5]-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S)-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, and CNMT.
  • the product benzylisoquinoline compound can be (5]-/V-methylcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, and CNMT and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (S)-coclaurine
  • the substrate can be a benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S)-coclaurine, wherein the enzymes are selected from NCS and 60MT.
  • the product benzylisoquinoline compound can be (5)-coclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S)-coclaurine, wherein the enzymes are selected from NCS and 60MT and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (5) -norcoclaurine
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5) -norcoclaurine
  • the enzyme is NCS and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
  • the product benzylisoquinoline compound can be (5)-norlaudanosoline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5)-norlaudanosoline
  • the enzyme can be norcoclaurine synthase (NCS)
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC) and monoamide oxidase (MAO).
  • the product benzylisoquinoline compound can be (S)-6-0-methyl-norlaudanosoline
  • the substrate can be a benzylisoquinoline compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound (5)-6-0-methyl-norlaudanosoline, wherein the enzymes are selected from norcoclaurine synthase (NCS) and 60MT.
  • NCS norcoclaurine synthase
  • the product benzylisoquinoline compound can be (S)-6-0-methyl-norlaudanosoline
  • the substrate can be a substrate benzylisoquinoline precursor compound
  • the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S)-6-0-methyl-norlaudanosoline
  • the enzymes can be selected from 60MT and norcoclaurine synthase (NCS)
  • the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC) and monoamide oxidase (MAO).
  • the host cell can further comprise an electron transfer facilitating protein.
  • the electron transfer facilitating protein can be a cytochrome P450 reductase (CPR).
  • CPR cytochrome P450 reductase
  • the cells include a cytochrome P450 reductase (CPR).
  • the CPR polypeptide may be naturally present in the host cell, or a nucleic acid expressing a CPR polypeptide, including SEQ.ID NO: 60 and SEQ.ID NO: 62, and including, a nucleic acid comprising nucleic acid sequences SEQ.ID NO: 59 and SEQ.ID NO: 61, may be introduced into the host cell, to thereby recombinantly produce the CPR polypeptide in the host cell.
  • the product upon the cell having produced the product benzylisoquinoline compound, the product can be further converted in the host cell to form a derivative benzylisoquinoline.
  • the product benzylisoquinoline compound can be thebaine, and the derivative benzylisoquinoline can be codeine, codeinone or morphine.
  • the conversion to form a derivative benzylisoquinoline can involve the performance of an enzyme catalyzed reaction by one or more of the enzymes T60DM, NISO, COR, and CODM.
  • the product benzylisoquinoline compound or the derivative benzylisoquinoline can be recovered, for example, by obtaining the medium and separating the product benzylisoquinoline compound or the derivative benzylisoquinoline from other medium constituents.
  • a variety of purification techniques and methodologies may be used, as will be known to those of skill in the art, for example chromatographical techniques, and in this manner a substantially pure product benzylisoquinoline compound or the derivative benzylisoquinoline may be obtained.
  • the host cell includes a benzylisoquinoline uptake permease.
  • a variety of benzylisoquinoline uptake permeases can be used.
  • the benzylisoquinoline uptake permease can a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:
  • SEQ.ID NO: 1 SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
  • the cell is selected or modulated to comprise a biosynthetic capability that can convert the substrate to form the product benzylisoquinoline compound.
  • the host cell can be any cell capable of producing a product benzylisoquinoline compound, including any microbial cell, plant cell or algal cell.
  • the benzylisoquinoline biosynthetic enzyme or the benzylisoquinoline precursor biosynthetic enzyme can be naturally present therein.
  • the host cell can be a cell that has been modulated to produce the product benzylisoquinoline compound, for example, the host cell can be a cell comprising one or more recombinant nucleic acids encoding one or more product benzylisoquinoline compounds, as is described further below.
  • the cell can be a cell, which, but for the presence of the substrate compound in the medium, is not capable of producing the product benzylisoquinoline compound.
  • the cell can be a cell, which, but for the presence of the substrate compound in the medium, produces substantially less of the product benzylisoquinoline compound, for example, at least about 2 times less, at least about 5 times less, or at least about 10 times less.
  • the cell can be a microbial cell.
  • the microbial cell can be a bacterial cell.
  • the bacterial cell can be an Escherichia coli cell.
  • the microbial cell can be a yeast cell.
  • the yeast cell can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cell.
  • the cell can be an algal cell.
  • the cell can be a plant cell.
  • the plant cell can be selected from a cell obtainable from plants belonging to the plant families of Eupteleaceae,
  • the host cells are grown to produce the benzylisoquinoline uptake permease and the product benzylisoquinoline compound.
  • Growth media and growth conditions can vary depending on the cell that is selected, as will be readily appreciated to those of ordinary skill in the art.
  • Example media include liquid culture media for the growth of yeast cells and bacterial cells including, but not limited to, Luria-Broth, Dulbecco-Eagle modified medium (DMEM] or Optimem. Further media and growth conditions can be found in Sambrook et ah, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.
  • the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
  • SEQ.ID NO: 1 SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
  • the host cells can be modulated to include a compatible biosynthetic enzyme complement to synthesize the product benzylisoquinoline compound from a substrate. Accordingly, in some embodiments, the host cell can be manipulated to obtain a cell comprising a chimeric nucleic acid sequence comprising as operably linked components:
  • (B) a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • SEQ.ID NO: 23 SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37, SEQ.ID NO: 39, and SEQ.ID NO: 55;
  • nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56;
  • nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and
  • the host cell can be manipulated to obtain a cell comprising a chimeric nucleic acid sequence comprising as operably linked components:
  • the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
  • (B) a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
  • B a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
  • Nucleic acid sequences capable of controlling expression in cells that may be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells.
  • promoters obtained from bacterial cells are used when a bacterial host is selected in accordance herewith, while a fimgal promoter will be used when a fimgal host cell is selected, a plant promoter will be used when a plant cell is selected, and so on.
  • Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.
  • the chimeric nucleic acid sequences comprising a promoter capable of controlling expression in host cell linked to a nucleic acid sequence encoding a benzylisoquinoline uptake permease or a benzylisoquinoline biosynthetic enzyme, can be integrated into a recombinant expression vector which ensures good expression in the host cell, wherein the expression vector is suitable for expression in a host cell.
  • suitable for expression in a host cell means that the recombinant expression vector comprises the chimeric nucleic acid sequence linked to genetic elements required to achieve expression in a cell.
  • the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell's genome, for example if a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome.
  • the expression vector may further contain a marker gene.
  • Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non- transformed cells, including all selectable and screenable marker genes.
  • a marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin.
  • Screenable markers that may be employed to identify transformants through visual inspection include b -glucuronidase (GUS] (U.S. Pat. Nos. 5,268,463 and 5,599,670] and green fluorescent protein (GFP] (Niedz et al, 1995, Plant Cell Rep., 14: 403]
  • One host cell that conveniently may be used is Escherichia coli.
  • the preparation of the £. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gelelectrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR] and other methodologies.
  • PCR Polymerase Chain Reaction
  • a wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector.
  • vectors with a replication system functional in E. coli are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc.
  • these cloning vectors contain a marker allowing selection of transformed cells.
  • Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in £. coli by preparing competent cells, electroporation or using other well known methodologies to a person of skill in the art.
  • £. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested.
  • Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells. Further, general guidance with respect to the preparation of recombinant vectors and growth of recombinant organisms may be found in, for example: Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.
  • the substrate can be provided by cell for co-culturing with the host cell.
  • the cell for co-culturing can be any cell capable of growing and secreting a substrate for use of by the host cell, i.e. a substrate benzylisoquinoline compound or a substrate benzylisoquinoline precursor compound.
  • co-cultured it is meant that the host cell and cell for co- culturing are contacted, and mixed, for example in a co-culturing medium, in such a manner that the host cell and cell for co-culturing remain viable, at least for a sufficiently long period of time for the host cell to produce the product benzylisoquinoline compound.
  • Co-culturing of the host cell and cell for co-culturing can be accomplished, for example, by growing and culturing a host cell in a first liquid growth medium for a period of time and under conditions suitable for growing the host cell, and separately growing the cell for co-culturing for a period of time in in a second liquid growth medium and under conditions suitable for growing the cell for co-culturing. Thereafter the first and second liquid media, containing the host cell and cell for co-culturing, respectively can be mixed to thereby contact the host cell and cell for co-culturing.
  • the cells from one of the cultures can be harvested from the liquid medium and the harvested cells can be admixed into the other culture.
  • the substrate can be produced by the cell for co-culturing and secreted in the co-culturing medium to thereby thereafter become available for use by the host cell.
  • the benzylisoquinoline uptake permease included in the host cell may facilitate uptake of the first benzylisoquinoline compound by the host cell.
  • the host cell and cell for co-culturing can be cells belonging to the same biological species.
  • the host cell and cell for co-culturing can be microbial cells.
  • the host cell and cell for co-culturing can be bacterial cells.
  • the host cell and cell for co-culturing can be yeast cells.
  • the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
  • the host cell and cell for co-culturing can be algal cells.
  • the host cell and cell for co-culturing can be plant cells.
  • the cell for co-culturing can include a benzylisoquinoline uptake permease.
  • the benzylisoquinoline uptake permease can be expressed recombinantly in the cell for co-culturing, and can include any benzylisoquinoline uptake permease set forth herein.
  • the present disclosure provides, in at least one embodiment, a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds, the host cell comprising a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell, and the host cell capable of producing the benzylisoquinoline uptake permease and a product benzylisoquinoline compound when provided with a substrate compound.
  • the present disclosure provides, in at least one embodiment, a mixture of cells comprising host cells recombinantly expressing a benzylisoquinoline uptake permease and having a benzylisoquinoline enzyme complement to biosynthetically produce a product benzylisoquinoline compound, and cells for co-culturing with the host cells, wherein the cells for co-culturing are capable of secreting a substrate that can be converted by the host cells when co cultured to form the product benzylisoquinoline compound.
  • the methods of the present disclosure may be used to make a variety of benzylisoquinoline compounds.
  • the obtained benzylisoquinoline compounds may be formulated for use as a pharmaceutical drug, therapeutic agent or medicinal agent.
  • the present disclosure further includes a pharmaceutical composition comprising a benzylisoquinoline compound prepared in accordance with the methods of the present disclosure.
  • Pharmaceutical drug preparations comprising a benzylisoquinoline compound in accordance with the present disclosure can comprise vehicles, excipients and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like. These vehicles, excipients and auxiliary substances are generally pharmaceutical agents that may be administered without undue toxicity.
  • compositions include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, benzoates, and the like. It is also preferred, although not required, that the preparation will contain a pharmaceutically acceptable excipient that serves as a stabilizer.
  • suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, sorbitol, inositol, dextran, and the like.
  • suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, glycine, polyethylene glycols (PEGs], and combinations thereof.
  • the pharmaceutical composition may be formulated for oral and intravenous administration and other routes of administration as desired. Dosing may vary and may be optimized using routine experimentation.
  • the present disclosure further provides, in an embodiment, a use of a benzylisoquinoline compound prepared in accordance with any one of the methods of the present disclosure to prepare a pharmaceutical composition comprising the benzylisoquinoline compound.
  • the benzylisoquinoline compounds of the present disclosure further may be used as precursor or feedstock material for the production of derivative benzylisoquinoline compounds.
  • thebaine made in accordance the disclosure can be used as a precursor to make codeinone
  • codeinone made in accordance with the present disclosure can be used as a precursor to make codeine
  • codeine can be used as a precursor to make morphine.
  • the benzylisoquinoline compounds made in accordance with the present disclosure can be used to make a wide variety of derivative benzylisoquinoline compounds.
  • the benzylisoquinoline compounds can be used to formulate pharmaceutical drugs, as hereinbefore described.
  • the present disclosure provides methods for treating a patient with a pharmaceutical composition comprising a benzylisoquinoline compound prepared in accordance with the present disclosure. Accordingly, the present disclosure further provides a method for treating a patient with a benzylisoquinoline compound prepared according to the methods of the present disclosure, the method comprising administering to the patient a pharmaceutical composition comprising a benzylisoquinoline compound, wherein the pharmaceutical composition is administered in an amount sufficient to ameliorate a medical condition in the patient. [000304] In further embodiments, the present disclosure provides a use of a pharmaceutical composition comprising a benzylisoquinoline compound prepared according to the present disclosure to treat a patient.
  • the present disclosure provides a pharmaceutical composition comprising a benzylisoquinoline compound prepared according to the present disclosure for use in treating a patient.
  • the treatment can comprise use of the pharmaceutical composition to the patient in an amount sufficient to ameliorate a medical condition in the patient.
  • SEQ.ID NO: 1 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 2 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 3 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 4 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 5 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 6 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 7 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 8 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 9 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 10 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 11 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 12 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 13 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 14 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 15 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 16 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 17 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 18 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 19 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 20 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 21 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 22 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
  • SEQ.ID NO: 23 sets forth a polynucleotide sequence encoding a 60MT polypeptide.
  • SEQ.ID NO: 24 sets forth a deduced amino acid sequence of a 60MT polypeptide.
  • SEQ.ID NO: 25 sets forth a polynucleotide sequence encoding a CNMT polypeptide.
  • SEQ.ID NO: 26 sets forth a deduced amino acid sequence of a CNMT polypeptide.
  • SEQ.ID NO: 27 sets forth a polynucleotide sequence encoding an NMCH polypeptide.
  • SEQ.ID NO: 28 sets forth a deduced amino acid sequence of an NMCH polypeptide.
  • SEQ.ID NO: 29 sets forth a polynucleotide sequence encoding a 4 ⁇ MT polypeptide.
  • SEQ.ID NO: 30 sets forth a deduced amino acid sequence of a 4 ⁇ MT polypeptide.
  • SEQ.ID NO: 31 sets forth a polynucleotide sequence encoding a REPI polypeptide.
  • SEQ.ID NO: 32 sets forth a deduced amino acid sequence of a REPI polypeptide.
  • SEQ.ID NO: 33 sets forth a polynucleotide sequence encoding a SalSyn polypeptide.
  • SEQ.ID NO: 34 sets forth a deduced amino acid sequence of a SalSyn polypeptide.
  • SEQ.ID NO: 35 sets forth a polynucleotide sequence encoding a SalR polypeptide.
  • SEQ.ID NO: 36 sets forth a deduced amino acid sequence of a SalR polypeptide.
  • SEQ.ID NO: 37 sets forth a polynucleotide sequence encoding a SalAT polypeptide.
  • SEQ.ID NO: 38 sets forth a deduced amino acid sequence of a SalAT polypeptide.
  • SEQ.ID NO: 39 sets forth a polynucleotide sequence encoding a TS polypeptide.
  • SEQ.ID NO: 40 sets forth a deduced amino acid sequence of a TS polypeptide.
  • SEQ.ID NO: 41 sets forth a polynucleotide sequence encoding a NISO polypeptide.
  • SEQ.ID NO: 42 sets forth a deduced amino acid sequence of a NISO polypeptide.
  • SEQ.ID NO: 43 sets forth a polynucleotide sequence encoding a COR polypeptide.
  • SEQ.ID NO: 44 sets forth a deduced amino acid sequence of a COR polypeptide.
  • SEQ.ID NO: 45 sets forth a polynucleotide sequence encoding a CODM polypeptide.
  • SEQ.ID NO: 46 sets forth a deduced amino acid sequence of a CODM polypeptide.
  • SEQ.ID NO: 47 sets forth a polynucleotide sequence encoding a TYR polypeptide.
  • SEQ.ID NO: 48 sets forth a deduced amino acid sequence of a TYR polypeptide.
  • SEQ.ID NO: 49 sets forth a polynucleotide sequence encoding a TYDC polypeptide.
  • SEQ.ID NO: 50 sets forth a deduced amino acid sequence of a TYDC polypeptide.
  • SEQ.ID NO: 51 sets forth a polynucleotide sequence encoding a DODC polypeptide.
  • SEQ.ID NO: 52 sets forth a deduced amino acid sequence of a DODC polypeptide.
  • SEQ.ID NO: 53 sets forth a polynucleotide sequence encoding a MAO polypeptide.
  • SEQ.ID NO: 54 sets forth a deduced amino acid sequence of a MAO polypeptide.
  • SEQ.ID NO: 55 sets forth a polynucleotide sequence encoding an NCS polypeptide.
  • SEQ.ID NO: 56 sets forth a deduced amino acid sequence of an NCS polypeptide.
  • SEQ.ID NO: 57 sets forth a polynucleotide sequence encoding a T60DM polypeptide.
  • SEQ.ID NO: 58 sets forth a deduced amino acid sequence of a T60DM polypeptide.
  • SEQ.ID NO: 59 sets forth a polynucleotide sequence encoding a CPR polypeptide.
  • SEQ.ID NO: 60 sets forth a deduced amino acid sequence of a CPR polypeptide.
  • SEQ.ID NO: 61 sets forth a polynucleotide sequence encoding a CPR polypeptide.
  • SEQ.ID NO: 62 sets forth a deduced amino acid sequence of a CPR polypeptide.
  • SEQ.ID NO: 63 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 2]
  • SEQ.ID NO: 64 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 2 plus c-myc tag]
  • SEQ.ID NO: 65 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 10).
  • SEQ.ID NO: 66 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 10 plus c-myc tag).
  • SEQ.ID NO: 67 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 8).
  • SEQ.ID NO: 68 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 8 plus c-myc tag).
  • SEQ.ID NO: 69 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 12).
  • SEQ.ID NO: 70 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 12 plus c-myc tag).
  • SEQ.ID NO: 71 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 14).
  • SEQ.ID NO: 72 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 14 plus c-myc tag).
  • SEQ.ID NO: 73 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 16).
  • SEQ.ID NO: 74 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 16 plus c-myc tag]
  • SEQ.ID NO: 75 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 18).
  • SEQ.ID NO: 76 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 18 plus c-myc tag).
  • SEQ.ID NO: 77 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 20).
  • SEQ.ID NO: 78 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 20 plus c-myc tag).
  • SEQ.ID NO: 79 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 22).
  • SEQ.ID NO: 80 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 24 plus c-myc tag).
  • SEQ.ID NO: 81 sets forth a polynucleotide sequence encoding a c-myc - tag.
  • SEQ.ID NO: 82 sets forth an amino acid sequence of a c- myc-tag.
  • Example 1 Method of making veast cells comprising benzylisoquinoline biosynthetic complement and a precursor benzylisoquinoline
  • Yeast strains with chromosome-integrated genes encoding enzymes involved in benzylisoquinoline alkaloid biosynthesis were constructed using a USER cloning system.
  • USER (uracil -specific excision reaction) -based cloning has been used for the integration of multiple genes into the yeast genome owing to its relatively straightforward application and independence from the enzyme-based ligation of DNA fragments.
  • Transient expression constructs were individually transformed to the platform yeast strains with chromosome-integrated BIA biosynthetic genes using the LiAc/PEG/single- stranded carrier DNA (ssDNA) transformation method. Each yeast strain transiently expressing a candidate gene was inoculated in SD-drop out medium overnight. The overnight cultures were then diluted into a SD-drop out medium containing 2% (w/v) galactose and 200 mM of the BIA suitable for conversion by the baseline yeast strain and/or the transient expression construct. Yeast cultures were grown in the presence of suitable substrates for 24 h.
  • yeast cells were removed from their culture medium by centrifugation and 5 pL of supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS) analysis.
  • MS mass spectrometry
  • Alkaloids produced in yeast were characterized by high- resolution MS n analysis.
  • alkaloids were injected by HPLC for electrospray ionization (ESI) prior to analysis by LTQ-Orbitrap-XL. Operation was conducted using LTQ Tune Plus v. 2.5.5 SP1 and Xcalibur v. 2.1.0.1140, with additional analyses using QualBrowser feature of Xcalibur.
  • Example 2 Production in veast cells of fSVreticuline using L-DOPA as a substrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21.
  • Each of the transformed strains grown in a liquid medium comprising L-DOPA and (SJ-reticuline product was measured in the media. The results are show in FIG. 7.
  • Example 3 Production in yeast cells of (5)-reticuline using dopamine as a substrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21.
  • Each of the transformed strains grown in a liquid medium comprising dopamine and (S]-reticuline product was measured in the media. The results are show in FIG. 8.
  • Example 4 Production in yeast cells of (5)-reticuline using norlaudanosoline as a substrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21.
  • Each of the transformed strains grown in a liquid medium comprising norlaudanosoline and (S]-reticuline product was measured in the media. The results are show in FIG. 9.
  • Example 5 Production in yeast cells of thebaine. co-cultured with a yeast cell strain using L-DOPA as a substrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permease, notably a nucleic acid construct comprising SEQ.ID NO: 1.
  • the yeast cell strain was co-cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS.
  • the cells of both strains were mixed and grown in a liquid medium comprising L-DOPA and thebaine product was measured in the media. The results are shown in FIG. 10.
  • Example 6 Production in yeast cells of thehaine. co-cultured with a yeast cell strain using dopamine as a substrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1.
  • the yeast cell strain was co- cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS.
  • the cells of both strains were mixed and grown in a liquid medium comprising dopamine and thebaine product was measured in the media. The results are shown in FIG. 11.
  • Example 7 Production in yeast cells of thebaine. co-cultured with a yeast cell strain using norlaudanosoline as a suhstrate
  • a yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4 ⁇ MT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1.
  • the yeast cell strain was co- cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS.
  • the cells of both strains were mixed and grown in a liquid medium comprising norlaudanosoline and thebaine product was measured in the media. The results are shown in FIG. 12.
  • Yeast Saccharomyces cerevisiae
  • CEN.PK102-5B was transformed with a galactose-inducible, transient expression construct based on pESC-HIS.
  • This plasmid had been modified with the addition of a gene conferring antibiotic resistance to G418 (KanMX; Walker et al. 2003, FEMS Yeast Research 4: 339-347] as described (Dastmalchi et al. 2018, Nature Chemical Biology, in press].
  • Codon-optimized coding sequences for BUP1 through BUP6 including C-terminal c- myc epitope tags (SEQ.ID NO: 81; SEQ.ID NO: 82] were synthesized at GenScript USA (www.genscript.com] and subcloned to MCS1 ofthe modified pESC-HIS using BamHl and SacII restrictions sites.
  • Codon optimized sequences used were: SEQ.ID NO: 63 (BUP1]; SEQ.ID NO: 65 (BUP2]; SEQ.ID NO: 67 (BUP3]; SEQ.ID NO: 69 (BUP4]; SEQ.ID NO: 71 (BUP5]; and SEQ.ID NO: 73 (BUP6] Codon optimized sequence including the c-myc epitope tag were: SEQ.ID NO: 64 (BUP1]; SEQ.ID NO: 66 (BUP2]; SEQ.ID NO: 68 (BUP3]; SEQ.ID NO: 70 (BUP4]; SEQ.ID NO: 72 (BUP5]; and SEQ.ID NO: 74 (BUP6] Following transformation, four individual colonies were selected per clone for yeast uptake assays.
  • Uptake assays were conducted as follows: yeast strains transiently expressing one of BUP1 (SEQ.ID NO: 2], BUP2 (SEQ.ID NO: 10], BUP3 (SEQ.ID NO: 8], BUP4 (SEQ.ID NO: 12], BUP5 (SEQ.ID NO: 14], BUP6 (SEQ.ID NO: 16], BUP8 (SEQ.ID NO: 20], BUP9 (SEQ.ID NO: 22] or no gene ("empty vector” controls] were inoculated in 200 m ⁇ SD-drop out medium containing 2% glucose overnight in a 96-well format, using a Fisherbrand Incubating Microplate Shaker (Fisher Scientific] The overnight cultures were then diluted with 300 m ⁇ SD-drop out medium containing 2% (w/v] galactose and 1 mM L-DOPA or dopamine, or 200 mM of alkaloid substrate.
  • yeast cultures were grown for 24 h post-induction at 30°C unless otherwise noted. Following culturing, yeast cells were gently pelleted and washed 2X with media. Alkaloids in yeast cell pellets were extracted twice with 500 m ⁇ of 100% methanol. Insoluble matter was centrifuged, supernatant was pooled, dried, and resuspended in 200 m ⁇ of 50:50 water:ACN.
  • Plasmids hosting BUPs or no gene (controls] were individually transformed to the platform yeast strains with chromosome-integrated BIA biosynthetic genes using the LiAc/PEG/single-stranded carrier DNA (ssDNA] transformation method, and four resulting transformants were selected for each clone as biological replicates.
  • Yeast strains transiently expressing candidate BUP genes were inoculated in 200 m ⁇ SD- drop out medium containing 2% glucose overnight, in a 96-well format, using a Fisherbrand Incubating Microplate Shaker (Fisher Scientific] The overnight cultures were then diluted with 300 m ⁇ SD-drop out medium containing 2% (w v 1 ] galactose and 250 mM alkaloid substrate for bioconversion. For DOPA and dopamine feeding, concentrations of 1 mM were used in the media. Yeast cultures were grown for 24 h post-induction at 30°C.
  • Yeast cells were removed by centrifugation and 5 pL of supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS] analysis.
  • MS mass spectrometry
  • strain 2 The same was done for strain 2, except that this strain was transiently expressing modified pESC-HIS harboring both BUP1 (in MCS2) and Papaver somniferum THS (in MCS1) to support sufficient thebaine formation.
  • the overnight cultures of strains 1, 2 and 3 were then co-cultured into 300 mI of SD-drop out medium containing 2% (w/v) galactose and 200 ug/l of G418 and 1 mM of DOPA to allow bioconversion.
  • Yeast co-cultures were grown for 24 h post-induction at 30°C, followed by removal of cells by centrifugation. Five microliters of the media supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS) analysis.
  • MS mass spectrometry
  • Yeast media and cell extracts were analyzed by high-resolution LC-ESI- LTQ-Orbitrap-XL mass spectrometry.
  • Cell extracts were pre-processed prior to loading to remove protein by diluting 50:50 with methanol, centrifugation to pellet insolubles, and retrieval of supernatant for analysis. Samples were either loaded directly or diluted with methanol to ensure target alkaloids remained within linear ranges as indicated by standard curves using authentic standards.
  • High-resolution LC-ESI-LTQ-Orbitrap-XL mass spectrometry (MS) was performed using a modified version ofa method described previously (Chang etal. 2015, PlantPhysiol.
  • Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature, 400°C; source voltage, 3 kV; sheath gas, 60 au, auxiliary gas, 20 au; capillary temperature, 380°C; capillary voltage, 6 V; tube lens, 45 V.
  • LTQ-Orbitrap-XL (Thermo Scientific] instrumentation was performed as three scan events in data-dependent, parallel detection mode. The first scan consisted of high-resolution FTMS from 150 to 450 m/z with ion injection time of 500 ms and scan time of approximately 1.5 s.
  • the second and third scans (approximately 0.5 s each] collected CID spectra in the ion trap, where the parent ions represented the first- and second-most abundant alkaloid masses, respectively, as determined by fast Fourier transform preview using a parent ion mass list corresponding to exact masses of known alkaloids. Dynamic-exclusion and reject-ion-mass-list features were enabled. External and internal calibration procedures ensured ⁇ 2 ppm error. Exact mass, retention times, and CID spectra of authentic standards were used to identify alkaloids, and quantification was performed using standard curves. The Quan Browser feature of Thermo X-Calibur v. 3.1 was employed for automated peak identification and quantification.
  • Results are show in FIG. 14A-H and FIG. 15. Shown in FIG. 14A-H is the production of various alkaloid compounds in yeast, notably reticuline (FIG. 14A- C], salutardine (FIG. 14D-E], thebaine (FIG. 14D-F], codeine (FIG. 14G], neopine (FIG. 14G] and morphine (FIG. 14H], respectively, (y-axes] using yeast strain 1 (FIG. 14A-C], strain 2 (FIG. 14D-F] and strain 3 (FIG.
  • FIG. 15A-D Shown in FIG. 15A-D are the production of various alkaloid compounds (y-axes], notably reticuline, salutardine, thebaine and codeine (x-axes] by co-culturing yeast strains 1 and 2 (FIG 15A and FIG. 15C] and strains 1, 2 and 3 (FIG. 15B and FIG. 15D] .
  • Alkaloid substrates that were used were L-DOPA (FIG. 15A and FIG.

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Abstract

Disclosed are methods for converting a substrate into a product benzylisoquinoline compound in a biosynthetic system involving the culturing of cells in a medium. The cells comprise a benzylisoquinoline uptake permease and a biosynthetic enzyme complement to produce the product benzylisoquinoline compound. Related compositions are also disclosed.

Description

TITLE: BENZYL1SOOU1NOL1NE UPTAKE PERMEASES AND METHODS OF USING
RELATED APPLICATION
[0001] This Patent Cooperation Treaty Application claims the benefit under
35 USC § 119 (e] from U.S. Provisional Patent Application No 62/599,113, filed on December 15, 2017, which is incorporated by reference herein in its entirety.
FIELD OF THE DISCLOSURE
[0002] The methods and systems disclosed herein relate to a class of chemical compounds known as alkaloids and methods of making alkaloids. In particular, the methods and systems disclosed herein relate to a class of alkaloids known as benzylisoquinolines, and to methods and systems involving the use of benzylisoquinoline uptake permeases to produce benzylisoquinolines.
BACKGROUND OF THE DISCLOSURE
[0003] The following paragraphs are provided by way of background to the present disclosure.
[0004] Alkaloids are a class of nitrogen containing organic chemical compounds that are naturally produced by opium poppy {Papaver somniferum), and a range of other plant species belonging to the Papaveraceae family of plants, as well as other plant families including, for example the Lauraceae, Annonaceae, Euphorbiaceae and the Moraceae. The interest of the art in alkaloid compounds is well established and can be explained by the pharmacological properties of these compounds, as well as their utility as feedstock materials in the manufacture of pharmaceutical compounds.
[0005] The manufacture of alkaloid compounds can involve the conversion of precursor alkaloid compounds into one or more intermediary alkaloid compounds to yield a desired alkaloid compound, for example a desired benzylisoquinoline. In biosynthetic production systems, enzymes can catalyze the conversion reaction of precursor alkaloid compounds into intermediate alkaloid compounds, or into a desired product alkaloid. However in many biosynthetic production systems, alkaloid compounds are not efficiently converted into the desired products, for example, due to substrate inhibition, or they can be converted into products other than the desired alkaloids products, each of which results into low alkaloid product yields. Thus, the yields of biosynthetic production systems are frequently lower than desired, while costs are higher than desired. [0006] There exists therefore a need in the art for improved processes to produce alkaloid compounds. In particular there is a need in the art for improved processes to produce benzylisoquinoline compounds.
SUMMARY OF THE DISCLOSURE
[0007] The following paragraphs are intended to introduce the reader to the more detailed description, not to define or limit the claimed subject matter of the present disclosure.
[0008] In one aspect, the present disclosure relates to alkaloid compounds, notably a class of alkaloid compounds known as benzylisoquinolines.
[0009] In another aspect, the present disclosure relates to biosynthetic systems for making benzylisoquinoline compounds.
[00010] Accordingly, in one aspect, the present disclosure provides, in at least one embodiment, a method of producing a product benzylisoquinoline compound in a host cell, the method comprising:
(a) providing a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds;
(b) introducing a chimeric nucleic acid into the host cell, the chimeric nucleic acid comprising as operably linked components (i) a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii) a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell; and
(c) growing the host cell in a medium to produce the benzylisoquinoline uptake permease, and the medium comprising a substrate for the biosynthetic conversion of the substrate into the product benzylisoquinoline compound by the host cell enzyme complement.
[00011] In some embodiments, the product benzylisoquinoline compound can be (5]-norcoclaurine, (5]-norlaudanosoline, (S)-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S)-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?)-reticuline, salutaridine, salutaridinol, or thebaine.
[00012] In some embodiments, the substrate can be a substrate benzylisoquinoline compound. [00013] In some embodiments, the substrate benzylisoquinoline compound can be (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S]-coclaurine, (5]-/V-methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (£}- reticuline, (/?}-reticuline, salutaridine, or salutaridinol.
[00014] In some embodiments, the substrate can be a substrate benzylisoquinoline precursor compound.
[00015] In some embodiments, the substrate benzylisoquinoline precursor compound can be L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydroxy- phenylacetaldehyde (4-HPAA]
[00016] In some embodiments, the enzyme complement can comprise one or more benzylisoquinoline biosynthetic enzymes, and the enzymes can be norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0-acetyltransferase (SalAT], or thebaine synthase (TS]
[00017] In some embodiments, the host cell further can have a benzylisoquinoline precursor enzyme complement.
[00018] In some embodiments, the benzylisoquinoline precursor enzyme complement can comprise one or more benzylisoquinoline precursor biosynthetic enzymes, and the enzymes can be tyrosine hydroxylase (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00019] In some embodiments, the substrate can be converted into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
[00020] In some embodiments, the substrate can be converted into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
[00021] In some embodiments, the product benzylisoquinoline can be thebaine, the substrate can be a substrate benzylisoquinoline compound and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS]
[00022] In some embodiments, the product benzylisoquinoline compound can be thebaine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00023] In some embodiments, the product benzylisoquinoline compound can be salutaridinol, the substrate can be a substrate benzylisoquinoline compound and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridinol, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O- acetyltransferase (SalAT]
[00024] In some embodiments, the product benzylisoquinoline compound can be salutaridinol, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridinol, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O-acetyltransferase (SalAT], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00025] In some embodiments, the product benzylisoquinoline compound can be salutaridine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn]
[00026] In some embodiments, the product benzylisoquinoline compound can be salutaridine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00027] In some embodiments, the product benzylisoquinoline compound can be (R] -reticuline, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (R] -reticuline, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’ -0-methyl transferase (4ΌMT], or reticuline epimerase (REPI]
[00028] In some embodiments, the product benzylisoquinoline compound can be (R] -reticuline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (R] -reticuline, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’ -O-methyl transferase (4ΌMT], or reticuline epimerase (REPI], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00029] In some embodiments, the product benzylisoquinoline compound can be (5] -reticuline, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (5] -reticuline, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], or 4’-0-methyltransferase (4ΌMT]
[00030] In some embodiments, the product benzylisoquinoline compound can be (5] -reticuline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5] -reticuline, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-0- methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase or (4ΌMT], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00031] In some embodiments, the product benzylisoquinoline compound can be (S]-3'-hydroxy-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-3’-hydroxy-/V-methylcoclaurine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase, or (NMCH] [00032] In some embodiments, the product benzylisoquinoline compound can be (5]-3’-hydroxy-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S]-3’-hydroxy-/V-methylcoclaurine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine, or 3’-hydroxylase (NMCH] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00033] In some embodiments, the product benzylisoquinoline compound can be (S]-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-/V-methylcoclaurine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O- methyltransferase (60MT], or coclaurine-/V-methyltransferase (CNMT]
[00034] In some embodiments, the product benzylisoquinoline compound can be (S]-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- /V-methylcoclaurine, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], or coclaurine-iV-methyltransferase (CNMT], and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00035] In some embodiments, the product benzylisoquinoline compound can be (5]-coclaurine, the substrate can be a benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-coclaurine, wherein the enzymes are selected from norcoclaurine synthase (NCS], and 6-O-methyltransferase (60MT] [00036] In some embodiments, the product benzylisoquinoline compound can be (S]-coclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- coclaurine, and the enzymes can be selected from norcoclaurine synthase (NCS] or 6- O-methyltransferase (60MT] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00037] In some embodiments, the product benzylisoquinoline compound can be (5] -norcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norcoclaurine, and the enzyme can be norcoclaurine synthase (NCS] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00038] In some embodiments, the product benzylisoquinoline compound can be (5]-norlaudanosoline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norlaudanosoline, and the enzyme can be norcoclaurine synthase (NCS] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00039] In some embodiments, the product benzylisoquinoline compound can be (S]-6-0-methyl-norlaudanosoline, the substrate can be a benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound (5]-6-0-methyl- norlaudanosoline, wherein the enzymes are selected from norcoclaurine synthase (NCS] and 60MT. [00040] In some embodiments, the product benzylisoquinoline compound can be (S]-6-0-methyl-norlaudanosoline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-6-0-methyl-norlaudanosoline, and the enzymes can be selected from 60MT or norcoclaurine synthase (NCS] and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00041] In some embodiments, the host cell can further include an electron transfer facilitating protein.
[00042] In some embodiments, the electron transfer facilitating protein can be a cytochrome P450 reductase (CPR]
[00043] In some embodiments, the substrate can be produced in the medium by a cell for co-culturing with the host cell, wherein the co-cultured cell is capable of secreting the substrate into the medium.
[00044] In some embodiments, the cell for co-culturing can be a cell that does not comprise the benzylisoquinoline biosynthetic enzyme complement to produce the product benzylisoquinoline compound.
[00045] In some embodiments, the host cell can be a cell that does not produce the substrate compound.
[00046] In some embodiments, the benzylisoquinoline uptake permease can be a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code; (d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f).
[00047] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
[00048] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37; SEQ.ID NO: 39; and SEQ.ID NO: 55
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a]; (e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (0, (d), (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
[00049] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
[00050] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 47, SEQ.ID NO: 49, SEQ.ID NO: 51, and SEQ.ID NO: 53;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a]; (e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (0, (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
[00051] In some embodiments, the product benzylisoquinoline compound can be further converted in the host cell to form a derivative benzylisoquinoline.
[00052] In some embodiments, the derivative benzylisoquinoline can be neopinone, codeine, codeinone or morphine.
[00053] In some embodiments, the conversion to form a derivative benzylisoquinoline can involve the performance of an enzyme-catalyzed reaction by one or more of the enzymes T6-0-demethylase (T60DM], neopinone isomerase (NISO], codeinone reductase (COR], and codeinone-O-demethylase (CODM]
[00054] In some embodiments, the method can further include a step comprising recovering the product benzylisoquinoline compound or the derivative benzylisoquinoline.
[00055] In some embodiments, the host cell can be a microbial cell.
[00056] In some embodiments, the host cell can be a bacterial cell.
[00057] In some embodiments, the host cell can be a yeast cell.
[00058] In some embodiments, the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
[00059] In some embodiments, the host cell can be an algal cell.
[00060] In some embodiments, the host cell can be a plant cell.
[00061] In some embodiments, the host cell and cell for co-culturing can be cells belonging to the same biological species.
[00062] In some embodiments, the host cell and cell for co-culturing can be microbial cells. [00063] In some embodiments, the host cell and cell for co-culturing can be bacterial cells.
[00064] In some embodiments, the host cell and cell for co-culturing can be yeast cells.
[00065] In some embodiments, the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
[00066] In some embodiments, the host cell and cell for co-culturing can be algal cells.
[00067] In some embodiments, the host cell and cell for co-culturing can be plant cells.
[00068] In some embodiments, the cell for co-culturing can comprise a benzylisoquinoline uptake permease.
[00069] In another aspect, the present disclosure provides, in at least one aspect, a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds, the host cell comprising a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell, and the host cell capable of producing the benzylisoquinoline uptake permease and a product benzylisoquinoline compound when provided with a substrate compound.
[00070] In some embodiments, the product benzylisoquinoline compound can be (5]-norcoclaurine, (5]-norlaudanosoline, (S]-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, salutaridinol, or thebaine.
[00071] In some embodiments, the substrate can be a substrate benzylisoquinoline compound.
[00072] In some embodiments, the substrate benzylisoquinoline compound can be (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S]-coclaurine, (5]-/V-methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, or salutaridinol.
[00073] In some embodiments, the substrate can be a substrate benzylisoquinoline precursor compound. [00074] In some embodiments, the substrate benzylisoquinoline precursor compound can be L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydrox- phenylacetaldehyde (4-HPAA]
[00075] In some embodiments, the enzyme complement can comprise one or more benzylisoquinoline biosynthetic enzymes, wherein the enzymes can be norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0-acetyltransferase (SalAT], or thebaine synthase (TS]
[00076] In some embodiments, the host cell further can have a benzylisoquinoline precursor enzyme complement.
[00077] In some embodiments, the benzylisoquinoline precursor enzyme complement can comprise one or more benzylisoquinoline precursor biosynthetic enzymes, wherein the enzymes can be (TYR], dihydroxyphenyl alanine decarboxylase DODC, tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[00078] In some embodiments, the substrate can be converted by the host cell into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
[00079] In some embodiments, the substrate can be converted by the host cell into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
[00080] In some embodiments, the benzylisoquinoline uptake permease can be a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79; (b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f).
[00081] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
[00082] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37, SEQ.ID NO: 39, and SEQ.ID NO: 55;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a]; (c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40 or SEQ.ID NO: 56;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
[00083] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline precursor biosynthetic enzymes in the second cell.
[00084] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 47, SEQ.ID NO: 49, SEQ.ID NO: 51, and SEQ.ID NO: 53;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a]; (c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 53, or SEQ.ID NO: 54; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
[00085] In some embodiments, the host cell can be a microbial cell.
[00086] In some embodiments, the host cell can be a bacterial cell.
[00087] In some embodiments, the host cell can be a yeast cell.
[00088] In some embodiments, the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
[00089] In some embodiments, the host cell can be an algal cell.
[00090] In some embodiments, the host cell can be a plant cell.
[00091] In another aspect, the present disclosure provides, in at least one embodiment, a mixture of cells comprising host cells recombinantly expressing benzylisoquinoline uptake permease and having a benzylisoquinoline enzyme complement to biosynthetically produce a product benzylisoquinoline compound, and cells for co-culturing with the host cells, wherein the cells for co-culturing are capable of secreting a substrate that can be converted by the host cells when co cultured to form the product benzylisoquinoline compound.
[00092] In some embodiments, the cells for co-culturing can be cells that do not include the benzylisoquinoline enzyme complement to produce the benzylisoquinoline product. [00093] In some embodiments, the host cells do not produce the substrate.
[00094] In some embodiments, the host cells and cells for co-culturing can be cells belonging to the same biological species.
[00095] In some embodiments, the host cells or cells for co-culturing can be microbial cells.
[00096] In some embodiments, the host cells or cells for co-culturing can be bacterial cells.
[00097] In some embodiments, the host cells or cells for co-culturing can be yeast cells.
[00098] In some embodiments, the yeast cells can be Saccharomyces cerevisiae cell or Yarrowia lipolytica cells.
[00099] In some embodiments, the host cells or cells for co-culturing can be algal cells.
[000100] In some embodiments, the host cells or cells for co-culturing can be plant cells.
[000101] In some embodiments, the cells for co-culturing can comprise a benzylisoquinoline uptake permease.
[000102] In another aspect, the present disclosure provides, in at least one embodiment, a use of a cell according to the present disclosure to convert a substrate and form a product benzylisoquinoline compound.
[000103] In another aspect, the present disclosure provides, in at least one embodiment, a use of a mixture of cells comprising host cells and cells for co-culturing with the host cells according to the present disclosure to convert a substrate and form a product benzylisoquinoline compound.
[000104] In another aspect, the present disclosure provides, in at least one embodiment, a product benzylisoquinoline compound produced in accordance with any one of the methods of the present disclosure.
[000105] Other features and advantages will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[000106] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect. The figures are not intended to limit the present disclosure.
[000107] FIGS. 1A, IB and 2C show prototype structures of benzylisoquinoline compounds.
[000108] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H depict the chemical structures of some benzylisoquinoline compounds, notably (5]-norcoclaurine (FIG. 2 A], (5]- coclaurine (FIG. 2B], (5]-/V-methylcoclaurine (FIG. 2C], (S]-3’-hydroxy-/V- methylcoclaurine (FIG. 2D], (5]-reticuline (FIG. 2E], (7?]-reticuline (FIG. 2F], (5]- norlaudanosoline (FIG. 2G], and (S]-6-0-methyl-norlaudanosoline (FIG. 2H]
[000109] FIGS. 3A, 3B, 2C, 3D, 3E, 3F and 3G depict the chemical structures of some other benzylisoquinoline compounds, notably salutaridine (FIG. 3A], salutaridinol (FIG. 3B], thebaine (FIG. 3C], codeinone (FIG. 3D], codeine (FIG. 3E], morphine (FIG. 3F] and neopinone (FIG. 3G]
[000110] FIG. 4A, 4B, 4C, 4D, 4E and 4F depict the chemical structures of some benzylisoquinoline precursor compounds, notably L-tyrosine (FIG. 4A], L- dihydroxyphenyl alanine (L-DOPA] (FIG. 4B], dopamine (FIG. 4C], tyramine (FIG. 4D], 4-hydroxyphenylacetaldehyde (4-HPAA] (FIG. 4E] and 3,4- hydroxyphenylacetaldehyde (3, 4-HPAA]
[000111] FIG. 5 depicts some chemical reactions involving the conversion of benzylisoquinoline compounds.
[000112] FIG. 6 depicts some chemical reactions involving the conversion of benzylisoquinoline precursor compounds.
[000113] FIG. 7 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising L-DOPA as a substrate.
[000114] FIG. 8 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising dopamine as a substrate. [000115] FIG. 9 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound (5]-reticuline in a yeast strain grown in a medium comprising norlaudanosoline as a substrate.
[000116] FIG. 10 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising L-DOPA as a substrate.
[000117] FIG. 11 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising dopamine as a substrate.
[000118] FIG. 12 depicts certain experimental results obtained, notably the production of the benzylisoquinoline compound thebaine in a yeast strain co cultured with another yeast strain and in a medium comprising norlaudanosoline as a substrate.
[000119] FIG. 13A-F depicts certain experimental results obtained, notably uptake of alkaloid compounds (y-axes] by Saccharomyces cerevisiae transformed with one of six plasmids, allowing expression of BUP1 (13A], BUP2 (13B], BUP3 (13C], BUP4 (13D], BUP5 (13E], BUP6 (13F], respectively. Substrates provided exogenously in the media and measured post-incubation in cellular extract are indicated on the x-axes. Black bars and grey-shaded bars represent empty vector and BUP-expressing strains, respectively. Standard error was calculated using four biological replicates representing four individual transformants.
[000120] FIG. 14A-H depict certain experimental results obtained, notably production of alkaloid compounds by three different Saccharomyces cerevisiae strains (strain 1 (14A-C], strain 2 (14D-F], strain 3 (14G-H]] transformed with one of nine plasmids, allowing expression of BUP1, BUP2, BUP3, BUP4, BUP5, BUP6, BUP7, BUP8, and BUP 9, respectively (x-axes]. Exogenously fed substrate compounds are: DOPA (FIG. 14A], dopamine (FIG. 14B], norlaudanosoline (FIG. 14C], (5]- reticuline (FIG. 14D], (7?]-reticuline (FIG. 14E], salutaridine (FIG. 14F], thebaine (FIG. 14G] and codeine (FIG. 14H] Alkaloids detected post-incubation (y-axes] are: reticuline (FIG. 14A-C], salutaridine (FIG. 14D-E], thebaine (FIG. 14D-F], codeine (FIG. 14G], neopine (FIG. 14G] and morphine (FIG. 14H] For comparison, dotted lines indicate levels of alkaloid product detectable in control assays. Standard error was calculated using four biological replicates representing four individual transformants.
[000121] FIG. 15 depicts certain experimental results obtained, notably production of alkaloid compounds by 2 different combinations of co-cultured Saccharomyces cerevisiae strains: strain 1 and strain 2 (FIG. 15A and FIG. 15C] and strain 1, strain 2 and strain 3 (FIG. 15B and FIG. 15D). Alkaloids produced are reticuline, salutaridine, thebaine and codeine (FIG. 15A and FIG. 15B] and salutaridine, thebaine and codeine (FIG. 15C and FIG. 15D] (x-axes). Substrates used are L-DOPA (FIG. 15A and FIG. 15B] or S-reticuline (FIG. 15C and FIG. 15D). Strains were transformed with BUP 1 (right hand side of each of FIGS. 15A-15D] or with empty vector (left hand side of each of FIGS. 15A-15D], as indicated.
[000122] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice.
DETAILED DESCRIPTION
[000123] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
[000124] As used herein and in the claims, the singular forms, such "a”, "an” and "the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, "comprise,” "comprises” and "comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.
[000125] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
[000126] As used herein and in the claims, the singular forms, such "a”, "an” and "the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, "comprise,” "comprises” and "comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term "or” is inclusive unless modified, for example, by "either”.
[000127] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub combinations of ranges and specific embodiments therein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about.” The term "about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range, as will be readily recognized by context. Furthermore any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed ( e.g . a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5] Similarly, other terms of degree such as "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.
[000128] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
[000129] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Terms and definitions
[000130] The terms“benzylisoquinoline”, or“benzylisoquinoline compound”, as may be used interchangeably herein are chemical compounds having the prototype structure set forth in FIGS. 1A, IB or 1C. It is noted that certain ring closure reactions of compounds having the prototype structure shown in FIG. 1A can lead to the formation of compounds having the prototype structure shown in FIG. IB or FIG. 1C. Benzylisoquinoline compounds are further intended to include, without limitation, (S]-norcoclaurine, (S]-coclaurine, (5]-/V-methylcoclaurine, (S]-3’-hydroxy-/V- methylcoclaurine, (S]-reticuline, (7?]-reticuline, salutaridine, salutaridinol, thebaine, codeinone, codeine, and morphine.
[000131] The term "benzylisoquinoline precursor compound” refers to a chemical compound that can be converted into a benzylisoquinoline compound and includes, L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4- hydroxy-phenylacetaldehyde (3,4-HPAA] and 4-hydrox-phenylacetaldehyde (4- HPAA] Benzylisoquinoline precursor compounds include compounds that can be directly converted into benzylisoquinoline compound, or compounds that can be converted into benzylisoquinoline compound via another precursor benzylisoquinoline compound.
[000132] The term "(S]-norcoclaurine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2A.
[000133] The term "(S]-coclaurine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2B.
[000134] The term "(S]-/V-methylcoclaurine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2C.
[000135] The term "(S]-3’-hydroxy-/V-methylcoclaurine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2D.
[000136] The term "(S]-reticuline”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2E.
[000137] The term "(7?]-reticuline”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2F.
[000138] The term "(S]-norlaudanosoline”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2G.
[000139] The term "(5]-6-0-methyl-norlaudanosoline”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2H.
[000140] The term "salutaridine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3A.
[000141] The term "salutaridinol”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3B.
[000142] The term "thebaine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3C.
[000143] The term "codeinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3D.
[000144] The term "codeine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3E.
[000145] The term "morphine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3F. [000146] The term "neopinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3G.
[000147] The term "L-tyrosine”, as used herein, refers to a chemical compound having the chemical structure set forth in (FIG. 4A]
[000148] The terms "L-dihydroxyphenyl alanine” or "L-DOPA”, as may be used interchangeable herein, refers to a chemical compound having the chemical structure set forth in (FIG. 4B],
[000149] The term dopamine, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 4C.
[000150] The term "tyramine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 4D.
[000151] The terms "4-hydroxy-phenylacetaldehyde” or "4-HPAA”, as may be interchangeably used herein, refer to a chemical compound having the structure set forth in FIG. 4E.
[000152] The terms "3, 4-hydroxy-phenylacetaldehyde” or "3, 4-HPAA”, as may be interchangeably used herein, refer to a chemical compound having the structure set forth in FIG. 4F.
[000153] The term "benzylisoquinoline biosynthetic enzyme”, as used herein, refers to a polypeptide capable of facilitating the chemical conversion of a first benzylisoquinoline compound into a second benzylisoquinoline compound, or the chemical conversion of a benzylisoquinoline precursor compound into a benzylisoquinoline compound in a single chemical step. Benzylisoquinoline biosynthetic enzymes include norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’- hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0- acetyltransferase (SalAT], and thebaine synthase (TS]
[000154] The term "benzylisoquinoline precursor enzyme complement”, as used herein, refers to one or more benzylisoquinoline precursor biosynthetic enzymes, which together when provided with a substrate molecule, can produce a compound that can act as a substrate compound for a benzylisoquinoline biosynthetic enzyme. Benzylisoquinoline precursor biosynthetic enzymes that can be included in a benzylisoquinoline precursor enzyme complement include tyrosine hydroxylase (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
[000155] The terms "6-0-methyltransferase” or "60MT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any 6-0-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 24, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any 6-0-methyltransferase polypeptide set forth herein, but for the use of synonymous codons.
[000156] The terms "coclaurine-/V-methyltransferase” or "CNMT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any coclaurine-/V-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 26, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any coclaurine-methyltransferase polypeptide set forth herein, but for the use of synonymous codons.
[000157] The terms "(S]-/V-methylcoclaurine 3’-hydroxylase” or "NMCH”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any (5]-/V-methylcoclaurine 3’-hydroxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 28, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide set forth herein, but for the use of synonymous codons.
[000158] The terms "4’-0-methyltransferase” or "4ΌMT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any 4’-0-methyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 30, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any 4’-0-methyltransferase set forth herein, but for the use of synonymous codons.
[000159] The terms "reticuline epimerase” or "REPI”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any reticuline epimerase polypeptide set forth herein, including, for example, SEQ.ID NO: 32, or (if) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any reticuline epimerase polypeptide set forth herein, but for the use of synonymous codons.
[000160] The terms "salutaridine synthase” or "SalSyn”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridine synthase polypeptide set forth herein, including, for example, SEQ.ID NO: 34, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutaridine synthase polypeptide set forth herein, but for the use of synonymous codons.
[000161] The terms "salutaridine reductase” or "SalR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridine reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 36, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutaridine reductase polypeptide set forth herein, but for the use of synonymous codons.
[000162] The terms "salutaridinol-7-0-acetyltransferase” or "SalAT”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any salutaridinol-7-0-acetyltransferase polypeptide set forth herein, including, for example, SEQ.ID NO: 38, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any salutardinol-7-O-acetyl transferase polypeptide set forth herein, but for the use of synonymous codons.
[000163] The terms "thebaine synthase” or "TS”, as maybe used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any thebaine synthase polypeptide set forth herein, including, for example, SEQ.ID NO: 40, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any thebaine synthase polypeptide set forth herein, but for the use of synonymous codons.
[000164] The terms "neopinone isomerase” or "NISO”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any neopinone isomerase polypeptide set forth herein, including, for example, SEQ.ID NO: 42, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any neopinone isomerase polypeptide set forth herein, but for the use of synonymous codons.
[000165] The terms "codeinone reductase” or "COR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeinone reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 44, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeinone reductase polypeptide set forth herein, but for the use of synonymous codons.
[000166] The terms "codeine-O-demethylase” or "CODM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeine-O-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 46, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeine-O-demethylase polypeptide set forth herein, but for the use of synonymous codons. [000167] The terms "T6-0-demethylase” or "T60DM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any T6-0-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 58, or (if) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any T6-0-demethylase polypeptide set forth herein, but for the use of synonymous codons.
[000168] The terms "cytochrome P450 reductase " or "CPR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any set cytochrome P450 reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 60, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any cytochrome P450 reductase polypeptide set forth herein, but for the use of synonymous codons.
[000169] The terms "benzylisoquinoline uptake permease”, "BUP”, "purine permease” or "PUP” are synonyms and may be used interchangeably herein. The terms "purine permease” or "PUP” were used in the priority U.S. Provisional Patent Application No 62/599,113 but the terms "benzylisoquinoline uptake permease” or "BUP” are now preferred. All of the terms "benzylisoquinoline uptake permease”, "BUP”, "purine permease” or "PUP” refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any benzylisoquinoline uptake permease polypeptide set forth herein, including, for example. SEQ.ID NO: 2, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any benzylisoquinoline uptake permease polypeptide set forth herein, but for the use of synonymous codons.
[000170] The terms "tyrosine hydroxylase”, or "TYR” refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any tyrosine hydroxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 48, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tyrosine hydroxylase polypeptide set forth herein, but for the use of synonymous codons.
[000171] The terms "tyrosine decarboxylase” or "TYDC”, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any tyrosine decarboxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 50, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tyrosine decarboxylase polypeptide set forth herein, but for the use of synonymous codons.
[000172] The terms "dihydroxyphenyl alanine decarboxylase” or "DODC”, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any dihydroxyphenyl alanine decarboxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 52, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any dihydroxyphenyl alanine decarboxylase set forth herein, but for the use of synonymous codons.
[000173] The terms "monoamide oxidase” or "MAO”, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any monoamide oxidase polypeptide set forth herein, including, for example, SEQ.ID NO: 54, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any monoamide oxidase polypeptide set forth herein, but for the use of synonymous codons.
[000174] The terms "norcoclaurine synthase” or "NCS””, refers to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any norcoclaurine synthase polypeptide set forth herein, including, for example, SEQ.ID NO: 56, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any norcoclaurine polypeptide set forth herein, but for the use of synonymous codons.
[000175] The terms "nucleic acid sequence encoding 6 -0-methyl transferase”, and "nucleic acid sequence encoding a 6-0-methyltransferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a 6-0-methyltransferase polypeptide, including, for example, SEQ.ID NO: 23. Nucleic acid sequences encoding a 6-0-methyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the 6-0-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any 6-0-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000176] The terms "nucleic acid sequence encoding coclaurine-iV- methyltransferase”, and "nucleic acid sequence encoding a coclaurine-iV- methyltransferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a coclaurine-iV-methyltransferase polypeptide, including, for example, SEQ.ID NO: 25. Nucleic acid sequences encoding a coclaurine-/V-methyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the coclaurine-/V-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any coclaurine-iV-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000177] The terms "nucleic acid sequence encoding (S]-/V-methylcoclaurine 3’- hydroxylase”, and "nucleic acid sequence encoding a (5] -/V-methylcoclaurine 3’- hydroxylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide, including, for example, SEQ.ID NO: 27. Nucleic acid sequences encoding a (S]-/V-methylcoclaurine 3’-hydroxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the (5]-/V-methylcoclaurine 3’-hydroxylase polypeptide sequences set forth herein; or (ii] hybridize to any (S]-/V-methylcoclaurine 3’-hydroxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons. [000178] The terms "nucleic acid sequence encoding 4’ -O-methyl transferase”, and "nucleic acid sequence encoding a 4’-0-methyltransferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a 4’-0-methyltransferase polypeptide, including, for example, SEQ.ID NO: 29. Nucleic acid sequences encoding a 4’ -O-methyl transferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the 4’-0-methyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any 4’-0-methyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000179] The terms "nucleic acid sequence encoding reticuline epimerase”, and "nucleic acid sequence encoding a reticuline epimerase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a reticuline epimerase polypeptide, including, for example, SEQ.ID NO: 31. Nucleic acid sequences encoding a reticuline epimerase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the reticuline epimerase polypeptide sequences set forth herein; or (ii] hybridize to any reticuline epimerase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000180] The terms "nucleic acid sequence encoding salutaridine synthase”, and "nucleic acid sequence encoding a salutaridine synthase polypeptide”, as maybe used interchangeably herein, refer to any and all nucleic acid sequences encoding a salutaridine synthase polypeptide, including, for example, SEQ.ID NO: 33. Nucleic acid sequences encoding a salutaridine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridine synthase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000181] The terms "nucleic acid sequence encoding salutaridine reductase”, and "nucleic acid sequence encoding a salutaridine reductase polypeptide”, as maybe used interchangeably herein, refer to any and all nucleic acid sequences encoding a salutaridine reductase polypeptide, including, for example, SEQ.ID NO: 35. Nucleic acid sequences encoding a salutaridine reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridine reductase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridine reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000182] The terms "nucleic acid sequence encoding salutaridinol-7-0- acetyltransferase”, and "nucleic acid sequence encoding a salutaridinol-7-0- acetyltransferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a salutaridinol-7-0-acetyltransferase polypeptide, including, for example, SEQ.ID NO: 37. Nucleic acid sequences encoding a salutaridinol-7-0-acetyltransferase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the salutaridinol-7-0-acetyltransferase polypeptide sequences set forth herein; or (ii] hybridize to any salutaridinol-7-0-acetyltransferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000183] The terms "nucleic acid sequence encoding thebaine synthase”, and "nucleic acid sequence encoding a thebaine synthase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a thebaine synthase polypeptide, including, for example, SEQ.ID NO: 39. Nucleic acid sequences encoding a thebaine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the thebaine synthase polypeptide sequences set forth herein; or (ii] hybridize to any thebaine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000184] The terms "nucleic acid sequence encoding neopinone isomerase”, and "nucleic acid sequence encoding a neopinone isomerase polypeptide”, as maybe used interchangeably herein, refer to any and all nucleic acid sequences encoding a neopinone isomerase polypeptide, including, for example, SEQ.ID NO: 41. Nucleic acid sequences encoding a neopinone isomerase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the neopinone isomerase polypeptide sequences set forth herein; or (ii] hybridize to any neopinone isomerase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000185] The terms "nucleic acid sequence encoding codeinone reductase”, and "nucleic acid sequence encoding a codeinone reductase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a codeinone reductase polypeptide, including, for example, SEQ.ID NO: 43. Nucleic acid sequences encoding a codeinone reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeinone reductase polypeptide sequences set forth herein; or (ii] hybridize to any codeinone reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000186] The terms "nucleic acid sequence encoding codeine-O-demethylase”, and "nucleic acid sequence encoding a codeine-O-demethylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a codeine-O-demethylase polypeptide, including, for example, SEQ.ID NO: 45. Nucleic acid sequences encoding a codeine-O-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeine-O-demethylase polypeptide sequences set forth herein; or (ii] hybridize to any codeine-O-demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000187] The terms "nucleic acid sequence encoding T6-0-demethylase”, and "nucleic acid sequence encoding a T6-0-demethylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a codeine-O-demethylase polypeptide, including, for example, SEQ.ID NO: 57. Nucleic acid sequences encoding a T6-0-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the T6-0-demethylase polypeptide sequences set forth herein; or (ii] hybridize to any T6-0-demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000188] The terms "nucleic acid sequence encoding cytochrome P450 reductase”, and "nucleic acid sequence encoding a cytochrome P450 reductase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a cytochrome P450 reductase polypeptide, including, for example, SEQ.ID NO: 59. Nucleic acid sequences encoding a cytochrome P450 reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the cytochrome P450 reductase polypeptide sequences set forth herein; or (ii] hybridize to any cytochrome P450 reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000189] The terms "nucleic acid sequence encoding a benzylisoquinoline uptake permease”, "nucleic acid sequence encoding a benzylisoquinoline uptake permease polypeptide”, "nucleic acid sequence encoding a purine permease”, and nucleic acid sequence encoding a purine permease polypeptide, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide, including, for example, SEQ.ID NO: 1. Nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the benzylisoquinoline uptake permease polypeptide sequences set forth herein; or (ii] hybridize to any benzylisoquinoline uptake permease nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000190] The terms "nucleic acid sequence encoding tyrosine hydroxylase”, and "nucleic acid sequence encoding a tyrosine hydroxylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a tyrosine hydroxylase polypeptide, including, for example, SEQ.ID NO: 47. Nucleic acid sequences encoding a benzylisoquinoline uptake permease polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the tyrosine hydroxylase polypeptide sequences set forth herein; or (ii] hybridize to any tyrosine hydroxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000191] The terms "nucleic acid sequence encoding tyrosine decarboxylase”, and "nucleic acid sequence encoding a tyrosine decarboxylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a tyrosine decarboxylase polypeptide, including, for example, SEQ.ID NO: 49. Nucleic acid sequences encoding a tyrosine decarboxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the tyrosine decarboxylase polypeptide sequences set forth herein; or (ii] hybridize to any tyrosine decarboxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000192] The terms "nucleic acid sequence encoding dihydroxyphenyl alanine decarboxylase”, and "nucleic acid sequence encoding a dihydroxyphenyl alanine decarboxylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a dihydroxyphenyl alanine decarboxylase polypeptide, including, for example, SEQ.ID NO: 51. Nucleic acid sequences encoding a dihydroxyphenyl alanine decarboxylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the dihydroxyphenyl alanine decarboxylase polypeptide sequences set forth herein; or (ii] hybridize to any dihydroxyphenyl alanine decarboxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons. [000193] The terms "nucleic acid sequence encoding monoamine oxidase”, and "nucleic acid sequence encoding a monoamine oxidase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a monoamine oxidase polypeptide, including, for example, SEQ.ID NO: 53. Nucleic acid sequences encoding a monoamine oxidase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the monoamine oxidase polypeptide sequences set forth herein; or (ii] hybridize to any monoamine oxidase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000194] The terms "nucleic acid sequence encoding norcoclaurine synthase”, and "nucleic acid sequence encoding a norcoclaurine synthase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a norcoclaurine synthase polypeptide, including, for example, SEQ.ID NO: 55. Nucleic acid sequences encoding a norcoclaurine synthase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the norcoclaurine synthase polypeptide sequences set forth herein; or (ii] hybridize to any norcoclaurine synthase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000195] The terms "nucleic acid”, or "nucleic acid sequence”, as used herein, refer to a sequence of nucleoside or nucleotide monomers, consisting of naturally occurring bases, sugars and intersugar (backbone] linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acids of the present disclosure may be deoxyribonucleic nucleic acids (DNA] or ribonucleic acids (RNA] and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The nucleic acids may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. A sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, nucleic acid sequence, a nucleotide sequence or a nucleoside sequence. [000196] The term "polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all polypeptides comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequence constituting the polypeptide having such reference SEQ.ID NO, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the polypeptide having such reference SEQ.ID NO, but for the use of synonymous codons. A sequence of amino acid residues may be referred to as an amino acid sequence, or polypeptide sequence.
[000197] The term "nucleic acid sequence encoding a polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all nucleic acid sequences encoding a polypeptide having such reference SEQ.ID NO. Nucleic acid sequences encoding a polypeptide, in conjunction with a reference SEQ.ID NO, further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the polypeptide having such reference SEQ.ID NO; or (ii) hybridize to any nucleic acid sequences encoding polypeptides having such reference SEQ.ID NO under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[000198] By the term "substantially identical” it is meant that two amino acid sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. In order to determine the percentage of identity between two amino acid sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al, Nucleic Acids Res., 1984, 12: 387] BLASTP, BLASTN and FASTA (Altschul et al, J. Mol. Biol., 1990:215:403] A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22]: 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
[000199] By "at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 ( e.g . 20, 25, 30, 40 or 50] nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (LoglO [Na+]]+0.41(% (G+C]-600/l], or similar equation]. Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate
(SSC]/5xDenhardt's solution/1.0% SDS at Tm (based on the above equation] -5° C, followed by a wash of 0.2xSSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3xSSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.
[000200] The term "functional variant”, as used herein in reference to polynucleotides or polypeptides, refers to polynucleotides or polypeptides capable of performing the same function as a noted reference polynucleotide or polypeptide. Thus, for example, a functional variant of the polypeptide set forth in SEQ.ID NO: 2, refers to a polypeptide capable of performing the same function as the polypeptide set forth in SEQ.ID NO: 2. Functional variants include modified a polypeptide wherein, relative to a noted reference polypeptide, the modification includes a substitution, deletion or addition of one or more amino acids. In some embodiments, substitutions are those that result in a replacement of one amino acid with an amino acid having similar characteristics. Such substitutions include, without limitation (i] glutamic acid and aspartic acid; (i] alanine, serine, and threonine; (iii] isoleucine, leucine and valine, (iv] asparagine and glutamine, and (v] tryptophan, tyrosine and phenylalanine. Functional variants further include polypeptides having retained or exhibiting an enhanced benzylisoquinoline biosynthetic bioactivity.
[000201] The term "chimeric”, as used herein in the context of nucleic acids, refers to at least two linked nucleic acids which are not naturally linked. Chimeric nucleic acids include linked nucleic acids of different natural origins. For example, a nucleic acid constituting a microbial promoter linked to a nucleic acid encoding a plant polypeptide is considered chimeric. Chimeric nucleic acids also may comprise nucleic acids of the same natural origin, provided they are not naturally linked. For example a nucleic acid constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid encoding a polypeptide obtained from that same cell- type, but not normally linked to the nucleic acid constituting the promoter. Chimeric nucleic acids also include nucleic acids comprising any naturally occurring nucleic acids linked to any non-naturally occurring nucleic acids.
[000202] The terms "substantially pure” and "isolated”, as may be used interchangeably herein describe a compound, e.g., a benzylisoquinoline, polynucleotide or a polypeptide, which has been separated from components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction] in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides, by chromatography, gel electrophoresis or HPLC analysis.
[000203] The term "recovered” as used herein in association with an enzyme, protein, alkaloid, or a benzylisoquinoline, refers to a more or less pure form of the enzyme, protein alkaloid or benzylisoquinoline.
General Implementation
[000204] As hereinbefore mentioned, the present disclosure relates to alkaloids. The current disclosure further relates to certain nucleic acids and polypeptides that can be used to make alkaloids. The herein provided methods and compositions are useful in that they facilitate a novel and efficient means of making certain alkaloid compounds, notably benzylisoquinolines. The methods and compositions can yield substantial quantities of product benzylisoquinoline compounds, including codeinone, codeine, morphine, and other benzylisoquinoline compounds. The current disclosure involves the use of benzylisoquinoline uptake permeases to facilitate the production of benzylisoquinoline compounds. In an embodiment, the present disclosure provides a method of making benzylisoquinoline compounds in a host cell comprising a benzylisoquinoline uptake permease. The benzylisoquinoline uptake permease can be recombinantly expressed in the host cell. The product benzylisoquinoline compound can be produced from a substrate benzylisoquinoline compound or a precursor thereof. The substrate benzylisoquinoline compound or the precursor of the substrate benzylisoquinoline compound can be included in the growth medium for the host cell. In an embodiment, the disclosure provides methods of making benzylisoquinoline compounds involving co-culturing of a host cell comprising a benzylisoquinoline uptake permease, and a cell for co-culturing. The cells for co-culturing comprise a biosynthetic capacity to produce substrate benzylisoquinoline compound, or optionally substrate benzylisoquinoline precursor compounds, which can be used as a substrate by the host cell. It has been found by the inventors that benzylisoquinoline compounds can be unexpectedly efficiently produced in cells comprising benzylisoquinoline uptake permeases. The cells may be used as a source whence the benzylisoquinoline compounds can economically be extracted. The benzylisoquinoline compounds produced in accordance with the present disclosure are useful inter alia in the manufacture of pharmaceutical compositions.
[000205] Initially several example methods involving the growth of host cells to produce product benzylisoquinoline compounds from substrate compounds will be described. Thereafter an example method involving culturing of a host cell with a co cultured cell producing substrate will be described.
[000206] Thus, the present disclosure provides, in at least one aspect, and in at least one embodiment, a method of producing a product benzylisoquinoline compound in a host cell, the method comprising:
(a] providing a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds;
(b] introducing a chimeric nucleic acid into the host cell, the chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell; and
(c] growing the host cell in a medium to produce the benzylisoquinoline uptake permease, and the medium comprising a substrate for the biosynthetic conversion of the substrate into the product benzylisoquinoline compound by the host cell enzyme complement.
In accordance herewith, in one embodiment, a variety of compounds can be used as substrates to provide to the host cell for the biosynthetic production of a product benzylisoquinoline compound by the conversion of the substrate into a variety of product benzylisoquinoline compounds.
[000207] In some embodiments, the substrate can be an alkaloid compound.
[000208] In some embodiments, the substrate can be a substrate benzylisoquinoline compound.
[000209] In some embodiments, the substrate can be a substrate precursor benzylisoquinoline compound.
[000210] Referring now to FIG. 5, shown therein is an example biosynthetic pathway showing the conversion of certain benzylisoquinoline compounds into other benzylisoquinoline compounds in a specified order. Thus, as can be appreciated from FIG. 5, for example, (S]-norcoclaurine can be a substrate benzylisoquinoline compound that can be converted into the product benzylisoquinoline compound (5]- coclaurine; or the substrate benzylisoquinoline compound (S]-coclaurine can be converted into the product benzylisoquinoline compound (S]-/V-methylcoclaurine. In some embodiments, a substrate benzylisoquinoline compound can any benzylisoquinoline compound that can be converted into another benzylisoquinoline compound, namely the product benzylisoquinoline compound, in a single chemical step, i.e. a step forming no or no substantial amounts of free stable intermediate compounds. In some embodiments, a substrate benzylisoquinoline compound can any benzylisoquinoline compound that can be converted into another benzylisoquinoline compound, namely the product benzylisoquinoline compound, in two or more chemical steps, for example, 3, 4, 5, 6, or 7 steps, and each step forming a stable intermediate compound, notably a stable intermediate benzylisoquinoline compound. Those of skill in the art will be familiar with other benzylisoquinoline compounds and other benzylisoquinoline biosynthetic pathways and thus will be able to identify other substrate benzylisoquinoline compounds and product benzylisoquinoline compounds.
[000211] In one embodiment, a variety of substrate benzylisoquinoline precursor compounds can be used to provide to the host cell for the biosynthetic production of a product benzylisoquinoline compound, by the initial conversion of the substrate benzylisoquinoline precursor compound to a substrate benzylisoquinoline compound, and the subsequent conversion of the substrate benzylisoquinoline compound into a product benzylisoquinoline compounds.
[000212] Referring now to FIG. 6, shown therein is an example biosynthetic pathway showing the conversion of certain alkaloid compounds, notably substrate benzylisoquinoline precursor compounds into other substrate benzylisoquinoline precursor compounds, in a specified order. Thus, as can be appreciated from FIG. 6, for example, the substrate benzylisoquinoline precursor compound L-tyrosine can be converted into another substrate benzylisoquinoline precursor compound, L-DOPA; or the substrate benzylisoquinoline precursor compound L-DOPA can be converted into another substrate benzylisoquinoline precursor compound, dopamine. In some embodiments, a substrate benzylisoquinoline precursor compound can be any compound, that can be converted into another substrate benzylisoquinoline precursor compound, in a single chemical step, i.e. a step forming no or no substantial amounts of free stable intermediate compounds. Those of skill in the art will be familiar with other substrate benzylisoquinoline precursor compounds and benzylisoquinoline biosynthetic pathways and thus will be able to identify other substrate benzylisoquinoline precursor compounds.
[000213] The conversion from a substrate benzylisoquinoline compound into a product benzylisoquinoline compound can be catalyzed, in different embodiments, by benzylisoquinoline biosynthetic enzymes, including norcoclaurine synthase (NCS), 6-O-methyltransferase (60MT), coclaurine-/V-methyltransferase (CNMT), (S)- /V-methylcoclaurine 3’-hydroxylase (NMCH), 4’-0-methyltransferase (4ΌMT), reticuline epimerase (REPI), salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridinol-7-O-acetyltransferase (SalAT), or by a thebaine synthase (TS).
[000214] The conversion of a substrate benzylisoquinoline precursor compound into another substrate benzylisoquinoline precursor compound can be catalyzed, in different embodiments, by a benzylisoquinoline precursor enzyme complement. The benzylisoquinoline precursor enzyme complement can include one or more of the following benzylisoquinoline precursor enzymes: tyrosine hydroxylase (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC), and monoamide oxidase (MAO]
[000215] Next, example methods are described involving, in different examples, various possible combinations of substrates and enzymes present in the biosynthetic benzylisoquinoline enzyme complement, or, optionally, in the benzylisoquinoline precursor enzyme complement, all of which can be used to make a specific product benzylisoquinoline compound, namely salutaridine. From his description it will become clear to those of skill in the art how, using methods of the present disclosure, the product benzylisoquinoline compound salutaridine can be made. Furthermore, those of skill in the art will readily be able to employ and adjust the methods to make other product benzylisoquinoline compounds, and it is emphasized that the methods provided herein are by no means limited in their application to make salutaridine. By using the methods of the present disclosure other product benzylisoquinoline compounds can be made including, for example, (5) -norcoclaurine, (5]- norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (S)-coclaurine, ( S]-N - methylcoclaurine, (5]-3’-hydroxy-/V-methylcoclaurine, (S]-reticuline, (/?}-reticuline, salutaridine, salutaridinol and thebaine. Depending on the desired benzylisoquinoline product a variety of substrates can be selected and combined with a cell which has a compatible biosynthetic enzyme capacity to convert the substrate into the product benzylisoquinoline compound, in a similar manner as various substrates can be selected and combined with a cell with a compatible biosynthetic enzyme capacity to convert these substrates into salutaridine, as hereinafter described.
[000216] Thus, referring now to FIG. 5, in one example embodiment, salutaridine can be made using (7?]-reticuline as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzyme SalSyn.
[000217] Continuing to refer to FIG. 5, in one example embodiment, salutaridine can be made using (S]-reticuline as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes REPI and SalSyn.
[000218] Continuing to refer to FIG. 5, in one example embodiment, salutaridine can be made using (5]-3’-hydroxy-/V-methylcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes 4ΌMT, REPI and SalSyn.
[000219] Continuing to refer to FIG. 5, in one example embodiment, salutaridine can be made using (S]-/V-methylcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NMCH, 4ΌMT, REPI and SalSyn.
[000220] Continuing to refer to FIG. 5, in one example embodiment, salutaridine can be made using (S]-coclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000221] Continuing to refer to FIG. 5, in one example embodiment, salutaridine can be made using (5]-norcoclaurine as a substrate benzylisoquinoline compound, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000222] Referring now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using dopamine and 4-HPAA as a substrate benzylisoquinoline precursor compounds, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000223] Referring now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using dopamine and 3, 4-HPAA as a substrate benzylisoquinoline precursor compounds, and using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000224] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-DOPA and dopamine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme MAO.
[000225] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-DOPA and tyramine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme MAO.
[000226] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR, DODC and MAO. [000227] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-DOPA as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes DODC and MAO.
[000228] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using dopamine and L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYR and MAO.
[000229] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using tyramine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000230] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme TYDC.
[000231] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using dopamine and L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzyme TYDC. [000232] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using tyramine and dopamine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn.
[000233] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, DODC, and MAO.
[000234] Continuing to refer now to FIG. 6 in conjunction with FIG. 5, in one example embodiment, salutaridine can be made using L-tyrosine as a substrate benzylisoquinoline precursor compounds, using a cell comprising a benzylisoquinoline enzyme complement comprising the benzylisoquinoline biosynthetic enzymes, NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI and SalSyn, the cell further comprising benzylisoquinoline precursor enzyme complement comprising the benzylisoquinoline precursor enzymes TYDC, TYR, and DODC.
[000235] It will now be clear from the foregoing that the product benzylisoquinoline salutaridine can be made in accordance with the methods of present disclosure using a variety of substrate benzylisoquinoline compounds or substrate precursor benzylisoquinoline compounds in combination with a cell including a compatible benzylisoquinoline enzyme complement and, optionally, a benzylisoquinoline precursor enzyme complement. As noted, other example product benzylisoquinoline compounds that can be made in accordance herewith are (5)- norcoclaurine, (S)-norlaudanosoline, (S)-6-0-methyl-norlaudanosoline, (5)- coclaurine, (S)-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (Sj- reticuline, (R)-reticuline, salutaridine, salutaridinol and thebaine Thus the present disclosure further includes methods to make these benzylisoquinoline compounds.
[000236] Accordingly, in some embodiments, referring to FIG. 5, the product benzylisoquinoline can be thebaine, the substrate can be a benzylisoquinoline compound and the enzyme complement can comprise one or more enzymes capable of converting the thebaine precursor compound into thebaine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, SalSyn, SalR, SalAT and TS.
[000237] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be thebaine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, SalSyn, SalR, SalAT and TS and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000238] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be salutaridinol, the substrate can be a substrate benzylisoquinoline compound and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridinol, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, SalSyn, and SalR.
[000239] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be salutaridinol, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridinol, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, SalSyn, and SalR, and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000240] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be salutaridine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, and SalSyn.
[000241] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be salutaridine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridine, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT, REPI, and SalSyn, and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000242] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be (R)-reticuline, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (R)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT and REPI.
[000243] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (R)-reticuline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (R)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, 4ΌMT and REPI, and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000244] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be (5]-reticuline, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound into (S)-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, and 4ΌMT.
[000245] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (5]-reticuline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-reticuline, wherein the enzymes are selected from NCS, 60MT, CNMT, NMCH, and 4ΌMT, and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO. [000246] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be (S)-3’-hydroxy-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (5]-3’-hydroxy-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, CNMT, and NMCH.
[000247] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (S)-3’-hydroxy-/V- methylcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-3’- hydroxy-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, CNMT, and NMCH, and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000248] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be (5]-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S)-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, and CNMT.
[000249] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (5]-/V-methylcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]-/V-methylcoclaurine, wherein the enzymes are selected from NCS, 60MT, and CNMT and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000250] In some embodiments, referring to FIG. 5, the product benzylisoquinoline compound can be (S)-coclaurine, the substrate can be a benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S)-coclaurine, wherein the enzymes are selected from NCS and 60MT.
[000251] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (5)-coclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S)-coclaurine, wherein the enzymes are selected from NCS and 60MT and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000252] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (5) -norcoclaurine, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5) -norcoclaurine, wherein the enzyme is NCS and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes TYR, DODC, TYDC and MAO.
[000253] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (5)-norlaudanosoline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5)-norlaudanosoline, and the enzyme can be norcoclaurine synthase (NCS) and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC) and monoamide oxidase (MAO).
[000254] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (S)-6-0-methyl-norlaudanosoline, the substrate can be a benzylisoquinoline compound, and the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline compound (5)-6-0-methyl-norlaudanosoline, wherein the enzymes are selected from norcoclaurine synthase (NCS) and 60MT. [000255] In some embodiments, referring to FIG. 5 in conjunction with FIG. 6, the product benzylisoquinoline compound can be (S)-6-0-methyl-norlaudanosoline, the substrate can be a substrate benzylisoquinoline precursor compound, the enzyme complement can comprise one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S)-6-0-methyl-norlaudanosoline, and the enzymes can be selected from 60MT and norcoclaurine synthase (NCS) and the cell can further have a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR), dihydroxyphenyl alanine decarboxylase (DODC), tyrosine decarboxylase (TYDC) and monoamide oxidase (MAO).
[000256] In some embodiments, the host cell can further comprise an electron transfer facilitating protein.
[000257] In some embodiments, the electron transfer facilitating protein can be a cytochrome P450 reductase (CPR). In particular, in embodiments hereof involving the use of the enzymes REPI or SalSyn, it is preferred that the cells include a cytochrome P450 reductase (CPR). The CPR polypeptide may be naturally present in the host cell, or a nucleic acid expressing a CPR polypeptide, including SEQ.ID NO: 60 and SEQ.ID NO: 62, and including, a nucleic acid comprising nucleic acid sequences SEQ.ID NO: 59 and SEQ.ID NO: 61, may be introduced into the host cell, to thereby recombinantly produce the CPR polypeptide in the host cell.
[000258] In some embodiments, upon the cell having produced the product benzylisoquinoline compound, the product can be further converted in the host cell to form a derivative benzylisoquinoline.
[000259] In some embodiments, the product benzylisoquinoline compound can be thebaine, and the derivative benzylisoquinoline can be codeine, codeinone or morphine.
[000260] Referring to FIG. 5 again, In some embodiments, the conversion to form a derivative benzylisoquinoline can involve the performance of an enzyme catalyzed reaction by one or more of the enzymes T60DM, NISO, COR, and CODM.
[000261] In some embodiments, the product benzylisoquinoline compound or the derivative benzylisoquinoline can be recovered, for example, by obtaining the medium and separating the product benzylisoquinoline compound or the derivative benzylisoquinoline from other medium constituents. A variety of purification techniques and methodologies may be used, as will be known to those of skill in the art, for example chromatographical techniques, and in this manner a substantially pure product benzylisoquinoline compound or the derivative benzylisoquinoline may be obtained.
[000262] Turning now to the host cells that can be used in accordance with the present disclosure. Initially it is noted that in accordance herewith the host cell includes a benzylisoquinoline uptake permease. A variety of benzylisoquinoline uptake permeases can be used. In some embodiments, the benzylisoquinoline uptake permease can a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f).
[000263] Furthermore, as hereinbefore noted, the cell is selected or modulated to comprise a biosynthetic capability that can convert the substrate to form the product benzylisoquinoline compound. [000264] The host cell can be any cell capable of producing a product benzylisoquinoline compound, including any microbial cell, plant cell or algal cell.
[000265] In some embodiments, the benzylisoquinoline biosynthetic enzyme or the benzylisoquinoline precursor biosynthetic enzyme can be naturally present therein.
[000266] In some embodiments, the host cell can be a cell that has been modulated to produce the product benzylisoquinoline compound, for example, the host cell can be a cell comprising one or more recombinant nucleic acids encoding one or more product benzylisoquinoline compounds, as is described further below.
[000267] In some embodiments, the cell can be a cell, which, but for the presence of the substrate compound in the medium, is not capable of producing the product benzylisoquinoline compound.
[000268] In some embodiments, the cell can be a cell, which, but for the presence of the substrate compound in the medium, produces substantially less of the product benzylisoquinoline compound, for example, at least about 2 times less, at least about 5 times less, or at least about 10 times less.
[000269] In some embodiments, the cell can be a microbial cell.
[000270] In some embodiments, the microbial cell can be a bacterial cell.
[000271] In some embodiments, the bacterial cell can be an Escherichia coli cell.
[000272] In some embodiments, the microbial cell can be a yeast cell.
[000273] In some embodiments, the yeast cell can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cell.
[000274] In some embodiments, the cell can be an algal cell.
[000275] In some embodiments, the cell can be a plant cell.
[000276] In some embodiments, the plant cell can be selected from a cell obtainable from plants belonging to the plant families of Eupteleaceae,
Lardizabalaceae, Circaeasteraceae, Menispermaceae, Berberidaceae, Ranunculaceae, and Papaveraceae (including those belonging to the subfamilies of Pteridophylloideae, Papaveroideae and Fumarioideae], and further including plants belonging to the genus Argemone, including Argemone mexicana (Mexican Prickly Poppy], plants belonging to the genus Berbehs, including Berberis thunbergii (Japanese Barberry], plants belonging to the genus Chelidonium, including Chelidonium majus (Greater Celandine], plants belonging to the genus Cissampelos, including Cissampelos mucronata (Abuta], plants belonging to the genus Cocculus, including Cocculus trilobus (Korean Moonseed], plants belonging to the genus Corydalis, including Corydalis chelanthifolia (Ferny Fumewort], Corydalis cava; Corydalis ochotenis; Corydalis ophiocarpa; Corydalis platycarpa; Corydalis tuberosa; and Cordyalis bulbosa, plants belonging to the genus Eschscholzia, including Eschscholzia californica (California Poppy], plants belonging to the genus Glaucium, including Glaucium flavum (Yellowhorn Poppy], plants belonging to the genus Hydrastis, including Hydrastis canadensis (Goldenseal], plants belonging to the genus Jeffersonia, including Jeffersonia diphylla (Rheumatism Root], plants belonging to the genus Mahonia, including Mahonia aquifolium (Oregon Grape], plants belonging to the genus Menispermum, including Menispermum canadense (Canadian Moonseed], plants belonging to the genus Nandina, including Nandina domestica (Sacred Bamboo], plants belonging to the genus Nigella, including Nigella sativa (Black Cumin], plants belonging to the genus Papaver, including Papaver bracteatum (Persian Poppy], Papaver somniferum, Papaver cylindricum , Papaver decaisnei, Papaver fug ax, Papaver nudicale, Papaver oreophyllum, Papaver orientale, Papaver paeonifolium, Papaver persicum, Papaver pseudo-orientale, Papaver rhoeas, Papaver rhopalothece, Papaver armeniacum, Papaver setigerum, Papaver tauricolum, and Papaver triniaefolium, plants belonging to the genus Sanguinaria, including Sanguinaria canadensis (Bloodroot], plants belonging to the genus Stylophorum, including Stylophorum diphyllum (Celandine Poppy], plants belonging to the genus Thalictrum, including Thalictrum flavum (Meadow Rue], plants belonging to the genus Tinospora, including Tinospora cordifolia (Heartleaf Moonseed], plants belonging to the genus Xanthoriza, including Xanthoriza simplicissima (Yellowroot] and plants belonging to the genus Romeria including Romeria carica.
[000277] In accordance herewith, the host cells are grown to produce the benzylisoquinoline uptake permease and the product benzylisoquinoline compound. Growth media and growth conditions can vary depending on the cell that is selected, as will be readily appreciated to those of ordinary skill in the art. Example media include liquid culture media for the growth of yeast cells and bacterial cells including, but not limited to, Luria-Broth, Dulbecco-Eagle modified medium (DMEM] or Optimem. Further media and growth conditions can be found in Sambrook et ah, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.
[000278] In some embodiments, the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline uptake permease selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c), (d), (e) or (f).
[000279] As noted, in some embodiments, the host cells can be modulated to include a compatible biosynthetic enzyme complement to synthesize the product benzylisoquinoline compound from a substrate. Accordingly, in some embodiments, the host cell can be manipulated to obtain a cell comprising a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
[000280] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37, SEQ.ID NO: 39, and SEQ.ID NO: 55;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the second cell. [000281] In some embodiments, the host cell can be manipulated to obtain a cell comprising a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
[000282] In some embodiments, the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 47, SEQ.ID NO: 49, SEQ.ID NO: 51, and SEQ.ID NO: 53;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell. [000283] A variety of techniques and methodologies to manipulate cells to introduce nucleic acid sequences in cells and attain expression exists and are well known to the skilled artisan and can, for example be found in Sambrook et ai, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.
[000284] Nucleic acid sequences capable of controlling expression in cells that may be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells. Generally, promoters obtained from bacterial cells are used when a bacterial host is selected in accordance herewith, while a fimgal promoter will be used when a fimgal host cell is selected, a plant promoter will be used when a plant cell is selected, and so on. Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.
[000285] In accordance with the present disclosure, the chimeric nucleic acid sequences comprising a promoter capable of controlling expression in host cell linked to a nucleic acid sequence encoding a benzylisoquinoline uptake permease or a benzylisoquinoline biosynthetic enzyme, can be integrated into a recombinant expression vector which ensures good expression in the host cell, wherein the expression vector is suitable for expression in a host cell. The term "suitable for expression in a host cell” means that the recombinant expression vector comprises the chimeric nucleic acid sequence linked to genetic elements required to achieve expression in a cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication and the like. In preferred embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell's genome, for example if a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome.
[000286] Pursuant to the present disclosure, the expression vector may further contain a marker gene. Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non- transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable markers that may be employed to identify transformants through visual inspection include b -glucuronidase (GUS] (U.S. Pat. Nos. 5,268,463 and 5,599,670] and green fluorescent protein (GFP] (Niedz et al, 1995, Plant Cell Rep., 14: 403]
[000287] One host cell that conveniently may be used is Escherichia coli. The preparation of the £. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gelelectrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR] and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc. Typically, these cloning vectors contain a marker allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in £. coli by preparing competent cells, electroporation or using other well known methodologies to a person of skill in the art. £. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells. Further, general guidance with respect to the preparation of recombinant vectors and growth of recombinant organisms may be found in, for example: Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.
[000288] As hereinbefore noted, in one embodiment, the substrate can be provided by cell for co-culturing with the host cell. The cell for co-culturing can be any cell capable of growing and secreting a substrate for use of by the host cell, i.e. a substrate benzylisoquinoline compound or a substrate benzylisoquinoline precursor compound. By the term "co-cultured” it is meant that the host cell and cell for co- culturing are contacted, and mixed, for example in a co-culturing medium, in such a manner that the host cell and cell for co-culturing remain viable, at least for a sufficiently long period of time for the host cell to produce the product benzylisoquinoline compound.
[000289] Co-culturing of the host cell and cell for co-culturing can be accomplished, for example, by growing and culturing a host cell in a first liquid growth medium for a period of time and under conditions suitable for growing the host cell, and separately growing the cell for co-culturing for a period of time in in a second liquid growth medium and under conditions suitable for growing the cell for co-culturing. Thereafter the first and second liquid media, containing the host cell and cell for co-culturing, respectively can be mixed to thereby contact the host cell and cell for co-culturing. In other embodiments, the cells from one of the cultures can be harvested from the liquid medium and the harvested cells can be admixed into the other culture. It is further also possible to harvest host cells and cells for co-culturing from the respective liquid media, and mix the cells in a third liquid medium. Upon establishing suitable co-culturing conditions, the substrate can be produced by the cell for co-culturing and secreted in the co-culturing medium to thereby thereafter become available for use by the host cell. Without wishing to be bound by theory, the inventors believe that the benzylisoquinoline uptake permease included in the host cell may facilitate uptake of the first benzylisoquinoline compound by the host cell.
[000290] In some embodiments, the host cell and cell for co-culturing can be cells belonging to the same biological species.
[000291] In some embodiments, the host cell and cell for co-culturing can be microbial cells.
[000292] In some embodiments, the host cell and cell for co-culturing can be bacterial cells.
[000293] In some embodiments, the host cell and cell for co-culturing can be yeast cells.
[000294] In some embodiments, the yeast cells can be Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
[000295] In some embodiments, the host cell and cell for co-culturing can be algal cells.
[000296] In some embodiments, the host cell and cell for co-culturing can be plant cells.
[000297] In some embodiments, the cell for co-culturing can include a benzylisoquinoline uptake permease. The benzylisoquinoline uptake permease can be expressed recombinantly in the cell for co-culturing, and can include any benzylisoquinoline uptake permease set forth herein. [000298] In another aspect, the present disclosure provides, in at least one embodiment, a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds, the host cell comprising a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell, and the host cell capable of producing the benzylisoquinoline uptake permease and a product benzylisoquinoline compound when provided with a substrate compound.
[000299] In another aspect, the present disclosure provides, in at least one embodiment, a mixture of cells comprising host cells recombinantly expressing a benzylisoquinoline uptake permease and having a benzylisoquinoline enzyme complement to biosynthetically produce a product benzylisoquinoline compound, and cells for co-culturing with the host cells, wherein the cells for co-culturing are capable of secreting a substrate that can be converted by the host cells when co cultured to form the product benzylisoquinoline compound.
[000300] It will be clear form the foregoing that the methods of the present disclosure may be used to make a variety of benzylisoquinoline compounds. The obtained benzylisoquinoline compounds may be formulated for use as a pharmaceutical drug, therapeutic agent or medicinal agent. Thus the present disclosure further includes a pharmaceutical composition comprising a benzylisoquinoline compound prepared in accordance with the methods of the present disclosure. Pharmaceutical drug preparations comprising a benzylisoquinoline compound in accordance with the present disclosure can comprise vehicles, excipients and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like. These vehicles, excipients and auxiliary substances are generally pharmaceutical agents that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, benzoates, and the like. It is also preferred, although not required, that the preparation will contain a pharmaceutically acceptable excipient that serves as a stabilizer. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, glycine, polyethylene glycols (PEGs], and combinations thereof. The pharmaceutical composition may be formulated for oral and intravenous administration and other routes of administration as desired. Dosing may vary and may be optimized using routine experimentation.
[000301] In another aspect, the present disclosure further provides, in an embodiment, a use of a benzylisoquinoline compound prepared in accordance with any one of the methods of the present disclosure to prepare a pharmaceutical composition comprising the benzylisoquinoline compound.
[000302] The benzylisoquinoline compounds of the present disclosure further may be used as precursor or feedstock material for the production of derivative benzylisoquinoline compounds. Thus, for example, as has been described herein, thebaine made in accordance the disclosure can be used as a precursor to make codeinone, codeinone made in accordance with the present disclosure can be used as a precursor to make codeine, and codeine can be used as a precursor to make morphine. It will be clear to those of skill in the art that the benzylisoquinoline compounds made in accordance with the present disclosure can be used to make a wide variety of derivative benzylisoquinoline compounds. Upon finishing synthesis the benzylisoquinoline compounds can be used to formulate pharmaceutical drugs, as hereinbefore described.
[000303] In further embodiments, the present disclosure provides methods for treating a patient with a pharmaceutical composition comprising a benzylisoquinoline compound prepared in accordance with the present disclosure. Accordingly, the present disclosure further provides a method for treating a patient with a benzylisoquinoline compound prepared according to the methods of the present disclosure, the method comprising administering to the patient a pharmaceutical composition comprising a benzylisoquinoline compound, wherein the pharmaceutical composition is administered in an amount sufficient to ameliorate a medical condition in the patient. [000304] In further embodiments, the present disclosure provides a use of a pharmaceutical composition comprising a benzylisoquinoline compound prepared according to the present disclosure to treat a patient. In other embodiments, the present disclosure provides a pharmaceutical composition comprising a benzylisoquinoline compound prepared according to the present disclosure for use in treating a patient. The treatment can comprise use of the pharmaceutical composition to the patient in an amount sufficient to ameliorate a medical condition in the patient.
SUMMARY OF SEQUENCES
[000305] SEQ.ID NO: 1 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000306] SEQ.ID NO: 2 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000307] SEQ.ID NO: 3 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000308] SEQ.ID NO: 4 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000309] SEQ.ID NO: 5 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000310] SEQ.ID NO: 6 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000311] SEQ.ID NO: 7 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000312] SEQ.ID NO: 8 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000313] SEQ.ID NO: 9 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000314] SEQ.ID NO: 10 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000315] SEQ.ID NO: 11 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000316] SEQ.ID NO: 12 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide. [000317] SEQ.ID NO: 13 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000318] SEQ.ID NO: 14 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000319] SEQ.ID NO: 15 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000320] SEQ.ID NO: 16 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000321] SEQ.ID NO: 17 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000322] SEQ.ID NO: 18 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000323] SEQ.ID NO: 19 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000324] SEQ.ID NO: 20 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000325] SEQ.ID NO: 21 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide.
[000326] SEQ.ID NO: 22 sets forth a deduced amino acid sequence of a benzylisoquinoline uptake permease polypeptide.
[000327] SEQ.ID NO: 23 sets forth a polynucleotide sequence encoding a 60MT polypeptide.
[000328] SEQ.ID NO: 24 sets forth a deduced amino acid sequence of a 60MT polypeptide.
[000329] SEQ.ID NO: 25 sets forth a polynucleotide sequence encoding a CNMT polypeptide.
[000330] SEQ.ID NO: 26 sets forth a deduced amino acid sequence of a CNMT polypeptide.
[000331] SEQ.ID NO: 27 sets forth a polynucleotide sequence encoding an NMCH polypeptide.
[000332] SEQ.ID NO: 28 sets forth a deduced amino acid sequence of an NMCH polypeptide. [000333] SEQ.ID NO: 29 sets forth a polynucleotide sequence encoding a 4ΌMT polypeptide.
[000334] SEQ.ID NO: 30 sets forth a deduced amino acid sequence of a 4ΌMT polypeptide.
[000335] SEQ.ID NO: 31 sets forth a polynucleotide sequence encoding a REPI polypeptide.
[000336] SEQ.ID NO: 32 sets forth a deduced amino acid sequence of a REPI polypeptide.
[000337] SEQ.ID NO: 33 sets forth a polynucleotide sequence encoding a SalSyn polypeptide.
[000338] SEQ.ID NO: 34 sets forth a deduced amino acid sequence of a SalSyn polypeptide.
[000339] SEQ.ID NO: 35 sets forth a polynucleotide sequence encoding a SalR polypeptide.
[000340] SEQ.ID NO: 36 sets forth a deduced amino acid sequence of a SalR polypeptide.
[000341] SEQ.ID NO: 37 sets forth a polynucleotide sequence encoding a SalAT polypeptide.
[000342] SEQ.ID NO: 38 sets forth a deduced amino acid sequence of a SalAT polypeptide.
[000343] SEQ.ID NO: 39 sets forth a polynucleotide sequence encoding a TS polypeptide.
[000344] SEQ.ID NO: 40 sets forth a deduced amino acid sequence of a TS polypeptide.
[000345] SEQ.ID NO: 41 sets forth a polynucleotide sequence encoding a NISO polypeptide.
[000346] SEQ.ID NO: 42 sets forth a deduced amino acid sequence of a NISO polypeptide.
[000347] SEQ.ID NO: 43 sets forth a polynucleotide sequence encoding a COR polypeptide.
[000348] SEQ.ID NO: 44 sets forth a deduced amino acid sequence of a COR polypeptide. [000349] SEQ.ID NO: 45 sets forth a polynucleotide sequence encoding a CODM polypeptide.
[000350] SEQ.ID NO: 46 sets forth a deduced amino acid sequence of a CODM polypeptide.
[000351] SEQ.ID NO: 47 sets forth a polynucleotide sequence encoding a TYR polypeptide.
[000352] SEQ.ID NO: 48 sets forth a deduced amino acid sequence of a TYR polypeptide.
[000353] SEQ.ID NO: 49 sets forth a polynucleotide sequence encoding a TYDC polypeptide.
[000354] SEQ.ID NO: 50 sets forth a deduced amino acid sequence of a TYDC polypeptide.
[000355] SEQ.ID NO: 51 sets forth a polynucleotide sequence encoding a DODC polypeptide.
[000356] SEQ.ID NO: 52 sets forth a deduced amino acid sequence of a DODC polypeptide.
[000357] SEQ.ID NO: 53 sets forth a polynucleotide sequence encoding a MAO polypeptide.
[000358] SEQ.ID NO: 54 sets forth a deduced amino acid sequence of a MAO polypeptide.
[000359] SEQ.ID NO: 55 sets forth a polynucleotide sequence encoding an NCS polypeptide.
[000360] SEQ.ID NO: 56 sets forth a deduced amino acid sequence of an NCS polypeptide.
[000361] SEQ.ID NO: 57 sets forth a polynucleotide sequence encoding a T60DM polypeptide.
[000362] SEQ.ID NO: 58 sets forth a deduced amino acid sequence of a T60DM polypeptide.
[000363] SEQ.ID NO: 59 sets forth a polynucleotide sequence encoding a CPR polypeptide.
[000364] SEQ.ID NO: 60 sets forth a deduced amino acid sequence of a CPR polypeptide. [000365] SEQ.ID NO: 61 sets forth a polynucleotide sequence encoding a CPR polypeptide.
[000366] SEQ.ID NO: 62 sets forth a deduced amino acid sequence of a CPR polypeptide.
[000367] SEQ.ID NO: 63 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 2]
[000368] SEQ.ID NO: 64 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 2 plus c-myc tag]
[000369] SEQ.ID NO: 65 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 10).
[000370] SEQ.ID NO: 66 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 10 plus c-myc tag).
[000371] SEQ.ID NO: 67 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 8).
[000372] SEQ.ID NO: 68 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 8 plus c-myc tag).
[000373] SEQ.ID NO: 69 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 12).
[000374] SEQ.ID NO: 70 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 12 plus c-myc tag).
[000375] SEQ.ID NO: 71 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 14).
[000376] SEQ.ID NO: 72 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 14 plus c-myc tag).
[000377] SEQ.ID NO: 73 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 16). [000378] SEQ.ID NO: 74 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 16 plus c-myc tag]
[000379] SEQ.ID NO: 75 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 18).
[000380] SEQ.ID NO: 76 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 18 plus c-myc tag).
[000381] SEQ.ID NO: 77 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 20).
[000382] SEQ.ID NO: 78 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 20 plus c-myc tag).
[000383] SEQ.ID NO: 79 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 22).
[000384] SEQ.ID NO: 80 sets forth a polynucleotide sequence encoding a benzylisoquinoline uptake permease polypeptide (codon optimized SEQ.ID NO: 24 plus c-myc tag).
[000385] SEQ.ID NO: 81 sets forth a polynucleotide sequence encoding a c-myc - tag.
[000386] SEQ.ID NO: 82 sets forth an amino acid sequence of a c- myc-tag.
[000387] Hereinafter are provided examples of specific implementations for performing the methods of the present disclosure, as well as implementations representing the compositions of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.
EXAMPLES
Example 1 - Method of making veast cells comprising benzylisoquinoline biosynthetic complement and a precursor benzylisoquinoline
Figure imgf000072_0001
biosynthetic enzvme complement.
[000388] Yeast strains with chromosome-integrated genes encoding enzymes involved in benzylisoquinoline alkaloid biosynthesis were constructed using a USER cloning system. USER (uracil -specific excision reaction) -based cloning has been used for the integration of multiple genes into the yeast genome owing to its relatively straightforward application and independence from the enzyme-based ligation of DNA fragments. Multiple PCR products of various BIA biosynthetic genes (DODC, MAO, NCS, 60MT, CNMT and 4ΌMT) and Gall/GallO promoter regions were simultaneous cloned to the USER cloning vectors initially nicked with As/SI and Nb.Bsml and then transformed into yeast cells using the LiAc/PEG/single-stranded carrier DNA (ssDNA) transformation method. The high-copy number pESC-Ura (or, alternatively, pESC-Leu or pESC-His) vector was used to express benzylisoquinoline uptake permease (BUP) genes using the GallO promoter. PCR-amplified candidate genes from cDNA using primers flanked with Spel and Notl restriction sites were ligated to the pESC-Ura vector to generate transient expressing constructs. Transient expression constructs were individually transformed to the platform yeast strains with chromosome-integrated BIA biosynthetic genes using the LiAc/PEG/single- stranded carrier DNA (ssDNA) transformation method. Each yeast strain transiently expressing a candidate gene was inoculated in SD-drop out medium overnight. The overnight cultures were then diluted into a SD-drop out medium containing 2% (w/v) galactose and 200 mM of the BIA suitable for conversion by the baseline yeast strain and/or the transient expression construct. Yeast cultures were grown in the presence of suitable substrates for 24 h.
[000389] After fermentation, yeast cells were removed from their culture medium by centrifugation and 5 pL of supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS) analysis. Alkaloids produced in yeast were characterized by high- resolution MSn analysis. For MSn experiments, alkaloids were injected by HPLC for electrospray ionization (ESI) prior to analysis by LTQ-Orbitrap-XL. Operation was conducted using LTQ Tune Plus v. 2.5.5 SP1 and Xcalibur v. 2.1.0.1140, with additional analyses using QualBrowser feature of Xcalibur. Internal calibration, external calibration, tuning, and general operations were performed using routine procedures optimized for alkaloid detection, with the exception that ESI, rather than heated HESI, was employed with reduced flow rates to minimize heat-induced degradation of analytes during ionization. Data acquisition for MS2 was performed using a single scan event, involving CID conducted in linear ion trap (IT) with normalized collision energy (NCE) of 35%, and detection by FTMS with resolution of 60,000 FWHM and scan range m/z 90-340. Data acquisition for MS3 was performed by first identifying the most intense ions in MS2, optimizing NCE for CID -based fragmentation of these ions (15-35%], and adjusting FTMS scan ranges for detection of MS3 ions. To ensure sufficient MS3 detection by FTMS, individual runs comprised of only one scan event were conducted for each MS2 ion subjected to CID analysis. Error was maintained at < 2 ppm to allow prediction of elemental formulae for all ions. Compound identity was based on comparisons with authentic standards and MSn assignments available in the literature.
Example 2 - Production in veast cells of fSVreticuline using L-DOPA as a substrate
[000390] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21. Each of the transformed strains grown in a liquid medium comprising L-DOPA and (SJ-reticuline product was measured in the media. The results are show in FIG. 7.
Example 3 - Production in yeast cells of (5)-reticuline using dopamine as a substrate
[000391] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21. Each of the transformed strains grown in a liquid medium comprising dopamine and (S]-reticuline product was measured in the media. The results are show in FIG. 8.
Example 4 - Production in yeast cells of (5)-reticuline using norlaudanosoline as a substrate
[000392] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding different benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 19, and SEQ.ID NO: 21. Each of the transformed strains grown in a liquid medium comprising norlaudanosoline and (S]-reticuline product was measured in the media. The results are show in FIG. 9.
Example 5 - Production in yeast cells of thebaine. co-cultured with a yeast cell strain using L-DOPA as a substrate
[000393] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permease, notably a nucleic acid construct comprising SEQ.ID NO: 1. The yeast cell strain was co-cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS. The cells of both strains were mixed and grown in a liquid medium comprising L-DOPA and thebaine product was measured in the media. The results are shown in FIG. 10.
Example 6 - Production in yeast cells of thehaine. co-cultured with a yeast cell strain using dopamine as a substrate
[000394] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1. The yeast cell strain was co- cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS. The cells of both strains were mixed and grown in a liquid medium comprising dopamine and thebaine product was measured in the media. The results are shown in FIG. 11.
Example 7 - Production in yeast cells of thebaine. co-cultured with a yeast cell strain using norlaudanosoline as a suhstrate
[000395] A yeast cell strain with genomically integrated genes encoding DODC, MAO, NCS, 60MT, CNMT and 4ΌMT was transformed with an expression construct comprising a nucleic acid encoding a benzylisoquinoline uptake permeases, notably a nucleic acid construct comprising SEQ.ID NO: 1. The yeast cell strain was co- cultured with a yeast strain containing genomically integrated genes encoding REPI, SalSyn, CPR2, SalR, SalAT and TS. The cells of both strains were mixed and grown in a liquid medium comprising norlaudanosoline and thebaine product was measured in the media. The results are shown in FIG. 12. Example 8 - Uptake of alkaloid compounds bv veast cells comprising BUP polypeptides
[000396] Yeast ( Saccharomyces cerevisiae) strain CEN.PK102-5B was transformed with a galactose-inducible, transient expression construct based on pESC-HIS. This plasmid had been modified with the addition of a gene conferring antibiotic resistance to G418 (KanMX; Walker et al. 2003, FEMS Yeast Research 4: 339-347] as described (Dastmalchi et al. 2018, Nature Chemical Biology, in press]. Codon-optimized coding sequences for BUP1 through BUP6 including C-terminal c- myc epitope tags (SEQ.ID NO: 81; SEQ.ID NO: 82] were synthesized at GenScript USA (www.genscript.com] and subcloned to MCS1 ofthe modified pESC-HIS using BamHl and SacII restrictions sites. Codon optimized sequences used were: SEQ.ID NO: 63 (BUP1]; SEQ.ID NO: 65 (BUP2]; SEQ.ID NO: 67 (BUP3]; SEQ.ID NO: 69 (BUP4]; SEQ.ID NO: 71 (BUP5]; and SEQ.ID NO: 73 (BUP6] Codon optimized sequence including the c-myc epitope tag were: SEQ.ID NO: 64 (BUP1]; SEQ.ID NO: 66 (BUP2]; SEQ.ID NO: 68 (BUP3]; SEQ.ID NO: 70 (BUP4]; SEQ.ID NO: 72 (BUP5]; and SEQ.ID NO: 74 (BUP6] Following transformation, four individual colonies were selected per clone for yeast uptake assays. Uptake assays were conducted as follows: yeast strains transiently expressing one of BUP1 (SEQ.ID NO: 2], BUP2 (SEQ.ID NO: 10], BUP3 (SEQ.ID NO: 8], BUP4 (SEQ.ID NO: 12], BUP5 (SEQ.ID NO: 14], BUP6 (SEQ.ID NO: 16], BUP8 (SEQ.ID NO: 20], BUP9 (SEQ.ID NO: 22] or no gene ("empty vector” controls] were inoculated in 200 mΐ SD-drop out medium containing 2% glucose overnight in a 96-well format, using a Fisherbrand Incubating Microplate Shaker (Fisher Scientific] The overnight cultures were then diluted with 300 mΐ SD-drop out medium containing 2% (w/v] galactose and 1 mM L-DOPA or dopamine, or 200 mM of alkaloid substrate. Yeast cultures were grown for 24 h post-induction at 30°C unless otherwise noted. Following culturing, yeast cells were gently pelleted and washed 2X with media. Alkaloids in yeast cell pellets were extracted twice with 500 mΐ of 100% methanol. Insoluble matter was centrifuged, supernatant was pooled, dried, and resuspended in 200 mΐ of 50:50 water:ACN.
[000397] The results are shown in the graphs of FIG. 13A-F. Shown in each graph is the uptake (y-axes] of the alkaloid compounds noted on the x-axes (z.e. DOPA, dopamine, norcoclaurine, (S]-reticuline, (R]-reticuline, salutaridine, thebaine, codeine, morphine, noscapine and papaverine], in yeast cells transformed with BUP1 (SEQ.ID NO: 2] (FIG. 13A], BUP2 (SEQ.ID NO: 10] (FIG. 13B], BUP3 (SEQ.ID NO: 8] (FIG. 13C], BUP4 (SEQ.ID NO: 12] (FIG. 13D], BUP5 (SEQ.ID NO: 14] (FIG. 13E] and BUP 6 (SEQ.ID NO: 16] (FIG. 13F], respectively. Uptake for each alkaloid compound is compared to yeast cells transformed with a vector not including a BUP protein (empty vector controls].
Example 9 - Synthesis of alkaloid compounds by yeast cells comprising BUP polypeptides
[000398] Parent yeast ( Saccharomyces cerevisiae) strain CEN.PK102-5B was selected for engineering through genomic integration. Integration cassettes were assembled based on a series of plasmids first reported by Mikkelsen et al. (2012] Metab. Eng. 14: 104, and improved upon by Jensen et al. (2014] FEMS Yeast Res. 14: 238. These plasmids were generously provided as a gift by Dr. Irina Borodina (Technical University of Denmark], Dr. Uffe Hasbro Mortensen (Technical University of Denmark] and Dr. Barbara Ann Halkier (University of Copenhagen] Construct assembly, genomic integration, and fermentation were performed as described previously (Morris et al. 2016, Methods Enzymol. 575: 143] Briefly, coding sequences for biosynthetic genes were codon-optimized and synthesized at GenScript USA (www.genscript.com], followed by subcloning to integration vectors using standard, USER-based strategy (Salomonsen et al. 2014, Methods in Molecular Biology 1116: 59-72] Each integration vector hosted two biosynthetic genes, under control of a bi-directional, inducible promoter system (Gal 1/Gal 10] Three yeast strains (strain 1, strain 2 and strain 3] were prepared as follows:
(i] three plasmids were used to engineer strain 1, capable of producing (5]- reticuline, containing (1] Pseudomonas putida L-DOPA decarboxylase (DODC] plus Homo sapiens monoamine oxidase-A (MAO-A]; (2] Papaver somniferum norcoclaurine synthase (NCS] plus Papaver somniferum norcoclaurine 6-0-methyltransferase (60MT]; and (3] Papaver somniferum coclaurine /V-methyltransferase (CNMT] plus Papaver somniferum 3’-hydroxy- /V-methylcoclaurine 4’-0-methyltransferase (4ΌMT], respectively;
(ii] three plasmids were used to engineer strain 2, capable of producing thebaine, containing (1] Papaver somniferum /V-terminally truncated (D32] (5] -reticuline epimerase /V-terminally fused to the initial 45 amino acids of Artemisa annua germacrene A hydroxylase (GAO-REPI] plus /V-terminally truncated (D9] Papaver somniferum salutaridine synthase (SalSyn]; (2] Papaver somniferum salutaridine reductase (SalR] plus Papaver somniferum salutaridinol 7-O-acetyltransferase (SalAT]; and (3] Papaver somniferum thebaine synthase (THS] plus Papaver somniferum cytochrome P450 reductase 2 (CPR2], respectively; and
(iii] two plasmids were used to engineer strain 3, capable of producing codeine, containing (1] Papaver somniferum thebaine 6-0-demethylase (T60DM] plus Papaver somniferum codeinone reductase 1.3 (C0R1.3]; and (2] Papaver somniferum codeine O-demethylase, respectively. For strain 3, a second version lacking CODM (but still integrated for T60DM and C0R1.3] was used for metabolic conversion assays.
Step-wise, genomic integration of these plasmids to strains 1, 2, and 3 was done according to Morris et al. (2016], Methods Enzymol. 575: 143. Each of these engineered strains were then transformed with a transient expression construct with no gene ("empty vector” control] or one of BUP1 through BUP9 (BUP1 (SEQ.ID NO: 2], BUP2 (SEQ.ID NO: 10], BUP3 (SEQ.ID NO: 8], BUP4 (SEQ.ID NO: 12], BUP5 (SEQ.ID NO: 14], BUP6 (SEQ.ID NO: 16], BUP8 (SEQ.ID NO: 20], BUP9 (SEQ.ID NO: 22] The transient expression construct was a modified version of the galactose-inducible plasmid pESC-HIS, exhibiting an additional selection marker (KanMX] for antibiotic G418 resistance (Dastmalchi et al. 2018. Nature Chemical Biology, in Press] Plasmids hosting BUPs or no gene (controls] were individually transformed to the platform yeast strains with chromosome-integrated BIA biosynthetic genes using the LiAc/PEG/single-stranded carrier DNA (ssDNA] transformation method, and four resulting transformants were selected for each clone as biological replicates. Yeast strains transiently expressing candidate BUP genes were inoculated in 200 mΐ SD- drop out medium containing 2% glucose overnight, in a 96-well format, using a Fisherbrand Incubating Microplate Shaker (Fisher Scientific] The overnight cultures were then diluted with 300 mΐ SD-drop out medium containing 2% (w v1] galactose and 250 mM alkaloid substrate for bioconversion. For DOPA and dopamine feeding, concentrations of 1 mM were used in the media. Yeast cultures were grown for 24 h post-induction at 30°C. Yeast cells were removed by centrifugation and 5 pL of supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS] analysis. [000399] Saccharomyces cerevisiae strains 1 and 3 with chromosome-integrated BIA biosynthetic genes and transient expression of BUP1 in modified pESC-HIS were individually inoculated in 200 mΐ SD-drop out medium containing 200 pg/l of G418 and 2% glucose and cultured overnight at 30°C. The same was done for strain 2, except that this strain was transiently expressing modified pESC-HIS harboring both BUP1 (in MCS2) and Papaver somniferum THS (in MCS1) to support sufficient thebaine formation. The overnight cultures of strains 1, 2 and 3 were then co-cultured into 300 mI of SD-drop out medium containing 2% (w/v) galactose and 200 ug/l of G418 and 1 mM of DOPA to allow bioconversion. Yeast co-cultures were grown for 24 h post-induction at 30°C, followed by removal of cells by centrifugation. Five microliters of the media supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS) analysis.
[000400] Yeast media and cell extracts were analyzed by high-resolution LC-ESI- LTQ-Orbitrap-XL mass spectrometry. Cell extracts were pre-processed prior to loading to remove protein by diluting 50:50 with methanol, centrifugation to pellet insolubles, and retrieval of supernatant for analysis. Samples were either loaded directly or diluted with methanol to ensure target alkaloids remained within linear ranges as indicated by standard curves using authentic standards. High-resolution LC-ESI-LTQ-Orbitrap-XL mass spectrometry (MS) was performed using a modified version ofa method described previously (Chang etal. 2015, PlantPhysiol. 169, 1127- 1140), with the exception that liquid chromatography was carried out using an UltiMate 3000 HPLC (Thermo Fisher Scientific) equipped with a Poroshell 120 SB- C18 column (Agilent Technologies) instead of an Accela HPLC system (Thermo Fisher Scientific) equipped with a Zorbax C18 column (Agilent Technologies). Five microliters of samples were fractionated at a flow rate of 0.5 mL min 1 and a gradient of Solvent A (10 mM ammonium acetate, pH 5.5, 5% ACN) and Solvent B (100% ACN) as follows: 100% to 80% Solvent A over 5 min, 80% to 50% over 3 min, 50% to 0% over 3 min, isocratic at 0% for 2 min, 0% to 100% over 0.1 min, and isocratic at 100% for 1.9 min. Total run time was 15 min, but data were collected for only 10 min. Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature, 400°C; source voltage, 3 kV; sheath gas, 60 au, auxiliary gas, 20 au; capillary temperature, 380°C; capillary voltage, 6 V; tube lens, 45 V. LTQ-Orbitrap-XL (Thermo Scientific] instrumentation was performed as three scan events in data-dependent, parallel detection mode. The first scan consisted of high-resolution FTMS from 150 to 450 m/z with ion injection time of 500 ms and scan time of approximately 1.5 s. The second and third scans (approximately 0.5 s each] collected CID spectra in the ion trap, where the parent ions represented the first- and second-most abundant alkaloid masses, respectively, as determined by fast Fourier transform preview using a parent ion mass list corresponding to exact masses of known alkaloids. Dynamic-exclusion and reject-ion-mass-list features were enabled. External and internal calibration procedures ensured < 2 ppm error. Exact mass, retention times, and CID spectra of authentic standards were used to identify alkaloids, and quantification was performed using standard curves. The Quan Browser feature of Thermo X-Calibur v. 3.1 was employed for automated peak identification and quantification.
[000401] Results are show in FIG. 14A-H and FIG. 15. Shown in FIG. 14A-H is the production of various alkaloid compounds in yeast, notably reticuline (FIG. 14A- C], salutardine (FIG. 14D-E], thebaine (FIG. 14D-F], codeine (FIG. 14G], neopine (FIG. 14G] and morphine (FIG. 14H], respectively, (y-axes] using yeast strain 1 (FIG. 14A-C], strain 2 (FIG. 14D-F] and strain 3 (FIG. 14 G-H], respectively, in each case transformed with BUP1 (SEQ.ID NO: 2], BUP2 (SEQ.ID NO: 10], BUP3 (SEQ.ID NO: 8], BUP4 (SEQ.ID NO: 12], BUP5 (SEQ.ID NO: 14], BUP6 (SEQ.ID NO: 16], BUP8 (SEQ.ID NO: 20], BUP9 (SEQ.ID NO: 22] (x-axes]. Substrate alkaloid were varied as follows: DOPA (FIG. 14A], dopamine (FIG. 14B], norlaudanosoline (FIG. 14C], (5] -reticuline (FIG. 14D], (R] -reticuline (FIG. 14E], salutaridine (FIG. 14F], thebaine (FIG. 14G] and codeine (FIG. 14H] Production of each alkaloid compound is compared to yeast cells transformed with vectors not including BUP proteins (empty vector controls] [000402] Shown in FIG. 15A-D are the production of various alkaloid compounds (y-axes], notably reticuline, salutardine, thebaine and codeine (x-axes] by co-culturing yeast strains 1 and 2 (FIG 15A and FIG. 15C] and strains 1, 2 and 3 (FIG. 15B and FIG. 15D] . Alkaloid substrates that were used were L-DOPA (FIG. 15A and FIG. 15B] and (S] -reticuline (FIG. 15C and FIG. 15D] Production of each alkaloid compound is detected in strains transformed with BUP1 (SEQ.ID NO: 2] (right hand side of each FIG. 15A-D], and compared with (empty vector controls] (left hand side of each FIG. 15A-D]

Claims

1. A method of producing a product benzylisoquinoline compound in a host cell, the method comprising:
(a] providing a host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds;
(b] introducing a chimeric nucleic acid into the host cell, the chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell; and
(c] growing the host cell in a medium to produce the benzylisoquinoline uptake permease, and the medium comprising a substrate for the biosynthetic conversion of the substrate into the product benzylisoquinoline compound by the host cell enzyme complement.
2. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-norcoclaurine, (5]-norlaudanosoline, (S]-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, salutaridinol, or thebaine.
3. The method of claim 1, wherein the substrate is a substrate benzylisoquinoline compound.
4. The method of claim 3, wherein the substrate benzylisoquinoline compound is (5]-norcoclaurine, (5]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, or salutaridinol.
5. The method of claim 1, wherein the substrate is a substrate benzylisoquinoline precursor compound.
6. The method of claim 5, wherein, the substrate benzylisoquinoline precursor compound can be L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydroxy- phenylacetaldehyde (4-HPAA]
7. The method of claim 1, wherein the enzyme complement comprises one or more benzylisoquinoline biosynthetic enzymes selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (5]- /V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-0-acetyltransferase (SalAT], or thebaine synthase (TS]
8. The method of claim 7, wherein the host cell further has a benzylisoquinoline precursor enzyme complement.
9. The method of claim 8, wherein the benzylisoquinoline precursor enzyme complement comprises one or more benzylisoquinoline precursor biosynthetic enzymes selected from tyrosine hydroxylase (TYR], (dihydroxyphenyl alanine decarboxylase] DODC, tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
10. The method of any one of claims 1 to 9, wherein the substrate is converted into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
11. The method of any one of claims 1 to 9, wherein, the substrate is converted into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
12. The method of claim 1, wherein the product benzylisoquinoline is thebaine, the substrate is a substrate benzylisoquinoline compound and the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS]
13. The method of claim 1, wherein, the product benzylisoquinoline compound is thebaine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into thebaine, the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS], and wherein the cell has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
14. The method of claim 1, wherein the product benzylisoquinoline compound is salutaridinol, the substrate is a substrate benzylisoquinoline compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridinol, and the enzymes can be selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-iV- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0- methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O-acetyltransferase (SalAT]
15. The method of claim 1, wherein the product benzylisoquinoline compound is salutaridinol, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridinol, the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], or salutaridinol-7-O- acetyltransferase (SalAT], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more ofthe enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
16. The method of claim 1, wherein the product benzylisoquinoline compound is salutaridine, the substrate is a substrate benzylisoquinoline compound, and the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound into salutaridine, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn]
17. The method of claim 1, wherein the product benzylisoquinoline compound is salutaridine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into salutaridine, the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], reticuline epimerase (REPI], or salutaridine synthase (SalSyn], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
18. The method of claim 1, wherein the product benzylisoquinoline compound is (R] -reticuline, the substrate is a substrate benzylisoquinoline compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound into (R] -reticuline, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4ΌMT], or reticuline epimerase (REPI]
19. The method of claim 1, wherein the product benzylisoquinoline compound is (7?] -reticuline, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (7?] -reticuline, the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurineTV-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’-0-methyltransferase (4ΌMT], or reticuline epimerase (REPI], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
20. The method of claim 1, wherein the product benzylisoquinoline compound is (5] -reticuline, the substrate is a substrate benzylisoquinoline compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound into (5] -reticuline, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-iV- methyltransferase (CNMT], (S]7V-methylcoclaurine 3’-hydroxylase (NMCH], or 4'-0- methyltransferase (4ΌMT]
21. The method of claim 1, wherein the product benzylisoquinoline compound is (5] -reticuline, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5] -reticuline, the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurineTV-methyltransferase (CNMT], (S]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4’ -O-methyl transferase, or (4ΌMT], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
22. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-3’-hydroxy-/V-methylcoclaurine, the substrate is a substrate benzylisoquinoline compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound to (5]-3’-hydroxy-/V- methylcoclaurine, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], ( S)-N - methylcoclaurine 3’-hydroxylase, or (NMCH]
23. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-3’-hydroxy-/V-methylcoclaurine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- 3’-hydroxy-/V-methylcoclaurine, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], coclaurine-/V-methyltransferase (CNMT], (5]-/V-methylcoclaurine, or 3’-hydroxylase (NMCH], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], (tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
24. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-/V-methylcoclaurine, the substrate is a substrate benzylisoquinoline compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound to (S]-/V-methylcoclaurine, and the enzymes are selected from norcoclaurine synthase (NCS], 6-O-methyltransferase (60MT], or coclaurine-/V-methyltransferase (CNMT]
25. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-/V-methylcoclaurine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into ( S]-N - methylcoclaurine, the enzymes are selected from norcoclaurine synthase (NCS], 6 0 methyltransferase (60MT], or coclaurine-/V-methyltransferase (CNMT], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], (tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
26. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-coclaurine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (S]-coclaurine, the enzymes are selected from norcoclaurine synthase (NCS], or 6-O-methyltransferase (60MT], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
27. The method of claim 1, wherein the product benzylisoquinoline compound is (5] -norcoclaurine, the substrate is a benzylisoquinoline compound, and the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound to (5] -norcoclaurine, wherein the enzyme is norcoclaurine synthase (NCS]
28. The method of claim 1, wherein the product benzylisoquinoline compound is (5] -norcoclaurine, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norcoclaurine, the enzyme is norcoclaurine synthase (NCS] and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], (tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
29. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-norlaudanosoline, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- norlaudanosoline, the enzyme is norcoclaurine synthase (NCS], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more of the enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
30. The method of claim 1, wherein the product benzylisoquinoline compound is (S]-6-0-methyl-norlaudanosoline, the substrate is a benzylisoquinoline compound, and the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline compound (5]-6-0-methyl-norlaudanosoline, wherein the enzymes are selected from norcoclaurine synthase (NCS] and 60MT.
31. The method of claim 1, wherein, the product benzylisoquinoline compound is (S]-6-0-methyl-norlaudanosoline, the substrate is a substrate benzylisoquinoline precursor compound, the enzyme complement comprises one or more enzymes capable of converting the substrate benzylisoquinoline precursor compound into (5]- 6-O-methyl-norlaudanosoline, the enzymes are selected from 60MT and norcoclaurine synthase (NCS], and the cell further has a benzylisoquinoline precursor enzyme complement comprising one or more ofthe enzymes (TYR], dihydroxyphenyl alanine decarboxylase (DODC], tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
32. The method of any one of claims 1 to 19, wherein the host cell further comprises an electron transfer facilitating protein.
33. The method of claim 32, wherein the electron transfer facilitating protein is a cytochrome P450 reductase (CPR]
34. The method of any one of claims 1 to 33, wherein the substrate is produced in the medium by a cell for co-culturing with the host cell, wherein the co-cultured cell is capable of secreting the substrate into the medium.
35. The method of claim 34, wherein the cell for co-culturing is a cell that does not comprise the benzylisoquinoline biosynthetic enzyme complement to produce the product benzylisoquinoline compound.
36. The method of claims 34, wherein the host cell is a cell that does not produce the substrate compound.
37. The method of any one of claims 1 to 36, wherein the benzylisoquinoline uptake permease is a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f).
38. The method of any one of claims 1 to 36, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
39. The method of any one of claims 1 to 36, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37; SEQ.ID NO: 39; and SEQ.ID NO: 55;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and (g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in
(a), (b), (c), (d), (e) or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
40. The method of any one of claims 1 to 36, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
41. The method of any one of claims 1 to 36, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 47, SEQ.ID NO: 49, SEQ.ID NO: 51, and SEQ.ID NO: 53;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54; (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (0, (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
42. The method of any one of claims 1 to 41, wherein the product benzylisoquinoline compound is further converted in the host cell to form a derivative benzylisoquinoline.
43. The method of claim 42, wherein the derivative benzylisoquinoline is codeine, codeinone or morphine.
44. The method of claim 42, wherein the conversion to form a derivative benzylisoquinoline involves the performance of an enzyme-catalyzed reaction by one or more of the enzymes T6-0-demethylase (T60DM], neopinone isomerase (NISO], codeinone reductase (COR], and codeinone-O-demethylase (CODM]
45. The method of any one of claims 1 to 44, wherein the method further includes a step comprising recovering the product benzylisoquinoline compound or the derivative benzylisoquinoline.
46. The method of any one of claims 1 to 45, wherein the host cell is a microbial cell.
47. The method of any one of claims 1 to 45, wherein the host cell is a bacterial cell.
48. The method of any one of claims 1 to 45, wherein the host cell is a yeast cell.
49. The method of claim 45, wherein the yeast cells are Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
50. The method of any one of claims 1 to 45, wherein the host cell is an algal cell.
51. The method of any one of claims 1 to 45, wherein the host cell is a plant cell.
52. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are cells belonging to the same biological species.
53. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are microbial cells.
54. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are bacterial cells.
55. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are yeast cells.
56. The method of claim 55, wherein the yeast cells are Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
57. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are algal cells.
58. The method of any one of claims 34 to 36, wherein the host cell and cell for co-culturing are plant cells.
59. The method of any one of claims 34 to 36 wherein the cell for co-culturing comprises a benzylisoquinoline uptake permease.
60. A host cell having an enzyme complement to biosynthetically produce benzylisoquinoline compounds, the host cell comprising a chimeric nucleic acid comprising as operably linked components (i] a nucleic acid sequence encoding a benzylisoquinoline uptake permease; and (ii] a nucleic acid sequence capable of controlling expression of the benzylisoquinoline uptake permease in the host cell, and the host cell capable of producing the benzylisoquinoline uptake permease and a product benzylisoquinoline compound when provided with a substrate compound.
61. The host cell of claim 57, wherein the product benzylisoquinoline compound is (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, salutaridinol, or thebaine.
62. The host cell of claim 60, wherein the substrate is a substrate benzylisoquinoline compound.
63. The host cell of claim 62, wherein the substrate benzylisoquinoline compound is (S]-norcoclaurine, (S]-norlaudanosoline, (5]-6-0-methyl-norlaudanosoline, (5]- coclaurine, (S]-/V-methylcoclaurine, (5]-3'-hyd roxy-/V-methylcoclaurine, (£}- reticuline, (7?] -reticuline, salutaridine, or salutaridinol.
64. The host cell of claim 60, wherein the substrate is a substrate benzylisoquinoline precursor compound.
65. The host cell of claim 64, wherein the substrate benzylisoquinoline precursor compound is L-tyrosine, L-dihydroxyphenyl alanine (L-DOPA], dopamine, tyramine, 3,4-hydroxy-phenylacetaldehyde (3,4-HPAA], or 4-hydrox-phenylacetaldehyde (4- HPAA]
66. The host cell of claim 60, wherein the enzyme complement comprises one or more benzylisoquinoline biosynthetic enzymes, wherein the enzymes are selected from norcoclaurine synthase (NCS], 6-0-methyltransferase (60MT], coclaurine-/V- methyltransferase (CNMT], (5]-/V-methylcoclaurine 3’-hydroxylase (NMCH], 4 0 methyltransferase (4ΌMT], reticuline epimerase (REPI], salutaridine synthase (SalSyn], salutaridine reductase (SalR], salutaridinol-7-O-acetyltransferase (SalAT], or thebaine synthase (TS]
67. The host cell of claim 66, wherein, the host cell further has a benzylisoquinoline precursor enzyme complement.
68. The host cell of claim 67, wherein the benzylisoquinoline precursor enzyme complement can comprise one or more benzylisoquinoline precursor biosynthetic enzymes, wherein the enzymes can be (TYR], dihydroxyphenyl alanine decarboxylase DODC, tyrosine decarboxylase (TYDC], and monoamide oxidase (MAO]
69. The host cell of any one claims 60 to 68, wherein the substrate is converted by the host cell into a product benzylisoquinoline compound in a single enzymatically catalyzed chemical step.
70. The host cell of any one of claims 60 to 68, wherein, the substrate is converted by the host cell into a product benzylisoquinoline compound in two or more enzymatically catalyzed chemical steps.
71. The host cell of any of claims 60 to 70, wherein the benzylisoquinoline uptake permease is a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 1, SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID. NO: 7, SEQ.ID NO: 9, SEQ.ID NO: 11, SEQ.ID NO: 13, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID. NO: 19, SEQ.ID NO: 21, SEQ. ID NO: 63, SEQ.ID NO: 65, SEQ.ID NO: 67, SEQ.ID NO: 69, SEQ.ID NO: 71, SEQ.ID NO: 73, SEQ.ID NO: 75, SEQ.ID NO: 77, and SEQ.ID NO: 79;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code; (d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2 SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2 SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID. NO: 8, SEQ.ID NO: 10, SEQ.ID NO: 12, SEQ.ID NO: 14 SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID. NO: 20, or SEQ.ID NO: 22; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f).
72. The host cell of any of claims 60 to 70, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
73. The host cell of any of claims 60 to 70, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 27, SEQ.ID. NO: 29, SEQ.ID NO: 31, SEQ.ID NO: 33, SEQ.ID NO: 35, SEQ.ID NO: 37, SEQ.ID NO: 39, and SEQ.ID NO: 55;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a];
(c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code; (d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID. NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, SEQ.ID NO: 36, SEQ.ID NO: 38, SEQ.ID NO: 40, or SEQ.ID NO: 56; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (0, (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline biosynthetic enzyme in the host cell.
74. The host cell of any of claims 60 to 70, wherein the host cell can comprise a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme; and
(B] a second nucleic acid sequence capable of controlling expression of the benzylisoquinoline precursor biosynthetic enzymes in the second cell.
75. The host cell of any of claims 60 to 70, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components:
(A] a first nucleic acid sequence encoding a benzylisoquinoline precursor biosynthetic enzyme selected from the group of nucleic acid sequences consisting of:
(a] SEQ.ID NO: 47, SEQ.ID NO: 49, SEQ.ID NO: 51, and SEQ.ID NO: 53;
(b] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a]; (c] a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a] but for the degeneration of the genetic code;
(d] a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a];
(e] a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO: 50, SEQ.ID NO: 52, or SEQ.ID NO: 54;
(f] a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 48, SEQ.ID NO:
50, SEQ.ID NO: 52, or SEQ.ID NO: 54; and
(g] a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a], (b], (c], (d], (e] or (f); and
(B] a second nucleic acid sequence capable of controlling the expression of the benzylisoquinoline precursor biosynthetic enzyme in the second cell.
76. The host cell of any one of claims 60 to 75, wherein the host cell is a microbial cell.
77. The host cell of any one of claims 60 to 75, wherein the host cell is a bacterial cell.
78. The host cell of any one of claims 60 to 75, wherein the host cell is a yeast cell.
79. The host cell of claim 78, wherein the yeast cells are Saccharomyces cerevisiae cells, or Yarrowia lipolytica cells.
80. The host cell of any one of claims 60 to 75, wherein the host cell is an algal cell.
81. The host cell of any one of claims 60 to 75, wherein the host cell is a plant cell.
82. A mixture of cells comprising host cells recombinantly expressing benzylisoquinoline uptake permease and having a benzylisoquinoline enzyme complement to biosynthetically produce a product benzylisoquinoline compound, and cells for co-culturing with the host cells, wherein the cells for co-culturing are capable of secreting a substrate that can be converted by the host cells when co cultured to form the product benzylisoquinoline compound.
83. The mixture of cells of claim 82, wherein the cells for co-culturing are cells that do not include the benzylisoquinoline enzyme complement to produce the benzylisoquinoline product.
84. The mixture of cells of claim 82, wherein the host cells do not produce the substrate.
85. The mixture of cells of claim 82, wherein the host cells and cells for co- culturing are cells belonging to the same biological species.
86. The mixture of cells of claim 82, wherein the host cells or cells for co-culturing are microbial cells.
87. The mixture of cells of claim 82, wherein the host cells or cells for co-culturing are bacterial cells.
88. The mixture of cells of claim 82, wherein the host cells or cells for co-culturing care yeast cells.
89. The mixture of cells of claim 88, wherein the yeast cells are Saccharomyces cerevisiae cells or Yarrowia lipolytica cells.
90. The mixture of cells of claim 82, wherein the host cells or cells for co-culturing are algal cells.
91. The mixture of cells of claim 82, wherein the host cells or cells for co-culturing are plant cells.
92. The mixture of cells according to claim 82, wherein the cells for co-culturing comprise benzylisoquinoline uptake permease.
93. A use of a cell of any one of claims 60 to 81 to convert a substrate and form a product benzylisoquinoline compound.
94. A use of a mixture of cells of claims 82 to 92 comprising host cells and cells for co-culturing with the host cells according to the present disclosure to convert a substrate and form a product benzylisoquinoline compound.
95. Ause of a product benzylisoquinoline compound produced in accordance with any one of claims 1 to 59.
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