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US-20260125725-A1 - Compositions And Methods For Making Benzylisoquinoline Alkaloids, Morphinan Alkaloids, Thebaine, And Derivatives Thereof

US20260125725A1US 20260125725 A1US20260125725 A1US 20260125725A1US-20260125725-A1

Abstract

Disclosed herein are methods that may be used for the synthesis of benzylisoquinoline alkaloids (“BIAs”) such as alkaloid morphinan. The methods disclosed can be used to produce thebaine, oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, and/or buprenorphine. Compositions and organisms useful for the synthesis of BIAs, including thebaine synthesis polypeptides, purine permeases, and polynucleotides encoding the same, are provided.

Inventors

  • Peter James FACCHINI
  • Xue Chen
  • Jeffrey C. COLBECK
  • Joseph E. Tucker

Assignees

  • ANTHEIA, INC.

Dates

Publication Date
20260507
Application Date
20250422

Claims (20)

  1. 1 . A genetically modified cell comprising a polynucleotide encoding for a heterologous enzyme capable of converting salutaridinol-7-O-acetate to thebaine.
  2. 2 . The genetically modified cell of claim 1 , wherein said heterologous enzyme capable of converting salutaridinol-7-O-acetate to thebaine is a thebaine synthesis polypeptide.
  3. 3 - 5 . (canceled)
  4. 6 . The genetically modified cell of claim 1 , wherein the polynucleotide encodes for an amino acid sequence substantially identical to SEQ ID NO. 6 or SEQ ID NO. 31.
  5. 7 . The genetically modified cell of claim 1 , further comprising one or more nucleic acids encoding for an enzyme capable of catalyzing one or more of the reactions: a) a sugar to L-tyrosine; b) L-tyrosine to L-DOPA; c) L-DOPA to Dopamine; d) Dopamine to (S)-Norcoclaurine; e) (S)-Norcoclaurine to (S)/(R)-Reticuline; f) (R)-Reticuline to Salutardine; g) Salutardine to Salutaridinol; h) Salutaridinol to Salutaridinol-7-O-acetate; or i) thebaine to oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, buprenorphine, or any combination thereof.
  6. 8 . The genetically modified cell of claim 1 , wherein the cell is a plant cell, fungus, yeast, or bacterium.
  7. 9 . The genetically modified cell of claim 1 , wherein the cell is a Papaver somniferum cell, Saccharomyces cerevisiae, Yarrowia lipolytica , or Escherichia coli.
  8. 10 . The genetically modified cell of claim 1 , wherein said cell is a eukaryote.
  9. 11 . The genetically modified cell of claim 1 , wherein said cell further comprises an additional heterologous polynucleotide that is substantially identical to any one of SEQ ID NOs. 18, or 20 to 30.
  10. 12 . The genetically modified cell of claim 11 , wherein said additional heterologous polynucleotide encodes for an amino acid sequence that is substantially identical to any one of SEQ ID NOs. 5, or 7 to 17.
  11. 13 . The genetically modified cell of claim 1 , further comprising a purine permease.
  12. 14 . The genetically modified cell of claim 13 , wherein said purine permease is encoded by a nucleotide sequence that is substantially identical to SEQ ID NO. 36.
  13. 15 . The genetically modified cell of claim 13 , wherein said purine permease is encoded by a nucleotide sequence that is substantially identical to SEQ ID NO. 38.
  14. 16 . The genetically modified cell of claim 13 , wherein said purine permease is encoded by a nucleotide sequence that is substantially identical to any one of SEQ ID NOs. 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 64.
  15. 17 . The genetically modified cell of claim 13 , wherein said purine permease comprises an amino acid sequence that is substantially identical to SEQ ID NO. 35.
  16. 18 . The genetically modified cell of claim 13 , wherein said purine permease comprises an amino acid sequence that is substantially identical to SEQ ID NO. 37.
  17. 19 . The genetically modified cell of claim 13 , wherein said purine permease comprises an amino acid sequence that is substantially identical to SEQ ID NO. 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63.
  18. 20 . A method of producing a benzylisoquinoline alkaloid (BIA) comprising: a) contacting a cell with a substrate, wherein said cell has a polynucleotide encoding for a heterologous enzyme capable of converting salutaridinol-7-O-acetate to thebaine, and the cell is capable of catalyzing one or more reactions of: a sugar to L-tyrosine; L-tyrosine to L-DOPA; L-DOPA to Dopamine; Dopamine to (S)-Norcoclaurine; (S)-Norcoclaurine to (S)/(R)-Reticuline; (R)-Reticuline to Salutardine; Salutardine to Salutaridinol; Salutaridinol to Salutaridinol-7-O-acetate; and b) growing the cell under conditions permitting the host cell to convert a substrate into a BIA.
  19. 21 - 41 . (canceled)
  20. 42 . A method of making a BIA outside of a cell comprising, a) contacting a substrate that is capable of being converted by one or more enzymes, wherein the one or more enzymes comprise a tyrosine hydroxylase (TYR); DOPA decarboxylase (DODC); norcoclaurine synthase (NCS); 6-O-Methyltransferase (6OMT); coclaurine N-methyltransferase (CNMT), cytochrome P450 N-methylcoclaurine hydroxylase (NMCH), and 4-O-methyltransferase (4OMT); cytochrome P450 reductase (CPR), salutaridine synthase (SAS); salutaridine reductase (SalR); salutaridinol-7-O-acetyltransferase (SalAT); purine permease (PUP); or any combination thereof, b) contacting the product of a) with one or more of a thebaine synthesis polypeptide; a codeine O-demethylase (CODM); a thebaine 6-O-demethylase (T6ODM); a codeinone reductase (COR); or any combination thereof.

Description

CROSS-REFERENCE This application is a continuation of U.S. application Ser. No. 17/496,540, filed Oct. 7, 2021, which is a continuation of U.S. application Ser. No. 16/312,776, filed on Dec. 21, 2018, which is a 371 of International Application No. PCT/US2017/039589, filed on Jun. 27, 2017 which claims the priority benefit of U.S. Provisional Patent Application Ser. Nos. 62/355,022, filed Jun. 27, 2016; 62/433,431, filed Dec. 13, 2016; 62/438,540, filed Dec. 23, 2016; 62/438,601, filed Dec. 23, 2016; 62/438,702, filed Dec. 23, 2016; 62/438,588, filed Dec. 23, 2016; 62/438,695, filed Dec. 23, 2016; 62/469,006, filed Mar. 9, 2017; and 62/514,104, filed on Jun. 2, 2017, all of which are hereby incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The contents of the electronic sequence listing 2025 Jun. 27 Sequence_Listing_ST26 062074-507C02US.xml; Size: 110,129 bytes; and Date of Creation: Jun. 27, 2025. BACKGROUND OF THE DISCLOSURE The following paragraphs are provided by way of background and not an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art. Thebaine, a chemical compound also known as paramorphine and codeine methyl enol ether, belongs to the class of compounds known as benzylisoquinoline alkaloids (“BIAs”), and within that class, to a BIA subclass of compounds known as morphinan alkaloids, and has long been recognized as a useful feedstock compound in the manufacture of therapeutic agents, including, for example, morphine and codeine. Other agents that can be manufactured can include but are not limited to oripavine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone and neopinone. It is known that thebaine in planta is produced from a precursor compound named salutaridine via intermediate morphinan alkaloids named salutaridinol and salutaridinol 7-O-acetate. Currently thebaine may be harvested from natural sources, such as opium poppy capsules (see e.g., U.S. Pat. Appl. Pub. No 2002/0106761; see also e.g., Poppy, the genus Papaver, 1998, pp 113, Harwood Academic Publishers, Editor: Bernáth, J.). Alternatively, thebaine may be prepared synthetically. The latter may be achieved by a reaction sequence starting with ketalization of iodoisovanillin (see e.g., Rinner, U. and Hudlicky, T., 2012, Top. Cur. Chem. 309; 33-66; Stork, G., 2009, J. Am. Chem. Soc. 131 (32) pp 11402-11406). The existing manufacturing methods for BIAs, including thebaine and other morphinan alkaloids and their derivatives, suffer from low yields and/or are expensive. Some of the known methodologies for the manufacture of thebaine exist in the production of undesirable quantities of morphinan alkaloid by-products (see e.g., Rinner, U., and Hudlicky, J., 2012, Top. Cur. Chem. 209: 33-66). No methods exist to commercially biosynthetically manufacture BIAs, including thebaine and other morphinan alkaloids and their derivatives. Therefore, there is a need for improved methods for the synthesis of BIAs including thebaine and other morphinan alkaloids and their derivatives. INCORPORATION BY REFERENCE All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls. SUMMARY This application discloses certain alkaloids belonging to the class of benzylisoquinoline alkaloids (“BIAs”), morphinan alkaloids, and their derivatives, as well as to methods of making such BIAs, including morphinan alkaloids (and their derivatives). For example, one BIA that can be made is thebaine. BIAs, such as morphinan alkaloids, more specifically thebaine, can be produced from sugar (or other substrates). Disclosed herein is a cell (e.g., a microorganism) that comprises one or more nucleic acids encoding for heterologous enzymes that can perform any one of the following reactions: i) sugar to 1-tyrosine; ii) 1-tyrosine to 1-DOPA; iii) 1-DOPA to dopamine; iv) dopamine to (S)-norcoclaurine; v) (S)-norcoclaurine to (S)/(R)-reticuline; vi) (R)-reticuline to salutardine; vii) salutardine to salutaridinol; viii) salutaridinol to salutaridinol-7-O-acetate; ix) salutaridinol-7-O-acetate to thebaine; x) thebaine to oripavine; morphinone; morphine; codeine; codeinone; and/or neopinone. In some instances, the enzyme that converts salutaridinol-7-O-acetate to thebaine is a thebaine synthesis polypeptide (a.k.a. thebaine synthase or BetV1). In some instances, a purine permease is also present within the cell. The method of converting a sugar (or other substrate) into a BIA, such as thebaine, can also be pe