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EP-4289959-B1 - COMPOSITIONS AND METHODS FOR MAKING (R)-RETICULINE AND PRECURSORS THEREOF

EP4289959B1EP 4289959 B1EP4289959 B1EP 4289959B1EP-4289959-B1

Inventors

  • FACCHINI, PETER JAMES
  • FARROW, Scott Cameron
  • BEAUDOIN, Guillaume Arthur Welch

Dates

Publication Date
20260513
Application Date
20141203

Claims (15)

  1. A method of making (R) -reticuline in a yeast cell comprising: (a) providing (S) -reticuline; (b) contacting the (S) -reticuline with an enzyme mixture capable of converting ( S)- reticuline to (R) -reticuline under conditions that permit the conversion of (S) -reticuline to (R) -reticuline in a yeast cell, wherein the enzyme mixture comprises a first polypeptide capable of oxidizing (S)- reticuline to form 1,2-dehydroreticuline and a second polypeptide capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible, and wherein the first polypeptide is a CYP450 polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 219-321, or 338 and the second polypeptide is an AKR polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340.
  2. A recombinant expression vector comprising as operably linked components: (a) a nucleic acid sequence capable of controlling expression in a yeast cell; and (b) a nucleic acid sequence encoding: (i) a CYP450 polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 219-321, or 338 and that is capable of oxidizing (S) -reticuline to form 1,2-dehydroreticuline; and (ii) an AKR polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340 and that is capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible; wherein the expression vector is suitable for expression in the yeast cell.
  3. A yeast host cell comprising a chimeric nucleic acid comprising as operably linked components: (a) a nucleic acid sequence encoding a CYP450 polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 219-321, or 338 and that is capable of oxidizing (S) -reticuline to form 1,2-dehydroreticuline; and (b) a nucleic acid sequence encoding a AKR polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340 and that is capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible.
  4. A method for preparing (R) -reticuline from (S) -reticuline comprising: (a) providing a chimeric nucleic acid comprising as operably linked components: (i) a first nucleic acid sequence encoding a cytochrome P450 (CYP450) polypeptide that is at least 85% identical to SEQ ID NO: 219-321, or 338; (ii) a second nucleic acid sequence encoding an aldo-ketoreductase (AKR) polypeptide that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340; and (iii) one or more nucleic acid sequences capable of controlling expression in a yeast cell; and (b) introducing the chimeric nucleic acid into a yeast cell and growing the yeast cell to produce the CYP450 polypeptide and AKR polypeptide and to produce (R)- reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible; and (c) recovering (R) -reticuline.
  5. The method of claim 1 or claim 4, the recombinant expression vector of claim 2, or the yeast host cell of claim 3, wherein: (a) the CYP450 polypeptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 219-321, or 338, or has the amino acid sequence of SEQ ID NO: 219-321, or 338; and/or (b) the AKR polypeptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 59-115, 329, 330, or 340, or has the amino acid sequence of SEQ ID NO: 59-115, 329, 330, or 340.
  6. A method of making (R) -reticuline in a yeast cell comprising: (a) providing (S) -reticuline; (b) contacting the (S) -reticuline with an enzyme mixture capable of converting (S)- reticuline to (R) -reticuline under conditions that permit the conversion of (S) -reticuline to (R) -reticuline in a yeast cell, wherein the enzyme mixture comprises a first CYP450 polypeptide capable of oxidizing (S) -reticuline to form 1,2-dehydroreticuline and a second AKR polypeptide capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible, the first polypeptide is a CYP450 polypeptide and the second polypeptide is an AKR polypeptide, the first and second polypeptide are a fusion polypeptide, and the fusion polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 323.
  7. A recombinant expression vector comprising as operably linked components: (a) a nucleic acid sequence capable of controlling expression in a yeast cell; and (b) a nucleic acid sequence encoding: (i) a CYP450 polypeptide that is capable of oxidizing (S)- reticuline to form 1,2-dehydroreticuline; and (ii) an AKR polypeptide and that is capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible; wherein the expression vector is suitable for expression in the yeast cell, and the CYP450 and AKR polypeptides are operably linked to form a fusion polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 323.
  8. A yeast host cell comprising a chimeric nucleic acid comprising as operably linked components: (a) a nucleic acid sequence encoding a CYP450 polypeptide that is capable of oxidizing (S) -reticuline to form 1,2-dehydroreticuline; and (b) a nucleic acid sequence encoding a AKR polypeptide that is capable of reducing 1,2-dehydroreticuline to form (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible, wherein the nucleic acid sequences are operably linked in order to produce a fusion polypeptide comprising CYP450 and AKR, and the fusion polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 323.
  9. The method of claim 6, the recombinant expression vector of claim 7, or the yeast host cell of claim 8, wherein the chimeric nucleic acid or recombinant expression vector is capable of capable of hybridizing under at least moderately stringent hybridization conditions to the nucleic acid sequence of SEQ ID NO: 322.
  10. A method for preparing (R) -reticuline from (S) -reticuline comprising: (a) providing a chimeric nucleic acid comprising as operably linked components: (i) a first nucleic acid sequence encoding a cytochrome P450 (CYP450) polypeptide; (ii) a second nucleic acid sequence encoding an aldo-ketoreductase (AKR) polypeptide; and (iii) one or more nucleic acid sequences capable of controlling expression in a yeast cell, wherein the first and second nucleic acid sequences are operably linked in order to produce a fusion polypeptide comprising CYP450 and AKR, and the fusion polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 323; and (b) introducing the chimeric nucleic acid into a yeast cell and growing the yeast cell to produce the fusion polypeptide and to produce (R) -reticuline, wherein the conversion of 1,2-dehydroreticuline to (R) -reticuline is reversible, or partially reversible; and (c) recovering (R)- reticuline.
  11. The method of any one of claims 4, 5, and 10, wherein the recovered (R) -reticuline is used to make salutaridine, codeine, morphine, thebaine, papaverine, noscapine, codamine, laudine, laudanosine, (+)-pallidine, (-)-isoboldine, or (-)-corytuberine.
  12. The method of any one of claims 1, 4, 5, 6, 9, 10, and 11, the recombinant expression vector of claim 2 or claim 7, or the yeast host cell of claim 3 or claim 8, wherein the expression vector or nucleic acid sequence comprises a yeast promoter, terminator, and/or enhancer.
  13. The method of any one of claims 1, 4, 5, 6, 9, 10, 11, and 12, comprising providing a precursor compound of (S) -reticuline, or a benzylisoquinoline derivative capable of being converted by the yeast cell into (R) -reticuline.
  14. The method of any one of claims 1, 4, 5, 6, 9, 10, 11, and 12, comprising providing (S) -reticuline, or a benzylisoquinoline derivative capable of being converted by the yeast cell into (R) -reticuline, to the yeast cell as part of the cell's growth medium, preferably comprising providing (S) -reticuline to the yeast cell as part of the cell's growth medium.
  15. The method of any one of claims 6 and 9-14, the recombinant expression vector of any one of claims 7, 9, and 12, or the yeast host cell of any one of claims 8, 9, and 12, wherein: the fusion polypeptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 323, or has the amino acid sequence of SEQ ID NO: 323.

Description

FIELD OF THE DISCLOSURE The compositions and methods disclosed herein relate to secondary metabolites and processes for manufacturing the same. More particularly, the present disclosure relates to (R)-Reticuline and certain precursors thereof and methods and compositions for manufacturing (R)-Reticuline and such precursors. BACKGROUND OF THE DISCLOSURE The following paragraphs are provided by way of background to the present disclosure. They are not however an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art. The biochemical pathways of living organisms are commonly classified as being either part of primary metabolism or part of secondary metabolism. Pathways that are part of a living cell's primary metabolism are involved in catabolism for energy production or in anabolism for building block production for the cell. Secondary metabolites, on the other hand, are produced by living cells without having any obvious anabolic or catabolic function. It has however long been recognized that many secondary metabolites are useful in many respects, including for example as therapeutic agents or natural deterrents. The secondary metabolite (R)-Reticuline is produced by opium poppy (Papaver somniferum) and other members of the plant families Papaveraceae, Lauraceae, Annonaceae, Euphorbiaceae and Moraceae, and may be used as a source material for producing the pharmaceutically active compounds including morphine and codeine. It is known that (R)-Reticuline in planta is produced from (S)-Reticuline. However it is not clear which genes and polypeptides are involved in catalyzing the conversion reaction(s). Currently (R)-Reticuline may be harvested from natural sources, such as opium poppy. Alternatively (R)-Reticuline may be prepared synthetically. The existing manufacturing methods for (R)-Reticuline however suffer from low yields of (R)-Reticuline and/or are expensive. No methods exist to biosynthetically make (R)-Reticuline from (S)-reticuline. There exists therefore in the art a need for improved methods for the synthesis of (R)-Reticuline. Desgagné-Penix et al. (BMC Plant Biol, 2010, 10:252) describes transcriptome and proteome analyses of genes involved in the alkaloid metabolism in poppy cell cultures, in particular in the biosynthetic pathway that produces sanguinarine and morphine. WO 00/58333 A1 relates to codeinone reductase, an enzyme involved in the metabolism of (R)-reticuline in alkaloid poppy plants. WO 2015/173590 A1 describes a cytochrome P450 polypeptide from Papaver somniferum. Farrow et al. (Nature Chemical Biology, 2015, vol. 11, no. 9, pages 728-732) describes stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy, Unterlinner et al. (The Plant Journal, 1995, 18: 465-475) describes the cloning and functional expression of a codeinone reductase from Papaver somniferum. GB 1 408 729 A describes sulfilimine compounds. SUMMARY OF THE DISCLOSURE The following paragraphs are intended to introduce the reader to the more detailed description that follows and not to define or limit the claimed subject matter of the present disclosure. The present disclosure relates to the secondary metabolite (R)-Reticuline and certain precursors thereof, as well as to methods of making (R)-Reticuline and certain precursors thereof. Accordingly, the invention provides a method of making (R)-reticuline in a yeast cell comprising: (a) providing (S)-reticuline;(b) contacting the (S)-reticuline with an enzyme mixture capable of converting (S)-reticuline to (R)-reticuline under conditions that permit the conversion of (S)-reticuline to (R)-reticuline in a yeast cell, wherein the enzyme mixture comprises a first polypeptide capable of oxidizing (S)-reticuline to form 1,2-dehydroreticuline and a second polypeptide capable of reducing 1,2-dehydroreticuline to form (R)-reticuline,wherein the conversion of 1,2-dehydroreticuline to (R)-reticuline is reversible, or partially reversible,and wherein the first polypeptide is a CYP450 polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 219-321, or 338 and the second polypeptide is an AKR polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340.The invention also provides a recombinant expression vector comprising as operably linked components: (a) a nucleic acid sequence capable of controlling expression in a yeast cell; and(b) a nucleic acid sequence encoding: (i) a CYP450 polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 219-321, or 338 and that is capable of oxidizing (S) reticuline to form 1,2-dehydroreticuline; and(ii) an AKR polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 59-115, 329, 330, or 340 and that is capable of reducing 1,2-dehydroreticuline to form (R)-reticuline, wherein the conversion of 1,2-dehydroreti