Compositions and Methods For Making Alkaloid Morphinans

Abstract

Methods that may be used for the manufacture of a class of chemical compounds known as morphinans, including neopine, are provided. Compositions useful for the synthesis of morphinans, including neopine, are also provided.

1 . An isolated nucleic acid sequence encoding a polypeptide comprising SEQ. ID. NO: 2 or a polypeptide substantially identical thereto, wherein SEQ. ID NO: 2 is capable of, in a first morphinan having a mono-unsaturated C 7 -C 8  bond and a saturated C 8 -C 14  bond:
 (i) saturating the C 7 -C 8  bond; and   (ii) unsaturating the C 8 -C 14  bond, to form a second morphinan having a saturated C 7 -C 8  and a mono-unsaturated C 8 -C 14  bond.   
     
     
         2 . The isolated nucleic acid sequence according to  claim 1  encoding the polypeptide substantially identical to SEQ ID NO:2, wherein the polypeptide comprises at least 16, at least 15, at least 14, at least 13, at least 12, at least 11, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2, or at least 1 of the following amino acids in the following positions in SEQ ID NO:2 (1) AA25=valine; (2) AA29=glutamic acid; (3) AA58=threonine (4) AA178=threonine; (5) AA180=asparagine; (6) AA181=serine; (7) AA219=valine; (8) AA 220=glycine; (9) AA222=alanine; (10) AA225=threonine; (11) AA230=histidine; (12) AA268=alanine; (13) AA285=aspartic acid; (14) AA 301=alanine; (15) AA318=glycine; (16) AA319=glutamic acid; and (17) AA320=valine. 
     
     
         3 . The isolated nucleic acid sequence according to  claim 1  encoding the polypeptide substantially identical to SEQ ID NO:2, wherein the polypeptide comprises the following amino acid residues in the following positions in SEQ ID NO: 2 (1) AA25=valine; (2) AA29=glutamic acid; (3) AA58=threonine (4) AA178=threonine; (5) AA180=asparagine; (6) AA181=serine; (7) AA219=valine; (8) AA 220=glycine; (9) AA222=alanine; (10) AA225=threonine; (11) AA230=histidine; (12) AA268=alanine; (13) AA285=aspartic acid; (14) AA 301=alanine; (15) AA318=glycine; (16) AA319=glutamic acid; and (17) AA320=valine. 
     
     
         4 . The isolated nucleic acid sequence according to  claim 1 , wherein the nucleic acid sequence comprises SEQ. ID NO: 1. 
     
     
         5 . The isolated nucleic acid sequence according to  claim 1  wherein the polypeptide substantially identical to SEQ ID NO:2 has at least 98% identity with SEQ ID NO:2. 
     
     
         6 . The isolated nucleic acid sequence according to  claim 1  wherein the polypeptide substantially identical to SEQ ID NO:2 has at least 99% identity with SEQ ID NO:2. 
     
     
         7 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (V) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (VI) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a methoxy group, R 2  is a hydroxyl group and R 2′  a hydrogen atom. 
       
     
     
         8 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (V) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (VI) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a hydroxyl group, R 2  is a hydroxyl group and R 2′  is a hydrogen atom. 
       
     
     
         9 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (VII) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (VIII) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a methoxy group, R 2  is a hydroxyl group and R 2′  is a hydrogen atom. 
       
     
     
         10 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (VII) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (VIII) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a hydroxyl group, R 2  is a hydroxyl group and R 2′  is a hydrogen atom. 
       
     
     
         11 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (IX) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (X) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a methoxy group, R 2  is a hydroxyl group and R 2′  is a hydrogen atom. 
       
     
     
         12 . The isolated nucleic acid sequence according to  claim 1 , wherein the first morphinan has the chemical formula (IX) 
       
         
           
           
               
               
           
         
         and wherein the second morphinan has the chemical formula (X) 
       
       
         
           
           
               
               
           
         
         and wherein in the first and second morphinan R 1  is a hydroxyl group, R 2  is a hydroxyl group and R 2′  is a hydrogen atom. 
       
     
     
         13 . A chimeric nucleic acid sequence comprising as operably linked components:
 (i) a nucleic acid sequence according to  claim 1 ; and   (ii) one or more nucleic acid sequences capable of controlling expression in a host cell.   
     
     
         14 . A recombinant expression vector comprising the chimeric nucleic acid sequence according to  claim 13 . 
     
     
         15 . A host cell comprising the chimeric nucleic acid sequence according to  claim 13 . 
     
     
         16 . A host cell according to  claim 15 , wherein the host cell is a microbial cell. 
     
     
         17 . A host cell according to  claim 16 , wherein the host cell is a plant cell. 
     
     
         18 . A host cell according to  claim 17  wherein the microbial cell is a yeast cell. 
     
     
         19 . A host cell according to  claim 16  wherein the microbial cell is an  Escherichia coli  cell. 
     
     
         20 . A host cell according to  claim 17  wherein the plant cell is a  Papaver  cell.