Methods For Making and Using Polynucleotide Sequences in the Synthesis of Alkaloid Compounds

Abstract

Novel methods that may be used for the manufacture of plant alkaloid compounds and novel polynucleotide compounds are provided. The plant alkaloid compounds are useful as medicinal compounds.

1 . A method of making an alkaloid compound, comprising:
 (a) providing a first alkaloid compound;   (b) contacting the alkaloid compound with an O-methyltransferase under reaction conditions that permit methylation of the alkaloid compound to form a second alkaloid compound;   wherein the first alkaloid compound has the chemical formula:   
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3  and R 4  each independently or simultaneously represent a hydroxyl group or a methoxy group, with the proviso that R 2 , R 3  and R 4  do not each simultaneously represent a methoxy group, and wherein represents a hydrogen atom or a methyl group; 
         and wherein the second alkaloid compound has the chemical formula: 
       
       
         
           
           
               
               
           
         
         and wherein R 1 , R 2 , R 3  and R 4  each independently or simultaneously represent a hydroxyl group or a methoxy group, with the proviso that at least one of R 1 , R 2  R 3  and R 4  represents a methoxy group, and wherein R 5  represents a hydrogen atom or a methyl group. 
       
     
     
         2 . The method according to  claim 1 , wherein the O-Methyltransferase comprises SEQ.ID NO: 1592 or SEQ.ID NO: 1593, or a sequence that is at least 80% identical thereto. 
     
     
         3 . The method according to  claim 1 , wherein in the first alkaloid compound R 1 , R 2 , R 3 , and R 4  represent a hydroxyl group, and R 5  represents a hydrogen atom, and wherein in the second alkaloid compound R 1  represents a methoxy group, R 2 , R 3  and R 4  represent a hydroxyl group and R 5  represents a hydrogen atom. 
     
     
         4 . The method according to  claim 2 , wherein in the first alkaloid compound R 1 , R 2 , R 3 , and R 4  represent a hydroxyl group, and R 5  represents a hydrogen atom, and wherein in the second alkaloid compound R 1  represents a methoxy group, R 2 , R 3  and R 4  represent a hydroxyl group and R 5  represents a hydrogen atom. 
     
     
         5 . The method according to  claim 1 , wherein the method is performed in in vitro. 
     
     
         6 . The method according to  claim 1 , wherein the method is performed in vivo in a cell, wherein the cell has been transformed with a nucleic acid sequence encoding the O-methyl-transferase. 
     
     
         7 . The method according to  claim 6 , wherein the nucleic acid sequence encoding the O-methyltransferase is a polypeptide comprising SEQ.ID NO: 1592 or SEQ.ID NO: 1593, or a sequence that is at least 80% identical thereto. 
     
     
         8 . The method according to  claim 7 , wherein in the first alkaloid compound R 1 , R 2 , R 3 , and R 4  represent a hydroxyl group, and R 5  represents a hydrogen atom, and wherein in the second alkaloid compound R 1  represents a methoxy group, R 2 , R 3  and R 4  represent a hydroxyl group, and R 5  represents a hydrogen atom. 
     
     
         9 . The method according to  claim 6 , wherein the cell is a microbial cell. 
     
     
         10 . The method according to  claim 6 , wherein the cell is a plant cell. 
     
     
         11 . The method according to  claim 9 , wherein the microbial cell is a bacterial cell or a yeast cell. 
     
     
         12 . The method according to  claim 11 , wherein the bacterial cell is  Escherichia coli  cell. 
     
     
         13 . The method according to  claim 12 , wherein the yeast cell is a  Saccharomyces cerevisiae  cell. 
     
     
         14 . A cell for the performance of the method according to  claim 6 , wherein the cell is transformed with SEQ.ID NO: 1592 or 1593 or a sequence that is at least 80% identical thereto. 
     
     
         15 . A cell for the performance of the method according to  claim 8 , wherein the cell is transformed with SEQ.ID NO: 1592 or 1593 or a sequence that is at least 80% identical thereto. 
     
     
         16 . The cell according to  claim 14 , wherein the cell is a microbial cell. 
     
     
         17 . The cell according to  claim 14 , wherein the cell is a plant cell. 
     
     
         18 . The cell according to  claim 16 , wherein the microbial cell is a bacterial cell or a yeast cell. 
     
     
         19 . The cell according toc  claim 18 , wherein the bacterial cell is an  Escherichia coli  cell. 
     
     
         20 . The cell according to  claim 18 , wherein the yeast cell is a  Saccharomyces cerevisiae  cell.