O-Methyltransferases of Tetrahydrobenzylisoquinoline Alkaloid Biosynthesis in Papaver Somniferum

Abstract

The present invention relates to methyl transfer enzymes involved in alkaloid biosynthesis in opium poppy. More particularly, the invention relates to proteins having (R,S)-reticuline 7-O-methyltransferase activity, to proteins having (R,S)-norcoclaurine 6-O-methyltransferase activity and to derivatives and analogues of these proteins. The invention also relates to nucleic acid molecules encoding the proteins, and their derivatives and analogues, and to their use in the production of methylated catechols and tetrahydrobenzylisoquinolines.

1 .- 18 . (canceled) 
     
     
         19 . An isolated or purified protein comprising the amino acid sequence of  FIG. 13  (SEQ ID NO: 21) or a variant of the amino acid sequence of  FIG. 13  (SEQ ID NO: 21), said variant having at least 70% identity with the PSOMT2 amino acid sequence of  FIG. 13  (SEQ ID NO: 21) over a length of at least 300 amino acids and, said variant having 0-methyltransferase activity. 
     
     
         20 . The protein according to  claim 19  wherein the variant has at least 80% identity with the PSOMT2 amino acid sequence of  FIG. 13  (SEQ ID NO: 21) over a length of at least 300 amino acids. 
     
     
         21 . The protein according to  claim 19  wherein the variant has at least 80% identity with the PSOMT2 amino acid sequence of  FIG. 13  (SEQ ID NO: 21) over a length of at least 300 amino acids. 
     
     
         22 . The protein according to  claim 21 , wherein the variant has at least 95% identity with the PSOMT2 amino acid sequence of  FIG. 13  (SEQ ID NO: 21) over a length of at least 300 amino acids and wherein said variant contains one or more conserved amino acid motifs, said motifs comprising amino acid sequence LVDVGGG, PXXDAXXMK, XGKVI, DLPHV, HVGGDMF, or GKERT, wherein X represents any amino acid. 
     
     
         23 . The protein according to  claim 22 , wherein the variant has at least 97% identity with the PSOMT2 amino acid sequence of  FIG. 13  (SEQ ID NO: 21) over a length of at least 300 amino acids. 
     
     
         24 . The protein according to  claim 23  comprising the PSOMT2 amino acid sequence illustrated in  FIG. 3  (SEQ ID NO: 3). 
     
     
         25 . The protein according to  claim 23  comprising the PSOMT2a amino acid sequence illustrated in  FIG. 13  (SEQ ID NO: 13). 
     
     
         26 . A protein or peptide comprising of a fragment of the protein illustrated in  FIG. 16  (SEQ ID NO: 25), said fragment having a length of 100 to 345 amino acids, including the portion spanning at least one of positions 93, 150, 233, 245 and 274, wherein X 93 , X 150 , X 233 , X 245 , X 274  are chosen from the following amino acids: 
       
         
           
                 
                 
                 
               
                     
                   X 93 : 
                   Pro, Val 
                 
                     
                     
                 
                     
                   X 150 : 
                   Val, Glu 
                 
                     
                     
                 
                     
                   X 233 : 
                   Ser, Pro 
                 
                     
                     
                 
                     
                   X 245 : 
                   Ala, Gly; 
                 
                     
                     
                 
                     
                   X 274 : 
                   Gly, Val, 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       with the proviso that X 93  is not Pro when X 150 , X 233 , X 245 , X 274  have the following meanings: Xaa 150  is Glu, Xaa 233 . is Ser, Xaa 245  is Ala and Xaa 274  is Gly, said protein or peptide having O-methyltransferase activity. 
     
     
         27 . The protein or peptide according to  claim 26 , comprising or consisting of a fragment of the PSOMT2 protein illustrated in  FIG. 3  (SEQ ID NO: 3), said fragment having a length of 100 to 345 amino acids, including the portion spanning at least one of positions 93, 150, 233, 245 and 274 of the sequence illustrated in  FIG. 3  (SEQ ID NO: 3). 
     
     
         28 . The protein or peptide according to  claim 26 , comprising or consisting of a fragment of the PSOMT2a protein illustrated in  FIG. 13  (SEQ. ID n° 21), said fragment having a length of 100 to 345 amino acids, including the portion spanning at least one of positions 93, 150, 233, 245 and 274 of the sequence illustrated in  FIG. 13  (SEQ ID NO: 21). 
     
     
         29 . The protein or peptide according to  claim 27  having 150 to 300 amino acids. 
     
     
         30 . The protein according to  claim 19 , which is a dimer comprising two protein sub-units, each sub-unit being chosen from any one of proteins as defined in  claim 19 . 
     
     
         31 . The protein according to  claim 19 , having (R,S)-norcoclaurine 6-O-methyltransferase activity. 
     
     
         32 . An isolated nucleic acid molecule encoding a protein according to  claim 19 . 
     
     
         33 .- 45 . (canceled) 
     
     
         46 . A cell transformed or transfected by a nucleic acid molecule according to  claim 19 . 
     
     
         47 . The cell according to  claim 46  which is a prokaryotic cell. 
     
     
         48 . (canceled) 
     
     
         49 . Cell according to  claim 46  which is a eukaryotic cell. 
     
     
         50 . Cell according to  claim 49  which is a yeast cell. 
     
     
         51 .- 64 . (canceled) 
     
     
         65 . Method for the production of methylated tetrahydrobenzylisoquinolines, said method comprising the steps of:
 i) contacting in vitro a protein having norcoclaurine 6-O-methyltransferase activity with a substrate chosen from (R,S)-norcoclaurine, (R,S)-isoorientaline, (R)-norprotosinomenine and (S)-norprotosinomenine at pH 6.0 to 9.0, wherein the protein having norcoclaurine 6-O-methyltransferase activity is a protein according to  claim 19 ;   ii) recovering the methylated tetrahydrobenzylisoquinolines thus produced.   
     
     
         66 . (canceled) 
     
     
         67 . A method for producing a protein having norcoclaurine 6-O-methyltransferase activity, said method comprising
 i) transforming or transfecting a cell with a nucleic acid molecule according to  claim 32 , in conditions permitting the expression of the protein having norcoclaurine 6-O-methyltransferase activity,   ii) propagating the said cell, and   iii) recovering the thus-produced protein having norcoclaurine 6-O-methyltransferase activity.