Cytochrome P450 reductases from poppy plants

Abstract

The present invention relates to production of alkaloids from poppy plants and in particular togenes encoding enzymes in the alkaloid pathway, to proteins encoded by the gens, to plants transformed or transfected with the genes and to methods of altering alkaloid content or blend of poppy plants.

1 . An isolated and purified polynucleotide encoding a cytochrome P-450 reductase enzyme from an alkaloid poppy plant, or a variant, fragment or analog thereof.  
     
     
         2 . A polynucleotide according to  claim 1 , selected from the group consisting of genomic DNA, cDNA, or synthetic DNA.  
     
     
         3 . A polynucleotide according to  claim 1  selected from the group consisting of: (a) the polynucleotide sequences set out in  FIGS. 6, 9  or  10 ; and (b) complementary sequences of the polynucleotide sequences which hybridize under stringent conditions to the polynucleotide sequences defined in (a).  
     
     
         4 . A polynucleotide according to  claim 1  lacking the native leader sequences or any of the 5′ or 3′ untranslated regions of the polynucleotide.  
     
     
         5 . A polynucleotide according to  claim 4 , wherein the native leader sequences or any of the 5′ or 3′ untranslated regions are replaced with exogenous control/regulatory sequences which regulate optimised/enhanced expression of the polynucleotide in an expression system.  
     
     
         6 . A polynucleotide according to  claim 1  which encodes cytochrome P-450 reductase enzyme of  Papaver somniferum  or  Eschscholzia californica.    
     
     
         7 . A polynucleotide according to  claim 6  which encodes cytochrome P-450 reductase enzyme of  Papaver somniferum.    
     
     
         8 . A polynucleotide according to  claim 6  which encodes cytochrome P-450 reductase enzyme of  Eschscholzia californica.    
     
     
         9 . A polynucleotide according to  claim 2 , which is a synthetic polynucleotide comprising one or more codons preferred for expression in plant cells.  
     
     
         10 . An isolated and purified polynucleotide which codes for prokaryotic or eukaryotic expression of a cytochrome P-450 reductase enzyme from an alkaloid poppy plant, or a variant, analog or fragment thereof, wherein the polynucleotide is expressed in an environment selected from the group consisting of the extracellular environment, an intracellular membranous compartment, intracellular cytoplasmic compartment or combinations thereof.  
     
     
         11 . A polynucleotide according to  claim 10 , comprising a nucleotide sequence which directs expression of the cytochrome P-450 reductase enzyme with respect to a particular cellular compartment or the extracellular environment.  
     
     
         12 . An isolated and purified polynucleotide having a sequence which is complementary to all or part of the sequence of a polynucleotide according to any one of claims  1  or  11 .  
     
     
         13 . A recombinant DNA construct comprising the polynucleotide according to  claim 1  or  claim 11 .  
     
     
         14 . A DNA construct according to  claim 13 , which is a viral or plasmid vector.  
     
     
         15 . A DNA construct according to  claim 13 , capable of directing prokaryotic or eukaryotic expression of the polynucleotide encoding a cytochrome P-450 reductase enzyme.  
     
     
         16 . A DNA construct according to  claim 13  comprising a promoter suitable to control the expression of the polynucleotide.  
     
     
         17 . A DNA construct according to  claim 16 , wherein the promoter is endogenous.  
     
     
         18 . A DNA construct according to  claim 16 , wherein the promoter is derived from cauliflower mosaic virus or subterranean clover mosaic virus.  
     
     
         19 . An isolated and purified cytochrome P-450 reductase enzyme, being a product of prokaryotic or eukaryotic expression of the polynucleotide of  claim 1  or  11 .  
     
     
         20 . An enzyme according to  claim 19 , being a product of yeast cell expression.  
     
     
         21 . An enzyme according to  claim 19 , being a product of bacterial cell expression.  
     
     
         22 . An-enzyme according to  claim 19 , being a product of animal cell expression.  
     
     
         23 . An enzyme according to  claim 22 , being a product of insect cell expression.  
     
     
         24 . An enzyme according to  claim 19 , being a product of plant cell expression.  
     
     
         25 . An enzyme according to  claim 24 , wherein the plant cell is an alkaloid poppy plant cell.  
     
     
         26 . An enzyme according to  claim 25 , wherein the alkaloid poppy is  Papaver somniferum.    
     
     
         27 . An enzyme according to  claim 25 , wherein the alkaloid poppy is  Eschscholzia californica.    
     
     
         28 . An enzyme according to  claim 19 , which is a variant incorporating amino acid deletions, substitutions, additions or combinations thereof wherein the variant retains one or more of the biological properties of cytochrome P-450 reductase enzyme.  
     
     
         29 . A cell transformed or transfected with a polynucleotide according to  claim 1  or  11 .  
     
     
         30 . A cell according to  claim 29 , which is a plant cell.  
     
     
         31 . A cell according to  claim 30 , wherein the plant cell is derived from an alkaloid poppy plant.  
     
     
         32 . A cell according to  claim 31  wherein the poppy plant is  Papaver somniferum    
     
     
         33 . A cell according to  claim 31 , wherein the poppy plant is  Eschscholzia californica.    
     
     
         34 . A cell according to  claim 29 , which is a bacterial cell.  
     
     
         35 . A cell according to  claim 29 , which is an animal cell.  
     
     
         36 . A cell according to  claim 29 , which is a yeast cell.  
     
     
         37 . A callus transformed or transfected with a polynucleotide according to  claim 1  or  11 .  
     
     
         38 . A plant transformed or transfected with a polynucleotide according to  claim 1  or  11 , wherein the plant exhibits altered expression of the cytochrome P-450 reductase enzyme.  
     
     
         39 . A plant according to  claim 38 , wherein the altered expression is overexpression of the cytochrome P-450 reductase enzyme.  
     
     
         40 . A plant according to  claim 38 , wherein the altered expression is reduced expression of the cytochrome P-450 reductase enzyme.  
     
     
         41 . A plant according to  claim 38 , which is an alkaloid poppy plant.  
     
     
         42 . A plant according to  claim 41 , wherein the plant has a higher or different alkaloid content when compared to a plant which has not been so transformed or transfected.  
     
     
         43 . A plant according to  claim 41 , wherein the alkaloid poppy plant is  Papaver somniferum.    
     
     
         44 . A plant according to  claim 41 , wherein the alkaloid poppy plant is  Eschscholzia californica.    
     
     
         45 . A method for preparing plants which overexpress a cytochrome P-450 reductase enzyme, comprising transfecting or transforming a plant cell, a plant part or a plant with the polynucleotide according to  claim 1  or  11 .  
     
     
         46 . A method according to  claim 45 , wherein the plant is an alkaloid poppy plant.  
     
     
         47 . A method according to  claim 46 , wherein the poppy plant is  Papaver somniferum.    
     
     
         48 . A method according to  claim 46 , wherein the poppy plant is  Eschscholzia californica.    
     
     
         49 . A method of altering the yield or type of alkaloid in a plant comprising transforming or transfecting a plant cell a plant part or a plant with a polynucleotide encoding a cytochrome P-450 reductase enzyme or a variant analog or fragment thereof, or with a polynucleotide which binds under stringent conditions to the polynucleotide encoding said enzyme.  
     
     
         50 . A method of increasing the yield of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or a plant with a polynucleotide encoding a cytochrome P-450 reductase enzyme or a variant, analog or fragment thereof, wherein the enzyme is overexpressed in said plant.  
     
     
         51 . A method of altering type or blend of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or a plant with a polynucleotide encoding a cytochrome P-450 reductase enzyme or a variant, analog or fragment thereof, or with a polynucleotide which binds under-stringent conditions to the polynucleotide encoding said enzyme.  
     
     
         52 . A stand of stably reproducing alkaloid poppies transformed or transfected with a polynucleotide according to  claim 1  or  11 , having altered expression of the cytochrome P-450 reductase enzyme.  
     
     
         53 . A stand of stably reproducing alkaloid poppies transformed or transfected with a polynucleotide according to  claim 1  or  11 , having a higher or different alkaloid content when compared to a plant which has not been so transformed or transfected.  
     
     
         54 . A stand of stably reproducing alkaloid poppies according to  claim 52 , wherein the alkaloid poppy is  Papaver somniferum.    
     
     
         55 . Straw of stably reproducing poppies according to  claim 52  having a higher or different alkaloid content when compared to the straw obtained from an alkaloid poppy which has not been transformed or transfected.  
     
     
         56 . A concentrate of straw according to  claim 55 , having a higher or different alkaloid content when compared to the concentrate of straw obtained from an alkaloid poppy which has not been transformed or transfected.  
     
     
         57 . An alkaloid when isolated from the straw according to  claim 55 .  
     
     
         58 . An alkaloid according to  claim 57 , selected from the group consisting of morphine, codeine, oripavine and thebaine.  
     
     
         59 - 61 . (canceled)