Modulation of coffee flavour precursor levels in green coffee grains

Abstract

The present invention relates to isolated polynucleotides encoding cysteine proteinases; cysteine proteinase inhibitors; and aspartic endoproteinases. The invention also relates to a transformed host cell, preferably a plant cell, in which over- or under-expression of these polynucleotides result in altered levels of coffee flavour precursor levels, specifically, amino group-containing molecules such as amino acids, peptides and proteins, in green coffee grains.

1 . An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having cysteine proteinase activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No. 2 have at least 70%, sequence identity based on the ClustalW alignment method; or the complement of the nucleotide sequence, wherein the complement contains the same number of nucleotides as the nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.  
     
     
         2 . The polynucleotide of  claim 1 , wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No. 2 have at least 85%, sequence identity based on the ClustalW alignment method.  
     
     
         3 . The polynucleotide of  claim 1 , wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID No. 1.  
     
     
         4 . The polynucleotide of  claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID No. 2.  
     
     
         5 . An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having cysteine proteinase inhibitor activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence are selected from the group consisting of SEQ ID Nos. 4, 10, 12 and 14 and have at least 80%, sequence identity based on the ClustalW alignment method; or the complement of the nucleotide sequence, wherein the complement contains the same number of nucleotides as the nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.  
     
     
         6 . The polynucleotide of  claim 5 , wherein the amino acid sequence of the polypeptide and the amino acid sequence selected have at least 85% sequence identity based on the ClustalW alignment method.  
     
     
         7 . The polynucleotide of  claim 5 , wherein the nucleotide sequence comprises the nucleotide sequence selected from the group consisting of SEQ ID Nos. 3, 9, 11 and 13.  
     
     
         8 . The polynucleotide of  claim 5 , wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID Nos. 4, 10, 12 and 14.  
     
     
         9 . An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having aspartic endoproteinase activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence are selected from the group consisting of SEQ ID No. 6 of and 8, have at least 75% sequence identity based on the ClustalW alignment method, or the complement of the nucleotide sequence, wherein the complement contains the same number of nucleotides as the nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.  
     
     
         10 . The polynucleotide of  claim 9 , wherein the amino acid sequence of the polypeptide and the amino acid sequence have at least 85% sequence identity based on the ClustalW alignment method.  
     
     
         11 . The polynucleotide of  claim 9 , wherein the nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID No. 5 or 7.  
     
     
         12 . The polynucleotide of  claim 9 , wherein the polypeptide comprises the amino acid sequence of SEQ ID No.  
     
     
         13 . An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having cysteine proteinase activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No. 16 have at least 70% sequence identity based on the ClustalW alignment method; or the complement of the nucleotide sequence, wherein the complement contains the same number of nucleotides as the nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.  
     
     
         14 . The polynucleotide of  claim 13 , wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No. 16 have at least 85% sequence identity based on the ClustalW alignment method.  
     
     
         15 . The polynucleotide of  claim 13 , wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID No. 15.  
     
     
         16 . The polynucleotide of  claim 13 , wherein the polypeptide comprises the amino acid sequence of SEQ ID No. 16.  
     
     
         17 . A vector comprising the polynucleotide of claim to  1 .  
     
     
         18 . A non-native recombinant DNA construct comprising the polynucleotide of  claim 1  operably linked to a regulatory sequence.  
     
     
         19 . A method for transforming a cell comprising transforming a cell with the polynucleotide of  claim 1 .  
     
     
         20 . A cell comprising the non-native recombinant DNA construct of  claim 18 .  
     
     
         21 . The cell of  claim 20 , which is selected from the group consisting of a prokaryotic cell, an eukaryotic cell and a plant cell.  
     
     
         22 . A transgenic plant comprising the cell of  claim 20 .  
     
     
         23 . A method for modulating coffee flavour precursor levels in green coffee grains, the method comprising introducing into the coffee plant the non-native recombinant DNA construct of  claim 18 .  
     
     
         24 . A vector comprising the polynucleotide of  claim 5 .  
     
     
         25 . A non-native recombinant DNA construct comprising the polynucleotide of  claim 5  operably linked to a regulatory sequence.  
     
     
         26 . A method for transforming a cell comprising transforming a cell with the polynucleotide of  claim 5 .  
     
     
         27 . A cell comprising the non-native recombinant DNA construct of  claim 25 .  
     
     
         28 . The cell of  claim 27 , which is selected from the group consisting of a prokaryotic cell, an eukaryotic cell and a plant cell.  
     
     
         29 . A transgenic plant comprising the cell of  claim 25 .  
     
     
         30 . A method for modulating coffee flavour precursor levels in green coffee grains, the method comprising introducing into the coffee plant the non-native recombinant DNA construct of  claim 25 .  
     
     
         31 . A vector comprising the polynucleotide of  claim 9 .  
     
     
         32 . A non-native recombinant DNA construct comprising the polynucleotide of  claim 9  operably linked to a regulatory sequence.  
     
     
         33 . A method for transforming a cell comprising transforming a cell with the polynucleotide of  claim 9 .  
     
     
         34 . A cell comprising the non-native recombinant DNA construct of  claim 32 .  
     
     
         35 . The cell of  claim 34 , which is selected from the group consisting of a prokaryotic cell, an eukaryotic cell and a plant cell.  
     
     
         36 . A transgenic plant comprising the cell of  claim 34 .  
     
     
         37 . A method for modulating coffee flavour precursor levels in green coffee grains, the method comprising introducing into the coffee plant the non-native recombinant DNA construct of  claim 32 .  
     
     
         38 . A vector comprising the polynucleotide of  claim 13 .  
     
     
         39 . A non-native recombinant DNA construct comprising the polynucleotide of  claim 13  operably linked to a regulatory sequence.  
     
     
         40 . A method for transforming a cell comprising transforming a cell with the polynucleotide of  claim 13 .  
     
     
         41 . A cell comprising the non-native recombinant DNA construct of  claim 39 .  
     
     
         42 . The cell of  claim 41 , which is selected from the group consisting of a prokaryotic cell, an eukaryotic cell and a plant cell.  
     
     
         43 . A transgenic plant comprising the cell of  claim 39 .  
     
     
         44 . A method for modulating coffee flavour precursor levels in green coffee grains, the method comprising introducing into the coffee plant the non-native recombinant DNA construct of  claim 39.