Increasing levels of nicotinic alkaloids in plants

Abstract

Four genes, A622, NBB1, PMT, and QPT, can be influenced for increasing nicotinic alkaloid levels in Nicotiana plants, as well as for synthesizing nicotinic alkaloids in non-nicotine producing plants and cells. In particular, overexpressing one or more of A622, NBB1, PMT, and QPT may be used to increase nicotine and nicotinic alkaloid levels in tobacco plants. Non-nicotine producing cells can be engineered to produce nicotine and related compounds by overexpressing A622 and NBB1.

1 - 26 . (canceled) 
     
     
         27 . A cell modified to express a A662 enzyme and a NBB1 enzyme, the cell comprising an isolated nucleic molecule encoding A662 and NBB1, wherein:
 (a) the isolated nucleic acid molecule encoding A662 comprises at least one of:
 (i) the nucleotide sequence set forth in SEQ ID NO: 3; and 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and 
   (b) the isolated nucleic acid molecule encoding NBB1 comprises at least one of:
 (i) the nucleotide sequence set forth in SEQ ID NO: 1; and 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. 
   
     
     
         28 . The cell of  claim 27 , wherein the cell is selected from the group consisting of bacteria, yeast, filamentous fungi, algae, green plants, insect, and mammalian cells. 
     
     
         29 . The cell of  claim 28 , wherein:
 (a) the yeast cell is a  Saccharomyces  cerivisae or  Pichia pastoris  cell; and/or   (c) the filamentous fungi cell is an  Aspergillus  or  Trichoderma  cell; and/or   (d) the bacteria cell is an  Escherichia coli  cell.   
     
     
         30 . The cell of  claim 28 , wherein the mammalian cell is a Chinese hamster ovary cell (CHO), a fertilized oocyte, or an embryonic stem cell. 
     
     
         31 . The cell of  claim 28 , wherein the algae cell is a  Chlamydomonas reinhardtii  cell. 
     
     
         32 . The cell of  claim 27 , wherein the cell further produces a nicotinic alkaloid. 
     
     
         33 . The cell of  claim 32 , wherein the nicotinic alkaloid is nicotine. 
     
     
         34 . A cell modified to express a NBB1 enzyme, the cell comprising an isolated nucleic molecule encoding NBB1, wherein the isolated nucleic acid molecule encoding NBB1 comprises at least one of:
 (a) the nucleotide sequence set forth in SEQ ID NO: 1; and   (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.   
     
     
         35 . The cell of  claim 34 , wherein the cell is selected from the group consisting of bacteria, yeast, filamentous fungi, algae, green plants, insect, and mammalian cells. 
     
     
         36 . The cell of  claim 35 , wherein:
 (a) the yeast cell is a  Saccharomyces  cerivisae or  Pichia pastoris  cell; and/or   (b) the filamentous fungi cell is an  Aspergillus  or  Trichoderma  cell; and/or   (c) the bacteria cell is an  Escherichia coli  cell.   
     
     
         37 . The cell of  claim 35 , wherein the mammalian cell is a Chinese hamster ovary cell (CHO), a fertilized oocyte, or an embryonic stem cell. 
     
     
         38 . The cell of  claim 35 , wherein the algae cell is a  Chlamydomonas reinhardtii  cell. 
     
     
         39 . The cell of  claim 34 , wherein the cell further produces a nicotinic alkaloid. 
     
     
         40 . The cell of  claim 39 , wherein the nicotinic alkaloid is nicotine. 
     
     
         41 . A cell modified to express a A662 enzyme and a NBB1 enzyme, the cell comprising an isolated nucleic molecule encoding A662 and an isolated nucleic molecule encoding NBB1, wherein:
 (a) the isolated nucleic acid molecule encoding A662 comprises at least one of:
 (i) the nucleotide sequence set forth in SEQ ID NO: 3; and 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and 
   (b) the isolated nucleic acid molecule encoding NBB1 comprises at least one of:
 (i) the nucleotide sequence set forth in SEQ ID NO: 1; and 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. 
   
     
     
         42 . The cell of  claim 41 , wherein the cell is selected from the group consisting of bacteria, yeast, filamentous fungi, algae, green plants, insect, and mammalian cells. 
     
     
         43 . The cell of  claim 42 , wherein:
 (a) the yeast cell is a  Saccharomyces  cerivisae or  Pichia pastoris  cell; and/or   (b) the filamentous fungi cell is an  Aspergillus  or  Trichoderma  cell; and/or   (c) the bacteria cell is an  Escherichia coli  cell; and/or   (d) the mammalian cell is a Chinese hamster ovary cell (CHO), a fertilized oocyte, or an embryonic stem cell.   
     
     
         44 . The cell of  claim 42 , wherein the algae cell is a  Chlamydomonas reinhardtii  cell. 
     
     
         45 . The cell of  claim 41 , wherein the cell further produces a nicotinic alkaloid. 
     
     
         46 . The cell of  claim 45 , wherein the nicotinic alkaloid is nicotine.