Monoterpene synthases from grand fir (abies grandis)

Abstract

cDNAs encoding gymnosperm monoterpene synthases have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a monoterpene synthase of the invention. Thus, systems and methods are provided for the recombinant expression of recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:  
     
         1 . An isolated nucleic acid molecule that hybridizes under stringent conditions to a probe selected from the group consisting of the portion of SEQ ID NO: 3 extending from nucleotide 1560 to nucleotide 1694 and the portion of SEQ ID NO: 5 extending from nucleotide 1180 to nucleotide 1302.  
     
     
         2 . An isolated nucleic acid molecule of  claim 1  that hybridizes under stringent conditions to the portion of SEQ ID NO: 3 extending from nucleotide 1560 to nucleotide 1694.  
     
     
         3 . An isolated nucleic acid molecule of  claim 1  that hybridizes under stringent conditions to the portion of SEQ ID NO: 5 extending from nucleotide 1180 to nucleotide 1302.  
     
     
         4 . An isolated nucleic acid molecule of  claim 1  encoding a (−)-camphene synthase.  
     
     
         5 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm (−)-camphene synthase.  
     
     
         6 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir (−)-camphene synthase.  
     
     
         7 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 65.  
     
     
         8 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 64:  
     
     
         9 . An isolated nucleic acid molecule of  claim 1  encoding a (−)-β-phellandrene synthase.  
     
     
         10 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm (−)-β-phellandrene synthase.  
     
     
         11 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir (−)-β-phellandrene synthase.  
     
     
         12 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 67.  
     
     
         13 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 66.  
     
     
         14 . An isolated nucleic acid molecule of  claim 1  encoding a terpinolene synthase.  
     
     
         15 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm terpinolene synthase.  
     
     
         16 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir terpinolene synthase.  
     
     
         17 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 78.  
     
     
         18 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 77.  
     
     
         19 . An isolated nucleic acid molecule of  claim 1  encoding a (−)-limonene/(−)-α-pinene synthase.  
     
     
         20 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm (−)-limonene/(−)-α-pinene synthase.  
     
     
         21 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir (−)-limonene/(−)-α-pinene synthase.  
     
     
         22 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 69.  
     
     
         23 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 68.  
     
     
         24 . An isolated nucleic acid molecule of  claim 1  encoding a myrcene synthase.  
     
     
         25 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm myrcene synthase.  
     
     
         26 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir myrcene synthase.  
     
     
         27 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 2.  
     
     
         28 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 1.  
     
     
         29 . An isolated nucleic acid molecule of  claim 1  encoding a (−)-limonene synthase.  
     
     
         30 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm (−)-limonene synthase.  
     
     
         31 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir (−)-limonene synthase.  
     
     
         32 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 6.  
     
     
         33 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 5.  
     
     
         34 . An isolated nucleic acid molecule of  claim 1  encoding a (−)-pinene synthase.  
     
     
         35 . An isolated nucleic acid molecule of  claim 1  encoding a gymnosperm (−)-pinene synthase.  
     
     
         36 . An isolated nucleic acid molecule of  claim 1  encoding a Grand fir (−)-pinene synthase.  
     
     
         37 . An isolated nucleic acid molecule of  claim 1  which encodes the amino acid sequence of SEQ ID NO: 4.  
     
     
         38 . An isolated nucleic acid molecule of  claim 1  having the sequence of SEQ ID NO: 3.  
     
     
         39 . An isolated (−)-camphene synthase protein.  
     
     
         40 . An isolated gymnosperm (−)-camphene synthase protein of  claim 39 .  
     
     
         41 . An isolated Grand fir (−)-camphene synthase protein of  claim 39 .  
     
     
         42 . An isolated (−)-camphene synthase protein of  claim 39 , said isolated (−)-camphene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 65.  
     
     
         43 . An isolated (−)-β-phellandrene synthase protein.  
     
     
         44 . An isolated gymnosperm (−)-β-phellandrene synthase protein of  claim 43 .  
     
     
         45 . An isolated Grand fir (−)-β-phellandrene synthase protein of  claim 43 .  
     
     
         46 . An isolated (−)-β-phellandrene synthase protein of  claim 43 , said isolated (−)-β-phellandrene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 67.  
     
     
         47 . An isolated terpinolene synthase protein.  
     
     
         48 . An isolated gymnosperm terpinolene synthase protein of  claim 47 .  
     
     
         49 . An isolated Grand fir terpinolene synthase protein of  claim 47 .  
     
     
         50 . An isolated terpinolene synthase protein of  claim 47 , said isolated terpinolene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 78.  
     
     
         51 . An isolated (−)-limonene/(−)-α-pinene synthase protein.  
     
     
         52 . An isolated gymnosperm (−)-limonene/(−)-α-pinene synthase protein of  claim 51 .  
     
     
         53 . An isolated Grand fir (−)-limonene/(−)-α-pinene synthase protein of  claim 51 .  
     
     
         54 . An isolated (−)-limonene/(−)-α-pinene synthase protein of  claim 51 , said isolated (−)-limonene/(−)-α-pinene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 69.  
     
     
         55 . An isolated myrcene synthase protein.  
     
     
         56 . An isolated gymnosperm myrcene synthase protein of  claim 55 .  
     
     
         57 . An isolated Grand fir myrcene synthase protein of  claim 55 .  
     
     
         58 . An isolated myrcene synthase protein of  claim 55 , said isolated myrcene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 2.  
     
     
         59 . An isolated (−)-limonene synthase protein.  
     
     
         60 . An isolated gymnosperm (−)-limonene synthase protein of  claim 59 .  
     
     
         61 . An isolated Grand fir (−)-limonene synthase protein of  claim 59 .  
     
     
         62 . An isolated (−)-limonene synthase protein of  claim 59 , said isolated (−)-limonene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 6.  
     
     
         63 . An isolated (−)-pinene synthase protein.  
     
     
         64 . An isolated gymnosperm (−)-pinene synthase protein of  claim 63 .  
     
     
         65 . An isolated Grand fir (−)-pinene synthase protein of  claim 63 .  
     
     
         66 . An isolated (−)-pinene synthase protein of  claim 63 , said isolated (−)-pinene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 4.  
     
     
         67 . A replicable expression vector comprising a nucleic acid sequence encoding a monoterpene synthase selected from the group consisting of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase.  
     
     
         68 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a (−)-camphene synthase.  
     
     
         69 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a (−)-β-phellandrene synthase.  
     
     
         70 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a terpinolene synthase.  
     
     
         71 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a (−)-limonene/(−)-α-pinene synthase.  
     
     
         72 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a myrcene synthase.  
     
     
         73 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a (−)-limonene synthase.  
     
     
         74 . A replicable expression vector of  claim 67  comprising a nucleic acid sequence encoding a (−)-pinene synthase.  
     
     
         75 . A host cell comprising a vector of  claim 68 .  
     
     
         76 . A host cell comprising a vector of  claim 69 .  
     
     
         77 . A host cell comprising a vector of  claim 70 .  
     
     
         78 . A host cell comprising a vector of  claim 71 .  
     
     
         79 . A host cell comprising a vector of  claim 72 .  
     
     
         80 . A host cell comprising a vector of  claim 73 .  
     
     
         81 . A host cell comprising a vector of  claim 74 .  
     
     
         82 . A method of enhancing the production of a monoterpene synthase in a suitable host cell comprising introducing into the host cell an expression vector of  claim 67  under conditions enabling expression of the monoterpene synthase in the host cell.  
     
     
         83 . The method of  claim 82  wherein said host cell is a plant cell.  
     
     
         84 . The method of  claim 83  wherein said cell is from a plant selected from the group consisting of Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee, tree nuts, spinach, culinary herbs, berries, stone fruit and citrus.  
     
     
         85 . The method of  claim 83  wherein said plant cell is a seed cell.  
     
     
         86 . The method of  claim 83  wherein said plant cell is a leaf cell.  
     
     
         87 . Seed comprising a cell produced by the method of  claim 85 .  
     
     
         88 . Seed of  claim 87  selected from the group consisting of seed of Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee and tree nuts.  
     
     
         89 . Oil extracted from the seed of  claim 88 .  
     
     
         90 . Meal extracted from the seed of  claim 88.