Novel sesquiterpene synthase gene and protein
AssigneeUniversity of Kentucky Research Foundation
InventorsJoe Chappell, Bryan Greenhagen
Life of this patent
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Abstract
The invention relates to sesquiterpene synthases and methods for their production and use. Particularly, the invention provides nucleic acids comprising the nucleotide sequence of citrus valencene synthase (CVS) which codes for at least one CVS. The invention further provides nucleic acids comprising the nucleotide sequence coding for amino acid residues forming the tier 1 and tier 2 domains of CVS. The invention also provides for methods of making and using the nucleic acids and amino acids of the current invention.
Claims71
An isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO 1.
An isolated nucleic acid that encodes the amino acid sequence of SEQ ID NO:
- 4.
An isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity.
An isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity.
An isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch.
An isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5 and SEQ ID NO.6.
An isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5.
An isolated nucleic acid that encodes a protein that includes amino acid residues 264 to 530 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity.
An isolated polypeptide of SEQ ID NO:
- 4.
An isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity.
An isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity.
An isolated polypeptide encoded by an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch, wherein the isolated polypeptide has sesquiterpene synthase activity.
An isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7 and SEQ ID NO. 8.
An isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7.
An isolated polypeptide that includes amino acid residues 264 to 530 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity.
A vector comprising a nucleic acid sequence operably linked to at least one control sequence, wherein the nucleic acid sequence is selected from the group consisting of:
- (a) an isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO 1;
- (b) an isolated nucleic acid that encodes the amino acid sequence of SEQ ID NO:
- 4;
- (c) an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (d) an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (e) an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch;
- (f) an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5 and SEQ ID NO. 6;
- (g) an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5;
- and
- (h) an isolated nucleic acid that encodes a protein that includes amino acid residues 264 to 530 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity.
A non-human multicellular organism that is modified to harbor a nucleic acid selected from the group consisting of:
- (a) an isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO 1;
- (b) an isolated nucleic acid that encodes the amino acid sequence of SEQ ID NO:
- 4;
- (c) an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (d) an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (e) an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch;
- (f) an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5 and SEQ ID NO. 6;
- (g) an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5;
- and
- (h) an isolated nucleic acid that encodes a protein that includes amino acid residues 264 to 530 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity;
- such that the nucleic acid can be expressed in the non-human multicellular organism.
A method of producing a sesquiterpene comprising the steps of:
- (a) reacting an isolated polypeptide having sesquiterpene synthase enzymatic activity with farnesyl pyrophosphate under conditions in which the enzymatic activity catalyzes the formation of a sesquiterpene;
- and
- (b) isolating the sesquiterpene formed in step (a);
- wherein the isolated polypeptide is selected from the group consisting of:
- (i) an isolated polypeptide of SEQ ID NO:
- 4;
- (ii) an isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (iii) an isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (iv) an isolated polypeptide encoded by an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch, wherein the isolated polypeptide has sesquiterpene synthase activity;
- (v) an isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7 and SEQ ID NO. 8;
- (vi) an isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7;
- and
- (vii) an isolated polypeptide that includes amino acid residues 264 to 528 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity.
A method of producing nootkatone comprising:
- (a) reacting an isolated polypeptide having valencene synthase enzymatic activity with farnesyl pyrophosphate under conditions in which the enzymatic activity catalyzes the formation of valencene;
- and
- (b) reacting the valencene formed in step (a) by regio-specific hydroxylation and then oxidation to form nootkatone;
- and
- (c) isolating the nootkatone produced.
- wherein the isolated polypeptide is selected from the group consisting of:
- (i) an isolated polypeptide of SEQ ID NO:
- 4;
- (ii) an isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has valencene synthase activity;
- (iii) an isolated polypeptide that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has valencene synthase activity;
- (iv) an isolated polypeptide encoded by an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch, wherein the isolated polypeptide has valencene synthase activity;
- (v) an isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has valencene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7 and SEQ ID NO. 8;
- (vi) an isolated polypeptide that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has valencene synthase activity, wherein the sequence of the protein includes SEQ ID NO. 7;
- and
- (vii) an isolated polypeptide that includes amino acid residues 264 to 528 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has valencene synthase activity.
An isolated nucleic acid that is an antisense nucleic acid, wherein the antisense nucleic acid is selected from the group consisting of:
- (a) an antisense nucleic acid that is the complement of an isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO 1;
- (b) an antisense nucleic acid that is the complement of isolated nucleic acid that encodes the amino acid sequence of SEQ ID NO:
- 4;
- (c) an antisense nucleic acid that is the complement of an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 264, 273, 403, 404, 441, 445, 522, 526, 528, and 529 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (d) an antisense nucleic acid that is the complement of an isolated nucleic acid encoding a protein that has the amino acid sequence of SEQ ID NO. 4 with 0 to 20 conservative amino acid substitutions, with the proviso that a conservative amino acid substitution is not made at amino acids 13, 264, 266, 270, 273, 298, 302, 372, 376, 401, 402, 403, 404, 407, 437, 441, 442, 445, 446, 449, 514, 517, 518, 521, 522, 528, 529, and 530 in the protein that has the amino acid sequence of SEQ ID NO:
- 4, wherein the encoded protein specifically binds farnesyl pyrophosphate in a substantially hydrophobic pocket and has sesquiterpene synthase activity;
- (e) an antisense nucleic acid that is the complement of an isolated nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:
- 1 under stringent conditions with no more than about a 5% mismatch;
- (f) an antisense nucleic acid that is the complement of an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5 and SEQ ID NO. 6;
- (g) an antisense nucleic acid that is the complement of an isolated nucleic acid that encodes a protein that is at least 500 amino acids in length, that specifically binds farnesyl pyrophosphate in a hydrophobic pocket, and that has sesquiterpene synthase activity, wherein the nucleic acid includes SEQ ID NO. 5;
- and
- (h) an antisense nucleic acid that is the complement of an isolated nucleic acid that encodes a protein that includes amino acid residues 264 to 530 of SEQ ID NO. 4, wherein the protein specifically binds farnesyl pyrophosphate in a hydrophobic pocket and has sesquiterpene synthase activity.
Dependencies are inferred from the claim text.