Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof
AssigneesThe University of British Columbia, The University of Western Australia
InventorsCarl Joerg BOHLMANN, Maria Luisa DIAZ CHAVEZ, Jessie MONIODIS
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Abstract
Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells.
Claims37
1-173. (canceled)
A host cell, comprising a nucleic acid molecule encoding a cytochrome P450 reductase or a catalytically active portion thereof, wherein:
- (a) the encoded cytochrome P450 reductase or catalytically active portion thereof exhibits at least 80% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:
- 12 or 13 or a corresponding catalytically active portion thereof;
- (b) the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor;
- and
- (c) the nucleic acid molecule is heterologous to the host cell.
A host cell, comprising:
- (a) a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
- (i) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:
- 50;
- (ii) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene;
- (b) a nucleic acid molecule encoding a cytochrome P450 reductase or catalytically active portion thereof, wherein the encoded cytochrome P450 reductase or catalytically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:
- 12-15, or a sequence of amino acids that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:
- 12-15;
- and
- (c) a nucleic acid molecule encoding a santalene synthase, wherein the encoded santalene synthase comprises the sequence of amino acids set forth in any of SEQ ID NO:
- 17, 52 or 53 or a sequence of amino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NO:
- 17, 52 or 53 or a catalytically active fragment thereof, wherein:
- at least one of the nucleic acid molecules set forth in (a) or (b) is heterologous to the host cell.
An isolated nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
- (a) the nucleic acid molecule is cDNA;
- (b) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 70% sequence identity to SEQ ID NO:50 or a corresponding catalytically active portion thereof;
- and
- (c) the encoded cytochrome P450 oxidase or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene.
An isolated nucleic acid molecule encoding a cytochrome P450 reductase or a catalytically active portion thereof, wherein:
- (a) the nucleic acid molecule is cDNA;
- (b) the encoded cytochrome P450 reductase or catalytically active portion thereof exhibits at least 80% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:
- 12 or 13;
- and
- (c) the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor.
A nucleic acid molecule that is a fusion protein, comprising a sequence of nucleic acids encoding at least two polypeptides selected from among nucleic acids encoding:
- (a) a Santalum album cytochrome P450 oxidase or a catalytically active fragment thereof, wherein the cytochrome P450 oxidase or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene;
- (b) a cytochrome P450 reductase or a catalytically active fragment thereof, wherein the cytochrome P450 reductase catalyzes the transfer of two electrons from NADPH to the cytochrome P450 oxidase electron acceptor;
- and
- (c) a santalene synthase or catalytically active fragment thereof, wherein the santalene synthase catalyzes the formation of a santalene and/or bergamotene terpene substrate of the cytochrome P450 wherein:
- the encoded cytochrome P450 oxidase or catalytically active fragment thereof comprises the sequence of amino acids set forth in SEQ ID NO:
- 6-9 or 73-77, or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a cytochrome P450 oxidase polypeptide set forth in SEQ ID NO:
- 6-9 or 73-77;
- and
- the santalene synthase or catalytically active fragment thereof comprises the sequence of amino acids set forth in any of SEQ ID NO:
- 17, 52 or 53 or a sequence of amino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NO:
- 17, 52 or 53 or a catalytically active fragment thereof.
Dependencies are inferred from the claim text.