# Patent

## Title
Cytochrome P450 and cytochrome P450 reductase polypeptides, encoding nucleic acid molecules and uses thereof

## 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.

## Background
RELATED APPLICATIONS
Benefit of priority is claimed to U.S. Provisional Application Ser. No. 61/796,129, filed Nov. 1, 2012, entitled “CYTOCHROME P450 AND CYTOCHROME P450 REDUCTASE POLYPEPTIDES, ENCODING NUCLEIC ACID MOLECULES AND USES THEREOF” and to U.S. Provisional Application Ser. No. 61/956,086, filed May 31, 2013, entitled “CYTOCHROME P450 AND CYTOCHROME P450 REDUCTASE POLYPEPTIDES, ENCODING NUCLEIC ACID MOLECULES AND USES THEREOF.” The subject matter of each of the above-noted applications is incorporated by reference in its entirety.


INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY
An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file is 301 kilobytes in size, and titled 229SEQPC1.txt.
FIELD OF THE INVENTION
Provided are cytochrome P450 santalene oxidases, cytochrome P450 bergamotene oxidases and cytochrome P450 reductases, nucleic acid molecules encoding the P450 santalene oxidases, cytochrome P450 bergamotene oxidases and cytochrome P450 reductases, and methods for producing products whose synthesis includes reactions catalyzed by the cytochrome P450 santalene and bergamotene oxidases. Included among the products are santalols and bergamotols and precursors and derivatives thereof.
BACKGROUND
Sandalwood (Santalum album) is a slow-growing hemi-parasitic tropical tree of great economic value found growing in southern India, Sri Lanka, eastern Indonesia and northern Australia. The timber is highly sought after for its fine grain, high density and excellent carving properties. Sandalwood heartwood has a unique fragrance imparted by the resins and essential oils, including santalols, santalenes and other sesquiterpenoids, in the heartwood. In general, Santalum album heartwood contains up to 6% dry weight sesquiterpene oils. Sandalwood oil predominantly contains the sesquiterpene alcohols α-santalol, β-santalol, Z-α-trans-bergamotol and epi-β-santalol, and additionally includes α-santalene, β-santalene, α-bergamotene, epi-β-santalene, β-bisabolene, α-curcumene, β-curcumene and γ-curcumene. Sandalwood oil has a soft, sweet-woody and animal-balsamic odor that is imparted from the terpenoid β-santalol and is highly valued. Sandalwood oil has been obtained by distillation of the heartwood of Santalum species and is used as a perfume ingredient, in incenses and traditional medicine and in pesticides.
Centuries of over-exploitation has led to the demise of sandalwood in natural stands. Large plantations are being established throughout northern Australia to satisfy demand and conserve remaining reserves. In addition, there is great variation in the amount of heartwood oil produced, even under near-identical growing conditions, due to genetic and environmental factors, such as climate and local conditions. Generally, the price and availability of plant natural extracts depend upon the abundance, oil yield and geographical origins of the plants.
Although chemical approaches to generate santalols and the other sesquiterpenoids in sandalwood oil have been attempted, the highly complex structures of these compounds have rendered economically viable synthetic processes for their preparation in large quantities unattainable. Thus, there is a need for efficient, cost-effective syntheses of santalols and other sesquiterpenoids that impart the highly sought after sandalwood fragrance for use in the fragrance industry.
Thus, among the objects herein, is the provision of methods for the production of santalols and other sesquiterpenoids and the resulting products of the methods.
SUMMARY
Provided herein are nucleic acid molecules encoding cytochrome P450 polypeptides or catalytically active fragments thereof and the encoded polypeptides, and host cells containing such nucleic acid molecules or encoded polypeptides. For example, the encoded cytochrome P450 polypeptide or catalytically active fragment or portion thereof exhibits at least 65%, 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, such as at least 90% sequence identity to SEQ ID NO:50. Also provided are nucleic acid molecules encoding cytochrome P450 reductase polypeptides or catalytically active fragments thereof and the encoded polypeptides, and host cells containing such nucleic acid molecules or encoded polypeptides. For example, the encoded cytochrome reductase polypeptide or catalytically active fragment thereof exhibits 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 or 13, such as at least 90% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13. Any of the nucleic acid molecules provided herein can be cDNA or can be an isolated or purified nucleic acid molecule. Among the nucleic acid molecules and polypeptides provided herein are a set of CYP450s that exhibit santalene/bergamotene oxidase activity, which provide for, among other things, metabolic engineering of sandalwood oil biosynthesis, improvement of sandalwood plantations, and conservation of native sandalwood forests.
In particular, among the host cells provided herein are host cells that are engineered to contain heterologous nucleic acid encoding any of the cytochrome P450 polypeptides provided herein, whereby the host cells are capable of producing one or more of α-santalol from α-santalene, β-santalol from β-santalene, epi-β-santalol from epi-β-santalene and α-trans-bergamotol from α-trans-bergamotene, such as one or more of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol or (E)-α-trans-bergamotol. For example, the host cells are also engineered to also contain a santalene synthase as described herein to produce a santalene and/or bergamotene terpene substrate of the encoded cytochrome P450 polypeptide. The host cells also can be engineered to also contain heterologous nucleic acid encoding a cytochrome P450 reductase, such as any provided herein. The host cells is a prokaryotic cell or an eukaryotic cell, such as a bacteria, yeast, insect, plant or mammalian cell. For example, the host cell is a Saccharomyces genus cell, a Pichia genus cell or an Escherichia coli cell. In particular examples herein, the host cell is a Saccharomyces cerevisiae cell. The host cell produces or is modified to produce or overexpress an acyclic pyrophosphate terpene precursor, such as farnesyl diphosphate.
For example, provided herein are isolated Santalum album cytochrome P450 polypeptides or catalytically active fragments thereof, including cytochrome P450 santalene oxidases or catalytically active fragments thereof and cytochrome P450 bergamotene oxidases or catalytically active fragments thereof. Also provided herein are nucleic acid molecules encoding the cytochrome P450 santalene oxidases and cytochrome P450 bergamotene oxidases or catalytically active fragments thereof. Also provided are modified forms thereof.
Also provided are nucleic acid molecules encoding cytochrome P450 reductase polypeptides, including modified cytochrome P450 reductase polypeptides. Provided herein are isolated Santalum album cytochrome P450 reductase polypeptides, and host cells containing the polypeptides, where the polypeptides are heterologous to the host cell. Provided are nucleic acid molecules encoding a fusion protein containing a cytochrome P450 enzyme and a second moiety such as a synthase or catalytically active portion thereof.
Also provided are nucleic acid molecules encoding fusion proteins containing a Santalum album santalene synthase and/or a cytochrome P450 santalene oxidase or bergamotene oxidase and/or a cytochrome P450 reductase, or catalytically active fragments of any of the enzymes. Exemplary of the nucleic acid molecules encoding fusion proteins are nucleic acid molecules encoding a fusion protein containing: a santalene synthase and a cytochrome P450 santalene oxidase; a santalene synthase and a bergamotene oxidase; a cytochrome P450 santalene oxidase and a cytochrome P450 reductase; and a cytochrome P450 bergamotene oxidase and a cytochrome P450 reductase or catalytically active fragments of any the preceding enzymes. The encoded proteins and host cells containing the nucleic acids and/or the proteins are provided.
Also provided herein are methods for producing any of the encoded cytochrome P450 polypeptides or catalytically active fragments thereof, including methods for producing a cytochrome P450 reductase polypeptide. Also provided herein are methods for production of a santalol, bergamotol and/or mixtures thereof by contacting the cytochrome P450 santalene oxidases and/or cytochrome P450 bergamotene oxidases with a substrate therefor from which these products are produced. The methods can be performed in vitro with isolated reagents or partially isolated reagents or in vivo in a host cell that encodes the enzymes, and optionally a synthase and/or other substrate.
For example, provided herein are isolated Santalum album cytochrome P450 santalene oxidases or catalytically active fragments thereof. The provided isolated Santalum album cytochrome P450 santalene oxidases catalyze the hydroxylation or monooxygenation of santalene and/or bergamotene. In one example, the provided isolated Santalum album cytochrome P450 santalene oxidases catalyze the formation of a santalol from a santalene and/or a bergamotol from a bergamotene. For example, the isolated Santalum album cytochrome P450 santalene oxidases catalyze the formation of α-santalol from α-santalene, β-santalol from β-santalene, epi-β-santalol from epi-β-santalene and/or Z-α-trans-bergamotol from α-trans-bergamotene. For example, the isolated Santalum album cytochrome P450 santalene oxidases catalyze the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol or (E)-α-trans-bergamotol. Also provided herein are isolated cytochrome P450 santalene oxidases that are members of the CYP76 family.
Provided herein are isolated nucleic acid molecules encoding a Santalum album cytochrome P450 santalene oxidase polypeptide or a catalytically active fragment thereof. For example, provided herein are isolated nucleic acid molecules (and host cells containing the nucleic acid molecules, which are heterologous to the host cells) encoding a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NO:7, 74, 75, 76 or 77; or a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids that has at least 96% sequence identity to a cytochrome P450 santalene oxidase whose sequence is set forth in SEQ ID NO:7, 74, 75, 76 or 77. In another example provided herein are isolated nucleic acid molecules encoding a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids that has at least 50% sequence identity to a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO:7, 74, 75, 76 or 77. The cytochrome P450 santalene oxidase polypeptide catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene. For example, the encoded cytochrome P450 santalene oxidase polypeptide exhibits at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO:7, 74, 75, 76 or 77.
Also provided herein are isolated nucleic acid molecules encoding a cytochrome P450 santalene oxidase or a catalytically active fragment thereof selected from among nucleic acid molecules having a sequence of nucleic acids set forth in SEQ ID NO:3, 68, 69, 70 or 71; a sequence of nucleic acids having at least 98% sequence identity to a sequence of nucleic acids set forth in SEQ ID NO:3, 68, 69, 70 or 71; and degenerates thereof. In a particular example, the isolated nucleic acid molecule has the sequence of nucleotides set forth SEQ ID NO:3, 68, 69, 70 or 71. In some examples, the isolated nucleic acid molecules encode a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NO:7, 74, 75, 76 or 77. The provided isolated nucleic acid molecules encode cytochrome P450 santalene oxidase polypeptides that catalyze the formation of a santalol, such as a α-santalol, β-santalol or epi-β-santalol, from a santalene, such as a α-santalene, β-santalene or epi-β-santalene, and/or catalyze the hydroxylation or monooxygenation of santalene. In some examples, the encoded cytochrome P450 santalene oxidase polypeptide catalyzes the formation of Z-α-trans-bergamotol from α-trans-bergamotene. Also provided herein are cytochrome P450 santalene oxidase polypeptides encoded by any of the isolated nucleic acid molecules provided herein.
For example, provided herein are isolated Santalum album cytochrome P450 bergamotene oxidases or catalytically active fragments thereof. The provided isolated Santalum album cytochrome P450 bergamotene oxidases or catalytically active fragments thereof catalyze the hydroxylation or monooxygenation of bergamotene and/or catalyze the formation of a bergamotol from a bergamotene. For example, the isolated Santalum album cytochrome P450 bergamotene oxidases catalyze the formation of Z-α-trans-bergamotol or (E)-α-trans-bergamotol from α-trans-bergamotene. In some examples, the isolated Santalum album cytochrome P450 bergamotene oxidases do not catalyze the hydroxylation of a santalene. In other examples, the isolated Santalum album cytochrome P450 bergamotene oxidases catalyze the hydroxylation of a santalene. Also provided herein are isolated Santalum album cytochrome P450 bergamotene oxidases that are members of the CYP76 family.
Provided herein are isolated nucleic acid molecules encoding a Santalum album cytochrome P450 bergamotene oxidase polypeptide or a catalytically active fragment thereof. For example, provided herein are isolated nucleic acid molecules encoding a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 or 73; or a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids that has at least 96% sequence identity to a cytochrome P450 polypeptide set forth in SEQ ID NO:6, 8, 9 or 73. In another example, provided herein are isolated nucleic acid molecules encoding a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids that has at least 50% sequence identity to a cytochrome P450 bergamotene oxidase polypeptide set forth in SEQ ID NO:6, 8, 9 or 73. The cytochrome P450 bergamotene oxidase polypeptide catalyzes the hydroxylation or monooxygenation of bergamotene. For example, the encoded cytochrome P450 bergamotene oxidase polypeptide exhibits at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 or 73.
Also provided herein are isolated nucleic acid molecules encoding a cytochrome P450 bergamotene oxidase polypeptide or a catalytically active fragment thereof having a sequence of nucleic acids set forth in any of SEQ ID NOS:2, 4, 5 or 67; a sequence of nucleic acids having at least 98% sequence identity to a sequence of nucleic acids set forth in any of SEQ ID NOS: 2, 4, 5 or 67; and degenerates thereof. In a particular example, the isolated nucleic acid molecule has sequence of nucleic acids set forth in SEQ ID NO:2, 4, 5 or 67. In some examples, the isolated nucleic acid molecule encodes a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 or 73. The provided isolated nucleic acid molecules encode a cytochrome P450 bergamotene oxidase polypeptide that catalyzes the formation of a bergamotol, such as Z-α-trans-bergamotol, from a bergamotene, such as α-trans-bergamotene, and/or catalyzes the hydroxylation or monooxygenation of bergamotene, such as α-trans-bergamotene. In some examples, the encoded cytochrome P450 bergamotene oxidase does not catalyze the hydroxylation of a santalene. Also provided herein are cytochrome P450 bergamotene oxidase polypeptides encoded by any of the isolated nucleic acid molecules provided herein.
Also provided herein are isolated nucleic acid molecules encoding a Santalum album cytochrome P450 polypeptide or catalytically active fragments thereof having a sequence of nucleic acids set forth in SEQ ID NO:1 or 72; a sequence of nucleic acids having at least 99% sequence identity to a sequence of nucleic acids set forth in SEQ ID NO:1 or 72; and degenerates thereof. Also provided herein are isolated nucleic acid molecules encoding a cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:50 or 78; or having a sequence of amino acids having at least 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO:50 or 78. Also provided herein are Santalum album cytochrome P450 polypeptides encoded by any of the isolated nucleic acid molecules provided herein.
Also provided herein are nucleic acid molecules encoding a cytochrome P450 polypeptide or catalytically active fragments thereof having one or more heterologous domains or portions thereof from one or more cytochrome P450s. The domain is selected from among helix A, β strand 1-1, β strand 1-2, helix B, β strand 1-5, helix B′, helix C, helix D, β strand 3-1, helix E, helix F, helix G, helix H, β strand 5-1, β strand 5-2, helix I, helix J, helix J′, helix K, β strand 1-4, β strand 2-1, β strand 2-2, β strand 1-3, Heme domain, helix L, β strand 3-3, β strand 4-1, β strand 4-2 and β strand 3-2. In some examples, the heterologous domain or a contiguous portion thereof replaces all or a contiguous portion of the corresponding native domain of the cytochrome P450 polypeptide not containing the heterologous domain. For example, the encoded modified cytochrome P450 polypeptide contains all of a heterologous domain of a different cytochrome P450. In other examples, the encoded modified cytochrome P450 polypeptide has at least 50%, 60%, 70%, 80%, 90%, or 95% of contiguous amino acids of a heterologous domain from one or more different cytochrome P450s.
Provided herein are isolated Santalum album cytochrome P450 reductases or catalytically active fragments thereof. For example, provided herein are isolated Santalum album cytochrome P450 reductases that catalyze the transfer of two electrons from NADPH to an electron acceptor, that is a cytochrome P450, heme oxygenase, cytochrome b5 or squalene epoxidase. In particular examples, the electron acceptor is a cytochrome P450.
Also provided herein are isolated nucleic acid molecules encoding a Santalum album cytochrome P450 reductase polypeptide or catalytically active fragments thereof. For example, provided herein are isolated nucleic acid molecules encoding a cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12 or 13; or encoding a cytochrome P450 reductase polypeptide having a sequence of amino acids that has at least 80% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13. In another example, provided herein is an isolated nucleic acid molecule encoding a cytochrome P450 reductase polypeptide that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO:12 or 13.
Also provided herein are isolated nucleic acid molecule having a sequence of nucleic acids set forth in SEQ ID NO:10 or 11; a sequence of nucleic acids having at least 95% sequence identity to a sequence of nucleic acids set forth in SEQ ID NO:10 or 11; and degenerates thereof. For example, provided herein is are isolated nucleic acid molecules having a sequence of nucleic acids set forth in SEQ ID NO:10 or 11. In some examples, the isolated nucleic acid molecules of encode cytochrome P405 reductase polypeptides having a sequence of amino acids that has at least 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13. In a particular example, the isolated nucleic acid molecule encodes a cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12 or 13. The provided nucleic acid molecules encode a cytochrome P450 reductase polypeptides catalyze the transfer of two electrons from NADPH to an electron acceptor, such as a cytochrome P450, heme oxygenase, cytochrome b5 or squalene epoxidase. In a particular example, the electron acceptor is a cytochrome P450. Also provided herein are cytochrome P450 reductase polypeptides encoded by the nucleic acid molecules.
Also provided herein are nucleic acid molecule encoding a modified Santalum album cytochrome P450 reductase polypeptide or catalytically active fragments thereof. For example, provided here are nucleic acid molecules encoding modified cytochrome P450 reductase polypeptides that contain at least one amino acid replacement, addition or deletion compared to the cytochrome P450 reductase polypeptide not containing the modification. In some examples, the encoded modified cytochrome P450 reductase polypeptide is N- or C-terminally truncated. For example, provide herein are nucleic acid molecules encoding a modified cytochrome P450 reductase polypeptide that is N-terminally truncated. For example, the nucleic acid molecule encodes a modified cytochrome P450 reductase polypeptide that has a sequence of amino acids set forth in SEQ ID NO:14 or 15; or has 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 SEQ ID NO:14 or 15. Also provided herein are nucleic acid molecules having a sequence of nucleic acids set forth in SEQ ID NO:63 or 64; a sequence of nucleic acids having at least 95% sequence identity to a sequence of nucleic acids set forth in SEQ ID NO:63 or 64; and degenerates thereof. The provided nucleic acid molecules encode a cytochrome P450 reductase polypeptides catalyze the transfer of two electrons from NADPH to an electron acceptor, such as a cytochrome P450, heme oxygenase, cytochrome b5 or squalene epoxidase. In a particular example, the electron acceptor is a cytochrome P450. Also provided herein are cytochrome P450 reductase polypeptides encoded by the nucleic acid molecules.
Provided herein are nucleic acid molecules encoding a fusion protein containing a Santalum album santalene synthase or a catalytically active fragment thereof and/or a cytochrome P450 santalene oxidase or bergamotene oxidase or a catalytically active fragment thereof and/or a cytochrome P450 reductase or a catalytically active fragment thereof.
Provided herein are nucleic acid molecules encoding a fusion protein containing santalene synthase and a cytochrome P450 santalene oxidase or a catalytically active fragment thereof. The full-length santalene synthase is encoded by a sequence of nucleotides set forth in any of SEQ ID NOS:58-60 and the cytochrome P450 santalene oxidase is encoded b any nucleic acid molecule provided herein that encodes a cytochrome P450 santalene oxidase. In another example, provided herein are nucleic acid molecules encoding a santalene synthase and a cytochrome P450 santalene oxidase. The santalene synthase has a sequence of amino acids set forth in any of SEQ ID NOS: 17, 52 and 53, and the cytochrome P450 santalene oxidase has a sequence of amino acids set forth in any of SEQ ID NOS:7, 73, 74, 75 and 76.
Provided herein are nucleic acid molecules encoding a fusion protein containing santalene synthase and a cytochrome P450 bergamotene oxidase or a catalytically active fragment thereof. The santalene synthase has a sequence of nucleotides set forth in any of SEQ ID NOS:58-60 and the cytochrome P450 bergamotene oxidase is any nucleic acid molecule provided herein that encodes a cytochrome P450 bergamotene oxidase. In another example, provided herein are nucleic acid molecules encoding a santalene synthase and a cytochrome P450 bergamotene oxidase. The santalene synthase has a sequence of amino acids set forth in any of SEQ ID NOS: 17, 52 and 53 and the cytochrome P450 bergamotene oxidase has a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 and 73.
Provided herein are nucleic acid molecules encoding a fusion protein containing a cytochrome P450 or a catalytically active fragment thereof and a cytochrome P450 reductase or a catalytically active fragment thereof, where the cytochrome P450 is any nucleic acid molecule provided herein that encodes a cytochrome P450 oxidase and the cytochrome P450 reductase is any nucleic acid molecule provided herein that encodes a cytochrome P450 reductase. For example, provided herein are nucleic acid molecules encoding a cytochrome P450 that has a sequence of amino acids set forth in any of SEQ ID NOS:6-9 and 73-78 and a cytochrome P450 reductase that has a sequence of amino acids set forth in any of SEQ ID NOS:12-15.
In some examples, in the nucleic acid molecules provided herein encoding a fusion protein, the santalene synthase and/or cytochrome P450 santalene oxidase or bergamotene oxidase and/or cytochrome P450 reductase are linked directly. In other examples, in the nucleic acid molecules provided herein encoding a fusion protein, the santalene synthase and/or cytochrome P450 santalene oxidase or bergamotene oxidase and/or cytochrome P450 reductase are linked via a linker.
Also provided herein are vectors containing any nucleic acid molecule provided herein, including nucleic acid molecules encoding cytochrome P450s, such as santalene oxidases and bergamotene oxidases, cytochrome P450 reductases, modified cytochrome P450 reductases and fusion proteins. In some examples, the vector is a prokaryotic vector, a viral vector, or an eukaryotic vector. For example, the vector is a yeast vector. Also provided herein are cells containing any vector provided herein. Also provided herein are cells containing any nucleic acid molecule provided herein, including nucleic acid molecules encoding cytochrome P450s, such as santalene oxidases and bergamotene oxidases, cytochrome P450 reductases, modified cytochrome P450 reductases and fusion proteins. In some examples, the cell is a prokaryotic cell or an eukaryotic cell. In other examples, the cells is selected from among a bacteria, yeast, insect, plant or mammalian cell. In an example, the cell is a yeast cell. Included among yeast cells is a Saccharomyces genus cell and a Pichia genus cell. For example, the cell is a Saccharomyces cerevisiae cell. In another example, the cell is an Escherichia coli cell. Thus, provided are of recombinant cells, including yeast cells, for production of santalols and bergamotol.
The cells can include nucleic acid encoding a synthase, such as santalene synthase, such as a Santalum album synthase, to catalyze production of a substrate for the P450 enzymes provided herein.
Also provided herein are cells that express a cytochrome P450 santalene oxidase polypeptide, a cytochrome P450 bergamotene oxidase polypeptide, a cytochrome P450 reductase polypeptide and/or a fusion protein containing a Santalum album santalene synthase and/or a cytochrome P450 santalene oxidase or bergamotene synthase and/or a cytochrome P450 reductase. Also provided herein are transgenic plants containing any vector provided herein. In some examples, the transgenic plant is a tobacco plant.
Provided herein are methods for producing a cytochrome P450 polypeptide, by: introducing a nucleic acid molecule provided herein that encodes a cytochrome P450 polypeptide or any vector provided herein that encodes a cytochrome P450 polypeptide into a cell; culturing the cell under conditions suitable for expression of the cytochrome P450 polypeptide encoded by the nucleic acid or vector; and, optionally isolating the cytochrome P450 polypeptide.
Provided herein are methods for producing a cytochrome P450 reductase polypeptide, by: introducing a nucleic acid molecule provided herein that encodes a cytochrome P450 reductase polypeptide or any vector provided herein that encodes a cytochrome P450 reductase polypeptide into a cell; culturing the cell under conditions suitable for expression of the cytochrome P450 reductase polypeptide encoded by the nucleic acid or vector; and, optionally isolating the cytochrome P450 reductase polypeptide.
Provided herein are methods for production of a santalol, bergamotol and/or mixtures thereof, by: (a) contacting a santalene and/or bergamotene with a cytochrome P450 santalene oxidase or bergamotene oxidase under conditions suitable for the formation of a santalol, bergamotol and/or mixtures thereof; and (b) optionally isolating the santalol, bergamotol and/or mixtures thereof. In some examples, step (a) is effected in vitro or in vivo. For example, step (a) is effected in vivo in a cell transformed with a nucleic acid molecule or vector encoding a cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide, whereby the cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide encoded by the nucleic acid molecule or vector is expressed; and the cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide catalyzes the formation of santalol and/or bergamotol from santalene and/or bergamotene.
Provided herein is a host cell containing a nucleic acid molecule encoding a cytochrome P450 or cytochrome P450 polypeptide provided herein. The nucleic acid molecule and cytochrome P450 polypeptide is heterologous to the cell. In some examples, the host cell further contains nucleic acid encoding a synthase that produces a terpene substrate of a cytochrome P450. In some examples, the synthase is heterologous to the host cell. In particular examples, the terpene synthase is a santalene synthase, such as a terpene synthase that catalyzes the formation of santalene and/or bergamotene. For example, the terpene synthase has a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 and 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 NOS:17, 52 and 53. In some examples, the host cell is a prokaryotic cell or an eukaryotic cell that is selected from among a bacteria, yeast, insect, plant or mammalian cell. In a particular example, the host cell is a yeast cell that is a Saccharomyces genus cell or a Pichia genus cell. For example, the host cell is a Saccharomyces cerevisiae cell. In other examples, the host cell is an Escherichia coli cell. In some examples, the host cell produces an acyclic pyrophosphate terpene precursor, such as farnesyl diphosphate. In particular examples, the host cell produces farnesyl diphosphate natively or is modified to produce more farnesyl diphosphate compared to an unmodified cell. Also provided herein is a method for production of a santalol, bergamotol and/or mixtures thereof, said method including the steps of culturing any of the host cell provided herein under conditions suitable for the formation of a santalol, bergamotol and/or mixtures thereof; and optionally isolating the santalol, bergamotol and/or mixtures thereof.
Provided herein are methods for production of a santalol, bergamotol and/or mixtures thereof, by: (a) contacting an acyclic pyrophosphate terpene precursor with a santalene synthase under conditions suitable for the formation of a santalene and/or bergamotene; (b) contacting the resulting santalene and/or bergamotene with a cytochrome P450 santalene oxidase or bergamotene oxidase under conditions suitable for the formation of a santalol, bergamotol and/or mixture thereof to produce a santalol, bergamotol or mixture thereof; and (c) optionally isolating the santalene and bergamotene produced in step (a) or the santalol, bergamotol, and/or mixtures thereof produced in step (b). In some examples, step (a) and/or step (b) is/are performed in vitro or in vivo. For example, step (a) is performed in vivo in a cell transformed with a nucleic acid molecule encoding a santalene synthase, whereby the santalene synthase encoded by the nucleic acid molecule is expressed; and the santalene synthase catalyzes the formation of santalene and bergamotene from the acyclic pyrophosphate terpene precursor; and/or step (b) is effected in vivo in a cell transformed with a nucleic acid molecule or vector encoding a cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide, whereby the cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide encoded by the nucleic acid molecule or vector is expressed; and the cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide catalyzes the formation of santalol and/or bergamotol from santalene and/or bergamotene. In such examples, the acyclic pyrophosphate terpene precursor can be a farnesyl pyrophosphate. In
In any of the methods provided herein, the call can be a prokaryotic cell or an eukaryotic cell that is selected from among a bacteria, yeast, insect, plant or mammalian cell. In some examples, the cell is a yeast cell that is a Saccharomyces genus cell or a Pichia genus cell, such as a Saccharomyces cerevisiae cell. In some examples, the cell is modified to produce more FPP compared to an unmodified cell. In some examples of the methods, the cell is modified to produce a santalene synthase. For example, the cell is modified to produce a santalene synthase that has a sequence of amino acids set forth in SEQ ID NO:17, 52 or 53 or a synthase having at least 80%, 85%, 90%, 95% sequence identity therewith.
In some examples of the methods provided herein the santalene or bergamotene is an α-santalene, β-santalene, epi-β-santalene or α-trans-bergamotene. In some examples, the santalol or bergamotol is an α-santalol, β-santalol, epi-β-santalol or α-trans-bergamotol. In some examples, the santalol or bergamotol is an (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol or (E)-α-trans-bergamotol. In further examples of the provided methods, the santalene, bergamotene, santalol, bergamotol or mixtures thereof are isolated by extraction with an organic solvent and/or column chromatography.
In some examples of the provided methods, santalene and/or bergamotene is contacted with a cytochrome P450 santalene oxidase that is: a cytochrome P450 santalene oxidase polypeptide provided herein; a cytochrome P450 santalene oxidase polypeptide provided herein encoded by any nucleic acid molecule provided herein; a nucleic acid molecule provided herein that encodes a cytochrome P450 santalene oxidase; or a vector provided herein that encodes a cytochrome P450 santalene oxidase, whereby santalol and/or bergamotol are produced.
In some examples of the provided methods, bergamotene is contacted with a cytochrome P450 bergamotene oxidase that is: a cytochrome P450 bergamotene oxidase polypeptide provided herein; a cytochrome P450 bergamotene oxidase polypeptide provided herein encoded by any nucleic acid molecule provided herein; a nucleic acid molecule provided herein that encodes a cytochrome P450 bergamotene oxidase; or a vector provided herein that encodes a cytochrome P450 bergamotene oxidase, whereby bergamotol is produced.
Also provided herein are methods for production of a santalol, bergamotol and/or mixtures thereof. Each of steps (a) and (b) can be effected simultaneously or sequentially. In one example, steps (a) and (b) are effected simultaneously with a nucleic acid molecule encoding a fusion polypeptide containing a santalene synthase and a cytochrome P450 santalene oxidase or bergamotene oxidase; or a fusion polypeptide containing a santalene synthase and a cytochrome P450 santalene oxidase or bergamotene oxidase. In particular examples, santalene and/or bergamotene is contacted with a nucleic acid molecule provided herein that encodes a fusion polypeptide; or a fusion polypeptide encoded by a nucleic acid molecule provided herein.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the chemical structures of (Z)-α-santalol (1), (E)-α-santalol (9), (Z)-β-santalol (2), (E)-β-santalol (10), (E)-epi-βp-santalol (3), (Z)-epi-β-santalol (11), (Z)-α-trans-bergamotol (4), (E)-α-trans-bergamotol (12), α-santalene (5), β-santalene (6), epi-β-santalene (7) and α-trans-bergamotene (8).
FIGS. 2A-2B depict the alignment of the santalene oxidase set forth in SEQ ID NO:7 with the bergamotene oxidases set forth in SEQ ID NOS:6, 8 and 9. A “*” means that the aligned residues are identical, a “:” means that aligned residues are not identical, but are similar and contain conservative amino acids residues at the aligned position, and a “.” means that the aligned residues are similar and contain semi-conservative amino acid residues at the aligned position.
FIGS. 3A-3C depict the alignment of Santalum album cytochrome P450 reductases set forth in SEQ ID NOS:12 and 13 with Arabidopsis thaliana cytochrome P450 reductases set forth in SEQ ID NOS:46 and 58. A “*” means that the aligned residues are identical, a “:” means that aligned residues are not identical, but are similar and contain conservative amino acids residues at the aligned position, and a “.” means that the aligned residues are similar and contain semi-conservative amino acid residues at the aligned position.
FIG. 4 depicts the neighbor joining phylogeny of the predicted protein sequences of SaCYP76F38v1 (SaCYP76-G5), SaCYP76F39v1 (SaCYP76-G10), SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12) and cytochrome P450 enzymes for terpenoid metabolism, as described in Example 4.
FIGS. 5A-5B depict the alignment of the santalene oxidase set forth in SEQ ID NO:7 and the bergamotene oxidase set forth in SEQ ID NO:6 with cytochrome P450BM-3 set forth in SEQ ID NO:66. A “*” means that the aligned residues are identical, a “:” means that aligned residues are not identical, but are similar and contain conservative amino acids residues at the aligned position, and a “.” means that the aligned residues are similar and contain semi-conservative amino acid residues at the aligned position.
FIGS. 6A-6D depict the GC-MS chromatogram of products extracted from in vivo assays with SaCYP76F38v1 (SaCYP76-G5) (FIG. 6A), SaCYP76F37v1 (SaCYP76-G11) (FIG. 6B), SaCYP76F38v2 (SaCYP76-G12) (FIG. 6C) and empty vector (FIG. 6D) as described in Example 10. The peaks are identified in Table 13.
FIG. 7 depicts the total ion chromatogram of S. album oil extract. The peaks are identified in Table 13.
FIGS. 8A-8C depict the GC-MS chromatogram of S. album native oil (FIG. 8A) and of products extracted from in vivo assays with SaCYP76F39v1 (SaCYP76-G10) (FIG. 8B) and empty vector (FIG. 8C) as described in Example 10. The peaks are identified in Table 11.
FIGS. 9A-9B depict the GC-MS chromatogram of S. album native oil (FIG. 9A) and of products extracted from in vitro assays with SaCYP76F39v1 (SaCYP76-G10) (FIG. 9B) as described in Example 11. The peaks are identified in Table 11.
FIG. 10 depicts the neighbor-joining phylogeny of the protein sequences of the S. album CYP76Fs and related terpene-modifying cytochrome P450, as described in Example 4. The highlighted CYP76Fs indicated those in clade I (marked with I) and clade II (marked with II).
FIGS. 11A-11B depict the GC-MS analysis (extracted ion chromatograms) of products formed in vivo in yeast cells expressing SaSSY, SaCPR2 and SaCYP76F39v1 (SaCYP76-G10) (FIG. 11A) and empty vector (FIG. 11B). The peaks are identified in Table 12. Peaks marked with the symbol (*) correspond to farnesol which is also produced in yeast cells without SaCYP76F. Peaks in FIG. 11A marked with the symbol (#) represent yeast in vivo modifications of santalols (see FIGS. 12A and 12B).
FIGS. 12A-12B depict the GC-MS analysis (extracted ion chromatograms) of sesquiterpenols of natural sandalwood oil sample before (FIG. 12A) and after (FIG. 12B) overnight incubation with yeast cells, which do not contain a SaCYP76F gene. Peaks in FIG. 12B marked with the symbol (#) represent yeast in vivo modifications of santalols independent of SaCYP76F. The peaks are identified in Table 12.
FIGS. 13A-13D depict the GC-MS analysis (extracted ion chromatograms) of compounds formed in vivo in yeast cells expressing SaSSy, SaCPR2 and SaCYP76F39v2 (SaCYP76-G15) (FIG. 13A), SaCYP76F40 (SaCYP76-G16) (FIG. 13B), SaCYP76F41 (SaCYP76-G17) (FIG. 13C), or SaCYP76F42 (SaCYP76-G13) (FIG. 13D). The peaks are identified in Table 12. Peaks marked with the symbol (*) correspond to farnesol which is produced in yeast cells without SaCYP76F. Peaks marked with the symbol (#) represent yeast in vivo modifications of santalols independent of SaCYP76F.
FIGS. 14A-14D depict the GC-MS analysis (extracted ion chromatograms) of compounds formed in vivo in yeast cells expressing SaSSy, SaCPR2 and SaCYP76F38v1 (SaCYP76-G5) (FIG. 14A), SaCYP76F38v2 (SaCYP76-G12) (FIG. 14B), SaCYP76F37v1 (SaCYP76-G11) (FIG. 14C), SaCYP76F37v2 (SaCYP76-G14) (FIG. 14D), or SaCYP76F43 (SaCYP76-G18) (FIG. 14E). The peaks are identified in Table 12. Peaks marked with the symbol (*) correspond to farnesol which is produced in yeast cells without SaCYP76F. Peaks marked with the symbol (#) represent yeast in vivo modifications of santalols independent of SaCYP76F.
FIGS. 15A-15C depicts the GC-MS analysis (extracted ion chromatograms) of products formed in vitro with SaCYP76F39v1 (SaCYP76-G10) and a sesquiterpene mixture of α-, β- and epi-β-santalene and α-trans-bergamotene (FIG. 15A). FIG. 15B depicts the GC-MS analysis (extracted ion chromatograms) of authentic S. album oil. FIG. 15C depicts the GC-MS analysis (extracted ion chromatograms) from control assays performed with microsomes isolated from yeast cells transformed with an empty vector. The peaks are identified in Table 12.
FIGS. 16A-16E depict the GC-MS analysis (extracted ion chromatograms) of products formed in vitro with a sesquiterpene mixture of α-, β- and epi-β-santalene and α-trans-bergamotene as the substrate and clade I SaCYP76F cDNAs SaCYP76F39v2 (SaCYP76-G15) (FIG. 16A); SaCYP76F40 (SaCYP76-G16) (FIG. 16B); SaCYP76F41 (SaCYP76-G17) (FIG. 16C); SaCYP76F42 (SaCYP76-G13) (FIG. 16D); or empty vector as control (FIG. 16E). The peaks are identified in Table 12.
FIGS. 17A-17E depict the GC-MS analysis (extracted ion chromatograms) of products formed in vitro with a sesquiterpene mixture of α-, β- and epi-β-santalene and α-trans-bergamotene as the substrate and clade II SaCYP76F cDNAs SaCYP76F38v1 (SaCYP76-G5) (FIG. 17A); SaCYP76F38v2 (SaCYP76-G12) (FIG. 17B); SaCYP76F37v1 (SaCYP76-G11) (FIG. 17C); SaCYP76F37v2 (SaCYP76-G14) (FIG. 17D); or empty vector as control (FIG. 17E). The peaks are identified in Table 12.
FIG. 18 depicts the reduced CO-difference spectra of isolated microsomes containing S. album CYP76F proteins. CO-difference spectra of microsomal fractions from S. cerevisiae harboring a cytochrome P450 or an empty vector are shown. Concentration of SaCYP76F proteins are given based on an extinction coefficient of 91,000 M−1 cm−1.
FIGS. 19A-19D depict the GC-MS analysis (extracted ion chromatograms) of a sesquiterpene mixture produced with a recombinant yeast strain expressing SaSSy (FIG. 19A) and fractions separated by TLC (FIGS. 19B-19D). The sesquiterpene mixture and fractions were prepared as described in Example 9. The peaks correspond to: α-santalene, peak 1; α-exo-bergamotene, peak 2; epi-β-santalene, peak 3; and β-santalene, peak 4.
FIGS. 20A-20G depict the GC-MS analysis (extracted ion chromatograms) of products formed in vitro with SaCYP76F39v1 (SaCYP76-G10) or SaCYP76F37v1 (SaCYP76-G11) using partially purified substrates. FIGS. 20A-20C depict product profiles in assays with SaCYP76F39v1 (SaCYP76-G10) using α-santalene (FIG. 20A), α-exo-bergamotene (FIG. 20B), or epi-β-santalene and β-santalene (FIG. 20C) as the substrates. FIGS. 20D-20F depict product profiles in assays with SaCYP76F37v1 (SaCYP76-G11) using α-santalene (FIG. 20D), α-exo-bergamotene (FIG. 20E), or epi-β-santalene and β-santalene (FIG. 20F) as the substrates. FIG. 20G depicts the extracted ion chromatogram for authentic Santalum album oil. The peaks are identified in Table 12.
FIGS. 21A-21C depict the alignment of the S. album cytochrome P450s set forth in SEQ ID NOS:6-9 and 73-78. Horizontal arrows indicate the proline region (a), oxygen binding motif (b) and heme binding motif (c). Boxes indicate the substrate recognition sites (SRS) regions originally described by Gotoh (1992) J Biol Chem 267:83-90. A “*” means that the aligned residues are identical, a “:” means that aligned residues are not identical, but are similar and contain conservative amino acids residues at the aligned position, and a “.” means that the aligned residues are similar and contain semi-conservative amino acid residues at the aligned position.



DETAILED DESCRIPTION










Outline
















A.
Definitions


B.
Overview










1.
Biosynthesis of Terpenoids










a.
Santalols



b.
Bergamotols










2.
Cytochrome P450 Enzymes










a.
Structure



b.
Activity










3.
Cytochrome P450 Reductases










a.
Structure



b.
Activity








C.
Cytochrome P450 polypeptides and encoding nucleic acid molecules










1.
Cytochrome P450 santalene oxidase polypeptides




Modified cytochrome P450 santalene oxidase polypeptides



2.
Cytochrome P450 bergamotene oxidase polypeptides




Modified cytochrome P450 bergamotene oxidase polypeptides



3.
Additional modifications










a.
Truncated polypeptides



b.
Polypeptides with altered activities or properties



c.
Domain swaps



d.
Fusion proteins








D.
Cytochrome P450 reductase polypeptides and encoding nucleic acid molecules










1.
Cytochrome P450 reductase polypeptides



2.
Modified cytochrome P450 reductase polypeptides



3.
Additional modifications










a.
Truncated polypeptides



b.
Polypeptides with altered activities or properties



c.
Domain swaps



d.
Fusion proteins








E.
Methods for producing modified cytochrome P450 and cytochrome P450 reductase



polypeptides and encoding nucleic acid molecules


F.
Expression of cytochrome P450 and cytochrome P450 reductase polypeptides and



encoding nucleic acid molecules










1.
Isolation of nucleic acid encoding Santalum album cytochrome P450 and




cytochrome P450 reductase polypeptides



2.
Generation of modified nucleic acids



3.
Vectors and Cells



4.
Expression systems










a.
Prokaryotic cells



b.
Yeast cells



c.
Plants and plant cells



d.
Insects and insect cells



e.
Mammalian cells



f.
Exemplary host cells










5.
Purification



6.
Fusion proteins








G.
Methods for producing terpenoids and methods for detecting such products and the



activity of the cytochrome P450 and cytochrome P450 reductase polypeptides










1.
Synthesis of Santalols and Bergamotols










a.
Oxidation of Santalenes and Bergamotenes



b.
Conversion of acyclic pyrophosphate terpene precursors










2.
Methods for production










a.
Exemplary cells



b.
Culture of cells



c.
Isolation and assays for detection and identification










3.
Production of sandalwood oil



4.
Assays for detecting enzymatic activity of cytochrome P450 and cytochrome




P450 reductase polypeptides










a.
Methods for determining the activity of cytochrome P450 polypeptides



b.
Methods for determining the activity of cytochrome P450 reductase




polypeptides








H.
Examples









A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, an acyclic pyrophosphate terpene precursor is any acyclic pyrophosphate compound that is a precursor to the production of at least one terpene, including, but not limited, farnesyl-pyrophosphate (FPP), geranyl-pyrophosphate (GPP) and geranylgeranyl-pyrophosphate (GGPP). Acyclic pyrophosphate terpene precursors are thus substrates for terpene synthases.
As used herein, a terpene is an unsaturated hydrocarbon based on the isoprene unit (C5H8), and having a general formula C5xH8x, such as C10H16. Reference to a terpene includes acyclic, monocyclic and polycyclic terpenes. Terpenes include, but are not limited to, monoterpenes, which contain 10 carbon atoms; sesquiterpenes, which contain 15 carbon atoms; diterpenes, which contain 20 carbon atoms, and triterpenes, which contain 30 carbon atoms. Reference to a terpene also includes stereoisomers of the terpene.
As used herein, a terpenoid is a chemically modified terpene. In one example, a terpenoid is a terpene that has been chemically modified by addition of a hydroxyl group, such as a santalol or bergamotol. Reference to a terpenoid includes acyclic, monocyclic and polycyclic terpenoids, including monoterpenoids, sesquiterpenoids and diterpenoids. Reference to a terpenoid also includes stereoisomers of the terpenoid.
As used herein, a terpene synthase is a polypeptide capable of catalyzing the formation of one or more terpenes from a pyrophosphate terpene precursor. In some examples, a terpene synthase catalyzes the formation of one or more terpenes from an acyclic pyrophosphate terpene precursor, for example, FPP, GPP or GGPP, including, but not limited to, santalene synthase. In other examples, a terpene synthase catalyzes the formation of one or more terpenes from an acyclic pyrophosphate terpene precursor, including, but not limited to, santalene synthase.
As used herein, “cytochrome P450,” “cytochrome P450 oxidase,” “cytochrome P450 polypeptide,” “cytochrome P450 oxidase polypeptide” or “CYP” is a polypeptide capable of catalyzing the monooxygenation of any terpene precursor, including monoterpenes, sesquiterpenes and diterpenes. A cytochrome P450 can catalyze the monooxygenation of a terpene or a mixture of terpenes, resulting in the production one or more terpenoids.
For purposes herein, cytochrome P450 oxidases provided herein are enzymes with cytochrome P450 oxidase activity and have greater than or greater than about or 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, when aligned with the cytochrome P450 oxidase sequence set forth in SEQ ID NO:50. Reference to a cytochrome P450 oxidase includes any cytochrome P450 oxidase polypeptide including, but not limited to, a recombinantly produced polypeptide, synthetically produced polypeptide and a cytochrome P450 oxidase polypeptide extracted or isolated from cells or plant matter, including, but not limited to, heartwood of a sandalwood tree. Exemplary of cytochrome P450 oxidase polypeptides include those isolated from Santalum album. Reference to a cytochrome P450 oxidase includes cytochrome P450 oxidase from any genus or species, and included allelic or species variants, variants encoded by splice variants, and other variants thereof, including polypeptides that have at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the cytochrome P450 oxidase set forth in SEQ ID NO:50 when aligned therewith. Cytochrome P450 oxidase also includes catalytically active fragments thereof that retain cytochrome P450 oxidase activity.
As used herein, “cytochrome P450 santalene oxidase” or “cytochrome P450 santalene oxidase polypeptide” is a polypeptide capable of catalyzing the formation of a santalol from a santalene, for example, capable of catalyzing the monooxygenation or hydroxylation of a santalene. A cytochrome P450 santalene oxidase polypeptide can produce one or a mixture of santalols from one or a mixture of santalenes. A cytochrome P450 santalene oxidase polypeptide is also capable of catalyzing the formation of a bergamotol from a bergamotene. For example, a cytochrome P450 santalene oxidase catalyzes the formation of α-santalol from α-santalene, β-santalol from β-santalene, epi-β-santalol from epi-β-santalene and/or Z-α-trans-bergamotol or E-α-trans-bergamotol from α-trans-bergamotene.
For purposes herein, cytochrome P450 santalene oxidases provided herein are enzymes with cytochrome P450 santalene oxidase activity and have greater than or greater than about or 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, when aligned with the cytochrome P450 santalene oxidase sequence set forth in SEQ ID NO:7, 74, 75, 76 or 77. Reference to a cytochrome P450 santalene oxidase includes any cytochrome P450 santalene oxidase polypeptide including, but not limited to, a recombinantly produced polypeptide, synthetically produced polypeptide and a cytochrome P450 santalene oxidase polypeptide extracted or isolated from cells or plant matter, including, but not limited to, heartwood of a sandalwood tree. Exemplary of cytochrome P450 santalene oxidase polypeptides include those isolated from Santalum album. Reference to a cytochrome P450 santalene oxidase includes cytochrome P450 santalene oxidase from any genus or species, and included allelic or species variants, variants encoded by splice variants, and other variants thereof, including polypeptides that have at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the cytochrome P450 santalene oxidase set forth in SEQ ID NO:7, 74, 75, 76 or 77 when aligned therewith. Cytochrome P450 santalene oxidase also includes catalytically active fragments thereof that retain cytochrome P450 santalene oxidase activity.
As used herein, “cytochrome P450 santalene oxidase activity” or “santalene oxidase activity” refers to the ability to catalyze the formation of one or more santalols from one or more santalenes. That is, cytochrome P450 santalene oxidases catalyze the monooxygenation or hydroxylation of santalenes. Cytochrome P450 santalene oxidases also catalyze the hydroxylation of bergamotene. For example, cytochrome P450 santalene oxidases catalyze the formation of α-santalol from α-santalene, β-santalol from β-santalene, epi-β-santalol from epi-β-santalene and/or Z-α-trans-bergamotol from α-trans-bergamotene. Methods to assess santalol or bergamotol formation from a reaction of a santalene or bergamotene are well known in the art and described herein. The production of a santalol or bergamotol can be assessed by methods such as, for example, gas chromatography-mass spectrometry (GC-MS) (see Examples below). A cytochrome P450 exhibits cytochrome P450 santalene oxidase activity or the ability to catalyze the formation of santalols or bergamotol from santalenes and bergamotene if the amount of santalols and bergamotol produced from the reaction is at least or at least about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total amount of terpenoids produced in the reaction.
As used herein, “cytochrome P450 bergamotene oxidase” or “cytochrome P450 bergamotene oxidase polypeptide” is a polypeptide capable of catalyzing the monooxygenation or hydroxylation of a bergamotene. For example, a cytochrome P450 bergamotene oxidase catalyzes the formation of Z-α-trans-bergamotol or E-α-trans-bergamotol from α-trans-bergamotene.
For purposes herein, cytochrome P450 bergamotene oxidases provided herein are enzymes with cytochrome P450 bergamotene oxidase activity and have greater than or greater than about or 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, when aligned with the cytochrome P450 bergamotene oxidase sequence set forth in SEQ ID NO:6, 8, 9 or 73. Reference to a cytochrome P450 bergamotene oxidase includes any cytochrome P450 bergamotene oxidase polypeptide including, but not limited to, a recombinantly produced polypeptide, synthetically produced polypeptide and a cytochrome P450 bergamotene oxidase polypeptide extracted or isolated from cells or plant matter, including, but not limited to, heartwood of a sandalwood tree. Exemplary of cytochrome P450 bergamotene oxidase polypeptides include those isolated from Santalum album. Reference to a cytochrome P450 bergamotene oxidase includes cytochrome P450 bergamotene oxidase from any genus or species, and included allelic or species variants, variants encoded by splice variants, and other variants thereof, including polypeptides that have at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the cytochrome P450 bergamotene oxidase set forth in SEQ ID NO: 6, 8, 9 or 73 when aligned therewith. Cytochrome P450 bergamotene oxidase also includes catalytically active fragments thereof that retain cytochrome P450 bergamotene oxidase activity.
As used herein, “cytochrome P450 bergamotene oxidase activity” or “bergamotene oxidase activity” refers to the ability catalyze the formation of bergamotols from bergamotenes That is, cytochrome P450 bergamotene oxidases catalyze the monooxygenation or hydroxylation of bergamotene. For example, cytochrome P450 bergamotene oxidases catalyze the formation of Z-α-trans-bergamotol from α-trans-bergamotene. Methods to assess bergamotol formation from a reaction of a bergamotene are well known in the art and described herein. The production of a bergamotol can be assessed by methods such as, for example, gas chromatography-mass spectrometry (GC-MS) (see Examples below). A cytochrome P450 exhibits cytochrome P450 bergamotene oxidase activity or the ability to catalyze the formation of bergamotol from bergamotene if the amount of bergamotol produced from the reaction is at least or at least about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total amount of terpenoids produced in the reaction.
As used herein, α-santalol is a sesquiterpenoid having the following structure or isomers or stereoisomers thereof:




As used herein, β-santalol is a sesquiterpenoid having the following structure or isomers or stereoisomers thereof:




As used herein, epi-β-santalol is a sesquiterpenoid having the following structure or isomers or stereoisomers thereof:




As used herein, Z-α-trans-bergamotol or Z-α-exo-bergamotol is a sesquiterpenoid having the following structure or isomers or stereoisomers thereof:




As used herein, E-α-trans-bergamotol or E-α-exo-bergamotol is a sesquiterpenoid having the following structure or isomers or stereoisomers thereof:




As used herein, α-santalene is a sesquiterpene having the following structure or isomers or stereoisomers thereof:




As used herein, β-santalene is a sesquiterpene having the following structure or isomers or stereoisomers thereof:




As used herein, epi-β-santalene is a sesquiterpene having the following structure or isomers or stereoisomers thereof:




As used herein, α-trans-bergamotene or α-exo-bergamotene is a sesquiterpene having the following structure or isomers or stereoisomers thereof:




As used herein, “cytochrome P450 reductase” or “CPR” is a polypeptide capable of catalyzing the transfer of two electrons from NADPH to an electron acceptor, such as a cytochrome P450. For purposes herein, cytochrome P450 reductases provided herein are enzymes with cytochrome P450 reductase activity and have greater than or greater than about or 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, when aligned with the cytochrome P450 reductase sequence set forth in SEQ ID NO:12 or 13. Reference to a cytochrome P450 reductase includes any cytochrome P450 reductase polypeptide including, but not limited to, a recombinantly produced polypeptide, synthetically produced polypeptide and a cytochrome P450 reductase polypeptide extracted or isolated from cells or plant matter, including, but not limited to, heartwood of a sandalwood tree. Exemplary of cytochrome P450 reductase polypeptides include those isolated from Santalum album. Reference to a cytochrome P450 reductase includes a cytochrome P450 reductase from any genus or species, and included allelic or species variants, variants encoded by splice variants, and other variants thereof, including polypeptides that have at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the cytochrome P450 reductase set forth in SEQ ID NO:12 or 13 when aligned therewith. Cytochrome P450 reductase also includes catalytically active fragments thereof that retain cytochrome P450 reductase activity.
As used herein, “cytochrome P450 reductase activity” refers to the ability to catalyze the transfer of two electrons from NADPH to an electron acceptor, such as a cytochrome P450. Methods to assess cytochrome P450 reductase activity are well known in the art and described herein. For example, cytochrome P450 reductase activity can be determined by reduction of an artificial electron receptor, such as cytochrome c.
As used herein, “wild type” or “native” with reference to a cytochrome P450 or cytochrome P450 reductase refers to a cytochrome P450 polypeptide or cytochrome P450 reductase polypeptide encoded by a native or naturally occurring cytochrome P450 gene or cytochrome P450 reductase gene, including allelic variants, that are present in an organism, including a plant, in nature. Reference to wild type cytochrome P450 or cytochrome P450 reductase without reference to a species is intended to encompass any species of a wild type cytochrome P450 or cytochrome P450 reductase.
As used herein, species variants refer to variants in polypeptides among different species, including different sandalwood species, such Santalum album, Santalum australocaledonicum, Santalum spicatum and Santalum murrayanum. Generally, species variants share at least or at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity. Corresponding residues between and among species variants can be determined by comparing, generally one-by-one to the same reference sequence, and aligning each sequence with the reference sequence to maximize the number of matching nucleotides or amino acid residues. The position of interest is then given the number assigned in the reference nucleic acid molecule or polypeptide. Alignment can be effected manually or by eye, particularly, where sequence identity is greater than 80%. To determine sequence identity among a plurality of variants, alignments are effected one-by-one against the same reference polypeptide.
As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term “allelic variant” also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wild type form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically, have at least 80%, 90% or greater amino acid identity with a wild type and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wild type and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wild type and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations n proteins among members of the same species.
As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
As used herein, a “modified cytochrome P450” or “modified cytochrome P450 polypeptide” or “modified CYP” refers to a cytochrome P450 polypeptide that has one or more amino acid differences compared to an unmodified or wild type cytochrome P450 polypeptide. The one or more amino acid differences can be amino acid mutations such as one or more amino acid replacements (substitutions), insertions or deletions, or can be insertions or deletions or replacements of entire domains or portions thereof, and any combination thereof. Modification can be effected by any mutational protocol, including gene shuffling methods. Typically, a modified cytochrome P450 polypeptide has one or more modifications in primary sequence compared to an unmodified cytochrome P450 polypeptide. For example, a modified cytochrome P450 polypeptide provided herein can have at least 1, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or more amino acid differences compared to an unmodified cytochrome P450 polypeptide. Typically, the modified cytochrome P450 polypeptide will have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid replacements, but can include more, particularly when domains or portions thereof are swapped. Any modification is contemplated as long as the resulting polypeptide has at least one cytochrome P450 activity associated with the wild type cytochrome P450, such as, for example, catalytic activity, monooxygenase activity, and/or the ability to catalyze the formation of a terpenoid from a terpene. Generally, the resulting cytochrome P450 polypeptide will have at least 50% sequence identity with the wild type cytochrome P450 polypeptide provided herein.
As used herein, a “modified cytochrome P450 santalene oxidase” or “modified cytochrome P450 santalene oxidase polypeptide” refers to a cytochrome P450 santalene oxidase polypeptide that has one or more amino acid differences compared to an unmodified or wild type cytochrome P450 santalene oxidase polypeptide. The one or more amino acid differences can be amino acid mutations such as one or more amino acid replacements (substitutions), insertions or deletions, or can be insertions or deletions or replacements of entire domains or portions thereof, and any combination thereof. Modification can be effected by any mutational protocol, including gene shuffling methods. Typically, a modified cytochrome P450 santalene oxidase polypeptide has one or more modifications in primary sequence compared to an unmodified cytochrome P450 santalene oxidase polypeptide. For example, a modified cytochrome P450 santalene oxidase polypeptide provided herein can have at least 1, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or more amino acid differences compared to an unmodified cytochrome P450 santalene oxidase polypeptide. Typically, the modified cytochrome P450 santalene oxidase polypeptide will have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid replacements, but can include more, particularly when domains or portions thereof are swapped. Any modification is contemplated as long as the resulting polypeptide has at least one cytochrome P450 santalene oxidase activity associated with the wild type cytochrome P450 santalene oxidase, such as, for example, catalytic activity, the ability to catalyze the formation of santalols or bergamotols from santalenes or bergamotenes. Generally, the resulting cytochrome P450 polypeptide santalene oxidase will have at least 50% sequence identity with the wild type cytochrome P450 santalene oxidase polypeptide provided herein.
As used herein, a “modified cytochrome P450 bergamotene oxidase” or “modified cytochrome P450 bergamotene oxidase polypeptide” refers to a cytochrome P450 bergamotene oxidase polypeptide that has one or more amino acid differences compared to an unmodified or wild type cytochrome P450 bergamotene oxidase polypeptide. The one or more amino acid differences can be amino acid mutations such as one or more amino acid replacements (substitutions), insertions or deletions, or can be insertions or deletions or replacements of entire domains or portions thereof, and any combination thereof. Modification can be effected by any mutational protocol, including gene shuffling methods. Typically, a modified cytochrome P450 bergamotene oxidase polypeptide has one or more modifications in primary sequence compared to an unmodified cytochrome P450 bergamotene oxidase polypeptide. For example, a modified cytochrome P450 bergamotene oxidase polypeptide provided herein can have at least 1, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or more amino acid differences compared to an unmodified cytochrome P450 polypeptide. Typically, the modified cytochrome P450 bergamotene oxidase polypeptide will have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid replacements, but can include more, particularly when domains or portions thereof are swapped. Any modification is contemplated as long as the resulting polypeptide has at least one cytochrome P450 bergamotene oxidase activity associated with the wild type cytochrome P450 bergamotene oxidase polypeptide, such as, for example, catalytic activity, the ability to catalyze the formation of bergamotols from bergamotenes. Generally, the resulting cytochrome P450 polypeptide bergamotene oxidase will have at least 50% sequence identity with the wild type cytochrome P450 bergamotene oxidase polypeptide provided herein.
As used herein, a “modified cytochrome P450 reductase” or “modified CPR” refers to a cytochrome P450 polypeptide that has one or more amino acid differences compared to an unmodified or wild type cytochrome P450 reductase polypeptide. The one or more amino acid differences can be amino acid mutations such as one or more amino acid replacements (substitutions), insertions or deletions, or can be insertions or deletions or replacements of entire domains or portions thereof, and any combination thereof. Modification can be effected by any mutational protocol, including gene shuffling methods. Typically, a modified cytochrome P450 reductase polypeptide has one or more modifications in primary sequence compared to an unmodified cytochrome P450 reductase polypeptide. For example, a modified cytochrome P450 reductase polypeptide provided herein can have at least 1, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or more amino acid differences compared to an unmodified cytochrome P450 reductase polypeptide. Typically, the modified cytochrome P450 reductase polypeptide will have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid replacements, but can include more, particularly when domains or portions thereof are swapped. Any modification is contemplated as long as the resulting polypeptide has at least one cytochrome P450 reductase activity associated with the wild type cytochrome P450 reductase, such as, for example, catalytic activity, the ability to transfer two electrons to an electron receptor, such as a cytochrome P450. Generally, the resulting cytochrome P450 reductase polypeptide will have at least 50% sequence identity with the wild type cytochrome P450 reductase polypeptide provided herein.
As used herein, corresponding residues refers to residues that occur at aligned loci. Related or variant polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods such as manual alignments and those produced by the numerous alignment programs available (for example, BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides, one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. For example, corresponding residues between a cytochrome P450 santalene oxidase synthase and cytochrome P450 bergamotene oxidase synthase are shown in FIGS. 2A-2B and 21A-21C and corresponding residues between Arabidopsis thaliana cytochrome P450 reductases and Santalum album cytochrome P450 reductases are shown in FIG. 3A-3C.
As used herein, domain or region (typically a sequence of at least three or more, generally 5 or 7 or more amino acids) refers to a portion of a molecule, such as a protein or the encoding nucleic acids, that is structurally and/or functionally distinct from other portions of the molecule and is identifiable. A protein can have one, or more than one, distinct domains. For example, a domain can be identified, defined or distinguished by homology of the sequence therein to related family members, such as other terpene synthases. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain a plurality of domains. Such domains are known, and can be identified by, those of skill in the art. For exemplification herein, definitions are provided, but it is understood that it is well within the skill in the art to recognize particular domains by name. If needed, appropriate software can be employed to identify domains. For example, as discussed above, corresponding domains in different cytochrome P450s or cytochrome P450 reductases can be identified by sequence alignments, such as using tools and algorithms well known in the art (for example, BLASTP).
As used herein, a functional domain refers to those portions of a polypeptide that is recognized by virtue of a functional activity, such as catalytic activity. A functional domain can be distinguished by its function, such as by catalytic activity, or an ability to interact with a biomolecule, such as substrate binding or metal binding. In some examples, a domain independently can exhibit a biological function or property such that the domain independently or fused to another molecule can perform an activity, such as, for example catalytic activity or substrate binding.
As used herein, a structural domain refers to those portions of a polypeptide chain that can form an independently folded structure within a protein made up of one or more structural motifs.
As used herein, “heterologous” with respect to an amino acid or nucleic acid sequence refers to portions of a sequence that is not present in a native polypeptide or encoded by a polynucleotide. For example, a portion of amino acids of a polypeptide, such as a domain or region or portion thereof, for a cytochrome P450 santalene oxidase synthase is heterologous thereto if such amino acids is not present in a native or wild type cytochrome P450 santalene oxidase synthase (e.g. as set forth in SEQ ID NO:7), or encoded by the polynucleotide encoding therefor. Polypeptides containing such heterologous amino acids or polynucleotides encoding therefor are referred to as “chimeric polypeptides” or “chimeric polynucleotides,” respectively. As used herein, the phrase “a property of the modified cytochrome P450 is improved compared to the first cytochrome P450” refers to a desirable change in a property of a modified cytochrome P450 compared to a cytochrome P450 that does not contain the modification(s). Typically, the property or properties are improved such that the amount of a desired terpenoid produced from the reaction of a terpene substrate with the modified cytochrome P450 synthase is increased compared to the amount of the desired terpenoid produced from the reaction of a substrate with a cytochrome P450 synthase that is not so modified. Exemplary properties that can be improved in a modified cytochrome P450 synthase include, for example, terpenoid production, catalytic activity, product distribution, substrate specificity, regioselectivity and stereoselectivity. One or more of the properties can be assessed using methods well known in the art to determine whether the property had been improved (i.e. has been altered to be more desirable for the production of a desired terpenoid or terpenoids).
As used herein, terpenoid production (also referred to as terpenoid yield) refers to the amount (in weight or weight/volume) of terpenoid produced from the reaction of a terpene with a cytochrome P450. Reference to total terpenoid production refers to the total amount of all terpenoids produced from the reaction, while reference to particular terpenoid production refers to the amount of a particular terpenoid (e.g. β-santalol and α-santalol), produced from the reaction.
As used herein, an improved terpenoid production refers to an increase in the total amount of terpenoid (i e improved total terpenoid production) or an increase in the particular amount of terpenoid resulting from the reaction of a terpene with a modified cytochrome P450 compared to the amount produced from the reaction of the same terpene with a cytochrome P450 that is not so modified. The amount of terpenoid (total or particular) produced from the reaction of a terpene with a cytochrome P450 can be increased by at least or at least about 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the amount of terpenoid produced from the reaction of the same terpene under the same conditions with a cytochrome P450 that is not so modified.
As used herein, substrate specificity refers to the preference of a cytochrome P450 for one target substrate over another, such as one terpene (e.g. β-santalene, α-santalene, epi-β-santalene or α-trans-bergamotene) over another. Substrate specificity can be assessed using methods well known in the art, such as those that calculate kcat/Km. For example, the substrate specificity can be assessed by comparing the relative Kcat/Km, which is a measure of catalytic efficiency, of the enzyme against various substrates (e.g. β-santalene, α-santalene, epi-β-santalene or α-trans-bergamotene).
As used herein, altered substrate specificity refers to a change in substrate specificity of a modified cytochrome P450 polypeptide (such as a modified cytochrome P450 santalene oxidase polypeptide or cytochrome P450 bergamotene oxidase polypeptide) compared to a cytochrome P450 that is not so modified (such as, for example, a wild type cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase). The specificity (e.g. kcat/Km) of a modified cytochrome P450 polypeptide for a substrate, such as β-santalene, α-santalene, epi-β-santalene or α-trans-bergamotene, can be altered by at least or at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the specificity of a starting cytochrome P450 for the same substrate.
As used herein, “improved substrate specificity” refers to a change or alteration in the substrate specificity to a more desired specificity. For example, an improved substrate specificity can include an increase in substrate specificity of a modified cytochrome P450 polypeptide for a desired substrate, such as β-santalene, α-santalene, epi-β-santalene or α-trans-bergamotene. The specificity (e.g. kcat/Km) of a modified cytochrome P450 polypeptide for a substrate, such as β-santalene, α-santalene, epi-β-santalene or α-trans-bergamotene, can be increased by at least or at least about 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the specificity of a cytochrome P450 that is not so modified. As used herein, “product distribution” refers to the relative amounts of different terpenoids produced from the reaction between a terpene, such as β-santalene, and a cytochrome P450, including the cytochrome P450 polypeptides provided herein. The amount of a produced terpenoid can be depicted as a percentage of the total products produced by the cytochrome P450. For example, the product distribution resulting from reaction of β-santalene with a cytochrome P450 santalene oxidase can be 90% (weight/volume) β-santalol and 10% (weight/volume) other compounds. Methods for assessing the type and amount of a terpenoid in a solution are well known in the art and described herein, and include, for example, gas chromatography-mass spectrometry (GC-MS) (see Examples below).
As used herein, an altered product distribution refers to a change in the relative amount of individual terpenoids produced from the reaction between a terpene, such as β-santalene, and a cytochrome P450, such as cytochrome P450 santalene oxidase. Typically, the change is assessed by determining the relative amount of individual terpenoids produced from the terpene using a first cytochrome P450 (e.g. wild type cytochrome P450) and then comparing it to the relative amount of individual terpenoids produced using a second cytochrome P450 (e.g. a modified cytochrome P450). An altered product distribution is considered to occur if the relative amount of any one or more terpenoids is increased or decreased by at least or by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more.
As used herein, an improved product distribution refers to a change in the product distribution to one that is more desirable, i.e. contains more desirable relative amounts of terpenoids. For example, an improved product distribution can contain an increased amount of a desired terpenoid and/or a decreased amount of a terpenoid that is not so desired. The amount of desired terpenoid in an improved production distribution can be increased by at least or by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more. The amount of a terpenoid that is not desired in an improved production distribution can be decreased by at least or by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more.
As used herein, nucleic acids or nucleic acid molecules include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.
As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”). The term nucleotides is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.
As used herein, heterologous nucleic acid is nucleic acid that is not normally produced in vivo by the cell in which it is expressed or that is produced by the cell but is at a different locus or expressed differently or that mediates or encodes mediators that alter expression of endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Heterologous nucleic acid can be endogenous, but is nucleic acid that is expressed from a different locus or altered in its expression. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell or in the same way in the cell in which it is expressed. Heterologous nucleic acid, such as DNA, also can be referred to as foreign nucleic acid, such as DNA. Thus, heterologous nucleic acid or foreign nucleic acid includes a nucleic acid molecule not present in the exact orientation or position as the counterpart nucleic acid molecule, such as DNA, is found in a genome. It also can refer to a nucleic acid molecule from another organism or species (i.e., exogenous).
Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which the nucleic acid is expressed is herein encompassed by heterologous nucleic acid; heterologous nucleic acid includes exogenously added nucleic acid that also is expressed endogenously. Examples of heterologous nucleic acid include, but are not limited to, nucleic acid that encodes traceable marker proteins, such as a protein that confers drug resistance, nucleic acid that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and nucleic acid, such as DNA, that encodes other types of proteins, such as antibodies. Antibodies that are encoded by heterologous nucleic acid can be secreted or expressed on the surface of the cell in which the heterologous nucleic acid has been introduced.
As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length.
As used herein, the amino acids that occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.
As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids in which the α-carbon has a side chain).
In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. §§1.821-1.822, abbreviations for the amino acid residues are shown in Table 1:







TABLE 1







Table of Correspondence











SYMBOL













1-Letter
3-Letter
AMINO ACID






Y
Tyr
Tyrosine



G
Gly
Glycine



F
Phe
Phenylalanine



M
Met
Methionine



A
Ala
Alanine



S
Ser
Serine



I
Ile
Isoleucine



L
Leu
Leucine



T
Thr
Threonine



V
Val
Valine



P
Pro
Proline



K
Lys
Lysine



H
His
Histidine



Q
Gln
Glutamine



E
Glu
Glutamic acid



Z
Glx
Glu and/or Gln



W
Trp
Tryptophan



R
Arg
Arginine



D
Asp
Aspartic acid



N
Asn
Asparagine



B
Asx
Asn and/or Asp



C
Cys
Cysteine



X
Xaa
Unknown or other









All amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.
As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.
As used herein, “non-natural amino acid” refers to an organic compound containing an amino group and a carboxylic acid group that is not one of the naturally-occurring amino acids listed in Table 1. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art and can be included in a modified cytochrome P450 polypeptide or cytochrome P450 reductase polypeptide provided herein.
As used herein, modification is in reference to modification of the primary sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements and rearrangements of amino acids and nucleotides. For purposes herein, amino acid replacements (or substitutions), deletions and/or insertions, can be made in any of the cytochrome P450s or cytochrome P450 reductases provided herein. Modifications can be made by making conservative amino acid replacements and also non-conservative amino acid substitutions as well as by insertions, domain swaps and other such changes in primary sequence. For example, amino acid replacements that desirably or advantageously alter properties of the cytochrome P450 or cytochrome P450 reductase can be made. For example, amino acid replacements can be made to the cytochrome P450 santalene oxidase such that the resulting modified cytochrome P450 santalene oxidase can produce more β-santalol from a mixture of santalenes and bergamotenes compared to an unmodified cytochrome P450 santalene oxidase. For example, amino acid replacements can be made to the cytochrome P450 bergamotene oxidase such that the resulting cytochrome P450 bergamotene oxidase can produce more bergamotol from a mixture of santalenes and bergamotenes compared to an unmodified cytochrome P450 bergamotene oxidase. Modifications also can include post-translational modifications or other changes to the molecule that can occur due to conjugation or linkage, directly or indirectly, to another moiety, but when such modifications are contemplated they are referred to as post-translational modifications or conjugates or other such term as appropriate. Methods of modifying a polypeptide are routine to those of skill in the art, and can be performed by standard methods, such as site directed mutations, amplification methods, and gene shuffling methods.
As used herein, amino acid replacements or substitutions contemplated include, but are not limited to, conservative substitutions, including, but not limited to, those set forth in Table 2. Suitable conservative substitutions of amino acids are known to those of skill in the art and can be made generally without altering the conformation or activity of the polypeptide. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Conservative amino acid substitutions are made, for example, in accordance with those set forth in Table 2 as follows:









TABLE 2






Original residue
Conservative substitution








Ala (A)
Gly; Ser



Arg (R)
Lys



Asn (N)
Gln; His



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



Gly (G)
Ala; Pro



His (H)
Asn; Gln



Ile (I)
Leu; Val



Leu (L)
Ile; Val



Lys (K)
Arg; Gln; Glu



Met (M)
Leu; Tyr; Ile



Phe (F)
Met; Leu; Tyr



Ser (S)
Thr



Thr (T)
Ser



Trp (W)
Tyr



Tyr (Y)
Trp; Phe



Val (V)
Ile; Leu; Met









Other conservative substitutions also are contemplated and can be determined empirically or in accord with known conservative substitutions.

As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5′ to 3′ direction, encodes the sequence of amino acids of the specified polypeptide.
As used herein, “primary sequence” refers to the sequence of amino acid residues in a polypeptide.
As used herein, “similarity” between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. “Identity” refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).
As used herein, “at a position corresponding to” or recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. For purposes herein, alignment of a cytochrome P450 santalene oxidase sequence is to the amino acid sequence set forth in SEQ ID NO:7. For purposes herein, alignment of a cytochrome P450 bergamotene oxidase sequence is to the amino acid sequence set forth in any of SEQ ID NOS:6, 8 or 9, and in particular SEQ ID NO:6. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). FIGS. 2A-2B, 3A-3C, 5A-5B and 21A-21C exemplify exemplary alignments and identification of exemplary corresponding residues for replacement.
As used herein, “sequence identity” refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. The alignment can be local or global. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical residues/length of the shortest sequence×100. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g. terminal gaps are not penalized). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions/length of the total aligned sequence×100.
As used herein, a “global alignment” is an alignment that aligns two sequences from beginning to end, aligning each letter in each sequence only once. An alignment is produced, regardless of whether or not there is similarity or identity between the sequences. For example, 50% sequence identity based on “global alignment” means that in an alignment of the full sequence of two compared sequences each of 100 nucleotides in length, 50% of the residues are the same. It is understood that global alignment also can be used in determining sequence identity even when the length of the aligned sequences is not the same. The differences in the terminal ends of the sequences will be taken into account in determining sequence identity, unless the “no penalty for end gaps” is selected. Generally, a global alignment is used on sequences that share significant similarity over most of their length. Exemplary algorithms for performing global alignment include the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplary programs for performing global alignment are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/), and the program available at deepc2.psi.iastate.edu/aat/align/align.html.
As used herein, a “local alignment” is an alignment that aligns two sequence, but only aligns those portions of the sequences that share similarity or identity. Hence, a local alignment determines if sub-segments of one sequence are present in another sequence. If there is no similarity, no alignment will be returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, 50% sequence identity based on “local alignment” means that in an alignment of the full sequence of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length has 50% of the residues that are the same in the region of similarity or identity.
For purposes herein, sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier or manually. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical,” or other similar variations reciting a percent identity, can be determined using known computer algorithms based on local or global alignment (see e.g., wikipedia.org/wiki/Sequence_alignment_software, providing links to dozens of known and publicly available alignment databases and programs). Generally, for purposes herein sequence identity is determined using computer algorithms based on global alignment, such as the Needleman-Wunsch Global Sequence Alignment tool available from NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)); and program from Xiaoqui Huang available at deepc2.psi.iastate.edu/aat/align/align.html. Generally, when comparing nucleotide sequences herein, an alignment with penalty for end gaps is used. Local alignment also can be used when the sequences being compared are substantially the same length.
As used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10/100 amino acid difference (approximately 90% identity). Differences also can be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Depending on the length of the compared sequences, at the level of homologies or identities above about 85-90%, the result reasonably independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.
As used herein, the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art, but that those of skill can assess such.
As used herein, an aligned sequence refers to the use of homology (similarity and/or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by about or 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.
As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound can, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell of tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as proteolytic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
As used herein, substantially free of cellular material includes preparations of cytochrome P450s, cytochrome P450 reductases, terpenes or terpenoid products in which the cytochrome P450, cytochrome P450 reductase, terpene or terpenoid product is separated from cellular components of the cells from which it is isolated or produced. In one embodiment, the term substantially free of cellular material includes preparations of cytochrome P450s, cytochrome P450 reductases, terpenes or terpenoid products having less that about or less than 30%, 20%, 10%, 5% or less (by dry weight) of non-cytochrome P450s, cytochrome P450 reductases, terpenes or terpenoid products, including cell culture medium. When the cytochrome P450 or cytochrome P450 reductase is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the cytochrome P450 or cytochrome P450 reductase preparation.
As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of cytochrome P450 or cytochrome P450 reductase proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of cytochrome P450 or cytochrome P450 reductase proteins having less than about or less than 30% (by dry weight), 20%, 10%, 5% or less of chemical precursors or non-synthase chemicals or components.
As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.
As used herein, production by recombinant methods by using recombinant DNA methods refers to the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
As used herein, vector (or plasmid) refers to discrete DNA elements that are used to introduce heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as bacterial artificial chromosomes, yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
As used herein, expression refers to the process by which nucleic acid is transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression can, if an appropriate eukaryotic host cell or organism is selected, include processing, such as splicing of the mRNA.
As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells. Viral vectors include, but are not limited to, adenoviral vectors, retroviral vectors and vaccinia virus vectors. As used herein, operably or operatively linked when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates downstream of the promoter and upstream of any transcribed sequences. The promoter is usually the domain to which the transcriptional machinery binds to initiate transcription and proceeds through the coding segment to the terminator.
As used herein, a “chimeric protein” or “fusion protein” refers to a polypeptide operatively-linked to a different polypeptide. For example, a polypeptide encoded by a nucleic acid sequence containing a coding sequence from one nucleic acid molecule and the coding sequence from another nucleic acid molecule in which the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, the protein is produced containing the two proteins. The two molecules can be adjacent in the construct or separated by a linker polypeptide that contains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide. A chimeric or fusion protein provided herein can include one or more santalene synthase polypeptides, or a portion thereof, and/or one or more cytochrome P450 polypeptides, or a portion thereof, and/or one or more cytochrome P450 reductase polypeptides and/or one or more other polypeptides, for any one or more of a transcriptional/translational control signals, signal sequences, a tag for localization, a tag for purification, a protein for identification, part of a domain of an immunoglobulin G, and/or a targeting agent. A chimeric cytochrome P450 polypeptide or cytochrome P450 reductase polypeptide also includes those having their endogenous domains or regions of the polypeptide exchanged with another polypeptide. These chimeric or fusion proteins include those produced by recombinant means as fusion proteins, those produced by chemical means, such as by chemical coupling, through, for example, coupling to sulfhydryl groups, and those produced by any other method whereby at least one polypeptide (i.e. cytochrome P450 or cytochrome P450 reductase), or a portion thereof, is linked, directly or indirectly via linker(s) to another polypeptide.
As used herein, the term assessing or determining includes quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a product, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect.
As used herein, recitation that a polypeptide “consists essentially” of a recited sequence of amino acids means that only the recited portion, or a fragment thereof, of the full-length polypeptide is present. The polypeptide can optionally, and generally will, include additional amino acids from another source or can be inserted into another polypeptide
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to polypeptide, comprising “an amino acid replacement” includes polypeptides with one or a plurality of amino acid replacements.
As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5%” means “about 5%” and also “5%.”
As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, an optional step of isolating a terpene means that the terpene is isolated or is not isolated, or, an optional stop of isolating a terpenoid means that the terpenoid is isolated or is not isolated.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.
B. Overview
Provided herein are cytochrome P450 enzymes from Santalum album, and variants and modified forms thereof, for production of santalols and other sesquiterpenoids. Such cytochrome P450s catalyze the biosynthetic production of santalols or bergamotols from santalenes and bergamotenes, both of which can be generated biosynthetically from farnesyl pyrophosphate by the enzyme santalene synthase (see, WO 2011/000026 and Jones et al. (2011) J Biol Chem 286:17445-17454. Also provided herein are cytochrome P450 reductases from Santalum album, and variants and modified forms thereof. Also provided herein are methods of making santalols and other sesquiterpenoids from farnesyl diphosphate and/or santalenes and bergamotene. The provided cytochrome P450 enzymes provide for production of these valuable products, including santalols and bergamotols, in commercially useful quantities and in a cost effective and energy efficient manner.
1. Biosynthesis of Terpenoids
Terpenes are a large and diverse class of organic compounds that are produced by a variety of plants from acyclic pyrophosphate isoprene precursors such as geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). Terpenes are named based on the number of isoprene (C5H8) units they contain. For example, monoterpenes are derived from GPP and contain 10 carbons, sesquiterpenes are derived from FPP and contain 15 carbons and diterpenes are derived from GGPP and contain 20 carbons. Terpenes that have been chemically modified are referred to as terpenoids or isoprenoids. Terpenes and terpenoids are the primary constituents of essential oils of plants and are widely used as flavor additives for food, fragrances in perfumery and in traditional and alternative medicine.
Santalols and bergamotol are sesquiterpenoids that occur in plants, including the heartwood of Santalum species, including Santalum album (Indian Sandalwood, White Sandalwood, Chandana), Santalum austrocaledonicum (Australian Sandalwood) and Santalum spicatum. Bergamotol can additionally be found in plants such as orchids. Santalols and bergamotol are the oxidation products of santalenes and bergamotene, respectively. In S. album, about 90% of the essential oil is composed of the sesquiterpene alcohols (Z)-α-, (Z)-β-, and (Z)-epi-β-santalol and (Z)-α-exo-bergamotol. The α- and β-santalols are the most important contributors to sandalwood oil fragrance. (Z)-α-Santalol and (Z)-β-santalol are the major components of authentic S. album oil.
The P450 enzymes provided herein can be employed to produce the sesquiterpene alcohols important for the sandalwood oil fragrance. Santalenes and bergamotene are synthesized biosynthetically from the acyclic pyrophosphate precursor FPP by the terpene synthase santalene synthase (see WO 2011/000026 and Jones et al. (2011) J Biol Chem 286:17445-17454). Santalene synthase is known to produce a mixture of santalenes (i.e. α-, β-, epi-β-santalene and α-exo-bergamotene). Exemplary of santalene synthases are Santalum album santalene synthase (SaSSY) set forth in SEQ ID NO:16 and encoding the amino acid sequence set forth in SEQ ID NO:17; Santalum austrocaledonicium santalene synthase (SauSSY, Genbank Accession Nos. HQ343277 or AD087001) set forth in SEQ ID NO:59 and encoding the sequence of amino acids set forth in SEQ ID NO:52; or Santalum spicatum santalene synthase (SspiSSy, Genbank Accession No. HQ343278 or AD087002) set forth in SEQ ID NO: 60 and encoding the sequence of amino acids set forth in SEQ ID NO:53.
The cytochrome P450 oxidase polypeptides provided herein are found to catalyze the formation of one or more of an α-santalol from α-santalene, β-santalol from β-santalene, epi-β-santalol from epi-β-santalene and/or α-trans-bergamotol from α-trans-bergamotene. Hydroxylation or monooxygenation of terpene substrates by the cytochrome P450 oxidase is generally performed in the presence of a cytochrome reductase. For example, Santalum album cytochrome reductases (SaCPR) provided herein are included in biosynthesis to supply electrons from NADPH to the cytochrome P450. Thus, the pathways for biosynthesis of santalols and bergamotols, including components of sandalwood oil, can be metabolically engineered in host cells by transforming nucleic acid encoding a cytochrome P450 oxidase and cytochrome P450 reductase provided herein in combination with a nucleic acid molecule encoding a santalene synthase.
a. Santalols
In particular, santalols responsible for the fragrance of sandalwood oil include α-santalols (1 and 9), β-santalols (2 and 10) and epi-β-santalols (3 and 11) (see FIG. 1). (Z)-α-Santalol (Z-α-santalol; (Z)-5-(1R,2S,6S)-2,3-dimethyltricyclol[2.2.1.02,6]heptan-3-yl)-2-methylpent-2-en-1-ol; 1) and (E)-α-Santalol ((E)-5-((1R,2s,6S)-2,3-dimethyltricyclo[2.2.1.02,6]heptan-3-yl)-2-methylpent-2-en-1-ol; 9) are synthesized biosynthetically by oxidation of the sesquiterpene α-santalene 5 (see FIG. 1). (Z)-β-Santalol (Z-ββ-santalol; (Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylene-bicyclo[2.2.1]heptan-2-yl]pent-2-en-1-ol; 2) and (E)-β-Santalol ((E)-2-methyl-5-((1S,2R,4R)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol; 10) are synthesized biosynthetically by oxidation of the sesquiterpene β-santalene 6 (see FIG. 1). (E)-epi-β-Santalol ((E)-2-methyl-5-[(1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol; 3) and (Z)-epi-β-Santalol ((Z)-2-methyl-5-((1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol; 11) are synthesized biosynthetically by oxidation of the sesquiterpene epi-β-santalene 7 (see FIG. 1).




b. Bergamotol
(Z)-α-trans-Bergamotol ((Z)-α-exo-bergamotol; cis-α-trans-bergamotol; (2Z)-5-[(1S,5S,6R)-2,6-dimethylbicyclo[3.3.1]hept-2-en-6-yl]-2-methyl-2-penten-1-ol; 4) and (E)-α-trans-Bergamotol ((E)-α-exo-bergamotol; (E)-5-((1S,5S,6R)-2,6-dimethylbicyclo[3.1.1]hept-2-en-6-yl)-2-methylpent-2-en-1-ol; 12) are sesquiterpenoids found in sandalwood oil that are synthesized biosynthetically by oxidation of the sesquiterpene α-trans-bergamotene 8 (see FIG. 1).




2. Cytochrome P450 Enzymes
Cytochromes P450 (CYPs) are a superfamily of hemoproteins, or heme-thiolate proteins, that catalyze the singular insertions of oxygen into a diverse range of hydrophobic substrates, often with high regio- and stereoselectivity. Cytochrome P450s are ubiquitous proteins that participate in metabolizing a wide range of compounds. As such, P450s are widespread in nature and are involved in processes such as detoxifying xenobiotics, catabolism of unusual carbon sources and biosynthesis of secondary metabolites. CYPs are noted for their broad substrate specificities and use of oxygen without the need for phosphorylation of adenosine diphosphate (ADP). They can mediate monooxygenations, hydroxylations at nitrogen and sulfur heteroatoms, epoxidations, dehalogenations, deaminations and dealkylations. Particular reactions catalyzed by CYPs include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups.
Typically, cytochrome P450s are monooxygenases, incorporating one oxygen atom into a substrate. In general, monooxygenations require one or two additional proteins to transfer electrons from NAD(P)H to the heme iron and CYPs are placed in groups or classes based on their electron transfer partner. Class I CYPs, common in bacterial and eukaryotic mitochondrial P450 systems, use a FAD-containing reductase and an iron-sulfer redoxin or ferrodoxin. The FAD-containing reductase transfers electrons from NAD(P)H to the ferrodoxin which in turn reduces the CYP. Class II cytochrome P450s are the most common CYPs in eukaryotes and plants, and also include microsomal and bacterial P450 systems. Class II CYPs use a NADPH:Cytochrome P450 reductase (or cytochrome P450 reductase) to transfer electrons from NAD(P)H to a cytochrome P450. Numerous other classes exist that exploit other electron transfer chains.
Cytochrome P450s are named using a systematic nomenclature that includes the root symbol CYP followed by number designating the family, a letter designating the subfamily and a number representing the individual gene, for example, CYP76-G5. Families share greater than 40% amino acid sequence identity and subfamilies share greater than 55% amino acid sequence identity.
Plant cytochrome P450 gene families are very large. For example, total genome sequence examination reveals at least 272 predicted cytochrome P450 genes in Arabidopsis and at least 455 unique cytochrome P450 genes in rice (see, e.g., Nelson et al. (2004) Plant Physiol. 135(2):756-772). Plant CYPs can be localized to the endoplasmic reticulum (ER) and to chloroplasts. In plants, CYPs include a wide range of hydroxylases, epoxidases, peroxidases and oxygenases that largely are based upon Class II monooxygenations. Plant p450s participate in biochemical pathways that include, for example, the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates (see, e.g., Chapple (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:311-343).
a. Structure
While sequence conservation among cytochrome P450s is relatively low, their general topography and structural fold are highly conserved. There are only 3 absolutely conserved residues among all CYPs, namely the glutamic acid and arginine of the ExxR motif (SEQ ID NO:54) and the heme-binding cysteine. Conserved structural nodules are important for structure and function, and variable regions involved in substrate recognition dictate individual properties (see, e.g., Werck-Reichhart and Feyereisen (2000) Genome Biology 1(6)3000.1-3000.9, Sirim et al. (2010) BMC Structural Biology 10:34 and Baudry et al. (2006) Prot Eng Design & Selection 19:343-353).
Cytochrome P450s typically contain a helices, designated A through L, and β-pleated sheets, designated 1 through 5, contained within a β domain that is associated with substrate recognition and composed predominately of β sheets and an α domain that contains the catalytic center and is predominantly α helices. The structural regions are as follows, from N-terminus to C-terminus: helix A, β strand 1-1, β strand 1-2, helix B, β strand 1-5, helix B′, helix C, helix C′, helix D, β strand 3-1, helix E, helix F, helix G, helix H, β strand 5-1, β strand 5-2, helix I, helix J, helix J′, helix K, β strand 1-4, β strand 2-1, β strand 2-2, β strand 1-3, helix K′, helix K′′, Heme domain, helix L, β strand 3-3, β strand 4-1, β strand 4-2 and β strand 3-2 (see, e.g., Werck-Reichhart and Feyereisen (2000) Genome Biology 1(6)3000.1-3000.9).
Cytochrome P450s are anchored to the endoplasmic reticulum (ER) or chloroplast in plants via a transmembrane helix near the N-terminus of the protein (Chapple (1998) Annu Rev Plant Physiol Plant Mol Biol 49:311-343). The transmembrane helix is typically followed by hinge region containing a series of basic amino acid residues and a proline-rich region containing the consensus sequence (P/I)PGPx(G/P)xP (SEQ ID NO:55). This hinge region allows for optimal orientation of the enzyme in relation to the membrane. Deletion of the proline hinge region resulted in complete loss of activity (Szczesna-Skorupa et al. (1993) Arch Biochem Biophys 304:170-175) and mutation of proline residues to alanine disrupted structure so as to eliminate heme incorporation (Yamazaki et al. (1993) J Biochem 114:652-657).
The conserved CYP core region is composed of a coil termed the ‘meander’, a four-helix bundle (helices D, E, I and L), helices J and K and two sets of β-sheets (Werck-Reichhart and Feyereisen (2000) Genome Biology 1(6)3000.1-3000.9). The core region contains the heme-binding loop containing the P450 consensus sequence GRRxCP(A/G) (SEQ ID NO:56) located on the proximal face of the heme just before helix L, with an absolutely conserved cysteine that serves as the 5th ligand for the heme iron. The active site for catalysis is the iron-protoporphryin IX (heme) with the thiolate of the conserved cysteine residue as the fifth ligand; the final coordination site is left to bind and activate molecular oxygen (Groves et al., 1995 In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ed: Ortiz de Montellano) Plenum Press, New York, N.Y., pp. 3-48). The core region also contains the central part of helix I containing the threonine-containing binding pocket for the oxygen molecule required in catalysis having a consensus sequence (A/G)Gx(D/E)T(T/S) (SEQ ID NO:57) which also corresponds to the proton-transfer groove. Finally, the core region contains the absolutely conserved ExxR motif (SEQ ID NO:54) in helix K on the proximal side of heme (see, e.g., Werck-Reichhart and Feyereisen (2000) Genome Biology 1(6)3000.1-3000.9). The proximal face of the enzyme is involved in redox partner recognition and electron transfer to active site. Protons flow into active site channel from distal face. The substrate access channel is located in close contact with the membrane between the F-G loop, A helix and β strands 1-1 and 1-2.
Cytochrome P450 substrate recognitions sites (SRS) are diverse and include SRS1, the loop region between B and C helices; SRS2, the C-terminal end of the F helix; SRS3, part of the FG loop and N-terminal end of the G helix; SRS4, helix I containing SRS4 extending over the pyrrole ring B in the active site; SRS5, the loop between the K helix and strand 4 of β-sheet 1; and SRS6, the β turn in β-sheet 4.
b. Function
Cytochrome P450s catalyze regiospecific and stereospecific oxidation of non-activated hydrocarbons at physiological temperatures. Cytochrome P450s activate molecular oxygen using an iron-heme center and use a redox electron shuttle to support the oxidation reaction. The general reaction for hydroxylation by the cytochrome P450 system is,

RH+NADPH+H++O2→ROH+NADP++H2O,

where R represents a substrate compound. As noted, typically, cytochrome P450s are monooxygenases, catalyzing the insertion of one of the atoms of molecular oxygen into a substrate, with the second oxygen being reduced to water. Catalysis involves 1) substrate binding; 2) one-electron reduction of the complex to a ferrous state; 3) binding of molecular oxygen to give the superoxide complex; and 4) a second reduction leading to a short lived activated oxygen species. The activated oxygen attacks the substrate resulting, typically, in monooxygenation of the substrate. Other reactions catalyzed by CYPs include dealkylation, dehydration, dehydrogenation, isomerization, dimerization, carbon-carbon bond cleavage and reduction.

3. Cytochrome P450 Reductase
Cytochrome P450 reductases (NADPH:cytochrome P450 reductase; NADPH-cytochrome P450 oxidoreductase; NADPH:ferrihemoprotein oxidoreductase; NADPH:P450 oxidoreductase; CPR; CYPOR; EC 1.6.2.4) are multidomain enzymes of the diflavin reductase family required for electron transfer from NAD(P)H to cytochrome P450s, heme oxygenases, cytochrome b5 and squalene epoxidases (Louerat-Orieu et al (1998) Eur J Biochem 258:1040-1049). Plants are known to contain multiple isoforms of cytochrome P450 reductases (see, Ro et al. (2002) Plant Physiology 130:1837-1851; Mizutani and Ohta (1998) Plant Physiology 116:357-367). Generally, at least one CPR is constitutively expressed and the other CPRs are enhanced by environmental stresses such as UV light and pathogen infection. In addition, plant cytochrome P450 reductases can be localized to the ER or to the chloroplast, with the location determined by the corresponding partner cytochrome P450 enzyme.
a. Structure
Cytochrome P450 reductases share amino acid sequence homology (about 30% up to about 90%) among different species, including as bacteria, yeast, fungi, plants, fish, insects and mammals (Louerat-Orieu et al (1998) Eur J Biochem 258:1040-1049). Cytochrome P450 reductases contain two functional domains, a hydrophobic N-terminal single α-helical membrane anchoring domain (amino acids 1-95 of SEQ ID NO:12) and a hydrophilic C-terminal catalytic domain (amino acids 96-704 of SEQ ID NO:12) (Wang et al. (1997) Proc Natl Acad Sci USA 94:8111-8416). The N-terminal domain contains a hydrophobic membrane anchoring domain (amino acids 40-60 of SEQ ID NO:12) that anchors the protein to a membrane, for example, to the ER or chloroplast in plants, thus ensuring the CPR and the CYP are spatially related to allow for electron transfer. The N-terminal domain is not necessary for activity, as the C-terminal soluble domain alone is capable transferring electrons to cytochrome c or other electron acceptors. The C-terminal soluble domain contains two structural domains, a N-terminal flavin mononucleotide (FMN) domain (amino acids of 101-244 SEQ ID NO:12) and a C-terminal flavin adenine dinucleotide (FAD) domain (amino acids 301-704 of SEQ ID NO:12) (Dym and Eisenberg (2001) Protein Science 10:1712-1728). The FMN domain is homologous to flavodoxin that allows for binding to flavin cofactor FMN. The FAD domain that contains binding domains for flavin cofactor FAD and for NADPH, and additionally contains residues necessary for catalytic activity. The FMN and FAD domains are joined by a connecting domain (amino acids 245-300 of SEQ ID NO:12) that is responsible for the relative orientation of the FMN and FAD domains ensuring proper alignment of the two flavin cofactors necessary for efficient electron transfer.
The N-terminal FMN domain has an antiparallel β-structure while the C-terminal NAD(P) subdomain has the typology typical of pyridine dinucleotide-binding folds. The FMN domain contains a five-stranded β-sheet flanked by five α-helices, with the FMN positioned at the C-terminal side of the β-sheet. The core of the FAD binding domain is an anti-parallel flattened β-barrel and the NADP(H) binding domain is a parallel five-stranded β-sheet flanked by α-helices. The connecting domain is composed mainly of α-helices. The structural regions are as follows, from N-terminus to C-terminus: α-helix A; β-strand 1; α-helix B; β-strand 2; α-helix C; β-strand 3; α-helix D; β-strand 4; α-helix E; β-strand 5; α-helix F; β-strand 6; β-strand 7; β-strand 8, β-strand 9; β-strand 10; α-helix G; β-strand 11; β-strand 12; β-strand 12′; α-helix H; α-helix I; α-helix J; α-helix K; α-helix M; β-strand 13; β-strand 14; β-strand 15; α-helix N; β-strand 16; β-strand 16′; β-strand 17; α-helix O; β-strand 18; α-helix P; β-strand 10; α-helix Q; α-helix R; β-strand 20; α-helix S; α-helix T; and β-strand 21.
Cytochrome P450 reductases contain conserved cofactor and substrate binding domains, including FMN-, FAD-, NADPH-binding regions and cytochrome c- and cytochrome P450-binding sites. The P450 and cytochrome c binding sites contains amino acids 232-240 of SEQ ID NO12. The FMN domain contains binding regions for the FMN pyrophosphate (amino acids 98-119 of SEQ ID NO:12) and the FMN isoalloxazine ring (amino acids 161-214 of SEQ ID NO:12). The FAD domain contains binding regions for the FAD pyrophosphate (amino acids 317-353 of SEQ ID NO:12) and the FAD isoalloxazine ring (amino acids 482-505 of SEQ ID NO:12). The FAD binding pocket includes amino acids 344, 482, 484, 485, 500-502, 516-519 and 704 of SEQ ID NO:12 and the FAD binding motif includes amino acids 482, 484 and 485 of SEQ ID NO:12. The FAD domain also contains binding regions for the NADPH ribose and pyrophosphate (amino acids 555-576 of SEQ ID NO:12) and the NADPH nicotinamide (amino acids 651-668 of SEQ ID NO:12). The NADPH binding pocket includes amino acid residues 324, 502, 204, 560, 561, 595, 595, 624, 625, 630, 632 634, 659, 663 and 666 of SEQ ID NO:12. Amino acid residues Ser485, Cys657, Asp702 and Trp704 of SEQ ID NO:12 are the catalytic residues involved in hydride transfer (Hubbard et al. (2001) J Biol Chem 276:29163-29170). Amino acid residues 516, 519 and 522 of SEQ ID NO:12 are involved in the phosphate binding motif (Dym and Eisenberg (2001) Protein Science 10:1712-1728). The βαβ structure motif is formed from amino acid residues 557, 560-563 and 565 of SEQ ID NO:12 (Dym and Eisenberg (2001) Protein Science 10:1712-1728).
b. Function
Cytochrome P450 reductases shuttle two electrons from NAD(P)H to cytochrome P450 through the flavin cofactors FAD and FMN. FAD receives a hydride anion from the two electron donor NAD(P)H and passes the electrons one at a time to FMN. FMN then donates the electrons to the cytochrome P450. Cytochrome P450 uses the electrons, as described above, for the hydroxylation of various substrates.
C. Cytochrome P450 Polypeptides And Nucleic Acid Molecules Encoding The Cytochrome P450 Polypeptides
Provided herein are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides and cytochrome P450 bergamotene oxidase polypeptides. Also provided herein are nucleic acid molecules that encode any of the cytochrome P450 polypeptides provided herein. The cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of α-santalol, β-santalol or epi-β-santalol from α-santalene, β-santalene or epi-β-santalene, respectively, including, the production of β-santalol from β-santalene. The cytochrome P450 santalene oxidase polypeptides provided herein are also capable of catalyzing the formation of α-trans-bergamotol from α-trans-bergamotene. In some examples, the nucleic acid molecules that encode the cytochrome P450 santalene oxidase polypeptides are those that are the same as or substantially the same as those that are isolated from the sandalwood tree Santalum album. In other example, the nucleic acid molecules and encoded cytochrome P450 santalene oxidase polypeptides are variants of those isolated from the sandalwood tree Santalum album. The cytochrome P450 bergamotene oxidase polypeptides provided herein catalyze the formation of α-trans-bergamotol from α-trans-bergamotene. In some examples, the nucleic acid molecules that encode the cytochrome P450 bergamotene oxidase polypeptides are those that are the same as those that are isolated from the sandalwood tree Santalum album. In other examples, the nucleic acid molecules and encoded cytochrome P450 bergamotene oxidase polypeptides are variants of those isolated from the sandalwood tree Santalum album. 
Also provided herein are modified cytochrome P450 polypeptides and nucleic acid molecules that encode any of the modified cytochrome P450 polypeptides provided herein. The modifications can be made in any region of a cytochrome P450 polypeptide, including a cytochrome P450 santalene oxidase polypeptide or cytochrome P450 bergamotene oxidase polypeptide, provided the resulting modified cytochrome P450 polypeptide retains at least retains the catalytic activity of the unmodified cytochrome P450 polypeptide. For example, modifications can be made to a cytochrome P450 santalene oxidase polypeptide provided the resulting modified cytochrome P450 santalene oxidase polypeptide retains cytochrome P450 santalene oxidase activity (i.e., the ability to catalyze the hydroxylation of a santalene, namely α-santalene, β-santalene or epi-β-santalene). In another example, modifications can be made to a cytochrome P450 bergamotene oxidase polypeptide provided the resulting modified cytochrome P450 bergamotene oxidase polypeptide retains cytochrome P450 bergamotene oxidase activity (i.e., the ability to catalyze the hydroxylation of a bergamotene, namely α-trans-bergamotene).
The modifications can include, but are not limited to, codon optimization of the nucleic acids and/or changes that results in a single amino acid modification in the encoded polypeptide, such as single or multiple amino acid replacements (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions, including swaps of regions or domains of the polypeptide. In some examples, entire or partial domains or regions, such as any domain or region described herein below, are exchanged with corresponding domains or regions or portions thereof from another cytochrome P450 polypeptide. Exemplary of modifications are amino acid replacements, including single or multiple amino acid replacements. For example, modified cytochrome P450 polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120 or more modified positions compared to the cytochrome P450 polypeptide not containing the modification.
Provided herein are cytochrome P450 polypeptides from the CYP76 family. Provided herein is a CYP76 cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:50. Also provided herein are cytochrome P450 polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 polypeptide set forth in SEQ ID NO:50. For example, the cytochrome P450 polypeptides provided herein can exhibit at least or at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a cytochrome P450 polypeptide set forth in SEQ ID NO:50, providing the resulting cytochrome P450 polypeptide at least retains cytochrome P450 monooxygenase activity (i.e., the ability to catalyze the hydroxylation or monooxygenation of a terpene). Also provided herein are modified cytochrome P450 polypeptides from the CYP76 family. In particular, modified cytochrome P450 polypeptides provided herein contain amino acid replacements or substitutions, additions or deletions, truncations or combinations thereof with reference to the cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:50. It is within the level of one of skill in the art to make such modifications in cytochrome P450 polypeptides or any variant thereof and test each for cytochrome P450 activity described herein, such as monooxygenase activity.
Also provided herein are CYP76 nucleic acid molecules that have a sequence of amino acids set forth in SEQ ID NO:1, or degenerates thereof, that encode a cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:50. The CYP76 nucleic acid molecule set forth in SEQ ID NO:1 can be used to design primers that are used to identify and/or clone additional CYP proteins. Also provided herein are nucleic acid molecules encoding a cytochrome P450 polypeptide having at least 85% sequence identity to a sequence of nucleotides set forth in SEQ ID NO:1. For example, the nucleic acid molecules provided herein can exhibit at least or about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence of nucleotides set forth in SEQ ID NO:1, so long as the encoded cytochrome P450 polypeptide at least retains cytochrome P450 monooxygenase activity (i.e., the ability to catalyze the hydroxylation of a terpene). Also provided herein are degenerate sequences of the sequence set forth in SEQ ID NO:1 encoding a cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:50. Percent identity can be determined by one skilled in the art using standard alignment programs.
Provided herein are cytochrome P450 SaCYP76F39v1 (CYP76-G10), SaCYP76F42 (CYP76-G13), SaCYP76F39v2 (CYP76-G15), SaCYP76F40 (CYP76-G16) and SaCYP76F41 (CYP76-G17) polypeptides. Provided herein are cytochrome P450 santalene oxidase polypeptides having a sequence of amino acids set forth in SEQ ID NO:7, 74, 75, 76 or 77. Also provided herein are cytochrome P450 santalene oxidase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77. For example, the cytochrome P450 santalene oxidase polypeptides provided herein can exhibit at least or at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO: 7, 73, 74, 75 or 76, providing the resulting cytochrome P450 santalene oxidase polypeptides at least retain cytochrome P450 santalene oxidase activity (i.e., the ability to catalyze the hydroxylation of a santalene, namely α-santalene, β-santalene or epi-β-santalene). Percent identity can be determined by one skilled in the art using standard alignment programs.
Provided herein are cytochrome P450 SaCYP76F38v1 (CYP76-G5), SaCYP76F37v1 (CYP76-G11), SaCYP76F38v2 (CYP76-G12) and SaCYP76F37v2 (CYP76-G14) polypeptides. Provided herein are cytochrome P450 bergamotene oxidase polypeptides having a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 or 73. Also provided herein are cytochrome P450 bergamotene oxidase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 bergamotene oxidase polypeptide set forth in SEQ ID NO:6, 8, 9 or 73. For example, the cytochrome P450 bergamotene oxidase polypeptides provided herein can exhibit at least or at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a cytochrome P450 bergamotene oxidase polypeptide set forth in SEQ ID NO:6, 8, 9 or 73, providing the resulting cytochrome P450 bergamotene oxidase polypeptide at least retains cytochrome P450 bergamotene oxidase activity (i.e., the ability to catalyze the hydroxylation of a bergamotene). Percent identity can be determined by one skilled in the art using standard alignment programs.
Also provided herein is cytochrome P450 SaCYP76F43 (CYP76-G18) polypeptide. Provided herein is a cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:78. Also provided herein are cytochrome P450 polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 polypeptide set forth in SEQ ID NO:78. For example, the cytochrome P450 polypeptides provided herein can exhibit at least or at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a cytochrome P450 polypeptide set forth in SEQ ID NO:78, providing the resulting cytochrome P450 polypeptide at least retains cytochrome P450 monooxygenase activity (i.e., the ability to catalyze the hydroxylation or monooxygenation of a terpene). In particular, modified cytochrome P450 polypeptides provided herein contain amino acid replacements or substitutions, additions or deletions, truncations or combinations thereof with reference to the cytochrome P450 polypeptide having a sequence of amino acids set forth in SEQ ID NO:78. It is within the level of one of skill in the art to make such modifications in cytochrome P450 polypeptides or any variant thereof and test each for cytochrome P450 activity described herein, such as monooxygenase activity.
Also, in some examples, provided herein are catalytically active fragments of cytochrome P450 polypeptides. In some examples, the active fragments of the cytochrome P450 polypeptides, including the cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase polypeptides, are modified as described above. Such fragments retain one or more properties of a full-length cytochrome P450 polypeptide, including full-length santalene oxidase or cytochrome P450 bergamotene oxidase polypeptides. Typically, the active fragments exhibit cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase activity (i.e., catalyze the formation of santalols and bergamotols, respectively).
The cytochrome P450s provided herein, including the cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase polypeptides provided herein, can contain other modifications, for example, modifications not in the primary sequence of the polypeptide, including post-translational modifications. For example, modification described herein can be a cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase that is a fusion polypeptide or chimeric polypeptide, including hybrids of different cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase polypeptides or different cytochrome P450 monooxygenases (e.g. contain one or more domains or regions from another cytochrome P450 monooxygenases) and also synthetic cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon the sequence of known polypeptides.
The cytochrome P450 santalene oxidase polypeptides or cytochrome P450 bergamotene oxidase polypeptides provided herein can be used to catalyze the production of santalols and bergamotols, respectively. Typically, the cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of santalols from santalenes, e.g., they catalyze the hydroxylation of santalenes. In some examples, the cytochrome P450 santalene oxidases also catalyze the formation of bergamotols from bergamotenes. Typically the cytochrome P450 bergamotene oxidase polypeptides provided herein catalyze the formation of bergamotol from bergamotene, e.g., they catalyze the hydroxylation of bergamotene. Reactions can be performed in vivo, such as in a host cell into which the nucleic acid has been introduced. At least one of the polypeptides will be heterologous to the host. Reactions also can be performed in vitro by contacting with enzyme with the appropriate substrate under appropriate conditions.
Also provided herein are nucleic acid molecules encoding a santalene synthase and a cytochrome P450 santalene oxidase. Also provided herein are nucleic acid molecules encoding a santalene synthase and a cytochrome P450 bergamotene oxidase. In such examples, expression of the nucleic acid molecule in a suitable host, for example, a bacterial or yeast cell, results in expression of santalene synthase and the cytochrome P450 oxidase. Such cells can be used to produce the santalene synthases and the cytochrome P450 oxidases and/or to perform reactions in vivo to produce santalols and bergamotols. For example, santalols and bergamotols can be generated in a host cell from farnesyl diphosphate (FPP), particularly a yeast cell that overproduces the acyclic terpene precursor FPP. In some examples, a nucleic acid molecule encoding a farnesyl diphosphate synthase, such as a Santalum album farnesyl diphosphate synthase, can also be expressed in the suitable host, for example, a bacterial or yeast cell, resulting in over-expression of FPP.
Also provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 polypeptide and a cytochrome P450 reductase polypeptide. For example, provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 santalene oxidase polypeptide and a cytochrome P450 reductase polypeptide. In another example, provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 bergamotene oxidase polypeptide and a cytochrome P450 reductase polypeptide. The nucleic acid molecules can be in the same vector or plasmid or on different vectors or plasmids. In such examples, expression of the nucleic acid molecule in a suitable host, for example, a bacterial or yeast cell, results in expression of santalene synthase and the cytochrome P450 oxidase. Such cells can be used to produce the santalene synthases and the cytochrome P450 oxidases and/or to perform reactions in vivo to produce santalols and bergamotols. For example, santalols and bergamotols can be generated in a host cell from farnesyl diphosphate (FPP), particularly a yeast cell that overproduces the acyclic terpene precursor FPP.
1. Cytochrome P450 Santalene Oxidase Polypeptides
Provided herein are cytochrome P450 santalene oxidase polypeptides. Also provided herein are nucleic acid molecules that encode any of the cytochrome P450 santalene oxidase polypeptides provided herein. The cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of catalyze the formation of terpenoids found in sandalwood oil, including α-santalols, β-santalols, epi-β-santalols and α-trans-bergamotols. The cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of santalols from santalenes. In some examples, the cytochrome P450 santalene oxidase polypeptides provided herein also catalyze the formation of bergamotols from bergamotene. For example, the cytochrome P450 santalene oxidase polypeptides catalyze the formation of α-santalol from α-santalene, β-santalol from β-santalene and/or epi-β-santalol from epi-β-santalene (e.g., the cytochrome P450 santalene oxidase polypeptides catalyze the hydroxylation of α-santalene, β-santalene and/or epi-β-santalene). In a particular example, the cytochrome P450 santalene oxidase polypeptides catalyze the formation of (E)-α-santalol from α-santalene, (Z)-α-santalol from α-santalene, (E)-β-santalol from β-santalene, (Z)-β-santalol from β-santalene, (E)-epi-β-santalol from epi-β-santalene and/or (Z)-epi-β-santalol from epi-β-santalene. In some examples, the cytochrome P450 santalene oxidase polypeptides provided herein also catalyze the formation of (Z)-α-trans-bergamotol and/or (E)-α-trans-bergamotol from α-trans-bergamotene. In a particular example, the cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and/or (E)-α-trans-bergamotol. In particular, the cytochrome P450 santalene oxidase polypeptides produce (Z) and (E) stereoisomers of α- and β-santalol in ratios of approximately 1:5 and 1:4, respectively. The cytochrome P450 santalene oxidase polypeptides exhibit narrow substrate specificity, preferring α-santalene or β-santalene. In some examples, the cytochrome P450 santalene oxidase polypeptides also converted the substrate α-bisabolol.
In some examples, the cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of terpenoids found in sandalwood oil, including α-santalol, β-santalol, epi-β-santalol and α-trans-bergamotol, from the terpene reaction products of the acyclic precursor farnesyl pyrophosphate and a santalene synthase. For example, the cytochrome P450 santalene oxidase polypeptides provided herein catalyze the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and/or (E)-α-trans-bergamotol from the terpene reaction products of the acyclic precursor FPP and a santalene synthase, such as Santalum album santalene synthase (SaSSY; SEQ ID NO:16). The cytochrome P450 santalene oxidase polypeptides catalyze the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and/or (E)-α-trans-bergamotol in different ratios from those of authentic sandalwood oil (see Example 11 and FIGS. 15A and 15B). For example, the main products formed with SaCYP76F39v1 (SaCYP76-G10) were (E)-α-santalol and (E)-β-santalol while the main compounds of sandalwood oil are (Z)-α-santalol and (Z)-β-santalol (see FIGS. 15A and 15B).
For example, provided herein are cytochrome P450 santalene oxidase polypeptides that have a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 and 77. Also provided herein are cytochrome P450 santalene oxidase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 and 77. For example, the cytochrome P450 santalene oxidase polypeptides provided herein can exhibit at least at or about or 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more amino acid sequence identity to a cytochrome P450 santalene oxidase polypeptide set in any of SEQ ID NOS:7, 74, 75, 76 and 77, provided the cytochrome P450 santalene oxidase polypeptides exhibit cytochrome P450 santalene oxidase activity (i.e. catalyze the formation of santalols from santalenes and/or bergamotols from bergamotene). Percent identity can be determined by one skilled in the art using standard alignment programs.
Provided herein are cytochrome P450 santalene oxidases designated SaCYP76F39v1 (CYP76-G10), SaCYP76F39v2 (CYP76-G15), SaCYP76F40 (CYP76-G16), SaCYP76F41 (CYP76-G17) and SaCYP76F42 (CYP76-G13) that have a sequence of amino acids set forth in SEQ ID NOS:7, 74, 75, 76 and 77, respectively. Also provided herein are active fragments of cytochrome P450 santalene oxidase polypeptides having a sequence of amino acids set forth in any of SEQ ID NO:7, 74, 75, 76 and 77. Such fragments retain one or more properties of a cytochrome P450 santalene oxidase polypeptide. Typically, the active fragments exhibit cytochrome P450 santalene oxidase activity (i.e. the ability to catalyze the formation of santalols from santalenes).
Also provided herein are nucleic acid molecules that have a sequence of amino acids set forth in any of SEQ ID NOS:3, 68, 69, 70 and 71, or degenerates thereof, that encode a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NOS:7, 74, 75, 76 and 77, respectively. Also provided herein are nucleic acid molecules encoding cytochrome P450 santalene oxidase polypeptides having at least 85% sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:3, 68, 69, 70 and 71. For example, the nucleic acid molecules provided herein can exhibit at least or about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:3, 68, 69, 70 and 71, so long as the encoded cytochrome P450 santalene oxidase polypeptides exhibits cytochrome P450 santalene oxidase activity (i.e. the ability to catalyze the formation of santalols from santalenes). Also provided herein are degenerate sequences of the sequence set forth in any of SEQ ID NOS:3, 68, 69, 70 and 71 encoding cytochrome P450 santalene oxidase polypeptides having a sequence of amino acids set forth in SEQ ID NO:7, 74, 75, 76 and 77, respectively. Percent identity can be determined by one skilled in the art using standard alignment programs.
In some examples, the nucleic acid molecules that encode the cytochrome P450 santalene oxidase polypeptides are isolated from the sandalwood tree Santalum album. In other examples, the nucleic acid molecules and encoded cytochrome P450 santalene oxidase polypeptides are variants of those isolated from the sandalwood tree Santalum album. 
In a particular example, the SaCYP76F39v1 (CYP76-G10) polypeptide having a sequence of amino acids set forth in SEQ ID NO:7 catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol in in vivo assays in yeast expressing a santalene synthase (see Example 10.B.2) and in in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate (see Example 11.B.2.a.ii). In in vivo assays, (E)-β-santalol, (E)-α-santalol and (Z)-β-santalol were the major products (see FIG. 11A). In in vitro assays, (E)-β-santalol and (E)-α-santalol were the major products (see FIG. 15A). In yet other examples, in in vitro assays with either α-santalene, α-trans-bergamotene, or epi-β-santalene and β-santalene, the SaCYP76F39v1 (CYP76-G10) polypeptide catalyzed the formation of (Z)- and (E)-α-santalol, (Z)- and (E)-α-trans-bergamotol, and (Z)- and (E)-epi-β-santalol and (Z)- and (E)-β-santalol, respectively (see Example 11.C. and FIGS. 20A-20C). The kinetic properties of the SaCYP76F39v1 (CYP76-G10) polypeptide for α- and β-santalene as substrates are described in Example 12 below.
In another example, the SaCYP76F39v2 (CYP76-G15) polypeptide having a sequence of amino acids set forth in SEQ ID NO:74 catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol in in vivo assays in yeast expressing a santalene synthase (see Example 10.B.3 and FIG. 13A). In in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate, the SaCYP76F39v2 (CYP76-G15) polypeptide catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol (see Example 11.B.3.b and FIG. 16A) with (E)-α-santalol and (E)-β-santalol as the major products.
In another example, the SaCYP76F40 (CYP76-G16) polypeptide having a sequence of amino acids set forth in SEQ ID NO:75 catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol in in vivo assays in yeast expressing a santalene synthase (see Example 10.B.3 and FIG. 13B). In in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate, the SaCYP76F40 (CYP76-G16) polypeptide catalyzed the formation of (E)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol (see Example 11.B.3.b and FIG. 16B) with (E)-α-trans-bergamotol and (E)-β-santalol as the major products.
In another example, the SaCYP76F41 (CYP76-G17) polypeptide having a sequence of amino acids set forth in SEQ ID NO:76 catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol and (E)-α-trans-bergamotol in in vivo assays in yeast expressing a santalene synthase (see Example 10.B.3 and FIG. 13C). In in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate, the SaCYP76F41 (CYP76-G17) polypeptide catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol (see Example 11.B.3.b and FIG. 16C) with (E)-α-santalol as the major product.
In another example, the SaCYP76F42 (CYP76-G13) polypeptide having a sequence of amino acids set forth in SEQ ID NO:77 catalyzed the formation of (Z)-α-santalol, (Z)-β-santalol, (E)-epi-β-santalol and (E)-α-trans-bergamotol in in vivo assays in yeast expressing a santalene synthase (see Example 10.B.3 and FIG. 13D). In in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate, the SaCYP76F42 (CYP76-G13) polypeptide catalyzed the formation of (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol (see Example 11.B.3.b and FIG. 16D) with (E)-α-trans-bergamotol as the major product.
Modified Cytochrome P450 Santalene Oxidase Polypeptides
Also provided herein are modified cytochrome P450 santalene oxidase polypeptides. The modifications, which typically are amino acid insertions, deletions and/or substitutions, can be effected in any region of a cytochrome P450 santalene oxidase polypeptide provided the resulting modified cytochrome P450 santalene oxidase polypeptides at least retain cytochrome P450 santalene oxidase activity. For example, modifications can be made in any region of a cytochrome P450 santalene oxidase provided the resulting modified cytochrome P450 santalene oxidase at least retains cytochrome P450 santalene oxidase activity (i.e., the ability to catalyze the formation of santalols from santalenes).
The modifications can be a single amino acid modification, such as single amino acid replacements (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions. In some examples, entire or partial domains or regions, such as any domain or region described herein below, are exchanged with corresponding domains or regions or portions thereof from another cytochrome P450 polypeptide. Exemplary of modifications are amino acid replacements, including single or multiple amino acid replacements. For example, modified cytochrome P450 santalene oxidase polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120 or more modified positions compared to the cytochrome P450 santalene oxidase polypeptide not containing the modification. For example, the modifications described herein can be in a cytochrome P450 santalene oxidase polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 and 77 or any variant thereof, including any that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77. Based on this description, it is within the level of one of skill in the art to generate a cytochrome P450 santalene oxidase polypeptide containing any one or more of the described mutations, and test each for cytochrome P450 santalene oxidase activity described herein.
Also, in some examples, provided herein are modified active fragments of cytochrome P450 santalene oxidase polypeptides, that contain any of the modifications provided herein. Such fragments retain on or more properties of a cytochrome P450 santalene oxidase. Typically, the cytochrome P450 santalene oxidase polypeptides exhibit santalene oxidase (i.e., the ability to hydrolyze santalene and/or bergamotene).
Modifications in a cytochrome P450 santalene oxidase also can be made to a cytochrome P450 santalene oxidase polypeptide that also contains other modifications, including modifications of the primary sequence and modifications not in the primary sequence of the polypeptide. For example, modification described herein can be in a cytochrome P450 santalene oxidase polypeptide that is a fusion polypeptide or chimeric polypeptide, including hybrids of different cytochrome P450 santalene oxidase polypeptides with different cytochrome P450 polypeptides (e.g. contain one or more domains or regions from another cytochrome P450s) and also synthetic cytochrome P450 santalene oxidase polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon the sequence of known polypeptides.
In some examples, the modifications are amino acid replacements. In further examples, the modified cytochrome P450 santalene oxidase polypeptides provided herein contain one or more modifications in a domain. As described elsewhere herein, the modifications in a domain or structural domain can be by replacement of corresponding heterologous residues from another cytochrome P450 polypeptide.
To retain cytochrome P450 santalene oxidase activity, modifications typically are not made at those positions necessary for cytochrome P450 santalene oxidase activity, i.e., in the catalytic center or in conserved residues. For example, generally modifications are not made a position corresponding to Glu367, Arg370, Gly445, Arg446, Arg447, Ile448, Cys449, Pro450 or Gly451 with reference to a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77.
The modified cytochrome P450 santalene oxidase polypeptides can contain two or more modifications, including amino acid replacements or substitutions, insertions or deletions, truncations or combinations thereof. Generally, multiple modifications provided herein can be combined by one of skill in the art so long as the modified cytochrome P450 santalene oxidase polypeptide retains cytochrome P450 santalene oxidase activity.
Also provided herein are nucleic acid molecules that encode any of the modified cytochrome P450 santalene oxidase polypeptides provided herein. In particular examples, the nucleic acid sequence can be codon optimized, for example, to increase expression levels of the encoded sequence. The particular codon usage is dependent on the host organism in which the modified polypeptide is expressed. One of skill in the art is familiar with optimal codons for expression in bacteria or yeast, including for example E. coli or Saccharomyces cerevisiae. For example, codon usage information is available from the Codon Usage Database available at kazusa.or.jp.codon (see Richmond (2000) Genome Biology, 1:241 for a description of the database). See also, Forsburg (2004) Yeast, 10:1045-1047; Brown et al. (1991) Nucleic Acids Research, 19:4298; Sharp et al. (1988) Nucleic Acids Research, 12:8207-8211; Sharp et al. (1991) Yeast, 657-78. In examples herein, nucleic acid sequences provided herein are codon optimized based on codon usage in Saccharomyces cerevisiae. 
The modified polypeptides and encoding nucleic acid molecules provided herein can be produced by standard recombinant DNA techniques known to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed or random mutagenesis of encoding nucleic acid molecules, or solid phase polypeptide synthesis methods. For example, as described herein, nucleic acid molecules encoding a cytochrome P450 santalene oxidase polypeptide can be subjected to mutagenesis, such as random mutagenesis of the encoding nucleic acid, by error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling, or other recombinant methods. The nucleic acid encoding the polypeptides then can be introduced into a host cell to be expressed heterologously. Hence, also provided herein are nucleic acid molecules encoding any of the modified polypeptides provided herein. In some examples, the modified cytochrome P450 santalene oxidase polypeptides are produced synthetically, such as using solid phase or solutions phase peptide synthesis.
2. Cytochrome P450 Bergamotene Oxidase Polypeptides
Provided herein are cytochrome P450 bergamotene oxidase polypeptides. Also provided herein are nucleic acid molecules that encode any of the cytochrome P450 bergamotene oxidase polypeptides provided herein. The cytochrome P450 bergamotene oxidase polypeptides provided herein catalyze the formation of bergamotols from bergamotenes. Typically the cytochrome P450 bergamotene oxidase polypeptides catalyze the formation of (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol from α-trans-bergamotene (e.g. the cytochrome P450 bergamotene oxidase polypeptides catalyze the hydroxylation of α-trans-bergamotene). In particular examples, the cytochrome P450 bergamotene oxidase polypeptides catalyze the formation of (E)-α-trans-bergamotol from α-trans-bergamotene. In some examples, the cytochrome P450 bergamotene oxidase polypeptides additionally catalyze the formation of minor amounts of (E)-α-santalol and (E)-β-santalol. The cytochrome P450 bergamotene oxidase polypeptides exhibit narrow substrate specificity, preferring α-santalene or β-santalene. In some examples, the cytochrome P450 bergamotene oxidase polypeptides also converted the substrate trans-nerolidol.
For example, provided herein are cytochrome P450 bergamotene oxidase polypeptides that have a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 and 73. Also provided herein are cytochrome P450 bergamotene oxidase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 and 73. For example, the cytochrome P450 bergamotene oxidase polypeptides provided herein can exhibit at least at or about or 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more amino acid sequence identity to a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 and 73, provided the cytochrome P450 bergamotene oxidase polypeptides exhibit cytochrome P450 bergamotene oxidase activity (i.e. catalyze the formation of bergamotols from bergamotenes). Percent identity can be determined by one skilled in the art using standard alignment programs.
Provided herein are cytochrome P450 bergamotene oxidases designated SaCYP76F38v1 (CYP76-G5), SaCYP76F37v1 (CYP76-G11), SaCYP76F38v2 (CYP76-G11) and SaCYP76F37v2 (CYP76-G14), that have a sequence of amino acids set forth in SEQ ID NOS: 6, 8, 9 and 73, respectively. Also provided herein are active fragments of cytochrome P450 bergamotene oxidase polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS: 6, 8, 9 and 73. Such fragments retain one or more properties of a cytochrome P450 bergamotene oxidase polypeptide. Typically, the active fragments exhibit cytochrome P450 bergamotene oxidase activity (i.e. the ability to catalyze the hydroxylation of bergamotenes from bergamotols).
In particular examples, the cytochrome P450 bergamotene oxidases provided herein having a sequence of amino acids set forth in SEQ ID NOS: 6, 8, 9 and 73 catalyzed the formation of (E)-α-trans-bergamotol, (E)-α-santalol and (E)-β-santalol in in vitro assays with a mixture of α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene as the substrate. In such examples, (E)-α-trans-bergamotol was the major product, and (E)-α-santalol and (E)-β-santalol were minor products (see Example 11.B.3.b and FIGS. 17A-17D). In another example, in in vitro assays with either α-santalene, α-trans-bergamotene, or epi-β-santalene and β-santalene, the cytochrome P450 bergamotene oxidase provided herein having a sequence of amino acids set forth in SEQ ID NO:8 catalyzed the formation of (E)-α-santalol, (E)-α-trans-bergamotol or (E)-β-santalol, respectively (see Example 11.0 and FIGS. 20D-20F). In yet other examples, the cytochrome P450 bergamotene oxidases provided herein having a sequence of amino acids set forth in SEQ ID NOS: 6, 8, 9 and 73 catalyzed the formation of (E)-α-trans-bergamotol in in vivo assays in yeast that express santalene synthase (see Example 10.C.2 and FIGS. 14A-14D). The kinetic properties of the SaCYP76F37v1 (SaCYP76-G11) polypeptide for α- and β-santalene as substrates are described in Example 12 below.
Also provided herein are nucleic acid molecules that have a sequence of amino acids set forth in any of SEQ ID NOS:2, 4, 5 and 67, or degenerates thereof, that encode a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NOS:6, 8, 9 and 73, respectively. Also provided herein are nucleic acid molecules encoding a cytochrome P450 bergamotene oxidase polypeptide having at least 85% sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:2, 4, 5 and 67. For example, the nucleic acid molecules provided herein can exhibit at least or about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:2, 4, 5 and 67, so long as the encoded cytochrome P450 bergamotene oxidase polypeptide exhibits cytochrome P450 bergamotene oxidase activity (i.e. the ability to catalyze the formation of bergamotols from bergamotene). Also provided herein are degenerate sequences of the sequence set forth in any of SEQ ID NOS:2, 4, 5 and 67 encoding a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 and 73, respectively. Percent identity can be determined by one skilled in the art using standard alignment programs.
In some examples, the nucleic acid molecules that encode the cytochrome P450 bergamotene oxidase polypeptides are isolated from the sandalwood tree Santalum album. In other examples, the nucleic acid molecules and encoded cytochrome P450 bergamotene oxidase polypeptides are variants of those isolated from the sandalwood tree Santalum album. 
Modified Cytochrome P450 Bergamotene Oxidase Polypeptides
Provided herein are modified cytochrome P450 bergamotene oxidase polypeptides. The modifications, which typically are amino acid insertions, deletions and/or substitutions, can be effected in any region of a cytochrome P450 bergamotene oxidase polypeptide provided the resulting modified cytochrome P450 bergamotene oxidase polypeptides at least retain cytochrome P450 bergamotene oxidase activity. For example, modifications can be made in any region of a cytochrome P450 bergamotene oxidase provided the resulting modified cytochrome P450 bergamotene oxidase at least retains cytochrome P450 bergamotene oxidase activity (i.e., the ability to catalyze the formation of a bergamotol from a bergamotene).
The modifications can be a single amino acid modification, such as single amino acid replacements (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions. In some examples, entire or partial domains or regions, such as any domain or region described herein below, are exchanged with corresponding domains or regions or portions thereof from another cytochrome P450 bergamotene oxidase polypeptide. Exemplary of modifications are amino acid replacements, including single or multiple amino acid replacements. For example, modified cytochrome P450 bergamotene oxidase polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120 or more modified positions compared to the cytochrome P450 polypeptide not containing the modification. For example, the modifications described herein can be in a cytochrome P450 bergamotene oxidase polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 or 73 or any variant thereof, including any that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 and 73. Based on this description, it is within the level of one of skill in the art to generate a cytochrome P450 bergamotene oxidase polypeptide containing any one or more of the described mutations, and test each for cytochrome P450 bergamotene oxidase activity described herein.
Also, in some examples, provided herein are modified active fragments of cytochrome P450 bergamotene oxidase polypeptides that contain any of the modifications provided herein. Such fragments retain on or more properties of a cytochrome P450 bergamotene oxidase. Typically, the modified cytochrome P450 bergamotene oxidase polypeptides exhibit bergamotene oxidase activity (i.e., the ability to hydrolyze bergamotene).
Modifications in a cytochrome P450 bergamotene oxidase polypeptide that also contains other modifications, including modifications of the primary sequence and modifications not in the primary sequence of the polypeptide. For example, modification described herein can be in a cytochrome P450 bergamotene oxidase polypeptide that is a fusion polypeptide or chimeric polypeptide, including hybrids of different cytochrome P450 bergamotene oxidase polypeptides with different cytochrome P450 polypeptides (e.g. contain one or more domains or regions from another cytochrome P450s) and also synthetic cytochrome P450 bergamotene oxidase polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon the sequence of known polypeptides.
In some examples, the modifications are amino acid replacements. In further examples, the modified cytochrome P450 bergamotene oxidase polypeptides provided herein contain one or more modifications in a domain. As described elsewhere herein, the modifications in a domain or structural domain can be by replacement of corresponding heterologous residues from another cytochrome P450 polypeptide.
To retain cytochrome P450 bergamotene oxidase activity, modifications typically are not made at those positions necessary for cytochrome P450 activity, i.e., in the catalytic center or in conserved residues. For example, generally modifications are not made a position corresponding to Glu367, Arg370, Gly445, Arg446, Arg447, Ile448, Cys449, Pro450 or Gly451 with reference to a sequence of amino acids set forth in SEQ ID NO:6, 8, 9 or 73.
The modified cytochrome P450 bergamotene oxidase polypeptides can contain two or more modifications, including amino acid replacements or substitutions, insertions or deletions, truncations or combinations thereof. Generally, multiple modifications provided herein can be combined by one of skill in the art so long as the modified cytochrome P450 bergamotene oxidase polypeptide retains cytochrome P450 bergamotene oxidase activity.
Also provided herein are nucleic acid molecules that encode any of the modified cytochrome P450 bergamotene oxidase polypeptides provided herein. In particular examples, the nucleic acid sequence can be codon optimized, for example, to increase expression levels of the encoded sequence. The particular codon usage is dependent on the host organism in which the modified polypeptide is expressed. One of skill in the art is familiar with optimal codons for expression in bacteria or yeast, including for example E. coli or Saccharomyces cerevisiae. For example, codon usage information is available from the Codon Usage Database available at kazusa.or.jp.codon (see Richmond (2000) Genome Biology, 1:241 for a description of the database). See also, Forsburg (2004) Yeast, 10:1045-1047; Brown et al. (1991) Nucleic Acids Research, 19:4298; Sharp et al. (1988) Nucleic Acids Research, 12:8207-8211; Sharp et al. (1991) Yeast, 657-78. In examples herein, nucleic acid sequences provided herein are codon optimized based on codon usage in Saccharomyces cerevisiae. 
The modified polypeptides and encoding nucleic acid molecules provided herein can be produced by standard recombinant DNA techniques known to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed or random mutagenesis of encoding nucleic acid molecules, or solid phase polypeptide synthesis methods. For example, as described herein, nucleic acid molecules encoding a cytochrome P450 bergamotene oxidase polypeptide can be subjected to mutagenesis, such as random mutagenesis of the encoding nucleic acid, by error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling, or other recombinant methods. The nucleic acid encoding the polypeptides then can be introduced into a host cell to be expressed heterologously. Hence, also provided herein are nucleic acid molecules encoding any of the modified polypeptides provided herein. In some examples, the modified cytochrome P450 bergamotene oxidase polypeptides are produced synthetically, such as using solid phase or solutions phase peptide synthesis.
3. Additional Modifications
Provided herein are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, that contain additional modifications. For example, modified cytochrome P450 polypeptides include, for example, truncated cytochrome P450 polypeptides, cytochrome P450 polypeptides having altered activities or properties, chimeric cytochrome P450 polypeptides, cytochrome P450 polypeptides containing domain swaps, cytochrome P450 fusion proteins, or cytochrome P450 polypeptides having any modification described elsewhere herein.
a. Truncated Polypeptides
Also provided herein are truncated cytochrome P450 polypeptides. The truncated cytochrome P450 polypeptides can be truncated at the N-terminus or C-terminus, so long as the truncated cytochrome P450 polypeptides retain the catalytic activity of a cytochrome P450, such as cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase activity. Typically, the truncated cytochrome P450 santalene oxidase polypeptides exhibit santalene oxidase activity (i.e., the ability to catalyze the hydroxylation of a santalene, namely α-santalene, β-santalene or epi-β-santalene). Typically, the truncated cytochrome P450 bergamotene oxidase polypeptides exhibit bergamotene oxidase activity (i.e., the ability to catalyze the hydroxylation of a bergamotene). In some examples, the cytochrome P450 polypeptides, including the cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, are truncated at the C-terminus. In other examples, the cytochrome P450 polypeptides, including the cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, are truncated at the N-terminus.
In some examples, the cytochrome P450 polypeptides, including the cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, are truncated at the N-terminus, C-terminus or both termini of a cytochrome P450 polypeptide provided herein, such as truncation of a sequence of amino acids set forth in any of SEQ ID NOS:6-9. In other examples, any of the modified cytochrome P450 polypeptides provided herein are truncated. The modified cytochrome P450 polypeptides can be truncated at their N-terminus, C-terminus, or both termini. For example, any cytochrome P450 polypeptide provided herein can be truncated by at or about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more amino acid residues at the N-terminus, provided the cytochrome P450 polypeptide retains cytochrome P450 activity. In other examples, any cytochrome P450 polypeptide provided herein can be truncated by at or about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more amino acid residues at the C-terminus, provided the cytochrome P450 polypeptide retains cytochrome P450 activity.
b. Polypeptides with Altered Activities or Properties
The modified cytochrome P450 polypeptides provided herein can also exhibit changes in activities and/or properties. The modified cytochrome P450 polypeptides can exhibit, for example, improved properties, such as increased catalytic activity, increased selectivity, increased substrate specificity, increased substrate binding, increased stability, and/or increased expression in a host cell, and altered properties, such as altered product distribution and altered substrate specificity. Such improved or altered activities can result in increased production of santalols and/or bergamotols.
In some examples, the modified cytochrome P450 polypeptides have altered substrate specificity. For example, the substrate specificity of a modified cytochrome P450 polypeptide can be altered by at least or at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the unmodified cytochrome P450 polypeptide. For example, a modified cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase polypeptide can catalyze the monooxygenation of a terpene substrate that is not a santalene or bergamotene. In such examples, the modified cytochrome P450 polypeptides catalyze the formation of terpenoids other than santalols or bergamotols from any suitable terpene substrate. For example, the modified cytochrome P450 polypeptides can produce one or more different monoterpenoids, sesquiterpenoids or diterpenoids other than santalols and bergamotols.
In some examples, the modified cytochrome P450 polypeptides have an altered terpenoid product distribution. In some examples, altered product distribution results in an increased amount of a desired terpenoid product, and thus product distribution is improved compared to the product distribution of the unmodified cytochrome P450. In other examples, altered product distribution results in an decreased amount of a desired terpenoid product, and thus the product distribution of the modified cytochrome P450 is decreased compared to that of the unmodified cytochrome P450. In one example, the modified cytochrome P450 santalene oxidase produces a different ratio of terpenoid products compared to the unmodified cytochrome P450 santalene oxidase. For example, the amount of a terpenoid produced by the modified cytochrome P450 can be increased or decreased by at least or at least about or 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to the amount of a different terpenoid produced by the unmodified cytochrome P450. For example, the amount of a terpenoid produced by the modified cytochrome P450 santalene oxidase, such as, for example, a β-santalol, can be increased by at least or at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to the amount of a different terpenoid produced by the unmodified cytochrome P450 santalene oxidase, such as, for example, an α-santalol. In some examples, the modified cytochrome P450 santalene oxidases produce more β-santalol than any other terpenoid compound. In another example, the modified cytochrome P450 bergamotene oxidase produces a different ratio of terpenoid products compared to the unmodified cytochrome P450 bergamotene oxidase. For example, the amount of a terpenoid produced by the modified cytochrome P450 bergamotene oxidase, such as, for example, a α-trans-bergamotol, can be increased by at least or at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to the amount of a different terpenoid produced by the unmodified cytochrome P450 bergamotene oxidase.
In some examples, the modified cytochrome P450 polypeptide exhibits a similar, increased and/or improved activity compared to the unmodified cytochrome P450 polypeptide. For example, a modified cytochrome P450 polypeptide exhibits increased terpenoid production compared to an unmodified cytochrome P450 polypeptide. The increased terpenoid production can be an increase in the total amount of terpenoids produced by the modified cytochrome P450 polypeptide or can be an increase in the amount of a particular terpenoid produced by the modified cytochrome P450 polypeptide. For example, the total terpenoid production of a modified cytochrome P450 polypeptide can be increased by at least or at least about 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to an unmodified cytochrome P450 polypeptide. In some examples, the total terpenoid production of a modified cytochrome P450 polypeptide is at least or about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more compared to an unmodified cytochrome P450 polypeptide. In another example, the production of a particular terpenoid by a modified cytochrome P450 polypeptide is increased by at least or at least about 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to an unmodified cytochrome P450 polypeptide. In some examples, a modified cytochrome P450 polypeptide produces at least or about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more of a particular terpenoid product compared to the unmodified cytochrome P450 polypeptide.
In some examples, the modified cytochrome P450 polypeptide exhibits improved substrate specificity compared to the unmodified cytochrome P450 polypeptide. Substrate specificity of the modified cytochrome P450 polypeptide can be increased by at least or at least about 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the substrate specificity of the unmodified cytochrome P450 polypeptide. For example, the modified cytochrome P450 polypeptide can exhibit increased substrate specificity for a terpene, such as a santalene, compared to a different terpene, such as a bergamotene. In such examples, increased specificity for a santalene results in increased production of santalols and decreased production of a bergamotol.
In some examples, the modified cytochrome P450 polypeptide, such as a modified cytochrome P450 santalene oxidase polypeptide, exhibits similar or increased or improved santalene oxidase activity compared to the unmodified cytochrome P450 santalene oxidase polypeptide. For example, the modified cytochrome P450 santalene oxidase polypeptide can exhibit increased specificity or selectivity for oxidation of α-santalene, β-santalene and/or epi-β-santalene compared to the unmodified cytochrome P450 santalene oxidase polypeptide. In some instances of such examples, the modified cytochrome P450 santalene oxidase selectively monooxygenates β-santalene compared to the unmodified cytochrome P450 santalene oxidase. In other examples, the modified cytochrome P450 santalene oxidase polypeptide exhibits reduced selectivity for oxidation of bergamotene compared to the unmodified cytochrome P450 santalene oxidase. For example, the modified cytochrome P450 santalene oxidase exhibits a decrease in activity towards oxidation of bergamotene of at least or at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the unmodified cytochrome P450 santalene oxidase.
In some examples, the modified cytochrome P450 polypeptide, such as a modified cytochrome P450 bergamotene oxidase polypeptide, exhibits similar or increased or improved bergamotene oxidase activity compared to the unmodified cytochrome P450 bergamotene oxidase polypeptide. For example, the modified cytochrome P450 bergamotene oxidase polypeptide can exhibit increased specificity or selectivity for oxidation of α-trans-bergamotene compared to the unmodified cytochrome P450 bergamotene oxidase.
c. Domain Swaps
Provided herein are modified cytochrome P450 polypeptides that are chimeric polypeptides containing a swap (deletion and insertion) by deletion of amino acid residues of one of more domains or regions therein or portions thereof and insertion of a heterologous sequence of amino acids. In some examples, the heterologous sequence is a randomized sequence of amino acids. In other examples, the heterologous sequence is a contiguous sequence of amino acids for the corresponding domain or region or portion thereof from another cytochrome P450. The heterologous sequence that is replaced or inserted generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids. In examples where the heterologous sequence is from a corresponding domain or a portion thereof of another cytochrome P450, the heterologous sequence generally includes at least 50%, 60%, 70%, 80%, 90%, 95% or more contiguous amino acids of the corresponding domain or region or portion. In such an example, adjacent residues to the heterologous corresponding domain or region or portion thereof also can be included in a modified cytochrome P450 polypeptide provided herein.
In one example of swap mutants provided herein, at least one domain or region or portion thereof of a cytochrome P450 polypeptide is replaced with a contiguous sequence of amino acids for the corresponding domain or region or portions thereof from another cytochrome P450 polypeptide. In some examples, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains or regions or portions thereof are replaced with a contiguous sequence of amino acids for the corresponding domain or region or portions thereof from another cytochrome P450 polypeptide.
Any domain or region or portion thereof of a cytochrome P450 polypeptide can be replaced with a heterologous sequence of amino acids, such as heterologous sequence from the corresponding domain or region from another cytochrome P450. A domain or region can be a structural domain or a functional domain. One of skill in the art is familiar with domains or regions in cytochrome P450s. Functional domains include, for example, the catalytic domain or a portion thereof. A structural domain can include all or a portion of helix A, β strand 1-1, β strand 1-2, helix B, β strand 1-5, helix B′, helix C, helix C′, helix D, β strand 3-1, helix E, helix F, helix G, helix H, β strand 5-1, β strand 5-2, helix I, helix J, helix J′, helix K, β strand 1-4, β strand 2-1, β strand 2-2, β strand 1-3, helix K′, helix K′′, Heme domain, helix L, β strand 3-3, β strand 4-1, β strand 4-2 and β strand 3-2. One of skill in the art is familiar with various cytochrome P450s and can identify corresponding domains or regions or portions of amino acids thereof.
Typically, the resulting modified cytochrome P450 polypeptides exhibit cytochrome P450 monooxygenase activity and the ability to produce santalols and/or bergamotols from santalenes and bergamotenes. For example, the modified cytochrome P450 santalene oxidase polypeptides exhibit 50% to 5000%, such as 50% to 120%, 100% to 500% or 110% to 250% of the santalol production from santalene compared to the cytochrome P450 santalene oxidase not containing the modification (e.g. the amino acid replacement or swap of amino acid residues of a domain or region) and/or compared to wild type cytochrome P450 santalene oxidase set forth in SEQ ID NO:7, 74, 75, 76 or 77. Typically, the modified cytochrome P450 santalene oxidase polypeptides exhibit increased santalol production from santalene compared to the cytochrome P450 santalene oxidase not containing the modification, such as compared to the cytochrome P450 santalene oxidase set forth in SEQ ID NO:7, 74, 75, 76 or 77. For example, the modified cytochrome P450 santalene oxidase polypeptides can produce santalols from santalenes in an amount that is at least or about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% of the amount of santalols produced from santalenes by wild type cytochrome P450 santalene oxidase synthase not containing the modification under the same conditions. For example, the santalol production is increased at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more.
In another example, the modified cytochrome P450 bergamotene oxidase polypeptides exhibit 50% to 5000%, such as 50% to 120%, 100% to 500% or 110% to 250% of the bergamotol production from bergamotene compared to the cytochrome P450 bergamotene oxidase not containing the modification (e.g. the amino acid replacement or swap of amino acid residues of a domain or region) and/or compared to wild type cytochrome P450 bergamotene oxidase set forth in SEQ ID NO:6, 8, 9 or 73. Typically, the modified cytochrome P450 bergamotene oxidase polypeptides exhibit increased bergamotol production from bergamotene compared to the cytochrome P450 bergamotene oxidase not containing the modification, such as compared to the cytochrome P450 bergamotene oxidase set forth in SEQ ID NO:6, 8, 9 or 73. For example, the modified cytochrome P450 bergamotene oxidase polypeptides can produce bergamotol from bergamotene in an amount that is at least or about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% of the amount of bergamotol produced from bergamotene by wild type cytochrome P450 bergamotene oxidase synthase not containing the modification under the same conditions. For example, the bergamotol production is increased at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more.
In particular examples herein, modified cytochrome P450 polypeptides provided herein are swap mutants whereby all or a portion of one or more structural domains is replaced with a corresponding structural domain of another cytochrome P450 polypeptide. Table 3 below identifies structural domains of cytochrome P450 santalene oxidase (SEQ ID NO:7) and cytochrome P450 bergamotene oxidase (SEQ ID NO:6) based on alignment of the cytochrome P450 polypeptides with cytochrome P450BM-3, a class II microsomal P450 (SEQ ID NO:66; Accession No. 2HPD; Ravichandran et al. (1993) Science 261:731-736; see also FIGS. 5A-5B). Hence, the corresponding domain can be identified in other cytochrome P450 polypeptides.







TABLE 3







Structural Domains












santalene oxidase
bergamotene oxidase



structure
(SEQ ID NO: 7)
(SEQ ID NO: 6)






helix A
54-65
54-65



β strand 1-1
67-74
67-74



β strand 1-2
75-82
75-82



helix B
83-91
83-91



β strand 1-5
95-98
95-98



helix B′
101-108
101-108



helix C
124-133
124-133



helix D
149-164
149-164



β strand 3-1
170-173
170-173



helix E
174-189
174-189



helix F
204-218
204-218



helix G
238-265
238-265



helix H
278-285
278-285



β strand 5-1
287-290
287-290



β strand 5-2
291-294
291-294



helix I
297-329
297-329



helix J
330-343
330-343



helix J′
351-358
351-358



helix K
359-371
359-371



β strand 1-4
376-382
376-382



β strand 2-1
383-389
383-389



β strand 2-2
391-397
391-397



β strand 1-3
398-402
398-402



helix K′
403-410
403-410



Heme domain
444-451
444-451



helix L
452-469
452-469



β strand 3-3
470-474
470-474



β strand 4-1
481-485
481-485



β strand 4-2
487-491
487-491



β strand 3-2
493-500
493-500









Any methods known in the art for generating chimeric polypeptides can be used to replace all or a contiguous portion of a domain or a cytochrome P450 with all or a contiguous portion of the corresponding domain of a second cytochrome P450 (see, U.S. Pat. Nos. 5,824,774, 6,072,045, 7,186,891 and 8,106,260, and U.S. Pat. Pub. No. 20110081703). Also, gene shuffling methods can be employed to generate chimeric polypeptides and/or polypeptides with domain or region swaps.
For example, corresponding domains or regions of any two cytochrome P450s can be exchanged using any suitable recombinant method known in the art, or by in vitro synthesis. Exemplary of recombinant methods is a two stage overlapping PCR method, such as described herein. In such methods, primers that introduce mutations at a plurality of codon positions in the nucleic acids encoding the targeted domain or portion thereof in the first cytochrome P450 can be employed. The mutations together form the heterologous region (i.e. the corresponding region from the second cytochrome P450). Alternatively, for example, randomized amino acids can be used to replace particular domains or regions. It is understood that primer errors, PCR errors and/or other errors in the cloning or recombinant methods can result in errors such that the resulting swapped or replaced region or domain does not exhibit an amino acid sequence that is identical to the corresponding region from the second cytochrome P450 reductase.
In an exemplary PCR-based method, the first stage PCR uses (i) a downstream primer that anneals downstream of the region that is being replaced with a mutagenic primer that includes approximately fifteen nucleotides (or an effective number to effect annealing, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 nucleotides or more) of homologous sequence on each side of the domain or region to be exchanged or randomized flanking the region to be imported into the target gene, and (ii) an upstream primer that anneals upstream of the region that is being replaced together with an opposite strand mutagenic primer that also includes approximately fifteen nucleotides (or an effective number to effect annealing, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 nucleotides or more) of homologous sequence on each side of the domain or region to be exchanged or randomized flanking the region to be imported into the target gene. If a replacement in which a domain or region of a first cytochrome P450 gene is replaced with the corresponding domain or region from a second cytochrome P450 is being performed, nucleotides in the mutagenic primers between the flanking regions from the first cytochrome P450 contain codons for the corresponding region of the second cytochrome P450. In instances where the amino acids in a domain or region are to be randomized, nucleotides of the mutagenic primers between the flanking regions from the first cytochrome P450 contains random nucleotides. An overlapping PCR is then performed to join the two fragments, using the upstream and downstream oligo. The resulting PCR product then can be cloned into any suitable vector for expression of the modified cytochrome P450.
Further, any of the modified cytochrome P450 polypeptides containing swap mutations herein can contain one or more further amino acid replacements as described herein above.
d. Additional Variants
Cytochrome P450 polypeptides provided herein can be modified by any method known to one of skill in the art for generating protein variants, including, but not limited to, DNA or gene shuffling, error prone PCR, overlap PCR or other recombinant methods. In one example, nucleic acid molecules encoding any cytochrome P450 polypeptide or variant cytochrome P450 polypeptide provided herein can be modified by gene shuffling. Gene shuffling involves one or more cycles of random fragmentation and reassembly of at least two nucleotide sequences, followed by screening to select nucleotide sequences encoding polypeptides with desired properties. The recombination can be performed in vitro (see Stemmer et al. (1994) Proc Natl Acad Sci USA 91:10747-10751; Stemmer et al. (1994) Nature 370:389-391; Cramieri et al. (1998) Nature 391:288-291; U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458) or in vivo (see, International Pat. Pub. No. WO199707205). The nucleic acid molecules encoding the polypeptides then can be introduced into a host cell to be expressed heterologously and tested for their cytochrome P450 activity by any method described in section G below.
e. Fusion or Chimeric Proteins
Nucleic acid molecules provided herein include fusion or chimeric nucleic acid molecules that contain a santalene synthase and a cytochrome P450 polypeptide. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that is capable of catalyzing the formation of a santalol, such as an α-santalol, β-santalol or epi-β-santalol, from FPP that contains any santalene synthase and cytochrome P450 santalene oxidase polypeptide provided herein. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53 and a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO:7, 74, 75, 76 or 77. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53 and a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO:7, 74, 75, 76 or 77. Also provided herein are nucleic acid molecules encoding a fusion polypeptide that is capable of catalyzing the formation of a bergamotol, such as an α-trans-bergamotol, from FPP that contains any santalene synthase and cytochrome P450 santalene bergamotene polypeptide provided herein. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53 and a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53 and a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73. The fusion polypeptides can be linked directly or via a linker.
Nucleic acid molecules provided herein include fusion or chimeric nucleic acid molecules that contain a cytochrome P450 polypeptide and a cytochrome P450 reductase. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. In another example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. The fusion polypeptides can be linked directly or via a linker.
Nucleic acid molecules provided herein include fusion or chimeric nucleic acid molecules that contain a santalene synthase, cytochrome P450 polypeptide and a cytochrome P450 reductase. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53, a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53, a cytochrome P450 santalene oxidase polypeptide set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. In another example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53, a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53, a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8, 9 or 73 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. The fusion polypeptides can be linked directly or via a linker.
In another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 and/or a cytochrome P450 reductase, such that, when expressed in a host cell, a bacterial or yeast host cell, a santalene synthase, a cytochrome P450 and/or a cytochrome P450 reductase are expressed. In one example, provided herein is a nucleic acid molecule that encodes a santalene synthase and a cytochrome P450 santalene oxidase. In another example, provided herein is a nucleic acid molecule that encodes a santalene synthase and a cytochrome P450 bergamotene oxidase. In yet another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 santalene oxidase and a cytochrome P450 reductase. In another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 bergamotene oxidase and a cytochrome P450 reductase. Further, when the host cell is capable of producing FPP, the encoded polypeptides catalyze the production of santalols and/or bergamotols.
Other examples of fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, GFP fusion or CBP fusion, and a sequence for directing protein secretion and/or membrane association.
D. Cytochrome P450 Reductase Polypeptides And Encoding Nucleic Acid Molecules
Provided herein are cytochrome P450 reductase polypeptides. Also provided herein are nucleic acid molecules that encode any of the cytochrome P450 reductase polypeptides provided herein. The cytochrome P450 reductase polypeptides provided herein transfer two electrons from NADPH to a cytochrome P450. In some examples, the nucleic acid molecules that encode the cytochrome P450 reductase polypeptides are those that are the same as those that are isolated from the sandalwood tree Santalum album. In other examples, the nucleic acid molecules and encoded cytochrome P450 reductase polypeptides are variants of those isolated from the sandalwood tree Santalum album. 
Also provided herein are modified cytochrome P450 reductase polypeptides and nucleic acid molecules that encode any of the modified cytochrome P450 reductase polypeptides provided herein. The modifications can be made in any region of a cytochrome P450 reductase polypeptide provided the cytochrome P450 reductase polypeptide at least retains the CPR catalytic activity of the unmodified cytochrome P450 reductase polypeptide. For example, modifications can be made to a cytochrome P450 reductase polypeptide provided that the cytochrome P450 reductase polypeptide retains CPR activity (i.e., the ability to transfer two electrons from NADPH to a cytochrome P450).
The modifications can include codon optimization of the nucleic acids and/or changes that result in a single amino acid modification in the encoded polypeptide, such as single amino acid replacement (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions, including swaps of domains or regions of the polypeptide. In some examples, entire or partial domains or regions, such as any domain or region described herein, are exchanged with corresponding domains or regions or portions thereof from another cytochrome P450 reductase polypeptide. Exemplary of modifications are amino acid replacements, including single or multiple amino acid replacements. For example, modified cytochrome P450 reductase polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120 or more modified positions compared to the cytochrome P450 reductase polypeptide not containing the modification.
Provided herein are cytochrome P450 reductase polypeptides having a sequence of amino acids set forth in SEQ ID NO:12 or 13. Also provided herein are cytochrome P450 reductase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13. For example, the cytochrome P450 reductase polypeptides provided herein can exhibit at least at or at least about or 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13, provided that the resulting cytochrome P450 reductase polypeptide at least retains CPR activity (i.e., the ability to transfer two electrons from NADPH to a cytochrome P450). Percent identity can be determined by one skilled in the art using standard alignment programs.
Also, in some examples, provided herein are catalytically active fragments of cytochrome P450 reductase polypeptides. In some examples, the active fragments of cytochrome P450 reductase polypeptides are modified as described above. Such fragments retain one or more properties of a full-length cytochrome P450 reductase polypeptide. Typically, the active fragments exhibit CPR activity (i.e., the ability to transfer two electrons from NADPH to a cytochrome P450).
The cytochrome P450 reductase polypeptides provided herein can contain other modifications, for example, modifications not in the primary sequence of the polypeptide, including post-translational modifications. For example, modification described herein can be a cytochrome P450 reductase polypeptide that is a fusion polypeptide or chimeric polypeptide, including hybrids of different cytochrome P450 reductase polypeptides (e.g. contain one or more domains or regions from another cytochrome P450 reductase polypeptide) and also synthetic cytochrome P450 reductase polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon the sequence of known polypeptides.
The cytochrome P450 reductase polypeptides provided herein can be used to transfer two electrons from NADPH to a cytochrome P450. Reactions can be performed in vivo, such as in a host cell into which the nucleic acid has been introduced. At least one of the polypeptides will be heterologous to the host. Reactions can also be performed in vitro by contacting with enzyme the appropriate substrate under appropriate conditions.
Also provided herein are nucleic acid molecules encoding a cytochrome P450 polypeptide and a cytochrome P450 reductase polypeptide. For example, provided herein are nucleic acid molecules encoding a cytochrome P450 santalene oxidase polypeptide and a cytochrome P450 reductase polypeptide. In another example, nucleic acid molecules encoding a cytochrome P450 bergamotene synthase polypeptide and a cytochrome P450 reductase polypeptide. Also provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 polypeptide and a cytochrome P450 reductase polypeptide. For example, provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 santalene oxidase polypeptide and a cytochrome P450 reductase polypeptide. In another example, provided herein are nucleic acid molecules encoding a santalene synthase, cytochrome P450 bergamotene oxidase polypeptide and a cytochrome P450 reductase polypeptide. The nucleic acid molecules can be in the same vector or plasmid or on different vectors or plasmids. In such examples, expression of the nucleic acid molecule(s) in a suitable host, for example, a bacterial or yeast cell, results in expression of cytochrome P450 oxidase and cytochrome P450 reductase, or results in expression of santalene synthase, cytochrome P450 oxidase and cytochrome P450 reductase, depending on the included nucleic acid molecules. Such cells can be used to produce the santalene synthases, the cytochrome P450 oxidases and the cytochrome P450 reductases and/or to perform reactions in vivo to produce santalols and bergamotols. For example, santalols and bergamotols can be generated in a host cell from farnesyl diphosphate (FPP), particularly a yeast cell that overproduces the acyclic terpene precursor FPP. In some examples, a nucleic acid molecule encoding a farnesyl diphosphate synthase, such as a Santalum album farnesyl diphosphate synthase, can also be expressed in the suitable host, for example, a bacterial or yeast cell, resulting in over-expression of FPP.
1. Cytochrome P450 Reductase Polypeptides
Provided herein are cytochrome P450 reductase polypeptides. Also provided herein are nucleic acid molecules that encode any of the cytochrome P450 reductase polypeptides provided herein. The cytochrome P450 reductase polypeptides provided herein exhibit CPR activity. Typically, the cytochrome P450 reductase polypeptides provided herein the ability to transfer two electrons from NADPH to a cytochrome P450.
For example, provided herein are cytochrome P450 reductase polypeptides that have a sequence of amino acids set forth in SEQ ID NO:12 or 13. Also provided herein are cytochrome P450 reductase polypeptides that exhibit at least 60% amino acid sequence identity to a cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12 or 13. For example, the cytochrome P450 reductase polypeptides provided herein can exhibit at least at or about or 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more amino acid sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13, provided the cytochrome P450 reductase polypeptides exhibit cytochrome P450 reductase activity (i.e. transfer two electrons from NADPH to a cytochrome P450). Percent identity can be determined by one skilled in the art using standard alignment programs.
Also provided herein are active fragments of cytochrome P450 reductase polypeptides having a sequence of amino acids set forth in SEQ ID NO:12 or 13. For example, provided herein are truncated cytochrome P450 reductase polypeptides having a sequence of amino acids set forth in SEQ ID NO:14 or 15. Such fragments retain one or more properties of a cytochrome P450 reductase polypeptide. Typically, the active fragments exhibit cytochrome P450 reductase activity (i.e. transfer two electrons from NADPH to a cytochrome P450).
Also provided herein are nucleic acid molecules that have a sequence of amino acids set forth in SEQ ID NO:10 or 11, or degenerates thereof, that encode a cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12 or 13, respectively. Also provided herein are nucleic acid molecules encoding a cytochrome P450 reductase polypeptide having at least 85% sequence identity to a sequence of nucleotides set forth in SEQ ID NO:10 or 11. For example, the nucleic acid molecules provided herein can exhibit at least or about at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence of nucleotides set forth in SEQ ID NO:10 or 11, so long as the encoded cytochrome P450 reductase polypeptide exhibits cytochrome P450 reductase activity (i.e. the ability to transfer two electrons from NADPH to a cytochrome P450). Also provided herein are degenerate sequences of the sequences set forth in SEQ ID NO:10 or 11 encoding a cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12 or 13, respectively. Percent identity can be determined by one skilled in the art using standard alignment programs.
In some examples, the nucleic acid molecules that encode the cytochrome P450 reductase polypeptides are isolated from the sandalwood tree Santalum album. In other examples, the nucleic acid molecules and encoded cytochrome P450 reductase polypeptides are variants of those isolated from the sandalwood tree Santalum album. 
2. Modified Cytochrome P450 Reductase Polypeptides
Provided herein are modified cytochrome P450 reductase polypeptides. The modifications can be made in any region of a cytochrome P450 reductase polypeptide provided the resulting modified cytochrome P450 reductase polypeptides at least retain cytochrome P450 reductase activity (e.g. the ability to transfer two electrons from NADPH to a cytochrome P450).
The modifications can be a single amino acid modification, such as single amino acid replacements (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions. In some examples, entire or partial domains or regions, such as any domain or region described herein below, are exchanged with corresponding domains or regions or portions thereof from another cytochrome P450 reductase polypeptide. Exemplary of modifications are amino acid replacements, including single or multiple amino acid replacements. For example, modified cytochrome P450 reductase polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, 120 or more modified positions compared to the cytochrome P450 reductase polypeptide not containing the modification.
The modifications described herein can be in any cytochrome P450 reductase polypeptide. For example, the modifications described herein can be in a cytochrome P450 reductase having a sequence of amino acids set forth in any of SEQ ID NOS:12-15 or any variant thereof, including any that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cytochrome P450 reductase having a sequence of amino acids set forth in any of SEQ ID NOS:12-15.
In particular, modified cytochrome P450 reductase polypeptides provided herein contain amino acid replacements or substitutions, additions or deletions, truncations or combinations thereof with reference to the cytochrome P450 reductase polypeptide having a sequence of amino acids set forth in SEQ ID NO:12. It is within the level of one of skill in the art to make such modifications in cytochrome P450 reductase polypeptides, such as any set forth in SEQ ID NOS:12-15 or any variant thereof. Based on this description, it is within the level of one of skill in the art to generate a cytochrome P450 reductase polypeptide containing any one or more of the described mutations, and test each for cytochrome P450 reductase activity described herein, such as the ability to transfer two electrons from NADPH to cytochrome P450.
Also, in some examples, provided herein are modified active fragments of cytochrome P450 reductase polypeptides that contain any of the modifications provided herein. Such fragments retain on or more properties of a cytochrome P450 reductase, such as the ability to transfer two electrons from NADPH to cytochrome P450. Modifications in a cytochrome P450 reductase polypeptide also can be made to a cytochrome P450 reductase polypeptide that also contains other modifications, including modifications of the primary sequence and modifications not in the primary sequence of the polypeptide. For example, modification described herein can be in a cytochrome P450 reductase polypeptide that is a fusion polypeptide or chimeric polypeptide with different cytochrome P450 reductase polypeptides (e.g. contain one or more domains or regions from another cytochrome P450 reductase s) and also synthetic cytochrome P450 reductase polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon the sequence of known polypeptides.
In some examples, the modifications are amino acid replacements. In further examples, the modified cytochrome P450 reductase polypeptides provided herein contain one or more modifications in a domain. For example, the modifications in a domain or structural domain can be by replacement of corresponding heterologous residues from another cytochrome P450 reductase polypeptide.
To retain cytochrome P450 reductase activity, modifications typically are not made at those positions necessary for cytochrome P450 reductase activity, i.e., in the catalytic center or in conserved residues. For example, generally modifications are not made a position corresponding to Ser485, Cys657, Asp702 and Trp704 with reference to a sequence of amino acids set forth in SEQ ID NO:12.
The modified cytochrome P450 reductase polypeptides provided herein can contain two or more modifications, including amino acid replacements or substitutions, insertions or deletions, truncations or combinations thereof. Generally, multiple modifications provided herein can be combined by one of skill in the art so long as the modified cytochrome P450 reductase polypeptide retains cytochrome P450 reductase activity.
Also provided herein are nucleic acid molecules that encode any of the modified cytochrome P450 reductase polypeptides provided herein. In particular examples, the nucleic acid sequence can be codon optimized, for example, to increase expression levels of the encoded sequence. The particular codon usage is dependent on the host organism in which the modified polypeptide is expressed. One of skill in the art is familiar with optimal codons for expression in bacteria or yeast, including for example E. coli or Saccharomyces cerevisiae. For example, codon usage information is available from the Codon Usage Database available at, for example, kazusa.or.jp.codon (see, e.g., Richmond (2000) Genome Biology, 1:241 for a description of the database). See also, Forsburg (2004) Yeast, 10:1045-1047; Brown et al. (1991) Nucleic Acids Research, 19:4298; Sharp et al. (1988) Nucleic Acids Research, 12:8207-8211; Sharp et al. (1991) Yeast, 657-78. In examples herein, nucleic acid sequences provided herein are codon optimized based on codon usage in Saccharomyces cerevisiae. 
The modified polypeptides and encoding nucleic acid molecules provided herein can be produced by standard recombinant DNA techniques known to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed or random mutagenesis of encoding nucleic acid molecules, or solid phase polypeptide synthesis methods. For example, as described herein, nucleic acid molecules encoding a cytochrome P450 reductase polypeptide can be subjected to mutagenesis, such as random mutagenesis of the encoding nucleic acid, by error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling, or other recombinant methods. The nucleic acid encoding the polypeptides then can be introduced into a host cell to be expressed heterologously. Hence, also provided herein are nucleic acid molecules encoding any of the modified polypeptides provided herein. In some examples, the modified cytochrome P450 reductase polypeptides are produced synthetically, such as using solid phase or solutions phase peptide synthesis.
3. Additional Modifications
Provided herein are cytochrome P450 reductase polypeptides that contain additional modifications. For example, modified cytochrome P450 reductase polypeptides include, for example, truncated cytochrome P450 reductase polypeptides, cytochrome P450 reductase polypeptides having altered activities or properties, chimeric cytochrome P450 reductase polypeptides, cytochrome P450 reductase polypeptides containing domain swaps, cytochrome P450 reductase fusion proteins, or cytochrome P450 reductase polypeptides having any modification described elsewhere herein.
a. Truncated Polypeptides
Also provided herein are truncated cytochrome P450 reductase polypeptides. The truncated cytochrome P450 reductase polypeptides can be truncated at the N-terminus or C-terminus, so long as the truncated cytochrome P450 reductase polypeptides retain the catalytic activity of a cytochrome P450 reductase, such as cytochrome P450 reductase activity. Typically, the truncated cytochrome P450 reductase polypeptides exhibit cytochrome P450 reductase activity (i.e., the ability to transfer two electrons from NADPH to cytochrome P450). In some examples, the cytochrome P450 reductase polypeptides are truncated at the C-terminus. In other examples, the cytochrome P450 reductase polypeptides are truncated at the N-terminus.
In some examples, the cytochrome P450 reductase polypeptides are truncated at the N-terminus, C-terminus or both termini of a cytochrome P450 reductase polypeptide provided herein, such as truncation of a sequence of amino acids set forth in any of SEQ ID NOS:12 or 13. In other examples, any of the modified cytochrome P450 reductase polypeptides provided herein are truncated. The modified cytochrome P450 reductase polypeptides can be truncated at their N-terminus, C-terminus, or both termini. For example, any cytochrome P450 reductase polypeptide provided herein can be truncated by at or about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more amino acid residues at the N-terminus, provided the cytochrome P450 reductase polypeptide retains cytochrome P450 reductase activity. In other examples, any cytochrome P450 reductase polypeptide provided herein can be truncated by at or about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more amino acid residues at the C-terminus, provided the cytochrome P450 reductase polypeptide retains cytochrome P450 reductase activity. In some examples, cytochrome P450 reductases can be truncated by digestion with pancreatic steapsin or trypsin, which releases the N-terminal hydrophobic anchor.
For example, provided herein are truncated cytochrome P450 reductase polypeptides having a sequence of amino acids set forth in SEQ ID NO:14 or 15. Also provided herein are truncated cytochrome P450 reductase polypeptides having a sequence of amino acids having at least or at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a truncated cytochrome P450 reductase having a sequence of amino acids set forth in SEQ ID NO:14 or 15, provided the resulting cytochrome P450 reductase polypeptide at least retains cytochrome P450 reductase activity (i.e., the ability to transfer two electrons from NADPH to cytochrome P450). Also provided herein are nucleic acid molecules having a sequence of nucleotides set forth in SEQ ID NOS:63 or 64 that encode the truncated cytochrome P450 reductase polypeptides having a sequence of amino acids set forth in SEQ ID NO:14 or 15, respectively.
b. Polypeptides with Altered Activities or Properties
The modified cytochrome P450 reductase polypeptides provided herein can also exhibit changes in activities and/or properties. The modified cytochrome P450 reductase polypeptides can exhibit, for example, improved properties, such as increased catalytic activity, increased stability, and/or increased expression in a host cell. In other examples, the modified cytochrome P450 reductase polypeptide exhibits a similar, increased and/or improved activity compared to the unmodified cytochrome P450 reductase polypeptide.
c. Domain Swaps
Provided herein are modified cytochrome P450 reductase polypeptides that are chimeric polypeptides containing a swap (deletion and insertion) by deletion of amino acid residues of one of more domains or regions therein or portions thereof and insertion of a heterologous sequence of amino acids. In some examples, the heterologous sequence is a randomized sequence of amino acids. In other examples, the heterologous sequence is a contiguous sequence of amino acids for the corresponding domain or region or portion thereof from another cytochrome P450 reductase. The heterologous sequence that is replaced or inserted generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids. In examples where the heterologous sequence is from a corresponding domain or a portion thereof of another cytochrome P450 reductase, the heterologous sequence generally includes at least 50%, 60%, 70%, 80%, 90%, 95% or more contiguous amino acids of the corresponding domain or region or portion. In such an example, adjacent residues to the heterologous corresponding domain or region or portion thereof also can be included in a modified cytochrome P450 reductase polypeptide provided herein.
In one example of swap mutants provided herein, at least one domain or region or portion thereof of a cytochrome P450 reductase polypeptide is replaced with a contiguous sequence of amino acids for the corresponding domain or region or portions thereof from another cytochrome P450 reductase polypeptide. In some examples, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains or regions or portions thereof are replaced with a contiguous sequence of amino acids for the corresponding domain or region or portions thereof from another cytochrome P450 reductase polypeptide.
Any domain or region or portion thereof of a cytochrome P450 reductase polypeptide can be replaced with a heterologous sequence of amino acids, such as heterologous sequence from the corresponding domain or region from another cytochrome P450 reductase. A domain or region can be a structural domain or a functional domain. One of skill in the art is familiar with domains or regions in cytochrome P450 reductases. Functional domains include, for example, the catalytic domain or a portion thereof. A structural domain can include all or a portion of α-helix A; β-strand 1; α-helix B; β-strand 2; α-helix C; β-strand 3; α-helix D; β-strand 4; α-helix E; β-strand 5; α-helix F; β-strand 6; β-strand 7; β-strand 8, β-strand 9; β-strand 10; α-helix G; β-strand 11; β-strand 12; β-strand 12′; α-helix H; α-helix I; α-helix J; α-helix K; α-helix M; β-strand 13; β-strand 14; β-strand 15; α-helix N; β-strand 16; β-strand 16′; β-strand 17; α-helix O; β-strand 18; α-helix P; β-strand 10; α-helix Q; α-helix R; β-strand 20; α-helix S; α-helix T; and β-strand 21. One of skill in the art is familiar with various cytochrome P450s and can identify corresponding domains or regions or portions of amino acids thereof. Typically, the resulting modified cytochrome P450 reductase polypeptides exhibit cytochrome P450 reductase activity.
Any methods known in the art for generating chimeric polypeptides can be used to replace all or a contiguous portion of a domain or a cytochrome P450 reductase with all or a contiguous portion of the corresponding domain of a second cytochrome P450 reductase (see, U.S. Pat. Nos. 5,824,774, 6,072,045, 7,186,891 and 8,106,260, and U.S. Pat. Pub. No. 20110081703). Also, gene shuffling methods can be employed to generate chimeric polypeptides and/or polypeptides with domain or region swaps.
For example, corresponding domains or regions of any two cytochrome P450 reductases can be exchanged using any suitable recombinant method known in the art, or by in vitro synthesis. Exemplary of recombinant methods is a two stage overlapping PCR method, such as described herein. In such methods, primers that introduce mutations at a plurality of codon positions in the nucleic acids encoding the targeted domain or portion thereof in the first cytochrome P450 reductase can be employed; the mutations together form the heterologous region (i.e. the corresponding region from the second cytochrome P450 reductase). Alternatively, for example, randomized amino acids can be used to replace particular domains or regions. It is understood that primer errors, PCR errors and/or other errors in the cloning or recombinant methods can result in errors such that the resulting swapped or replaced region or domain does not exhibit an amino acid sequence that is identical to the corresponding region from the second cytochrome P450 reductase synthase.
In an exemplary PCR-based method, the first stage PCR uses (i) a downstream primer that anneals downstream of the region that is being replaced with a mutagenic primer that includes approximately fifteen nucleotides (or an effective number to effect annealing, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 nucleotides or more) of homologous sequence on each side of the domain or region to be exchanged or randomized flanking the region to be imported into the target gene, and (ii) an upstream primer that anneals upstream of the region that is being replaced together with an opposite strand mutagenic primer that also includes approximately fifteen nucleotides (or an effective number to effect annealing, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 nucleotides or more) of homologous sequence on each side of the domain or region to be exchanged or randomized flanking the region to be imported into the target gene. If a replacement in which a domain or region of a first cytochrome P450 reductase gene is replaced with the corresponding domain or region from a second cytochrome P450 reductase is being performed, nucleotides in the mutagenic primers between the flanking regions from the first cytochrome P450 reductase contain codons for the corresponding region of the second cytochrome P450 reductase. In instances where the amino acids in a domain or region are to be randomized, nucleotides of the mutagenic primers between the flanking regions from the first cytochrome P450 reductase contains random nucleotides. An overlapping PCR is then performed to join the two fragments, using the upstream and downstream oligo. The resulting PCR product then can be cloned into any suitable vector for expression of the modified cytochrome P450 reductase.
Further, any of the modified cytochrome P450 reductase polypeptides containing swap mutations herein can contain one or more further amino acid replacements as described herein above.
d. Additional Variants
Cytochrome P450 reductase polypeptides provided herein can be modified by any method known to one of skill in the art for generating protein variants, including, but not limited to, DNA or gene shuffling, error prone PCR, overlap PCR or other recombinant methods. In one example, nucleic acid molecules encoding any cytochrome P450 reductase polypeptide or variant cytochrome P450 reductase polypeptide provided herein can be modified by gene shuffling. Gene shuffling involves one or more cycles of random fragmentation and reassembly of at least two nucleotide sequences, followed by screening to select nucleotide sequences encoding polypeptides with desired properties. The recombination can be performed in vitro (see Stemmer et al. (1994) Proc Natl Acad Sci USA 91:10747-10751; Stemmer et al. (1994) Nature 370:389-391; Cramieri et al. (1998) Nature 391:288-291; U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458) or in vivo (see, International Pat. Pub. No. WO199707205). The nucleic acid molecules encoding the polypeptides then can be introduced into a host cell to be expressed heterologously and tested for their cytochrome P450 reductase activity by any method described in section G below.
e. Fusion or Chimeric Proteins
Nucleic acid molecules provided herein include fusion or chimeric nucleic acid molecules that contain a cytochrome P450 polypeptide and a cytochrome P450 reductase polypeptide. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that is capable of catalyzing the formation of a santalol or bergamotol, such as an α-santalol, β-santalol, epi-β-santalol or Z-α-trans-bergamotol, from santalenes or bergamotene that contains any cytochrome P450 polypeptide and any cytochrome P450 reductase polypeptide provided herein. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a cytochrome P450 polypeptide set forth in any of SEQ ID NOS:6-9 and a cytochrome P450 reductase polypeptide set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a cytochrome P450 polypeptide set forth in any of SEQ ID NOS:6-9 and a cytochrome P450 reductase polypeptide set forth in any of SEQ ID NOS:12-15. The fusion polypeptides can be linked directly or via a linker.
Nucleic acid molecules provided herein include fusion or chimeric nucleic acid molecules that contain a santalene synthase, cytochrome P450 polypeptide and a cytochrome P450 reductase. For example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53, a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO:7 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53, a cytochrome P450 santalene oxidase polypeptide set forth in SEQ ID NO:7 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. In another example, provided herein are nucleic acid molecules encoding a fusion polypeptide that contains a santalene synthase set forth in any of SEQ ID NOS:17, 52 or 53, a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8 or 9 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. Also provided herein are fusion polypeptides containing a santalene synthase set forth in any of SEQ ID NOS: 17, 52 or 53, a cytochrome P450 bergamotene oxidase polypeptide set forth in any of SEQ ID NOS:6, 8 or 9 and a cytochrome P450 reductase set forth in any of SEQ ID NOS:12-15. The fusion polypeptides can be linked directly or via a linker.
In another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 and/or a cytochrome P450 reductase, such that, when expressed in a host cell, a bacterial or yeast host cell, a santalene synthase, a cytochrome P450 and/or a cytochrome P450 reductase are expressed. In one another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 santalene oxidase and a cytochrome P450 reductase. In another example, provided herein is a nucleic acid molecule that encodes a santalene synthase, a cytochrome P450 bergamotene oxidase and a cytochrome P450 reductase. Further, when the host cell is capable of producing FPP, the encoded polypeptides catalyze the production of santalols and/or bergamotols.
Other examples of fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, GFP fusion or CBP fusion, and a sequence for directing protein secretion and/or membrane association.
E. Methods for Producing Modified Cytochrome P450 and Cytochrome P450 Reductase Polypeptides and Encoding Nucleic Acid Molecules
Provided are methods for producing modified cytochrome P450 and cytochrome P450 reductase polypeptides, including santalene oxidase and bergamotene oxidase polypeptides. The methods can be used to generate cytochrome P450s and cytochrome P450 reductases with desired properties, including, but not limited to, increased catalytic activity, increased selectivity, increased substrate specificity, increased substrate binding, increased stability, increased expression in a host cell, altered product distribution and/or altered substrate specificity. Modified cytochrome P450s and cytochrome P450 reductases can be produced using any method known in the art and, optionally, screened for the desired properties. In particular examples, modified cytochrome P450s and cytochrome P450 reductases with desired properties are generated by mutation in accord with the methods exemplified herein. Thus, provided herein are modified cytochrome P450s and cytochrome P450 reductases and nucleic acid molecules encoding the modified cytochrome P450s and cytochrome P450 reductases that are produced using the methods described herein.
Exemplary of the methods provided herein are those in which modified cytochrome P450s and cytochrome P450 reductases are produced by replacing one or more endogenous domains or regions of a first cytochrome P450 or cytochrome P450 reductase with the corresponding domain(s) or regions(s) from a second cytochrome P450 or cytochrome P450 reductase (i.e. heterologous domains or regions). In further examples, two or more endogenous domains or regions of a first cytochrome P450 or cytochrome P450 reductase are replaced with the corresponding heterologous domain(s) or regions(s) from two or more other cytochrome P450s or cytochrome P450 reductases, such as a second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth cytochrome P450s or cytochrome P450 reductases. Thus, the resulting modified cytochrome P450 or cytochrome P450 reductase can include heterologous domains or regions from 1, 2, 3, 4, 5, 6, 7, 8, 9 or more different cytochrome P450s or cytochrome P450 reductases. In further examples, the methods also or instead include replacing one or more domains or regions of a first cytochrome P450 or cytochrome P450 reductase synthase with randomized amino acid residues.
Any cytochrome P450 or cytochrome P450 reductase can be used in the methods provided herein. The first cytochrome P450 or cytochrome P450 reductase (i.e. the cytochrome P450 or cytochrome P450 reductase to be modified) can be of the same or different class as the second (or third, fourth, fifth, etc.) cytochrome P450 or cytochrome P450 reductase (i.e. the cytochrome P450(s) or cytochrome P450 reductase(s) from which the heterologous domain(s) or region(s) is derived).
In practicing the methods provided herein, all or a contiguous portion of an endogenous domain of a first cytochrome P450 or cytochrome P450 reductase can be replaced with all or a contiguous portion of the corresponding heterologous domain from a second cytochrome P450 or cytochrome P450 reductase. For example, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous amino acids from a domain or region in a first cytochrome P450 or cytochrome P450 reductase can be replaced with 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous amino acids from the corresponding region from a second cytochrome P450 or cytochrome P450 reductase. In some examples, one or more amino acid residues adjacent to the endogenous domain of the first cytochrome P450 or cytochrome P450 reductase also are replaced, and/or one or more amino acid residues adjacent to the heterologous domain also are used in the replacement. Further, the methods provided herein also include methods in which all or a contiguous portion of a first domain and all or a contiguous portion of a second adjacent domain are replaced with the corresponding domains (or portions thereof) from another cytochrome P450 or cytochrome P450 reductase.
Domains or regions that can be replaced include functional domains or structural domains. Exemplary domains or regions that can be replaced in a cytochrome P450 using the methods described herein include, but are not limited to, structural domains or regions corresponding to helix A, β strand 1-1, β strand 1-2, helix B, β strand 1-5, helix B′, helix C, helix C′, helix D, β strand 3-1, helix E, helix F, helix G, helix H, β strand 5-1, β strand 5-2, helix I, helix J, helix J′, helix K, β strand 1-4, β strand 2-1, β strand 2-2, β strand 1-3, helix K′, helix K′′, Heme domain, helix L, β strand 3-3, β strand 4-1, β strand 4-2 and β strand 3-2. Any one or more of these domains or regions, or a portion thereof, can be replaced with a corresponding domain from another cytochrome P450 using the methods provided herein. These domains are regions can be identified in any cytochrome P450 using methods well known in the art, such as, for example, by alignment using methods known to those of skill in the art (see, e.g., FIG. 5A-5B). Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTP) and others known to those of skill in the art. By aligning the sequences of the cytochrome P450 set forth in SEQ ID NO:50, and any other cytochrome P450, any of the domains or regions recited above can be identified in any cytochrome P450.
Exemplary domains or regions that can be replaced in a cytochrome P450 reductase using the methods described herein include, but are not limited to, structural domains or regions corresponding to α-helix A; β-strand 1; α-helix B; β-strand 2; α-helix C; β-strand 3; α-helix D; β-strand 4; α-helix E; β-strand 5; α-helix F; β-strand 6; β-strand 7; β-strand 8, β-strand 9; β-strand 10; α-helix G; β-strand 11; β-strand 12; β-strand 12′; α-helix H; α-helix I; α-helix J; α-helix K; α-helix M; β-strand 13; β-strand 14; β-strand 15; α-helix N; β-strand 16; β-strand 16′; β-strand 17; α-helix O; β-strand 18; α-helix P; β-strand 10; α-helix Q; α-helix R; β-strand 20; α-helix S; α-helix T; and β-strand 21. These domains are regions can be identified in any cytochrome P450 reductase using methods well known in the art, such as, for example, by alignment using methods known to those of skill in the art (see, e.g., FIGS. 3A-3C). Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTP) and others known to those of skill in the art. By aligning the sequences of the cytochrome P450 reductase set forth in SEQ ID NO:12, and any other cytochrome P450 reductase, any of the domains or regions recited above can be identified in any cytochrome P450 reductase.
In the methods provided herein, all or a contiguous portion of an endogenous domain of a first cytochrome P450 or cytochrome P450 reductase can be replaced with all or a contiguous portion of the corresponding heterologous domain from a second cytochrome P450 or cytochrome P450 reductase using an suitable recombinant method known in the art as discussed above in Sections C.4.c. and D.3.c.
F. Expression of Cytochrome P450 and Cytochrome P450 Reductase Polypeptides and Encoding Nucleic Acid Molecules
Cytochrome P450 and cytochrome P450 reductase polypeptides and active fragments thereof, including cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, can be obtained by methods well known in the art for recombinant protein generation and expression. Such cytochrome P450 santalene oxidase polypeptides can be used to produce santalols from santalenes in a host cell from which the cytochrome P450 santalene oxidase is expressed or in vitro following purification of the cytochrome P450 santalene oxidase polypeptide. Such cytochrome P450 bergamotene oxidase polypeptides can be used to produce bergamotols from bergamotenes in a host cell from which the cytochrome P450 bergamotene oxidase is expressed or in vitro following purification of the cytochrome P450 bergamotene oxidase polypeptide. Such cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides can be used to produce santalols or bergamotols from a suitable acyclic pyrophosphate precursor, such as FPP, in a host cell in which a santalene synthase and the cytochrome P450 are expressed. Any method known to those of skill in the art for identification of nucleic acids that encode desired genes can be used to obtain the nucleic acid encoding a cytochrome P450, such as a cytochrome P450 santalene oxidase or cytochrome P450 bergamotene oxidase, or cytochrome P450 reductase. For example, nucleic acid encoding unmodified or wild type cytochrome P450 polypeptides or cytochrome P450 reductase polypeptides can be obtained using well known methods from a plant source, such as Santalum album. Modified cytochrome P450 polypeptides or cytochrome P450 reductase polypeptides then can be engineered using any method known in the art for introducing mutations into unmodified or wild type cytochrome P450 polypeptides or cytochrome P450 reductase polypeptides, including any method described herein, such as random mutagenesis of the encoding nucleic acid by error-prone PCR, site-directed mutagenesis, overlap PCR, or other recombinant methods. The nucleic acids encoding the polypeptides then can be introduced into a host cell to be expressed heterologously.
In some examples, the cytochrome P450 polypeptides or cytochrome P450 reductase polypeptides provided herein, including cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides, are produced synthetically, such as using sold phase or solution phase peptide synthesis.
1. Isolation of Nucleic Acid Encoding Santalum album Cytochrome P450 and Cytochrome P450 Reductase Polypeptides
Nucleic acids encoding cytochrome P450s or cytochrome P450 reductases, such as cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase, can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening. In some examples, methods for amplification of nucleic acids can be used to isolate nucleic acid molecules encoding a cytochrome P450 or cytochrome P450 reductase polypeptide, including for example, polymerase chain reaction (PCR) methods. A nucleic acid containing material can be used as a starting material from which a cytochrome P450 or cytochrome P450 reductase-encoding nucleic acid molecule can be isolated. For example, DNA and mRNA preparations from Santalum species, including but not limited to Santalum album can be used to obtain cytochrome P450 or cytochrome P450 reductase genes. Nucleic acid libraries also can be used as a source of starting material. Primers can be designed to amplify a cytochrome P450 or cytochrome P450 reductase-encoding molecule, such as a cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase-encoding molecule. For example, primers can be designed based on known nucleic acid sequences encoding a cytochrome P450 such as those set forth in SEQ ID NOS:22-25. In another example, primers can be designed based on known nucleic acid sequences encoding a cytochrome P450 reductase such as those set forth in SEQ ID NOS:40-41. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a cytochrome P450 or cytochrome P450 reductase polypeptide. The nucleic acid molecules provided herein can be used to identify related nucleic acid molecules in other species.
Additional nucleotide sequences can be joined to a cytochrome P450 or cytochrome P450 reductase-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a cytochrome P450 or cytochrome P450 reductase-encoding nucleic acid molecule. Still further, nucleic acid encoding other moieties or domains also can be included so that the resulting synthase is a fusion protein. For example, nucleic acids encoding other enzymes, such as FPP synthase or santalene synthase, or protein purification tags, such as His or Flag tags.
2. Generation of Modified Nucleic Acid
Nucleic acid encoding a cytochrome P450 or cytochrome P450 reductase, such as a modified cytochrome P450 santalene oxidase polypeptides, modified cytochrome P450 bergamotene oxidase polypeptides or modified cytochrome P450 reductase polypeptides, can be prepared or generated using any method known in the art to effect mutation. Methods for modification include standard rational and/or random mutagenesis of encoding nucleic acid molecules (using e.g., error prone PCR, random site-directed saturation mutagenesis, DNA shuffling or rational site-directed mutagenesis, such as, for example, mutagenesis kits (e.g. QuikChange available from Stratagene)). In addition, routine recombinant DNA techniques can be used to generate nucleic acids encoding polypeptides that contain heterologous amino acid. For example, nucleic acid encoding chimeric polypeptides or polypeptides containing heterologous amino acid sequence, can be generated using a two-step PCR method, such as described above, and/or using restriction enzymes and cloning methodologies for routine subcloning of the desired chimeric polypeptide components.
Once generated, the nucleic acid molecules can be expressed in cells to generate modified cytochrome P450 or cytochrome P450 reductase polypeptides using any method known in the art. The modified cytochrome P450 or cytochrome P450 reductase polypeptides, such as modified cytochrome P450 santalene oxidase polypeptides, modified cytochrome P450 bergamotene oxidase polypeptides or modified cytochrome P450 reductase polypeptides, then can be assessed by screening for a desired property or activity, for example, for the ability to produce a terpenoid from a terpene substrate. In particular examples, modified cytochrome P450 or cytochrome P450 reductase polypeptides with desired properties are generated by mutation and screened for a property in accord with the examples exemplified herein. Typically, in instances where a modified cytochrome P450 santalene oxidase polypeptide is generated, the modified cytochrome P450 santalene oxidase polypeptides produce a santalol from a santalene. Typically, in instances where a modified cytochrome P450 bergamotene oxidase polypeptide is generated, the modified cytochrome P450 bergamotene oxidase polypeptides produce a bergamotol from a bergamotene.
3. Vectors and Cells
For recombinant expression of one or more of the cytochrome P450 or cytochrome P450 reductase polypeptides provided herein, including cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides, the nucleic acid containing all or a portion of the nucleotide sequence encoding the synthase can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. Depending upon the expression system used, the necessary transcriptional and translational signals also can be supplied by the native promoter for a cytochrome P450 or cytochrome P450 reductase gene, and/or their flanking regions. Thus, also provided herein are vectors that contain nucleic acid encoding any cytochrome P450 or cytochrome P450 reductase polypeptide provided herein. Exemplary vectors include but are not limited to pESC-LEU, pESC-LEU2d, and pYEDP60.
Cells, including prokaryotic and eukaryotic cells, containing the vector also are provided. Also provided are host cells containing nucleic acid molecules encoding cytochrome P450 polypeptides provided herein, including cytochrome P450 santalene oxidases, cytochrome P450 bergamotene oxidases and cytochrome P450 reductases. Such cells and host cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells. In particular examples, the cells or host cells are yeast cells, such as Saccharomyces cerevisiae or Pichia pastoris cells. In particular examples, the cells or host cells are Saccharomyces cerevisiae cells that express an acyclic pyrophosphate terpene precursor, such as farnesyl diphosphate (FPP). In some examples, the cells or host cells containing a cytochrome P450 provided herein can be modified to produce more FPP than an unmodified cell.
The cells are used to produce a cytochrome P450 or cytochrome P450 reductase polypeptide, such as cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides, by growing the above-described cells under conditions whereby the encoded cytochrome P450 or cytochrome P450 reductase is expressed by the cell. In some examples, the cytochrome P450 polypeptide, such as cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptide, are heterologous to the cell. In some instances, the expressed cytochrome P450 and/or cytochrome P450 reductases are purified. In other instances, the expressed cytochrome P450s and cytochrome P450 reductases, convert one or more santalenes or bergamotenes to one or more santalols or bergamotols in the host cell. In some examples, a santalene synthase, a cytochrome P450 santalene oxidase and a cytochrome P450 reductase are expressed thereby converting the acyclic pyrophosphate terpene precursor FPP to santalol. In other examples, a santalene synthase, a cytochrome P450 bergamotene oxidase and a cytochrome P450 reductase are expressed thereby converting the acyclic pyrophosphate terpene precursor FPP to bergamotol.
Any method known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate transcriptional/translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequences encoding a cytochrome P450 or cytochrome P450 reductase polypeptide or modified cytochrome P450 or cytochrome P450 reductase polypeptide, or domains, derivatives, fragments or homologs thereof, can be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. In one embodiment, the promoter is not native to the genes for a cytochrome P450 or cytochrome P450 reductase protein. Promoters that can be used include but are not limited to prokaryotic, yeast, mammalian and plant promoters. The type of promoter depends upon the expression system used, described in more detail below.
In one embodiment, a vector is used that contains a promoter operably linked to nucleic acids encoding a cytochrome P450 or cytochrome P450 reductase polypeptide or modified cytochrome P450 or cytochrome P450 reductase polypeptide, or a domain, fragment, derivative or homolog, thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Vectors and systems for expression of cytochrome P450 or cytochrome P450 reductase polypeptides are described.
4. Expression Systems
Cytochrome P450 or cytochrome P450 reductase polypeptides, including cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides (modified and unmodified) can be produced by any methods known in the art for protein production including in vitro and in vivo methods such as, for example, the introduction of nucleic acid molecules encoding the cytochrome P450 or cytochrome P450 reductase (e.g. cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase) into a host cell or host plant for in vivo production or expression from nucleic acid molecules encoding the cytochrome P450 or cytochrome P405 reductases (e.g. cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase) in vitro. Cytochrome P450 or cytochrome P450 reductase polypeptides such as cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase and modified cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides can be expressed in any organism suitable to produce the required amounts and forms of a synthase polypeptide. Expression hosts include prokaryotic and eukaryotic organisms such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.
Expression in eukaryotic hosts can include expression in yeasts such as those from the Saccharomyces genus (e.g. Saccharomyces cerevisiae) and Pichia genus (e.g. Pichia pastoris), insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as citrus, tobacco, corn, rice, algae, and lemna. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs.
Many expression vectors are available and known to those of skill in the art for the expression of a cytochrome P450 or cytochrome P450 reductase, such as cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase. Exemplary of expression vectors are those encoding a santalene synthase and a FPP synthase, including the vectors described in Example 7. The choice of expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells.
Cytochrome P450 or cytochrome P450 reductase polypeptides, including cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides and modified cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides, also can be used or expressed as protein fusions. For example, a fusion can be generated to add additional functionality to a polypeptide. Examples of fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, GFP fusion or CBP fusion, and a sequence for directing protein secretion and/or membrane association.
Methods of production of cytochrome P450 and cytochrome P450 reductase polypeptides, including cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase polypeptides, can include co-expression of an acyclic pyrophosphate terpene precursor, such as FPP, in the host cell. In some instances, the host cell naturally expresses FPP. Such a cell can be modified to express greater quantities of FPP (see e.g. U.S. Pat. Nos. 6,531,303, 6,689,593, 7,838,279 and 7,842,497). In other instances, a host cell that does not naturally produce FPP is modified genetically to produce FPP.
a. Prokaryotic Cells
Prokaryotes, especially E. coli, provide a system for producing large amounts of the cytochrome P450 and cytochrome P450 reductase polypeptides provided herein. Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. Exemplary expression vectors for transformation of E. coli cells, include, for example, the pGEM expression vectors, the pQE expression vectors, and the pET expression vectors (see, U.S. Pat. No. 4,952,496; available from Novagen, Madison, Wis.; see, also literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; pET 15b and pET19b (Novagen, Madison, Wis.), which contain a His-TagTM leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator; pACYC-Duet (Novagen, Madison, Wis.; SEQ ID NO:45).
Expression vectors for E. coli can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Exemplary prokaryotic promoters include, for example, the β-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) and the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful Proteins from Recombinant Bacteria”: in Scientific American 242:79-94 (1980)). Examples of inducible promoters include the lac promoter, the tip promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λPL promoter.
Cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and β-mercaptoethanol and denaturants (e.g., such as guanidine-HCl and urea) can be used to resolubilize the proteins. An alternative approach is the expression of cytochrome P450s and cytochrome P450 reductases in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases leading to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility. Typically, temperatures between 25° C. and 37° C. are used. Mutations also can be used to increase solubility of expressed proteins. Typically, bacteria produce aglycosylated proteins.
b. Yeast Cells
Yeast systems, such as, but not limited to, those from the Saccharomyces genus (e.g. Saccharomyces cerevisiae), Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris can be used to express the cytochrome P450s and cytochrome P450 reductases, such as cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides and modified cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, provided herein. Yeast expression systems also can be used to produce terpenes whose reactions are catalyzed by the synthases. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. In some examples, inducible promoters are used to regulate gene expression. Exemplary promoter sequences for expression of cytochrome P450 and cytochrome P450 reductase polypeptides in yeast include, among others, promoters for metallothionine, 3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem. 255:2073), or other glycolytic enzymes (Hess et al. (1968) J. Adv. Enzyme Reg. 7:149; and Holland et al. (1978) Biochem. 17:4900), such as enolase, glyceraldehyde phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et al. (1991) Gene, 107:285-195; and van den Berg et al. (1990) Bio/Technology, 8:135-139. Another alternative includes, but is not limited to, the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982), or a modified ADH1 promoter. Shuttle vectors replicable in yeast and E. coli can be constructed by, for example, inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
Yeast expression vectors can include a selectable marker such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of the transformed DNA. Exemplary vectors include pESC-Leu, pESC-Leu2D, pESC-His and pYEDP60. Proteins expressed in yeast are often soluble and co-expression with chaperonins, such as Bip and protein disulfide isomerase, can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisiae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site (e.g., the Kex-2 protease) can be engineered to remove the fused sequences from the polypeptides as they exit the secretion pathway.
Yeast naturally express the required proteins, including FPP synthase (ERG20; which can produce FPP) for the mevalonate-dependent isoprenoid biosynthetic pathway. Thus, expression of the cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides provided herein, in yeast cells can result in the production of sesquiterpenes, such as santalenes and bergamotenes from FPP, and santalols and bergamotols. Exemplary yeast cells for the expression of cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, include yeast modified to express increased levels of FPP. For example, yeast cells can be modified to produce less squalene synthase or less active squalene synthase (e.g. erg9 mutants; see e.g. U.S. Pat. Nos. 6,531,303 and 6,689,593). This results in accumulation of FPP in the host cell at higher levels compared to wild type yeast cells, which in turn can result in increased yields of sesquiterpenes and sesquiterpenoids (e.g. santalenes, bergamotenes, santalols and bergamotols). In another example, yeast cells can be modified to produce more FPP synthase by introduction of a FPP synthase gene, such as SaFPPS from Santalum album (SEQ ID NO:18). In some examples, the native FPP gene in such yeast can be deleted. Other modifications that enable increased production of FPP in yeast include, for example, but are not limited to, modifications that increase production of acetyl CoA, inactivate genes that encode enzymes that use FPP and GPP as substrate and overexpress HMG-CoA reductases, as described in U.S. Pat. No. 7,842,497. Exemplary modified yeast cells include, but are not limited to, YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63, his3-Δ200, leu2-Δ1), WAT11 (MATa, ade2-1, his3-11,-15; leu2-3,-112, ura3-1, canR, cyr+; containing chromosomally integrated Arabidopsis NADPH-dependent P450 reductase ATR1; see Pompon et al. (1995) Toxicol Lett 82-83:815-822; Ro et al. (2005) Proc Natl Acad Sci USA 102:8060-8065); and BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0; ATCC #201388), modified Saccharomyces cerevisiae strains CALI5-1 (ura3, leu2, his3, trp1, Δ erg9::HIS3, HMG2cat/TRP1::rDNA, dpp1, sue), ALX7-95 (ura3, his3, trp1, Δerg9::HIS3, HMG2cat/TRP1::rDNA, dpp1 sue), ALX11-30 (ura3, trp1, erg9def25, HMG2cat/TRP1::rDNA, dpp1, sue), which are known and described in one or more of U.S. Pat. Nos. 6,531,303, 6,689,593, 7,838,279, 7,842,497, and U.S. Pat. publication Nos. 20040249219 and 20110189717.
c. Plants and Plant Cells
Transgenic plant cells and plants can be used for the expression of cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides provided herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements, and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce proteins (see, for example, Mayfield et al. (2003) Proc Natl Acad Sci USA 100:438-442). Transformed plants include, for example, plants selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Medicago (alfalfa), Gossypium (cotton) and Brassica (rape). In some examples, the plant belongs to the species of Nicotiana tabacum, and is transformed with vectors that overexpress a cytochrome P450 and/or a cytochrome P450 reductase, such as described in U.S. Pat. Pub. No. 20090123984 and U.S. Pat. No. 7,906,710.
d. Insects and Insect Cells
Insects and insect cells, particularly a baculovirus expression system, can be used for expressing cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides provided herein (see, for example, Muneta et al. (2003) J. Vet. Med. Sci. 65(2):219-223). Insect cells and insect larvae, including expression in the haemolymph, express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typically, expression vectors use a promoter such as the polyhedrin promoter of baculovirus for high level expression. Commonly used baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda (see, e.g., Mizutani and Ohta (1998) Plant Physiology 116:357-367), Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems.
An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin.
e. Mammalian Expression
Mammalian expression systems can be used to express cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides provided herein and also can be used to produce terpenes whose reactions are catalyzed by the synthases. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha 1-antitrypsin, beta-globin, myelin basic protein, myosin light chain-2 and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase and thymidine kinase. Fusion with cell surface signaling molecules such as TCR-ζ and FcεRI-γ can direct expression of the proteins in an active state on the cell surface.
Many cell lines are available for mammalian expression including mouse, rat human, monkey, and chicken and hamster cells. Exemplary cell lines include, but are not limited to, BHK (i.e. BHK-21 cells), 293-F, CHO, CHO Express (CHOX; Excellgene), Balb/3T3, HeLa, MT2, mouse NS0 (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. One such example is the serum free EBNA-1 cell line (Pham et al. (2003) Biotechnol. Bioeng. 84:332-42).
f. Exemplary Host Cells
Exemplary host cells for expression of a cytochrome p450 polypeptide provided herein, such as a cytochrome P450 santalene oxidase, cytochrome P450 bergamotene oxidase or cytochrome P450 reductase, include prokaryotic and eukaryotic cells. Typically, the host cell produces an acyclic pyrophosphate terpene precursor. For example, the host cell produces farnesyl diphosphate. In some examples, the host cell can be a cell line that produces FPP as part of the mevalonate-dependent isoprenoid biosynthetic pathway (e.g. fungi, including yeast cells, and animal cells) or the mevalonate-independent isoprenoid biosynthetic pathway (e.g. bacteria and higher plants). In some examples, the host cell produces farnesyl diphosphate natively. In other examples, the host cell is modified to produce more farnesyl diphosphate compared to an unmodified cell. Exemplary host cells include bacteria, yeast, insect, plant and mammalian cells. In particular examples, the host cell is a yeast cell. For example, the yeast cell is a Saccharomyces genus cell, such as a Saccharomyces cerevisiae cell. In another example, the yeast cell is a Pichia genus cell, such as a Pichia pastoris cell. In other particular examples, the host cell is an Escherichia coli cell.
In particular examples, the host cell has been modified to overproduce FPP. Exemplary of such cells are modified yeast cells. For example, yeast cells that have been modified to produce less squalene synthase or less active squalene synthase (e.g. erg9 mutants; see e.g. U.S. Pat. Nos. 6,531,303 and 6,689,593) are useful in the methods provided herein to produce labdenediol diphosphate. Reduced squalene synthase activity results in accumulation of FPP in the host cell at higher levels compared to wild type yeast cells. Exemplary modified yeast cells include, but are not limited to, modified Saccharomyces cerevisiae strains YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63, his3-Δ200, leu2-Δ1), WAT11 (MATa, ade2-1, his3-11,-15; leu2-3,-112, ura3-1, canR, cyr+; containing chromosomally integrated Arabidopsis NADPH-dependent P450 reductase ATR1; see Pompon et al. (1995) Toxicol Lett 82-83:815-822; Ro et al. (2005) Proc Natl Acad Sci USA 102:8060-8065); and BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0; ATCC #201388). The use of such host cells for expression of a cytochrome P450 polypeptide provided herein allows for increased yields of the precursor FPP and thus allows for increased yields of santalenes and bergamotenes.
Provided herein are host cells containing any cytochrome P450 polypeptide or catalytically active fragment thereof provided herein. Provided herein are host cells containing a cytochrome P450 polypeptide or a catalytically active fragment thereof. In some examples, the host cell contains a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72. In other examples, the host cell contains a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72. In other examples, the host cell contains nucleic acid encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78. In yet other examples, the host cell contains nucleic acid encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78.
Provided herein are host cells containing a cytochrome P450 santalene oxidase or a catalytically active fragment thereof. In some examples, the host cell contains a cytochrome P450 santalene oxidase or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:3, 68, 69, 70 or 71. In other examples, the host cell contains a cytochrome P450 santalene oxidase or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:3, 68, 69, 70 or 71. In other examples, the host cell contains nucleic acid encoding a cytochrome P450 santalene oxidase or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77. In yet other examples, the host cell contains nucleic acid encoding a cytochrome P450 santalene oxidase or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:7, 74, 75, 76 or 77.
Provided herein are host cells containing a cytochrome P450 bergamotene oxidase or a catalytically active fragment thereof. In some examples, the host cell contains a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:2, 4, 5 or 67. In other examples, the host cell contains a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:2, 4, 5 or 67. In other examples, the host cell contains nucleic acid encoding a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 or 73. In yet other examples, the host cell contains nucleic acid encoding a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:6, 8, 9 or 73.
In some examples, any of the host cells provided herein containing a cytochrome P450 or catalytically active fragment thereof can further contain a terpene synthase. Provided herein are host cells containing a cytochrome P450 or catalytically active fragment thereof and a terpene synthase. In such examples, the terpene synthase can be a santalene synthase. For example, the terpene synthase is a santalene synthase having a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a santalene synthase having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a nucleic acid molecule encoding a santalene synthase. The encoding nucleic acid molecule has a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60, or a nucleic acid molecule encoding a santalene synthase. The nucleic acid molecule has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% percent identity to a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60.
Provided herein are host cells containing a cytochrome P450 or catalytically active fragment thereof and a santalene synthase having a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a santalene synthase having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a nucleic acid molecule encoding a santalene synthase. The nucleic acid molecule has a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60, or a nucleic acid molecule encoding a santalene synthase. The nucleic acid molecule has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% percent identity to a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60. In such examples, the cytochrome P450 or catalytically active fragment thereof is a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78.
In one example, provided herein is a host cell that contains a cytochrome P450 polypeptide or catalytically active fragment thereof and a santalene synthase. In another example, provided herein is a host cell that contains a cytochrome P450 santalene oxidase or catalytically active fragment thereof and a santalene synthase. In yet another example, provided herein is a host cell that contains a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof and a santalene synthase. Also provided herein are host cells containing a cytochrome P450 or catalytically active fragment thereof and a terpene synthase that further contain a cytochrome P450 reductase or catalytically active fragment thereof. In such examples, the terpene synthase can be a santalene synthase. For example, the terpene synthase is a santalene synthase having a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a santalene synthase having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:17, 52 or 53, or a nucleic acid molecule encoding a santalene synthase. The nucleic acid molecule has a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60, or a nucleic acid molecule encoding a santalene synthase. The nucleic acid molecule has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% percent identity to a sequence of nucleotides set forth in any of SEQ ID NOS:16, 59 or 60. In such examples, the cytochrome P450 reductase or catalytically active fragment thereof is a cytochrome P450 reductase or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:10 or 11, or a cytochrome P450 reductase or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:10 or 11, or a nucleic acid molecule encoding a cytochrome P450 reductase or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:12-15 or a nucleic acid molecule encoding a cytochrome P450 reductase or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:12-15. In such examples, the cytochrome P450 or catalytically active fragment thereof is a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78.
In one example, provided herein is a host cell containing a cytochrome P450 polypeptide or catalytically active fragment thereof, a santalene synthase and a cytochrome P450 reductase or catalytically active fragment thereof. In another example, provided herein is a host cell containing a cytochrome P450 santalene oxidase or catalytically active fragment thereof, a santalene synthase and a cytochrome P450 reductase or catalytically active fragment thereof. In yet another example, provided herein is a host cell containing a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof, a santalene synthase and a cytochrome P450 reductase or catalytically active fragment thereof.
Provided herein are host cells containing a cytochrome P450 reductase or a catalytically active fragment thereof. In some examples, the host cell contains a cytochrome P450 reductase or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:10 or 11. In other examples, the host cell contains a cytochrome P450 reductase or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:10 or 11. In other examples, the host cell contains nucleic acid encoding a cytochrome P450 reductase or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:12-15. In yet other examples, the host cell contains nucleic acid encoding a cytochrome P450 reductase or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:12-15.
In some examples, the host cell containing a cytochrome P450 reductase or catalytically active fragment thereof further contains a cytochrome P450 or catalytically active fragment thereof. For example, provided herein are host cells containing a cytochrome P450 reductase or a catalytically active fragment thereof and a cytochrome P450 or catalytically active fragment thereof. In such examples, the cytochrome P450 or catalytically active fragment thereof is a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a cytochrome P450 polypeptide or catalytically active fragment thereof has a sequence of nucleic acids that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of nucleotides set forth in any of SEQ ID NOS:1-5 and 67-72, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78, or a nucleic acid molecule encoding a cytochrome P450 polypeptide or catalytically active fragment thereof that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent sequence identity to a sequence of amino acids set forth in any of SEQ ID NOS:6-9, 50 and 73-78.
In one example, provided herein is a host cell containing a cytochrome P450 polypeptide or catalytically active fragment thereof and a cytochrome P450 reductase or catalytically active fragment thereof. In another example, provided herein is a host cell containing a cytochrome P450 santalene oxidase or catalytically active fragment thereof and a cytochrome P450 reductase or catalytically active fragment thereof. In yet another example, provided herein is a host cell containing a cytochrome P450 bergamotene oxidase or catalytically active fragment thereof and a cytochrome P450 reductase or catalytically active fragment thereof.
5. Purification
Methods for purification of cytochrome P450s and cytochrome P450 reductases, such as cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, from host cells depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary the proteins can be extracted and further purified using standard methods in the art.
Cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography and ionic exchange chromatography. Expression constructs also can be engineered to add an affinity tag such as a myc epitope, GST fusion or His6 and affinity purified with myc antibody, glutathione resin, and Ni-resin, respectively, to a protein. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques.
6. Fusion Proteins
Fusion proteins containing a cytochrome P450s and cytochrome P450 reductases, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides, and one or more other polypeptides also are provided. Linkage of a cytochrome P450 or cytochrome P450 reductase polypeptide with another polypeptide can be effected directly or indirectly via a linker. In one example, linkage can be by chemical linkage, such as via heterobifunctional agents or thiol linkages or other such linkages. Fusion also can be effected by recombinant means. Fusion of a cytochrome P450 or cytochrome P450 reductase, such as a cytochrome P450 santalene oxidase polypeptide, cytochrome P450 bergamotene oxidase polypeptide and cytochrome P450 reductase polypeptide, to another polypeptide can be to the N- or C-terminus of the cytochrome P450 santalene oxidase polypeptide, cytochrome P450 bergamotene oxidase polypeptide and cytochrome P450 reductase polypeptide.
A fusion protein can be produced by standard recombinant techniques. For example, DNA fragments coding for the different polypeptide sequences can be ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A cytochrome P450 santalene oxidase polypeptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the cytochrome P450 santalene oxidase protein. A cytochrome P450 bergamotene oxidase polypeptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the cytochrome P450 bergamotene oxidase protein. In some examples, a cytochrome P450 polypeptide-encoding nucleic acid can be cloned into such an expression vector such that the cytochrome P450 is linked in frame to a santalene synthase polypeptide-encoding nucleic acid. For example, a cytochrome P450 santalene oxidase or bergamotene oxidase polypeptide-encoding nucleic acid can be cloned into such an expression vector such that the cytochrome P450 santalene oxidase or bergamotene oxidase is linked in frame to a santalene synthase polypeptide-encoding nucleic acid. The cytochrome P450 and santalene synthases can be linked directly, without a linker, or alternatively, linked indirectly in-frame with a linker.
G. Methods for Producing Terpenoids and Methods For Detecting Such Products and The Activity of the Cytochrome P450 and Cytochrome P450 Reductase Polypeptides
The cytochrome P450 polypeptides provided herein can be used to, and assessed for their ability to, produce terpenoids, including monoterpenoids, sesquiterpenoids and diterpenoids, from any suitable terpene substrate, including monoterpenes, sesquiterpenes and diterpenes. Typically, the cytochrome P450 santalene oxidases provided herein produce santalols from santalenes and the cytochrome P450 bergamotene oxidases provided herein produce bergamotols from bergamotenes. Any method known to one of skill in the art can be used to produce terpenoids catalyzed by the cytochrome P450 polypeptides provided herein. The ability of the cytochrome P450 polypeptides provided herein to catalyze the formation of terpenoids from terpene substrates can be assessed using these methods. Terpenoid products analyzed by GC-MS and can be identified based on matches of the MS fragmentation patterns with entries in the NIST and Wiley libraries (for example, as described in Example 6 below).
The cytochrome P450 reductase polypeptides provided herein can be used to, and assessed for their ability to, transfer two electrons from NADPH to any suitable electron receptor, including cytochrome P450s, cytochrome c, heme oxygenases, cytochrome b5 and squalene epoxidases.
Other activities and properties of the cytochrome P450 and cytochrome P450 reductase polypeptides, such as the cytochrome P450 santalene oxidases, cytochrome P450 bergamotene oxidases and cytochrome P450 reductases provided herein, also can be assessed using methods and assays well known in the art. In addition to assessing the activity of the cytochrome P450 and cytochrome P450 reductase polypeptides and their ability to catalyze the formation of terpenoids, the kinetics of the reaction, increased substrate specificity, altered substrate utilization and/or altered product distribution (as compared to another cytochrome P450 and cytochrome P450 reductase polypeptide) can be assessed using methods well known in the art. For example, the amount and type of terpenoids produced from santalenes or bergamotenes by the santalene oxidase and bergamotene oxidase polypeptides provided herein can be assessed by gas chromatography methods (e.g. GC-MS), such as those described in Example 6, and compared to the MS fragmentation patterns with entries in the NIST and Wiley libraries (see Example 6). Products can also be identified by comparison with compounds of authentic sandalwood oil.
Provided below are methods for the production of santalols, including (Z)-α-santalol, (E)-α-santalol, (Z)-β-santalol, (E)-β-santalol, (Z)-epi-β-santalol and (E)-epi-β-santalol, and (E)-α-trans-bergamotol and (Z)-α-trans-bergamotol, where production of the santalols and bergamotols is catalyzed by the cytochrome P450 and cytochrome P450 reductase polypeptides provided herein. Also provided herein are methods for assessing the activity of the cytochrome P450 and cytochrome P450 reductase polypeptides provided herein.
1. Synthesis of Santalols and Bergamotols
The cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides provided herein can be used to catalyze the formation of santalols and bergamotols from the terpene substrates santalenes and bergamotenes. In some examples, the cytochrome P450 santalene oxidases are expressed in cells that produce or overexpress a santalene synthase and FPP, such that santalols are produced as described elsewhere herein. In other examples, the cytochrome P450 bergamotene oxidases are expressed in cells that produce of overexpress a santalene synthase, such that bergamotols are produced as described elsewhere herein. In other examples, the cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides provided herein are expressed and purified form any suitable host cells, such as any described in Section E. The purified cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides are then combined in vitro with santalenes and bergamotenes to produce santalols and bergamotols.
a. Oxidation of Santalenes and Bergamotenes
In some examples, the cytochrome P450 santalene oxidase polypeptides provided herein are overexpressed and purified as described in Section E above. The cytochrome P450 santalene oxidase is then incubated with one or more terpene substrates, including α-santalene, β-santalene, epi-β-santalene and/or α-trans-bergamotene, and one or more of α-santalol, β-santalol and epi-β-santalol, and α-trans-bergamotol, such as (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol, are produced. Alternatively, the cytochrome P450 santalene oxidase polypeptides provided herein expressed in host cells that also produce terpene substrates, including α-santalene, β-santalene, epi-β-santalene and/or α-trans-bergamotene, resulting in the production of one or more of α-santalol, β-santalol and epi-β-santalol, and α-trans-bergamotol, such as (E)-α-santalol, (Z)-α-santalol, (E)-β-santalol, (Z)-β-santalol, (E)-epi-β-santalol, (Z)-epi-β-santalol, (Z)-α-trans-bergamotol and (E)-α-trans-bergamotol. Production of santalols and bergamotols and quantification of the amount of product are then determined using any method provided herein, such as gas chromatography-mass spectroscopy (e.g. GC-MS), gas chromatography-flame ionization detection (GC-FID) and liquid chromatography-mass spectroscopy (LC-MS). Mass spectrometry patterns can be compared to the MS fragmentation patterns with entries in the NIST and Wiley libraries, such as described in Example 6, or by comparison with known terpenoids in sandalwood oil.
In other examples, the cytochrome P450 bergamotene oxidase polypeptides provided herein are overexpressed and purified as described in Section E above. The cytochrome P450 bergamotene oxidase is then incubated with one or more terpene substrates, including α-santalene, β-santalene, epi-β-santalene and/or α-trans-bergamotene, and one or more of (E)-α-trans-bergamotol or (Z)-α-trans-bergamotol is produced. In some examples, small amounts of α-santalol, β-santalol and/or epi-β-santalol are also produced. Alternatively, the cytochrome P450 bergamotene oxidase polypeptides provided herein expressed in host cells that also produce terpene substrates, including α-santalene, β-santalene, epi-β-santalene and/or α-trans-bergamotene, resulting in the production of (E)-α-trans-bergamotol or (Z)-α-trans-bergamotol. In some examples, small amounts of α-santalol, β-santalol and/or epi-β-santalol are also produced. Production of bergamotols and quantification of the amount of product are then determined using any method provided herein, such as gas chromatography-mass spectroscopy (e.g. GC-MS), gas chromatography-flame ionization detection (GC-FID) and liquid chromatography-mass spectroscopy (LC-MS). Mass spectrometry patterns can be compared to the MS fragmentation patterns with entries in the NIST and Wiley libraries, such as described in Example 6, or by comparison with known terpenoids in sandalwood oil.
b. Conversion of Acyclic Pyrophosphate Terpene Precursors
In some examples, terpenoids can be generated biosynthetically from acyclic pyrophosphate terpene precursors, such as geranyl pyrophosphate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, by expression of a cytochrome P450 monooxygenase in a host cell that produces the acyclic pyrophosphate terpene precursor and a terpene synthase. Suitable host cells are described in Section E above. In one example, santalols and bergamotols are generated biosynthetically by expression of a cytochrome P450 santalene oxidase in a host cell that produces FPP and santalene synthase (see Example 10). In another example, bergamotols are generated biosynthetically by expression of a cytochrome P450 bergamotene oxidase in a host cell that produces FPP and santalene synthase (see Example 10). Production of santalols and bergamotols and quantification of the amount of products are then determined using any method provided herein, such as gas chromatography-mass spectroscopy (e.g. GC-MS), gas chromatography-flame ionization detection (GC-FID) and liquid chromatography-mass spectroscopy (LC-MS). Mass spectrometry patterns can be compared to the MS fragmentation patterns with entries in the NIST and Wiley libraries, such as described in Example 6, or by comparison with known terpenoids in sandalwood oil.
In another example, terpenoids can be generated from acyclic pyrophosphate terpene precursors by 1) incubating an acyclic pyrophosphate terpene precursor with a terpene synthase and 2) incubating the reaction products with a cytochrome P450 monooxygenase. In some examples, the reaction products of the acyclic pyrophosphate terpene precursor with the terpene synthase are isolated. In other examples, the cytochrome P450 monooxygenase is added directly to the first reaction mixture without previous purification. The two steps can be performed simultaneously or sequentially. Terpenoids produced by the reaction can be identified and quantified using any method provided herein, such as gas chromatography-mass spectroscopy (e.g. GC-MS), gas chromatography-flame ionization detection (GC-FID) and liquid chromatography-mass spectroscopy (LC-MS). Mass spectrometry patterns can be compared to the MS fragmentation patterns with entries in the NIST and Wiley libraries, such as described in Example 6, or by comparison with known terpenoids in sandalwood oil.
2. Methods for Production
a. Exemplary Cells
Santalols and bergamotols can be produced by expressing a cytochrome P450 synthase polypeptide and/or a cytochrome P450 reductase polypeptide provided herein in a cell line that produces FPP as part of the mevalonate-dependent isoprenoid biosynthetic pathway (e.g. fungi, including yeast cells, and animal cells) or the mevalonate-independent isoprenoid biosynthetic pathway (e.g. bacteria and higher plants). In particular examples, santalols are produced by expressing a cytochrome P450 santalene oxidase polypeptide provided herein and a santalene synthase polypeptide in a cell line that has been modified to overproduce FPP. In other examples, bergamotols are produced by expressing a cytochrome P450 bergamotene oxidase polypeptide provided herein and a santalene synthase polypeptide in a cell line that has been modified to overproduce FPP. Exemplary of such cells are modified yeast cells. For example, yeast cells that have been modified to produce less squalene synthase or less active squalene synthase (e.g. erg9 mutants; see e.g. U.S. Pat. Nos. 6,531,303 and 6,689,593) are useful in the methods provided herein to produce labdenediol diphosphate. Reduced squalene synthase activity results in accumulation of FPP in the host cell at higher levels compared to wild type yeast cells, thus allowing for increased yields of santalenes and bergamotenes. Exemplary modified yeast cells include, but are not limited to, modified Saccharomyces cerevisiae strains YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63, his3-Δ200, leu2-Δ1), WAT11 (MATa, ade2-1, his3-11,-15; leu2-3,-112, ura3-1, canR, cyr+; containing chromosomally integrated Arabidopsis NADPH-dependent P450 reductase ATR1; see Pompon et al. (1995) Toxicol Lett 82-83:815-822; Ro et al. (2005) Proc Natl Acad Sci USA 102:8060-8065); and BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0; ATCC #201388).
b. Culture of Cells
In exemplary methods, a cytochrome P450 provided herein is expressed in a host cell line that has been modified to overexpress farnesyl diphosphate and a santalene synthase, whereby upon expression of the cytochrome P450, farnesyl diphosphate is converted to santalols and bergamotols. In other exemplary methods, a cytochrome P450 provided herein and a santalene synthase are expressed in a host cell line that has been modified to overexpress farnesyl diphosphate whereby upon expression of both proteins, farnesyl diphosphate is converted to santalols or bergamotols. The cytochrome P450 and santalene synthase can be expressed separately, or together, as a fusion protein described elsewhere herein. cytochrome P450 and santalene synthase can be expressed simultaneously or sequentially. The host cell is cultured using any suitable method well known in the art. In some examples, such as for high throughput screening of cell expressing various cytochrome P450s, the cells expressing the cytochrome P450 are cultured in individual wells of a 96-well plate. In other examples where the host cell is yeast, the cell expressing the cytochrome P450 polypeptides, santalene synthase and FPP is cultured using fermentation methods such as those described below.
A variety of fermentation methodologies can be used for the production of santalols and bergamotols from yeast cells expressing the cytochrome P450 polypeptides provided herein. For example, large scale production can be effected by either batch or continuous fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur without further addition of nutrients. Typically, the concentration of the carbon source in a batch fermentation is limited, and factors such as pH and oxygen concentration are controlled. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells typically modulate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die.
A variation on the standard batch system is the Fed-Batch system, which is similar to a typical batch system with the exception that nutrients are added as the fermentation progresses. Fed-Batch systems are useful when catabolite repression tends to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Also, the ability to feed nutrients will often result in higher cell densities in Fed-Batch fermentation processes compared to Batch fermentation processes. Factors such as pH, dissolved oxygen, nutrient concentrations, and the partial pressure of waste gases such as CO are generally measured and controlled in Fed-Batch fermentations.
Production of the santalols or bergamotols also can be accomplished with continuous fermentation. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. This system generally maintains the cultures at a constant high density where cells are primarily in their log phase of growth. Continuous fermentation allows for modulation of any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by the medium turbidity, is kept constant. Continuous systems aim to maintain steady state growth conditions and thus the cell loss due to the medium removal must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art.
Following cell culture, the cell culture medium then can be harvested to obtain the produced santalols and bergamotols.
c. Isolation and Assays for Detection and Identification
The santalols and bergamotols produced using the methods above with the cytochrome P450 polypeptides provided herein can be isolated and assessed by any method known in the art. In one example, the cell culture medium is extracted with an organic solvent to partition any terpenes or terpenoids produced into the organic layer. Production of santalols and/or bergamotols can be assessed and/or the santalols and/or bergamotols isolated from other products using any method known in the art, such as, for example, gas chromatography or column chromatography. For example, the organic layer can be analyzed by GC-MS.
The quantity of santalols and/or bergamotols produced can be determined by any known standard chromatographic technique useful for separating and analyzing organic compounds. For example, santalol and/or bergamotol production can be assayed by any known chromatographic technique useful for the detection and quantification of hydrocarbons, such as santalol and/or bergamotol and other terpenoids, including, but not limited to, gas chromatography mass spectrometry (GC-MS), gas chromatography using a flame ionization detector (GC-FID), capillary GC-MS, high performance liquid chromatography (HPLC) and column chromatography. Typically, these techniques are carried out in the presence of known internal standards which are used to quantify the amount of the terpenoid produced. For example, terpenoids, including sesquiterpenoids, such as santalol and/or bergamotol, can be identified by comparison of retention times and mass spectra to those of authentic standards in gas chromatography with mass spectrometry detection. Typical standards include, but are not limited to, santalols and/or bergamotols. In other examples, quantification can be achieved by gas chromatography with flame ionization detection based upon calibration curves with known amounts of authentic standards and normalization to the peak area of an internal standard. These chromatographic techniques allow for the identification of any terpene present in the organic layer, including, for example, other terpenoids produced by the cytochrome P450s.
In some examples, kinetics of santalol and/or bergamotol production can be determined by synthase assays in which radioactive isoprenoid substrates, such as 3H FPP or 14C FPP, are used with varying concentrations of synthase. The products are extracted into an organic layer and radioactivity is measured using a liquid scintillation counter. Kinetic constants are determined from direct fits of the Michaelis-Menton equation to the data.
3. Production of Sandalwood Oil
The cytochrome P450 santalene oxidase and cytochrome P450 bergamotene oxidase polypeptides provided herein can be used to produce sandalwood oil. For example, the cytochrome P450 santalene oxidases can be expressed in cells that produce or overexpress a santalene synthase, such that santalols and bergamotol, including α-santalol, β-santalol and epi-β-santalol, and Z-α-trans-bergamotol, are produced as described elsewhere herein. The terpenoid products can be compared to those found in authentic sandalwood oil from S. album by GC-MS analysis, for example, as described in Example 8.
4. Assays for Detecting Enzymatic Activity of Cytochrome P450 and Cytochrome P450 Reductase Polypeptides
a. Methods for Determining the Activity of Cytochrome P450 Polypeptides
One of skill in the art is familiar with methods and assays to detect the enzymatic activity of cytochrome P450 polypeptides. Cytochrome P450 polypeptides can be expressed in yeast or purified from microsomal membrane fractions. Cytochrome P450 monooxygenase activity can be determined in vitro by incubation of a cytochrome P450 polypeptide with various monoterpene, sesquiterpene and diterpene substrates, as described in Example 11. Reaction products, including ratios of the products, can be determined by any method known to one of skill in the art, including GC-MS, GC-FID, LC-MS, comparison to known standards, and proton and carbon nuclear magnetic resonance (NMR). Alternatively, activity can be determined in vivo by addition of terpene substrates to yeast cultures of the cytochrome P450s and identifying products as described above. Total P450 content in microsomes can be quantified by CO differential absorption spectroscopy (see Guengerich et al. (2009) Nat Protoc 4:1245-1251 and Example 8).
Enzyme kinetics can be determined in vitro in the presence of NADPH and CPR. In such assays, CPR is included in limited amounts, e.g., 0.1 U, for determination of enzyme activity and 5 milliunits for determination relative activities and kinetic parameters. Assays can be performed over a range of substrate concentrations and product formation can be determined by GC-MS. Add terpene directly to yeast cultures
b. Methods for Determining the Activity of Cytochrome P450 Reductase Polypeptides
One of skill in the art is familiar with methods and assays to detect the enzymatic activity of cytochrome P450 reductase polypeptides. In one example, CPR activity can be determined using an assay that detects for C4H (cinnamate 4-hydroxylase) activity, for example, as described in Ro et al. (2001) Plant Physiology 126:317-329. C4H is a heme-thiolate protein that catalyzes the formation of p-coumarate from cinnamic acid. This assay can be used in vivo by expression of the cytochrome P450 reductase in yeast cells in the presence of C4H (see also, Ro et al. (2002) Plant Physiology 130:1837-1851). C4H activity is determined by detection of p-coumaric acid formation by HPLC (Mizutani et al. (1993) Plant Cell Physiology 34:481-488).
In order to assess CPR activity in vitro, CPRs can be purified from yeast microsomal fractions, such as described in Pompon et al. ((1996) Methods Enzymol 272:51-64) and Example 8 below. Total P450 content in microsomes can be quantified by CO differential absorption spectroscopy (Omura and Sato (1964) J Biol Chem 239:2370-2378; Mizutani and Ohta (1998) Plant Physiology 116:357-367). FAD and FMN content can be determined as described in Faeder and Siegel (1973) Anal Biochem 53:332-336. CPR activity in vitro can be assessed by a variety of assays known to one of skill in the art. For example, activity can be determined using the C4H assay described above. In another example, activity is determined by measuring reduction of an artificial electron receptor, such as cytochrome c or oxidized ferricyanide (Xia et al. (2011)J Biol Chem 286:16246-16260; Hamdane et al. (2009)J Biol Chem 284:11374-11384; Shen et al. (1989)J Biol Chem 264:7584-7589). Formation of reduced cytochrome c is measured using a spectrophotometer and calculating the rate of reduction from A550 change using an extinction coefficient (Σ=21 mM−1 cm−1) (Imai (1976) J Biochem 80:267-276). Another assay that be used to detect CPR is the ethoxycoumarin O-de-ethylase activity reporter assay in P450 2B4 reconstituted systems (Louerat-Orieu et al (1998) Eur J Biochem 258:1040-1049).
The subcellular membrane localization site, e.g., whether the CPR is located in the ER or the chloroplast, of a cytochrome P450 reductase polypeptide can be determined by expressing CPR with GFP-fused to its C-terminus in Arabidopsis under the control of cauliflower mosaic virus 35S promoter (see, Ro et al. (2002) Plant Physiology 130:1837-1851). Independently transformed T1 and T2 seedlings are then screened for the presence of GFP by fluorescence microscopy and confocal microscopy (see Ro et al. (2002) Plant Physiology 130:1837-1851) or by immunoblot analysis of microsomal proteins of seedlings. The functionality of the CPR in the GFP-CPR fusions can be verified using the C4H assay.
G. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLE 1
Cloning and Sequencing of Santalum album cDNA
In this example, RNA was extracted from wood samples of Sandalwood (Santalum album) trees and cDNA was generated and sequenced.
A. Isolation and Extraction of S. album RNA
Several 25 mm holes were drilled into the lower stems of mature Santalum album trees growing on land managed by the Forest Products Commission of Western Australia. Wood samples from the heartwood-sapwood transition zone were collected and frozen immediately in liquid nitrogen. RNA was extracted from 10 g tissue using a protocol modified from Kolosova et al., (2004) BioTechniques 36:821-824. After precipitation with LiC1, RNA was stored at −80° C. until cDNA synthesis.
B. Generation of S. album cDNA Library
S. album xylem total RNA (1.4 μg) was reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) at 42° C. for 1 hour using the SMART-Creator kit with the pDNR-LIB vector (Clontech; SEQ ID NO:20). The ligation mixture was transformed by electroporation into 25 μL of phage resistant electrocompetent E. coli cells and Sanger sequenced at the Genome Sciences Centre, Vancouver, Canada.
C. 454 Pyr Sequencing and Sanger Sequencing
Two cDNA libraries from Santalum album cores were prepared and sequenced with Sanger technologies generating 11,520 paired end sequences. One plate of 454 Titanium sequencing was done on both libraries and generated 902,111 reads. Assembly was effected using the 454 and Sanger sequences with Newbler assembler v2.6 (454 Life Sciences, Roche Diagnostics) with default parameters. This generated 31,461 contigs (isotigs).
EXAMPLE 2
Identification of Nucleic Acid Encoding S. album Cytochrome P450 Polypeptides
Cytochrome P450 encoding genes were identified by comparing the assembled sequences (from Example 1) against a set of known plant P450 encoding genes from the CYP76 families of P450 proteins using a BLASTx search (blast.ncbi.nlm.nih.gov; Altschul et al. (1990) J Mol Biol 215:403-410).
Table 4 below provides a summary of 7 isotigs identified in the BLASTx search (blast.ncbi.nlm.nih.gov; Altschul et al. (1990) J Mol Biol 215:403-410), including the isotig, lowest E-value, the gene ID of the match in the P450 database, the CYP450 family and the number of reads. The E-value (Expect Value) describes the number of matches expected to occur randomly with a given score. In general, the smaller E-value, the more likely the match is significant.







TABLE 4







Summary of CYP450 transcripts















Identity to






Lowest E-
Gene ID






value
of the match in






with match
the P450 database

Number




in P450
CrCYP76B6
CYP450
of


#
Query
data base
(CAC80883)
Family
reads















1
isotig05182
8.34E−142
71%
SaCYP76
910


2
isotig05183
2.68E−145
71%
SaCYP76
763


3
isotig05184
1.61E−78
52%
SaCYP76
470


4
isotig06871
1.23E−126
83%
SaCYP76
110


5
isotig06872
9.19E−156
83%
SaCYP76
118


6
isotig14788
1.53E−93
86%
SaCYP76
11


7
isotig29133
1.49E−52
60%
SaCYP76
1









Transcripts from this family were the most abundant in the EST database and cluster into four different groups. Group 1 is represented by 3 isotigs (numbers 1-3 in Table 4) with a total of 2,143 reads including 1,107 unique sequences generating a final assembled sequence of 1917 base pairs (bp) with an open reading frame (ORF) of 1530 bp. Group 2 is represented by 2 isotigs (numbers 4-5 in Table 4), had 228 reads with 140 unique reads generating an assembled sequence of 1776 bp and an ORF of 1530 bp. Group 3 (number 6 in Table 7) was represented by 11 reads generating a partial sequence of 1200 bp. Group 4 (number 7 in Table 7) is a singleton of 277 bp with several stop codons along the sequence.
EXAMPLE 3
Isolation of Cytochrome P450 Encoding cDNA
Group 1 and Group 2 cDNA molecules (numbers 1-5 in the table above) of the CYP76 family identified in Example 2, were selected for cDNA isolation.
A. Cloning of Members of the CYP76 Family
Full-length cDNA molecules were amplified by polymerase chain reaction (PCR) with Phusion Hot Start II DNA Polymerase (Thermo Scientific) of S. album cDNA (set forth in SEQ ID NO:1) prepared as described in Example 1 using gene specific primers designed according to the ORF of Group 1 and Group 2 (set forth in Table 5 below). PCR conditions were as follows:
98° C. for 3 min;
2 cycles of: 98° C. for 10 sec, Tm −2° C. for 20 sec, 72° C. for 30 sec;
30 cycles of: 98° C. for 10 sec, Tm for 20 sec, 72° C. for 30 sec;
Final extension at 72° C. for 7 min
with a Tm of 55° C. for Isogroup 1 and a Tm of 52° C. for Isogroup 2. The PCR products were gel purified and cloned into the pJET1.2 vector (Fermentas, SEQ ID NO:21) according to the manufacturer's instructions. E. coli α-Select chemically competent cells (Bioline) were used for cloning and plasmid propagation. All constructs were verified by DNA sequencing.







TABLE 5







Primers for amplification of cytochrome 


P450 cDNA











SEQ




ID


Primer
Sequence
NO





Isogroup 1 Forward
ATGGACTTCTTAAGTTTTATCCTGTTTG
22


 


Isogroup 1 Reverse
TTACCCCCGGATCGGGACAG
23


 


Isogroup 2 Forward
ATGGACTTCTTAAGTTGTATCCTG
24


 


Isogroup 2 Reverse
TTACCCCCGGATTGGGACAG
25









Amplification with primers for Isogroup 1 resulted in a single unique cDNA clone designated SaCYP76F38v1 (SaCYP76-G5) Amplification with primers from Isogroup 2 resulted in 3 different cDNA clones designated: SaCYP76F39v1 (SaCYP76-G10), SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12). A second amplification with primers from Isogroup 2 resulted in 6 additional different cDNA clones, designated SaCYP76F37v2 (SaCYP76-G14), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17), SaCYP76F42 (SaCYP76-G13) and SaCYP76F43 (SaCYP76-G18). The SEQ ID NOS of the sequences of the nucleic acids and the encoded amino acids are set forth in Table 6 below. The translated amino acid sequences encoded by the 10 isolated cDNA molecules share between 93% and 99% identity (see Table 7 below) and between 1.0 and 6.6% divergence. Pair distances were prepared with ClustalW (slow/accurate, Gonnet weight matrix) (ebi.ac.uk/clustalw; European Bioinformatics Institute).







TABLE 6







Cytochrome P450 Polypeptides












Nucleic
Amino




acid
acid




SEQ
SEQ



Cytochrome P450
ID NO
ID NO














SaCYP76F38v1 (SaCYP76-G5)
2
6



SaCYP76F39v1 (SaCYP76-G10)
3
7



SaCYP76F37v1 (SaCYP76-G11)
4
8



SaCYP76F38v2 (SaCYP76-G12)
5
9



SaCYP76F37v2 (SaCYP76-G14)
67
73



SaCYP76F39v2 (SaCYP76-G15)
68
74



SaCYP76F40 (SaCYP76-G16)
69
75



SaCYP76F41 (SaCYP76-G17)
70
76



SaCYP76F42 (SaCYP76-G13)
71
77



SaCYP76F43 (SaCYP76-G18)
72
78
















TABLE 7







Percent amino acid identity for cytochrome P450s from the CYP76 family









SaCYP76


















F38v1
F39v1
F37v1
F38v2
F37v2
F39v2
F40
F41
F42
F43





















SaCYP76F38v1
100
94
97
99
98
93
94
96
95
96


SaCYP76F39v1

100
95
94
95
99
98
96
95
95


SaCYP76F37v1


100
98
99
95
94
95
94
95


SaCYP76F38v2



100
99
94
94
96
95
95


SaCYP76F37v2




100
95
93
95
94
95


SaCYP76F39v2





100
98
95
94
95


SaCYP76F40






100
97
96
95


SaCYP76F41







100
97
94


SaCYP76F42








100
96


SaCYP76F43









100









EXAMPLE 4
Sequence and Phylogenetic Analysis of SaCYP76 Proteins
A BLASTx search of the deduced amino acid sequences against the GenBank non-redundant protein database (blast.ncbi.nlm.nih.gov; Altschul et al. (1990) J Mol Biol 215:403-410) identified a putative cytochrome P450 from Vitis vinifera (GenBank Accession No. XP_002281735; SEQ ID NO:26) that has 62% to 64% sequence identity to the S. album CYPs and a CYP76B6 geraniol hydroxylase from Catharanthus roseus (GenBank Accession No. CAC80883; Collu et al. (2001) FEBS Lett 308:215-220; SEQ ID NO:27) that has 54% to 55% sequence identity to the S. album CYPs. Protein alignment of the full length protein sequences was made with ClustalW (ebi.ac.uk/clustalw; European Bioinformatics Institute).
Phylogenetic trees were constructed with MEGA version 4 (Centre for Evolutionary Medicine and Informatics; Tamura et al., 2007 Mol Biol Evol 24:1596-1599) employing the neighbor joining (NJ) method with default parameters. Bootstrap (500 replications) confidence values over 50% are displayed at branch points. The neighbor-joining phylogeny of the predicted protein sequences of the initial four S. album CYP clones SaCYP76F38v1 (SaCYP76-G5), SaCYP76F39v1 (SaCYP76-G10), SaCYP76F37v1 (SaCYP76-G11) SaCYP76F38v2 (SaCYP76-G12) and cytochrome P450 enzymes for terpenoid metabolism in other species is set forth in FIG. 4. The SaCYP76 genes, which form a separate cluster in this phylogeny, are most closely related to the CYP76B cluster that includes geraniol/nerol hydroxylases from different species. Accession numbers of the amino acid sequences included in the phylogeny in FIG. 4, in addition to the S. album CYP76 P450 clones SaCYP76F38v1 (SaCYP76-G5), SaCYP76F39v1 (SaCYP76-G10), SaCYP76F37v1 (SaCYP76-G11) SaCYP76F38v2 (SaCYP76-G12) provided herein, included: Helianthus tuberosus CYP76B1 (CAA71178; SEQ ID NO:28); Catharanthus roseus CYP76B6 (CAC80883; SEQ ID NO:27); Swertia mussotii CYP76B6 (ACZ48680; SEQ ID NO:29); Persea americana CYP71A1 (P24465; SEQ ID NO:30); Mentha x piperita CYP71A32 (Q947B7; SEQ ID NO:31); Artemisia annua CYP71AV1 (ABB82944; SEQ ID NO:32); Cichorium intybus CYP71AV8 (ADM86719; SEQ ID NO:33); Lactuca sativa CYP71BL1 (AEI59780; SEQ ID NO:34); Nicotiana tabacum CYP71D20 (Q94FM7; SEQ ID NO:35); Mentha x piperita CYP71D13 (Q9XHE7; SEQ ID NO:36); Mentha spicata CYP71D18 (Q6WKZ1; SEQ ID NO:37); Catharanthus roseus CYP72A1 (Q05047; SEQ ID NO:38); and Oryza sativa CYP76M7 (AK105913; SEQ ID NO:39).
A second neighbor joining phylogenetic tree was constructed with all 10 S. album CYP76F proteins and related terpene-modifying cytochrome P450s members of the CYP71 clan, using Picea sitchensis PsCYP720B4 (ADR78276; SEQ ID NO:79) as an outgroup. The phylogenetic tree is set forth in FIG. 10. The S. album CYP76F proteins fell into two separate clades and were closest to the CYP76B cluster of other species. Clade I santalene/bergamotene oxidases included SaCYP76F39v1 (SaCYP76-G10), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13). Clade II bergamotene oxidases included SaCYP76F37v1 (SaCYP76-G11), SaCYP76F37v2 (SaCYP76-G14), SaCYP76F38v1 (SaCYP76-G5) and SaCYP76F38v2 (SaCYP76-G12). Accession numbers of the amino acid sequences for other terpene-modifying CYPs included in the phylogenetic tree in FIG. 10, in addition to the S. album CYP76 P450 clones, include CaCYP76B4 Camptotheca acuminate putative geraniol-10-hydroxylase (AES93118; SEQ ID NO:80); CrCYP76B6 Catharanthus roseus geraniol 10-hydroxylase (Q8VWZ7; SEQ ID NO:81); SmCYP76B4 Swertia mussotii geraniol 10-hydroxylase (D1MI46; SEQ ID NO:82); OsCYP76M7 Oryza sativa entcassadiene C11a-hydroxylase (NP_001047185; SEQ ID NO:83); MpCYP71A32 Mentha x piperita menthofuran synthase (Q947B7; SEQ ID NO:84); PaCYP71A1 Persea americana (P24465; SEQ ID NO:85); CiCYP71AV8 Cichoriium intybus valencene oxidase (ADM86719; SEQ ID NO:86); MpCYP71D13 Mentha x gracilis (−)-limonene-3-hydroxylase (AY281027; SEQ ID NO:87); NtCYP71D20 Nicotiana tabacum 5-epi-aristocholene-1,3-dihydroxylase (AF368376; SEQ ID NO:88); and GaCYP706B1 Gossypium arboretum (+)-delta-cadinene-8-hydroxylase (AAK60517; SEQ ID NO:89).
EXAMPLE 5
Cytochrome P450 Reductase
Cytochrome P450 reductase encoding genes were identified by comparing the assembled sequences with a set of known plant cytochrome P450 reductases from Arabidopsis (CAB58575.1 (SEQ ID NO:58) and CAB58576.1 (SEQ ID NO:46)). Full length cDNA genes SaCPR1 and SaCPR2 were amplified by polymerase chain reaction (PCR) with Phusion Hot Start II DNA Polymerase (Thermo Scientific) of S. album cDNA prepared as described in Example 1 with gene specific primers designed according to the ORF of the cytochrome P450 reductase (set forth in Table 8).







TABLE 8







Primers for PCR of cytochrome P450 reductase 


genes













SEQ





ID


Primer
Sequence
Tm
NO





SaCPR1 
ATG AGT TCG AGC TCG GAG CTA TG
57
40


Forward





 


SaCPR1 
TCA CCA CAC ATC CCG TAA ATA CCT
57
41


Reverse
TC




 


SaCPR2 
ATG CAA TTG AGC TCC GTC AAG
58
61


Forward





 


SaCPR2 
TCA CCA CAC ATC CCG TAA ATA CCT
58
62


Reverse
TCC









PCR conditions were as follows:

98° C. for 3 min;
2 cycles of: 98° C. for 10 sec, Tm −2° C. for 20 sec, 72° C. for 30 sec;
30 cycles of: 98° C. for 10 sec, Tm for 20 sec, 72° C. for 30 sec;
Final extension at 72° C. for 7 min
The PCR products were gel purified and cloned directly into the pET28b(+) vector (SEQ ID NO:51) or first cloned into pJET vector and then subcloned into expression vectors. E. coli α-Select chemically competent cells (Bioline) were used for cloning and plasmid propagation. All constructs were verified by DNA sequencing. PCR amplification resulted in two S. album cytochrome P450 reductase (CPR) clones designated CPR1 and CPR2, having nucleic acid sequences set forth in SEQ ID NOS:10 and 11, respectively, encoding the proteins set forth in SEQ ID NO:12 and 13. The two CPR nucleic acid sequences share 70% sequence identity and the two CPR proteins share 82% sequence identity.
The web-based BlastX program (Altschul et al., (1990) J. Mol. Biol. 215:403-410) was then used to compare the sequence of the identified with sequences in the GenBank database. The CPR sequences share 79% sequence homology with the Vitis vinifera predicted cytochrome P450 reductase-like protein (Genbank Accession No. XP_002270732; SEQ ID NO:42), 78% sequence homology with the Gossypium hirsutum cytochrome P450 reductase (Genbank Accession No. ACN54324; SEQ ID NO:43) and 75% sequence homology with the Artemisia annua cytochrome P450 reductase (Genbank Accession No. ABI98819; SEQ ID NO:44).
Truncated CPRs were generated containing amino acids 44-692 of SEQ ID NO:12 (truncated protein sequence set forth in SEQ ID NO:14; nucleic acid sequence set forth in SEQ ID NO:63) and amino acids 61-704 of SEQ ID NO:13 (truncated protein sequence set forth in SEQ ID NO:15; nucleic acid sequence set forth in SEQ ID NO:64).
Activity of recombinant SaCPR was assayed using the Cytochrome C Reductase (NADPH) assay kit (Sigma).
EXAMPLE 6
Gas Chromatography-Mass Spectrometry Analysis
Gas chromatography-mass spectrometry (GC-MS) analysis was used to analyze the oxidation products of the S. album cytochrome P450s and S. album oil.
A. SGE Solgel-Wax Capillary Column
GC-MS analysis was performed on a Agilent 6890A/5973N GC-MS system containing a SGE Solgel-Wax capillary column (30 m×0.25 mm ID×0.25 μm thickness) in SIM-scan mode (scan: m/z 40-400; SIM: m/z 93, 94, 119, 136, 122, 202 and 204 [dwell time 50]. Volumes of 2 μL samples were injected in pulsed splitless mode at 250° C. with a column flow of 1 mL/min helium and 50 psi pulse pressure for 0.5 min with the following program: 40° C. for 2 min, ramp of 8° C. per min to 100° C., 15° C. per min to 250° C., hold 5 min.
Alternatively, the following program was also used to analyze the products of S. album SaCYP76F39v1 (SaCYP76-G10) and S. album oil: volumes of 2 μL samples were injected in pulsed splitless mode at 250° C. with a column flow of 0.8 mL/min helium and 10 psi pulse pressure for 0.05 min with the following program: 40° C. for 3 min, 10° C. per min to 100° C., 2° C. per min to 250° C., hold 10 min.
Product identification was based on best match of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil and Kovats index values.
B. HP5 and DB-Wax Fused Silica Column
GC-MS analysis was performed on a Agilent 7890A/5975C GC-MS system operating in electron ionization selected ion monitoring (SIM)-scan mode. Samples were analyzed on an HP5 (non-polar; 30 m×0.25 mm ID×0.25 μm thickness) and a DB-Wax fused silica column (polar; 30 m×0.25 mm ID×0.25 μm thickness). In both cases, the injector was operated in pulsed splitless mode at with the injector temperature maintained at 250° C. Helium gas was used as the carrier gas with a flow rate of 0.8 mL/min and pulsed pressure set at 25 psi for 0.5 min. Scan range: m/z 40-500; SIM: m/z 93, 94, 105, 107, 119, 122 and 202 [dwell time 50 msec].
The oven program for the HP5 column was:
40° C. for 3 min, ramp of 10° C. per min to 130° C., 2° C. per min to 180° C., 50° C. per min to 300° C., hold 300° C. for 10 min.
The oven program for the DB-wax column was:
40° C. for 3 min, ramp of 10° C. per min to 130° C., 2° C. per min to 200° C., 50° C. per min to 250° C., hold 250° C. for 15 min.
Chemstation software was used for data acquisition and processing. Compounds were identified by comparison of mass spectral with authentic samples and the NIST/EPA/NIH mass spectral library v2.0 and by comparison of retention indices with those appearing in Valder et al. (2003) J Essent Oil Res 15:178-186 and Sciarrone et al. (2011) J Chromatogr A 1218:5374.
EXAMPLE 7
Expression in Bacteria and Yeast
The S. album FPP synthase, santalene synthase, cytochrome P450 SaCYP76F38v1 (SaCYP76-G5) and cytochrome P450 reductase genes were cloned into a pCDF-Duet (Novagen) and pACYC-Duet (Novagen) bacterial expression vectors. Genes encoding the full length S. album cytochrome CYP76F P450s, cytochrome P450 reductase, santalene synthase and farnesyl diphosphate synthase were cloned into various yeast expression vectors to allow expression in the Saccharomyces cerevisiae yeast strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; ATCC #201388).
A. Bacterial Expression Vectors
Genes encoding FPP synthase (SEQ ID NO:18) and santalene synthase (SEQ ID NO:16), previously characterized from S. album (see, International PCT application No. WO2011000026 and Jones et al. (2011)J Biol Chem 286:17445-17454), were cloned into the bacterial expression vector pCDF-Duet (Novagen, SEQ ID NO:65) generating pCDF-Duet:SaFPPS:SaSSy. Genes encoding SaCPR (SEQ ID NO:11) and SaCYP76F38v1 (SaCYP76-G5) gene (SEQ ID NO:2) were cloned into the bacterial expression vector pACYC-Duet (Novagen, SEQ ID NO:45) generating pACYC-Duet:SaCPR:SaCYP76F38v1. These expression vectors are dual expression vectors that allow co-expression of two target genes via two multiple cloning sites.
pCDF-Duet:SaFPPS:SaSSy, which has a streptomycin selectable marker, was transformed into chemically competent C41 (DE3) E. coli cells (Avidis). These cells were grown up and rendered chemically competent again using calcium chloride, and transformed with the pACYC-Duet:SaCPR:SaCYP76F38v1, which has a chloramphenicol selectable marker. Both antibiotics were used to select for colonies containing both duet vectors. These colonies were grown overnight in a rich media (terrific broth) at 16° C. and protein expression was initiated through the addition of IPTG. Cytochrome P450 protein expression was supplemented with 5-amino-levulinic acid to aid in porphyrin synthesis, and evidenced by a reddening of the cell pellet.
B. Generation of Yeast Expression Vectors
1. S. album Cytochrome P450s
The S. album CYP76F full length cDNAs identified in Table 6 above were sub-cloned into the yeast expression vector pYeDP60 (Cullin and Pompon (1988) Gene 65:203-217; Pompon et al. (1996) Methods Enzymol 272:51-64; Abecassis et al. (2003) Methods Mol Biol 231:165-173) following the uracil-excision (USER) cloning technique of Hamann and Moller (2007) Protein Expr Purif56:121-127. The pYeDP60 vector contains a URA marker. The resulting constructs are set forth in Table 9 below.
2. S. album Santalene Synthase and Farnesyl Diphosphate Synthase
Santalene synthase encoding cDNA (SaSSY, SEQ ID NO:16) and farnesyl diphosphate synthase encoding cDNA (SaFPPS, SEQ ID NO:18) were cloned into the NotI-Bgl II and BamHI-XhoI sites, respectively, of the galactose inducible expression vectors pESC-LEU (Stratagene, SEQ ID NO:47) or pESC-LEU2d (see, Ro et al. (2008) BMC Biotechnology 8:83) by in-Fusion Cloning (Clontech) following the manufacturer's instructions. Additional vectors were generated containing only the SaSSy gene (SEQ ID NO:16). The pESC-LEU and pESC-LEU2d vectors contain a LEU marker and the pESC-LEU2d vector is a high copy number vector containing a deletion in the Leu2 promoter. The resulting constructs are set forth in Table 9 below.
3. Cytochrome P450 Reductase
Cytochrome P450 reductase encoding cDNA (SaCPR, SEQ ID NO:11), identified in Example 3, was cloned into the EcoRi-NotI sites of pESC-HIS vector (Stratagene, SEQ ID NO:49) by in-Fusion Cloning (Clontech) following the manufacturer's instructions. The resulting constructs are summarized in Table 9 below.







TABLE 9







Yeast expression vectors









Construct ID
Marker
Description (MCS = multiple cloning site)





pESC-LEU:SaG1:SaG2
-LEU
MCS1 contains S. album Santalene Synthase




(SaSSY)




MCS2 contains S. album FPPS (SaFPPS)


pESC-LEU2d:SaG1:SaG2
-LEU
MCS1 contains S. album Santalene Synthase




(SaSSY)




MCS2 contains S. album FPPS (SaFPPS)


pESC-LEU:SaSSY
-LEU
MCS1 contains S. album Santalene Synthase




(SaSSY)


pESC-LEU2d:SaSSY
-LEU
MCS1 contains S. album Santalene Synthase




(SaSSY)


pESC-His:SaCPR
-HIS
MCS1 contains S. album cytochrome P450




reductase (SaCPR)


pYEDP60:F38v1
-URA
pYEDP60 contains S. album SaCYP76F38v1




(SaCYP76-G5)


pYEDP60:F39v1
-URA
pYEDP60 contains S. album SaCYP76F39v1




(SaCYP76-G10)


pYEDP60:F37v1
-URA
pYEDP60 contains S. album SaCYP76F37v1




(SaCYP76-G11)


pYEDP60:F38v2
-URA
pYEDP60 contains S. album SaCYP76F38v2




(SaCYP76-G12)


pYEDP60:F37v2
-URA
pYEDP60 contains S. album SaCYP76F37v2




(SaCYP76-G14)


pYEDP60:F39v2
-URA
pYEDP60 contains S. album SaCYP76F39v2




(SaCYP76-G15)


pYEDP60:F40
-URA
pYEDP60 contains S. album SaCYP76F40




(SaCYP76-G16)


pYEDP60:F41
-URA
pYEDP60 contains S. album SaCYP76F41




(SaCYP76-G17)


pYEDP60:F42
-URA
pYEDP60 contains S. album SaCYP76F42




(SaCYP76-G13)


pYEDP60:F43
-URA
pYEDP60 contains S. album SaCYP76F43




(SaCYP76-G18)









C. Yeast Transformation and Expression

All constructs were transformed into the Saccharomyces cerevisiae yeast strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; ATCC #201388) using the LiC1 method as described in Gietz et al. (1992) Nucleic Acids Res 20:1425. Transformed yeast were selected on plates with appropriate synthetic drop-out selection medium and grown at 30° C. for 48 hours.
1. Expression of Santalene Synthase
Production of santalenes and bergamotene was evaluated using constructs encoding the S. album santalene synthase. Yeast cells expressing the high copy number construct pESC-LEU2d:SaSSY produced about twice the amount of santalenes and bergamotene as determined by GC-MS (as described in Example 6A) compared to yeast cells expressing the pESC-LEU:SaSSY construct. No differences were observed between the cells expressing the santalene synthase in the presence or absence of farnesyl diphosphate synthase, indicating that FPP produced by yeast enzymes was accessible for S. album santalene synthase to produce santalenes and bergamotene. The high copy number construct pESC-LEU2d:SaSSY was used for further experiments.
2. Expression of Santalene Synthase and Cytochrome P450 Reductase
The pESC-LEU2d:SaSSY construct encoding santalene synthase and the pESC-His:SaCPR construct encoding S. album cytochrome P450 reductase (SaCPR) were co-transformed into the yeast strain BY4741. SaCPR was included to supply electrons from NADPH to the CYP450.
EXAMPLE 8
Microsome Preparation
In order to purify the S. album cytochrome P450 enzymes for use in in vitro assays, microsomes were prepared. Microsomes contain fragmented endoplasmic reticulum (ER) which contains cytochrome P450. Thus, purification of microsomes results in concentrated and isolated cytochrome P450. CO spectra of recombinant P450s encoded by the S. album CYP76F P450s was measured according to Guengerich et al. (2009) Nat Protoc 4:1245-1251.
Microsome membranes were prepared from 250 mL yeast cultures according to Pompom et al. (1996) Methods Enzymol 2(71):51-64. In brief, a 5 mL overnight culture was used to inoculate 50 mL of SD-selective media starting at an OD600 of 0.2 and grown at 30° C., 170 rpm for 24 hours. A volume of 200 mL YPDE medium (1% yeast extract, 2% bacto-peptone, 5% ethanol, 2% dextrose) was inoculated with the 50 mL culture and incubated for another 24 hours at 30° C., 170 rpm. Cells were collected by centrifugation for 10 min at 1,000×g and induced with 2% galactose in 250 mL YP medium at 30° C., 170 rpm for 12-16 hours. For microsome isolation, yeast cells were pelleted by centrifugation at 2,000×g for 10 min, washed once with 5 mL TEK (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM KCl) and resuspended in TES2 buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 600 mM Sorbitol, 5 mM DTT and 0.25 mM PMSF). All subsequent steps were performed at 4° C. Yeast cell walls were disrupted mechanically using acid-washed glass beads (425-600 μm, Sigma) and vigorous manual shaking for 3×30 sec. The cell homogenate was centrifuged at 10,000×g for 15 min followed by ultracentrifugation of the supernatant at 100,000×g for 1 hour to collect membranes. Microsomes were resuspended and homogenized in a buffer containing 50 mM Tris-HCl buffer pH 7.5, 1 mM EDTA and 30% (v/v) glycerol, and used directly for enzyme assays or stored at −80° C.
Microsome preparations for all ten S. album CYP76Fs except SaCYP76F43 (SaCYP76-G18) displayed characteristic P450 CO difference spectra (see FIG. 18). The P450 content of the microsomal preparations ranged from 0.2 to 1.6 μM. Microsome preparations were screened for P450 activity as described in Example 11 below.
EXAMPLE 9
Generation and Isolation of Sesquiterpene Olefins
The sesquiterpene olefins α-santalene, β-santalene, epi-β-santalene and α-trans-bergamotene are not commercially available but can be produced by expression of S. album santalene synthase (SaSSY; SEQ ID NO:16) in yeast as described in Jones et al. (2011)J Biol Chem 286:17445-17454.
A sesquiterpene oil containing α-santalene, β-santalene, epi-β-santalene and α-trans-bergamotene was produced in an industrial scale fermentation. The mixture was separated using silver nitrate impregnated TLC plates according to Daramwar et al. (Analyst 137:4564-4570 (2012)). Fractions were scraped from the TLC plates and the sesquiterpenes were eluted with pentane followed by GC-MS analysis for purity. The extracted ion chromatograms are shown in FIGS. 19A-19D for the oil containing α-santalene, β-santalene, epi-β-santalene and α-trans-bergamotene (FIG. 19A), α-santalene (peak 1, FIG. 19B), α-trans-bergamotene (peak 2, FIG. 19C) and epi-β-santalene and β-santalene (peaks 3 and 4, FIG. 19D). The isolated sesquiterpenes were used in in vitro assays in Example 11 below.
EXAMPLE 10
Functional Characterization of S. album Cytochrome P450 Activity in S. cerevisiae 
The S. cerevisiae yeast host strain containing active santalene synthase and cytochrome P450 reductase described in Example 7.C.2. was used to express the S. album cytochrome CYP76F P450s identified in Example 2 above. Activity was assessed by measurement of in vivo formation of oxidation products as described in Section A below. Each S. album CYP76F in a pYeDP60 vector was transformed individually into the yeast host cell expressing santalene synthase and CPR. A control strain was generated that contained the empty pYeDP60 vector.
A. In Vivo P450 Assays in Yeast
For in vivo assays, yeast were grown overnight at 30° C. in 5 mL of 2% dextrose and minimal selective media. The next day, a 50 mL culture was initiated at a starting OD600 of 0.2 and grown at 30° C. with shaking at 170 rpm until the culture reached an OD600 of 0.6-0.8. Protein expression was initiated by transfer into minimal selective media with 2% galactose and grown for about 14-16 hours. Yeast cells were harvested by centrifugation at 1,000×g for 10 min and washed once with 5 mL sterile ddH2O. Cells were extracted twice with 2 mL hexane:ethyl acetate (85:15) using about 250 μL acid-washed glass beads (425-600 μm, Sigma) and vortexing for 1 min. Pooled extracts were transferred to a clean test-tube containing anhydrous Na2SO4 and evaporated under a gentle stream of N2 gas to about 200 μL. The samples were transferred to a GC glass vial for GC-MS analysis (as described in Example 6) or stored at −80° C.
B. Clade I Santalum album P450s
Clade I S. album P450s SaCYP76F39v1 (SaCYP76-G10), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) were assayed for their activity in vivo with GC-MS analysis as described in Example 6A or 6B.
1. SaCYP76F39v1 (SaCYP76-G10) with GC-MS Analysis as Described in Example 6A
Co-expression of santalene synthase and SaCYP76F39v1 (SaCYP76-G10) resulted in the detection of 11 product peaks identified as α-, β- and epi-β-santalol and α-trans-bergamotol (see FIGS. 8A-8B and Table 11 below). Nine (9) of the 11 products were also detected in the S. album oil, albeit in different ratios, as shown in FIGS. 8A and 8B. The products were identified based on matches of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil and Kovats index values (See FIG. 8A and Table 11). The main components of S. album oil are α-santalol, Z-α-trans-bergamotol, E-cis,epi-β-santalol and trans-β-santalol whereas the main products from SaCYP76F39v1 (SaCYP76-G10) are cis-α-santalol, α-santalol and trans-β-santalol. These differences can be due to different physiological conditions, such as pH, under which the SaSSy and SaP450 enzymes are active in the yeast cells and in the trees, or they can be due to changes in the ratios of products over time. The products monitored in yeast were formed and accumulated over a period of hours, while oil extracted from trees is potentially the product of years of accumulation. Farnesol (labeled #), which is produced by yeast independent of the expression of santalene synthase, and dodecanoic acid (labeled *), which is extracted from yeast, were also observed (see FIGS. 8B and 8C).







TABLE 11







Terpenoids identified in in vivo assay with SaCYP76F39v1


(SaCYP76-G10) and S. album oil












Reten-


Compounds



tion
Retention
Products detected from
detected in


Peak
Time
Index1
SaCYP76F39v1
S. album oil














 1
32.23
2169
unknown isomer of
traces





α-trans-bergamotol



 2
35.2
2214
unknown
Yes


 3
35.8
2228
unknown isomer of α-santalol
No


 4
38.5
2294
cis-α-santalol
Yes


 5a
39.1
2308
unknown isomer of α-santalol
No


 5b
39.1
2308
α-trans-bergamotol
Yes


 6
40.0
2331
unknown isomer of α-santalol
Yes


 7
40.4
2341
unknown isomer of
Yes





α-trans-bergamotol



 8
41.1
2359
Epi-β-santalol
Yes


 9
41.7
2374
β-santalol
Yes


10
42.7
2399
unknown isomer of β-santalol
Yes


11
43.2
2412
unknown isomer of β-santalol
Yes





1Linear retention indices (LRI) measured on a SGE Solgel-Wax column






* Dodecanoic acid, extracted from yeast; # Farnesol, product of yeast.

2. SaCYP76F39v1 (SaCYP76-G10) with GC-MS Analysis as Described in Example 6B
Co-expression of santalene synthase and SaCYP76F39v1 (SaCYP76-G10) resulted in the detection of eight products identified as (Z)- and (E)-α-santalol (peaks 5 and 7), (Z)- and (E)-β-santalol (peaks 6 and 8), (Z)- and (E)-epi-β-santalol (peaks 9 and 11) and (Z)- and (E)-α-trans-bergamotol (peaks 10 and 12) (see FIG. 11A). Table 12 below sets forth the peak number, compound and linear retention indices for the DBwax column and the HP5 column. Product identification was based on best match of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil and Kovats index values. As shown in the figure, the product peak for (Z)-α-trans-bergamotol overlapped with a peak corresponding to (E,E)-farnesol, which was produced in yeast independent of SaCYP76F39v1 (SaCYP76-G10) (see FIG. 11B).
A fraction of the sesquiterpenols produced were modified to unidentified compounds (identified with hash tags (#) in FIG. 11A). When untransformed yeast cells were incubated with authentic sandalwood oil, the same unknown compounds were identified implying that these unidentified compounds are not direct products of SaCYP76F39v1 (SaCYP76-G10) but are produced by an endogenous activity of yeast converting sandalwood sesquiterpenols (see FIGS. 12A-12B).







TABLE 12







Retention indices of sesquiterpenes and sesquiterpenols












LRI1
LRI2


Peak
Compound
DBwax
HP5













1
α-santalene
1579
1423


2
α-trans-bergamotene
1592
1437


3
epi-β-santalene
1637
1450


4
β-santalene
1652
1463


5
(Z)-α-santalol
2343
1676


6
(Z)-α-trans-bergamotol
2353
1692


7
(E)-α-santalol
2382
1697


8
(E)-α-trans-bergamotol
2389
1711


9
(Z)-epi-β-santalol
2409
1703


10
(Z)-β-santalol
2423
1717


11
(E)-epi-β-santalol (tentative)
2452
1726


12
(E)-β-santalol
2465
1738





1Linear retention indices (LRI) measured on a DBwax column.


2Linear retention indices (LRI) measured on a HP5 column.






3. SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13)
SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) were assayed for their ability to oxidize sesquiterpenes using the in vivo assay described above with GC-MS analysis described in Example 6B. Co-expression of santalene synthase and SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) or SaCYP76F42 (SaCYP76-G13) gave product profiles with nearly identical ratios to those observed for SaCYP76F39v1 (SaCYP76-G10) (see Table 12 and FIGS. 13A-13D).
C. Clade II Santalum album P450s
Clade II S. album P450s SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5) were assayed for their activity in vivo with GC-MS analysis as described in Example 6A or 6B.
1. SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12) with GC-MS Analysis as Described in Example 6A
Co-expression of santalene synthase with SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) or SaCYP76F38v2 (SaCYP76-G12) in the recombinant yeast system resulted in virtually identical products (see FIGS. 6A, 6B and 6C and Table 13 below). The products were identified based on matches of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil (See FIG. 7 and Table 13). Peaks 1 and 7, which were observed for SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12), correspond to α-trans-bergamotol, possibly representing different isomers. Peaks 1 and 7 were also observed for S. album oil (see FIG. 7 and Table 13). A third peak (labeled #) with a retention time of approximately 18 minutes was identified as farnesol, which is produced by yeast independent of the expression of santalene synthase and SaCYP76, as observed by its expression in the control cells containing an empty vector (FIG. 6D).







TABLE 13







Terpenoids identified in in vivo assay with SaCYP76-F38v1,


-F37v1, -F38v2 and S. album oil













Compounds



Retention
Products detected from
detected in


Peak
Time
CYP76-F38v1, -F37v1, -F38v2
S. album oil





1
17.64
unknown isomer of α-trans-bergamotol
traces


4
18.00
cis-α-santalol
Yes


 5b
18.05
α-trans-bergamotol
Yes


7
18.15
unknown isomer of α-trans-bergamotol
Yes


8
18.40
Epi-β-santalol
Yes


9
18.50
β-santalol
Yes





1Linear retention indices (LRI) measured on a SGE Solgel-Wax column






# Farnesol, product of yeast.

2. SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) or SaCYP76F38v2 (SaCYP76-G12) or SaCYP76F37v2 (SaCYP76-G14)
SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) or SaCYP76F38v2 (SaCYP76-G12) or SaCYP76F37v2 (SaCYP76-G14) were assayed for their ability to oxidize sesquiterpenes using the in vivo assay described above with GC-MS analysis described in Example 6B. Co-expression of santalene synthase with SaCYP76F38v1 (SaCYP76-G5), SaCYP76F37v1 (SaCYP76-G11) or SaCYP76F38v2 (SaCYP76-G12) or SaCYP76F37v2 (SaCYP76-G14) in the recombinant yeast system resulted in mostly E-α-trans-bergamotene (peak 8 in Table 12) with only traces of (E)-α-santalol and (E)-β-santalol (peaks 7 and 12 in Table 12) (see Table 12 and FIGS. 14A-14D)
D. SaCYP76F43 (SaCYP76-G18)
SaCYP76F43 (SaCYP76-G18) was assayed for its ability to oxidize sesquiterpenes using the in vivo assay described above with GC-MS analysis described in Example 6B. No activity was observed after co-expression of santalene synthase with SaCYP76F43 (SaCYP76-G18) (see FIG. 14E).
E. SaCPR1 and SaCPR2
To test if SaCPR1 and SaCPR2, which are 70% identical at the protein level, could affect changes in the product profiles, both CPRs were tested as indicated in Example 6B with representative class I and class II SaCYP76Fs SaCYP76F39v1 and SaCYP76F38v1. No differences were observed in the products and relative abundances as compared to those described in Sections B.2. and C.2. above.
EXAMPLE 11
In Vitro Enzymatic Assays
Yeast microsomes containing a S. album cytochrome P450 and a cytochrome P450 reductase, generated in Example 8, were assayed for their ability to oxidize santalenes and bergamotene using either A) a coupled enzyme assay with the in vitro reaction products of SaSSy and FPP; B) an isolated mixture of santalenes and bergamotene as the substrate; or C) individual santalenes or bergamotene as the substrate.
A. Oxidation of Santalenes and Bergamotene Using a Coupled Enzyme Assay
Coupled enzyme assays with S. album santalene synthase (SaSSy) expressed in bacteria (Jones et al. (2011) J Biol Chem 286:17445-17454) were initiated with 50 μg of His6-tag purified SaSSy and 70 μM farnesyl pyrophosphate (FPP) in TPS buffer (25 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM DTT) in a volume of 450 μL. The assays were incubated for 30 min at 30° C. followed by the addition of 50 μL of the microsome preparation containing a S. album cytochrome P450 and a cytochrome P450 reductase and 0.8 mM NADPH. The reaction was incubated for an additional 1 hour at 30° C. and was stopped by extraction with 500 μL hexane/ethyl acetate (85:15). The organic layer was concentrated under a gentle stream of N2 gas to about 100 μL and analyzed by GC-MS analysis (as described in 6A above) or was stored at −80° C.
1. SaCYP76F38v1 (SaCYP76-G5)
The coupled enzyme assay was performed in vitro with SaCYP76F38v1 (SaCYP76-G5) and compared to the in vivo results to verify the utility of the assay. GC-MS analysis of the reaction products from the coupled assay showed the same two peaks identified in the in vivo assay in Example 8. In both assays, SaCYP76F38v1 (SaCYP76-G5) catalyzed the hydroxylation of bergamotene into Z-α-trans-bergamotol but did not catalyze the oxidation of any santalenes.
B. Oxidation of a Mixture of Santalenes and Bergamotene
S. album P450s were assayed for their sesquiterpene oxidase activities using a mixture of santalenes and bergamotene as the substrate.
1. Assays
Two different in vitro assays were used to screen the S. album CYP76Fs for sesquiterpene oxidase activity.
a. In Vitro Assay 1
Assays were performed in 400 μL reaction volumes containing 150 μL potassium phosphate buffer 100 mM (pH 7.5), 20 μL 20 mM NADPH, 1 μL of 25 mM santalene/bergamotene mixture [containing α-santalene, epi-β-santalene, β-santalene, α-bergamotene] and 80 pmol of the microsomes preparation (prepared as described in Example 8). The reactions were incubated at 30° C. for 1 hour and stopped by adding 500 μL hexane:ethyl acetate (85:15) followed by vortexing for 30 seconds. The organic layer was concentrated under a gentle stream of N2 gas to about 100 μL and analyzed by GC-MS analysis (as described in Example 6A above) or was stored at −80° C.
b. In Vitro Assay 2
Assays were performed in 400 μL reaction volumes containing 50 mM potassium phosphate pH 7.5, 0.8 mM NADPH and 40 μM of substrate. Enzyme reactions were initiated by adding 50 μL of the microsomes preparation (prepared in Example 8), incubated at 30° C. for 2 hours with shaking and stopped by adding 500 μL hexane. The organic layer was transferred to a new GC vial and concentrated under a gentle stream of N2 gas to about 100 μL and analyzed by GC-MS analysis (as described in Example 6B above).
2. Clade I Santalum album P450s
Microsomes containing clade I S. album P450s SaCYP76F39v1 (SaCYP76-G10), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) were assayed for their sesquiterpene oxidize activity using the assays set forth above using a mixture of santalenes and bergamotene as the substrate.
a. SaCYP76F39v1 (SaCYP76-G10)
The in vitro sesquiterpene oxidase activity Clade I S. album P450 SaCYP76F39v1 (SaCYP76-G10) was assessed using both assays described above.
i. Initial Experiment Using In Vitro Assay 1
Microsomes containing SaCYP76F39v1 (SaCYP76-G10) were assayed for their activity using the assay described in Section B.1.a. above. GC-MS analysis revealed eight different product peaks that were identified as santalols (see FIG. 9B, peaks correspond to those in Table 11 above). Product identification was based on best match of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil (FIG. 9A) and Kovats index values.
ii. Assay Using In Vitro Assay 2
Microsomes containing SaCYP76F39v1 (SaCYP76-G10) were assayed for their activity using the assay described in Section B.1.b above. GC-MS analysis of the reaction products revealed that SaCYP76F39v1 (SaCYP76-G10) catalyzed the hydroxylation of α-santalene, β-santalene, epi-β-santalene and α-trans-bergamotene, leading to 8 different compounds identified as (Z)- and (E)-α-santalol, (Z)- and (E)-β-santalol, (Z)- and (E)-epi-β-santalol and (Z)- and (E)-α-trans-bergamotol (see FIG. 15A and Table 12). The product profile was compared to an authentic sandalwood oil sample (see Table 12 and FIG. 15B), which showed identical retention times and mass spectra for all 8 compounds but in different ratios. SaCYP76F39v1 (SaCYP76-G10) produced (E)-α-santalol and (Z)-α-santalol in a ratio of approximately 5:1, and (E)-β-santalol and (Z)-β-santalol in a ratio of approximately 4:1. The main products formed with SaCYP76F39v1 (SaCYP76-G10) were (E)-α-santalol and (E)-β-santalol while the main compounds of sandalwood oil are (Z)-α-santalol and (Z)-β-santalol. No product was formed in the absence of NADPH or with microsomes from yeast carrying an empty vector (see FIG. 15C).
b. SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13)
Microsomes containing SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) were assayed for their activity using the assay described in Section B.1.b above. GC-MS analysis of the reaction products revealed that SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) gave product profiles similar to those observed for SaCYP76F39v1 (SaCYP76-G10) (see Table 12 and FIGS. 16A-16D). The major products observed for SaCYP76F40 (SaCYP76-G16) and SaCYP76F42 (SaCYP76-G13) were (E)-α-trans-bergamotol (or (E)-α-exo-bergamotol) and (E)-β-santalol.
3. Clade II S. album P450s
Microsomes containing clade II S. album P450s SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5) were assayed for their sesquiterpene oxidize activity using the assays set forth above using a mixture of santalenes and bergamotene as the substrate.
a. SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12)
Microsomes containing SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F38v2 (SaCYP76-G12) were assayed for their activity using the assay described in Section B.1.a. above. GC-MS analysis of the reaction products revealed one product peak that was absent in the control reaction (microsomes containing only vector control). The product peak was identified as Z-α-trans-bergamotol based on best match of its MS fragmentation pattern with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.) and by comparison with compounds of authentic S. album oil.
b. SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5)
Microsomes containing SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5) were assayed for their activity using the assay described in Section B1b. above. GC-MS analysis of the reaction products revealed that SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5) produced three compounds, which were identified as (E)-α-trans-bergamotol (or (E)-α-exo-bergamotol) as the major product, and (E)-α-santalol and (E)-β-santalol as minor products (see Table 12 and FIGS. 17A-17D).
4. SaCYP76F43 (SaCYP76-G18)
Microsomes containing SaCYP76F43 (SaCYP76-G18) were assayed for their activity using the assay described in Section B.1.b above using a mixture of santalenes and bergamotene as the substrate. No activity was observed (see FIG. 17E) possibly due to low expression in yeast as evidenced by the corresponding CO difference spectrum (see FIG. 18).
C. Oxidation of Individual Sesquiterpenes
Microsome preparations containing candidate P450 were assayed for their capacity to oxidize individual sesquiterpenes. The sesquiterpenes were isolated as described in Example 9 above. Three fractions containing mainly α-santalene, α-trans-bergamotene, or epi-β-santalene and β-santalene were used as individual substrates in assays containing clade I P450 SaCYP76F39v1 (SaCYP76-G10) or clade II P450 SaCYP76F37v1 (SaCYP76-G11). The assays were performed as described in Section B.1.b. above and products were identified by comparison to authentic standards (see Table 12 and FIG. 20G).
Reaction of SaCYP76F39v1 (SaCYP76-G10) with α-santalene produced (Z)- and (E)-α-santalol while only (E)-α-santalol was produced with SaCYP76F37v1 (SaCYP76-G11) (see FIG. 20A versus FIG. 20D). With α-trans-bergamotene, SaCYP76F39v1 (SaCYP76-G10) produced (Z)- and (E)-α-trans-bergamotol while only (E)-α-trans-bergamotol formation was observed for SaCYP76F37v1 (SaCYP76-G11) (see FIG. 20B versus FIG. 20E). SaCYP76F39v1 (SaCYP76-G10) gave four products (Z)- and (E)-epi-β-santalol and (Z)- and (E)-β-santalol in assays with epi-β-santalene and β-santalene whereas only (E)-β-santalol was detected in assays with SaCYP76F37v1 (SaCYP76-G11) (see FIG. 20C versus FIG. 20F). These results confirm the activities observed with microsome in vitro assays with the mixture of santalenes and bergamotene (Section B above).
SUMMARY OF RESULTS FROM EXAMPLES 10 and 11
Clade I S. album P450 Santalene/Bergamotene Oxidases
Clade I S. album P450s SaCYP76F39v1 (SaCYP76-G10), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) catalyzed the oxidation of santalenes and bergamotene producing the (Z) and (E) stereoisomers of α-, β- and epi-β-santalols and bergamotols. The P450 ratios of (Z) and (E) stereoisomers of α- and β-santalol were approximately 1:5 and 1:4, respectively. Thus SaCYP76F39v1 (SaCYP76-G10), SaCYP76F39v2 (SaCYP76-G15), SaCYP76F40 (SaCYP76-G16), SaCYP76F41 (SaCYP76-G17) and SaCYP76F42 (SaCYP76-G13) were identified as a santalene/bergamotene oxidases.
Clade II S. album P450 Bergamotene Oxidases
Clade II S. album P450s SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 (SaCYP76-G5) primarily catalyzed the oxidation of bergamotene into bergamotol, with (E)-α-trans-bergamotol as the major product and minor amounts of (E)-α-santalol and (E)-β-santalol observed. SaCYP76F37v1 (SaCYP76-G11), SaCYP76F38v2 (SaCYP76-G12), SaCYP76F37v2 (SaCYP76-G14) and SaCYP76F38v1 were identified as bergamotene oxidases.
EXAMPLE 12
Kinetic Properties
To test the kinetics of the clade I and clade II SaCYP76F enzymes, kinetic assays were performed with SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F39v1 (SaCYP76-G10) with α-santalene or β-santalene as the substrate. Assays were performed in 400 μL reaction volumes containing 50 mM potassium phosphate pH 7.5, 0.8 mM NADPH and substrate concentrations of 12 to 138 μM of α-santalene or β-santalene. Enzyme reactions were initiated by adding either 17 pmol of SaCYP7639v1 or 35 pmol of SaCYP7637v1, incubated at 30° C. for 20 minutes with shaking and stopped by adding 500 μL hexane. The organic layer was transferred to a new GC vial and concentrated under a gentle stream of N2 gas to about 100 μL and analyzed by GC-MS analysis (as described in Example 6B above). Kinetic data were evaluated using tools described in Hernandez and Ruiz ((1998) Bioinformatics. 14:227-228).
The apparent Km values, kcat values and kcat/Km values for SaCYP76F39v1 (SaCYP76-G10) and SaCYP76F37v1 (SaCYP76-G11) with α-santalene and β-santalene are set forth in Table 14 below.







TABLE 14





Kinetic constants for SaCYP76F39v1 and SaCYP76F37v1

















α-santalene














kcat
kcat/Km



P450
Km (μM)
(s−1)
(s−1 M−1)






SaCYP76F39v1
25.92 ± 0.11
1.12
4.3 × 104



(SaCYP76-G10)






SaCYP76F37v1
  133 ± 0.41
0.2 
1.5 × 103



(SaCYP76-G11)












β-santalene












P450
Km (μM)
kcat
kcat/Km






SaCYP76F39v1
34.82 ± 0.41
1.17
3.3 × 104



(SaCYP76-G10)






SaCYP76F37v1
  157 ± 0.17
0.13
8.1 × 102



(SaCYP76-G11)









EXAMPLE 13
Substrate Specificity
A. Substrate Specificity of Clade I and Clade II SaCYP76F Enzymes
To test the range of substrates used by the clade I and clade II SaCYP76F enzymes, yeast microsomes containing SaCYP76F37v1 (SaCYP76-G11) and SaCYP76F39v1 (SaCYP76-G10) were assayed for their ability to convert various sesquiterpenes, including the substrates α-santalene and β-santalene and 7 additional sesquiterpenes which resemble santalenes in the acyclic isoprenyl side chain, including α-curcumene, zingiberine, β-bisabolene, β-sesquiphellandrene, α-bisabolol, trans-β-farnesene and trans-nerolidol. Each substrate was tested using the in vitro assay described in Example 11.B.1.b above.
The results are shown in Table 15 below, which sets forth the substrates, including their structures, and the relative activities which represent the rate of product formation relative to product formation by SaCYP76F39v1 (SaCYP76-G10) with β-santalene. As shown in the table, SaCYP76F39v1 (SaCYP76-G10) and SaCYP76F37v1 (SaCYP76-G11) exhibited narrow substrate selectivity, with both preferring santalenes, including α-santalene or β-santalene, as substrates. SaCYP76F39v1 (SaCYP76-G10) efficiently converted only the two santalenes and had low activity with α-bisabolol. SaCYP76F39v1 (SaCYP76-G10) did not use α-curcumene, zingiberene, β-bisabolene, β-sesquiphellandrene, trans-β-farnesene or trans-nerolidol as a substrate. Similarly, SaCYP76F37v1 (SaCYP76-G11) was selectively active with the two santalenes and trans-nerolidol.







TABLE 15







Relative activities of SaCYP76F39v1 and SaCYP76F37v1 with various


sesquiterpene substrates.










SaCYP76F39v1
SaCYP76F37v1



(SaCYP76-G10)
(SaCYP76-G11)


Substrate
[%]*
[%]*













α-santalene




99.8
17.3


 


β-santalene




100
17.7


 


α-curcumene




0
0


 


zingiberene




0
0


 


β-bisabolene




0
0


 


β-sesquiphellandrene




0
0


 


α-bisabolol




9.4
0


 


trans-farnesene




0
0


 


trans-nerolidol




0
11.3





*Relative activities represent rate of product formation relative to product formation by SaCYP76F39v1 eith β-santalene.






B. Oxidation of Various Mono- and Sesquiterpenes Substrates

Yeast microsomes containing S. album cytochrome P450 SaCYP76F38v1 (SaCYP76-G5) and cytochrome P450 reductase were directly assayed for their capacity to oxidize different mono- and sesquiterpene substrates, including linalool, geraniol, nerol, nerolidol and bisabolol. The reaction mixtures contained 50 mM potassium phosphate, 0.8 mM NADPH and 60 to 80 μM of the terpene substrate in a total volume of 350 μL. Enzyme reactions were started by adding 50 μL of the microsome preparation, incubated at 30° C. for 1 hour with shaking and stopped by extraction with 500 μL of hexane/ethyl acetate (85:15). The organic layer was concentrated under a gentle stream of N2 gas to about 100 μL and analyzed by GC-MS analysis as described in Example 6. Results were compared to vector control. The reaction products were identified based on matches of the MS fragmentation patterns with entries in the NIST and Wiley libraries (Wiley Registry® 9th Edition/NIST 2011; Fred W. McLafferty, John Wiley & Sons, Inc.).
1. SaCYP76F38v1 (SaCYP76-G5)
Reaction of SaCYP76F38v1 (SaCYP76-G5) with linalool resulted in two products: Peak 1, retention time of at approximately 17.5 minutes and Peak 2, retention time of approximately 18.5 minutes. Linalool had a retention time of approximately 10.5. The best matches for the MS fragmentation patterns of Peaks 1 and 2 correspond to 3,8-dimethyl-1,7-octadien-6-ol and 8-hydroxylinalool, respectively. Reaction of SaCYP76F38v1 (SaCYP76-G5) with geraniol resulted in one product with a retention time of approximately 21 minutes. Geraniol had a retention time of approximately 14 minutes. The best match for this peak's MS fragmentation pattern corresponds to trans,trans-2,6-dimethyl-2,6-octadiene-1,8 diol. Reaction of SaCYP76F38v1 (SaCYP76-G5) with nerol resulted in one product with a retention time of approximately 20.8 minutes, whereas nerol had a retention time of approximately 13.4 minutes. The best match for this peak's MS fragmentation pattern corresponds to 2,6-dimethyl-2,6-octadiene-1,8 diol. Reaction of SaCYP76F38v1 (SaCYP76-G5) with nerolidol resulted in two products, with retention times of approximately 21.3 and 22.3 minutes, whereas nerolidol had a retention time of approximately 16.1 minute. Reaction of SaCYP76F38v1 (SaCYP76-G5) with bisabolol resulted in one product having a retention time of approximately 25.2 with bisabolol having a retention time of approximately 17.6. The MS fragmentation patterns of products formed by reaction of SaCYP76F38v1 (SaCYP76-G5) with nerolidol and bisabolol did not match with known substances in the MS fragmentation pattern databases.
2. SaCYP76F39v1 (SaCYP76-G10)
CYP76-G10 also catalyzed the hydroxylation of linalool, nerol and bisabolol in vitro. In each case, product formation was the same as that catalyzed by CYP76-G5 described above.
Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

## Claims

1. 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 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 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.

2. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof exhibits at least 96% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 13.

3. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a Santalum P450 reductase polypeptide.

4. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a Santalum album P450 reductase polypeptide.

5. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof comprises a sequence of amino acids that has at least 99% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13.

6. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof comprises a sequence of amino acids set forth in SEQ ID NO: 13.

7. The host cell of claim 1, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a catalytically active fragment comprising a sequence of amino acids set forth in SEQ ID NO: 15, or a sequence that exhibits at least 95% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 15.

8. The host cell of claim 1, wherein the electron acceptor is a cytochrome P450, heme oxygenase, cytochrome b5 or squalene epoxidase.

9. The host cell of claim 1, comprising a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
(a) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 95% sequence identity to SEQ ID NO: 50 or a corresponding catalytically active portion thereof;
(b) the encoded cytochrome P450 oxidase or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene; and
(c) the nucleic acid molecule is heterologous to the host cell.

10. The host cell of claim 9, wherein the encoded full-length cytochrome P450 oxidase polypeptide exhibits at least 99% sequence identity to SEQ ID NO: 50.

11. The host cell of claim 1, comprising a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
(a) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof comprises the sequence of amino acids set forth in SEQ ID NO: 7; or
(b) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof comprises a sequence of amino acids that exhibits at least 95% sequence identity to a cytochrome P450 oxidase polypeptide set forth in SEQ ID NO: 7; and
(c) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene.

12. The host cell of claim 11, wherein the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof exhibits at least 99% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 7.

13. The host cell of claim 11, wherein the cytochrome P450 oxidase polypeptide or catalytically active fragment thereof comprises the sequence of amino acids set forth in SEQ ID NO: 7.

14. The host cell of claim 1, 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 95% sequence identity to SEQ ID NO:7;
(ii) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof catalyzes the hydroxylation or monooxygenation of santalene and/or bergamotene; and
(iii) the nucleic acid molecule is heterologous to the host cell;

(b) a nucleic acid molecule encoding a cytochrome P450 reductase or catalytically active portion thereof, wherein:
(i) the nucleic acid molecule is heterologous to the host cell; and
(ii) the encoded cytochrome P450 reductase or catalytically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO: 13, or a sequence of amino acids that has at least 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13; and

(c) a nucleic acid molecule encoding a santalene synthase, wherein:
(i) the nucleic acid molecule is heterologous to the host cell; and
(ii) the encoded santalene synthase comprises the sequence of amino acids set forth in any of SEQ ID NO: 17 or a sequence of amino acids that is at least 95% identical to any of SEQ ID NO: 17 or a catalytically active fragment thereof.

15. 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 95% sequence identity to SEQ ID NO: 7;
(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: 13, or a sequence of amino acids that has at least 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13; 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 or a sequence of amino acids that is at least 95% identical to any of SEQ ID NO: 17 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.

16. The host cell of claim 11 or 15, wherein the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:7, or a sequence of amino acids that exhibits at least 96% sequence identity to a cytochrome P450 oxidase polypeptide set forth in SEQ ID NO: 7.

17. The host cell of claim 1 or 15, that is a prokaryotic cell or an eukaryotic cell.

18. The host cell of claim 1 of 15, that produces farnesyl diphosphate natively or is modified to produce more farnesyl diphosphate compared to an unmodified cell.

19. 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 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 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.

20. The nucleic acid molecule of claim 19, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof exhibits at least 96% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 13.

21. The nucleic acid molecule of claim 19, wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof comprises a sequence of amino acids set forth in SEQ ID NO: 13.

22. The nucleic acid molecule of claim 19, wherein the encoded cytochrome P450 reductase polypeptide is a catalytically active fragment comprising a sequence of amino acids set forth in SEQ ID NO: 15, or a sequence that exhibits at least 95% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 15.

23. The nucleic acid molecule of claim 19, wherein the electron acceptor is a cytochrome P450, heme oxygenase, cytochrome b5 or squalene epoxidase.

24. A modified cytochrome P450 reductase polypeptide or catalytically active portion thereof encoded by the nucleic acid molecule of claim 19; wherein the modified cytochrome P450 polypeptide is at least 95% identical to SEQ ID NO: 13, provided that the modified cytochrome P450 reductase sequence is not SEQ ID NO: 13.

25. A vector, comprising the nucleic acid molecule of claim 19.

26. A host cell, comprising the vector of claim 25.

27. A method for producing a cytochrome P450 reductase polypeptide or a catalytically active fragment thereof, comprising:
(a) culturing the cells of claim 1 or 15 under conditions suitable for expression of the cytochrome P450 reductase polypeptide; and
(b) optionally isolating the cytochrome P450 reductase polypeptide.

28. A method for producing a santalol, bergamotol and/or mixtures thereof, comprising:
(a) culturing a host cell of claim 15 under conditions suitable for the formation of a santalol, bergamotol and/or mixtures thereof; wherein the host cell of claim 15 expresses the nucleic acid molecules of part (a), (b) and (c); and
(b) optionally isolating the santalol, bergamotol and/or mixtures thereof.