Cytochrome p450 protein or active fragment thereof

Abstract

The present invention addresses the problem of providing a cytochrome P450 protein by identifying a gene of a Cupressaceae plant involved in the biosynthesis of ferruginol and encoding the cytochrome P450 protein with said gene. The present invention also addresses the problem of providing a new method for producing ferruginol on the basis of said gene.　Provided is a cytochrome P450 protein of an isolated CYP720 family or an active fragment thereof, the protein having an activity of oxidizing the carbon at position 12 of an abietane-type diterpene. The cytochrome P450 protein or an active fragment thereof is derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Cephalotaxaceae, Podocarpaceae, and Araucariaceae.

Description

Cytochrome P450 protein or its active fragment

[0001] The present invention relates to cytochrome P450 proteins or active fragments thereof, nucleic acids encoding these proteins or active fragments thereof, oxidizing agents for the 12th carbon in abietane diterpenes, transformed cells, methods for producing terpenoid compounds having a hydroxyl group at the 12th carbon, and the like.

[0002] Plants of the genus Salvia, which are perennial evergreen shrubs in the Lamiaceae family, are widely used worldwide as an important source of essential oils. Essential oil components extracted from Salvia include rosmarinic acid, a type of polyphenol, as well as abietane-type diterpene oxidation compounds such as carnosic acid, carnosol, and rosmanol. Carnosic acid and its oxidation compounds are known to have antioxidant, antibacterial, and anti-Alzheimer's effects, and various studies are being conducted to utilize these physiological activities (Non-Patent Document 1).

[0003] In general, to provide physiologically active substances stably and sustainably, it is necessary to identify the synthase genes involved in the biosynthesis of the physiologically active substances and then introduce these synthase genes into a biological production system to reconstitute the biosynthetic pathway.

[0004] In the biosynthesis of carnosic acid in the Lamiaceae genus, the gene involved in the reaction of carnosic acid biosynthesis using myrtilaradiene as a substrate has been identified, and it has previously been reported that carnosic acid is biosynthesized in transformed yeast into which this gene has been introduced. In transformed yeast into which a cytochrome P450 gene of the CYP76AH subfamily derived from the genus, ferruginol is biosynthesized using myrtilaradiene as a substrate, and carnosic acid is then biosynthesized through a series of oxidation reactions (Figure 1).

[0005] On the other hand, in the Cupressaceae family, ferruginol accumulates in the heartwood and is known to exhibit anti-termite activity, but no genes involved in the biosynthesis of ferruginol or carnosic acid have been reported in the Cupressaceae family, and the mechanism of their biosynthesis remains unknown.

[0006] Furthermore, the substrates for ferruginol biosynthesis are not known in the Cupressaceae family. As mentioned above, miltiradiene is known to be a substrate for ferruginol biosynthesis in the Cupressaceae genus, but miltiradiene biosynthesis has not been confirmed in conifers such as the Cupressaceae family. Therefore, it is suggested that the mechanism of ferruginol biosynthesis in the Cupressaceae family is different from that in the Cupressaceae genus.

[0007] Birtic S., et al., Phytochemistry 2015, 115: 9-19.

[0008] The objective of this study is to identify a gene involved in the biosynthesis of ferruginol in plants of the Cupressaceae family, and to provide a cytochrome P450 protein encoded by said gene, as well as a new method for producing ferruginol based on said gene.

[0009] In conifers of the Cupressaceae family, the heartwood formed in the center of the xylem is rich in phenolic abietane diterpenes such as ferruginol, so the ferruginol synthase gene involved in ferruginol biosynthesis may be highly expressed in the heartwood transition zone. Furthermore, based on the above-mentioned findings from the Lamiaceae genus, Atractylodes macrocarpa, it is predicted that the ferruginol synthase gene in the Cupressaceae family is a cytochrome P450 gene.

[0010] Based on this prediction, we searched for cytochrome P450 genes highly expressed in the heartwood transition zone of T. cryptomerioides, a species of Cupressaceae, using transcriptome data, and identified six genes as candidate genes for ferruginol synthase.

[0011] Next, we verified which of the six identified genes was the ferruginol synthase gene. As mentioned above, the substrate for ferruginol biosynthesis in the Cupressaceae family is unknown, but levopimaradiene, an abietane-based diterpene olefin similar to miltiradiene, has been widely identified in the Cupressaceae family. Therefore, it was speculated that levopimaradiene, rather than miltiradiene, may be the substrate for ferruginol biosynthesis in the Cupressaceae family.

[0012] Therefore, the inventors introduced each of the six candidate genes, along with the gene required for the biosynthesis of levopimaradiene, which can be a substrate for ferruginol biosynthesis, into yeast and cultured them, and evaluated the presence or absence of ferruginol synthesized from levopimaradiene in the cultured yeast.As a result, they found that among the six candidate genes, the CYP720 family cytochrome P450 protein has the activity of binding a hydroxy group to the 12th carbon of levopimaradiene to produce ferruginol.

[0013] Furthermore, the present inventors identified and evaluated the activity of genes homologous to the above-mentioned gene derived from Taiwan cedar in Chinese fir (C. lanceolata), which belongs to the closely related genus C. fortunei and the subfamily Cedrus, both of which belong to the Cupressaceae family. As a result, it was revealed that the cytochrome P450 proteins encoded by these homologous genes also have the activity to produce ferruginol.

[0014] The present invention is based on the above findings and provides the following: (1) An isolated cytochrome P450 protein of the CYP720 family or an active fragment thereof, which has the activity of oxidizing the 12-carbon atom of an abietane diterpene, and is derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebacterium, Podocarpaceae, and Araucaria. (2) The cytochrome P450 protein or active fragment thereof according to (1), comprising (a) an amino acid sequence set forth in any of SEQ ID NOS: 1 to 3, (b) an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in the amino acid sequence set forth in any of SEQ ID NOS: 1 to 3, or (c) an amino acid sequence having 89% or more amino acid identity to the amino acid sequence set forth in any of SEQ ID NOS: 1 to 3. (3) An isolated nucleic acid encoding the cytochrome P450 protein or active fragment thereof according to (1). (4) The nucleic acid according to (3), comprising (i) a base sequence set forth in any one of SEQ ID NOS: 4 to 6, (ii) a base sequence in which one or several bases are added, deleted, or substituted in the base sequence set forth in any one of SEQ ID NOS: 4 to 6, or (iii) a base sequence having 88% or more base identity to the base sequence set forth in any one of SEQ ID NOS: 4 to 6. (5) The nucleic acid according to (3), which is a gene expression vector. (6) An oxidizing agent for the 12th carbon of an abietane-type diterpene, comprising a cytochrome P450 protein of the CYP720 family or an active fragment thereof, and / or a nucleic acid encoding the cytochrome P450 protein or an active fragment thereof, derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucaria. (7) A transformed cell into which the nucleic acid according to any one of (3) to (6) has been introduced. (8) A transformed plant comprising the transformed cell according to (7).(9) A method for producing a terpenoid compound having a hydroxyl group at the 12th carbon, comprising a culture / cultivation step of culturing a transformed cell into which a nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Papaceae, and Araucariales has been introduced, or cultivating a transformed plant containing the transformed cell. (10) The method according to (9), wherein the transformed cell contains a gene encoding levopimaradiene synthase in an expressible state. (11) The method according to (9) or (10), further comprising an extraction step of extracting an extract containing the terpenoid compound having a hydroxyl group at the 12th carbon from the transformed cell or the transformed plant after the culture / cultivation step. (12) The method according to any one of (9) to (11), wherein the terpenoid compound having a hydroxyl group at the 12th carbon is ferruginol. (13) A method for producing a terpenoid compound having a hydroxyl group at the 12th carbon, comprising an oxidation step of oxidizing the 12th carbon of an abietane diterpene in the presence of a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Papaceae, and Araucaria, to obtain a terpenoid compound having a hydroxyl group at the 12th carbon. (14) The method according to (13), wherein the abietane diterpene is levopimaradiene or dehydroabietadiene, and the terpenoid compound having a hydroxyl group at the 12th carbon is ferruginol. (15) Use of a cytochrome P450 protein of the CYP720 family or an active fragment thereof, and / or a nucleic acid encoding the cytochrome P450 protein or an active fragment thereof, derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Papaceae, and Araucaria, in the oxidation of carbon at position 12 in abietane diterpenes. This specification incorporates the disclosure of Japanese Patent Application No. 2024-122532, from which the present application claims priority.

[0015] According to the present invention, a gene involved in the biosynthesis of ferruginol in plants of the Cupressaceae family is provided, as well as a cytochrome P450 protein encoded by the gene and a new method for producing ferruginol based on the gene.

[0016] Figure 1 shows the biosynthetic pathway of ferruginol and its derivatives in the genus Salvia in the Lamiaceae family. Figure 2 shows an example of an abietane-type diterpene. Figure 3 shows the biosynthetic reaction of ferruginol in the Cupressaceae family. Figure 4 shows the biosynthetic pathway of ferruginol reconstructed in transformed yeast. Figure 5 shows the results of detecting levopimaradiene and ferruginol in extracts of transformed yeast. Figure 5A shows the results for transformed yeast into which the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR vector were introduced. Figure 5B shows the results for transformed yeast into which the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR-TcFS vector were introduced. Figure 5C shows the results for the standard ferruginol. In the figure, the horizontal axis represents elution time (minutes), the vertical axis represents the total ion chromatogram (TIC), and the open arrowheads represent levopimaradiene and the closed arrowheads represent ferruginol. Figure 6 shows MS fragment data. Figure 6A shows the results for transformed yeast carrying the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR-TcFS vector. The upper spectrum shows the mass spectral data of the peak component indicated by the black arrow in Figure 5B, and the lower spectrum shows the spectrum obtained by searching the compound mass spectral library. Figure 6B shows the results for the standard ferruginol. The upper spectrum shows the mass spectral data of the peak component indicated by the black arrow in Figure 5C, and the lower spectrum shows the spectrum obtained by searching the compound mass spectral library. In the figure, the horizontal axis represents m / z (relative mass / charge number of the ion), and the vertical axis represents the detection intensity (abundance of the ion). Figure 7 shows the results of detecting levopimaradiene and ferruginol in the extract of transformed yeast. Figure 7A shows the results for transformed yeast into which the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR-ClFS vector were introduced. Figure 7B shows the results for transformed yeast into which the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR-CfFS vector were introduced. In the figure, the horizontal axis shows elution time (minutes), the vertical axis shows the total ion chromatogram (TIC), the open arrowheads show levopimaradiene, and the closed arrowheads show ferruginol.

[0017] 1. Cytochrome P450 Protein or Active Fragment Thereof 1-1. Overview A first aspect of the present invention is an isolated cytochrome P450 protein or active fragment thereof derived from a plant such as a conifer. The cytochrome P450 protein or active fragment thereof of this aspect belongs to the CYP720 family and has the activity of oxidizing the 12th carbon of abietane diterpenes such as levopimaradiene. Based on this activity, the cytochrome P450 protein or active fragment thereof of this aspect can synthesize terpenoid compounds having a hydroxyl group at the 12th carbon, such as ferruginol.

[0018] 1-2. Definitions of Terms Terms frequently used in this specification are defined below.

[0019] As used herein, the term "abietane diterpene" refers to a 20-carbon isoprenoid having a tricyclic abietane skeleton and consisting of four isoprene units. Specific examples of abietane diterpenes include levopimaradiene, abietadiene, neoabietadiene, palustradiene, miltiradiene, and dehydroabietadiene (Figure 2). Also included are dehydroabietadiene obtained by oxidation of levopimaradiene, abietadiene, neoabietadiene, palustradiene, or miltiradiene. The abietane diterpene of the present invention is preferably levopimaradiene or dehydroabietadiene.

[0020] As used herein, "a terpenoid compound having a hydroxyl group at the 12th carbon" refers to a terpenoid compound produced from the above-mentioned abietane diterpene, in which a hydroxyl group is bonded to the 12th carbon. A specific example of a terpenoid compound having a hydroxyl group at the 12th carbon is ferruginol. Note that, while dehydroabietadienes are obtained in the oxidation reactions of the above-mentioned levopimaradiene, abietadiene, neoabietadienes, palustradiene, and miltiradiene, ferruginol is produced in the reaction in which a hydroxyl group is bonded to the 12th carbon of dehydroabietadienes.

[0021] As used herein, "activity to oxidize the 12th carbon in an abietane diterpene" refers to the activity of catalyzing the reaction of replacing the hydrogen group at the 12th carbon in an abietane diterpene with a hydroxy group. Examples of this activity include the activity of catalyzing the reaction of producing ferruginol from dehydroabietadienes and the activity of catalyzing the reaction of producing ferruginol from levopimaradiene. In addition to the oxidation of the 12th carbon (replacement of the hydrogen group at the 12th carbon with a hydroxy group) in the reaction of producing ferruginol from levopimaradiene catalyzed by the ferruginol synthase discovered in the Examples described below, a dehydrogenation reaction also occurs simultaneously, in which a double bond is added to the six-membered ring containing the 12th carbon to form a benzene ring (Figure 3).

[0022] As used herein, "cytochrome P450" or "cytochrome P450 protein" refers to a reduced protoheme-containing protein enzyme that catalyzes a monooxygenation reaction, which uses an electron donor such as NAD(P)H and oxygen to attach an oxygen atom to a substrate and simultaneously generate water. Cytochrome P450 proteins are also called cytochrome P450s, CYPs, etc., and are also known as drug-metabolizing enzymes. Cytochrome P450 proteins are classified into various families based on similarity in their amino acid sequences. For example, the CYP720 family cytochrome P450 derived from the Cupressaceae family (the cytochrome P450 of the present invention) has the activity of synthesizing ferruginol using levopimaradiene as a substrate, while CYP76AH1 and CYP76AH24, which are cytochrome P450s of the CYP76AH subfamily derived from the Lamiaceae family's Atractylodes genus, have the activity of synthesizing ferruginol using miltiradiene as a substrate. Generally, cytochrome P450s are classified into families based on an amino acid sequence identity of 40% or more, and each family is further classified into subfamilies based on an amino acid sequence identity of 55% or more.

[0023] As used herein, the term "orthologous protein" refers to a protein encoded by a homologous gene that arose from a common ancestral gene when species diverged during the course of biological evolution.

[0024] As used herein, "isolated" refers to a state in which a biomolecule, such as a protein or nucleic acid, is separated from the naturally occurring cell, tissue, or individual from which it originates. An isolated biomolecule may or may not be purified, and may be mixed with other substances. Furthermore, the term "isolated" as used herein also includes a state in which the isolated biomolecule is contained in a cell, tissue, or individual, such as a transformed cell or transformed plant, other than the wild-type cell, tissue, or individual from which it originates.

[0025] As used herein, "several" means 2 or more, 3 or more, 4 or more, or 5 or more. There is no particular upper limit, but examples include 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. Exemplary ranges include 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3, or 2.

[0026] As used herein, "amino acid identity" refers to the percentage (%) of matching amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting appropriate gaps into one or both sequences as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW. "Nucleotide identity" is calculated in a similar manner.

[0027] As used herein, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of polypeptides.

[0028] As used herein, "stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form. "Highly stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form or are not formed at all. In general, the lower the salt concentration and the higher the temperature of the reaction conditions, the more stringent the conditions. For washing after hybridization, for example, washing is performed at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C with 0.1×SSC and 0.1% SDS. In addition, the stringency of hybridization can be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

[0029] 1-3. Configuration The cytochrome P450 protein or active fragment thereof of this embodiment is an isolated cytochrome P450 protein or active fragment thereof of the CYP720 family that has the activity of oxidizing the 12-carbon atom of an abietane diterpene. The cytochrome P450 protein or active fragment thereof of this embodiment is derived from any plant species belonging to the Cupressaceae, Taxaceae, Cephalotaxaceae, Podocarpaceae, or Araucariaceae families, preferably any plant species in the Cupressaceae family, and is isolated from a wild-type plant of the plant species from which it is derived. Note that, in this specification, Cupressaceae includes Taxodiaceae.

[0030] In the present invention, the Cupressaceae plant may be, for example, the subfamily Cunninghamioideae (e.g., the genus Cunninghamia); the subfamily Taiwanioideae (e.g., the genus Taiwania); the subfamily Sequoioideae (e.g., the genus Sequoia, Sequoiadendron, and Metasequoia); the subfamily Taxodioideae (e.g., the genus Taxodium, Glyptostrobus, and Cryptomeria); the subfamily Callitroideae (e.g., the genus Callitris and Neocallitris); ropsis, Actinostrobus, Widdringtonia, Diselma, Austrocedrus, Libocedrus, Pilgerodendron, Papuacedrus, Fitzroya); or Cupressoideae (e.g., Thuja, Fokienia, Thujopsis, Chamaecyparis, Tetraclinis, Platycladus, Microbiota decussata), Calocedrus, Xanthocyparis, Cupressus, Juniperus). Preferred cupressaceae plants are Chinese fir plants, Taiwan cedar plants, or Cryptomeria japonica plants, and specific examples include Taiwan cedar (T. cryptomerioides), Chinese fir (C. lanceolata (Lamb.) Hook), and willow cedar (C. fortunei).

[0031] In one embodiment, the cytochrome P450 protein or active fragment thereof of this aspect comprises or consists of the amino acid sequence set forth in any of SEQ ID NOS: 1 to 3. SEQ ID NOS: 1 shows the amino acid sequence of a wild-type CYP720 family cytochrome P450 protein derived from Taiwan cedar (sometimes referred to herein as "TcFS"). SEQ ID NOS: 2 shows the amino acid sequence of a wild-type CYP720 family cytochrome P450 protein derived from Chinese fir (sometimes referred to herein as "ClFS"). SEQ ID NOS: 3 shows the amino acid sequence of a wild-type CYP720 family cytochrome P450 protein derived from willow cedar (sometimes referred to herein as "CfFS").

[0032] In one embodiment, the cytochrome P450 protein or active fragment thereof of this aspect may be an orthologous protein or active fragment thereof of a cytochrome P450 protein consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3, in any plant species belonging to the Cupressaceae, Taxaceae, Corynebaceae, Papaceae, or Araucaria families. This orthologous protein or active fragment thereof preferably has the activity of oxidizing the 12th carbon of an abietane diterpene. Alternatively, the cytochrome P450 protein or active fragment thereof of this aspect may be a mutant protein or active fragment thereof derived from a cytochrome P450 protein consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3. This mutant protein or active fragment thereof preferably has the activity of oxidizing the 12th carbon of an abietane diterpene.

[0033] For example, the cytochrome P450 protein or active fragment thereof of this embodiment may comprise or consist of an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3 (e.g., an amino acid sequence in which 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted; an amino acid sequence in which 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been added; or an amino acid sequence in which 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been substituted with another amino acid).

[0034] Furthermore, for example, the cytochrome P450 protein or active fragment thereof of this embodiment comprises or consists of an amino acid sequence that has 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, or 87% or more, preferably 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3. The amino acid identity between SEQ ID NOs: 1 and 2 is 92.5%, the amino acid identity between SEQ ID NOs: 2 and 3 is 90.6%, and the amino acid identity between SEQ ID NOs: 1 and 3 is 89.1%.

[0035] As used herein, the term "active fragment" refers to a fragment of any of the above-mentioned cytochrome P450 proteins that has the activity of oxidizing the 12th carbon of an abietane diterpene, for example, a fragment having 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of a cytochrome P450 protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 3, or an activity equivalent to or greater than these. The amino acid length of the polypeptide constituting this fragment is not particularly limited, and may be, for example, a contiguous region having a length of 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, or 450 or more amino acids in any of the above-mentioned cytochrome P450 proteins.

[0036] The cytochrome P450 protein or active fragment thereof of this embodiment can be isolated from any plant of the Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, or Araucaria families (e.g., plants of the genus Cunninghamia, Cryptomeria japonica, or Cryptomeria japonica), or can be synthesized by known chemical synthesis methods based on any of the amino acid sequences described above. Alternatively, the cytochrome P450 protein or active fragment thereof can be biosynthesized by expressing a gene encoding the cytochrome P450 protein or active fragment thereof (e.g., the nucleic acid of the second embodiment described below) in a protein expression system based on any cell, such as Escherichia coli, yeast, insect cells, or mammalian cells.

[0037] Furthermore, when the cytochrome P450 protein or active fragment thereof of this embodiment is the mutant protein or active fragment thereof described above, the mutant protein can be obtained by any method of introducing a mutation into a gene. The mutation introduction method here may be a known method such as the Kunkel method or the gapped duplex method, or may be a method using a commercially available mutagenesis kit that utilizes site-directed mutagenesis (e.g., Diversify™ PCR Random Mutagenesis Kit (TaKaRa), GeneMorph II Random Mutagenesis Kit (Agilent), or the JBS Random Mutagenesis Kit series (Funakoshi)). Alternatively, methods such as treating nucleic acids or cells containing nucleic acids with mutagens (e.g., alkylating agents such as ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine) or irradiating nucleic acids or cells containing nucleic acids with ultraviolet light can also be used.

[0038] 1-4. Effects The cytochrome P450 protein of the present invention or an active fragment thereof can oxidize the carbon at position 12 of an abietane diterpene by allowing it to act on the diterpene. In addition to this oxidation activity (the activity of catalyzing a reaction that replaces the hydrogen group at carbon 12 with a hydroxy group), the cytochrome P450 protein of the present invention or an active fragment thereof may also have the activity of catalyzing a dehydrogenation reaction that adds a double bond to a six-membered ring containing the carbon at position 12 to form a benzene ring. For example, the cytochrome P450 protein of the present invention or an active fragment thereof can oxidize the carbon at position 12 of these abietane diterpenes by allowing it to act on levopimaradiene, abietadiene, neoabietadiene, palustradiene, mirtiradiene, or dehydroabietadiene. In particular, by allowing the cytochrome P450 protein of the present invention or an active fragment thereof to act on levopimaradiene, the carbon at position 12 in levopimaradiene is oxidized, and ferruginol can be produced, in which the six-membered ring containing the carbon at position 12 becomes a benzene ring.

[0039] 2. Nucleic Acid Encoding a Cytochrome P450 Protein or an Active Fragment Thereof 2-1. Configuration A second aspect of the present invention is an isolated nucleic acid encoding a cytochrome P450 protein or an active fragment thereof. The nucleic acid of this aspect encodes any of the cytochrome P450 proteins or active fragments thereof described in the first aspect above.

[0040] The nucleic acid of this embodiment is not particularly limited in its base sequence, as long as it is a nucleic acid such as a polynucleotide encoding any one of the cytochrome P450 proteins or active fragments thereof described in embodiment 1. For example, the nucleic acid of this embodiment may comprise (i) the base sequence set forth in any one of SEQ ID NOs: 4 to 6, (ii) a base sequence in which one or several bases have been added, deleted, or substituted in the base sequence set forth in any one of SEQ ID NOs: 4 to 6, or (iii) a base sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, or 87% or more, preferably 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more base identity to the base sequence set forth in any one of SEQ ID NOs: 4 to 6. SEQ ID NO: 4 shows the nucleotide sequence encoding a wild-type CYP720 family cytochrome P450 protein (TcFS) derived from Taiwan cedar. SEQ ID NO: 5 shows the nucleotide sequence encoding a wild-type CYP720 family cytochrome P450 protein (ClFS) derived from Chinese fir. SEQ ID NO: 6 shows the nucleotide sequence encoding a wild-type CYP720 family cytochrome P450 protein (CfFS) derived from willow cedar. The nucleotide identity between SEQ ID NO: 4 and SEQ ID NO: 5 is 93.0%, the nucleotide identity between SEQ ID NO: 5 and SEQ ID NO: 6 is 89.6%, and the nucleotide identity between SEQ ID NO: 4 and SEQ ID NO: 6 is 88.6%.

[0041] In one embodiment, the nucleic acid of this aspect may be codon-optimized. For example, the nucleic acid may be codon-optimized to match the codon usage frequency in a cell, such as yeast, into which the nucleic acid is to be introduced. Specific examples of codon-optimized nucleic acids include the nucleotide sequences shown in SEQ ID NOs: 9, 12, and 13, which are obtained by codon-optimizing the nucleotide sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, for yeast cells.

[0042] Furthermore, the nucleic acid of this embodiment may be DNA or RNA such as mRNA. When the nucleic acid of this embodiment is mRNA, the base sequence may be an mRNA containing, as a coding region, a base sequence in which thymine (T) in any of the base sequences exemplified above is substituted with uracil (U). In addition to the coding region, the mRNA corresponding to the nucleic acid of the present invention may also contain a cap structure at the 5' end, a poly(A) tail at the 3' end, a 5' untranslated region (5' UTR) upstream of the start codon, and / or a 3' untranslated region (3' UTR) downstream of the stop codon. The 5' UTR and / or 3' UTR may contain a sequence for regulating the amount of translation from the mRNA.

[0043] In one embodiment, the nucleic acid of this aspect is a gene expression vector. The gene expression vector comprises, in an expressible state, a gene encoding any of the cytochrome P450 proteins or active fragments thereof described in the first aspect. As used herein, the term "expressible state" refers to the placement of the gene to be expressed downstream of a promoter under the control of the promoter.

[0044] Vectors that can be used as the gene expression vector include, for example, expression vectors that utilize plasmids or viruses. The type of expression vector is not particularly limited and can be appropriately selected depending on the purpose (e.g., for cloning or gene expression) or the host into which it is introduced (e.g., Escherichia coli, yeast, insect cells, animal cells, plant cells, or plants). It may be a plasmid vector, a viral vector, or a recombinant vector. Specific examples of expression vectors include, but are not limited to, plasmid vectors such as the pBI series, pPZP series, pSMA series, pUC series, pBR series, pBluescript series (Agilent), and pTriEX series (Novagen); viral vectors such as cauliflower mosaic virus (CaMV), bean mosaic virus (BGMV), and tobacco mosaic virus (TMV); and binary vectors such as the pBI series.

[0045] Various promoters, such as overexpression promoters, constitutive promoters, site-specific promoters, time-specific promoters, and / or inducible promoters, can be used as promoters in the gene expression vectors described above. Specific examples of promoters operable in bacterial cells include the promoters of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis α-amylase gene, the Bacillus amyloliquefatiens BAN amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene; the PR or PL promoter of phage lambda; and the lac, trp, or tac promoter of Escherichia coli. Examples of promoters operable in yeast cells include promoters derived from yeast glycolytic pathway genes, the alcohol dehydrogenase gene promoter, the TPI1 promoter, and the GAL promoter. Examples of promoters operable in fungi include the ADH3 promoter and the tpiA promoter. Examples of promoters that can function in plant cells include the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene promoter (Pnos), the maize ubiquitin promoter, the rice actin promoter, the tobacco PR protein promoter, etc. Examples of promoters that can function in animal cells include the SV40 early promoter, the SV40 late promoter, the CMV promoter, etc. Examples of promoters that can function in insect cells include the polyhedrin promoter, the P10 promoter, the Autographa californica polyhedrosis basic protein promoter, the baculovirus immediate early gene 1 promoter, the baculovirus 39K delayed early gene promoter, etc.

[0046] The gene expression vector may also contain other genes, such as a terminator, an enhancer, a poly(A) addition signal, a 5'-UTR (untranslated region) sequence, an intron sequence, a ribosome binding sequence, a tagging or selection marker gene, a multicloning site, a nuclease recognition sequence, a replication origin, and / or an abietane diterpene synthesis-related enzyme gene shown below. The type of each enhancer is not particularly limited, as long as it can function in the cells into which the gene expression vector is introduced. Specific examples of enhancers include an enhancer region containing an upstream sequence in the CaMV 35S promoter, an SV40 enhancer, a CMV enhancer, etc. Specific examples of terminators include the nopaline synthase (NOS) gene terminator, the octopine synthase (OCS) gene terminator, the CaMV 35S terminator, the 3' terminator of Escherichia coli lipopolyprotein lpp, the trp operon terminator, the amyB terminator, the ADH1 gene terminator, etc. Specific examples of labeling or selection marker genes include drug resistance genes (e.g., tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, spectinomycin resistance gene, chloramphenicol resistance gene, or neomycin resistance gene), fluorescent or luminescent reporter genes (e.g., luciferase, β-galactosidase, β-glucuronidase (GUS), or green fluorescent protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase.

[0047] The nucleic acid of this embodiment can be isolated from any plant of the Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, or Araucariaceae families. For example, it can be isolated by amplifying the genomic DNA or cDNA of Taiwan cedar, Chinese fir, or Salix japonica as a template using a nucleic acid amplification method such as PCR. Alternatively, it can be synthesized based on any of the above-mentioned base sequences using known nucleic acid sequence synthesis methods such as chemical synthesis.

[0048] 2-2. Effect When the nucleic acid of this embodiment, for example, the above-described gene expression vector, is introduced into a eukaryotic cell such as an actinomycete, a bacterium such as Escherichia coli, or a yeast, any of the cytochrome P450 proteins or active fragments thereof described in the first embodiment can be expressed in the cell, and the activity of oxidizing the carbon at position 12 of an abietane diterpene such as levopimaradiene can be obtained in the cell.

[0049] That is, by using the nucleic acid of this embodiment as the above-mentioned gene expression vector, its manipulation and / or control can be facilitated, and therefore, by introducing this gene expression vector into cells, the carbon at position 12 of an abietane diterpene can be easily and efficiently oxidized.

[0050] 3. Oxidizing Agent for the 12th Carbon in Abietane Diterpenes and Composition for Oxidizing the 12th Carbon in Abietane Diterpenes The third aspect of the present invention is an oxidizing agent for the 12th carbon in abietane diterpenes and a composition for oxidizing the 12th carbon in abietane diterpenes.

[0051] In one embodiment, the oxidizing agent for the 12-carbon in the abietane diterpene of this aspect comprises or consists of a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebacterium, Podocarpus, and Araucaria. In this embodiment, the composition of the cytochrome P450 protein of the CYP720 family or an active fragment thereof is similar to that described in the first aspect, and therefore a detailed description thereof will be omitted here.

[0052] In one embodiment, the oxidizing agent for the 12-carbon of an abietane diterpene of this aspect comprises or consists of a nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof, derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebacterium, Podocarpus, and Araucaria. In this embodiment, the structure of the nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof is similar to that described in the second aspect, and therefore a detailed description thereof will be omitted here.

[0053] The composition for oxidizing the 12-carbon atom of an abietane diterpene of this embodiment contains, as an active ingredient, a cytochrome P450 protein of the CYP720 family or an active fragment thereof, and / or a nucleic acid encoding the cytochrome P450 protein or an active fragment thereof, which is derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucaria. Both active ingredients are similar to those described in the first and second embodiments, and therefore detailed description thereof will be omitted here.

[0054] The composition for oxidizing the 12-carbon atom of an abietane diterpene of this embodiment may contain a carrier in addition to the active ingredient. The carrier is a solvent and / or additive commonly used in the production of bioactive substances. Examples of solvents include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Examples of additives include excipients, binders, etc. Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol (PEG), kaolin, silicic acid, or combinations thereof. Examples of binders include starch paste, gelatin, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.

[0055] The present invention also provides use of any of the cytochrome P450 proteins of the CYP720 family or active fragments thereof described in the first aspect, and / or any of the nucleic acids described in the second aspect, in the oxidation of the 12-carbon atom in an abietane diterpene.

[0056] 4. Transformed Cells 4-1. Configuration The fourth aspect of the present invention is a transformed cell. The transformed cell of this aspect has any of the nucleic acids (e.g., gene expression vectors) described in the second aspect introduced therein, or has been transformed with any of the nucleic acids (e.g., gene expression vectors) described in the second aspect.

[0057] The type of transformed cell is not limited herein. The transformed cell may be any cell capable of carrying and / or replicating any of the nucleic acids (e.g., gene expression vectors) described in the second aspect. For example, the transformed cell may be a eukaryotic cell, a bacterium, or an archaea. The eukaryotic cell may be, for example, a yeast cell, a fungal cell, a plant cell, or an animal cell. Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of fungi include Aspergillus, Neurospora, Fusarium, and Trichoderma. Examples of plants include monocotyledonous plants (e.g., Poaceae) and dicotyledonous plants (e.g., Leguminosae or Brassicaceae). The plant may be of the Cupressaceae, Taxaceae, Corynebaceae, Papaceae, or Araucariaceae family, or may be a plant other than the Cupressaceae, Taxaceae, Corynebaceae, Papaceae, or Araucariaceae family. Examples of animal cells include insect cells (e.g., sf9 or sf21), algae cells, protozoan cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). The bacterium may be, for example, Escherichia coli or Bacillus subtilis.

[0058] Also provided is a transformed plant containing the transformed cell of this embodiment. The transformed plant of the present invention may be a clone obtained from a part of a plant body collected from the first generation of a transformed plant, for example, plant tissues such as the epidermis, phloem, parenchyma, xylem, or vascular bundle, or plant organs such as leaves, petals, stems, roots, or seeds, or plant cells by plant tissue culture, cuttings, grafting, or layering, or may be a clone newly generated from a vegetative propagation organ obtained by asexual reproduction from the first generation of a transformed plant, such as a rhizome, tuberous root, corm, or runner. For example, somatic embryos induced by dedifferentiation treatment from the first generation of a transformed plant or a clone derived therefrom are also included in the transformed plant of the present invention.

[0059] The plant species from which the above-mentioned transformed plants are derived is not particularly limited and may be either angiosperms or gymnosperms. Angiosperms include both dicotyledonous and monocotyledonous plants. Specific examples of dicotyledonous plants include Brassicaceae, Fabaceae, Solanaceae, Rosaceae, Orchidaceae, Liliaceae, Rutaceae, Vitaceae, Asteraceae, Caryophyllaceae, and Theaceae. Examples of monocotyledonous plants include species belonging to the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, and sorghum). The plant species from which the transformed plants are derived may be Cupressaceae, Taxaceae, Corynebaceae, Papaceae, or Araucaria, or may be a species other than Cupressaceae, Taxaceae, Corynebaceae, Papaceae, and Araucaria.

[0060] The method for producing the transformed cell of this embodiment is not limited. For example, any of the nucleic acids described in the second embodiment (e.g., gene expression vectors) can be introduced into host cells by methods known in the art (e.g., Agrobacterium method, PEG-calcium phosphate method, electroporation, liposome method, particle gun method, microinjection method). The nucleic acid introduced into the cell may be incorporated into the genomic DNA of the transformed cell, or may be permanently or transiently maintained outside the genome as a plasmid or the like. The introduced nucleic acid can be confirmed by nucleic acid amplification methods such as PCR.

[0061] In one embodiment, the transformed cell of this embodiment contains, in addition to any of the nucleic acids described in the second embodiment, a cytochrome P450 reductase gene in an expressible state. As used herein, "cytochrome P450 reductase" refers to an enzyme that has the function of donating electrons to cytochrome P450. In an oxidation reaction catalyzed by cytochrome P450, electrons are donated to oxygen molecules via a heme molecule contained in cytochrome P450, resulting in an active state in which the substrate can be oxidized. After the oxidation reaction, the heme molecule loses electrons and becomes inactive, preventing the next oxidation reaction from occurring. Therefore, in normal cells, cytochrome P450 reductase functions to donate electrons from NAD(P)H to cytochrome P450. Based on this function, cytochrome P450 is regenerated into an active state in which it can oxidize the substrate, thereby improving metabolic turnover. Therefore, it is preferable that the transformed cell of this embodiment contains a cytochrome P450 reductase gene in an expressible state. Although it is not necessary for normal cells such as eukaryotic cells to have endogenous cytochrome P450 reductase, the efficiency of the oxidation reaction by cytochrome P450 can be further increased by introducing an exogenous cytochrome P450 reductase gene. Furthermore, since yew is a coniferous tree and a species closely related to the Cupressaceae family, it is presumed that the electron transport mechanism is likely to proceed smoothly. A preferred example of a cytochrome P450 reductase is the yew (Taxus chinensis) cytochrome P450 reductase consisting of the amino acid sequence set forth in SEQ ID NO: 16. Further examples of cytochrome P450 reductases include ATR1 (GenBank Accession No. X66016) derived from Arabidopsis (Arabidopsis thaliana) and PcCPR (GenBank Accession No. KJ914574) derived from Pinus contorta.Specific examples of cytochrome P450 reductase genes include the yew (Taxus chinensis)-derived cytochrome P450 reductase gene (GenBank Accession No. AY959320; SEQ ID NO: 7) and the nucleotide sequence shown in SEQ ID NO: 8, which is the nucleotide sequence shown in SEQ ID NO: 7 codon-optimized for yeast.

[0062] In one embodiment, the transformed cell of this aspect contains, in addition to any one of the nucleic acids described in aspect 2, a levopimaradiene synthase gene in an expressible state. Specific examples of the levopimaradiene synthase gene include a gene encoding levopimaradiene synthase (GbLAS) derived from Ginkgo biloba, which produces levopimaradiene from geranylgeranyl diphosphate (GGDP), and which consists of the amino acid sequence shown in SEQ ID NO: 17 (GenBank Accession No. AF331704; SEQ ID NO: 10), and the nucleotide sequence shown in SEQ ID NO: 11, which is the nucleotide sequence shown in SEQ ID NO: 10 codon-optimized for yeast. Another example is a gene encoding levopimaradiene synthase PsLAS derived from Picea sitchensis, which consists of the amino acid sequence shown in SEQ ID NO: 18 (GenBank Accession No. HQ426170; SEQ ID NO: 19). PsLAS can synthesize a mixture of abietane-type diterpenes, mainly containing abietadiene, and can also synthesize levopimaradiene and / or dehydroabietadiene by isomerization and / or oxidation.

[0063] In one embodiment, the transformed cell of this aspect contains, in addition to any one of the nucleic acids described in the second aspect, a gene encoding a geranylgeranyl diphosphate synthase that has the activity of producing geranylgeranyl diphosphate (GGDP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in an expressible state. Specific examples of geranylgeranyl diphosphate synthases that have the activity of producing GGDP from IPP and DMAPP include wild-type geranylgeranyl diphosphate synthases, as well as the ERG20(F96C) protein (SEQ ID NO: 14), in which the 96th Phe residue in the amino acid sequence of yeast farnesyl diphosphate synthase (ERG20) shown in SEQ ID NO: 20 has been replaced with a Cys residue. An example of a gene encoding a geranylgeranyl diphosphate synthase that has the activity of producing GGDP from IPP and DMAPP is the gene encoding the ERG20(F96C) protein (SEQ ID NO: 15). In addition to the above, examples of geranylgeranyl diphosphate synthases include those having the activity of producing GGDP from FDP and DMAPP, and those having the activity of producing GGDP from GDP and GDP.

[0064] Preferably, the transformed cell of this embodiment contains, in an expressible state, a cytochrome P450 reductase gene and a levopimaradiene synthase gene in addition to any one of the nucleic acids according to embodiment 2. More preferably, the transformed cell of this embodiment contains, in addition to these genes, a gene encoding a geranylgeranyl diphosphate synthase that has the activity of producing GGDP from IPP and DMAPP.

[0065] In a further embodiment, the transformed cell of this aspect contains a gene encoding a geranylgeranyl diphosphate synthase or a wild-type farnesyl diphosphate synthase that does not contain the substitution mutation (i.e., the 96th Phe residue is not replaced with a Cys residue). A farnesyl diphosphate synthase that does not contain the substitution mutation (i.e., the 96th Phe residue is not replaced with a Cys residue) has the activity of producing farnesyl diphosphate (FDP) from IPP and DMAPP. A specific example of a wild-type farnesyl diphosphate synthase is yeast-derived farnesyl diphosphate synthase (ERG20) consisting of the amino acid sequence set forth in SEQ ID NO: 20. A specific example of a gene encoding a wild-type farnesyl diphosphate synthase is the yeast-derived farnesyl diphosphate synthase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 21. Since ordinary cells such as yeast cells contain a gene encoding a farnesyl diphosphate synthase that has the activity of producing FDP from IPP and DMAPP, it is usually not necessary to introduce an exogenous farnesyl diphosphate synthase gene into the cells. Furthermore, normal cells also contain a gene encoding geranylgeranyl diphosphate synthase (e.g., BTS1 in yeast), which has the activity of producing GGDP from FDP and DMAPP. Therefore, GGDP can be produced from IPP and DMAPP as starting materials even without introducing an exogenous farnesyl diphosphate synthase gene into the cells.

[0066] The transformed cell of this embodiment does not necessarily need to contain genes necessary for the biosynthesis of abietane diterpenes, such as a levopimaradiene synthase gene or a gene encoding geranylgeranyl diphosphate synthase. This is because, even if the transformed cell does not contain genes necessary for the biosynthesis of abietane diterpenes, it is possible to produce terpenoid compounds having a hydroxyl group at the 12th carbon by culturing the transformed cell in the presence of an abietane diterpene such as levopimaradiene. In this specification, the levopimaradiene synthase gene, geranylgeranyl diphosphate synthase gene, and farnesyl diphosphate synthase gene may be collectively referred to as "abietane diterpene synthesis-related enzyme genes."

[0067] In the transformed cell of this embodiment, the expression of the gene encoding the cytochrome P450 protein or an active fragment thereof described in the second embodiment and the gene encoding the cytochrome P450 reductase, levopimaradiene synthase, and / or geranylgeranyl diphosphate synthase may be controlled by the same promoter or by different promoters. The combination of different promoters may be, for example, a combination with different induction conditions or a combination with different expression intensities.

[0068] 4-2. Effect The transformed cell of this embodiment, i.e., a transformed cell into which any of the nucleic acids described in the second embodiment has been introduced or which has been transformed with any of the nucleic acids described in the second embodiment, can express any of the cytochrome P450 proteins or active fragments thereof described in the first embodiment in the transformed cell, thereby enabling the transformed cell to acquire the activity of oxidizing the carbon at position 12 of abietane diterpenes such as levopimaradiene.

[0069] 5. Method for Producing Terpenoid Compounds 5-1. Overview The fifth aspect of the present invention is a method for producing a terpenoid compound having a hydroxyl group at carbon atom 12. The specific steps of the method of this aspect differ between a method using transformed cells (in vivo method) and a method not using transformed cells (in vitro method), and therefore each step will be described below for each of the two cases.

[0070] 5-2. Method Using Transformed Cells (In Vivo Method) In this embodiment, the method using transformed cells (in vivo method) includes a culture / cultivation step as an essential step and an extraction step as an optional step.

[0071] (Cultivation / Cultivation Step) In the method using a transformed cell in this embodiment, the "cultivation / cultivation step" refers to a step of culturing a transformed cell into which a nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucariales has been introduced, or cultivating a transformed plant containing the transformed cell. The transformed cell cultured in this step into which a nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucariales has been introduced may be any of the transformed cells described in the fourth embodiment above.

[0072] The method for culturing the transformed cells in this step is not particularly limited as long as the transformed cells can produce a terpenoid compound having a hydroxyl group at the 12th carbon, and the culture conditions may be any conditions suitable for culturing the cells used as the transformed cells.

[0073] The medium used in this step can be any known medium capable of culturing cells used as transformed cells. Specific components that can be contained in the medium include carbon sources (e.g., glucose, glycerin, mannitol, fructose, lactose, etc.), nitrogen sources (e.g., inorganic nitrogen sources such as ammonium sulfate and ammonium chloride, and organic nitrogen sources such as casein hydrolysate, yeast extract, polypeptone, bactotryptone, and beef extract), inorganic salts (e.g., sodium diphosphate, potassium diphosphate, magnesium chloride, magnesium sulfate, and calcium chloride), vitamins (e.g., vitamin B1), and / or drugs (antibiotics such as ampicillin, tetracycline, and kanamycin). Specific examples of media include LB medium or M9 medium when the transformed cells are bacteria such as Escherichia coli, and YPD medium, YPG medium, YPM medium, YPDM medium, and SMM medium when the transformed cells are yeast. In addition to the above-mentioned components and components contained in these media, the medium used in this step may also contain abietane-type diterpenes, such as levopimaradiene, abietadiene, neoabietadiene, palustradiene, miltiradiene, and / or dehydroabietadiene, which serve as substrates for the reaction producing terpenoid compounds having a hydroxyl group at the 12th carbon.

[0074] The culture conditions in this step are not particularly limited, as long as the transformed cells are able to sufficiently express a cytochrome P450 protein of the CYP720 family or an active fragment thereof and the reaction to produce a terpenoid compound having a hydroxyl group at carbon 12 proceeds. Culture is typically carried out at a temperature of 10 to 45°C, with aeration and stirring as necessary, for several hours to several days or weeks. Further culture conditions can be found in, for example, Sambrook, J. et al. (1989) Molecular Cloning: a Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0075] In this step, the transformed plant is cultivated using a suitable medium for cultivation. For example, but not limited to, suitable culture soil or a medium for hydroponic cultivation can be used. The cultivation conditions are typically at a temperature of 10 to 45°C, 15 to 40°C, or 18 to 37°C, with aeration, irradiation, and / or agitation as necessary, for a period of several hours to several hundred hours, several days to several months, or several days to several years.

[0076] (Extraction step) In the method using transformed cells in this embodiment, the "extraction step" is a step of extracting an extract containing a terpenoid compound having a hydroxyl group at the 12th carbon from the transformed cells or transformed plants after the culture / cultivation step.

[0077] The extraction method for this step is not limited, and terpenoid compounds having a hydroxyl group at the 12th carbon may be extracted from the culture solution or culture product (e.g., culture supernatant or cultured transformed cells) after the culture / cultivation step, or from the cultivated transformed plant or seeds obtained from the plant. Specifically, the culture supernatant, transformed cells, transformed plant, or seeds is dried and / or pulverized, and components containing terpenoid compounds having a hydroxyl group at the 12th carbon are extracted from the powder using water, an organic solvent (e.g., ethyl acetate), a mixture thereof, or the like. Furthermore, if necessary, the extract may be concentrated to increase the concentration of terpenoid compounds having a hydroxyl group at the 12th carbon. Furthermore, if a purer terpenoid compound having a hydroxyl group at the 12th carbon is required, the extract may be separated and / or purified by HPLC or the like to obtain a fraction containing the desired terpenoid compound having a hydroxyl group at the 12th carbon.

[0078] The method of using a transformed cell in this embodiment can include, as an optional additional step, a transformation step of transforming a host cell with any of the nucleic acids (e.g., gene expression vectors) described in the second embodiment to obtain a transformed cell. In this transformation step, nucleic acids such as an expression vector encoding farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, levopimaradiene synthase, and / or cytochrome P450 reductase may be further introduced into the transformed cell, although it is preferable to further introduce nucleic acids such as an expression vector encoding levopimaradiene synthase into the transformed cell. For example, nucleic acids such as an expression vector encoding geranylgeranyl diphosphate synthase and cytochrome P450 reductase in addition to levopimaradiene synthase may be introduced into the transformed cell.

[0079] 5-3. Methods not using transformed cells (in vitro methods) In this embodiment, the methods not using transformed cells (in vitro methods) include an oxidation step as an essential step and an extraction step as an optional step.

[0080] (Oxidation step) In this embodiment, the "oxidation step" in the method that does not use transformed cells is a step of obtaining a terpenoid compound having a hydroxyl group at the 12th carbon of an abietane diterpene by oxidizing the 12th carbon in the presence of a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucariales (e.g., in the presence of the cytochrome P450 protein or an active fragment thereof and cytochrome P450 reductase).

[0081] The reaction conditions for this step are not limited as long as they allow the activity of a cytochrome P450 protein of the CYP720 family or an active fragment thereof to be obtained (e.g., conditions that allow the activity of the cytochrome P450 protein or an active fragment thereof and a cytochrome P450 reductase to be obtained). For example, a cytochrome P450 protein of the CYP720 family or an active fragment thereof can be reacted, optionally together with a cytochrome P450 reductase, in a buffer solution (e.g., 1 M potassium phosphate buffer (pH 7.2)) containing an abietane diterpene substrate such as levopimaradiene and an electron donor such as NADPH. The reaction temperature can be, for example, 25°C to 45°C or 30°C to 40°C, and the reaction time can be 10 minutes or more, 1 hour or more, or 6 hours or more, and / or 3 days or less, 24 hours or less, or 12 hours or less.

[0082] In one embodiment, the abietane-type diterpene is levopimaradiene or dehydroabietadiene, and the terpenoid compound having a hydroxyl group at the 12-carbon is ferruginol.

[0083] (Extraction Step) In this embodiment, the "extraction step" in the method that does not use transformed cells is a step of extracting an extract containing a terpenoid compound having a hydroxyl group at carbon atom 12 from the reaction solution after the oxidation step. The specific extraction method is not limited, and for example, a component containing a terpenoid compound having a hydroxyl group at carbon atom 12 is extracted as an extract from the reaction solution after the oxidation step using water, an organic solvent (ethyl acetate, etc.), a mixture thereof, or the like.

[0084] In this embodiment, the method not using a transformed cell can also optionally include, as a further optional step, a step of producing GGDP from IPP and DMAPP using geranylgeranyl diphosphate synthase and / or a step of producing levopimaradiene from GGDP using levopimaradiene synthase.

[0085] 5-4. Effects According to the method using transformed cells in this embodiment, ferruginol can be stably and sustainably produced in the transformed cells from abietane diterpenes such as levopimaradiene. For example, even biological species or cells that are not originally capable of biosynthesizing ferruginol can biosynthesize ferruginol, and by using a host that is easy to culture and has high proliferation ability, such as yeast, it becomes possible to produce ferruginol in large quantities, stably, and at low cost. According to the method of this embodiment, it is possible to provide highly pure ferruginol at a lower cost than when extracted from plants such as cedar.

[0086] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0087] Example 1: Extraction of candidate ferruginol synthase genes from Taiwan cedar (T. cryptomerioides) (Objective) In cupressaceae plants such as Taiwan cedar (T. cryptomerioides), the heartwood formed in the center of the xylem contains abundant phenolic abietane diterpenes such as ferruginol. In this example, with the aim of obtaining candidate genes for ferruginol synthase in Taiwan cedar, genes that are highly expressed in the heartwood transition zone compared to the sapwood zone of the stem tissue and that correspond to cytochrome P450 are extracted from transcriptome data of Taiwan cedar published in a previous paper (Ting-Feng Yeh, et al., Int. J. Mol. Sci., 2020, 21(3):960.).

[0088] (Methods and Results) (1) Analysis of transcriptome data for Taiwan cedar. Transcriptome sequence data (SRA accession: PRJNA589955) from stem tissue of Taiwan cedar was obtained from NCBI. Fastq data was processed using Trimmomatic software to remove adapter sequences and low-quality reads. This data was assembled using Trinity software, resulting in 421,669 unique contigs over 200 bases in length.

[0089] (2) Extraction of genes highly expressed in the heartwood transition zone compared to the sapwood zone. Using the contig sequences obtained in (1) above as templates, transcriptome reads from the sapwood and heartwood transition zones of stem tissues were mapped using Kallisto software. Based on the obtained TPM values, statistical analysis was performed to identify genes highly expressed in each tissue, with a p-value of 0.01 or less as a threshold. From the resulting gene group, genes highly expressed in the heartwood transition zone compared to the sapwood zone were selected, yielding 316 candidate genes.

[0090] (3) Extraction of cytochrome P450 genes: TransDecoder software was run on the 316 genes obtained in (2) above, and only contigs containing full-length cDNA sequences were selected. P450.pfam was obtained from the Pfam protein family database, and the contigs translated into amino acid sequences using TransDecoder were used to search for P450 proteins using HMMER software. As a result, six full-length cDNA sequences were obtained as candidate genes. One of these six candidate genes was a gene encoding a cytochrome P450 protein of the CYP720 family (hereinafter referred to as "TcFS") (hereinafter referred to as "TcFS gene").

[0091] Example 2: Identification of ferruginol synthase gene derived from Taiwan cedar based on activity evaluation (Objective) Each of the P450 proteins encoded by the six candidate genes obtained in Example 1 will be evaluated for the presence or absence of enzyme activity to convert levopimaradiene to ferruginol. Specifically, an expression vector containing each candidate gene (hereinafter referred to as "candidate gene expression vector") will be constructed and introduced into yeast, and the yeast will be cultured after introduction to verify whether ferruginol is produced.

[0092] In this example, to evaluate the presence or absence of the target enzyme activity (enzyme activity that converts levopimaradiene to ferruginol), the substrate levopimaradiene must be produced intracellularly in yeast. Within yeast cells, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are produced from glucose, followed by the production of 15-carbon farnesyl diphosphate (FDP) by endogenous farnesyl diphosphate synthase (hereinafter referred to as "ERG20"). By substituting ERG20 for its mutant form, ERG20(F96C), 20-carbon geranylgeranyl diphosphate (GGDP) can be produced from IPP and DMAPP. Therefore, expression vectors for expressing two enzyme proteins, ERG20(F96C), a biosynthetic enzyme for producing GGDP from IPP and DMAPP, and ginkgo-derived levopimaradiene synthase (hereinafter referred to as "GbLAS"), for producing levopimaradiene from GGDP, were introduced together with the above-mentioned candidate gene expression vectors (Figure 4).

[0093] (Methods and Results) (1) Construction of pESC-TchCPR Vector. The nucleic acid sequence (GenBank Accession No. AY959320; SEQ ID NO: 7) encoding the yew (Taxus chinensis)-derived cytochrome P450 reductase (hereinafter referred to as "TchCPR") was obtained from the NCBI database. This sequence was optimized for yeast codons to obtain an artificially synthesized gene (SEQ ID NO: 8; hereinafter referred to as "TchCPR gene"), which was then introduced into the PacI / SpeI site of the yeast expression vector pESC-LEU (Agilent). A vector for expressing the TchCPR gene in yeast (hereinafter referred to as "pESC-TchCPR vector") was constructed. In this example, a yew-derived cytochrome P450 reductase, which is closely related to the Cupressaceae family, was used to improve the metabolic turnover rate of cytochrome P450.

[0094] (2) Preparation of Candidate Gene Expression Vectors The six candidate genes obtained in Example 1 were optimized for yeast codons, and artificial synthetic genes were prepared by adding start and stop codons. Of the six artificial synthetic genes obtained, the base sequence of the artificial synthetic gene derived from the TcFS gene described above is shown in SEQ ID NO: 9. Next, each of the six artificial synthetic genes was introduced into the SalI / HindIII site of the pESC-TchCPR vector prepared in (1) above to prepare vectors (hereinafter sometimes referred to as "candidate gene expression vectors") for expressing the TchCPR gene and two genes from each candidate gene under the control of the Gal10 promoter and the Gal1 promoter, respectively, in yeast. Of the six candidate gene expression vectors prepared, the candidate gene expression vector containing the artificial synthetic gene derived from the TcFS gene described above is referred to as the "pESC-TchCPR-TcFS vector."

[0095] (3) Construction of pESC-ERG20(F96C)-GbLAS vector It is known that by substituting the 96th amino acid residue from Phe to Cys in the amino acid sequence of yeast farnesyl diphosphate synthase (ERG20), geranylgeranyl diphosphate (GGPP), a substrate for diterpene synthesis, is produced from DMAPP and IPP in the cell. Therefore, in order to overproduce GGPP in yeast cells, the ERG20 gene sequence was PCR-amplified using the genome of the yeast strain BY4742 as a template. Furthermore, the ERG20(F96C) gene (SEQ ID NO: 15) encoding ERG20(F96C) (SEQ ID NO: 14) into which a point mutation had been introduced to replace the 96th Phe residue in the ERG20 amino acid sequence with a Cys residue was introduced, was introduced into the PacI / SpeI site of the yeast expression vector pESC-URA (Agilent), thereby preparing the pESC-ERG20(F96C) vector for expressing the ERG20(F96C) gene in yeast.

[0096] Next, the nucleotide sequence (GenBank Accession No. AF331704; SEQ ID NO: 10) encoding ginkgo-derived levopimaradiene synthase (GbLAS; SEQ ID NO: 17) was optimized for yeast codons, and an initiation and termination codons were added to create an artificially synthesized gene (SEQ ID NO: 11; hereinafter, sometimes referred to as the "GbLAS gene"). This artificially synthesized gene was introduced into the SalI / HindIII site of the pERG20(F96C) expression vector described above, to create a vector for expressing the ERG20(F96C) gene and the GbLAS gene in yeast under the control of the Gal10 promoter and the Gal1 promoter, respectively (hereinafter, sometimes referred to as the "pESC-ERG20(F96C)-GbLAS vector").

[0097] (4) Preparation of Transformed Yeast. Using Frozen-EZ Yeast Transformation II (Zymo Research), the yeast strain BY4742 (NBRP yeast) (MATα his3-Δ1 leu2Δ lys2Δ ura3Δ) was transformed with the pESC-ERG20(F96C)-GbLAS vector and then further transformed with the candidate gene expression vector to produce a transformed yeast carrying both vectors. As a negative control, a transformed yeast carrying both the pESC-ERG20(F96C)-GbLAS vector and the pESC-TchCPR vector was prepared in the same manner.

[0098] (5) Evaluation of ferruginol production. Each transformed yeast prepared in (4) above was cultured in 2 mL of SC-Leu / Ura medium at 30°C with shaking at 220 rpm for 1 day. The entire cultured yeast was added to 50 mL of SC-Leu / Ura-glucose-galactose medium supplemented with glucose (2 mg / mL) and galactose (18 mg / mL). The cultured yeast was then cultured at 30°C with shaking at 220 rpm for 4 days. After the culture, the yeast was centrifuged at 3,000 g for 10 minutes to collect cells. 5 mL of ethyl acetate was added to the pellet and mixed, and the ethyl acetate extract was recovered. Another 5 mL of ethyl acetate was added to the pellet and mixed, and the ethyl acetate extract was recovered. Anhydrous sodium sulfate was added to the ethyl acetate extract, and the sample was dried and removed, and then subjected to GC-MS analysis. The identification of ferruginol in the analytical results was based on the analysis of authentic ferruginol (AS ONE Corporation).

[0099] The results of GC-MS analysis are shown in Figures 5 and 6. Among the six candidate gene-introduced yeast transformants, extracts from the transformant containing the candidate gene expression vector (pESC-TchCPR-TcFS vector) containing the synthetic gene based on the TcFS gene showed ferruginol (black arrowhead) at the same peak position as the authentic ferruginol (Figures 5B and 5C). Mass spectrometry analysis of the peaks indicated by the black arrows in Figures 5B and 5C revealed that these peaks corresponded to ferruginol (Figure 6). In contrast, extracts from the transformant containing the candidate gene expression vector containing the synthetic gene based on the remaining five candidate genes and the transformant containing the pESC-TchCPR vector showed only levopimaradiene (white arrowhead), but not ferruginol (Figure 5A).

[0100] These results demonstrate that TcFS (a cytochrome P450 protein of the CYP720 family derived from Taiwan cedar) has the activity to convert the 12th carbon of levopimaradiene to a hydroxyl group, producing ferruginol.

[0101] Example 3: Search for TcFS homologous genes in Chinese fir and willow cedar (C. lanceolata) (Objective) To identify genes homologous to the TcFS gene in Chinese fir (C. lanceolata), which belongs to the same Cupressaceae family as the genus Cryptomeria japonica and is closely related to the genus Cryptomeria japonica, and in the distantly related species Cryptomeria japonica (C. fortunei), which belongs to the genus Cryptomeria japonica in the subfamily Pinaceae.

[0102] (Methods and Results) (1) Identification of TcFS homologous genes in Chinese fir. Transcriptome sequence data from Chinese fir cone tissue (SRA accession numbers SRR10161401, SRR10161402, SRR10161403, and SRR10161404) were obtained from NCBI. Fastq data were processed using Trimmomatic software to remove adapter sequences and low-quality reads. The data were then assembled using Trinity software, yielding 370,894 unique contigs of 200 bases or more.

[0103] The resulting contigs were then run through TransDecoder software to extract only those contigs containing full-length cDNA sequences. Next, P450.pfam was retrieved from the Pfam protein family database, and the contigs translated into amino acid sequences using TransDecoder were searched for P450 proteins using HMMER software. As a result, 384 full-length cDNA sequences of P450s expressed in Chinese fir cones were extracted.

[0104] The 384 extracted sequences were searched for amino acid sequences highly homologous to the TcFS amino acid sequence using the Blastp program. As a result, a gene consisting of the base sequence shown in SEQ ID NO: 5 (hereinafter sometimes referred to as the "ClFS gene") was identified as the TcFS homologous gene in Chinese fir.

[0105] (2) Identification of TcFS homologous genes in C. fortunei. Transcriptome sequence data from C. fortunei needle tissue (SRA accession numbers PRJNA697258 and PRJNA720228) were obtained from NCBI. Fastq data were processed using Trimmomatic software to remove adapter sequences and low-quality reads. The data were then assembled using Trinity software, yielding 318,377 unique contigs of over 200 bases.

[0106] The resulting contigs were run through TransDecoder software to extract only those contigs containing full-length cDNA sequences. Next, P450.pfam was retrieved from the Pfam protein family database, and the contigs translated into amino acid sequences using TransDecoder were searched for P450 proteins using HMMER software. As a result, 322 full-length cDNA sequences of P450s expressed in willow cedar needles were extracted.

[0107] The 322 extracted sequences were searched for amino acid sequences highly homologous to the amino acid sequence of TcFS using the Blastp program. As a result, a gene consisting of the base sequence shown in SEQ ID NO: 6 (hereinafter sometimes referred to as the "CfFS gene") was identified as the TcFS homologous gene in willow cedar.

[0108] Example 4: Evaluation of ClFS and CfFS activity (Objective) To verify that the P450 protein encoded by the ClFS gene identified in Example 3 (hereinafter referred to as "ClFS") and the P450 protein encoded by CfFS (hereinafter referred to as "CfFS") have the activity to produce ferruginol from levopimaradiene.

[0109] (Methods and Results) (1) Preparation of ClFS Gene Expression Vector The nucleotide sequence of the ClFS gene was optimized for yeast codons, and an artificially synthesized gene was prepared by adding a start codon and a stop codon. The nucleotide sequence of the obtained artificially synthesized gene is shown in SEQ ID NO: 12. This artificially synthesized gene was introduced into the SalI / HindIII site of the TchCPR expression vector prepared in Example 2(1), thereby preparing a vector (hereinafter referred to as the "pESC-TchCPR-ClFS vector") for expressing the two genes, the TchCPR gene and the ClFS gene, under the control of the Gal10 promoter and the Gal1 promoter, respectively.

[0110] (2) Preparation of CfFS Gene Expression Vector The nucleotide sequence of the CfFS gene was optimized for yeast codons, and an artificially synthesized gene was prepared by adding a start codon and a stop codon. The nucleotide sequence of the obtained artificially synthesized gene is shown in SEQ ID NO: 13. This artificially synthesized gene was introduced into the SalI / HindIII site of the TchCPR expression vector prepared in Example 2(1), thereby preparing a vector (hereinafter referred to as "pESC-TchCPR-CfFS vector") for expressing the two genes, the TchCPR gene and the CfFS gene, under the control of the Gal10 promoter and the Gal1 promoter, respectively.

[0111] (3) Preparation of transformed yeast Using Frozen-EZ Yeast Transformation II (Zymo Research), the above-mentioned yeast strain BY4742 (NBRP yeast) was transformed with the pESC-ERG20(F96C)-GbLAS vector, and then further transformed with the pESC-TchCPR-ClFS vector or the pESC-TchCPR-CfFS vector to prepare transformed yeast carrying two expression vectors.

[0112] (4) Evaluation of ferruginol production ability The ferruginol production ability of the transformed yeast obtained in (3) above was evaluated using the same method as in (5) of Example 2. The results are shown in Figure 7. In both the transformed yeast into which the ClFS gene expression vector or the CfFS gene expression vector had been introduced, ferruginol (black arrowhead) was detected in addition to levopimaradiene (open arrowhead).

[0113] These results demonstrate that, like TcFS derived from Taiwan cedar, ClFS derived from Chinese fir and CfFS derived from willow cedar have the activity of converting the 12th carbon of levopimaradiene to a hydroxyl group to produce ferruginol. These results demonstrate that the activity of producing ferruginol is conserved in cytochrome P450 proteins of the CYP720 family derived from the Cupressaceae family. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An isolated cytochrome P450 protein of the CYP720 family, or an active fragment thereof, which has the activity of oxidizing the 12-carbon atom of an abietane diterpene, and which is derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucaria.

2. The cytochrome P450 protein or an active fragment thereof according to claim 1, comprising: (a) an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3; (b) an amino acid sequence in which one or more amino acids have been added, deleted, or substituted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3; or (c) an amino acid sequence having 89% or more amino acid identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3.

3. An isolated nucleic acid encoding the cytochrome P450 protein or active fragment thereof of claim 1.

4. The nucleic acid according to claim 3, comprising: (i) a base sequence shown in any one of SEQ ID NOs: 4 to 6; (ii) a base sequence in which one or several bases have been added, deleted, or substituted in any one of SEQ ID NOs: 4 to 6; or (iii) a base sequence having 88% or more base identity to any one of SEQ ID NOs: 4 to 6.

5. The nucleic acid of claim 3, which is a gene expression vector.

6. An oxidizing agent for the 12-carbon position of an abietane diterpene, the oxidizing agent comprising a cytochrome P450 protein of the CYP720 family or an active fragment thereof, and / or a nucleic acid encoding the cytochrome P450 protein or an active fragment thereof, which is derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucaria.

7. A transformed cell into which the nucleic acid according to claim 3 has been introduced.

8. A transformed plant comprising the transformed cell of claim 7.

9. A method for producing a terpenoid compound having a hydroxyl group at the 12th carbon, the method comprising a culture / cultivation step of culturing a transformed cell into which a nucleic acid encoding a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Carpinaceae, Podocarpaceae, and Araucariae has been introduced, or cultivating a transformed plant containing the transformed cell.

10. The method of claim 9, wherein the transformed cell contains a gene encoding levopimaradiene synthase in an expressible state.

11. The method according to claim 9, further comprising an extraction step of extracting an extract containing a terpenoid compound having a hydroxyl group at the 12th carbon from the transformed cells or transformed plants after the culture / cultivation step.

12. The method according to claim 9, wherein the terpenoid compound having a hydroxyl group at the 12th carbon is ferruginol.

13. A method for producing a terpenoid compound having a hydroxyl group at the 12th carbon, the method comprising an oxidation step of oxidizing the 12th carbon of an abietane diterpene in the presence of a cytochrome P450 protein of the CYP720 family or an active fragment thereof derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Carpinaceae, Podocarpaceae, and Araucaria, thereby obtaining a terpenoid compound having a hydroxyl group at the 12th carbon.

14. The method according to claim 13, wherein the abietane diterpene is levopimaradiene or dehydroabietadiene, and the terpenoid compound having a hydroxyl group at the 12th carbon is ferruginol.

15. Use of a cytochrome P450 protein of the CYP720 family or an active fragment thereof, and / or a nucleic acid encoding said cytochrome P450 protein or an active fragment thereof, derived from a plant selected from the group consisting of Cupressaceae, Taxaceae, Corynebaceae, Podocarpaceae, and Araucaria, in the oxidation of the 12-carbon of an abietane diterpene.

Images