Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.


Introduction
Perilla (Perilla frutescens Britton var. crispa W. Deane) is a common culinary herb in East and Southeast Asia, and types of perilla that principally contain perillaldehyde, a monoterpene (MT) compound, in their essential oils are used pharmaceutically in China and Japan. Essential oils of perilla can be classified into more than 10 types [1], which are roughly divided into two groups based on the structures of their constituents [2]: MT-type oils, and phenylpropene (PP)-type oils whose main constituents are elemicin, myristicin, dillapiole, and nothoapiole ( Figure 1). This biosynthetic pathway of perilla oil is genetically controlled [3,4], and the functions of each gene can be determined by cloning the enzymes that catalyze the relevant reaction steps in the biosynthetic pathway. The enzymes that catalyze the formation of MT-type oils, such as limonene synthase, geraniol synthase, linalool synthase, and perillaldehyde synthase, were previously characterized [5][6][7], whereas no PP-type synthases have been reported to date. Known phenylpropanoid volatile components include anethole in fennel, apiole in parsley, eugenol in clove, and myristicin in nutmeg (Figures 1 and 2), and their pharmacological actions and toxicities are well studied due to their use as pharmaceuticals and as ( Figures 1 and 2), and their pharmacological actions and toxicities are well studied due to their use as pharmaceuticals and as flavorings. For example, myristicin is used to treat rheumatism and anxiety in traditional medicine. Risk assessment of myristicin using the margin of exposure approach has been conducted because of its potential genotoxicity or carcinogenicity [8]. Dillapiole, which is found mainly in dill, has been reported to have cytotoxic effects [9]. However, only a few enzymes involved in the synthesis of these compounds have been isolated and reported, and these known enzymes catalyze the formation of compounds with simple side groups [10]. In the text, the oil types of each strain of perilla are represented as PP-em, PP-m, PP-md, PP-emd, and PP-mdn, meaning that each strain mainly contains elemicin + myristicin, myristicin, myristicin + dillapiole, elemicin + myristicin + dillapiole, and myristicin + dillapiole + nothoapiole, respectively. Perilla plants, whose major aromatic compound is perillaldehyde, are used as herbal medicine for Kampo prescriptions in Japan. Those perilla plants that are rich in perillaldehyde do not contain (E)-asarone ( Figure 2) [11]. (E)-Asarone is structurally similar to PP-type oil components [12,13] and is likely formed through a PP-type oil component pathway, although the details are currently unknown. However, the (Z)-asarone isomer was reported to be toxic [14] and (E)-asarone may be carcinogenic [15], and therefore it should always be certified that no perilla plants containing (E)-asarone are present in the plant material used to prepare the pharmaceutical formulation. In order to find plants including this toxic compound and remove them from medicine ingredients, purity tests using liquid chromatography, optimized to separate perillaldehyde and (E)-asarone, are defined in the Japanese Pharmacopoeia.
The main constituents of PP-type perilla oil are elemicin, myristicin, dillapiole, and nothoapiole, and are likely synthesized from phenylalanine ( Figure 1). The reaction steps to all four compounds are genetically controlled based on crossing experiments using pure strains developed by repeated self-pollination, and on detailed gas chromatography (GC) analyses of perilla essential oils [2,16]. Previous reports on the synthesis of alkaloids suggested that methoxy or methylenedioxy groups are formed in the synthetic pathways leading to PP-type oil components and may involve hydroxylation or cyclization reactions by cytochrome P450 (hereafter, "P450") [17]. P450 enzymes catalyze different reactions, such as oxidation, hydroxylation, epoxidation, and dealkylation, and are involved in the synthesis of many plant secondary metabolites. Their low substrate specificity and high reaction regio-and stereo-specificity are beneficial for generating useful compounds, making their catalytic mechanisms an attractive target of study.
Here we describe the isolation of a P450 enzyme involved in the synthesis of dillapiole in PP-type perilla oils using expressed sequence tag (EST) libraries of pure strains of perilla. The perilla strains have been developed and maintained for more than 30 years through repeated In the text, the oil types of each strain of perilla are represented as PP-em, PP-m, PP-md, PP-emd, and PP-mdn, meaning that each strain mainly contains elemicin + myristicin, myristicin, myristicin + dillapiole, elemicin + myristicin + dillapiole, and myristicin + dillapiole + nothoapiole, respectively.
Plants 2020, 9, x FOR PEER REVIEW 2 of 19 ( Figures 1 and 2), and their pharmacological actions and toxicities are well studied due to their use as pharmaceuticals and as flavorings. For example, myristicin is used to treat rheumatism and anxiety in traditional medicine. Risk assessment of myristicin using the margin of exposure approach has been conducted because of its potential genotoxicity or carcinogenicity [8]. Dillapiole, which is found mainly in dill, has been reported to have cytotoxic effects [9]. However, only a few enzymes involved in the synthesis of these compounds have been isolated and reported, and these known enzymes catalyze the formation of compounds with simple side groups [10]. In the text, the oil types of each strain of perilla are represented as PP-em, PP-m, PP-md, PP-emd, and PP-mdn, meaning that each strain mainly contains elemicin + myristicin, myristicin, myristicin + dillapiole, elemicin + myristicin + dillapiole, and myristicin + dillapiole + nothoapiole, respectively. Perilla plants, whose major aromatic compound is perillaldehyde, are used as herbal medicine for Kampo prescriptions in Japan. Those perilla plants that are rich in perillaldehyde do not contain (E)-asarone ( Figure 2) [11]. (E)-Asarone is structurally similar to PP-type oil components [12,13] and is likely formed through a PP-type oil component pathway, although the details are currently unknown. However, the (Z)-asarone isomer was reported to be toxic [14] and (E)-asarone may be carcinogenic [15], and therefore it should always be certified that no perilla plants containing (E)-asarone are present in the plant material used to prepare the pharmaceutical formulation. In order to find plants including this toxic compound and remove them from medicine ingredients, purity tests using liquid chromatography, optimized to separate perillaldehyde and (E)-asarone, are defined in the Japanese Pharmacopoeia.
The main constituents of PP-type perilla oil are elemicin, myristicin, dillapiole, and nothoapiole, and are likely synthesized from phenylalanine ( Figure 1). The reaction steps to all four compounds are genetically controlled based on crossing experiments using pure strains developed by repeated self-pollination, and on detailed gas chromatography (GC) analyses of perilla essential oils [2,16]. Previous reports on the synthesis of alkaloids suggested that methoxy or methylenedioxy groups are formed in the synthetic pathways leading to PP-type oil components and may involve hydroxylation or cyclization reactions by cytochrome P450 (hereafter, "P450") [17]. P450 enzymes catalyze different reactions, such as oxidation, hydroxylation, epoxidation, and dealkylation, and are involved in the synthesis of many plant secondary metabolites. Their low substrate specificity and high reaction regio-and stereo-specificity are beneficial for generating useful compounds, making their catalytic mechanisms an attractive target of study.
Here we describe the isolation of a P450 enzyme involved in the synthesis of dillapiole in PP-type perilla oils using expressed sequence tag (EST) libraries of pure strains of perilla. The perilla strains have been developed and maintained for more than 30 years through repeated Perilla plants, whose major aromatic compound is perillaldehyde, are used as herbal medicine for Kampo prescriptions in Japan. Those perilla plants that are rich in perillaldehyde do not contain (E)-asarone ( Figure 2) [11]. (E)-Asarone is structurally similar to PP-type oil components [12,13] and is likely formed through a PP-type oil component pathway, although the details are currently unknown. However, the (Z)-asarone isomer was reported to be toxic [14] and (E)-asarone may be carcinogenic [15], and therefore it should always be certified that no perilla plants containing (E)-asarone are present in the plant material used to prepare the pharmaceutical formulation. In order to find plants including this toxic compound and remove them from medicine ingredients, purity tests using liquid chromatography, optimized to separate perillaldehyde and (E)-asarone, are defined in the Japanese Pharmacopoeia.
The main constituents of PP-type perilla oil are elemicin, myristicin, dillapiole, and nothoapiole, and are likely synthesized from phenylalanine ( Figure 1). The reaction steps to all four compounds are genetically controlled based on crossing experiments using pure strains developed by repeated self-pollination, and on detailed gas chromatography (GC) analyses of perilla essential oils [2,16]. Previous reports on the synthesis of alkaloids suggested that methoxy or methylenedioxy groups are formed in the synthetic pathways leading to PP-type oil components and may involve hydroxylation or cyclization reactions by cytochrome P450 (hereafter, "P450") [17]. P450 enzymes catalyze different reactions, such as oxidation, hydroxylation, epoxidation, and dealkylation, and are involved in the synthesis of many plant secondary metabolites. Their low substrate specificity and high reaction regio-and stereo-specificity are beneficial for generating useful compounds, making their catalytic mechanisms an attractive target of study.
Here we describe the isolation of a P450 enzyme involved in the synthesis of dillapiole in PP-type perilla oils using expressed sequence tag (EST) libraries of pure strains of perilla. The perilla strains have been developed and maintained for more than 30 years through repeated self-pollination. The high purity of these strains allows for the selection of P450 sequences that may be involved in the biosynthesis of each oil component by comparing the expression levels of those sequences [18].

Isolation of a P450 Sequence from Perilla
P450-like sequences that are specifically expressed in perilla strains whose essential oils contain mainly dillapiole were identified. Contig 49487 was selected from our EST library because it had the largest difference in reads per kilobase of exon per million mapped read (RPKM) values of the sequences studied (Table 1). We PCR-amplified the selected contig using primers based on sequence information from the EST database, using complementary DNA (cDNA) as a template, then confirmed the size of the PCR product by agarose-gel electrophoresis. The size of contig 49487 was in accordance with the sequence information. The full-length sequence of contig 49487 was determined by RACE methods. The sequence Pf-49487 (GenBank Accession No. LC476554) encoded 505 amino acids (57 kD, Figure 3), and regions characteristic of P450s, such as a proline-rich region, an oxygen-binding pocket, and a heme-binding region, were conserved [19]. BLAST analysis showed that the amino acid sequence of Pf-49487 shared 56% identity with that of the Sesamum indicum premnaspirodiene oxygenase-like enzyme (predicted) (GenBank Accession No. JP653840) and this was the highest similarity among all entries in the database. Premnaspirodiene oxygenase is a hydroxylase classified as a member of the CYP71D family. Pf-49487 had a 49% amino acid sequence identity to the characterized Hyoscyamus muticus premnaspirodiene oxygenase (GenBank Accession No. EF569601) [20]. Cinnamate 4-hydroxylase from Arabidopsis thaliana (GenBank Accession No. NP180607) was a member of the P450 family that hydroxylates the aromatic ring of phenylpropanoid compounds and shared 28% sequence identity with Pf-49487 at the amino acid level. Based on the standardized P450 nomenclature system, Pf-49487, is in the CYP71D subfamily and was named CYP71D558. self-pollination. The high purity of these strains allows for the selection of P450 sequences that may be involved in the biosynthesis of each oil component by comparing the expression levels of those sequences [18].

Isolation of a P450 Sequence from Perilla
P450-like sequences that are specifically expressed in perilla strains whose essential oils contain mainly dillapiole were identified. Contig 49487 was selected from our EST library because it had the largest difference in reads per kilobase of exon per million mapped read (RPKM) values of the sequences studied (Table 1). We PCR-amplified the selected contig using primers based on sequence information from the EST database, using complementary DNA (cDNA) as a template, then confirmed the size of the PCR product by agarose-gel electrophoresis. The size of contig 49487 was in accordance with the sequence information. The full-length sequence of contig 49487 was determined by RACE methods. The sequence Pf-49487 (GenBank Accession No. LC476554) encoded 505 amino acids (57 kD, Figure 3), and regions characteristic of P450s, such as a proline-rich region, an oxygen-binding pocket, and a heme-binding region, were conserved [19]. BLAST analysis showed that the amino acid sequence of Pf-49487 shared 56% identity with that of the Sesamum indicum premnaspirodiene oxygenase-like enzyme (predicted) (GenBank Accession No. JP653840) and this was the highest similarity among all entries in the database. Premnaspirodiene oxygenase is a hydroxylase classified as a member of the CYP71D family. Pf-49487 had a 49% amino acid sequence identity to the characterized Hyoscyamus muticus premnaspirodiene oxygenase (GenBank Accession No. EF569601) [20]. Cinnamate 4-hydroxylase from Arabidopsis thaliana (GenBank Accession No. NP180607) was a member of the P450 family that hydroxylates the aromatic ring of phenylpropanoid compounds and shared 28% sequence identity with Pf-49487 at the amino acid level. Based on the standardized P450 nomenclature system, Pf-49487, is in the CYP71D subfamily and was named CYP71D558.  The following conserved regions of P450 are underlined: the proline-rich region, the oxygen-binding pocket, and the heme-binding region from upstream of the sequence. Black background indicates 100% amino acid identity among the three clones and gray background indicates greater than 50% amino acid identity. from upstream of the sequence. Black background indicates 100% amino acid identity among the three clones and gray background indicates greater than 50% amino acid identity.

Comparison of Pf-49487 Expression among Strains of Phenylpropene (PP)-Type Perilla
Quantitative reverse transcription-PCR (RT-PCR) was used to compare the Pf-49487 expression levels in various strains using RNAs isolated from different oil types of perilla: strain 12 of PP-m, strain 25 of PP-emd, and strain 5316 of PP-mdn (Figures 1 and 4). The difference in expression level among oil types is similar to the RPKM values in the EST library used for the isolation of Pf-49487, namely, strain 12: 0.0968, strain 25: 23.7, strain 5316: 56.7 (Table 1). These ratios are believed to be relevant to the dillapiole content (%) in oils, namely, strain 12: 0%, strain 25: 9.01%, strain 5316: 21.13% [4,16].

Heterologous Expression and Functional Analysis of Pf-49487
P450 enzymes with a bound carbon monoxide (CO) molecule show an absorption spectrum peak at ca. 450 nm, and heterologously expressed P450 enzymes with such an absorption maximum generally have high activity. The reduced CO-difference spectrum of the microsomal fraction of yeast harboring the Pf-49487 expression plasmid was measured and a peak at 448 nm was observed, suggesting that Pf-49487 has P450 activity ( Figure 5).

Heterologous Expression and Functional Analysis of Pf-49487
P450 enzymes with a bound carbon monoxide (CO) molecule show an absorption spectrum peak at ca. 450 nm, and heterologously expressed P450 enzymes with such an absorption maximum generally have high activity. The reduced CO-difference spectrum of the microsomal fraction of yeast harboring the Pf-49487 expression plasmid was measured and a peak at 448 nm was observed, suggesting that Pf-49487 has P450 activity ( Figure 5).
Plants 2020, 9, x FOR PEER REVIEW 4 of 19 from upstream of the sequence. Black background indicates 100% amino acid identity among the three clones and gray background indicates greater than 50% amino acid identity.

Comparison of Pf-49487 Expression among Strains of Phenylpropene (PP)-Type Perilla
Quantitative reverse transcription-PCR (RT-PCR) was used to compare the Pf-49487 expression levels in various strains using RNAs isolated from different oil types of perilla: strain 12 of PP-m, strain 25 of PP-emd, and strain 5316 of PP-mdn (Figures 1 and 4). The difference in expression level among oil types is similar to the RPKM values in the EST library used for the isolation of Pf-49487, namely, strain 12: 0.0968, strain 25: 23.7, strain 5316: 56.7 (Table 1). These ratios are believed to be relevant to the dillapiole content (%) in oils, namely, strain 12: 0%, strain 25: 9.01%, strain 5316: 21.13% [4,16].
The use of elemicin as a substrate resulted in lower activity than did the use of myristicin (Table 3). This result suggests that myristicin and not elemicin is the main substrate for Pf-49487.
We performed kinetic analyses of the Pf-49487 reaction using myristicin as substrate. The P450 concentration in the microsomal fraction was determined to be 1.03 µM from the reduced CO-difference spectrum and the Km, turnover number (kcat), and catalytic efficiency (kcat/Km) values were 9.1 µM, 15 min −1 , and 1.65 × 10 −6 min −1 M −1 , respectively. The Km value for the oxidization of premnaspirodiene by premnaspirodiene oxygenase (whose amino acid sequence is most similar to Pf-49487) was 14 µM, similar to that of Pf-49487 [20]. The optimum pH of Pf-49487 was approximately 7.5 (Figure 7), similar to that of premnaspirodiene oxygenase [20].

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Discussion
Pf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiole intermediate (compound 20). Pf-49487 catalyzes the hydroxylation of methyl eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and geraniol, which are MT-type oil components, as well as dillapiole and eugenol, which are phenylpropanoid compounds. These results indicate that Pf-49487 exhibits rather low substrate specificity and high regiospecificity. Premnaspirodiene oxygenase, whose sequence is most similar to that of Pf-49487, Plants 2020, 9, 577 8 of 18 has similar properties [20]. Other members of the CYP71D family also have high regiospecificity, namely the limonene hydroxylases CYP71D13 and CYP71D18, which hydroxylate the C3 and C6 positions, respectively [21].
The biosynthetic pathways of PP-type oil components have been inferred from the results of crossing experiments conducted with pure strains of perilla containing elemicin, myristicin, dillapiole, and nothoapiole as the main oil components, and the genetic control of each step in the formation of elemicin, dillapiole, and nothoapiole was in accordance with Mendelism [16] (Figure 1). Phenylpropanoids are believed to be synthesized via the shikimate pathway and the genes for methyl eugenol formation have been cloned [22]; consequently, methyl eugenol could be a precursor of PP-type oil components. However, neither methyl eugenol nor compound 5 have been detected in perilla oil whereas Pf-49487 produces compound 5 from methyl eugenol in vitro. On the other hand, compound 14 (a putative precursor of elemicin and myristicin) and compound 30 (a putative precursor of nothoapiole) were present in oil from strain 5316 (PP-mdn). However, compound 14 was not found in oil from strain 10 (PP-em) and strain 12 (PP-m), even though compound 14 is a likely intermediate of myristicin and thus was expected to be present in these oils. These findings indicate that the catalytic patterns and reactivities of enzymes may differ among oil types. Our identification of monohydrate intermediates of phenylpropanoid compounds in perilla oil suggests that O-methyltransferases or P450s are involved in perilla biosynthetic pathways and catalyze the generation of methoxy groups or methylenedioxy bridges from these monohydrate compounds. Our future studies will further explore these enzymes.

Plant Materials
All perilla plants used in this study were grown at the Experimental Station for Medicinal Plant Research, Graduate School of Pharmaceutical Sciences, Kyoto University. They have been bred and kept as pure lines through repeated self-pollination [18]. Strain numbers and oil types (Figure 1) of perilla used in this study were as follows: strain 10, PP-em; strain 12, PP-m; strain 16, PP-md; strain 25, PP-emd; strain 5316, PP-mdn; strain 5717, C.

Construction of the Expressed Sequence Tag (EST) Library and Cloning of Pf-49487
The EST library was constructed by the Kazusa DNA Research Institute as described previously [7]. P450-like sequences were selected from the EST library by BLAST analysis and the expression levels of these sequences were compared between different oil types. High expression level contigs in strains containing high levels of dillapiole (strains 16, 25, and 5316) were selected and their expression levels were compared using RPKM values.
We focused on contig 49487, which is most specifically expressed in dillapiole-containing perilla ( Table 1). PCR-amplification was performed for the target sequence, using cDNA as a template. cDNA was obtained by reverse transcription of RNA isolated from fresh young perilla leaves (strain 5316) using a RNeasy Plant Mini Kit (Qiagen), and reverse transcription was performed using RevTra Ace (Toyobo) with primer add2 (5 -CCACGCGTCGACTACTTTTTTTTTTTTTTT-3 ). Primers for PCR-amplification were designed based on sequence information from the EST database. The forward primer 49487-f1 (5 -TCCGTTCCGTTCCTTCAGAGATCTCGCG-3 ) and reverse primer 49487-r1 (5 -GGATGCCTTATCAGTTCAGTCATTGCC-3 ) were used for amplifying contig 49487. The reaction mixture contained 0.2 µM primer, 0.2 mM dNTPs, and 0.025 U µL −1 Blend Taq (Toyobo). The temperature program started at 94 • C for 30 s, followed by 30 cycles of 51 • C for 30 s, 72 • C for 60 s, and a final elongation at 72 • C for 60 s. The size of contig 49487 corresponded to the sequence information from the EST database and RACE methods were used to obtain its full-length sequence. 3 -RACE was performed with the primers add2 and 49487-f2 (5 -AGAAGGTCGGCACAATGGTCAGC TCCATC-3 ), and then nested PCR was performed with the primers amm (5 -GGCCACGCGTCGACTAC-3 ) Plants 2020, 9, 577 9 of 18 and 49487-f3 (5 -TGTAGGTCTGCGTTCGGCACGGTGTGCAAG-3 ) in the same reaction mixture as described above, with a temperature program starting at 94 • C for 30 s, followed by 30 cycles of 55 • C (52 • C for nested PCR) for 30 s, 72 • C for 60 s, and a final elongation at 72 • C for 60 s. The reaction products were electrophoresed in agarose gel, purified using NucleoSpin Gel and PCR clean-up (Macherey-Nagel), and ligated to the vector pTA2 (Toyobo). Sequences were confirmed using FASMAC. For 5 -RACE, reverse transcription was performed as described above with primer 49487-r2 (5 -GTAATTGTACCAGCAGACCTCTAGG-3 ) or primer 49487-r3 (5 -GGATCATTGTCTTTGAGGACT TCCTTC-3 ) and the reaction products were purified using NucleoSpin Gel and PCR clean-up. After the addition of poly C using 0.6 U µL −1 TdT (Invitrogen) and 0.2 mM dCTP (Toyobo), PCR was performed with the primers 5ann (5 -GGCCACGCGTCGACTAGTACGGG(I)(I)GGG(I)(I)GGG(I)(I)G-3 ) and 49487-r3 or 49487-r4 (5 -CGATGACACGAGGAACGAGTCGAC-3 ), and then nested PCR was performed with the primers amm and 49487-r3 in the same reaction mixture as described above, with a temperature program starting at 94 • C for 100 s, followed by 25 cycles (30 cycles for nested PCR) of 94 • C for 30 s, 50 • C (50.5 • C for nested PCR) for 30 s, 72 • C for 60 s, and a final elongation at 72 • C for 60 s.

Heterologous Expression of Pf-49487 in Saccharomyces cerevisiae
Pf-49487 was ligated into yeast expression vector pGYR-SpeI containing the S. cerevisiae NADPH-P450 reductase gene and a SpeI-cloning site [17]. The full-length sequence of Pf-49487 was amplified by PCR with the forward primer 49487-f4 (5 -ACTAGTATGGAGTCCGATCTCGCAACTG-3 ) and the reverse primer 49487-r5 (5 -ACTAGTTCACGGTGATGTCGGTTCAAATGG-3 ) in a reaction mixture containing 0.3 µM primer, 0.2 mM dNTPs, and 0.02 U µL −1 KOD-Plus (Toyobo) using a temperature program starting at 94 • C for 100 s, followed by 25 cycles of 94 • C for 15 s, 52.7 • C for 30 s, 68 • C for 90 s, and final elongation at 68 • C for 90 s. The amplified sequence was ligated into pTA2 and the nucleotide sequence was confirmed, then Pf-49487 was ligated into pGYR-SpeI and the vector was introduced into S. cerevisiae strain AH22 using the LiCl method. The recombinant yeast cells were cultivated, and the microsomal fraction was prepared as previously described [23]. The microsomal fraction was suspended in 100 mM HEPES/NaOH (pH 7.5) and stored at −80 • C until needed. The reduced CO-difference spectrum was measured with a UV1800 spectrophotometer (Shimadzu) and enzymatic activity was confirmed by the peak at 450 nm [24].

Enzymatic Assays and Gas Chromatography-Mass Spectrometry (GC-MS) Analyses
Enzymatic reactions were performed in 1-mL volumes in screw-capped glass tubes. Each reaction mixture was composed of 50 mM HEPES/NaOH (pH 7.5), 0.5 mM NADPH, 0.2 mM substrate, and 1.98 mg mL −1 enzyme preparation. After incubation at 30 • C for 15 min, 12 nmol of eugenol was added as an internal standard, and the reaction mixture was extracted three times with 2 mL of pentane. The pentane fractions were combined, dehydrated with MgSO 4 , concentrated under nitrogen, and analyzed by gas chromatography-mass spectrometry (GC-MS) instrument (6850GC/5975MSD, Agilent Technologies or GCMS-QP2020 NX, Shimadzu). The compounds were separated on a DB-WAX column (60 m × 0.25 mm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA) under the following conditions: injector, 180 • C; oven program starting at 100 • C, increasing at 5 • C min −1 to 220 • C, and holding at this temperature for 60 min. Helium was used as the carrier gas and column flow was 1.0 mL min −1 . The compounds were identified by comparing their retention times and mass spectra with authentic standards, synthesized samples, or a MS data library (NIST11 or NIST17; National Institute of Standards and Technology).
For kinetic analyses, 0.78 mg mL −1 enzyme preparations were incubated with myristicin concentrations ranging from 0.2 to 10 µM. The reaction mixtures were treated as described above and analyzed by GC-FID (G5000, Hitachi, Tokyo, Japan). Compounds were separated on an InertCap WAX column (60 m × 0.25 mm × 0.25 µm, GL Sciences) or a DB-WAX column (60 m × 0.25 mm × 0.25 µm, Agilent Technologies) under the following conditions: injector, 180 • C; FID, 220 • C; oven program starting at 100 • C, increasing at 5 • C min −1 to 220 • C, and holding at this temperature for 40 min. Assays were repeated independently three times. The P450 concentration in the enzyme preparation was determined from the reduced CO-difference spectrum using a differential absorption coefficient of 91 mM −1 cm −1 [24].
The optimum pH (ranging from 6.0 to 9.0) was analyzed using 0.2 mM myristicin and 1.65 mg mL −1 enzyme preparation. Assays were performed in the same manner as the kinetic analyses.

Quantitative RT-PCR
The differences in the expression levels of Pf-49487 between different perilla oil types were determined using quantitative RT-PCR. Total RNA was isolated from fresh young perilla leaves (strains 12, 25, and 5316) using the method described above. First-strand cDNAs were synthesized from 1 µg total RNA using ReverTra Ace and oligo(dT) primer (Takara), and were then purified using NucleoSpin Gel and PCR clean-up. Quantitative RT-PCR was performed with StepOnePlus (Applied Biosystems) using THUNDERBIRD SYBR qPCR Mix (Toyobo) following the manufacturers' protocols, with a temperature program starting at 95 • C for 60 s, followed by 40 cycles of 95 • C for 15 s, 60 • C for 60 s. The forward primer 49487-f (5 -TGGTGCCTCTCATAATGCTG-3 ) and the reverse primer 49487-r (5 -TCTGAAGGAACGGAACGGAG-3 ) were used to amplify Pf-49487, and the forward primer Histone-f (5 -TCACGAACAAGCCTTTGGAA-3 ) and the reverse primer Histone-r (5 -AAGCCTCACCGTTACCGTC-3 ) were used to provide histone transcripts as the internal control for PCR. Quantitation was performed using the 2 −∆∆CT method. Total RNA was extracted in duplicate and reactions were performed independently three times.

GC-MS Analysis of Perilla Oil
Perilla essential oils were obtained by extracting approximately 300 g of fresh perilla leaves (strains 10, 12, and 5316) with diethyl ether overnight at 4 • C. The oils were concentrated, dehydrated, and analyzed by GC-MS as described above. Because these analyses were performed to confirm the presence of monohydrate intermediates, the oils were highly concentrated; consequently, we focused on the retention times of the synthesized compounds ( Table 2). The chemical profiles of each perilla strain were previously reported [4,16].

Chemicals
Chemical reagents were purchased from Nacalai Tesque or Fujifilm Wako Pure Chemical. Compounds 5,8,14,20,23, and 30 were synthesized as described below (Figure 8). NMR, MS, and IR data are shown in Supplementary Data S1. Compound 31 was synthesized as previously reported [25].   [29] (515 mg, 2.65 mmol) was dissolved in decaline (0.5 mL) and heated at 200 • C under microwave irradiation for 12 h. After cooled to ambient temperature, the solution was directly purified by column chromatography (hexane/EtOAc 3:1) to give 2-allyl-4,5-dimethoxyphenol (8) (68 mg, 13%) as a pale-yellow solid along with a 91:9 mixture of the title compounds (411 mg, 73% and 7%, respectively) as a pale-yellow oil. 2-Allyl-3,4-dimethoxyphenol (9) was partially separated as a pale-yellow oil from the mixture by another column chromatography (hexane/  (11): To a solution of the crude 3-tert-butyldimethylsiloxy-4,5-dimethoxybenzaldehyde (10) in EtOH (24 mL) cooled in an ice-water bath, was portion-wise added NaBH 4 (104 mg, 2.75 mmol). After 15 min, water was added to the mixture, and most of EtOH was removed from the mixture by evaporation. The mixture was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo. The resulting residue was purified by column chromatography (hexane/EtOAc 3:1) to give the title compound (563 mg, 69% over 2 steps) as a white solid. Recrystallization from hexane gave colorless needles of mp 57-58 • C. The 13 C NMR was identical to that reported, while all the 1 H NMR chemical shifts differed by 0.18 ppm from the reported values [32]. 3.

Conclusions
An enzyme catalyzing a reaction in a proposed synthetic pathway of PP-type perilla oil components, namely, the conversion of myristicin into a monohydrate intermediate of dillapiole (compound 20) by regiospecific hydroxylation, was cloned. The potentially harmful to humans (E)-Asarone is structurally similar to PP-type oil components and has been identified in perilla plants grown in China [12,13]. The methoxy group of (E)-asarone may result from the hydroxylation of an intermediate. The position of the double bond in the side chain of (E)-asarone differs from that of PP-type oil components and thus Pf-49487 is unlikely to be involved in the biosynthesis of (E)-asarone. However, our findings regarding Pf-49487 may help elucidate the (E)-asarone biosynthetic pathway and aid in the quality assessment and safety evaluation of perilla products.
Little is known about the biosynthetic pathways of phenylpropanoid volatile components. Only a few enzymes involved in the synthesis of compounds such as anethole and eugenol have been reported, and enzymes involved in the synthesis of compounds such as apiole, myristicin, and asarone have not been characterized. These components are important plant flavor compounds and are also used as medicines and food supplements due to their pharmacological actions. Some of these compounds are genotoxic or carcinogenic [8]. Further elucidation of the biosynthetic mechanisms of phenylpropanoid volatile components may contribute to the genetic manipulation and enzymatic production of useful compounds.