Glucuronyltransferase, gene encoding the same, and method for using the same

Abstract

The purpose of the present invention is to identify a gene of a first glucuronyltransferase that catalyzes the transfer of glucuronic acid to the hydroxyl group at the 3-position of an oleanane-type triterpenoid.　Provided are first glucuronyltransferases having the target activity that are derived from plants of the leguminous family (soy beans, Glycyrrhiza uralensis and Lotus japonicus) and comprise respectively the base sequences represented by SEQ ID NOs: 2, 4 and 6.

Description

Technical Field

[0001] The present invention relates to an enzyme that transfers glucuronic acid to the hydroxyl group at the 3-position in oleanane-type triterpenoids, a gene encoding the enzyme, and a method for producing glycyrrhizin.

Background Art

[0002] Licorice (Glycyrrhiza uralensis) is a perennial herbaceous plant of the legume family. The roots and stolons of this plant are known as the important crude drug "licorice" in traditional Chinese medicine and are widely used worldwide. The main active ingredient of licorice is glycyrrhizin, an oleanane-type triterpenoid saponin (Non-Patent Document 1). Research on glycyrrhizin has been conducted from various aspects such as its pharmacognosy, pharmacological usefulness, and breeding studies.

[0003] In order to stably and continuously provide high-quality glycyrrhizin as a pharmaceutical by a biological production system, it is necessary to establish optimal production conditions, select glycyrrhizin-high-producing strains, or breed glycyrrhizin-high-producing plants by introducing synthetic enzyme genes, using genes involved in the biosynthetic system of glycyrrhizin and the expression levels of these genes as markers. For this purpose, it is essential to identify the gene group involved in the biosynthetic system of glycyrrhizin.

[0004] Glycyrrhizin is commonly contained in plants and is biosynthesized from β-amyrin, which belongs to oleanane-type triterpenoids, through two-step oxidation reactions and two-step glycosylation reactions. β-Amyrin is known to be a precursor substance that is a biosynthetic branch point between glycyrrhizin and soyasaponin I in the triterpenoid saponin biosynthetic system (Figure 1).

[0005] As shown in FIG. 2, the synthetic enzymes from β-amyrin to glycyrrhizin have hitherto included two oxidases, CYP88D6 (Patent Document 1) and CYP72A154 (Patent Document 2), which respectively catalyze each of the two-step oxidation reactions for biosynthesizing glycyrrhetinic acid, the aglycone (non-sugar part) of glycyrrhizin, from β-amyrin, and among the synthetic enzyme genes that catalyze the two-step glycosylation reaction for glycyrrhetinic acid obtained to biosynthesize glycyrrhizin, the glycosyltransferase UGT73P12 (Patent Document 3) that catalyzes the second step has been known. However, the glycosyltransferase that catalyzes the first-step glycosylation reaction of directly transferring glucuronic acid to glycyrrhetinic acid, that is, the glucuronic acid first transferase, has not been obtained at all so far despite many persons skilled in the art having tried to isolate it several times. Therefore, it has become a bottleneck in synthesizing glycyrrhizin from β-amyrin in a biological production system or an in vitro synthesis system, and a sufficient amount of glycyrrhizin could not be obtained stably and continuously.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to isolate a gene of a glucuronic acid first transferase that catalyzes the glucuronic acid transfer to the 3-position hydroxyl group of oleanane-type triterpenoids including glycyrrhetinic acid in order to solve the above problems, and to produce and provide a biological expression system capable of biosynthesizing a large amount of glycyrrhizin from β-amyrin in vivo or in cells using the gene expression system.

Means for Solving the Problems

[0009] In the biosynthetic pathway from β - amyrin to glycyrrhizin, in order to identify the last remaining glucuronic acid first transferase, although many persons skilled in the art attempted to isolate it from licorice, it could not be identified over a long period of time. Therefore, the inventors considered that there was some inhibitory factor in the isolation of the glucuronic acid first transferase from plants of the genus Glycyrrhiza, and attempted a strategy to isolate the glucuronic acid first transferase from plants other than plants of the genus Glycyrrhiza. In the leguminous plants to which the plants of the genus Glycyrrhiza belong, generally, there is a biosynthetic pathway from β - amyrin to soyasaponin I via soyasapogenol B as an intermediate product (Figure 1). Soyasapogenol B, which is an oleanane - type triterpenoid, has a hydroxyl group at the 3 - position, while the final product soyasaponin I has a glucuronic acid bound at the 3 - position. This is the same as the fact that glycyrrhetinic acid, which is also an intermediate and an oleanane - type triterpenoid, has a hydroxyl group at the 3 - position, while glucuronic acid is bound at the 3 - position of the final product glycyrrhizin. That is, the glucuronic acid first transferase that functions in the biosynthetic pathway from β - amyrin to glycyrrhizin may also function in the biosynthetic pathway from β - amyrin to soyasaponin I. Based on this hypothesis, the inventors attempted to identify a gene that may have glucuronic acid first transfer activity from soybean, which has a biosynthetic pathway from β - amyrin to soyasaponin I, and as a result, successfully isolated a cellulose synthase - like gene with an unknown specific function. As a result of verifying the sugar transfer activity of this enzyme using soyasapogenol B as a sugar acceptor substrate, it was revealed that glucuronic acid was transferred to the hydroxyl group at the 3 - position of soyasapogenol B. This enzyme activity was the same even when the sugar acceptor substrate was glycyrrhetinic acid. Thus, the inventors succeeded in identifying the glucuronic acid first transferase that could not be isolated so far by changing the source plant to soybean instead of plants of the genus Glycyrrhiza. The present invention is based on the research results and provides the following.

[0010] (1) A polypeptide having an activity of transferring glucuronic acid to the hydroxyl group at the 3 - position in an oleanane - type triterpenoid and containing any one of the amino acid sequences shown in the following (a) to (c), or a fragment thereof having the above activity. (a) An amino acid sequence represented by any one of SEQ ID NO: 1, 3, and 5, (b) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by any one of SEQ ID NO: 1, 3, and 5, or (c) An amino acid sequence having 80% or more identity with the amino acid sequence represented by any one of SEQ ID NO: 1, 3, and 5 A polypeptide. (2) The oleanane-type triterpenoid is selected from the group consisting of β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxoglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, medicagenic acid, soyasapogenol B, soyasapogenol A, hederagenin, camelliagenin, and psychogenic acid, and the polypeptide according to (1). (3) The polypeptide according to (1) or (2), which is derived from a plant of the family Fabaceae. (4) A polynucleotide encoding the polypeptide according to any one of (1) to (3). (5) The polynucleotide according to (4), which contains any one of the following base sequences (a) to (d). (a) A base sequence represented by any one of SEQ ID NO: 2, 4, and 6, (b) A base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by any one of SEQ ID NO: 2, 4, and 6, (c) A base sequence having 80% or more identity with the base sequence represented by any one of SEQ ID NO: 2, 4, and 6, or (d) A base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence represented by any one of SEQ ID NO: 2, 4, and 6 (6) A CSyGT expression vector containing the polynucleotide according to (4) or (5). (7) A transformant containing the polynucleotide according to (4) or (5) or the CSyGT expression vector according to (6), or its progeny that retains the polynucleotide or the CSyGT expression vector. (8) The transformant or its progeny according to (7), wherein the host is a Fabaceae plant. (9) The transformant or its progeny according to (7), wherein the host is yeast. (10) The polypeptide according to any one of (1) to (3), to which a sugar chain derived from yeast obtained from the transformant or its progeny according to (9) is added. (11) The polypeptide according to (10), wherein the sugar chain derived from yeast is a high-mannose type sugar chain. (12) A method for producing a polypeptide having an activity of transferring glucuronic acid to the 2-position hydroxy group of glucuronic acid in oleanane-type triterpenoid, the method comprising: culturing the transformant or its progeny according to (7) or (8); and extracting the polypeptide according to any one of (1) to (3) from the culture. (13) A genetically modified organism for glycyrrhizin production that can biosynthesize β-amyrin and contains all of the expression vectors shown in the following (A) to (D). (A) A CYP88D6 expression vector containing a polypeptide having an activity of oxidizing the 11-position in oleanane-type triterpenoid and containing any one of the amino acid sequences shown in the following (a) to (c): (a) The amino acid sequence shown in SEQ ID NO: 7 (b) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 7, or (c) An amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 7 (B) A CYP72A154 expression vector containing a polypeptide having an activity of oxidizing the 30-position in oleanane-type triterpenoid and containing any one of the amino acid sequences shown in the following (d) to (f): (d) The amino acid sequence shown in any one of SEQ ID NO: 9, 11, and 13 (e) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in any one of SEQ ID NO: 9, 11, and 13, or (f) An amino acid sequence having 80% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 9, 11, and 13 (C) A UGT73P12 expression vector comprising a polypeptide having an activity of transferring glucuronic acid to the 2-hydroxy group of glucuronic acid in oleanane-type triterpenoid monoglucuronide and containing any of the amino acid sequences shown in the following (g) to (i): (g) The amino acid sequence represented by SEQ ID NO: 15 (h) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 15, or (i) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15, and (D) The CSyGT expression vector according to (6) (14) The recombinant according to (13), wherein the host is a leguminous plant. (15) A method for producing glycyrrhizin from β-amyrin, the production method including a step of culturing the recombinant according to (13) or (14). This specification incorporates the disclosure of Japanese Patent Application No. 2019-190060, which is the basis of the priority of this application. [Effect of the Invention]

[0011] According to the present invention, a polypeptide having an activity of transferring glucuronic acid to the 3-hydroxy group in oleanane-type triterpenoid, a polynucleotide encoding the polypeptide, or a method using them can be provided. [Brief Description of the Drawings]

[0012]

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Mode for Carrying Out the Invention

[0013] 1. Glucuronosyltransferase 1 (CSyGT) 1-1. Overview The first aspect of the present invention relates to UDP-glucuronosyltransferase, a fragment thereof having UDP-glucuronosyltransferase activity, and a nucleic acid encoding the same. The UDP-glucuronosyltransferase of the present invention has an activity of catalyzing the glucuronidation reaction of the hydroxyl group at the 3-position of glycyrrhetinic acid, which is obtained through a two-step oxidation reaction from β-amyrin in the biosynthesis pathway specific to the genus Glycyrrhiza for synthesizing glycyrrhizin from β-amyrin that can be biosynthesized by many plants. By the UDP-glucuronosyltransferase of the present invention and the like, not only can glucuronic acid be glycosylated to the hydroxyl group at the 3-position of oleanane-type triterpenoids such as glycyrrhetinic acid, but also by combining with known enzymes involved in the biosynthesis pathway from β-amyrin to glycyrrhizin, it becomes possible to create a biological production system from β-amyrin to glycyrrhizin using plants other than the genus Glycyrrhiza as hosts. Thereby, high-quality glycyrrhizin can be stably and continuously provided.

[0014] 1-2. Definitions of Terms The following terms frequently used in this specification are defined. As used herein, the term "UDP-glucuronosyltransferase (CSyGT)" (often referred to as "CSyGT" in this specification) refers to an enzyme that catalyzes a glycosyltransferase reaction in which one glucuronic acid, which is a kind of sugar, is transferred to the hydroxyl group of an oleanane-type triterpenoid having a hydroxyl group at the 3-position carbon. The "first" as used herein means having the first glycosyltransferase activity in the two-step glycosyltransferase reaction at the hydroxyl group at the 3-position of the oleanane-type triterpenoid. The specific constitution of the UDP-glucuronosyltransferase will be described later.

[0015] As used herein, the term "a fragment thereof having UDP-glucuronosyltransferase activity" refers to an active fragment of the UDP-glucuronosyltransferase.

[0016] As used herein, the term "UDP-glucuronic acid first transfer activity" refers to the activity of catalyzing the glycosyltransferase reaction of the CSyGT, that is, the activity of transferring glucuronic acid to the hydroxyl group at the 3-position of the oleanane-type triterpenoid. Due to this activity, oleanane-type triterpenoid monoglucuronide is produced from oleanane-type triterpenoid. In the present specification, CSyGT and its fragments having UDP-glucuronic acid first transfer activity are collectively often referred to as "polypeptide having UDP-glucuronic acid first transfer activity" or "UDP-glucuronic acid first transfer enzyme etc. (CSyGT etc.)". Note that the UDP-glucuronic acid first transfer enzyme etc. in the present specification may be a glycoprotein to which different sugar chains are added. For example, both the UDP-glucuronic acid first transfer enzyme etc. to which a plant-derived sugar chain is added and the UDP-glucuronic acid first transfer enzyme etc. to which a yeast-derived sugar chain is added are included in the UDP-glucuronic acid first transfer enzyme etc. in the present specification.

[0017] The term "oleanane-type triterpenoid" refers to a C30 isoprenoid having a pentacyclic oleanane skeleton and consisting of six isoprene units. It corresponds to the aglycone of glycyrrhizin, which is the final target product in the present invention. Unless otherwise specified, the oleanane-type triterpenoid described in this specification means an oleanane-type triterpenoid having a hydroxyl group (OH group) at the 3-position carbon. Specific examples of oleanane-type triterpenoids include, but are not limited to, β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxoglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, medicagenic acid, soyasapogenol B, soyasapogenol A, hederagenin, camelliagenin, and psychogenic acid. All of these can serve as substrates for the glucuronic acid 1-transferase of the present invention. Through the glucuronic acid 1-transfer activity, β-amyrin-3-O-monoglucuronide, 11-oxo-β-amyrin-3-O-monoglucuronide, 30-hydroxy-11-oxo-β-amyrin-3-O-monoglucuronide, 30-hydroxy-β-amyrin-3-O-monoglucuronide, 24-hydroxy-β-amyrin-3-O-monoglucuronide, 11-deoxoglycyrrhetinic acid-3-O-monoglucuronide, glycyrrhetinic acid-3-O-monoglucuronide, oleanolic acid-3-O-monoglucuronide, medicagenic acid-3-O-monoglucuronide, soyasapogenol B-3-O-monoglucuronide, soyasapogenol A-3-O-monoglucuronide, hederagenin-3-O-monoglucuronide, camelliagenin-3-O-monoglucuronide, and psychogenic acid-3-O-monoglucuronide are biosynthesized from the above substrates.

[0018] In this specification, Fabaceae plants are not limited to Glycyrrhiza plants, but include all plant species belonging to Fabaceae in plant taxonomy. For example, plants of the genus Arachis, Cicer, Aspalathus, Dalbergia, Pterocarpus, Desmodium, Lespedeza, Uraria, Galegeae, Astragalus, Glycyrrhiza, Oxytropis, Augyrocytisus, Cytisus, Genista, Spartium, Hedysarum, Cyamopsis, Indigofera, Lotus, Lupinus, Wisteria, Cajanus, Canavalia, Erythrina, Glycine, Hardenbergia, Lablab, Mucuna, Phaseolus, Psophocarpus, Pueraria, Vigna, Robinia, Castanospermum, Maackia, Ormosia, Sophora, Styphnolobium, Medicago, Trigonella, Trifolium, Lathyrus, Lens, Pisum and Vicia are included.Plants of the genus Glycyrrhiza to which Glycyrrhiza uralensis belongs and plants of the genus Umtfaria which are related species thereof have a biosynthetic pathway of glycyrrhetinic acid which is a substrate of CSyGT in the biosynthesis of glycyrrhizin. Therefore, it is suitable as the leguminous plant of the present invention. Specific examples of plants of the genus Glycyrrhiza include G. glabra, G. inflata, G. aspera, G. eurycarpa, G. pallidiflora, G. yunnanensis, G. lepidota, G. echinata, and G. acanthocarpa, etc. Specific examples of plants of the genus Umtfaria include M. truncatula (M. truncatula; Talumtfaria), etc.

[0019] 1-3. Configuration The glucuronate first transferase (CSyGT) of the present invention is a polypeptide consisting of the amino acid sequence shown by any of SEQ ID NOs: 1, 3, and 5. These polypeptides respectively correspond to the wild-type CSyGT (GmCSyGT) derived from soybean (Glycine max), the wild-type CSyGT (GuCSyGT) derived from Glycyrrhiza uralensis (G. uralensis), and the wild-type CSyGT (LjCSyGT) derived from Lotus japonicus. GuCSyGT derived from Glycyrrhiza uralensis has 81% amino acid identity with GmCSyGT derived from soybean. Also, LjCSyGT derived from Lotus japonicus has 82% amino acid identity with GmCSyGT derived from soybean.

[0020] In addition to the aforementioned plant species, orthologs may exist in many other plant species, particularly Fabaceae plant species, for CSyGT of the present invention. The CSyGT of the present invention includes, in addition to such other-species wild-type CSyGT orthologs, homologous wild-type CSyGT paralogs and mutant CSyGTs having glucuronic acid first transfer activity. Examples of such other-species wild-type CSyGT orthologs and mutant CSyGTs include amino acid sequences in which one or more amino acids are deleted, substituted or added in the amino acid sequences shown by any of SEQ ID NOs: 1, 3, and 5, or polypeptides having amino acid identity of 80% or more, 82% or more, 85% or more, 87% 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 but less than 100% with respect to the amino acid sequences shown by any of SEQ ID NOs: 1, 3, and 5. In fact, for GmCSyGT of soybean, GuCSyGT and LjCSyGT are CSyGT orthologs of licorice and Lotus japonicus, respectively, and have amino acid identity of 80% or more as described above. In addition, mutant CSyGTs having glucuronic acid first transfer activity include, but are not limited to, specifically, mutants based on splicing variants, SNPs, etc.

[0021] As used herein, "a plurality of" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, "amino acid identity" refers to the ratio (%) of the number of identical amino acid residues to the total number of amino acid residues when the amino acid sequences of two polypeptides to be compared are aligned by appropriately inserting gaps into one or both as necessary so that the number of matching amino acid residues is maximized. Alignment of the two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, ClustalW, etc.

[0022] As used herein, the term "(amino acid) substitution" refers to substitution within a group of conservative amino acids having similar properties such as charge, side chain, polarity, aromaticity, etc. among the 20 types of amino acids that constitute natural proteins. For example, substitution within the group of uncharged polar amino acids having a low-polarity side chain (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the group of branched-chain amino acids (Leu, Val, Ile), the group of neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), the group of neutral amino acids having a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), the group of acidic amino acids (Asp, Glu), the group of basic amino acids (Arg, Lys, His), and the group of aromatic amino acids (Phe, Tyr, Trp) can be mentioned. Amino acid substitutions within these groups are preferred because it is known that they are less likely to cause changes in the properties of the polypeptide.

[0023] In this embodiment, the term "its active fragment" refers to a polypeptide fragment that contains a partial region of the glucuronosyltransferase 1 and retains the glucuronosyltransferase 1 activity. For example, a polypeptide fragment containing the substrate-binding site of the glucuronosyltransferase 1 can be mentioned. The substrates of the glucuronosyltransferase 1 of the present invention include the aforementioned oleanane-type triterpenoids. Preferably, it is glycyrrhetinic acid. The length of the amino acids of the polypeptide constituting this active fragment is not particularly limited. For example, in the polypeptides of the above (a) to (c), a continuous region of at least 10, 15, 20, 25, 30, 50, 100, or 150 amino acids may be sufficient.

[0024] In this specification, CSyGT and its active fragments are often collectively referred to as "CSyGT etc. (glucuronosyltransferase 1 etc.)".

[0025] According to the CSyGT etc. of the present invention, by using an oleanane-type triterpenoid as a sugar acceptor substrate and transferring glucuronic acid to the 3-position hydroxyl group by the glucuronosyltransferase 1 activity, an oleanane-type triterpenoid monoglucuronide can be obtained.

[0026] In the biosynthesis system of glycyrrhizin in licorice, the biosynthetic pathway from β-amyrin, which can be regarded as the starting material, to glycyrrhetinic acid has already been clarified, and artificial biosynthesis is also possible. Furthermore, the biosynthetic pathway in which one molecule of glucuronic acid is glycosyl-transferred to glycyrrhetinic acid to synthesize glycyrrhetinic acid monoglucuronide and then glucuronic acid is further glycosyl-transferred to synthesize glycyrrhizin has also been clarified. That is, in the biosynthetic pathway from β-amyrin to glycyrrhizin, only the pathway of glycosyl-transferring glucuronic acid to glycyrrhetinic acid to synthesize glycyrrhetinic acid monoglucuronide has been unknown until now. However, with the present invention, since all the biosynthetic pathways of glycyrrhizin in licorice have been clarified, in vitro synthesis from β-amyrin to glycyrrhizin becomes possible. In addition, since β-amyrin can be biosynthesized by many plant species other than licorice, an in vivo synthesis system using common plant species among them as hosts is also possible. Furthermore, if it is an organism that can biosynthesize a precursor of β-amyrin but does not contain β-amyrin, by using it together with the gene for synthesizing β-amyrin, an in vivo synthesis system using organisms other than plants as hosts is also possible.

[0027] 2. Glucuronic acid first transferase gene (CSyGT gene) and its active fragment 2-1. Overview The second aspect of the present invention relates to a polynucleotide encoding the polypeptide (such as CSyGT) described in the first aspect, that is, the glucuronic acid first transferase gene and its active fragment. The polynucleotide of the present invention enables the construction of the recombinant vector of the third aspect described below.

[0028] 2-2. Composition The "UDP-glucuronosyltransferase gene" (often referred to as the "CSyGT gene" in this specification) refers to a polynucleotide encoding CSyGT described in the first aspect. As long as it is a polynucleotide encoding CSyGT, its nucleotide sequence is not particularly limited. Preferably, it is a polynucleotide encoding a wild-type CSyGT containing the amino acid sequences shown in SEQ ID NO: 1, 3, and 5. For example, a polynucleotide encoding a wild-type GmCSyGT derived from soybean consisting of the amino acid sequence shown in SEQ ID NO: 1, specifically, for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2, that is, the wild-type GmCSyGT gene derived from soybean can be mentioned. Also, a polynucleotide encoding a wild-type GuCSyGT derived from licorice consisting of the amino acid sequence shown in SEQ ID NO: 3, specifically, for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4, that is, the wild-type GuCSyGT gene derived from licorice can be mentioned. And a polynucleotide encoding a wild-type LjCSyGT derived from Lotus japonicus consisting of the amino acid sequence shown in SEQ ID NO: 5, specifically, for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6, that is, the wild-type LjCSyGT gene derived from Lotus japonicus can be mentioned.

[0029] In addition, orthologs of other species of the wild-type CSyGT gene and mutant CSyGT genes that maintain enzyme activity are also included. Examples of such CSyGT genes include polynucleotides containing a nucleotide sequence in which one or more bases of the wild-type CSyGT gene are deleted, substituted, or added. Specifically, for example, a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence of any of the wild-type GmCSyGT gene derived from soybean (for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2), the wild-type GuCSyGT gene derived from licorice (for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4), and the wild-type LjCSyGT gene derived from Lotus japonicus (for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6).

[0030] Furthermore, polynucleotides are provided that include a base sequence having a base identity of 80% or more, 85% or more, 87% or more, 90% or more, 95% or more, or 99% or more, but less than 100%, with the wild-type CSyGT gene. Specifically, for example, a polynucleotide including a base sequence having a base identity of 80% or more, 85% or more, 87% or more, 90% or more, 95% or more, or 99% or more, but less than 100%, with any one of the base sequences of the wild-type GmCSyGT gene derived from soybean (for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2), the wild-type GuCSyGT gene derived from licorice (for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4), and the wild-type LjCSyGT gene derived from Lotus japonicus (for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6) is provided.

[0031] Also included are nucleotides that retain enzyme activity and are polynucleotides including a base sequence that hybridizes under highly stringent conditions with a nucleotide fragment consisting of a base sequence complementary to a partial base sequence of the wild-type CSyGT gene.

[0032] As used herein, "stringent conditions" means conditions under which non-specific hybridization is less likely to occur. "Highly stringent conditions" means conditions under which non-specific hybridization is even less likely to occur or does not occur. Generally, the higher the salt concentration and temperature of the reaction conditions, the more stringent the conditions. For example, in the washing after hybridization, the conditions are washing at 0.1× SSC and 0.1% SDS at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C. In addition, the stringency of hybridization can also be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

[0033] In this embodiment, the "active fragment" refers to a fragment of the CSyGT gene, and the polypeptide encoded by the fragment has CSyGT activity. Substantially, the polynucleotide encoding the active fragment of CSyGT described in the first embodiment corresponds. Therefore, the length of the base sequence of the polynucleotide constituting this active fragment, that is, the number of bases, may be three times the number of amino acid sequences in the active fragment of CSyGT described in the first embodiment.

[0034] According to the polynucleotide of the present invention or its active fragment, a recombinant vector capable of expressing CSyGT and its active fragment in a host cell can be constructed.

[0035] In this specification, the CSyGT gene and its active fragment are often collectively referred to as "CSyGT gene etc. (glucuronate 1-transferase gene etc.)".

[0036] The CSyGT gene etc. of this embodiment can be isolated from an appropriate plant, for example, a leguminous plant, using a known method. For example, based on the base sequence of the wild-type GmCSyGT gene derived from soybean shown in SEQ ID NO: 2, a primer pair having an appropriate base sequence length is designed. Specifically, for example, the primer pair shown in SEQ ID NO: 17 and 18 can be mentioned. The GmCSyGT gene can be obtained by performing a nucleic acid amplification reaction such as PCR using a nucleic acid derived from a soybean DNA library or a genomic DNA library as a template using the pair. In addition, the polynucleotide of the present invention can be obtained by hybridization from the above library etc. using a nucleic acid fragment consisting of a part of the base sequence shown in SEQ ID NO: 2 as a probe. These methods may refer to the methods described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0037] 3. Recombinant vector 3-1. Overview The third aspect of the present invention relates to a recombinant vector. The recombinant vector of the present invention contains the polynucleotide described in the second aspect, and can clone genes such as the CSyGT gene or express CSyGT etc. in a host cell. In this aspect, in particular, a CSyGT expression vector that expresses CSyGT etc. is preferably applied.

[0038] 3-2. Configuration The recombinant vector of the present invention can be constructed by introducing the polynucleotide described in the second aspect into an appropriate recombinant vector. The type of vector is not particularly limited. A vector for cloning (for transformation) or for gene expression etc. may be appropriately selected according to the purpose or according to the host to be introduced. A vector for plant transformation or an (gene) expression vector is particularly preferred.

[0039] In the present invention, the “(gene) expression vector” is a gene expression system that can transport a polynucleotide encoding an encapsulated polypeptide into a target plant cell and express the polypeptide. For example, an expression vector using a plasmid or a virus can be mentioned. In the present invention, a CSyGT expression vector that expresses CSyGT etc. incorporating the CSyGT gene etc. corresponds to this.

[0040] In the case of an expression vector using a plasmid (hereinafter often referred to as a “plasmid expression vector”), the plasmid includes, but is not limited to, for example, pPZP series, pSMA series, pUC series, pBR series, pBluescript series (Agilent Technologies), pTriEX TM series (TaKaRa), or a binary vector such as pBI series, pRI series or pGW series can be used.

[0041] In the case of an expression vector using a virus (hereinafter often referred to as a “virus expression vector”), viruses such as cauliflower mosaic virus (CaMV), common bean golden mosaic virus (BGMV), tobacco mosaic virus (TMV) etc. can be used.

[0042] The recombinant vector contains an expression regulatory region of a promoter and a terminator. In addition to this, it can also contain an enhancer, a polyA addition signal, a 5'-UTR (untranslated region) sequence, a labeling or selection marker gene, a multiple cloning site, an origin of replication, etc. Each type is not particularly limited as long as it can exert its function in the host cell. Those known in the art may be appropriately selected according to the host to be introduced. Preferably, it is the case where a plant cell or a plant is used as the host.

[0043] Promoters can use various promoters, for example, overexpression promoters, constitutive promoters, site-specific promoters, time-specific promoters, and / or inducible promoters. Specific examples of overexpression and constitutive promoters that are operable in plant cells include the 35S promoter derived from cauliflower mosaic virus (CaMV), the promoter Pnos of the nopaline synthase gene derived from the Ti plasmid, the ubiquitin promoter derived from maize, the actin promoter derived from rice, the PR protein promoter derived from tobacco, etc. The ribulose bisphosphate carboxylase small subunit (Rubisco ssu) promoter or histone promoter of various plant species can also be used. In addition, promoters that are operable in bacterial cells include the promoters of the maltogenic amylase gene of Bacillus stearothermophilus, the α-amylase gene of Bacillus licheniformis (B. licheniformis), the BAN amylase gene of Bacillus amyloliquefaciens (B. amyloliquefaciens), the alkaline protease gene of Bacillus subtillis (B. subtilis) or the xylosidase gene of Bacillus pumilus (B. pumilus), or the PR or PL promoters of phage lambda, the lac, trp or tac promoters of Escherichia coli, etc. Promoters that are operable in yeast host cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPI1 promoter, ADH2-4c promoter, etc. Promoters that are operable in fungi include the ADH3 promoter, tpiA promoter, etc.Examples of promoters that can function in animal cells include the SV40 early promoter, SV40 late promoter, CMV promoter, etc. Examples of promoters that can function in insect cells include the polyhedrin promoter, P10 promoter, basic protein promoter of Autographa californica polyhedrosis which is a baculovirus, baculovirus immediate early gene 1 promoter, baculovirus 39K delayed early gene promoter, etc.

[0044] Terminators include, for example, the terminator of the nopaline synthase (NOS) gene, the terminator of the octopine synthase (OCS) gene, the CaMV 35S terminator, the 3' terminator of the Escherichia coli lipoprotein lpp, the trp operon terminator, the amyB terminator, the terminator of the ADH1 gene, etc. There is no particular limitation as long as it is a sequence capable of terminating the transcription of the gene transcribed by the said promoter.

[0045] Examples of enhancers include the enhancer region containing the upstream sequence within the CaMV 35S promoter. There is no particular limitation as long as it can enhance the expression efficiency of nucleic acids encoding active peptides, etc.

[0046] Examples of selectable marker genes include drug resistance genes (for example, 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 (for example, luciferase, β - galactosidase, β - glucuronidase (GUS), or green fluorescent protein (GFP)), enzyme genes such as neomycin phosphotransferase II (NPT II), dihydrofolate reductase, etc.

[0047] According to the recombinant vector of the present invention, in addition to facilitating the manipulation and / or control of expression of the polynucleotide described in the second aspect, etc., it is possible to manipulate the expression of CSyGT, etc. in host cells.

[0048] 4. Transformant or its progeny 4-1. Overview The fourth aspect of the present invention relates to a transformant or its progeny. The transformant or its progeny of the present invention contains the polynucleotide described in the second aspect or the recombinant vector described in the third aspect in the cell, and can clone a CSyGT gene, etc., and / or express CSyGT, etc. According to the transformant of the present invention, it becomes possible to stably biosynthesize CSyGT, etc. in an in vivo expression system.

[0049] 4-2. Constitution As used herein, the "transformant" refers to a host transformed by the introduction of the polynucleotide described in the second aspect or the recombinant vector described in the third aspect.

[0050] The host to be transformed is not particularly limited. For example, bacteria such as Escherichia coli or Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Pichia pastoris, fungi such as Aspergillus, Neurospora, Fusarium or Trichoderma, monocotyledonous plants, dicotyledonous plants, or plant cells, mammalian cells, or insect cells (for example, sf9 or sf21) can be mentioned. Preferably, it is a leguminous plant or yeast.

[0051] The transformant of the present invention includes clone bodies having the same genetic information. For example, if the host is a unicellular microorganism that performs asexual reproduction such as Escherichia coli or yeast, clone bodies newly generated from the first generation of the transformant by division or budding are also included in the transformant of the present invention. Further, if the host is a plant, a part of the plant collected from the first generation of the transformant, for example, plant tissues such as epidermis, phloem, parenchyma, xylem or vascular bundle, plant organs such as leaves, petals, stems, roots or seeds, or clone bodies obtained from plant cells by plant tissue culture methods, cuttings, grafting or layering, or new clone bodies newly generated from vegetative propagation organs obtained by asexual reproduction from the first generation of the transformant such as rhizomes, tuberous roots, bulbs, runners, etc. are also included in the transformant of the present invention.

[0052] In addition to the polynucleotide according to the second aspect or the recombinant vector according to the third aspect, the transformant of the present invention may further have one or more other polynucleotides or other recombinant vectors. The other polynucleotide referred to herein means a polynucleotide other than the polynucleotide according to the second aspect. For example, the β-amyrin synthase gene, CYP88D6 or CYP72A154, or any oleanane-type triterpenoid monoglucuronide synthase gene is applicable. The other recombinant vector means a recombinant vector other than the recombinant vector according to the third aspect.

[0053] The transformant of the present invention can be prepared by introducing the above polynucleotide or recombinant vector into a suitable host.

[0054] The method for introducing the polynucleotide or recombinant vector can be a method known in the art, for example, the Agrobacterium method, the PEG-calcium phosphate method, the electroporation method, the liposome method, the particle gun method, or the microinjection method. The introduced polynucleotide may be integrated into the host genomic DNA or may exist in the state of the introduced polynucleotide (for example, remaining contained in a foreign vector). Furthermore, the introduced polynucleotide may be continuously maintained in the host cell as in the case of being integrated into the host genomic DNA or may be transiently retained.

[0055] After introducing the polynucleotide described in the second aspect or the recombinant vector described in the third aspect into the host by the above method, the presence or absence of the introduction of the target polynucleotide can be confirmed by the PCR method, the Southern hybridization method, the Northern hybridization method, the in situ hybridization, etc.

[0056] As used herein, "its progeny" means the progeny through sexual reproduction of the first generation of the transformant, which is a host that retains the polynucleotide described in the second aspect or the recombinant vector described in the third aspect of the present invention in an expressible state. Preferably, it refers to the host progeny that retains the polynucleotide described in the second aspect in the polynucleotide or the recombinant vector in an expressible state. For example, when the transformant is a plant, the seedlings of the transformant are applicable. The generation of the progeny is not limited.

[0057] According to the transformant of the present embodiment, by enhancing the expression of the introduced polynucleotide, the oleanane-type triterpenoid present in the host cell can be converted into an oleanane-type triterpenoid monoglucuronide. Further, by changing the host of the transformant, a glucuronic acid 1-transferase with different sugar chains added can be obtained. For example, when the host of the transformant is yeast, unlike the case where a leguminous plant is the host, a glucuronic acid 1-transferase with a high-mannose type sugar chain added is expressed. This is because the sugar chain addition reaction in yeast is different from that in plants (Strasser R. Glycobiology, 2016, 26(9): 926-939).

[0058] 5. Method for producing glucuronic acid 1-transferase and its active fragment (such as CSyGT) 5-1. Overview The fifth aspect of the present invention relates to a method for producing CSyGT etc., which includes culturing the transformant of the fourth aspect or its progeny and extracting from the culture a polypeptide having the glucuronic acid 1-transfer activity described in the first aspect, that is, CSyGT etc. According to the method for producing the polynucleotide of the present invention, by using the host as a biological production system, it becomes possible to stably and in large quantities obtain CSyGT etc.

[0059] 5-2. Method The production method of the present invention includes a culturing step and an extraction step as essential steps. Hereinafter, each step will be specifically described.

[0060] (1) Culturing step In this aspect, the "culturing step" is a step of culturing the transformant of the fourth aspect or its progeny. It is preferable to use a transformant or its progeny that can overexpress or constitutively express the polypeptide described in the first aspect for use in the present invention. For example, in the case of a transformant or its progeny having the recombinant vector described in the third aspect, it is preferable that the recombinant vector is an expression vector containing an overexpression promoter or a constitutive promoter. The transformant of the fourth aspect or its progeny may be any host, but is preferably a leguminous plant or yeast. By changing the host, even if the same glucuronosyltransferase 1 described in the first aspect is expressed, glycoproteins with different sugar chains added can be obtained.

[0061] For the medium used for culturing, a medium suitable for culturing the host may be appropriately used. Media known in the art can be used. For example, without limitation, in the case of culturing bacteria such as Escherichia coli as the host, LB medium or M9 medium, etc.; in the case of culturing yeast as the host, YPD medium, YPG medium, YPM medium, YPDM medium, SMM medium, etc.; in the case of culturing plants as the host, appropriate culture soil or hydroponic culture medium can be mentioned.

[0062] The medium may appropriately contain a carbon source (e.g., glucose, glycerin, mannitol, fructose, lactose, etc.), a nitrogen source (e.g., inorganic nitrogen such as ammonium sulfate, ammonium chloride, and organic nitrogen sources such as casein hydrolysate, yeast extract, polypeptone, bactotryptone, beef extract, etc.), inorganic salts (e.g., sodium diphosphate, potassium diphosphate, magnesium chloride, magnesium sulfate, calcium chloride, etc.), vitamins (vitamin B1, etc.), drugs (antibiotics such as ampicillin, tetracycline, kanamycin, etc.).

[0063] The culturing conditions are not particularly limited as long as they are appropriate for the expression of the polynucleotide, but usually, the culture is carried out for several hours to several hundred hours at a temperature of 10 to 45°C, 15 to 40°C, or 18 to 37°C, with aeration, irradiation, and / or stirring as necessary.

[0064] (2) Extraction step In this embodiment, the "extraction step" is a step of extracting CSyGT and the like from the culture obtained in the above-mentioned culture step.

[0065] As used herein, the "culture" refers to a culture supernatant or a cultured transformant. Not only inside the cells of the transformant, but also the culture supernatant may contain CSyGT and the like secreted from the transformant.

[0066] To recover the polypeptide described in the first aspect from the culture, the polypeptide present in the culture may be extracted by a known method and purified as necessary. For example, the target polypeptide can be obtained by using a solvent extraction method, salting-out method, solvent precipitation method, dialysis method, ultrafiltration method, gel electrophoresis method, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. alone or in appropriate combination. Specifically, reference may be made to the methods of Hayashi et al. (Hayashi et al., 1996, Phytochemistry, 42: 665-666) and Noguchi et al. (Noguchi et al., 2007, J. Biol. Chem., 282: 23581-23590) described above. In addition, based on the sugar chain specific to the host, the polypeptide described in the first aspect of the target can also be recovered. For example, when the transformant of the fourth aspect or its progeny is yeast, the expressed polypeptide of the present invention has high-mannose type sugar chains added, so it can also be extracted and purified using a mannose-binding lectin (for example, UDA lectin, BC2L-A lectin, etc.).

[0067] 6. Recombinant for glycyrrhizin production 6-1. Overview The sixth aspect of the present invention is a gene recombinant for glycyrrhizin production. The gene recombinant of the present invention contains an expression vector that expresses a set of four enzymes required for the biosynthetic pathway from β-amyrin to glycyrrhizin in Glycyrrhiza plants, that is, a set of four enzymes that catalyze two-step oxidation reactions and two-step glycosylation reactions. The gene recombinant of the present invention can biosynthesize glycyrrhizin from β-amyrin in living cells, and thus can be used as a biological production system for glycyrrhizin.

[0068] 6-2. Configuration 6-2-1. Expression vector included The gene recombinant of the present invention is characterized by at least including an expression vector that contains a polynucleotide encoding a set of four enzymes and / or their respective active fragments required for the biosynthetic pathway from β-amyrin to glycyrrhizin in a host cell. If necessary, it may further include an expression vector containing a polynucleotide encoding a β-amyrin synthase gene. The four enzymes are polypeptides that catalyze the first-stage oxidation reaction and the second-stage oxidation reaction of β-amyrin, and the first-stage glycosylation reaction and the second-stage glycosylation reaction of oleanane-type triterpenoids, respectively. The expression vectors containing each enzyme or its active fragment are shown and specifically described in the following (1) to (4). In (1) to (4), the four expression vectors are described separately, but the genes of each enzyme may be contained in different expression vectors, or two or more of them may be contained in the same expression vector.

[0069] (1) CYP88D6 expression vector The "CYP88D6 expression vector" contains a gene encoding a polypeptide having an activity of oxidizing the 11-position in oleanane-type triterpenoids, that is, CYP88D6 and its active fragments (often referred to as "CYP88D6 etc." in this specification) and its fragments (often referred to as "CYP88D6 gene etc." in this specification). Therefore, CYP88D6 etc. are expressed by the CYP88D6 expression vector in the gene recombinant.

[0070] As a specific example of the CYP88D6, although not limited thereto, CYP88D6 derived from Glycyrrhiza uralensis consisting of the amino acid sequence shown in SEQ ID NO: 7 can be mentioned. In addition, a polypeptide having the activity of the first oxidation step and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 7, or consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 7 is also exemplified.

[0071] Although not limited thereto, in the gene recombinant of the present invention, due to the catalytic activity etc. of CYP88D6 etc. expressed from the CYP88D6 expression vector, mainly endogenous or exogenous β-amyrin is used as a substrate, and its 11-position is oxidized to produce 11-oxo-β-amyrin. In addition, using 30-hydroxy-β-amyrin as a substrate, its 11-position can be oxidized to produce 30-hydroxy-11-oxo-β-amyrin. Furthermore, using 11-deoxoglutiric acid as a substrate, its 11-position can be oxidized to produce glutaric acid.

[0072] The constitution of the plasmid region in the CYP88D6 expression vector conforms to the expression vector in the recombinant vector described in the third aspect. Also, the recombinant vector described in Japanese Patent No. 5526323 may be used.

[0073] (2) CYP72A154 expression vector The "CYP72A154 expression vector" contains a gene encoding a polypeptide having an activity of oxidizing the 30-position in oleanane-type triterpenoids, that is, CYP72A154 and its active fragment (often referred to as "CYP72A154 etc." in this specification) and its fragment (often referred to as "CYP72A154 gene etc." in this specification). Therefore, in the gene recombinant, CYP72A154 etc. are expressed by the CYP72A154 expression vector.

[0074] Specific examples of the CYP72A154 include, but are not limited to, CYP72A154 derived from licorice (G. uralensis) having the amino acid sequence shown in SEQ ID NO: 9, CYP72A154 derived from licorice root (G. glabra) having the amino acid sequence shown in SEQ ID NO: 11, and CYP72A63 derived from Medicago truncatula having the amino acid sequence shown in SEQ ID NO: 13. In addition, polypeptides having the activity of the second oxidation step and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in any of SEQ ID NOs: 9, 11, and 13, or polypeptides consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 9, 11, and 13 are also exemplified.

[0075] Although not limited, in the gene recombinant of the present invention, due to the catalytic activity of CYP72A154 and the like expressed from the CYP72A154 expression vector, mainly using β-amyrin and 11-oxo-β-amyrin as substrates, the 30th position thereof can be oxidized to produce 30-hydroxy-β-amyrin and 30-hydroxy-11-oxo-β-amyrin, respectively. In addition, using 30-hydroxy-11-oxo-β-amyrin as a substrate, the 30th position thereof can be further oxidized to produce glycyrrhetinic acid.

[0076] The composition of the plasmid region in the CYP72A154 expression vector conforms to the expression vector in the recombinant vector described in the third aspect. In addition, the recombinant vector described in Japanese Patent No. 5771846 may be used.

[0077] (3) UGT73P12 recombinant vector The "UGT73P12 recombinant vector" contains a gene encoding a polypeptide, namely UGT73P12 and its active fragments (often referred to as "UGT73P12 etc." in this specification) and its fragments (often referred to as "UGT73P12 gene etc." in this specification), which has the activity of transferring glucuronic acid to the 2-position hydroxy group of glucuronic acid in oleanane-type triterpenoid monoglucuronide. Therefore, in the recombinant organism, UGT73P12 etc. are expressed by the UGT73P12 expression vector.

[0078] Specific examples of the above UGT73P12 include, but are not limited to, UGT73P12 derived from licorice (G. uralensis) consisting of the amino acid sequence shown in SEQ ID NO: 15. In addition, polypeptides having the second-stage glycosylation activity and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 15, or consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 15 are also exemplified.

[0079] The composition of the plasmid region in the UGT73P12 expression vector conforms to the expression vector in the recombinant vector described in the third aspect. In addition, the recombinant vector described in Japanese Patent No. 6344774 may also be used.

[0080] (4) CSyGT expression vector Since the "CSyGT expression vector" corresponds to the CSyGT expression vector in the CSyGT recombinant vector described in the third aspect, detailed description here is omitted.

[0081] 6-2-2. Recombinant organism for glycyrrhizin production The "recombinant for glycyrrhetinic acid production" of the present invention refers to a transformant into which an expression vector containing at least the above-mentioned four types of enzyme gene groups has been introduced, or its progeny that retains those enzyme gene groups. Therefore, except for the difference in the types of expression vectors included, its basic configuration may be the same as that of the transformant and its progeny described in the fourth aspect. However, since the present invention is a recombinant capable of biosynthesizing glycyrrhetin from β-amyrin intracellularly, it is preferable that the host can biosynthesize β-amyrin, which is the starting material in the biosynthetic system, intracellularly. The biosynthesis of β-amyrin in the host may be based on an endogenous synthesis system or an exogenous synthesis system. Since many plants can biosynthesize β-amyrin, which is an oleanane-type triterpenoid, when based on an endogenous synthesis system, the host of the present invention is preferably a plant. Preferably, it is a plant species with high β-amyrin synthetic ability, strong fertility, and easy cultivation. More preferably, it is a plant relatively closely related to licorice, that is, a leguminous plant. For example, species belonging to the genus Glycyrrhiza, species belonging to the genus Glycine, species belonging to the genus Miyakogusa, etc. can be mentioned. On the other hand, when the biosynthesis of β-amyrin is based on an exogenous synthesis system, the host may be a biological species that cannot biosynthesize β-amyrin itself. For example, by introducing an expression vector containing a β-amyrin synthase gene into yeast, the transformant of the yeast can be used as a host capable of biosynthesizing β-amyrin intracellularly.

[0082] According to the present invention, even a host that could not conventionally biosynthesize glycyrrhetin can biosynthesize glycyrrhetin as a metabolite using β-amyrin as a starting material.

[0083] 7. Method for producing glycyrrhetin 7-1. Overview The seventh aspect of the present invention is a method for producing glycyrrhetin. The production method of the present invention is characterized by using the recombinant for glycyrrhetinic acid production of the sixth aspect as a biological production system and producing glycyrrhetin from β-amyrin. According to the production method of the present invention, glycyrrhizin, which was conventionally expensive, can be stably and mass-produced without relying on extraction from licorice.

[0084] 7-2. Method The production method of the present invention includes a culturing step as an essential step and an "extraction step" as a selection step. (1) Culturing step The "culturing step" in this aspect may basically follow the culturing step described in the fifth aspect. When the recombinant is a plant, a known method for culturing plants may be applied. By this step, glycyrrhizin is produced in the recombinant for glycyrrhizin production of the sixth aspect. (2) Extraction step The "extraction step" in this aspect may basically follow the extraction step described in the fifth aspect. When the recombinant is a plant, the same method as the method for extracting glycyrrhizin from licorice can be used.

[0085] By the production method of the present invention, glycyrrhizin can be stably and mass-produced from various recombinants without relying on extraction from licorice.

Examples

[0086] In the following examples, an example of the present invention will be specifically described.

[0087] <Example 1: Isolation of the soybean-derived cellulose synthase-like gene Glyma.06G324300> Seeds of the soybean (Glycine max) cultivar "Williams 82" cultivated in a greenhouse were collected during ripening. Total RNA was prepared using the RNA extraction reagent RNeasy Plant Mini Kit (QIAGEN) according to the attached protocol. Using 200 ng of the obtained total RNA, first-strand cDNA was synthesized using the QuantiTech Reverse Transcription Kit (QIAGEN) according to the attached protocol. Using 1 μL of each 5-fold diluted first-strand cDNA as a template, oligonucleotide DNAs corresponding to the N-terminal and C-terminal portions of the polypeptide deduced from Glyma.06G324300 were used as the forward primer (SEQ ID NO: 17) and the reverse primer (SEQ ID NO: 18), respectively, and PCR was performed for 30 cycles at an annealing temperature of 55°C and a reaction temperature of 68°C using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.). Note that for the base sequence-specific recombination reaction (GATEWAY attB × attP reaction) required for cloning into pDONR TM 221 (Thermo Fisher Technologies), 12 bases (AAAAAGCAGGCT) were artificially added to the 5'-end of the forward primer, and 12 bases (AGAAAGCTGGGT) were artificially added to the 5'-end of the reverse primer. The DNA fragment amplified from the first-strand cDNA derived from seeds was cloned into pDONR TM 221 by a base sequence-specific recombination reaction (GATEWAY attB × attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies), and the polynucleotide sequences of the three independent clones obtained were determined. The sequence thus obtained was SEQ ID NO: 2, and the deduced polypeptide sequence was SEQ ID NO: 1.

[0088] <Example 2: Search for a Glyma.06G324300 homologous gene derived from licorice> From Glycyrrhiza uralensis, which is a leguminous plant the same as soybean and is known to biosynthesize glycyrrhizin, a Glyma.06G324300 homologous gene was searched as an orthologous gene candidate for Glyma.06G324300 by gene homology search. Using the BLAST homology search function in the Glycyrrhiza uralensis GDB (http: / / ngs-data-archive.psc.riken.jp / Gur-genome / index.pl), which is a genomic information database of Glycyrrhiza uralensis, a nucleotide sequence Glyur003152s00037491 encoding a protein showing high amino acid identity to Glyma.06G324300 was found. The polypeptide deduced from Glyur003152s00037491 showed 81% amino acid identity to Glyma.06G324300.

[0089] <Example 3: Isolation of Glyma.06G324300 Homologous Gene from Glycyrrhiza uralensis> Total RNA was prepared from the roots of Glycyrrhiza uralensis using the RNA extraction reagent PureLink Plant RNA Reagent (Thermo Fisher Scientific). Using 1 μg of the obtained total RNA, first-strand cDNA synthesis was performed according to the attached protocol using the SMART RACE cDNA amplification kit (Clontech). Using 2 μL of the first-strand cDNA as a template, oligonucleotides corresponding to the N-terminal and C-terminal of the polypeptide deduced from Glyur003152s00037491 were used as the forward primer (SEQ ID NO: 19) and the reverse primer (SEQ ID NO: 20), respectively, and PCR was performed for 30 cycles at an annealing temperature of 55°C and a reaction temperature of 72°C using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). Note that pENTR TMSince it is necessary for cloning into the / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), 4 bases (cacc) are artificially added to the 5'-end of the forward primer. The amplified DNA fragment was cloned into the pENTR TM / D-TOPO entry vector, and the nucleotide sequences of the 4 independent clones obtained were determined. The nucleotide sequence of the Glyma.06G324300 homologous gene of licorice obtained thereby is SEQ ID NO: 4, and the deduced polypeptide sequence therefrom is SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 had 82% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0090] <Example 4: Search for Glyma.06G324300 Homologous Gene Derived from Lotus japonicus> In the same manner as in Example 2, a Glyma.06G324300 homologous gene was searched as a candidate for the Glyma.06G324300 orthologous gene derived from Lotus japonicus. Using the BLAST homology search function in the genomic information database of Lotus japonicus, miyakogusa.jp (http: / / www.kazusa.or.jp / lotus / release1 / index.html), a nucleotide sequence Lj3g3v1981230 encoding a protein showing high amino acid identity to Glyma.06G324300 was found. The deduced polypeptide from Lj3g3v1981230 showed 81.4% amino acid identity to Glyma.06G324300.

[0091] <Example 5: Isolation of Glyma.06G324300 Homologous Gene Derived from Lotus japonicus> Using 1 μg of total RNA obtained from *Miyakogusa*, first-strand cDNA was synthesized according to the attached protocol using the SMART RACE cDNA amplification kit (Clontech) . Using 2 μl of the first-strand cDNA as a template, oligonucleotides corresponding to the N-terminal and C-terminal portions of the polypeptide predicted from Lj3g3v1981230 were used as the forward primer (SEQ ID NO: 37) and the reverse primer (SEQ ID NO: 38), respectively, and PCR was performed for 30 cycles at an annealing temperature of 55°C and a reaction temperature of 72°C using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). Since it is necessary for cloning into the pENTR TM / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), 4 bases (cacc) were artificially added to the 5'-end of the forward primer. The amplified DNA fragment was cloned into the pENTR TM / D-TOPO entry vector, and the polynucleotide sequences were determined for the two independent clones obtained. The nucleotide sequence of the *Miyakogusa* Glyma.06G324300 homologous gene thus obtained is SEQ ID NO: 6, and the polypeptide sequence deduced therefrom is SEQ ID NO: 5. SEQ ID NO: 5 had 82% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0092] <Example 6: Construction of Destination Vector for Yeast Expression> To examine the predicted glycosyltransferase activity of Glyma.06G324300 and its homologous proteins isolated in Examples 1, 3, and 5, expression vectors for each protein were constructed using a yeast expression system.

[0093] The yeast (Saccharomyces cerevisiae) INVSc1 strain used does not intrinsically contain UDP-glucuronic acid, which is the sugar donor substrate in the glycosylation reaction assumed for the candidate gene product. Therefore, the UDP-glucose dehydrogenase (UGD) gene, which synthesizes UDP-glucuronic acid using yeast-intrinsic UDP-glucose as a substrate, was introduced into a yeast expression destination vector. Specifically, using the cDNA of UGD from Arabidopsis thaliana (AtUGD2) as a template, oligonucleotide DNAs corresponding to the N-terminal and C-terminal portions of the polypeptide were used as the forward primer (SEQ ID NO: 21) and reverse primer (SEQ ID NO: 22), respectively, and PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) was used to perform 30 cycles of PCR at an annealing temperature of 55°C and a reaction temperature of 72°C. In addition, for the requirements of In-fusion cloning, 19 bases in total, including 15 bases upstream of the cloning position of the destination vector at the 5' end of the polynucleotide represented by SEQ ID NO: 23 (gggcggccgcactag) and 4 bases (aaaa), were artificially added to the forward primer. Also, 15 bases downstream of the cloning position of the destination vector were added to the 3' end of the polynucleotide represented by SEQ ID NO: 24 (atccatcgatactag) in the reverse primer. The destination vector pESC-HIS-GW, which was created by introducing Gateway cassette A (Thermo Fisher Technologies) into the SrfI restriction enzyme site within MCS2 of the pESC-HIS (registered trademark) yeast expression vector (Agilent Technologies), was treated with the SpeI restriction enzyme, mixed with the DNA fragment amplified from the cDNA, and the DNA fragment represented by SEQ ID NO: 25 was introduced into MCS1 within pESC-HIS-GW using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.) to obtain the destination vector pESC-HIS-AtUGD2-GW.

[0094] <Example 7: Construction of Yeast Expression Clones> The plasmid (entry clone) having the polynucleotide shown by SEQ ID NO: 2 prepared in Example 1 was mixed with the destination vector pESC-HIS-AtUGD2-GW prepared in Example 6, and by a base sequence-specific recombination reaction (GATEWAY attL × attR reaction) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Technologies), the DNA fragment shown by SEQ ID NO: 2 was transferred to pESC-HIS to obtain the yeast expression vector pESC-HIS-AtUGD2-Glyma.06G324300 of the gene shown by SEQ ID NO: 2. Also, yeast expression vectors pESC-HIS-AtUGD2-Glyur003152s00037491 and pESC-HIS-AtUGD2-Lj3g3v1981230 of the genes shown by SEQ ID NOs: 4 and 6 prepared in Examples 3 and 5 were obtained in the same manner as above, respectively.

[0095] <Example 8: Introduction into Yeast Strains Producing Glycyrrhetinic Acid and Soyasapogenol B> The expression vector pYES3-BAS of the β-amylase synthase (LjOSC1) gene of Japanese millet was introduced into the yeast INVScI strain (Thermo Fisher Technologies) (MATa his3D1 leu2 trp1-289 ura3-52 MATAlpha his3D1 leu2 trp1-289 ura3-52), and the co-expression vector pESC-CPR-CYP88D6 of the CYP88D6 gene and the cytochrome P450 reductase (LjCPR1) of Japanese millet, and the expression vector pDEST52-CYP72A63 of the CYP72A63 gene, which is an ortholog of the Glycyrrhiza glabra CYP72A154 gene in oil palm, were introduced and co-expressed to obtain a glycyrrhetinic acid-producing yeast strain (Figure 3(a)). At the same time, the expression vector pYES3-BAS of the β-amylase synthase gene, the co-expression vector pESC-CPR-CYP93E3 of the CYP93E3 gene and the cytochrome P450 reductase (LjCPR1) of Japanese millet, and the expression vector pDEST52-CYP72A566 of the CYP72A566 gene were introduced and co-expressed to obtain a soyasapogenol B-producing yeast (Figure 3(b)). The pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 7 were respectively introduced into these yeast strains. As a negative control, pESC-HIS-AtUGD2 corresponding to the empty vector was introduced into the glycyrrhetinic acid-producing yeast strain. Yeast transformation was performed according to the attached protocol using Frozen-EZ Yeast Transformation II (Zymo Research).

[0096] <Example 9: In Vivo Enzyme Assay Using Recombinant Yeast> The glychyrrhetinic acid-producing yeast strain harboring pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, pESC-HIS-AtUGD2-Lj3g3v1981230, or pESC-HIS-AtUGD2 for negative control obtained in Example 8 was cultured with shaking at 30 °C and 200 rpm for 24 hours using 1 mL of Yeast nitrogen base (YNB) medium (-Trp / -Leu / -Ura / -His) containing 2% glucose. Then, the culture solution was centrifuged at 3,000 g and 4 °C for 5 minutes to obtain a pellet of yeast cells. The obtained pellet of yeast cells was suspended in 1 mL of Yeast nitrogen base (YNB) medium (-Trp / -Leu / -Ura / -His), and then centrifuged again at 3,000 g and 4 °C for 5 minutes to obtain a pellet of yeast cells. The obtained pellet of yeast cells was suspended in 5 mL of Yeast nitrogen base (YNB) medium (-Trp / -Leu / -Ura / -His) containing 2% galactose and cultured with shaking at 30 °C and 200 rpm for 5 days. Then, a volume equivalent to 1 mL of glass beads (SIGMA) and 4 mL of 1-butanol were added to the culture solution. To disrupt the yeast cells, it was vigorously stirred with a strong shaker for 30 minutes, and the obtained liquid was centrifuged at 10,000 g and 4 °C for 10 minutes, and then the supernatant was recovered as a yeast metabolite extract. 4 mL of 1-butanol was newly added to the remaining liquid, and it was stirred again for 30 minutes. The obtained liquid was centrifuged at 10,000 g and 4 °C for 10 minutes, and then the supernatant was extracted. As a result, a metabolite extract (Sample A) derived from a glychyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 1 (Glyma.06G324300), a metabolite extract (Sample B) derived from a glychyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 3 (Glyur003152s00037491), a metabolite extract (Sample C) derived from a glychyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 5 (Lj3g3v1981230), and a metabolite extract (Sample D) derived from a glychyrrhetinic acid-producing yeast strain with only an empty vector that does not express any gene were obtained.

[0097] Soyasapogenol B-producing yeast strains harboring pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, pESC-HIS-AtUGD2-Lj3g3v1981230, or pESC-HIS-AtUGD2 for negative control were similarly cultured and metabolites were extracted. As a result, metabolite extracts (Sample E) from a soyasapogenol B-producing yeast strain expressing the polypeptide (Glyma.06G324300) shown in SEQ ID NO: 1, metabolite extracts (Sample F) from a soyasapogenol B-producing yeast strain expressing the polypeptide (Glyur003152s00037491) shown in SEQ ID NO: 3, metabolite extracts (Sample G) from a soyasapogenol B-producing yeast strain expressing the polypeptide (Lj3g3v1981230) shown in SEQ ID NO: 5, and metabolite extracts (Sample H) from a soyasapogenol B-producing yeast strain with only an empty vector that does not express any gene were obtained.

[0098] <Example 10: Analysis of Yeast Metabolite Extracts> Samples A, B, C, and D, and Samples E, F, G, and H obtained in Example 9 were evaporated using a rotary evaporator. The precipitate was suspended in 300 μL of methanol and then filtered using Millex-GV, 0.22 μm, PVDF, 4 mm (Merck) to obtain samples for LC-MS analysis.

[0099] LC-MS analysis was performed using an ACQUITY UPLC / TQD-MS (Waters Corp.). The column used was UPLC HSS C18 (2.1 mm x 150 mm, 1.7 μm) (Waters Corp.). The solvents were 0.1% acetic acid-acetonitrile: 0.1% acetic acid-water = 30:70 (0 - 5 minutes), 40:60 - 100:0 (5 - 28 minutes), 100:0 (28 - 31.5 minutes), and the flow rate was 0.2 mL / min for the analysis. MS was analyzed in SIM mode with the m / z values of each compound, glycyrrhetinic acid = 469.7, glycyrrhetinic acid monoglycoside = 631.9, glycyrrhetinic acid monoglucuronide = 645.8, glycyrrhizin = 821.9 as parameters. The identification of metabolites was determined by comparing the LC retention time and MS spectra with samples prepared by dissolving commercially available glycyrrhetinic acid monoglucuronide and glycyrrhizin in methanol at a concentration of 1 μM as standards.

[0100] The results are shown in FIGS. 4 and 5. FIG. 4 shows the results of the enzyme activity of Glyma.06G324300 or its homolog when glycyrrhetinic acid and glucuronic acid are used as substrates. From sample A in (a), one peak corresponding to glycyrrhetinic acid monoglucuronide was detected (black arrow). The retention time and mass spectrum of that peak were in good agreement with those of glycyrrhetinic acid monoglucuronide. Similarly, peaks corresponding to glycyrrhetinic acid monoglucuronide were detected from sample B in (b) and sample C in (c) (black arrows). The retention time and mass spectrum of each peak were in good agreement with those of glycyrrhetinic acid monoglucuronide. On the other hand, for sample D in (d) which was the negative control, no peak corresponding to glycyrrhetinic acid monoglucuronide was detected.

[0101] Figure 5 shows the results of the enzyme activity of Glyma.06G324300 or its homolog when soyasapogenol B and glucuronic acid are used as substrates. From sample E in (a), one peak corresponding to soyasapogenol B monoglucuronide was detected (black arrow). Peaks corresponding to soyasapogenol B monoglucuronide were also detected from sample F in (b) and sample G in (c) (black arrows). The retention time and mass spectrum of each peak were in good agreement with those of soyasapogenol B monoglucuronide. On the other hand, for sample H in (d) which is a negative control, no peak corresponding to soyasapogenol B monoglucuronide was detected.

[0102] <Example 11: Preparation of Transformed Yeast for Substrate Feeding Assay> The pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 8 were each introduced into the yeast INVScI strain. As a negative control, pESC-HIS-AtUGD2 corresponding to an empty vector was introduced into the same yeast INVScI strain. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

[0103] <Example 12: Substrate Feeding Assay Using Recombinant Yeast> The transformed yeast cells carrying pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, pESC-HIS-AtUGD2-Lj3g3v1981230, or pESC-HIS-AtUGD2 for negative control obtained in Example 11 were cultured with shaking at 30 °C and 200 rpm for 24 hours using 2 mL of Yeast nitrogen base (YNB) medium (-His) containing 2% glucose. Then, the culture broth was centrifuged at 3,000 g and 4 °C for 5 minutes to obtain a pellet of yeast cells. The obtained pellet of yeast cells was suspended in 2 mL of Yeast nitrogen base (YNB) medium (-His), and then centrifuged again at 3,000 g and 4 °C for 5 minutes to obtain a pellet of yeast cells. The obtained pellet of yeast cells was suspended in 10 mL of Yeast nitrogen base (YNB) medium (-His) containing 2% galactose and divided into two equal parts of 5 mL. To one sample, glycyrrhetinic acid with a final concentration of 5 μM was added, and to the other sample, soyasapogenol B with a final concentration of 5 μM was added (Figure 4). Then, the cells were cultured with shaking at 30 °C and 200 rpm for 10 days. One milliliter equivalent volume of glass beads (SIGMA) and 4 mL of 1-butanol were added to the culture broth. To disrupt the yeast cells, the mixture was vigorously stirred on a strong shaker for 30 minutes, and the resulting liquid was centrifuged at 10,000 g and 4 °C for 10 minutes, and then the supernatant was collected as the yeast feeding assay extract. Another 4 mL of 1-butanol was newly added to the remaining liquid and extracted again.As a result, a feeding assay extract (Sample I) obtained by adding glycyrrhetinic acid to transformed yeast expressing the polypeptide (Glyma.06G324300) shown in SEQ ID NO: 1, a feeding assay extract (Sample M) obtained by adding soyasapogenol B, a feeding assay extract (Sample J) obtained by adding glycyrrhetinic acid to transformed yeast expressing the polypeptide (Glyur003152s00037491) shown in SEQ ID NO: 3, a feeding assay extract (Sample N) obtained by adding soyasapogenol B, a feeding assay extract (Sample K) obtained by adding glycyrrhetinic acid to transformed yeast expressing the polypeptide (Lj3g3v1981230) shown in SEQ ID NO: 5, a feeding assay extract (Sample O) obtained by adding soyasapogenol B, a feeding assay extract (Sample L) obtained by adding glycyrrhetinic acid to transformed yeast with only an empty vector that does not express any gene, and a feeding assay extract (Sample P) obtained by adding soyasapogenol B were obtained.

[0104] <Example 13: Analysis of Substrate Feeding Assay Extracts> Samples I, J, K, L, M, N, O, and P obtained in Example 12 were evaporated using a rotary evaporator. The precipitate was suspended in 300 μL of methanol and then filtered using Millex-GV, 0.22 μm, PVDF, 4 mm (Merck) to obtain samples for LC-MS analysis.

[0105] The LC-MS analysis was carried out under the same conditions as in Example 10. For MS, in the SIM mode, for samples I, J, K, and L, the m / z values of each compound were used as parameters: glycyrrhetinic acid = 469.7, glycyrrhetinic acid monoglycoside = 631.9, glycyrrhetinic acid monoglucuronide = 645.8, and glycyrrhizin = 821.9. For samples M, N, O, and P, the m / z values of each compound were used as parameters: soyasapogenol B = 457.8, soyasapogenol B monoglycoside = 619.8, soyasapogenol B monoglucuronide = 633.8, and soyasapogenol B diglucuronide = 809.9. The identification of metabolites was determined by comparing the retention time of LC and the MS spectrum with samples prepared by dissolving commercially available glycyrrhetinic acid monoglucuronide, glycyrrhizin, and soyasapogenol B monoglucuronide in methanol at a concentration of 1 μM as standards.

[0106] The results when glycyrrhetinic acid was used as the sugar receptor substrate are shown in Figures 7 to 10. Figure 7 shows the results of the substrate feeding assay in sample I. In addition to the peak (white arrow) corresponding to glycyrrhetinic acid, which is the sugar receptor substrate, detected in (b), one peak (black arrow) that is considered to be glycyrrhetinic acid with one molecule of glucuronic acid added was detected in (c). The retention time and mass spectrum of that peak were in good agreement with those of glycyrrhetinic acid monoglucuronide.

[0107] From sample J shown in Figure 8 and sample K shown in Figure 9, peaks corresponding to glycyrrhetinic acid (white arrow) and glycyrrhetinic acid monoglucuronide (black arrow) were detected as shown in (b) and (c), respectively. The retention time and mass spectrum of each peak were in good agreement with those of glycyrrhetinic acid monoglucuronide.

[0108] On the other hand, in the negative control sample L shown in Figure 10, the peak (white arrow) corresponding to glycyrrhetinic acid, which is the sugar receptor substrate, was detected in (b), but no peak was detected at the position corresponding to glycyrrhetinic acid monoglucuronide (dashed arrow) in (c).

[0109] The results of the substrate feeding assay using soyasapogenol B as a sugar receptor substrate are shown in Fig. 11. In sample M containing Glyma.06G324300 derived from soybean shown in (b), sample N containing Glyur003152s00037491 derived from licorice shown in (c), and sample O containing Lj3g3v1981230 derived from Portulaca oleracea shown in (d), peaks corresponding to soyasapogenol B monoglucuronide, in which one molecule of glucuronic acid was added to soyasapogenol B, were detected (black arrows). The retention time and mass spectrum of the peaks were in good agreement with those of soyasapogenol B monoglucuronide. On the other hand, in the negative control sample P shown in (e), no peak corresponding to soyasapogenol B monoglucuronide was detected.

[0110] From the above results and the results obtained in Example 10, it was revealed that the novel enzyme Glyma.06G324300 derived from soybean obtained in Example 1, the novel enzyme Glyur003152s00037491 derived from licorice obtained in Example 3, and the novel enzyme Lj3g3v1981230 derived from Portulaca oleracea obtained in Example 5 have glucuronic acid first transfer activity to convert glycyrrhetinic acid to glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the 3-position hydroxy group of glycyrrhetinic acid. It was also demonstrated that they have glucuronic acid first transfer activity to convert soyasapogenol B to soyasapogenol B monoglucuronide by transferring glucuronic acid to the 3-position hydroxy group of soyasapogenol B. From the above, the obtained novel enzymes were identified as glucuronic acid first transfer enzymes that transfer glucuronic acid to the 3-position hydroxy group of oleanane-type triterpenoids.

[0111] <Example 14: Isolation of a functional deficiency mutant of the Glyma.06G324300 homologous gene of Portulaca oleracea> Based on the gene and protein sequence information of *Miyakogusa* and the expression database Lotus Base (https: / / lotus.au.dk / ), as a result of searching for mutant lines with LORE insertions in Lj3g3v1981230, 19 lines were hit. From among those lines, 2 lines (30006020, 30115796) were selected based on the LORE1 insertion position in Lj3g3v1981230 and the number of LORE1 insertions into other genes, and seeds were obtained from the distribution institution (Aarhus University, Denmark). Those seeds were sown, genomic DNA was extracted from a part of the developed cotyledons, and the insertion of LORE1 into Lj3g3v1981230 was confirmed by PCR. For PCR, 25 cycles of PCR were performed using GoTaq® Colorless Master Mix (Promega) at an annealing temperature of 60°C and a reaction temperature of 72°C. Forward primers (SEQ ID NO: 26 for 30006020, SEQ ID NO: 28 for 30115796), reverse primers (SEQ ID NO: 27 for 30006020, SEQ ID NO: 29 for 30115796), and P2 primer (SEQ ID NO: 30) were used for PCR.

[0112] <Example 15: Analysis of Triterpenoid Saponin Composition of a Functional Deficiency Mutant of the *Miyakogusa* Glyma.06G324300 Homologous Gene> The whole plants of the Glyma.06G324300 homologous gene function-deficient mutant lines (30006020, 30115796) of Lotus japonicus sown in Example 14 and grown for one month were freeze-dried, and then 10 times the dry weight of 80% methanol was added. The mixture was shaken at room temperature for 1 hour and centrifuged at 15,000 rpm for 5 minutes. The supernatant obtained by centrifugation was purified with a membrane filter with a pore size of 0.45 μm (GL Chromatodisk 4P, GL Sciences), and 2 μL of each extract was subjected to LC-PDA / MS / MS analysis. The apparatus used was Ultimate 3000SD HPLC / LTQ orbitrap discovery MS (both from Thermo Fisher Scientific). The extract was applied to a reverse-phase column (C30, Develosil C30-UG-3, Nomura Chemical), and saponins were eluted with a linear gradient of acetonitrile containing 0.1% (v / v) formic acid (20 - 80% / 60 minutes) at a flow rate of 0.15 ml / min. The eluate was detected by UV absorption and mass spectrometry (parent ion by orbitrap type, fragment ion by ion trap type). The eluate vaporized and positively ionized by electrospray ionization method was injected into the mass spectrometer. For the standard of analysis, soyasaponin Bb (m / z = 943.52) was used, and the annotation of each saponin molecule was performed based on the fragment pattern by MS / MS analysis.

[0113] As a result, in Figures 12-1 and 12-2, as shown in (b) and (c), saponins (Bb, βg, etc.) that are normally accumulated in the mutant homo were below the detection limit, and an abnormality was observed in the saponin composition. From this result, it became clear that the Glyma.06G324300 homologous gene (Lj3g3v1981230) of Lotus japonicus actually functions in vivo in the saponin biosynthesis system.

[0114] <Example 16: Construction of an expression vector for Lotus japonicus> Using the cloning vector containing the Glyma.06G324300 homologous gene derived from Glycyrrhiza glabra created in Example 4 as a template, a forward primer (SEQ ID NO: 31) and a reverse primer (SEQ ID NO: 32) that amplify from the start codon to the stop codon of SEQ ID NO: 4 were used, and PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) was used to perform 30 cycles of PCR at an annealing temperature of 60°C and a reaction temperature of 68°C. The amplified DNA fragment was cloned into pDONR TM 221 by a base sequence-specific recombination reaction (GATEWAY attB × attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies). For the three independent clones obtained, the polynucleotide sequence was determined and confirmed to be identical to SEQ ID NO: 4. The plasmid pDONR-Glyur003152s00037491 having the polynucleotide was obtained as an entry clone.

[0115] Subsequently, using the cloning vector containing the Glyma.06G324300 homologous gene derived from Medicago truncatula created in Example 5 as a template, a forward primer (SEQ ID NO: 33) and a reverse primer (SEQ ID NO: 34) that amplify from the start codon to the stop codon of SEQ ID NO: 6 were used, and PCR was performed in the same manner as above, and cloned into pDONR TM 221. For the three independent clones obtained, the polynucleotide sequence was determined and confirmed to be identical to SEQ ID NO: 6. The plasmid pDONR-Lj3g3v1981230 having the polynucleotide was obtained as an entry clone. Note that pDONR TMSince it is necessary for the sequence-specific recombination reaction (GATEWAY attB × attP reaction) during cloning into 221 (Thermo Fisher Technologies), a 12-base sequence (AAAAAGCAGGCT) shown in SEQ ID NO: 35 is added to the 5'-end of the forward primer, and a 12-base sequence (AGAAAGCTGGGT) shown in SEQ ID NO: 36 is added to the 5'-end of the reverse primer. The plasmid (entry clone) pDONR-Glyma.06g324300 having the polynucleotide shown in SEQ ID NO: 2 or the plasmid (entry clone) pDONR-Glyur003152s00037491 having the polynucleotide shown in SEQ ID NO: 4, prepared in Example 1, the plasmid (entry clone) pDONR-Lj3g3v1981230 having the polynucleotide shown in SEQ ID NO: 6 and the destination vector pG35NGw were mixed, and the DNA fragment shown in SEQ ID NO: 6 was transferred to pCAMBIA-G35NGw by a sequence-specific recombination reaction (GATEWAY attL × attR reaction) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Technologies) to obtain the vector pG35N-LjCSL for transformation of Medicago truncatula containing the Glyma.06G324300 homologous gene derived from Medicago truncatula shown in SEQ ID NO: 6. Also, in the same manner as above, the Glyma.06G324300 gene derived from soybean shown in SEQ ID NO: 2 and the Glyma.06G324300 homologous gene derived from licorice shown in SEQ ID NO: 4 were transferred to obtain the vectors pG35N-GmCSL and pG35N-GuCSL for transformation of Medicago truncatula, respectively.

[0116] <Example 17: Rescue experiment by introducing soybean Glyma.06G324300 and Glyma.06G324300 homologous genes of licorice and Medicago truncatula into Medicago truncatula mutants> The Glyma.06G324300 homologous gene was introduced into a Glyma.06G324300 homologous gene function-deficient mutant of Lotus japonicus according to the method described in Diaz et al., (2005) Induction of hairy roots for symbiotic gene expression studies. In Lotus japonicus Handbook, A.J. Marquez, ed (Dordrecht, The Netherlands: Springer), pp. 261-277. Seeds obtained from the mutant homozygous line of the Glyma.06G324300 homologous gene function-deficient mutant 30006020 of Lotus japonicus obtained in Example 14 were sterilized with hypochlorous acid (containing 0.02% Tween 20) with an available chlorine concentration of 2% for 20 minutes and then allowed to absorb water in sterile distilled water overnight. After removing the seed coats of the water-absorbed seeds, they were sown on a 0.8% water agar medium, covered with aluminum foil to block light, cultured at 25°C for 4 days, and then exposed to light for 1 day. The vector prepared in Example 16 was introduced into Agrobacterium (LBA1334), plated on the front of L medium, and cultured at 28°C for 1 day. Agrobacterium cultured for 1 day was suspended in 10 mL of sterile water, placed in a round sterile petri dish, and then the sprouts of the mutant homozygous of the Glyma.06G324300 homologous gene function-deficient mutant 30006020 of Lotus japonicus were immersed in it, and the hypocotyls were cut with a razor blade. The cut sprouts were arranged on the co-culture medium, covered with aluminum foil to block light, and co-cultured at 21°C for 4 days. After co-culture, the plants were arranged on HRE medium and grown for 2 weeks at 25°C for 16 hours of light period / 23°C for 8 hours of dark period. Plants with hairy roots were confirmed for GFP fluorescence under a fluorescence stereomicroscope.

[0117] <Example 18: Analysis of Triterpenoid Saponin Composition in Lotus japonicus Hairy Roots> The plants from which the hairy roots obtained in Example 17 were generated were transplanted into pots filled with vermiculite, B&D hydroponic solution (Diaz et al., 2005) was added, and they were grown for 1 month. The fully grown plants were freeze-dried and pulverized at 2500 rpm for 30 seconds using a multi-bead shocker (Yasui Kikai Co., Ltd.). 80% methanol in an amount 100 times the weight of the freeze-dried product was added, shaken at room temperature for 1 hour, centrifuged at 15 krpm for 5 minutes, and the supernatant was recovered. The supernatant was analyzed by LC-PDA / MS / MS using the method shown in Example 15. As a result, as shown in Fig. 13, the saponin that had disappeared in the mutant was restored in the transformed hairy roots. From this, it was considered that the Glyma.06G324300 homologous gene catalyzes the saponin synthesis reaction even in vivo.

[0118] <Example 19: Search and Isolation of Glyma.06G324300 Homologous Gene from Astragalus sinicus> From Astragalus sinicus, which is the same leguminous plant as soybean, a Glyma.06G324300 homologous gene was searched as a candidate for the orthologous gene of Glyma.06G324300 by gene homology search. One nucleotide sequence AsCSyGT encoding a protein showing high amino acid identity to Glyma.06G324300 was found from the integrated sequence dataset of RNA sequence data obtained from the roots, stems, and leaves of Astragalus sinicus. Using 1 μg of total RNA obtained from the stems of Astragalus sinicus, first-strand cDNA was synthesized using a SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μL of the first-strand cDNA as a template, oligonucleotide DNAs corresponding to the locations corresponding to the N-terminus and C-terminus of the polypeptide deduced from AsCSyGT were used as the forward primer (SEQ ID NO: 39) and the reverse primer (SEQ ID NO: 40), respectively, and PCR was performed for 30 cycles at an annealing temperature of 55 °C and a reaction temperature of 72 °C using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). Note that pENTR TMSince it is necessary for cloning into the / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), 4 bases (cacc) are artificially added to the 5' end of the forward primer. The amplified DNA fragment was cloned into pENTR TM / D-TOPO entry vector, and the polynucleotide sequences of two independent clones obtained were determined. The nucleotide sequence of the Glyma.06G324300 homologous gene of the radish thus obtained is SEQ ID NO: 41, and the polypeptide sequence deduced therefrom is SEQ ID NO: 42. SEQ ID NO: 42 had 77% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0119] <Example 20: Isolation of Glyma.06G324300 Homologous Gene from Soybean> The Glyma.06G324300 homologous gene was searched as a paralog gene candidate of Glyma.06G324300 by gene homology search from soybean. Using the BLAST homology search function in Soybase (https: / / soybase.org), which is a genomic information database of soybean, two base sequences, Glyma.04g255400 and Glyma.11g151800, encoding proteins showing high amino acid identity with Glyma.06G324300 were found. The two Glyma.06G324300 homologous genes, Glyma.04g255400 and Glyma.11g151800, were amplified by the method shown in Example 1 and cloned into pDONR TM 221 (Thermo Fisher Technologies). Oligo DNAs corresponding to the N-terminal and C-terminal portions of the polypeptide deduced from Glyma.04g255400 were used as the forward primer (SEQ ID NO: 43) and the reverse primer (SEQ ID NO: 44), respectively, and oligo DNAs corresponding to the N-terminal and C-terminal portions of the polypeptide deduced from Glyma.11g151800 were used as the forward primer (SEQ ID NO: 45) and the reverse primer (SEQ ID NO: 46), respectively. Note that pDONR TMSince it is necessary for the sequence-specific recombination reaction (GATEWAY attB × attP reaction) during cloning into 221 (Thermo Fisher Technologies), 12 bases (AAAAAGCAGGCT) are artificially added to the 5'-end of the forward primer, and 12 bases (AGAAAGCTGGGT) are artificially added to the 5'-end of the reverse primer. A DNA fragment amplified from the first-strand cDNA derived from seeds was cloned into pDONR by a sequence-specific recombination reaction (GATEWAY attB × attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies). TM Cloning into 221 was performed, and the polynucleotide sequences were determined for three independent clones obtained for each. The sequences thus obtained are SEQ ID NO: 47 and SEQ ID NO: 49, and the polypeptide sequences deduced therefrom are SEQ ID NO: 48 and SEQ ID NO: 50. SEQ ID NO: 48 and SEQ ID NO: 50 had 93.9% and 71.1% identity, respectively, to the amino acid sequence shown in SEQ ID NO: 1.

[0120] <Example 21: Introduction of Glycine max Glyma.06G324300 homologous genes of lotus and soybean into yeast strains producing glycyrrhetinic acid and soyasapogenol B> Using the method shown in Example 7, the yeast expression clones of the Glycine max Glyma.06G324300 homologous genes of lotus obtained in Example 19 and soybean obtained in Example 20, pESC-HIS-AsCSyGT, pESC-HIS-AtUGD2-Glyma04g255400, pESC-HIS-AtUGD2-Glyma.11g151800 were constructed, and using the method shown in Example 8, they were introduced into yeast strains producing glycyrrhetinic acid and soyasapogenol B, respectively.

[0121] <Example 22: In vivo enzyme assay using recombinant yeast into which Glycine max Glyma.06G324300 homologous genes of lotus and soybean have been introduced> By the method shown in Example 9, recombinant yeast was cultured and metabolites were extracted. As a result, metabolite extracts (Sample Q) from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (AsCSyGT) represented by SEQ ID NO: 42, metabolite extracts (Sample R) from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (Glyma04g255400) represented by SEQ ID NO: 48, and metabolite extracts (Sample S) from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (Glyma.11g151800) represented by SEQ ID NO: 50 were obtained. The same culturing and metabolite extraction were also performed for the soyasapogenol B-producing yeast strains carrying pESC-HIS-AsCSyGT, pESC-HIS-AtUGD2-Glyma04g255400, and pESC-HIS-AtUGD2-Glyma.11g151800. As a result, metabolite extracts (Sample T) from a soyasapogenol B-producing yeast strain expressing the polypeptide (AsCSyGT) represented by SEQ ID NO: 42, metabolite extracts (Sample U) from a soyasapogenol B-producing yeast strain expressing the polypeptide (Glyma04g255400) represented by SEQ ID NO: 48, and metabolite extracts (Sample V) from a soyasapogenol B-producing yeast strain expressing the polypeptide (Glyma.11g151800) represented by SEQ ID NO: 50 were obtained.

[0122] <Example 23: Analysis of Metabolite Extracts of Yeast into Which the Glyma.06G324300 Homologous Genes of Lotus and Soybean Were Introduced> Samples for LC-MS analysis were prepared and analyzed by the method shown in Example 10. The results are shown in FIGS. 14 and 15. From sample Q of (a) shown in FIG. 14, one peak corresponding to glycyrrhetinic acid monoglucuronide was detected (black arrow). The retention time and mass spectrum of that peak were in good agreement with those of glycyrrhetinic acid monoglucuronide. Similarly, peaks corresponding to glycyrrhetinic acid monoglucuronide were also detected from sample R of (b) and sample S of (c) (black arrows). The retention time and mass spectrum of each peak were in good agreement with those of glycyrrhetinic acid monoglucuronide. From sample T in (a) shown in Fig. 15, one peak corresponding to soyasapogenol B monoglucuronide was detected (black arrow). Peaks corresponding to soyasapogenol B monoglucuronide were also detected from sample U in (b) and sample V in (c) (black arrows). The retention times and mass spectra of each peak were in good agreement with those of soyasapogenol B monoglucuronide.

[0123] From the above results, it was revealed that the renge-derived AsCSyGT obtained in Example 19 and Glyma04g255400 and Glyma.11g151800 derived from soybean obtained in Example 20 have glucuronic acid first transfer activity to convert glycyrrhetinic acid to glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the 3-position hydroxy group of glycyrrhetinic acid. In addition, it was demonstrated that they also have glucuronic acid first transfer activity to convert soyasapogenol B to soyasapogenol B monoglucuronide by transferring glucuronic acid to the 3-position hydroxy group of soyasapogenol B.

[0124] <Example 24: Preparation of Transformed Yeast for Substrate Feeding Assay of Glyma.06G324300 Homologous Gene of Soybean> The pESC-HIS-AtUGD2-Glyma04g255400 and pESC-HIS-AtUGD2-Glyma.11g151800 obtained in Example 21 were each introduced into the yeast INVScI strain. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

[0125] <Example 25: Substrate Feeding Assay Using Transformed Yeast into Which Glyma.06G324300 Homologous Gene of Soybean was Introduced> The recombinant yeast obtained in Example 24 was cultured by the method shown in Example 12. The suspension of each obtained yeast cell was divided equally, and ursolic acid, an oleanane-type triterpenoid, or betulinic acid, a lupane-type triterpenoid, each having a final concentration of 5 μM, was added thereto. Thereafter, culture and metabolite extraction were performed again by the method shown in Example 12. As a result, a feeding assay extract obtained by adding ursolic acid, an oleanane-type triterpenoid, to the transformed yeast expressing the polypeptide represented by SEQ ID NO: 48 (Glyma04g255400), or the polypeptide represented by SEQ ID NO: 50 (Glyma.11g151800), and a feeding assay extract obtained by adding betulinic acid, a lupane-type triterpenoid, were obtained.

[0126] <Example 26: Analysis of Substrate Feeding Assay Extract Using Transformed Yeast into Which Glyma.06G324300 Homologous Gene of Soybean was Introduced> The sample obtained in Example 25 was analyzed under the same conditions as in Example 10. MS was performed in the SIM mode, and m / z of each assumed reaction product, ursolic acid monoglucuronide = 631 (FIG. 16, a), betulinic acid monoglucuronide = 631 (FIG. 17, a), were used as parameters for analysis. FIG. 16 shows the analysis results of a feeding assay extract (sample W) obtained by adding ursolic acid to the transformed yeast expressing the polypeptide represented by SEQ ID NO: 50 (Glyma.11g151800). A peak presumed to be ursolic acid monoglucuronide was detected from sample W shown in (b). On the other hand, in sample X as a negative control shown in (c), a peak presumed to be ursolic acid monoglucuronide was not detected.

[0127] FIG. 17 shows the analysis results of a feeding assay extract (sample Y) obtained by adding betulinic acid to the transformed yeast expressing the polypeptide represented by SEQ ID NO: 50 (Glyma.11g151800). A peak presumed to be betulinic acid monoglucuronide was detected from sample Y shown in (b). On the other hand, in sample Z as a negative control shown in (c), a peak presumed to be betulinic acid monoglucuronide was not detected.

[0128] From the above, the polypeptide (Glyma.11g151800) represented by SEQ ID NO: 50 is considered to be a glucuronic acid O-transferase that can transfer glucuronic acid not only to the 3-hydroxy group of oleanane-type triterpenoids but also to the 3-hydroxy group of ursane-type triterpenoids such as ursolic acid and β-boswellic acid, and lupane-type triterpenoids such as betulinic acid.

[0129] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A glucuronyltransferase comprising a polypeptide having an activity of transferring glucuronic acid to the 3-position hydroxy group in an oleanane-type triterpenoid, wherein the polypeptide comprises a polypeptide containing any one of the amino acid sequences represented by the following (a) or (b) or a fragment thereof having the above activity, the glucuronyltransferase: (a) an amino acid sequence represented by any one of SEQ ID NOs: 3 and 5, or (b) an amino acid sequence having 90% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 3 and 5.

2. The glucuronyltransferase according to claim 1, wherein the oleanane-type triterpenoid is selected from the group consisting of β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxoglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, medicagenic acid, soyasapogenol B, soyasapogenol A, hederagenin, camelliagenin, and psychogenic acid.

3. The glucuronyltransferase according to claim 1 or 2, which is derived from a plant of the family Fabaceae.

4. A recombinant for glycyrrhizin production that can biosynthesize β-amyrin and contains all of the following expression vectors (1) to (4): (1) A CYP88D6 expression vector having an activity of oxidizing the 11-position in an oleanane-type triterpenoid and comprising a polypeptide containing any one of the amino acid sequences represented by the following (a) or (b), (a) an amino acid sequence represented by SEQ ID NO: 7, or (b) an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 7, (2) A CYP72A154 expression vector having an activity of oxidizing the 30-position in an oleanane-type triterpenoid and comprising a polypeptide containing any one of the amino acid sequences represented by the following (c) or (d), (c) an amino acid sequence represented by any one of SEQ ID NOs: 9, 11, and 13, or (d) an amino acid sequence having 90% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 9, 11, and 13, (3) A UGT73P12 expression vector having an activity of transferring glucuronic acid to the 2-position hydroxy group of glucuronic acid in an oleanane-type triterpenoid monoglucuronide and comprising a polypeptide containing any one of the amino acid sequences represented by the following (e) or (f), (e) the amino acid sequence represented by SEQ ID NO: 15, or (f) an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 15, and (4) a CSyGT expression vector comprising a polypeptide having an activity of transferring glucuronic acid to the hydroxy group at the 3-position in an oleanane-type triterpenoid and comprising any one of the amino acid sequences represented by the following (g) or (h): (g) the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5, or (h) an amino acid sequence having 90% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, and 5. **Claim 5** The recombinant according to claim 4, wherein the host is a leguminous plant. **Claim 6** A method for producing glycyrrhizin from β-amyrin, the production method comprising the step of culturing the recombinant according to claim 4 or 5.

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