Glucuronyl transferase, gene encoding the enzyme and method of using the same

Abstract

The invention relates to the identification of a glucuronyl transferase gene that catalyzes the transfer of glucuronic acid to the hydroxyl group at position 3 of an oleanane-type triterpenoid compound. The invention also relates to the provision of a glucuronyl transferase gene having a desired activity and derived from plants of the family Leguminosae (soybean, licorice, and lotus root), and comprising the base sequences shown in SEQ ID NOs. 2, 4, and 6, respectively.

Description

Technical Field

[0001] The present invention relates to an enzyme for transferring glucuronic acid from the 3-hydroxyl group in an oleanane-type triterpenoid compound, a gene encoding the enzyme, and a method for producing glycyrrhizin. Background Art

[0002] Licorice (Glycyrrhiza uralensis) is a perennial herbaceous plant of the Leguminosae family. The root and stolon (stolon: underground stem) of the plant are known as "licorice", an important herbal medicine in traditional Chinese medicine, and are widely used in the world. The main active ingredient of licorice is glycyrrhizin, which is a saponin of oleanane-type triterpenoid compound (Non-Patent Document 1). Glycyrrhizin has been studied from various aspects such as its herbal medicine, pharmacological usefulness, and breeding research.

[0003] In order to stably and continuously provide high-quality glycyrrhizin as a drug through a biological production system, it is necessary to use genes involved in the biosynthesis system of glycyrrhizin and the expression levels of the genes as markers, and to breed glycyrrhizin high-producing plants by establishing the optimal production conditions, screening glycyrrhizin high-producing strains, or introducing synthase genes. Therefore, the identification of the gene group involved in the biosynthesis system of glycyrrhizin is indispensable.

[0004] Glycyrrhizin is present in plants and is biosynthesized through two-stage oxidation and two-stage glycosylation reactions using β-amyrin, which belongs to the oleanane-type triterpenoid compound, as a starting material. β-amyrin is known to be a precursor substance that serves as a biosynthetic branch point for glycyrrhizin and soyasaponin I in the triterpenoid compound saponin biosynthesis system ( Figure 1 ).

[0005] like Figure 2As shown, regarding the synthase for synthesizing glycyrrhizin from β-amyrin, it is known so far that two oxidases CYP88D6 (patent document 1) and CYP72A154 (patent document 2) catalyze the above-mentioned two-stage oxidation reaction for the biosynthesis of glycyrrhetinic acid as the aglycone (non-sugar part) of glycyrrhizin from β-amyrin, and the glycosyltransferase UGT73P12 (patent document 3) catalyzing the second stage in the synthase gene for biosynthesizing glycyrrhizin by catalyzing the above-mentioned two-stage glycosylation reaction of the obtained glycyrrhetinic acid. However, although a large number of technicians in this field have tried to separate it many times, it has not been obtained at all so far. Therefore, it has become a bottleneck in the synthesis of glycyrrhizin from β-amyrin using a biological production system and an in vitro (invitro) synthesis system, and a sufficient amount of glycyrrhizin cannot be obtained stably and continuously.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 5526323

[0009] Patent Document 2: Japanese Patent No. 5771846

[0010] Patent Document 3: Japanese Patent No. 6344774

[0011] Non-patent literature

[0012] Non-patent document 1: Gibson, MR, 1978, Lloydia-the journal of Natural Products, 41(4): 348-354 Summary of the invention

[0013] Problems to be solved by the invention

[0014] In order to solve the above-mentioned problems, the present invention isolates the gene of glucuronic acid transferase 1 that catalyzes the transfer of glucuronic acid to the 3-hydroxyl group of oleanane-type triterpenoid compounds represented by glycyrrhetinic acid, and uses the gene expression system to produce and provide a biological expression system capable of biosynthesizing glycyrrhizin in large quantities from β-amyrin in an individual or cell.

[0015] Means for solving problems

[0016] In order to identify the last remaining glucuronyl transferase I in the biosynthetic pathway from β-amyrin to glycyrrhizin, many people skilled in the art have tried to separate it from licorice, but have not been able to identify it along the entire length. Therefore, the present inventors considered that there were some obstacles in the separation of glucuronyl transferase I from Glycyrrhiza plants, and tried a strategy to separate glucuronyl transferase I from plants other than Glycyrrhiza plants. In the leguminous plants described in Glycyrrhiza plants, there is generally a pathway for biosynthesizing soyasaponin I from β-amyrin via soyasaponin B as an intermediate product ( Figure 1 ). Soysaponin B, which is an oleanane-type triterpenoid compound, has a hydroxyl group at the 3-position, but soysaponin I, which is the final product, has glucuronic acid bound to the 3-position. This is the same as the case where glycyrrhetinic acid, which is also an oleanane-type triterpenoid compound as an intermediate product, has a hydroxyl group at the 3-position, while glucuronic acid, which is the final product, has glucuronic acid bound to the 3-position. That is, the glucuronic acid transferase 1 that functions in the biosynthetic pathway from β-amyrin to glycyrrhizin may also function in the biosynthetic pathway from β-amyrin to soysaponin I. Based on this hypothesis, the inventors tried to identify genes that can have glucuronic acid transfer activity 1 from soybeans having a biosynthetic pathway from β-amyrin to soysaponin I, and as a result, they successfully isolated a cellulose synthase-like gene whose specific function was unknown. The sugar transfer activity of the enzyme was verified using soysaponin B as a sugar acceptor substrate, and the results showed that glucuronic acid was transferred to the hydroxy sugar at the 3-position of soysaponin B. The enzyme activity is the same even when the sugar acceptor substrate is glycyrrhizic acid. Thus, the inventors succeeded in identifying the previously unisolated first glucuronyl transferase by changing the source plant from a Glycyrrhiza plant to soybean. The present invention is based on the results of this study and provides the following (1) to (15).

[0017] (1) A polypeptide or a fragment thereof comprising any one of the amino acid sequences shown in (a) to (c) below, wherein the polypeptide or the fragment thereof has an activity of transferring glucuronic acid to the 3-hydroxyl group of an oleanane-type triterpenoid compound,

[0018] (a) the amino acid sequence represented by any one of SEQ ID NOs: 1, 3 and 5,

[0019] (b) an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of any one of SEQ ID NOs: 1, 3 and 5, or

[0020] (c) an amino acid sequence having 80% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 3 and 5.

[0021] (2) According to the polypeptide described in (1), the oleanane-type triterpenoid compound is selected from β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxyglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, alfalfa acid, soybean saponin B, soybean saponin A, ivy sapogenin, camellia sapogenin and bupleurogenin.

[0022] (3) The polypeptide according to (1) or (2), which is derived from a plant of the family Fabaceae.

[0023] (4) A polynucleotide encoding the polypeptide according to any one of (1) to (3).

[0024] (5) The polynucleotide according to (4), comprising any one of the base sequences shown in (a) to (d) below.

[0025] (a) the base sequence represented by any one of SEQ ID NOs: 2, 4 and 6,

[0026] (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 NOs: 2, 4 and 6,

[0027] (c) a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 2, 4 and 6, or

[0028] (d) a base sequence that hybridizes under highly stringent conditions to a complementary base sequence of the base sequence represented by any one of SEQ ID NOs: 2, 4, and 6

[0029] (6) A CSyGT expression vector comprising the polynucleotide described in (4) or (5).

[0030] (7) A transformant comprising the polynucleotide described in (4) or (5) or the CSyGT expression vector described in (6), or a progeny thereof, wherein the progeny retains the polynucleotide or the CSyGT expression vector.

[0031] (8) The transformant or progeny thereof according to (7), wherein the host is a plant of the family Fabaceae.

[0032] (9) The transformant or progeny thereof according to (7), wherein the host is yeast.

[0033] (10) The polypeptide according to any one of (1) to (3) obtained from the transformant according to (9) or its progeny, to which a yeast-derived sugar chain is added.

[0034] (11) The polypeptide according to (10), wherein the yeast-derived sugar chain is a high mannose sugar chain.

[0035] (12) A method for producing a polypeptide having the activity of transferring glucuronic acid to the 2-hydroxyl group of glucuronic acid in an oleanane-type triterpenoid compound, the method comprising: a step of culturing the transformant described in (7) or (8) or its progeny, and a step of extracting the polypeptide described in any one of (1) to (3) from the culture.

[0036] (13) A recombinant gene for producing glycyrrhizin, which is capable of biosynthesizing β-amyrin and comprises all of the expression vectors shown in (A) to (D) below.

[0037] (A) a CYP88D6 expression vector having an activity of oxidizing position 11 of an oleanane-type triterpenoid compound and comprising a polypeptide having any one of the amino acid sequences shown in (a) to (c) below,

[0038] (a) the amino acid sequence shown in SEQ ID NO: 7,

[0039] (b) an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of SEQ ID NO: 7, or

[0040] (c) an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 7,

[0041] (B) a CYP72A154 expression vector having an activity of oxidizing position 30 of an oleanane-type triterpenoid compound and comprising a polypeptide having any one of the amino acid sequences shown in (d) to (f) below,

[0042] (d) the amino acid sequence represented by any one of SEQ ID NOs: 9, 11 and 13,

[0043] (e) an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of any one of SEQ ID NOs: 9, 11 and 13, or

[0044] (f) an amino acid sequence having 80% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 9, 11 and 13,

[0045] (C) a UGT73P12 expression vector having an activity of transferring glucuronic acid from the 2-hydroxyl group of glucuronic acid in the monoglucuronide of an oleanane-type triterpenoid compound and comprising a polypeptide having any of the amino acid sequences shown in (g) to (i) below,

[0046] (g) the amino acid sequence represented by SEQ ID NO: 15,

[0047] (h) an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of SEQ ID NO: 15, or

[0048] (i) an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 15, and

[0049] (D) CSyGT expression vector described in (6)

[0050] (14) The genetically modified organism according to (13), wherein the host is a plant of the Leguminosae family.

[0051] (15) A method for producing glycyrrhizin from β-amyrin, the method comprising the step of culturing the recombinant gene according to (13) or (14).

[0052] This specification includes the disclosure of Japanese Patent Application No. 2019-190060 which is the basis of priority of the present application.

[0053] Effects of the Invention

[0054] According to the present invention, there can be provided a polypeptide having an activity of transferring glucuronic acid to the 3-hydroxyl group of an oleanane-type triterpenoid compound, a polynucleotide encoding the polypeptide, or a method of utilizing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The biosynthetic pathways of glycyrrhizin and soybean saponin I in the triterpenoid saponin biosynthesis system are shown.

[0056] Figure 2 The present invention shows the catalytic position of each oleanane-type triterpenoid compound produced as an intermediate in the biosynthetic pathway from β-amyrin to glycyrrhizin, the known enzymes (CYP88D6, CYP72A154, and UGT73P12) involved in each conversion reaction, and the first glucuronyl transferase CSyGT newly identified in the present specification.

[0057] Figure 3The diagram is a conceptual diagram showing triterpenoid compound-producing yeast strains used in the functional analysis of soybean Glyma.06G324300, and Glyma.06G324300 homologous proteins derived from licorice and Japanese lotus in the examples. The yeast strain (a) can produce glycyrrhetinic acid from 2,3-epoxysqualene in yeast cells by simultaneously expressing the β-amyrin synthase gene (from Japanese lotus), CYP88D6 (derived from licorice), and CYP72A63 (derived from Medicago truncatula). The yeast strain (b) can produce soybean saponin B from 2,3-epoxysqualene in yeast cells by simultaneously expressing the β-amyrin synthase gene (from Japanese lotus), CYP93E3 (derived from licorice), and CYP72A566 (derived from licorice).

[0058] Figure 4 : This is a diagram showing the results of metabolite analysis of glycyrrhetinic acid-producing yeast into which Glyma.06G324300 and homologous genes were introduced. Sample A in (a) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing soybean-derived Glyma.06G324300 in glycyrrhetinic acid-producing yeast, sample B in (b) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing licorice-derived Glyur003152s00037491 in glycyrrhetinic acid-producing yeast, and sample C in (c) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing Lj3g3v1981230 derived from Japanese lotus in glycyrrhetinic acid-producing yeast. The peak of the black arrow represents glycyrrhetinic acid monoglucuronide. In addition, sample D in (d) is the detection result of glycyrrhetinic acid-producing yeast into which an empty vector as a negative control was introduced.

[0059] Figure 5 The graphs show the results of metabolite analysis of soybean saponin B-producing yeast into which Glyma.06G324300 and homologous genes were introduced. Sample E in (a) is the detection result of soybean saponin B monoglucuronide produced by expressing soybean-derived Glyma.06G324300 in soybean saponin B-producing yeast, sample F in (b) is the detection result of soybean saponin B monoglucuronide produced by expressing licorice-derived Glyur003152s00037491 in soybean saponin B-producing yeast, and sample G in (c) is the detection result of soybean saponin B monoglucuronide produced by expressing Lj3g3v1981230 derived from Lotus japonicus in soybean saponin B-producing yeast. The peaks indicated by black arrows represent soybean saponin B monoglucuronide. Sample H in (d) is the detection result of soybean saponin B-producing yeast into which an empty vector as a negative control was introduced.

[0060] Figure 6This is a diagram showing the outline of a substrate feeding experiment. In order to supply UDP-glucuronic acid as a sugar donor substrate in cells, UDP-glucose dehydrogenase (AtUGD) of Arabidopsis thaliana and Glyma.06G324300 and its homologous genes were simultaneously expressed in yeast. (a) is a diagram showing the conversion reaction to monoglucuronide when glycyrrhetinic acid is added to yeast culture medium, and (b) is a diagram showing the conversion reaction to monoglucuronide when soybean saponin B is added to yeast culture medium.

[0061] Figure 7 The diagram shows the metabolite analysis results of soybean-derived Glyma.06G324300 introduced into yeast fed with glycyrrhetinic acid. (a) is a conceptual diagram of a hypothetical conversion reaction to glycyrrhetinic acid monoglucuronide when using Glyma.06G324300. (b) shows the detection results of glycyrrhetinic acid before conversion. The peak of the white arrow represents glycyrrhetinic acid. (c) shows the detection results of glycyrrhetinic acid monoglucuronide generated by Glyma.06G324300. The peak of the black arrow represents glycyrrhetinic acid monoglucuronide.

[0062] Figure 8 The figures show the metabolite analysis results of licorice-derived Glyur003152s00037491 introduced into yeast fed with glycyrrhetinic acid. (a) is a conceptual diagram of a hypothetical conversion reaction to glycyrrhetinic acid monoglucuronide when using Glyur003152s00037491. (b) shows the detection results of glycyrrhetinic acid before conversion. The peak of the white arrow represents glycyrrhetinic acid. (c) shows the detection results of glycyrrhetinic acid monoglucuronide generated by Glyur003152s00037491. The peak of the black arrow represents glycyrrhetinic acid monoglucuronide.

[0063] Fig. 9 The diagrams show the results of metabolite analysis of Lj3g3v1981230 derived from Japanese Lotus japonicus fed with glycyrrhizic acid. (a) is a conceptual diagram of a hypothetical conversion reaction to glycyrrhizic acid monoglucuronide when using Lj3g3v1981230. (b) shows the detection results of glycyrrhizic acid before conversion. The peak of the white arrow represents glycyrrhizic acid. (c) shows the detection results of glycyrrhizic acid monoglucuronide generated by Lj3g3v1981230. The peak of the black arrow represents glycyrrhizic acid monoglucuronide.

[0064] Fig.10 As Figures 7 to 9Figures showing the results of metabolite analysis of yeast fed with an empty vector and introduced with glycyrrhetinic acid as a negative control. (a) is a conceptual diagram of the hypothetical conversion reaction to glycyrrhetinic acid monoglucuronide when an empty vector is used. Since there is no enzyme activity, the hypothetical glycyrrhetinic acid monoglucuronide is not generated. (b) shows the detection results of glycyrrhetinic acid before the reaction. The peak of the white arrow represents glycyrrhetinic acid. (c) shows the detection results of glycyrrhetinic acid monoglucuronide after the reaction. The peak of the black arrow represents glycyrrhetinic acid monoglucuronide. The dotted arrow indicates the position of the peak when glycyrrhetinic acid monoglucuronide is generated.

[0065] Fig.11 The diagrams show the results of metabolite analysis of Glyma.06G324300 and its homologous gene introduced into yeast fed with soybean saponin B. (a) is a conceptual diagram of a hypothetical conversion reaction from soybean saponin B to soybean saponin B monoglucuronide. (b) shows the detection results of soybean saponin B monoglucuronide produced by soybean-derived Glyma.06G324300. The peak with a black arrow represents soybean saponin B monoglucuronide. (c) shows the detection results of soybean saponin B monoglucuronide produced by Glyur003152s00037491 derived from licorice. The peak with a black arrow represents soybean saponin B monoglucuronide. (d) shows the detection results of soybean saponin B monoglucuronide produced by Lj3g3v1981230 derived from Lotus japonicus. The peak with a black arrow represents soybean saponin B monoglucuronide. (e) is a negative control for the above (b) to (d).

[0066] Figure 12-1 The figures show the results of LC-PDA / MS / MS analysis of extracts of Glyma.06G324300 homologous gene functional defective mutants of Lotus japonicus. (a) shows the base peak ion chromatogram of the plant extract derived from the wild type (Gifu), and (b) and (c) show the base peak ion chromatograms of the plant extracts derived from the transposon insertion homozygous mutants (30006020 and 30115796, respectively).

[0067] Figure 12-2 The figures show the results of LC-PDA / MS / MS analysis of extracts of Glyma.06G324300 homologous gene functional defective mutants of Lotus japonicus. (a) shows the total ion chromatogram of the plant extract derived from the wild type (Gifu), and (b) and (c) show the total ion chromatograms of the plant extracts derived from the transposon insertion homozygous mutants (30006020 and 30115796, respectively).

[0068] Fig.13The figures show the results of LC-PDA / MS / MS analysis of hairy roots of mutants into which a Glyma.06G324300 homologous gene was introduced. (a) shows the total ion chromatogram of hairy root extracts of a strain into which an empty vector (pG35N_empty) was introduced into the wild type (Gifu), and (b) to (e) show the total ion chromatograms of hairy root extracts derived from transformants into which pG35N_empty, expression vectors pG35N-GmCSL, pG35N-GuCSL, and pG35N-LjCSL were introduced into the homozygous Glyma.06G324300 homologous gene functional defective mutant (30115796), respectively.

[0069] Fig.14 The diagram shows the results of metabolite analysis in glycyrrhizic acid-producing yeast into which the Chinese asparagus Glyma.06G324300 interspecies homologous gene and the soybean Glyma.06G324300 intraspecies homologous gene were introduced. Sample Q of (a) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing the AsCSyGT gene, which is an interspecific homologous gene of Glyma.06G324300 derived from Astragalus, in glycyrrhetinic acid-producing yeast, sample R of (b) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing the Glyma04g255400 gene, which is an intraspecific homologous gene of Glyma.06G324300 derived from soybean, in glycyrrhetinic acid-producing yeast, and sample S of (c) is the detection result of glycyrrhetinic acid monoglucuronide produced by expressing the Glyma.11g151800 gene, which is an intraspecific homologous gene of Glyma.06G324300 derived from soybean, in glycyrrhetinic acid-producing yeast. The peak indicated by the black arrow represents glycyrrhetinic acid monoglucuronide.

[0070] Fig.15 The diagram shows the results of metabolite analysis in soybean saponin B acid-producing yeast into which the interspecific homologous gene of Glyma.06G324300 of Astragalus sinensis and the intraspecific homologous gene of Glyma.06G324300 of soybean were introduced. Sample T of (a) is the detection result of soybean saponin B monoglucuronide produced by expressing the AsCSyGT gene derived from Astragalus sinensis in soybean saponin B-producing yeast, sample U of (b) is the detection result of soybean saponin B monoglucuronide produced by expressing the Glyma04g255400 gene derived from soybean in soybean saponin B-producing yeast, and sample V of (c) is the detection result of soybean saponin B monoglucuronide produced by expressing the Glyma.11g151800 gene derived from soybean in soybean saponin B-producing yeast. The peak indicated by the black arrow indicates soybean saponin B monoglucuronide.

[0071] Fig.16The diagrams show the results of metabolite analysis of soybean-derived Glyma.11g151800 gene introduced into yeast fed with ursolic acid. (a) is a conceptual diagram of a hypothetical conversion reaction to ursolic acid monoglucuronide when a test extract (sample W) is fed with Glyma.11g151800. (b) shows the detection results of ursolic acid monoglucuronide generated by Glyma.11g151800 when sample W is added. The peak of the black arrow represents ursolic acid monoglucuronide. (c) shows the detection results of ursolic acid monoglucuronide when sample X as a negative control is added.

[0072] Fig.17 The diagrams show the results of metabolite analysis of soybean-derived Glyma.11g151800 gene introduced into yeast fed with betulinic acid. (a) is a conceptual diagram of a hypothetical conversion reaction to betulinic acid monoglucuronide when a test extract (sample Y) is fed with Glyma.11g151800. (b) shows the detection results of betulinic acid monoglucuronide generated by Glyma.11g151800 when sample Y is added. The peak indicated by the black arrow represents betulinic acid monoglucuronide. (c) shows the detection results of betulinic acid monoglucuronide when sample Z as a negative control is added. DETAILED DESCRIPTION

[0073] 1. Glucuronyl transferase 1 (CSyGT)

[0074] 1-1. Overview

[0075] The first embodiment of the present invention relates to a glucuronyl transferase 1 and a fragment thereof having a glucuronyl transfer activity 1, and nucleic acids encoding them. The glucuronyl transferase 1 of the present invention has the activity of catalyzing the glycosylation reaction of glucuronic acid on the hydroxyl group at the 3rd position of glycyrrhizic acid obtained by β-amyrin through a two-stage oxidation reaction in the synthetic pathway unique to the genus Glycyrrhiza from β-amyrin that can be biosynthesized by a large number of plants. By the glucuronyl transferase 1 of the present invention, not only can glucuronic acid be transferred to the hydroxyl sugar at the 3rd position of oleanane-type triterpenoid compounds represented by glycyrrhizic acid, but also by combining with known enzymes involved in the biosynthetic pathway from β-amyrin to glycyrrhizic acid, a biological production system from β-amyrin to glycyrrhizic acid can be produced with plants other than the genus Glycyrrhiza as hosts. Thus, high-quality glycyrrhizic acid can be provided stably and continuously.

[0076] 1-2. Definition of terms

[0077] The following terms frequently used in this specification are defined.

[0078] In this specification, the so-called "glucuronyl transferase 1 (CSyGT)" (often expressed as "CSyGT" in this specification) refers to an enzyme that catalyzes a transsugar reaction of transferring one glucuronic acid, which is a type of sugar, to the hydroxyl group of an oleanane-type triterpenoid compound having a hydroxyl group at the carbon at the 3-position. The "1st" here means that it has the initial transsugar activity in the two-stage transsugar reaction in the hydroxyl group at the 3-position of the oleanane-type triterpenoid compound. The specific structure of the glucuronyl transferase 1 will be described later.

[0079] In the present specification, "a fragment thereof having a glucuronyl transferase I activity" refers to an active fragment of the above-mentioned glucuronyl transferase I.

[0080] In this specification, the so-called "glucuronic acid first transfer activity" refers to the activity of CSyGT in catalyzing the sugar transfer reaction, that is, the activity of transferring glucuronic acid to the hydroxyl group at the 3rd position of oleanane-type triterpenoids. Through this activity, oleanane-type triterpenoids generate oleanane-type triterpenoid monoglucuronide. In addition, in this specification, CSyGT and its fragments having glucuronic acid first transfer activity are often described as "polypeptides having glucuronic acid first transfer activity" or "glucuronic acid first transferase, etc. (CSyGT, etc.)". In addition, in this specification, glucuronic acid first transferase, etc. can be glycoproteins to which different sugar chains are added. For example, glucuronic acid first transferase, etc. to which sugar chains derived from plants are added, and glucuronic acid first transferase, etc. to which sugar chains derived from yeast are added are all included in the glucuronic acid first transferase, etc. in this specification.

[0081] The so-called "oleanane triterpenoids" refers to a C30 isoprenoid having a pentacrylic oleanane skeleton and containing 6 isoprene units. In the present invention, it is equivalent to the non-sugar part (aglycone) of glycyrrhizin, which is the final target. The oleanane triterpenoids described in this specification refer to oleanane triterpenoids having a hydroxyl group (OH group) at the carbon at position 3, unless otherwise specified. Specific examples of oleanane triterpenoids are not limited, but include β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxyglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, alfalfa acid, soybean saponin B, soybean saponin A, hederagenine, camellia sapogenin, and bupleurogenin. All of these can be substrates for the glucuronyl transferase I of the present invention. 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-deoxy ... Glycyrrhetinic acid-3-O-monoglucuronide, glycyrrhetinic acid-3-O-monoglucuronide, oleanolic acid-3-O-monoglucuronide, alfalfa acid-3-O-monoglucuronide, soyasapogenin B-3-O-monoglucuronide, soyasapogenin A-3-O-monoglucuronide, hederagenin-3-O-monoglucuronide, camelliasapogenin-3-O-monoglucuronide, and saikosapogenin-3-O-monoglucuronide.

[0082] In this specification, the Fabaceae plant is not limited to the Glycyrrhiza plant, and includes all plant species belonging to the Fabaceae in plant taxonomy. For example, the Arachis plant, the Cicer plant, the Aspalathus plant, the Dalbergia plant, the Pterocarpus plant, the Desmodium plant, the Lespedeza plant, the Uraria plant, the Galegeae plant, the Astragalus plant, the Glycyrrhiza plant, the Echinops plant, the Oxytropis, Augyrocytisus, Cytisus, Genista, Spartium, Hedysarum, Cyamopsis, Indigofera, Lotus, Lupinus, Wisteria, Cajanus, Canavalia, Erythrina, Glycine, Hardenbergia, Lablab, Mucuna, Phaseolus, Psophocarpus, Pueraria, Vigna, Robinia, Castanea Castanospermum, Maackia, Ormosia, Sophora, Styphnolobium, Medicago, Trigonella, Trifolium, Lathyrus, Lens, Pisum and Vicia. Licorice plants to which Glycyrrhiza uralensis belongs and Medicago, which is a relative thereof, have a biosynthetic pathway for glycyrrhetinic acid, which is a substrate for CSyGT, in the biosynthesis of glycyrrhizin. Therefore, the leguminous plants of the present invention are preferred.Specific examples of Glycyrrhiza plants include G. glabra (Glycyrrhiza glabra), G. inflata (Glycyrrhiza uralensis), G. aspera (Glycyrrhiza uralensis), G. eurycarpa (Glycyrrhiza eurycarpa), G. pallidiflora (Glycyrrhiza uralensis), G. yunnanensis (Glycyrrhiza yunnanensis), G. lepidota (Glycyrrhiza uralensis), G. echinata (Glycyrrhiza uralensis), and G. acanthocarpa (Glycyrrhiza uralensis). Specific examples of Medicago plants include M. truncatula (Medicago truncatula).

[0083] 1-3. Composition

[0084] The first glucuronyl transferase (CSyGT) of the present invention is a polypeptide consisting of an amino acid sequence shown in any one of sequence numbers 1, 3 and 5. These polypeptides correspond to wild-type CSyGT (GmCSyGT) derived from soybean (Glycine max), wild-type CSyGT (GuCSyGT) derived from licorice (G. uralensis; Ural licorice), and wild-type CSyGT (LjCSyGT) derived from Lotus japonicus. GuCSyGT derived from licorice has 81% amino acid identity with GmCSyGT derived from soybean. In addition, LjCSyGT derived from Lotus japonicus has 82% amino acid identity with GmCSyGT derived from soybean.

[0085] In addition to the aforementioned plant species, CSyGT may also exist in a large number of other plant species, particularly in Fabaceae plant species. In addition to such interspecific homologs of other wild-type CSyGT, the CSyGT of the present invention also includes intraspecific homologs of the same wild-type CSyGT having the first glucuronide transfer activity and mutant CSyGT. Examples of these interspecific homologs of other wild-type CSyGT and mutant CSyGT include amino acid sequences in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in any one of SEQ ID NOs. 1, 3 and 5, or polypeptides having an 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, and less than 100% with respect to the amino acid sequence shown in any one of SEQ ID NOs. 1, 3 and 5. In fact, with respect to soybean GmCSyGT, GuCSyGT and LjCSyGT are interspecific homologs of CSyGT of Glycyrrhiza uralensis and Lotus japonicus, respectively, but the amino acid identity is more than 80% as described above. In addition, the mutant CSyGT having the first glucuronic acid transfer activity is not limited, and specific examples include splicing mutants, mutants based on SNPs, etc.

[0086] In this specification, the term "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3. In addition, the term "amino acid identity" refers to the ratio (%) of the number of consistent amino acid residues in the total number of amino acid residues when the amino acid sequences of the two polypeptides being compared are aligned (aligned) by inserting appropriate gaps in one or both sides as required in a manner that maximizes the number of consistent amino acid residues. The alignment of the two amino acid sequences used to calculate the amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW.

[0087] In this specification, the so-called "(amino acid) replacement" refers to the replacement within the conservative amino acid group with similar properties such as charge, side chain, polarity, and aromaticity between the 20 amino acids constituting natural proteins. For example, there can be mentioned the replacement within the uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) with low polarity side chain, branched amino acid group (Leu, Val, Ile), neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acid group (Asn, Gln, Thr, Ser, Tyr, Cys) with hydrophilic side chain, acidic amino acid group (Asp, Glu), basic amino acid group (Arg, Lys, His), aromatic amino acid group (Phe, Tyr, Trp). It is known that as long as the amino acid replacement within these groups, the properties of the polypeptide are not easy to change, so it is preferred.

[0088] The so-called "active fragment" in this embodiment refers to a polypeptide fragment that contains a part of the region of glucuronyl transferase I and maintains the glucuronyl transfer activity of glucuronyl transferase I. For example, a polypeptide fragment containing the substrate binding site of glucuronyl transferase I can be mentioned. The substrate of the glucuronyl transferase I of the present invention can be listed as the aforementioned oleanane-type triterpenoid compounds. Glycyrrhizic acid is preferred. The length of the amino acids of the polypeptide constituting the active fragment is not particularly limited. For example, it can be a region of at least 10, 15, 20, 25, 30, 50, 100 or 150 amino acids in the above-mentioned polypeptides (a) to (c).

[0089] In addition, in this specification, CSyGT and active fragments thereof are often collectively expressed as "CSyGT etc. (glucuronosyl transferase I etc.)".

[0090] According to the CSyGT and the like of the present invention, oleanane triterpenoids can be used as sugar acceptor substrates, and glucuronic acid can be transferred to the hydroxy sugar at position 3 through the glucuronic acid first transfer activity to obtain oleanane triterpenoid monoglucuronide.

[0091] In the biosynthesis system of glycyrrhizin in licorice, the biosynthesis pathway from β-amyrin, which is an oleanane-type triterpenoid compound and can be said to be the origin material, to glycyrrhetinic acid has been clarified, and artificial biosynthesis can also be performed. Furthermore, the pathway for biosynthesis of glycyrrhizin by further transferring glucuronic acid to glycyrrhetinic acid monoglucuronide, which has transferred one molecule of glucuronic acid to glycyrrhetinic acid, has also been clarified. That is, in the biosynthesis pathway of glycyrrhizin from β-amyrin, the pathway for biosynthesis of glycyrrhizinic acid monoglucuronide by transferring glucuronic acid to glycyrrhetinic acid alone has been unknown so far. However, through the present invention, the biosynthesis pathway of glycyrrhizin in licorice has been clarified, and in vitro synthesis from β-amyrin to glycyrrhetin can be performed. In addition, β-amyrin can be biosynthesized by a large number of plant species other than licorice, so an in vivo synthesis system with a common plant species among them as a host becomes possible. Furthermore, if an organism that does not contain β-amyrin is used together with a gene for biosynthesizing β-amyrin, an in vivo synthesis system using organisms other than plants as hosts becomes possible.

[0092] 2. Glucuronyl transferase 1 gene (CSyGT gene) and its active fragments

[0093] 2-1. Overview

[0094] The second aspect of the present invention relates to a polynucleotide encoding the polypeptide (CSyGT, etc.) described in the first aspect, that is, a glucuronyl transferase 1 gene and an active fragment thereof. The recombinant vector of the third aspect described below can be constructed using the polynucleotide of the present invention.

[0095] 2-2. Composition

[0096] The so-called "glucuronyl transferase 1 gene" (often expressed as "CSyGT gene" in this specification) refers to a polynucleotide encoding CSyGT described in the first embodiment. As long as it is a polynucleotide encoding CSyGT, its base sequence is not particularly limited. It is preferably a polynucleotide encoding a wild-type CSyGT comprising the amino acid sequence shown in sequence numbers 1, 3, and 5. For example, as a polynucleotide encoding a soybean-derived wild-type GmCSyGT composed of the amino acid sequence shown in sequence number 1, specifically, for example, a polynucleotide composed of the base sequence shown in sequence number 2, that is, a soybean-derived wild-type GmCSyGT gene. In addition, as a polynucleotide encoding a licorice-derived wild-type GuCSyGT composed of the amino acid sequence shown in sequence number 3, specifically, for example, a polynucleotide composed of the base sequence shown in sequence number 4, that is, a licorice-derived wild-type GuCSyGT gene. Specific examples of the polynucleotide encoding the wild-type LjCSyGT derived from Lotus japonicus consisting of the amino acid sequence shown in SEQ ID NO: 5 include a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6, that is, the wild-type LjCSyGT gene derived from Lotus japonicus.

[0097] In addition, other interspecific homologs of the above-mentioned wild-type CSyGT gene and mutant CSyGT genes that maintain enzyme activity are also included. As examples of such CSyGT genes, polynucleotides containing base sequences in which one or more bases are deleted, replaced, or added to the wild-type CSyGT gene can be cited. Specifically, for example, a polynucleotide containing a base sequence in which one or more bases are deleted, replaced, or added to any of the base sequences of the wild-type GmCSyGT gene derived from soybean (for example, a polynucleotide consisting of the base sequence shown in sequence number 2), the wild-type GuCSyGT gene derived from licorice (for example, a polynucleotide consisting of the base sequence shown in sequence number 4), and the wild-type LjCSyGT gene derived from Japanese lotus (for example, a polynucleotide consisting of the base sequence shown in sequence number 6).

[0098] Furthermore, examples include polynucleotides comprising 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 and less than 100% amino acid identity with the wild-type CSyGT gene. Specifically, examples include polynucleotides having 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 and less than 100% with any of the base sequences of the wild-type GmCSyGT gene derived from soybean (e.g., a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2), the wild-type GuCSyGT gene derived from licorice (e.g., a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4), and the wild-type LjCSyGT gene derived from Lotus japonicus (e.g., a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6).

[0099] Furthermore, it also includes a polynucleotide having a base sequence that hybridizes to a nucleotide fragment consisting of a complementary base sequence to a partial base sequence of a wild-type CSyGT gene under highly stringent conditions and a nucleotide that retains enzyme activity.

[0100] In this specification, the so-called "stringent conditions" refer to conditions that are not easy to form non-specific hybrids. "High stringency conditions" refer to conditions that are more difficult to form or not formed by non-specific hybrids. Generally, the lower the salt concentration and the higher the temperature of the reaction conditions, the higher the stringency. For example, in the cleaning after hybridization, for example, the conditions of cleaning at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C and 0.1×SSC and 0.1% SDS are used. In addition, other conditions such as probe concentration, probe base length, hybridization time, etc. can be appropriately combined to increase the stringency of hybridization.

[0101] The so-called "active fragment" in this embodiment refers to a fragment of the CSyGT gene, and the polypeptide encoded by the fragment has CSyGT activity. In essence, it is equivalent to the polynucleotide encoding the active fragment of CSyGT described in the first embodiment. Therefore, the length of the base sequence of the polynucleotide constituting the active fragment, that is, the number of bases, is 3 times the number of amino acids in the active fragment of CSyGT described in the first embodiment.

[0102] The polynucleotide or active fragment thereof of the present invention can be used to construct a recombinant vector capable of expressing CSyGT and its active fragment in a host cell.

[0103] In addition, in this specification, the CSyGT gene and its active fragment are often collectively expressed as "CSyGT gene etc. (glucuronosyl transferase 1 gene etc.)".

[0104] The CSyGT gene of this scheme can be isolated from appropriate plants, such as leguminous plants, using known methods. For example, based on the base sequence of the wild-type GmCSyGT gene from soybean shown in sequence number 2, a primer pair with an appropriate base sequence length is designed. Specifically, for example, the primer pairs shown in sequence numbers 17 and 18 can be cited. The GmCSyGT gene can be obtained by using the primer pair and performing nucleic acid amplification reactions such as PCR using a nucleic acid derived from a soybean DNA library or a genomic DNA library as a template. In addition, the polynucleotide of the present invention can be obtained by hybridization from the above-mentioned library, etc., using a nucleic acid fragment composed of a part of the base sequence shown in sequence number 2 as a probe. These methods can refer to the methods described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0105] 3. Recombinant vector

[0106] 3-1. Overview

[0107] The third embodiment of the present invention relates to a recombinant vector. The recombinant vector of the present invention comprises the polynucleotide described in the second embodiment, and can clone the CSyGT gene, etc., or express CSyGT, etc. in a host cell. In this embodiment, a CSyGT expression vector that expresses CSyGT, etc. is particularly preferably used.

[0108] 3-2. Composition

[0109] The recombinant vector of the present invention can be constructed by introducing the polynucleotide described in the second embodiment into an appropriate recombinant vector. The type of vector is not particularly limited. Depending on the purpose or the host to be introduced, a vector for cloning (transformation) or gene expression can be appropriately selected. Plant transformation vectors or (gene) expression vectors are particularly preferred.

[0110] The so-called "(gene) expression vector" in the present invention is a gene expression system that can transport a polynucleotide encoding a polypeptide contained therein into a target plant cell to express the polypeptide. Examples thereof include expression vectors using plasmids or viruses. In the present invention, it is equivalent to a CSyGT expression vector expressing CSyGT, etc., into which a CSyGT gene, etc. is integrated.

[0111] When a plasmid expression vector is used (hereinafter often referred to as "plasmid expression vector"), the plasmid is not limited, and examples thereof include pPZP, pSMA, pUC, pBR, pBluescript (Agilent Technologies), pTriEX TMsystem (TaKaRa), or pBI system, pRI system or pGW system binary vector, etc.

[0112] When a viral expression vector (hereinafter often referred to as “viral expression vector”) is used, viruses such as cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV), and tobacco mosaic virus (TMV) can be used.

[0113] The above-mentioned recombinant vector comprises the expression regulation region of promoter and terminator. In addition, it can comprise enhancer, poly A addition signal, 5'-UTR (untranslated region) sequence, mark or screening marker gene, multiple cloning site, replication origin site etc. As long as the respective kind can play its function in host cell, it is not particularly limited. According to the host of importing, it is appropriate to select the known in the field. It is preferred to use plant cell or plant body as the host.

[0114] The promoter can use various promoters, for example, overexpression promoter, constitutive promoter, site-specific promoter, time-specific promoter, and / or inducible promoter. As a specific example of overexpression and constitutive promoters that can work in plant cells, the 35S promoter from cauliflower mosaic virus (CaMV), the promoter Pnos of the nopaline synthase gene from Ti plasmid, the ubiquitin promoter from corn, the actin promoter from rice, the PR protein promoter from tobacco, etc. can be cited. The small subunit (Rubiscossu) promoter of ribulose bisphosphate carboxylase of various plant species or histone promoters can also be used. In addition, as a promoter that can work in bacterial cells, there can be mentioned the promoter of the maltogenic amylase gene of Bacillus stearothermophilus (Bacillus stearothermophilus), the promoter of the α-amylase gene of Bacillus licheniformis (Bacillus licheniformis), the promoter of the BAN amylase gene of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the promoter of the alkaline protease gene of Bacillus subtillis (Bacillus subtilis) or the promoter of the xylosidase gene of Bacillus pumilus (Bacillus pumilus), or the PR or PL promoter of λ phage, the lac, trp or tac promoter of Escherichia coli, etc. As a promoter that can work in yeast host cells, there can be mentioned the promoter derived from the yeast glycolytic system gene, the alcohol dehydrogenase gene promoter, the TPI1 promoter, the ADH2-4c promoter, etc. As a promoter that can work in fungi, there can be mentioned the ADH3 promoter, the tpiA promoter, etc. Examples of promoters that can operate in animal cells include the SV40 early promoter, the SV40 late promoter, and the CMV promoter. Examples of promoters that can operate in insect cells include the polyhedrin promoter, the P10 promoter, the basic protein promoter of the baculovirus Autographa californica polyhedrosis (Autographa californica polyhedrosis virus), the baculovirus immediate early gene 1 promoter, and the baculovirus 39K delayed early gene promoter.

[0115] Examples of terminators include 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 lipopolyprotein lpp, the trp operon terminator, the amyB terminator, the terminator of the ADH1 gene, etc. The terminator is not particularly limited as long as it is a sequence that can terminate the transcription of the gene transcribed from the above promoter.

[0116] In the case of an enhancer, for example, an enhancer region including an upstream sequence in the CaMV 35S promoter can be mentioned, and it is not particularly limited as long as it can enhance the expression efficiency of a nucleic acid encoding an active peptide.

[0117] As screening marker genes, there can be listed 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)), neomycin phosphotransferase II (NPT II), dihydrofolate reductase and other enzyme genes.

[0118] The recombinant vector of the present invention not only facilitates the manipulation and / or control of the expression of the polynucleotide described in the second embodiment, but also enables manipulation of the expression of CSyGT and the like in host cells.

[0119] 4. Transformants or their progeny

[0120] 4-1. Overview

[0121] The fourth aspect of the present invention relates to a transformant or its progeny. The transformant of the present invention or its progeny contains the polynucleotide described in the second aspect or the recombinant vector described in the third aspect in the cell, and can express clones of the CSyGT gene, etc., and / or CSyGT, etc. The transformant of the present invention can stably biosynthesize CSyGT, etc. using an in vivo expression system.

[0122] 4-2. Composition

[0123] In the present specification, the term "transformant" refers to a host transformed by the introduction of the polynucleotide described in the second embodiment or the recombinant vector described in the third embodiment.

[0124] The transformed host is not particularly limited. For example, bacteria such as Escherichia coli or Bacillus subtilis, yeast such as budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe) or methanol assimilating yeast (Pichia pastoris), fungi such as Aspergillus, Neurospora, Fusarium or Trichoderma, monocots, dicots or plant cells, mammalian cells, or insect cells (for example, sf9 or sf21) can be cited. Preferably, leguminous plants or yeast.

[0125] Transformants of the present invention include clones with the same genetic information. For example, if the host is a unicellular microorganism such as Escherichia coli, yeast, etc. that reproduces asexually, clones newly generated by the first generation of transformants through division or budding, etc. are also included in the transformants of the present invention. In addition, if the host is a plant, clones obtained by plant tissue culture or cutting, grafting or layering of a part of the plant body collected by the first generation of transformants, such as plant tissues such as epidermis, phloem, parenchyma, xylem or vascular bundles, plant organs such as leaves, petals, stems, roots or seeds, or plant cells, or new clones newly generated by the vegetative propagation organs obtained by asexual reproduction of the first generation of transformants such as rhizomes, tubers, bulbs, runners, etc. are also included in the transformants of the present invention.

[0126] The transformant of the present invention may further have one or more other polynucleotides or other recombinant vectors in addition to the polynucleotides described in the second embodiment or the recombinant vectors described in the third embodiment. The so-called other polynucleotides here refer to polynucleotides other than the polynucleotides described in the second embodiment. For example, it corresponds to the β-amyrin synthase gene, CYP88D6 or CYP72A154, or any oleanane-type triterpenoid monoglucuronide synthase gene. In addition, the so-called other recombinant vectors refer to recombinant vectors other than the recombinant vectors described in the third embodiment.

[0127] The transformant of the present invention can be produced by introducing the above-mentioned polynucleotide or recombinant vector into an appropriate host.

[0128] The method for introducing the above-mentioned polynucleotide or recombinant vector can use methods known in the art, for example, Agrobacterium method, PEG-calcium phosphate method, electroporation method, liposome method, gene gun method, microinjection method. The introduced polynucleotide can be integrated into the host's genomic DNA, or it can exist in the state of the introduced polynucleotide (for example, always contained in an external vector). Furthermore, the introduced polynucleotide can be continuously maintained in the host cell as in the case of integration into the host's genomic DNA, or it can be temporarily maintained.

[0129] After the polynucleotide described in the second embodiment or the recombinant vector described in the third embodiment is introduced into the host by the above method, the presence of the introduction of the target polynucleotide can be confirmed by PCR, Southern blotting, Northern blotting, in situ hybridization, etc.

[0130] In this specification, the so-called "progeny thereof" refers to the progeny obtained by sexual reproduction of the first generation of the above-mentioned transformant, and refers to a host that maintains the polynucleotide described in the second embodiment of the present invention or the recombinant vector described in the third embodiment in a state that can be expressed. Preferably, it refers to the progeny of the host that maintains the polynucleotide described in the second embodiment of the above-mentioned polynucleotide or the polynucleotide described in the second embodiment of the above-mentioned recombinant vector in a state that can be expressed. For example, when the transformant is a plant, it is a plant body grown by germination of the seed of the transformant. The generation of the progeny is not limited.

[0131] According to the transformant of this scheme, the oleanane-type triterpenoid compound present in the host cell can be converted into the monoglucuronide of the oleanane-type triterpenoid compound by enhancing the expression of the introduced polynucleotide. In addition, it is possible to obtain a glucuronyl transferase 1 to which different sugar chains are added by changing the host of the transformant. For example, when the host of the transformant is yeast, unlike the case where the host is a leguminous plant, the glucuronyl transferase 1 to which a high mannose type sugar chain is 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).

[0132] 5. Method for producing glucuronyl transferase I and its active fragments (CSyGT, etc.)

[0133] 5-1. Summary

[0134] The fifth aspect of the present invention relates to a method for producing CSyGT, etc., comprising culturing the transformant of the fourth aspect or its progeny, and extracting the polypeptide having the first glucuronide transfer activity described in the first aspect, i.e., CSyGT, etc., from the culture. According to the method for producing a polynucleotide of the present invention, CSyGT, etc. can be obtained stably and in large quantities by using a host as a biological production system.

[0135] 5-2. Methods

[0136] The production method of the present invention includes a culture step and an extraction step as essential steps. Each step will be described in detail below.

[0137] (1) Cultivation process

[0138] In this embodiment, the "cultivation step" is a step of culturing the transformant of the fourth embodiment or its offspring. The transformant or its offspring used in the present invention is preferably a transformant or its offspring that can overexpress or constitutively express the polypeptide described in the first embodiment. For example, if it is a transformant or its offspring having a recombinant vector described in the third embodiment, it is preferred that the recombinant vector is an expression vector containing an overexpression promoter or a constitutive promoter. The transformant or its offspring of the fourth embodiment can use any host, preferably a leguminous plant or yeast. By changing the host, even if the same glucuronyl transferase 1 described in the first embodiment is expressed, glycoproteins with different sugar chains added can be obtained.

[0139] The medium used in the culture can be any medium suitable for the culture of the host. The medium can be a medium known in the art. For example, although not limited, when bacteria such as Escherichia coli are used as hosts for culture, LB medium or M9 medium can be cited, when yeast is used as a host for culture, YPD medium, YPG medium, YPM medium, YPDM medium, SMM medium, etc. can be cited, and when plants are used as hosts for culture, appropriate culture soil and hydroponic culture medium can be cited.

[0140] The culture medium appropriately contains a carbon source (for example, glucose, glycerol, mannitol, fructose, lactose, etc.), a nitrogen source (for example, inorganic nitrogen such as ammonium sulfate and ammonium chloride, organic nitrogen sources such as casein hydrolyzate, yeast extract, polypeptone, bacterial trypsin, beef extract, etc.), inorganic salts (for example, sodium diphosphate, potassium diphosphate, magnesium chloride, magnesium sulfate, calcium chloride, etc.), vitamins (vitamin B1, etc.), drugs (antibiotics such as ampicillin, tetracycline, kanamycin, etc.), etc.

[0141] The culture conditions are not particularly limited as long as they are suitable for the expression of the polynucleotide, and culture is usually performed at 10 to 45°C, 15 to 40°C, or 18 to 37°C for several to several hundred hours with aeration, irradiation, and / or stirring as necessary.

[0142] (2) Extraction process

[0143] The "extraction step" in this embodiment is a step of extracting CSyGT and the like from the culture obtained by the above-mentioned culturing step.

[0144] In this specification, the term "culture" refers to the culture supernatant or the transformed body after culture. The cells of the transformant naturally contain CSyGT and the like, and the supernatant may also contain CSyGT and the like secreted from the transformant.

[0145] In order to recover the polypeptide described in the first embodiment from the culture, the polypeptide present in the culture can be extracted by a known method and purified as needed. For example, the target polypeptide can be obtained by solvent extraction, salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. alone or in appropriate combination. Specifically, the method of Hayashi et al. (Hayashi et al., 1996, Phytochemistry, 42: 665-666) and the method of Noguchi et al. (Noguchi et al., 2007, J. Biol. Chem., 282: 23581-23590) can be referred to. In addition, the polypeptide described in the first embodiment as the target can also be recovered based on host-specific sugar chains. For example, when the transformant or its progeny in the fourth embodiment is yeast, since the expressed polypeptide of the present invention has high mannose sugar chains added, it can be extracted and purified using mannose-binding lectins (e.g., UDA lectin, BC2L-A lectin, etc.).

[0146] 6. Genetic recombinant for the production of glycyrrhizin

[0147] 6-1. Summary

[0148] The sixth embodiment of the present invention is a gene recombinant for producing glycyrrhizin. The gene recombinant of the present invention expresses an expression vector containing a group of enzymes required for the biosynthetic pathway from β-amyrin to glycyrrhizin in Glycyrrhiza plants, that is, four enzyme groups that catalyze two-stage oxidation reactions and two-stage glycosylation reactions. The gene recombinant of the present invention can biosynthesize glycyrrhizin from β-amyrin in biological cells, and thus can be used as a biological production system for glycyrrhizin.

[0149] 6-2. Composition

[0150] 6-2-1. Expression vectors included

[0151] The genetic recombinant of the present invention is characterized by at least containing an expression vector containing a polynucleotide encoding a group 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, an expression vector containing a polynucleotide encoding a β-amyrin synthase gene may be further contained. The above 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 triterpenoid compounds, respectively. The expression vectors containing each enzyme or its active fragment are shown in the following (1) to (4) and are specifically described. In addition, in (1) to (4), the above four expression vectors are recorded one by one, but the genes of each enzyme can be contained in different expression vectors, or two or more can be contained in the same expression vector.

[0152] (1) CYP88D6 expression vector

[0153] The "CYP88D6 expression vector" includes a gene and a fragment thereof (often referred to as "CYP88D6 gene, etc." in this specification) encoding a polypeptide having the activity of oxidizing position 11 in oleanane-type triterpenoid compounds. Therefore, CYP88D6, etc. is expressed in a genetically recombinant body through the CYP88D6 expression vector.

[0154] Specific examples of the above-mentioned CYP88D6 include, but are not limited to, CYP88D6 derived from licorice (G. uralensis) consisting of the amino acid sequence shown in SEQ ID NO: 7. In addition, polypeptides having the above-mentioned first-stage oxidation activity and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added to 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 can also be exemplified.

[0155] Although not limited, in the gene recombinant of the present invention, the catalytic activity of CYP88D6 expressed by the CYP88D6 expression vector can be used to mainly use endogenous or exogenous β-amyrin as a substrate and oxidize its 11-position to produce 11-oxo-β-amyrin. In addition, 30-hydroxy-β-amyrin can be used as a substrate and oxidized its 11-position to produce 30-hydroxy-11-oxo-β-amyrin. Furthermore, 11-deoxyglycyrrhetinic acid can be used as a substrate and oxidized its 11-position to produce glycyrrhetinic acid.

[0156] The structure of the plasmid region in the CYP88D6 expression vector is in accordance with the expression vector in the recombinant vector described in the above-mentioned embodiment 3. In addition, the recombinant vector described in Japanese Patent No. 5526323 can also be used.

[0157] (2) CYP72A154 expression vector

[0158] The "CYP72A154 expression vector" includes a gene and a fragment thereof (often referred to as "CYP72A154 gene, etc." in this specification) encoding a polypeptide having the activity of oxidizing position 30 in oleanane-type triterpenoid compounds. Therefore, in a genetically recombinant body, CYP72A154, etc. is expressed through the CYP72A154 expression vector.

[0159] Specific examples of the above-mentioned CYP72A154 include, but are not limited to, CYP72A154 derived from liquorice (G. uralensis) consisting of the amino acid sequence set forth in SEQ ID NO: 9, CYP72A154 derived from liquorice (G. glabra) consisting of the amino acid sequence set forth in SEQ ID NO: 11, and CYP72A63 derived from Medicago truncatula consisting of the amino acid sequence set forth in SEQ ID NO: 13. In addition, polypeptides having the above-mentioned second-stage oxidation activity and consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11 and 13, or consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in any one of SEQ ID NOs: 9, 11 and 13 can also be exemplified.

[0160] Although not limited, in the gene recombinant of the present invention, 30-hydroxy-β-amyrin and 30-hydroxy-11-oxo-β-amyrin can be produced respectively by oxidizing the 30-position of β-amyrin and 11-oxo-β-amyrin as substrates through the catalytic activity of CYP72A154 or the like expressed by the CYP72A154 expression vector. In addition, 30-hydroxy-11-oxo-β-amyrin can be produced by further oxidizing the 30-position of 30-hydroxy-11-oxo-β-amyrin as a substrate.

[0161] The structure of the plasmid region in the CYP72A154 expression vector is in accordance with the expression vector in the recombinant vector described in the above-mentioned embodiment 3. In addition, the recombinant vector described in Japanese Patent No. 5771846 can also be used.

[0162] (3) UGT73P12 recombinant vector

[0163] The "UGT73P12 recombinant vector" includes genes and fragments thereof (often referred to as "UGT73P12 gene, etc." in this specification) encoding polypeptides having the activity of transferring glucuronic acid to the 2-hydroxyl group of glucuronic acid in the monoglucuronide of oleanane-type triterpenoid compounds, i.e., UGT73P12 and its active fragments (often referred to as "UGT73P12, etc." in this specification). Therefore, in the gene recombinant body, UGT73P12, etc. are expressed through the UGT73P12 expression vector.

[0164] Specific examples of the UGT73P12 include, but are not limited to, UGT73P12 derived from Glycyrrhiza uralensis and consisting of the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO: 15, or consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 15 can also be exemplified.

[0165] The structure of the plasmid region in the UGT73P12 expression vector is in accordance with the expression vector in the recombinant vector described in the above-mentioned embodiment 3. In addition, the recombinant vector described in Japanese Patent No. 6344774 can also be used.

[0166] (4) CSyGT expression vector

[0167] The "CSyGT expression vector" corresponds to the CSyGT expression vector in the CSyGT recombinant vector described in the third embodiment above, and therefore a detailed description thereof will be omitted here.

[0168] 6-2-2. Genetic recombinant for producing glycyrrhizin

[0169] The so-called "genetic recombinant for producing glycyrrhetinic acid" of the present invention refers to a transformant into which an expression vector containing at least the above-mentioned four enzyme gene groups is introduced, or its offspring that maintains these enzyme gene groups. Therefore, if the types of the included expression vectors are excluded, its basic structure can be the same as the transformant and its offspring recorded in the above-mentioned fourth scheme. However, since the present invention is a genetic recombinant that can biosynthesize glycyrrhizin from β-amyrin in cells, it is preferred to use a host that can biosynthesize β-amyrin as the starting material in the above-mentioned biosynthesis system in cells. The biosynthesis of β-amyrin in the host can be based on an endogenous synthesis system or an exogenous synthesis system. β-amyrin, which is an oleanane-type triterpenoid compound, can be biosynthesized by a large number of plants, so in the case of an endogenous synthesis system, the host of the present invention is preferably a plant. It is preferably a plant species with high β-amyrin synthesis ability, strong fertility, and easy cultivation. More preferably, it is a plant that is relatively closely related to licorice, that is, a leguminous plant. For example, species belonging to the genus Glycyrrhiza, species belonging to the genus Glycine max, species belonging to the genus Lotus, etc. On the other hand, when the biosynthesis of β-amyrin is based on an exogenous synthesis system, the host itself may be a species that cannot biosynthesize β-amyrin. For example, by introducing an expression vector containing a β-amyrin synthase gene into yeast in advance, the transformant of the yeast can be used as a host capable of biosynthesizing β-amyrin in cells.

[0170] According to the present invention, even a host that has been unable to biosynthesize glycyrrhizin can now biosynthesize glycyrrhizin as a metabolite using β-amyrin as a starting material.

[0171] 7. Method for producing glycyrrhizin

[0172] 7-1. Summary

[0173] The seventh aspect of the present invention is a method for producing glycyrrhizin. The method of the present invention is characterized in that glycyrrhizin is produced from β-amyrin using the genetically modified product for producing glycyrrhizin of the sixth aspect as a bioproduction system.

[0174] According to the production method of the present invention, glycyrrhizin, which is conventionally expensive, can be produced stably and in large quantities without relying on extraction from licorice.

[0175] 7-2. Methods

[0176] The production method of the present invention includes a culturing step as an essential step, and further includes an "extraction step" as an optional step.

[0177] (1) Cultivation process

[0178] The "cultivation step" in this scheme can basically be in accordance with the cultivation step described in the fifth scheme. In the case where the genetically modified organism is a plant, a method for cultivating a known conditional plant can be applied. By this step, glycyrrhizin can be produced in the genetically modified organism for producing glycyrrhizin in the sixth scheme.

[0179] (2) Extraction process

[0180] The "extraction step" in this embodiment may basically follow the extraction step described in Embodiment 5. When the genetically modified organism is a plant, the same method as the method for extracting glycyrrhizin from licorice can be used.

[0181] The production method of the present invention makes it possible to produce glycyrrhizin from various genetic recombinants stably and in large quantities without relying on extraction from licorice.

[0182] Example

[0183] In the following examples, one example of the present invention is given and described in detail.

[0184] <Example 1: Isolation of soybean-derived cellulose synthase-like gene Glyma.06G324300>

[0185] Seeds of soybean (Glycine max) variety "Williams 82" grown 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, the first-strand cDNA was synthesized using the QuantiTech Reverse Transcription Kit (QIAGEN) according to the attached protocol. Using 1 μL of the first-strand cDNA diluted 5 times as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide inferred from Glyma.06G324300 were used as forward primers (sequence number 17) and reverse primers (sequence number 18), respectively, and PrimeSTAR GXL DNA Polymerase (タカラバイオ) was used to perform PCR for 30 cycles under the conditions of an annealing temperature of 55°C and a reaction temperature of 68°C. In addition, due to the introduction of pDONR TMIn order to meet the need for a base sequence-specific recombination reaction (GATEWAY attB×attP reaction) during cloning of pDONR 221 (ThermoFisher Technologies), the forward primer artificially added 12 bases (AAAAAGCAGGCT) at the 5' end, and the reverse primer artificially added 12 bases (AGAAAGCTGGGT) at the 5' end. The DNA fragment amplified from the first-strand cDNA of the seed was cloned into pDONR using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies) by a base sequence-specific recombination reaction (GATEWAY attB×attP reaction). TM The polynucleotide sequences of the three independent clones obtained were determined in 221. The sequence thus obtained is SEQ ID NO: 2, and the polypeptide sequence deduced therefrom is SEQ ID NO: 1.

[0186] <Example 2: Exploration of homologous genes of Glyma.06G324300 from licorice>

[0187] Glycyrrhiza uralensis, which is known to be a leguminous plant like soybean and biosynthesizes glycyrrhizin, was searched for as a candidate for interspecific homologous gene of Glyma.06G324300 by gene homology search. Using the BLAST homology search function in Glycyrrhiza uralensis GDB (http: / / ngs-data-archive.psc.riken.jp / Gur-genome / index.pl), which is a genomic information database of licorice, a base sequence Glyur003152s00037491 encoding a protein showing high amino acid identity with Glyma.06G324300 was found. The polypeptide deduced from Glyur003152s00037491 showed 81% amino acid identity with Glyma.06G324300.

[0188] <Example 3: Isolation of Glycyrrhiza uralensis-derived Glyma.06G324300 homologous genes>

[0189] Total RNA was prepared from the root of licorice using the RNA extraction reagent PureLink Plant RNA Reagent (Thermo Fisher Scientific). Using 1 μg of the obtained total RNA, the first-strand cDNA was synthesized using the SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μL of the first-strand cDNA as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide inferred from Glyur003152s00037491 were used as forward primers (SEQ ID NO: 19) and reverse primers (SEQ ID NO: 20), respectively, and PrimeSTAR Max DNA Polymerase (TAKARA Bio) was used to perform PCR for 30 cycles under the conditions of an annealing temperature of 55°C and a reaction temperature of 72°C. In addition, due to the addition of pENTR TM / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), the forward primer had 4 bases (cacc) artificially added to the 5' end. The amplified DNA fragment was cloned into pENTR TM / D-TOPO entry vector, the base sequence of the obtained 4 independent clones was determined. The base sequence of the Glycyrrhiza uralensis Glyma.06G324300 homologous gene obtained in this way is sequence number 4, and the polypeptide sequence deduced therefrom is sequence number 3. The amino acid sequence of sequence number 3 has 82% identity with the amino acid sequence shown in sequence number 1.

[0190] <Example 4: Search for homologous genes of Glyma.06G324300 from Lotus japonicus>

[0191] By the same method as in Example 2, a homologous gene of Glyma.06G324300 was searched as a candidate of an interspecific homologous gene of Glyma.06G324300 from Lotus japonicus. Using the BLAST homology search function in miyakogusa.jp (http: / / www.kazusa.or.jp / lotus / release1 / index.html), which is a database of genome information of Lotus japonicus, a base sequence Lj3g3v1981230 encoding a protein showing high amino acid identity with Glyma.06G324300 was found. The polypeptide deduced from Lj3g3v1981230 showed 81.4% amino acid identity with Glyma.06G324300.

[0192] <Example 5: Isolation of Glyma.06G324300 homologous genes from Lotus japonicus>

[0193] 1 μg of total RNA obtained from Lotus japonicus was used to synthesize the first-strand cDNA using the SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μl of the first-strand cDNA as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide inferred from Lj3g3v1981230 were used as forward primers (SEQ ID NO: 37) and reverse primers (SEQ ID NO: 38), respectively, and PrimeSTAR Max DNA Polymerase (Takara Bio) was used to perform PCR for 30 cycles under the conditions of annealing temperature of 55°C and reaction temperature of 72°C. In addition, due to the addition of pENTR TM / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), the forward primer had 4 bases (cacc) artificially added to the 5' end. The amplified DNA fragment was cloned into pENTR TM / D-TOPO entry vector, and the polynucleotide sequences of the two independent clones obtained were determined. The base sequence of the Glyma.06G324300 homologous gene of Lotus japonicus obtained in this way is SEQ ID NO: 6, and the polypeptide sequence deduced therefrom is SEQ ID NO: 5. SEQ ID NO: 5 has 82% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0194] <Example 6: Construction of destination vector for yeast expression>

[0195] In order to investigate the sugar transfer activity of Glyma.06G324300 isolated in Examples 1, 3 and 5 and proteins predicted to be homologous thereto, expression vectors of the respective proteins were constructed using a yeast expression system.

[0196] The yeast (Saccharomyces cerevisiae) INVSc1 strain used does not contain UDP-glucuronic acid, which is a sugar donor substrate in the hypothetical glycosylation reaction for the candidate gene product. Therefore, the UDP-glucose dehydrogenase (UGD) gene that synthesizes UDP-glucuronic acid using UDP-glucose in yeast as a substrate was introduced into the yeast expression destination vector. Specifically, the cDNA of Arabidopsis thaliana-derived UGD (AtUGD2) was used as a template, and oligo DNAs at the positions corresponding to the N-terminus and C-terminus of the polypeptide were used as forward primers (SEQ ID NO: 21) and reverse primers (SEQ ID NO: 22), respectively, and PrimeSTAR Max DNA Polymerase (Takara Bio) was used to perform PCR for 30 cycles under the conditions of annealing temperature 55°C and reaction temperature 72°C. In addition, due to the need for In-fusion cloning, the forward primer artificially added 15 bases upstream of the cloning position of the target vector and 4 bases (aaaa) totaling 19 bases at the 5' end of the polynucleotide shown in sequence number 23 (gggcggccgcactag). In addition, the reverse primer artificially added 15 bases downstream of the cloning position of the target vector at the 3' end of the polynucleotide shown in sequence number 24 (atccatcgatactag). The target vector pESC-HIS-GW prepared by introducing the SrfI restriction enzyme site in MCS2 of the pESC-HIS (registered trademark) yeast expression vector (Agilent Technologies) into Gateway cassette A (Thermo Fisher Technologies) was treated with SpeI restriction enzyme, mixed with the DNA fragment amplified from cDNA, and the DNA fragment shown in sequence number 25 was introduced into MCS1 in pESC-HIS-GW using In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio) to obtain the target vector pESC-HIS-AtUGD2-GW.

[0197] <Example 7: Construction of yeast expression clone>

[0198] The plasmid (entry clone) having the polynucleotide shown in sequence number 2 prepared in Example 1 was mixed with the destination vector pESC-HIS-AtUGD2-GW prepared in Example 6, and the DNA fragment shown in sequence number 2 was transferred to pESC-HIS by a base sequence-specific recombination reaction (GATEWAY attL×attR reaction) using Gateway LR ClonaseII Enzyme Mix (Thermo Fisher Technologies), thereby obtaining the yeast expression vector pESC-HIS-AtUGD2-Glyma.06G324300 for the gene shown in sequence number 2. In addition, the yeast expression vectors pESC-HIS-AtUGD2-Glyur003152s00037491 and pESC-HIS-AtUGD2-Lj3g3v1981230 for the genes shown in sequence numbers 4 and 6 prepared in Examples 3 and 5 were obtained respectively by the same method as above.

[0199] <Example 8: Introduction of yeast strains for producing glycyrrhetinic acid and soyasaponin B>

[0200] The expression vector pYES3-BAS for the β-amyrin synthase (LjOSC1) gene of Lotus japonicus, the vector pESC-CPR-CYP88D6 for simultaneous expression of the CYP88D6 gene and the cytochrome P450 reductase (LjCPR1) of Lotus japonicus, and the expression vector pDEST52-CYP72A63 for the CYP72A63 gene, which is a homolog of the Medicago truncatula species and the CYP72A154 gene of Licorice, were introduced into the yeast INVScI strain (Thermo Fisher Technologies) (MATa his3D1 leu2trp1-289ura3-52 MATAlpha his3D1 leu2 trp1-289 ura3-52) and simultaneously expressed the genes, thereby obtaining a glycyrrhetinic acid-producing yeast strain ( Figure 3 (a)). At the same time, the expression vector pYES3-BAS for the β-amyrin synthase gene, the expression vector pESC-CPR-CYP93E3 for the cytochrome P450 reductase (LjCPR1) of Japanese lotus, and the expression vector pDEST52-CYP72A566 for the CYP72A566 gene were introduced to express them simultaneously, thereby obtaining soybean saponin B-producing yeast ( Figure 3(b)). pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 7 were introduced into these yeast strains, respectively. As a negative control, pESC-HIS-AtUGD2, which is equivalent to an empty vector, was introduced into the glycyrrhetinic acid-producing yeast strain. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

[0201] <Example 9: In vivo enzyme assay using recombinant yeast>

[0202] The glycyrrhetinic acid-producing yeast strains containing pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, pESC-HIS-AtUGD2-Lj3g3v1981230 or pESC-HIS-AtUGD2 for negative control obtained in Example 8 were 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. The culture solution was then centrifuged at 3,000 g and 4°C for 5 minutes to obtain a pellet of yeast cells. After suspending the obtained yeast cell pellet in 1 mL of Yeastnitrogen base (YNB) medium (-Trp / -Leu / -Ura / -His), centrifuge again at 3,000g and 4°C for 5 minutes to obtain the yeast cell pellet. The obtained yeast cell pellet was suspended in 5 mL of Yeastnitrogen base (YNB) medium (-Trp / -Leu / -Ura / -His) containing 2% galactose and cultured with shaking at 30°C and 200rpm for 5 days. Then, glass beads (SIGMA) equivalent to a volume of 1 mL and 4 mL of 1-butanol were added to the culture solution. In order to break up the yeast cells, stir vigorously with a strong shaker for 30 minutes, centrifuge the obtained liquid at 10,000g and 4°C for 10 minutes, and recover the supernatant as a yeast metabolite extract. 4 mL of 1-butanol was newly added to the remaining liquid, and the mixture was stirred again for 30 minutes. The resulting liquid was centrifuged at 10,000 g and 4° C. for 10 minutes, and the supernatant was extracted. As a result, a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in sequence number 1 (Glyma.06G324300) (sample A), a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in sequence number 3 (Glyur003152s00037491) (sample B), a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in sequence number 5 (Lj3g3v1981230) (sample C), and a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain containing only an empty vector in which no gene was expressed (sample D) were obtained.

[0203] The soyasaponol B-producing yeast strains harboring pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, pESC-HIS-AtUGD2-Lj3g3v1981230, or pESC-HIS-AtUGD2 for a negative control were cultured and metabolites were extracted in the same manner. As a result, a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 1 (Glyma.06G324300) (sample E), a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 3 (Glyur003152s00037491) (sample F), a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 5 (Lj3g3v1981230) (sample G), and a metabolite extract derived from a soybean saponin B-producing yeast strain containing only an empty vector in which no gene is expressed (sample H) were obtained.

[0204] <Example 10: Analysis of Yeast Metabolite Extract>

[0205] 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 filtered using Millex-GV, 0.22 μm, PVDF, 4 mm (Merck) to prepare a sample for LC-MS analysis.

[0206] LC-MS analysis was performed using ACQUITY UPLC / TQD-MS (Waters Corp.). The column used was UPLC HSS C18 (2.1 mm×150 mm, 1.7 μm) (Waters Corp.), and the solvent was 0.1% acetic acid-acetonitrile: 0.1% acetic acid-water = 30:70 (0 to 5 minutes), 40:60-100:0 (5 to 28 minutes), 100:0 (28 to 31.5 minutes), and the flow rate was 0.2 mL / min. MS used SIM mode, and the m / z of each compound was analyzed as glycyrrhetinic acid = 469.7, glycyrrhetinic acid monoglycoside = 631.9, glycyrrhetinic acid monoglucuronide = 645.8, and glycyrrhizin = 821.9 as parameters. Metabolites were identified by comparing LC retention times and MS spectra using samples prepared by dissolving commercially available glycyrrhetinic acid monoglucuronide and glycyrrhizin in methanol at a concentration of 1 μM as standards.

[0207] The results are shown in Figure 4 and Figure 5 .

[0208] Figure 4 It is the result of the enzyme activity of Glyma.06G324300 or its homologs when glycyrrhetinic acid and glucuronic acid are used as substrates. From sample A of (a), a single peak (black arrow) corresponding to glycyrrhetinic acid monoglucuronide was detected. The retention time and mass spectrum of this peak are well consistent with those of glycyrrhetinic acid monoglucuronide. Similarly, peaks (black arrows) corresponding to glycyrrhetinic acid monoglucuronide were also detected from sample B of (b) and sample C of (c). The retention time and mass spectrum of each peak are well consistent with those of glycyrrhetinic acid monoglucuronide. On the other hand, no peak corresponding to glycyrrhetinic acid monoglucuronide was detected for sample D of (d) as a negative control.

[0209] Figure 5 The results of the enzyme activity of Glyma.06G324300 or its homologues when soybean saponin B and glucuronic acid are used as substrates. A single peak (black arrow) corresponding to soybean saponin B monoglucuronide was detected from sample E of (a). Peaks (black arrows) corresponding to soybean saponin B monoglucuronide were also detected from sample F of (b) and sample G of (c). The retention time and mass spectrum of each peak are well consistent with soybean saponin B monoglucuronide. On the other hand, no peak corresponding to soybean saponin B monoglucuronide was detected for sample H of (d) as a negative control.

[0210] <Example 11: Preparation of transformed yeast for substrate feeding test>

[0211] The pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 8 were introduced into the yeast INVScI strain, respectively. As a negative control, pESC-HIS-AtUGD2 equivalent 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.

[0212] <Example 12: Substrate feeding test using recombinant yeast>

[0213] The transformed yeasts holding 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 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 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 yeast cell pellet was suspended in 10 mL of Yeast nitrogen base (YNB) medium (-His) containing 2% galactose and divided into two equal parts of 5 mL each. Glycyrrhetinic acid was added to one sample at a final concentration of 5 μM, and soybean saponin B ( Figure 4 ). Then, culture at 30°C and 200 rpm for 10 days. Add glass beads (SIGMA) equivalent to 1 mL volume and 4 mL of 1-butanol to the culture solution. In order to break the yeast cells, vigorously stir for 30 minutes with a strong shaker, centrifuge the resulting liquid at 10,000g and 4°C for 10 minutes, and collect the supernatant as a yeast feeding test extract. Add 4 mL of 1-butanol to the remaining liquid and extract again. As a result, a feeding test extract (sample I) in which glycyrrhetinic acid was added to the transformed yeast expressing the polypeptide shown in sequence number 1 (Glyma.06G324300), a feeding test extract (sample M) in which soybean saponin B was added, a feeding test extract (sample J) in which glycyrrhetinic acid was added to the transformed yeast expressing the polypeptide shown in sequence number 3 (Glyur003152s00037491), a feeding test extract (sample N) in which soybean saponin B was added, a feeding test extract (sample K) in which glycyrrhetinic acid was added to the transformed yeast expressing the polypeptide shown in sequence number 5 (Lj3g3v1981230), a feeding test extract (sample O) in which glycyrrhetinic acid was added to the transformed yeast containing only an empty vector but no gene was expressed, and a feeding test extract (sample P) in which soybean saponin B was added were obtained.

[0214] <Example 13: Analysis of extracts from substrate feeding test>

[0215] 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 filtered using Millex-GV, 0.22 μm, PVDF, 4 mm (Merck) to prepare a sample for LC-MS analysis.

[0216] LC-MS analysis was performed under the same conditions as in Example 10, but the MS used the SIM mode, and in the case of samples I, J, K, and L, the m / z of each compound was glycyrrhetinic acid = 469.7, glycyrrhetinic acid monoglycoside = 631.9, glycyrrhetinic acid monoglucuronide = 645.8, and glycyrrhizin = 821.9 as parameters for analysis, and in the case of samples M, N, O, and P, the m / z of each compound was soyasapogenol B = 457.8, soyasapogenol B monoglycoside = 619.8, soyasapogenol B monoglucuronide = 633.8, and soyasapogenol B diglucuronide = 809.9 as parameters for analysis. Metabolites were identified by comparing LC retention times and MS spectra with samples obtained by dissolving commercially available glycyrrhetinic acid monoglucuronide, glycyrrhizin, and soyasapogenol B monoglucuronide in methanol at a concentration of 1 μM as standards.

[0217] The results when glycyrrhetinic acid was used as the sugar acceptor substrate are shown in Figures 7-10 . Figure 7 This is the result of the substrate feeding test in sample 1. A peak corresponding to glycyrrhetinic acid as a sugar acceptor substrate was detected from (b) (white arrow), and a single peak (black arrow) believed to be the addition of one molecule of glucuronic acid to glycyrrhetinic acid was detected from (c). The retention time and mass spectrum of this peak are well consistent with glycyrrhetinic acid monoglucuronide.

[0218] Depend on Figure 8 The samples J and Fig. 9 Sample K shown also detected peaks corresponding to glycyrrhetinic acid (white arrow) and glycyrrhetinic acid monoglucuronide (black arrow) as shown in (b) and (c), respectively. The retention time and mass spectrum of each peak were consistent with glycyrrhetinic acid monoglucuronide.

[0219] On the other hand, Fig.10 In the negative control sample L shown, a peak corresponding to glycyrrhetinic acid, which is a sugar acceptor substrate, was detected in (b) (white arrow), but no peak was detected at the position corresponding to glycyrrhetinic acid monoglucuronide in (c) (dashed arrow).

[0220] In addition, the results of the substrate feeding test when soybean saponin B was used as the sugar acceptor substrate are shown in Fig.11In the sample M containing soybean-derived Glyma.06G324300 shown in (b), the sample N containing licorice-derived Glyur003152s00037491 shown in (c), and the sample O containing Lj3g3v1981230 derived from Japanese lotus root shown in (d), a peak (black arrow) corresponding to soybean saponin B monoglucuronide was detected in which one molecule of glucuronic acid was added to soybean saponin B. The retention time and mass spectrum of the peak were well consistent with soybean saponin B monoglucuronide. On the other hand, in the negative control sample P shown in (e), no peak corresponding to soybean saponin B monoglucuronide was detected.

[0221] The above results and the results obtained in Example 10 indicate that the soybean-derived novel enzyme Glyma.06G324300 obtained in Example 1, the licorice-derived novel enzyme Glyur003152s00037491 obtained in Example 3, and the Japanese lotus-derived novel enzyme Lj3g3v1981230 obtained in Example 5 have the glucuronic acid first transfer activity of converting glycyrrhetinic acid into glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the 3-hydroxyl group of glycyrrhetinic acid. In addition, it was demonstrated that the enzyme has the glucuronic acid first transfer activity of converting soyasapogenol B into soyasapogenol B monoglucuronide by also transferring glucuronic acid to the 3-hydroxyl group of soyasapogenol B. Based on the above, the obtained novel enzyme was identified as the glucuronic acid first transferase that transfers glucuronic acid to the 3-hydroxyl group of oleanane-type triterpenoid compounds.

[0222] <Example 14: Isolation of a functionally defective mutant of the Glyma.06G324300 homologous gene of Lotus japonicus>

[0223] Based on the sequence information of genes and proteins of Japanese lotus and Lotus Base (https: / / lotus.au.dk / ), which is their expression database, mutant systems with LORE insertion were searched for Lj3g3v1981230, and 19 systems were hit. From these systems, two systems (30006020 and 30115796) were selected based on the insertion position of LORE1 in Lj3g3v1981230 and the number of LORE1 insertions in other genes. Seeds were obtained from the distribution agency (Denmark Aarhus University). The seeds were sown, genomic DNA was extracted from a part of the expanded cotyledons, and the insertion of LORE1 into Lj3g3v1981230 was confirmed by PCR. PCR was performed using GoTaq (registered trademark) Colorless Master Mix (Promega) at an annealing temperature of 60°C and a reaction temperature of 72°C for 25 cycles. PCR used a forward primer (30006020 is sequence number 26, 30115796 is sequence number 28), a reverse primer (30006020 is sequence number 27, 30115796 is sequence number 29), and a P2 primer (sequence number 30).

[0224] <Example 15: Analysis of triterpenoid saponin composition of Glyma.06G324300 homologous gene functional defective mutant of Lotus japonicus>

[0225] After sowing in Example 14, the whole plant body of the Glyma.06G324300 homologous gene functional defective mutant system (30006020, 30115796) of Japanese lotus was freeze-dried after 1 month, and then 80% methanol in an amount of 10 times the dry weight was added. Oscillating at room temperature for 1 hour, centrifuged at 15krpm for 5 minutes. The supernatant obtained by centrifugation was purified with a membrane filter with a pore size of 0.45μm (GLクロマトディスク4P, GLサイエンス), and 2μL of each extract was provided for LC-PDA / MS / MS analysis. The device used was Ultimate 3000SD HPLC / LTQ orbitrap discovery MS (both from Thermo Fisher Scientific). The extract was loaded onto a reverse phase column (C30, Develosil C30-UG-3, Nomura Chemical), and saponins were eluted using a linear gradient (20-80% / 60 minutes) of acetonitrile containing 0.1% (v / v) formic acid at a flow rate of 0.15 ml / min. The eluate was detected by UV absorption and mass spectrometry (using an electrostatic field orbital trap (Orbitrap) type for parent ions and an ion trap type for fragment ions). The eluate vaporized / positively ionized by electrospray ionization was injected into the mass spectrometer. Soybean saponin Bb (m / z=943.52) was used as the standard for analysis, and each saponin molecule was annotated by using the fragment pattern analyzed by MS / MS.

[0226] The result is Figure 12-1 and Figure 12-2 In the results, as shown in (b) and (c), saponins (Bb, βg, etc.) that are normally accumulated in the homozygous mutant (mutant homo) were below the detection limit, and abnormalities in the saponin composition were confirmed. This result indicates that the Glyma.06G324300 homologous gene (Lj3g3v1981230) of Lotus japonicus actually functions in the saponin biosynthesis system in vivo.

[0227] <Example 16: Construction of expression vector for Lotus japonicus>

[0228] Using the cloning vector containing the licorice-derived Glyma.06G324300 homologous gene prepared in Example 4 as a template, a forward primer (sequence number 31) and a reverse primer (sequence number 32) that amplify sequence number 4 from the start codon to the stop codon were used, and PCR was performed for 30 cycles using PrimeSTAR GXL DNA Polymerase (タカラバイオ) at an annealing temperature of 60°C and a reaction temperature of 68°C. The amplified DNA fragment was cloned into pDONR by a base sequence-specific recombination reaction (GATEWAY attB×attP reaction) using Gateway BP ClonaseII Enzyme Mix (Thermo Fisher Technologies). TM 221, the polynucleotide sequences of the three independent clones obtained were determined and confirmed to be identical to SEQ ID NO: 4. A plasmid pDONR-Glyur003152s00037491 containing the polynucleotide was obtained as an entry clone.

[0229] Next, using the cloning vector containing the Glyma.06G324300 homologous gene from Lotus japonicus prepared in Example 5 as a template, PCR was performed in the same manner as above using a forward primer (SEQ ID NO: 33) and a reverse primer (SEQ ID NO: 34) that amplifies sequence number 6 from the start codon to the stop codon, and cloned into pDONR TM 221, the polynucleotide sequences of the three independent clones obtained were determined and confirmed to be consistent with sequence number 6. The plasmid pDONR-Lj3g3v1981230 containing the polynucleotide was obtained as an entry clone. TMIn order to achieve the base sequence-specific recombination reaction (GATEWAY attB×attP reaction) during cloning of 221 (Thermo Fisher Technologies), the forward primer added the 12 bases shown in sequence number 35 (AAAAAGCAGGCT) at the 5' end, and the reverse primer added the 12 bases shown in sequence number 36 (AGAAAGCTGGGT) at the 5' end. The plasmid (entry clone) pDONR-Glyma.06g324300 having the polynucleotide shown in sequence number 2 or the plasmid (entry clone) pDONR-Glyur003152s00037491 having the polynucleotide shown in sequence number 4, and the plasmid (entry clone) pDONR-Lj3g3v1981230 having the polynucleotide shown in sequence number 6 prepared in Example 1 were mixed with the target vector pG35NGw, and the DNA fragment shown in sequence number 6 was transferred to pCAMBIA-G35NGw by a base sequence-specific recombination reaction (GATEWAY attL×attR reaction) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Technologies), thereby obtaining the Japanese lotus transformation vector pG35N-LjCSL containing the Glyma.06G324300 homologous gene derived from Japanese lotus shown in sequence number 6. In addition, the soybean-derived Glyma.06G324300 gene shown in SEQ ID NO: 2 and the licorice-derived Glyma.06G324300 homologous gene shown in SEQ ID NO: 4 were transferred by the same method as above to obtain Lotus japonicus transformation vectors pG35N-GmCSL and pG35N-GuCSL, respectively.

[0230] <Example 17: Rescue experiment by introducing homologous genes of soybean Glyma.06G324300, licorice and Lotus japonicus Glyma.06G324300 into Lotus japonicus mutants>

[0231] The Glyma.06G324300 homologous gene was introduced into the Glyma.06G324300 homologous gene functional defective mutant of Lotus japonicus according to the method described in Diaz et al., (2005) Induction of hairyroots for symbiotic gene expression studies. In Lotus japonicus Handbook, AJ Marquez, ed (Dordrecht, The Netherlands: Springer), pp. 261-277. Seeds obtained from the mutant homozygous system of the Glyma.06G324300 homologous gene functional defective mutant 30006020 of Lotus japonicus obtained in Example 14 were sterilized with hypochlorous acid (containing 0.02% Tween 20) with an effective chlorine concentration of 2% for 20 minutes and then allowed to absorb water in sterile distilled water overnight. After removing the seed coat of the seeds after absorbing water, inoculate them in a 0.8% water agar medium, shade them with aluminum foil, culture them at 25°C for 4 days, and then irradiate them with light for 1 day. The vector prepared in Example 16 was introduced into Agrobacterium (LBA1334), plated in front of the L medium, and cultured at 28°C for 1 day. After suspending the Agrobacterium cultured in 10 mL of sterile water for 1 day, adding it to a round sterile culture dish, soak the seedlings of the mutant homozygous system of the Glyma.06G324300 homologous gene functional defect mutant 30006020 of Japanese lotus, and cut the hypocotyl with a razor blade. The cut seedlings were placed in a co-culture medium, shaded with aluminum foil, and co-cultured at 21°C for 4 days. After co-culture, the plants were placed in HRE medium and grown for 2 weeks with a light period of 16 hours, 25°C / dark period of 8 hours, and 23°C. Plants that produced hairy roots were confirmed to have GFP fluorescence under a fluorescent stereomicroscope.

[0232] <Example 18: Analysis of the triterpenoid saponin composition of hairy roots of Lotus japonicus>

[0233] The plants with hairy roots obtained in Example 17 were transplanted into a pot filled with vermiculite, and B&D hydroponic solution (Diaz et al., 2005) was added to grow for 1 month. The fully grown plants were freeze-dried and crushed at 2500 rpm for 30 seconds using a multi-bead oscillator (Yasui Instruments Co., Ltd.). 80% methanol in an amount 100 times the weight of the freeze-dried material was added, and after shaking at room temperature for 1 hour, the mixture was centrifuged at 15krpm for 5 minutes to recover the supernatant. The supernatant was analyzed by LC-PDA / MS / MS using the method shown in Example 15. The results are as follows. Fig.13As shown, saponins that disappeared in the mutant were restored in the transformed hairy roots, suggesting that the Glyma.06G324300 homologous gene also catalyzes saponin synthesis in vivo.

[0234] <Example 19: Exploration and Isolation of Homologous Genes of Glyma.06G324300 from Astragalus membranaceus>

[0235] From Astragalus sinicus, a leguminous plant like soybean, a Glyma.06G324300 homologous gene was explored as a candidate for interspecific homologous gene of Glyma.06G324300 through gene homology search. From a sequence data set integrating RNA sequence data obtained from roots, stems, and leaves of Astragalus sinicus, a base sequence AsCSyGT encoding a protein showing high amino acid identity with Glyma.06G324300 was found. Using 1 μg of total RNA obtained from the stem of Astragalus sinicus, the first-strand cDNA was synthesized using SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μL of the first-strand cDNA as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide predicted by AsCSyGT were used as forward primers (SEQ ID NO: 39) and reverse primers (SEQ ID NO: 40), respectively, and PrimeSTAR Max DNA Polymerase (Takara Bio) was used to perform PCR for 30 cycles at an annealing temperature of 55°C and a reaction temperature of 72°C. TM / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies), the forward primer had 4 bases (cacc) artificially added to the 5' end. The amplified DNA fragment was cloned into pENTR TM / D-TOPO entry vector, the polynucleotide sequences of the two independent clones obtained were determined. The base sequence of the Glyma.06G324300 homologous gene of Astragalus membranaceus obtained in this way is sequence number 41, and the polypeptide sequence deduced therefrom is sequence number 42. Sequence number 42 has 77% identity with the amino acid sequence shown in sequence number 1.

[0236] <Example 20: Isolation of Glyma.06G324300 Homologous Genes from Soybean>

[0237] Glyma.06G324300 homologous genes were discovered as intraspecific homologous gene candidates of Glyma.06G324300 by gene homology search in soybean. Using the BLAST homology search function in Soybase (https: / / soybase.org), a soybean genome information database, two base sequences, Glyma.04g255400 and Glyma.11g151800, encoding proteins showing high amino acid identity with Glyma.06G324300 were discovered. 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-terminus and C-terminus of the polypeptide inferred by Glyma.04g255400 were used as forward primers (SEQ ID NO: 43) and reverse primers (SEQ ID NO: 44), and oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide inferred by Glyma.11g151800 were used as forward primers (SEQ ID NO: 45) and reverse primers (SEQ ID NO: 46). In addition, due to the addition of pDONR TM In order to meet the need for base sequence-specific recombination reaction (GATEWAY attB×attP reaction) during cloning of pDONR 221 (Thermo Fisher Technologies), the forward primer had 12 bases (AAAAAGCAGGCT) artificially added to the 5' end, and the reverse primer had 12 bases (AGAAAGCTGGGT) artificially added to the 5' end. The DNA fragment amplified from the first-strand cDNA of the seed was cloned into pDONR 221 (Thermo Fisher Technologies) by base sequence-specific recombination reaction (GATEWAY attB×attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies). TM 221, the polynucleotide sequences of the three independent clones obtained were determined. The sequences obtained are SEQ ID NOs: 47 and 49, and the polypeptide sequences deduced from them are SEQ ID NOs: 48 and 50. SEQ ID NOs: 48 and 50 have 93.9% and 71.1% identities, respectively, with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0238] <Example 21: Introduction of Glyma.06G324300 homologous genes of Astragalus sinensis and soybean into yeast strains producing glycyrrhetinic acid and soybean saponin B>

[0239] By the method shown in Example 7, pESC-HIS-AsCSyGT, pESC-HIS-AtUGD2-Glyma04g255400, and pESC-HIS-AtUGD2-Glyma.11g151800 were constructed as yeast expression clones of the Glyma.06G324300 homologous genes of the astragalus obtained in Example 19 and the soybean obtained in Example 20, and were introduced into the glycyrrhizic acid and soybean saponin B production yeast strains, respectively, by the method shown in Example 8.

[0240] <Example 22: In vivo enzyme assay using recombinant yeast into which Glyma.06G324300 homologous genes of Astragalus membranaceus and soybean were introduced>

[0241] The recombinant yeast was cultured and metabolites were extracted by the method shown in Example 9. As a result, a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (AsCSyGT) shown in sequence number 42 (sample Q), a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (Glyma04g255400) shown in sequence number 48 (sample R), and a metabolite extract derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide (Glyma.11g151800) shown in sequence number 50 (sample S) were obtained. The soybean saponin B-producing yeast strains holding pESC-HIS-AsCSyGT, pESC-HIS-AtUGD2-Glyma04g255400, and pESC-HIS-AtUGD2-Glyma.11g151800 were similarly cultured and metabolites were extracted. As a result, a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 42 (AsCSyGT) (sample T), a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 48 (Glyma04g255400) (sample U), and a metabolite extract derived from a soybean saponin B-producing yeast strain expressing the polypeptide shown in sequence number 50 (Glyma.11g151800) (sample V) were obtained.

[0242] <Example 23: Analysis of Metabolite Extracts from Yeast Introduced with Glyma.06G324300 Homologous Genes from Astragalus and Soybean>

[0243] The sample for LC-MS analysis was prepared by the method shown in Example 10 and analyzed. The results are shown in Fig.14 and Fig.15 .

[0244] Depend on Fig.14In the sample Q of (a) shown, a single peak (black arrow) corresponding to glycyrrhetinic acid monoglucuronide was detected. The retention time and mass spectrum of the peak are well consistent with those of glycyrrhetinic acid monoglucuronide. Similarly, peaks (black arrows) corresponding to glycyrrhetinic acid monoglucuronide were also detected from the sample R of (b) and the sample S of (c). The retention time and mass spectrum of each peak are well consistent with those of glycyrrhetinic acid monoglucuronide.

[0245] Depend on Fig.15 A single peak (black arrow) corresponding to soybean saponin B monoglucuronide was detected in sample T of (a). Peaks (black arrows) corresponding to soybean saponin B monoglucuronide were also detected in sample U of (b) and sample V of (c). The retention time and mass spectrum of each peak were consistent with soybean saponin B monoglucuronide.

[0246] The above results show that AsCSyGT derived from Astragalus obtained in Example 19 and Glyma04g255400 and Glyma.11g151800 derived from soybean obtained in Example 20 have the glucuronic acid first transfer activity of converting glycyrrhetinic acid into glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the 3-hydroxyl group of glycyrrhetinic acid. In addition, it was confirmed that they have the glucuronic acid first transfer activity of converting soyasaponin B into soyasaponin B monoglucuronide by transferring glucuronic acid to the 3-hydroxyl group of soyasaponin B.

[0247] <Example 24: Preparation of transformed yeast for substrate feeding test of soybean Glyma.06G324300 homologous gene>

[0248] The yeast INVScI strain was introduced with pESC-HIS-AtUGD2-Glyma04g255400 and pESC-HIS-AtUGD2-Glyma.11g151800 obtained in Example 21. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

[0249] <Example 25: Substrate feeding test using transformed yeast into which soybean Glyma.06G324300 homologous gene was introduced>

[0250] The recombinant yeast obtained in Example 24 was cultured by the method described in Example 12. The suspension of each yeast cell obtained was divided equally, and ursolic acid, an ursane-type triterpenoid compound, or betulinic acid, a lupine-type triterpenoid compound, was added to each of them at a final concentration of 5 μM. Then, the cells were cultured again and the metabolites were extracted by the method described in Example 12. As a result, a feeding test extract in which ursolic acid, an ursane-type triterpenoid compound, and a feeding test extract in which betulinic acid, a lupine-type triterpenoid compound, were added to the transformed yeast expressing the polypeptide described in sequence number 48 (Glyma04g255400) or the polypeptide described in sequence number 50 (Glyma.11g151800) were added was obtained.

[0251] <Example 26: Analysis of extracts from a substrate feeding test using transformed yeast into which a soybean Glyma.06G324300 homologous gene was introduced>

[0252] The sample obtained in Example 25 was analyzed under the same conditions as in Example 10. The MS was performed in SIM mode, with the m / z of each hypothetical reaction product being ursolic acid monoglucuronide = 631 ( Fig.16 , a), betulinic acid monoglucuronide = 631 ( Fig.17 , a) are analyzed as parameters. Fig.16 This is the analysis result of the feed test extract (sample W) to which ursolic acid was added to the transformed yeast expressing the polypeptide (Glyma.11g151800) shown in SEQ ID NO: 50. A peak estimated to be ursolic acid monoglucuronide was detected from sample W shown in (b). On the other hand, in sample X shown in (c) as a negative control, no peak estimated to be ursolic acid monoglucuronide was detected.

[0253] Fig.17 The results of analysis of the extract (sample Y) of the feeding test to which betulinic acid was added to the transformed yeast expressing the polypeptide (Glyma.11g151800) shown in SEQ ID NO: 50. A peak estimated to be betulinic acid monoglucuronide was detected from sample Y shown in (b). On the other hand, a peak estimated to be betulinic acid monoglucuronide was not detected in sample Z shown in (c), which was a negative control.

[0254] Based on the above considerations, the polypeptide shown in sequence number 50 (Glyma.11g151800) is a first glucuronyl transferase that can transfer glucuronic acid to the 3-hydroxyl group not only to oleanane-type triterpenoids, but also to ursane-type triterpenoids such as ursolic acid and β-boswellic acid, and lupenic acid-type triterpenoids such as betulinic acid.

[0255] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

1. Yeast or its progeny comprising the polynucleotide or CSyGT expression vector, The CSyGT is a glucuronyl transferase 1, The polynucleotide encodes a polypeptide having an activity of transferring glucuronic acid to the 3-hydroxyl group of glycyrrhetinic acid and consisting of the amino acid sequence shown in sequence number 3, The CSyGT expression vector comprises the polynucleotide, The progeny maintain the polynucleotide or the CSyGT expression vector. 2 . The yeast or its progeny according to claim 1 , wherein the polynucleotide comprises the base sequence shown in SEQ ID NO:

4.

3. Use of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 for producing glycyrrhetinic acid monoglucuronide from glycyrrhetinic acid and glucuronic acid.

4. A method for producing glycyrrhetinic acid monoglucuronide, comprising reacting glycyrrhetinic acid with glucuronic acid in the presence of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.

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