Glucuronyltransferase, gene encoding same and method for using same

Abstract

To identify glucuronosyltransferase 1 gene which catalyzes glucuronic acid transfer to the hydroxyl group at the 3-position in an oleanane-type triterpenoid.SOLUTION: A glucuronosyltransferase 1 gene having a desired activity, derived from a Fabaceae plant (soybean, Glycyrrhiza, and Lotus japonicus), and comprising nucleotide sequences represented by SEQ ID Nos: 2, 4, and 6, respectively, is provided.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Licorice (Glycyrrhiza uralensis) is a perennial herbaceous plant of the Fabaceae family. The roots and stolon (underground stem) of this plant are known as "licorice," an important herbal medicine in traditional Chinese medicine, and are widely used worldwide. The main active ingredient in licorice is glycyrrhizin, an oleanane-type triterpenoid saponin (Non-Patent Document 1). Research on glycyrrhizin is being conducted from various aspects, including its pharmacognostical and pharmacological usefulness, and breeding research.

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

[0004] Glycyrrhizin is commonly found in plants and is biosynthesized through two oxidation and two glycosylation reactions starting from β-amyrin, an oleanane-type triterpenoid. β-amyrin is known to be a precursor substance that is the branching point for the biosynthesis of glycyrrhizin and soyasaponin I in the triterpenoid saponin biosynthesis pathway (Figure 1).

[0005] As shown in Figure 2, the enzymes known to synthesize glycyrrhizin from β-amyrin include two oxidases, CYP88D6 (Patent Document 1) and CYP72A154 (Patent Document 2), which catalyze the two oxidation steps required for biosynthesis of glycyrrhetinic acid, the aglycone (non-sugar moiety) of glycyrrhizin, from β-amyrin, and the glycosyltransferase UGT73P12 (Patent Document 3), which catalyzes the second step of the two glycosylations of the resulting glycyrrhetinic acid. However, despite numerous attempts by many skilled in the art to isolate the glycosyltransferase that catalyzes the first glycosylations, i.e., glucuronosyltransferase 1, which directly transfers glucuronic acid to glycyrrhetinic acid, has not been isolated to date. This has been a bottleneck in synthesizing glycyrrhizin from β-amyrin in biological production systems or in vitro synthesis systems, making it impossible to obtain sufficient amounts of glycyrrhizin stably and sustainably. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5526323 [Patent Document 2] Patent No. 5771846 [Patent Document 3] Patent No. 6344774 [Non-patent literature]

[0007] [Non-Patent Document 1] Gibson, MR, 1978, Lloydia-the journal of Natural Products, 41(4): 348-354 Summary of the Invention [Problem to be solved by the invention]

[0008] In order to solve the above problems, the present invention aims to isolate the gene for glucuronosyltransferase 1, which catalyzes the transfer of glucuronidyl to the hydroxyl group at the 3-position of oleanane-type triterpenoids, including glycyrrhetinic acid, and to create and provide a biological expression system using this gene expression system that can biosynthesize large amounts of compounds from β-amyrin to glycyrrhizin within an individual or cells. [Means for solving the problem]

[0009] Many experts in the field have attempted to isolate the last remaining glucuronosyltransferase in the biosynthetic pathway from β-amyrin to glycyrrhizin from licorice, but have been unable to do so for a long time. Therefore, the present inventors suspected that there were some factors inhibiting the isolation of glucuronosyltransferase from plants in the genus Glycyrrhizin, and attempted a strategy to isolate glucuronosyltransferase from plants other than Glycyrrhizin. Leguminous plants, which include Glycyrrhizin, generally have a pathway for biosynthesis of soyasaponin I from β-amyrin via the intermediate product soyasapogenol B (Figure 1). Soyasapogenol B, an oleanane-type triterpenoid, has a hydroxyl group at the 3-position, while the final product, soyasaponin I, has a glucuronic acid bond at the 3-position. This is similar to the fact that glycyrrhetinic acid, an intermediate product and oleanane-type triterpenoid, has a hydroxyl group at position 3, while the final product, glycyrrhizin, has a glucuronic acid bond at position 3. In other words, glucuronosyltransferase I, which functions in the biosynthetic pathway from β-amyrin to glycyrrhizin, may also function in the biosynthetic pathway from β-amyrin to soyasaponin I. Based on this hypothesis, the present inventors attempted to identify genes with glucuronosyltransferase I activity from soybean, which has the biosynthetic pathway from β-amyrin to soyasaponin I, and succeeded in isolating a cellulose synthase-like gene with unknown function. Testing the glycosyltransferase activity of this enzyme using soyasapogenol B as a glycosyl acceptor substrate revealed that it transfers glucuronic acid to the hydroxyl group at position 3 of soyasapogenol B. This enzyme activity was similar when the glycosyl acceptor substrate was glycyrrhetinic acid. Thus, by changing the source plant from Glycyrrhiza to soybean, the present inventors succeeded in identifying a glucuronosyltransferase that had not previously been isolated. The present invention is based on these research results and provides the following:

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

[0011] According to the present invention, there can be provided a polypeptide having an activity of transferring glucuronic acid to the hydroxy group at the 3-position of an oleanane-type triterpenoid, a polynucleotide encoding the polypeptide, or a method utilizing the same. [Brief explanation of the drawings]

[0012] [Figure 1] The biosynthetic pathway of glycyrrhizin and soyasaponin I in the triterpenoid saponin biosynthesis system is shown. [Figure 2]The diagram shows each oleanane-type triterpenoid that occurs as an intermediate product in the biosynthetic pathway from β-amyrin to glycyrrhizin, the known enzymes involved in each conversion reaction (CYP88D6, CYP72A154, and UGT73P12), and the catalytic site of the newly identified glucuronosyltransferase CSyGT. [Figure 3] Schematic diagrams showing triterpenoid-producing yeast strains used in the examples for functional analysis of soybean Glyma.06G324300 and Glyma.06G324300 homologous proteins derived from licorice and Lotus japonicus. The yeast strain (a) can intracellularly produce glycyrrhetinic acid from endogenous 2,3-oxidosqualene by co-expressing the β-amyrin synthase gene (derived from Lotus japonicus), CYP88D6 (derived from licorice), and CYP72A63 (derived from Medicago sativa). The yeast strain (b) can intracellularly produce soyasapogenol B from endogenous 2,3-oxidosqualene by co-expressing the β-amyrin synthase gene (derived from Lotus japonicus), CYP93E3 (derived from licorice), and CYP72A566 (derived from licorice). [Figure 4] This figure shows the results of metabolite analysis of glycyrrhetinic acid-producing yeast into which Glyma.06G324300 and homologous genes were introduced. (a) Sample A shows the detection results of glycyrrhetinic acid monoglucuronide produced by expressing soybean-derived Glyma.06G324300, (b) Sample B shows the detection results of licorice-derived Glyur003152s00037491, and (c) Sample C shows the detection results of lotus-derived Lj3g3v1981230 in glycyrrhetinic acid-producing yeast. The peak indicated by the black arrow indicates glycyrrhetinic acid monoglucuronide. (d) Sample D shows the detection results of glycyrrhetinic acid-producing yeast into which an empty vector was introduced as a negative control. [Figure 5]This figure shows the results of metabolite analysis of soyasapogenol B-producing yeast cells transfected with Glyma.06G324300 and homologous genes. (a) Sample E expresses soybean-derived Glyma.06G324300, (b) Sample F expresses licorice-derived Glyur003152s00037491, and (c) Sample G expresses Lotus japonicus-derived Lj3g3v1981230 in soyasapogenol B-producing yeast cells. The peak indicated by the black arrow indicates soyasapogenol B monoglucuronide. (d) Sample H expresses soyasapogenol B-producing yeast cells transfected with an empty vector as a negative control. [Figure 6] This figure shows an outline of a substrate feeding experiment. To supply the sugar donor substrate UDP-glucuronic acid intracellularly, Arabidopsis thaliana UDP-glucose dehydrogenase (AtUGD), Glyma.06G324300, and its homologous genes were co-expressed in yeast. (a) shows the conversion reaction to monoglucuronide when glycyrrhetinic acid was added to the yeast culture medium, and (b) shows the conversion reaction when soyasapogenol B was added to the yeast culture medium. [Figure 7] Figures showing the results of metabolite analysis in yeast introduced with Glyma.06G324300 derived from soybeans and fed glycyrrhetinic acid. (a) Schematic diagram of the expected conversion reaction to glycyrrhetinic acid monoglucuronide when using Glyma.06G324300. (b) Shows the results of detection of glycyrrhetinic acid before conversion. The peak indicated by the white arrow represents glycyrrhetinic acid. (c) Shows the results of detection of glycyrrhetinic acid monoglucuronide produced by Glyma.06G324300. The peak indicated by the black arrow represents glycyrrhetinic acid monoglucuronide. [Figure 8]Figures showing the results of metabolite analysis in yeast introduced with Glyur003152s00037491 derived from licorice and fed with glycyrrhetinic acid. (a) Schematic diagram of the expected conversion reaction to glycyrrhetinic acid monoglucuronide when using Glyur003152s00037491. (b) Detection results for glycyrrhetinic acid before conversion. The peak indicated by the white arrow represents glycyrrhetinic acid. (c) Detection results for glycyrrhetinic acid monoglucuronide produced by Glyur003152s00037491. The peak indicated by the black arrow represents glycyrrhetinic acid monoglucuronide. [Figure 9] Figures showing the results of metabolite analysis in Lotus japonicus-derived Lj3g3v1981230-introduced yeast fed glycyrrhetinic acid. (a) Schematic diagram of the expected conversion reaction to glycyrrhetinic acid monoglucuronide when Lj3g3v1981230 is used. (b) Detection results for glycyrrhetinic acid before conversion. The peak indicated by the white arrow represents glycyrrhetinic acid. (c) Detection results for glycyrrhetinic acid monoglucuronide produced by Lj3g3v1981230. The peak indicated by the black arrow represents glycyrrhetinic acid monoglucuronide. [Figure 10] 7 to 9 show the results of metabolite analysis in the empty vector-introduced yeast fed with glycyrrhetinic acid, which are negative controls. (a) A conceptual diagram of the expected conversion reaction to glycyrrhetinic acid monoglucuronide when an empty vector is used. Due to the lack of enzyme activity, it is expected that glycyrrhetinic acid monoglucuronide will not be produced. (b) The results of detection of glycyrrhetinic acid before the reaction are shown. The peak indicated by the white arrow represents glycyrrhetinic acid. (c) The results of detection of glycyrrhetinic acid monoglucuronide after the reaction are shown. The peak indicated by the black arrow represents glycyrrhetinic acid monoglucuronide. The dashed arrow indicates the position of the peak when glycyrrhetinic acid monoglucuronide is produced. [Figure 11]Figure 1 shows the results of metabolite analysis of Glyma.06G324300 and its homologous gene-transfected yeast fed soyasapogenol B. (a) Schematic diagram of the assumed conversion reaction from soyasapogenol B to soyasapogenol B monoglucuronide. (b) Detection results of soyasapogenol B monoglucuronide produced by soybean-derived Glyma.06G324300. The peak indicated by the black arrow represents soyasapogenol B monoglucuronide. (c) Detection results of soyasapogenol B monoglucuronide produced by licorice-derived Glyur003152s00037491. The peak indicated by the black arrow represents soyasapogenol B monoglucuronide. (d) Detection results of soyasapogenol B monoglucuronide produced by Lotus japonicus-derived Lj3g3v1981230. The peak indicated by the black arrow indicates soyasapogenol B monoglucuronide. (e) Negative control for (b) to (d). [Figure 12-1] LC-PDA / MS / MS analysis of extracts from loss-of-function mutants of the Glyma.06G324300 homologous gene in Lotus japonicus (Lotus japonicus). (a) shows the base peak ion chromatogram of the wild-type (Gifu) plant extract, and (b) and (c) show the base peak ion chromatogram of the transposon insertion homozygous mutants (30006020 and 30115796, respectively). [Figure 12-2] Figure 1 shows the results of LC-PDA / MS / MS analysis of extracts from loss-of-function mutants of the Glyma.06G324300 homologous gene in 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 extract derived from the transposon insertion homozygous mutants (30006020 and 30115796, respectively). [Figure 13]LC-PDA / MS / MS analysis of loss-of-function mutant hairy roots into which a Glyma.06G324300 homologous gene had been introduced. (a) shows the total ion chromatograms of hairy root extracts from a wild-type (Gifu) strain into which an empty vector (pG35N_empty) had been introduced, and (b) to (e) show the total ion chromatograms of hairy root extracts from transformants into a Glyma.06G324300 homologous gene loss-of-function homozygous mutant (30115796) into which pG35N_empty and the expression vectors pG35N-GmCSL, pG35N-GuCSL, and pG35N-LjCSL had been introduced, respectively. [Figure 14] This figure shows the results of metabolite analysis in glycyrrhetinic acid-producing yeast cells transfected with the astragalus Glyma.06G324300 ortholog gene and the soybean Glyma.06G324300 paralog gene. Sample Q (a) expresses the Astragalus Glyma.06G324300 ortholog gene, the Astragalus Glyma.06G324300 Astragalus Glyma.04g255400 gene (b) expresses the soybean Glyma.06G324300 paralog gene, the soybean Glyma.06G324300 paralog gene, the soybean Glyma.11g151800 gene (c) expresses the soybean Glyma.06G324300 paralog gene. The black arrows indicate the peaks of glycyrrhetinic acid monoglucuronide. [Figure 15] This figure shows the results of metabolite analysis in soyasapogenol B acid-producing yeast cells transfected with the astragalus Glyma.06G324300 orthologous gene and the soybean Glyma.06G324300 paralogous gene. (a) Sample T expresses the astragalus-derived AsCSyGT gene; (b) Sample U expresses the soybean-derived Glyma04g255400 gene; (c) Sample V expresses the soybean-derived Glyma.11g151800 gene. The results show the detection of soyasapogenol B monoglucuronide produced by these expression in soyasapogenol B-producing yeast cells. The peaks indicated by black arrows indicate soyasapogenol B monoglucuronide. [Figure 16]Figures showing the results of metabolite analysis in yeast introduced with the soybean-derived Glyma.11g151800 gene and fed with sorbic acid. (a) Schematic diagram of the expected conversion reaction to sorbic acid monoglucuronide when using a Glyma.11g151800 feeding assay extract (sample W). (b) Shows the results of detection of sorbic acid monoglucuronide produced by Glyma.11g151800 when sample W was added. The peak indicated by the black arrow indicates sorbic acid monoglucuronide. (c) Shows the results of detection of sorbic acid monoglucuronide when the negative control sample X was added. [Figure 17] Figures showing the results of metabolite analysis in yeast introduced with the Glyma.11g151800 gene derived from soybean fed betulinic acid. (a) Schematic diagram of the expected conversion reaction to betulinic acid monoglucuronide when using a Glyma.11g151800 feeding assay extract (sample Y). (b) Detection results of betulinic acid monoglucuronide produced by Glyma.11g151800 when sample Y was added. The peak indicated by the black arrow indicates betulinic acid monoglucuronide. (c) Detection results of betulinic acid monoglucuronide when the negative control sample Z was added. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1. Glucuronyltransferase (CSyGT) 1-1. Overview A first aspect of the present invention relates to glucuronoyl transferases and fragments thereof having glucuronoyl transferase activity, as well as nucleic acids encoding them. The glucuronoyl transferases of the present invention catalyze the glycosylation of glucuronic acid to the 3-hydroxyl group of glycyrrhetinic acid, which is obtained via a two-step oxidation reaction from β-amyrin, a synthesis pathway specific to the genus Licorice, in which glycyrrhizin is biosynthesized from β-amyrin, which can be biosynthesized by many plants. The glucuronoyl transferases of the present invention not only enable the glycosylation of glucuronic acid to the 3-hydroxyl group of oleanane-type triterpenoids, including glycyrrhetinic acid, but also enable the creation of a biological production system from β-amyrin to glycyrrhizin using plants other than those of the genus Licorice as hosts, by combining them with known enzymes involved in the biosynthetic pathway from β-amyrin to glycyrrhizin. This allows for the stable and sustained provision of high-quality glycyrrhizin.

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

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

[0016] As used herein, "glucuronoyltransferase activity" refers to the activity of CSyGT to catalyze a glycosyltransferase reaction, i.e., the activity of transferring glucuronic acid to the hydroxyl group at the 3-position of an oleanane triterpenoid. This activity results in the production of an oleanane triterpenoid monoglucuronide from an oleanane triterpenoid. In this specification, CSyGT and fragments thereof having glucuronoyltransferase activity are often collectively referred to as "polypeptides having glucuronoyltransferase activity" or "glucuronoyltransferase (CSyGT, etc.)." In this specification, glucuronoyltransferase may be a glycoprotein having a different sugar chain attached thereto. For example, both a glucuronoyltransferase having a sugar chain attached thereto derived from a plant and a glucuronoyltransferase having a sugar chain attached thereto derived from a yeast are included in the glucuronoyltransferase as used herein.

[0017] "Oleanane-type triterpenoid" refers to a C30 isoprenoid having a pentacyclic oleanane skeleton and six isoprene units. It corresponds to the non-sugar portion (aglycone) of glycyrrhizin, the final target of the present invention. Unless otherwise specified, the oleanane-type triterpenoid described herein refers to an oleanane-type triterpenoid having a hydroxyl group (OH group) at the third carbon. Specific examples of oleanane-type triterpenoids include, but are not limited to, β-amyrin, 11-oxo-β-amyrin, 30-hydroxy-11-oxo-β-amyrin, 30-hydroxy-β-amyrin, 24-hydroxy-β-amyrin, 11-deoxoglycyrrhetinic acid, glycyrrhetinic acid, oleanolic acid, medicagenic acid, soyasapogenol B, soyasapogenol A, hederagenin, camelliagenin, and saicogenin. All of these can be substrates for the glucuronyl transferase of the present invention. The glucuronyl transferase activity converts the substrates into β-amyrin-3-O-monoglucuronide, 11-oxo-β-amyrin-3-O-monoglucuronide, 30-hydroxy-11-oxo-β-amyrin-3-O-monoglucuronide, 30-hydroxy-β-amyrin-3-O-monoglucuronide, 24-hydroxy-β-amyrin-3-O-monoglucuronide, 11-deoxoglycyrrhetinic acid ... The glucuronides glycyrrhetinic acid-3-O-monoglucuronide, oleanolic acid-3-O-monoglucuronide, medicagenic acid-3-O-monoglucuronide, soyasapogenol B-3-O-monoglucuronide, soyasapogenol A-3-O-monoglucuronide, hederagenin-3-O-monoglucuronide, camelliagenin-3-O-monoglucuronide, and saikogenin-3-O-monoglucuronide are biosynthesized.

[0018] As used herein, the term "Fabaceae" is not limited to plants of the genus Licorice, but includes all plant species belonging to the botanical family Fabaceae, such as plants of the genus Arachis, chickpea, Aspalathus, Dalbergia, Pterocarpus, Desmodium, Lespedeza, Uraria, Galegeae, Astragalus, Glycyrrhiza, and Scutellaria. Oxytropis plants, Augyrocytisus plants, Cytisus plants, Genista plants, Spartium plants, Hedysarum plants, Cyamopsis plants, Indigofera plants, Lotus plants, Lupinus plants, Wisteria plants, Cajanus plants Plants, Canavalia plants, Erythrina plants, Glycine plants, Hardenbergia plants, Lablab plants, Mucuna plants, Phaseolus plants, Psophocarpus plants, Pueraria plants, Vigna plants, Robinia plants, Castanospermum plants nospermum plants, Maackia plants, Ormosia plants, Sophora plants, Styphnolobium plants, Medicago plants, Trigonella plants, Trifolium plants, Lathyrus plants, Lens plants, Pisum plants and Vicia plants.Plants of the genus Glycyrrhiza, to which licorice (G. uralensis) belongs, and its closely related species, the genus Medicago, possess a biosynthetic pathway for glycyrrhetinic acid, which serves as a substrate for CSyGT in the biosynthesis of glycyrrhizin, and are therefore suitable as legumes for the present invention. Specific examples of plants of the genus Glycyrrhiza include G. glabra, G. inflata, G. aspera, G. eurycarpa, G. pallidiflora, G. yunnanensis, G. lepidota, G. echinata, and G. acanthocarpa, and specific examples of plants of the genus Medicago include M. truncatula.

[0019] 1-3.Configuration The glucuronosyltransferase (CSyGT) of the present invention is a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 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), and wild-type CSyGT (LjCSyGT) derived from lotus (Lotus japonicus), respectively. GuCSyGT derived from licorice shares 81% amino acid identity with GmCSyGT derived from soybean. LjCSyGT derived from lotus japonicus shares 82% amino acid identity with GmCSyGT derived from soybean.

[0020] In addition to the aforementioned plant species, CSyGT may have orthologs in many other plant species, particularly in legume (Fabaceae) plant species. The CSyGT of the present invention encompasses not only wild-type CSyGT orthologs from other species, but also wild-type CSyGT paralogs and mutant CSyGTs of the same species that have glucuronosyltransferase activity. Examples of wild-type CSyGT orthologs from other species and mutant CSyGTs include amino acid sequences in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NOs: 1, 3, or 5, or polypeptides that have 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but less than 100%, amino acid identity to the amino acid sequence set forth in SEQ ID NOs: 1, 3, or 5. In fact, GuCSyGT and LjCSyGT are orthologues of the soybean GmCSyGT and the licorice and lotus CSyGT, respectively, and share over 80% amino acid identity as mentioned above. Furthermore, mutant CSyGTs with glucuronidyl transferase activity include, but are not limited to, splicing variants and mutants based on SNPs.

[0021] As used herein, "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. Furthermore, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting appropriate gaps into one or both of them as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW.

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

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

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

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

[0026] In the biosynthetic pathway of glycyrrhizin in licorice, the pathway from β-amyrin, an oleanane-type triterpenoid that can be considered the origin material, to glycyrrhetinic acid has already been elucidated, making artificial biosynthesis possible. Furthermore, the pathway by which glycyrrhizin is biosynthesized by further transglycosylating glucuronic acid to glycyrrhetinic acid monoglucuronide, which is formed by the transglycosylation of one molecule of glucuronic acid to glycyrrhetinic acid, has also been elucidated. In other words, in the biosynthetic pathway from β-amyrin to glycyrrhizin, the only pathway previously unknown was the transglycosylation of glucuronic acid to glycyrrhetinic acid to form glycyrrhetinic acid monoglucuronide. However, with the present invention, the entire biosynthetic pathway of glycyrrhizin in licorice has now been elucidated, enabling in vitro synthesis from β-amyrin to glycyrrhizin. Furthermore, since β-amyrin can be biosynthesized in numerous plant species other than licorice, an in vivo synthesis system using common plant species as hosts is also possible. Furthermore, if an organism does not contain β-amyrin but can biosynthesize a precursor to β-amyrin, it may be possible to use it together with a gene that biosynthesizes β-amyrin to create an in vivo synthesis system using organisms other than plants as hosts.

[0027] 2. Glucuronyltransferase gene (CSyGT gene) and its active fragment 2-1. Overview The second aspect of the present invention relates to a polynucleotide encoding the polypeptide (CSyGT, etc.) described in the first aspect, i.e., a glucuronosyltransferase gene and an active fragment thereof. The polynucleotide of the present invention enables the construction of a recombinant vector according to the third aspect described below.

[0028] 2-2.Configuration The term "glucuronosyltransferase gene" (often referred to herein as "CSyGT gene") refers to a polynucleotide encoding the CSyGT described in the first aspect. The nucleotide sequence of the polynucleotide is not particularly limited as long as it encodes CSyGT. A preferred polynucleotide is a wild-type CSyGT encoding the amino acid sequence shown in SEQ ID NO: 1, 3, or 5. Examples of such polynucleotides include a polynucleotide encoding soybean-derived wild-type GmCSyGT consisting of the amino acid sequence shown in SEQ ID NO: 1, specifically a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2, i.e., a soybean-derived wild-type GmCSyGT gene. Other examples include a polynucleotide encoding licorice-derived wild-type GuCSyGT consisting of the amino acid sequence shown in SEQ ID NO: 3, specifically a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4, i.e., a licorice-derived wild-type GuCSyGT gene. Finally, examples of such polynucleotides include a Lotus japonicus-derived wild-type LjCSyGT consisting of the amino acid sequence shown in SEQ ID NO: 5, specifically a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6, i.e., a Lotus japonicus-derived wild-type LjCSyGT gene.

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

[0030] Further examples include polynucleotides containing a nucleotide sequence that has 80% or more, 85% or more, 87% or more, 90% or more, 95% or more, or 99% or more but less than 100% amino acid identity to the wild-type CSyGT gene. Specific examples include polynucleotides containing a nucleotide sequence that has 80% or more, 85% or more, 87% or more, 90% or more, 95% or more, or 99% or more but less than 100% amino acid identity to the nucleotide sequence of any of the wild-type GmCSyGT gene derived from soybean (e.g., a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2), the wild-type GuCSyGT gene derived from licorice (e.g., a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4), and the wild-type LjCSyGT gene derived from Lotus japonicus (e.g., a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6).

[0031] Also included is a polynucleotide that contains a base sequence that hybridizes under highly stringent conditions with a nucleotide fragment consisting of a base sequence complementary to a partial base sequence of the wild-type CSyGT gene, and that retains enzyme activity.

[0032] As used herein, "stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form. "Highly stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form or are not formed at all. Generally, the lower the salt concentration and the higher the temperature of the reaction conditions, the more stringent the conditions. For example, post-hybridization washing conditions include washing with 0.1×SSC and 0.1% SDS at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C. In addition, the stringency of hybridization can be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

[0033] In this embodiment, the term "active fragment thereof" refers to a fragment of the CSyGT gene, the polypeptide encoded by which has CSyGT activity. This essentially refers to a 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, i.e., the number of bases, may be three times the number of amino acids in the active fragment of CSyGT described in the first embodiment.

[0034] The polynucleotide of the present invention or an active fragment thereof can be used to construct a recombinant vector capable of expressing CSyGT and an active fragment thereof in a host cell.

[0035] In this specification, the CSyGT gene and its active fragments are often collectively referred to as "CSyGT gene, etc. (glucuronosyltransferase gene, etc.)."

[0036] The CSyGT gene of this embodiment can be isolated from an appropriate plant, such as a legume, using known methods. For example, a primer pair having an appropriate nucleotide sequence length is designed based on the nucleotide sequence of the wild-type GmCSyGT gene derived from soybean shown in SEQ ID NO: 2. Specific examples include the primer pair shown in SEQ ID NOs: 17 and 18. The GmCSyGT gene can be obtained by performing a nucleic acid amplification reaction such as PCR using the pair and nucleic acids derived from a soybean DNA library or genomic DNA library as a template. Furthermore, the polynucleotide of the present invention can be obtained by hybridization from the library or the like using a nucleic acid fragment consisting of a portion of the nucleotide sequence shown in SEQ ID NO: 2 as a probe. For these methods, please refer to the methods described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0037] 3. Recombinant Vector Overview The third aspect of the present invention relates to a recombinant vector. The recombinant vector of the present invention comprises the polynucleotide described in the second aspect above and is capable of cloning a CSyGT gene or expressing CSyGT or the like in a host cell. In this aspect, a CSyGT expression vector that expresses CSyGT or the like is particularly preferably used.

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

[0039] In the present invention, a "(gene) expression vector" refers to a gene expression system that can deliver a polynucleotide encoding a polypeptide encapsulated therein into a target plant cell and express the polypeptide. Examples include expression vectors that utilize a plasmid or a virus. In the present invention, a CSyGT expression vector that expresses CSyGT or the like, into which a CSyGT gene or the like has been incorporated, corresponds to this.

[0040] In the case of an expression vector using a plasmid (hereinafter often referred to as a "plasmid expression vector"), the plasmid may be, but is not limited to, a pPZP series, a pSMA series, a pUC series, a pBR series, a pBluescript series (Agilent Technologies), a pTriEX series, or the like. TM system (TaKaRa), or pBI, pRI or pGW binary vectors, etc. can be used.

[0041] In the case of expression vectors that utilize viruses (hereinafter often referred to as "viral expression vectors"), viruses that can be used include cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV), and tobacco mosaic virus (TMV).

[0042] The recombinant vector contains expression regulatory regions of a promoter and a terminator. It may also contain other components such as an enhancer, a poly(A) addition signal, a 5'-UTR (untranslated region) sequence, a marker or selectable marker gene, a multicloning site, and a replication origin. The type of each component is not particularly limited as long as it can function in the host cell. Any component known in the art may be appropriately selected depending on the host to be introduced. Preferably, the host is a plant cell or a plant body.

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

[0044] Examples of terminators include the nopaline synthase (NOS) gene terminator, the octopine synthase (OCS) gene terminator, the CaMV 35S terminator, the 3' terminator of Escherichia coli lipopolyprotein lpp, the trp operon terminator, the amyB terminator, the ADH1 gene terminator, etc. There are no particular limitations on the sequence as long as it is capable of terminating transcription of the gene transcribed by the promoter.

[0045] The enhancer may be, for example, an enhancer region containing an upstream sequence in the CaMV 35S promoter, and is not particularly limited as long as it can enhance the expression efficiency of a nucleic acid encoding an active peptide.

[0046] Examples of selectable marker genes include drug resistance genes (e.g., tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, spectinomycin resistance gene, chloramphenicol resistance gene, or neomycin resistance gene), fluorescent or luminescent reporter genes (e.g., luciferase, β-galactosidase, β-glucuronidase (GUS), or green fluorescent protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase.

[0047] The recombinant vector of the present invention not only facilitates the manipulation and / or control of expression of the polynucleotide described in the second aspect, but also allows the manipulation of the expression of CSyGT and the like in host cells.

[0048] 4. Transformants or their progeny Overview A 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 according to the second aspect or the recombinant vector according to the third aspect in its cells, and is capable of cloning the CSyGT gene and / or expressing CSyGT and the like. The transformant of the present invention enables stable biosynthesis of CSyGT and the like in an in vivo expression system.

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

[0050] The host to be transformed is not particularly limited. Examples include bacteria such as Escherichia coli or Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris, fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma, monocotyledonous plants, dicotyledonous plants, or plant cells, mammalian cells, or insect cells (e.g., sf9 or sf21). Leguminous plants or yeasts are preferred.

[0051] The transformants of the present invention include clones having the same genetic information. For example, if the host is a unicellular microorganism that reproduces asexually, such as Escherichia coli or yeast, clones newly generated from the first generation of transformants by division or budding are also included in the transformants of the present invention. Furthermore, if the host is a plant, clones obtained from parts of the plant collected from the first generation of transformants, such as plant tissues such as the epidermis, phloem, parenchyma, xylem, or vascular bundles, plant organs such as leaves, petals, stems, roots, or seeds, or plant cells by plant tissue culture, cuttings, grafting, or layering, or clones newly generated from vegetative propagation organs obtained by asexual reproduction from the first generation of transformants, such as rhizomes, tuberous roots, corms, and runners, are also included in the transformants of the present invention.

[0052] The transformant of the present invention may further comprise one or more other polynucleotides or other recombinant vectors in addition to the polynucleotide described in the second aspect or the recombinant vector described in the third aspect. The other polynucleotides referred to here refer to polynucleotides other than the polynucleotides described in the second aspect. Examples include the β-amyrin synthase gene, CYP88D6 or CYP72A154, or any oleanane-type triterpenoid monoglucuronide synthase gene. Furthermore, the other recombinant vectors refer to recombinant vectors other than the recombinant vector described in the third aspect.

[0053] The transformant of the present invention can be prepared by introducing the above-mentioned polynucleotide or recombinant vector into an appropriate host.

[0054] The polynucleotide or recombinant vector can be introduced using methods known in the art, such as the Agrobacterium method, the PEG-calcium phosphate method, electroporation, liposome method, particle gun method, or microinjection method. The introduced polynucleotide may be integrated into the genomic DNA of the host, or may exist in the state of the introduced polynucleotide (e.g., as contained in a foreign vector). Furthermore, the introduced polynucleotide may be maintained continuously in the host cell, as if it were integrated into the genomic DNA of the host, or may be maintained transiently.

[0055] After introducing the polynucleotide according to the second aspect or the recombinant vector according to the third aspect into a host by the method described above, whether or not the polynucleotide of interest has been introduced can be confirmed by PCR, Southern hybridization, Northern hybridization, in situ hybridization, or the like.

[0056] As used herein, the term "progeny" refers to a host that is a descendant of the first generation of the transformant through sexual reproduction and that retains the polynucleotide according to the second aspect of the present invention or the recombinant vector according to the third aspect in an expressible state. Preferably, the term refers to a host progeny that retains the polynucleotide according to the second aspect of the polynucleotide or the recombinant vector in an expressible state. For example, when the transformant is a plant, the term refers to a seedling of the transformant. The progeny may be any generation.

[0057] The transformant of this embodiment can convert oleanane-type triterpenoids present in host cells into oleanane-type triterpenoid monoglucuronides by enhancing the expression of the introduced polynucleotide. Furthermore, by changing the host of the transformant, glucuronosyltransferase with a different sugar chain attached can be obtained. For example, when the host of the transformant is yeast, glucuronosyltransferase with a high-mannose sugar chain attached is expressed, unlike when a legume plant is used as the host. This is because the glycosylation reaction in yeast differs from that in plants (Strasser R. Glycobiology, 2016, 26(9): 926-939).

[0058] 5. Method for producing glucuronosyltransferase and its active fragments (CSyGT, etc.) Overview A fifth aspect of the present invention relates to a method for producing CSyGT or the like, which comprises culturing the transformant of the fourth aspect or its progeny and extracting the polypeptide having glucuronyl transferase activity described in the first aspect, i.e., CSyGT or the like, from the culture. According to the method for producing a polynucleotide of the present invention, CSyGT or the like can be obtained stably and in large quantities by using a host as a biological production system.

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

[0060] (1)Culture process In this aspect, the "culturing step" is a step of culturing the transformant of the fourth aspect or its progeny. The transformant or its progeny used in the present invention is preferably a transformant or its progeny capable of overexpressing or constitutively expressing the polypeptide described in the first aspect. For example, in the case of a transformant or its progeny having the recombinant vector described in the third aspect, the recombinant vector is preferably an expression vector comprising an overexpression promoter or a constitutive promoter. The transformant or its progeny of the fourth aspect may use any host, but is preferably a legume or yeast. By changing the host, glycoproteins with different sugar chains can be obtained even when the same glucuronosyltransferase described in the first aspect is expressed.

[0061] The medium used for culturing may be a medium suitable for culturing the host. Any medium known in the art can be used. Examples include, but are not limited to, LB medium or M9 medium when culturing bacteria such as Escherichia coli as a host; YPD medium, YPG medium, YPM medium, YPDM medium, SMM medium when culturing yeast as a host; and appropriate culture soil or hydroponic medium when culturing a plant as a host.

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

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

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

[0065] As used herein, the term "culture" refers to a culture supernatant or a cultured transformant. CSyGT and the like secreted from the transformant may be contained not only in the cells of the transformant but also in the culture supernatant.

[0066] To recover the polypeptide described in the first aspect from the culture, the polypeptide present in the culture can be extracted by a known method and purified as necessary. 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, or the like, either alone or in combination. Specifically, the method of Hayashi et al. (Hayashi et al., 1996, Phytochemistry, 42: 665-666) or the method of Noguchi et al. (Noguchi et al., 2007, J. Biol. Chem., 282: 23581-23590) can be used. Alternatively, the target polypeptide described in the first aspect can be recovered based on a sugar chain specific to the host. For example, when the transformant of the fourth aspect or its progeny is yeast, the expressed polypeptide of the present invention has a high-mannose sugar chain attached thereto, and therefore can also be extracted and purified using a mannose-binding lectin (e.g., UDA lectin, BC2L-A lectin, etc.).

[0067] 6. Genetically modified organisms for producing glycyrrhizin Overview A sixth aspect of the present invention is a genetic recombinant for producing glycyrrhizin. The genetic recombinant of the present invention comprises an expression vector that expresses a set of enzymes required for the biosynthetic pathway from β-amyrin to glycyrrhizin in Glycyrrhiza plants, i.e., a set of four enzymes that catalyze a two-step oxidation reaction and a two-step glycosidation reaction. The genetic recombinant of the present invention is capable of biosynthesizing glycyrrhizin from β-amyrin in biological cells, and can therefore be used as a biological production system for glycyrrhizin.

[0068] 6-2.Configuration 6-2-1. Expression vector included The genetic recombinant of the present invention is characterized by comprising, in a host cell, at least an expression vector containing a polynucleotide encoding a set of four enzymes and / or their active fragments required for the biosynthetic pathway from β-amyrin to glycyrrhizin. If necessary, the genetic recombinant may further comprise an expression vector containing a polynucleotide encoding a β-amyrin synthase gene. The four enzymes are polypeptides that catalyze the first and second oxidation reactions of β-amyrin and the first and second glycosidation reactions of oleanane-type triterpenoids. Expression vectors containing each enzyme or its active fragment are shown below in (1) to (4) and are described in detail below. Note that although the four expression vectors are described separately in (1) to (4), the genes for each enzyme may be contained in different expression vectors, or two or more types may be contained in the same expression vector.

[0069] (1) CYP88D6 expression vector A "CYP88D6 expression vector" contains a gene and a fragment thereof (often referred to herein as a "CYP88D6 gene, etc.") that encodes a polypeptide having the activity of oxidizing position 11 of oleanane-type triterpenoids, i.e., CYP88D6 and active fragments thereof (often referred to herein as "CYP88D6, etc."). Therefore, CYP88D6, etc. is expressed in the genetic recombinant organism by the CYP88D6 expression vector.

[0070] Specific examples of the CYP88D6 include, but are not limited to, CYP88D6 derived from licorice (G. uralensis) and consisting of the amino acid sequence shown in SEQ ID NO: 7. Other examples include polypeptides that have the activity of the first oxidation step and consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence that has 80% or more identity to the amino acid sequence shown in SEQ ID NO: 7.

[0071] Although not limited thereto, the genetic recombinant of the present invention can produce 11-oxo-β-amyrin by oxidizing the 11th position of endogenous or exogenous β-amyrin as a substrate, mainly by the catalytic activity of CYP88D6 expressed from a CYP88D6 expression vector. It can also produce 30-hydroxy-11-oxo-β-amyrin by oxidizing the 11th position of 30-hydroxy-β-amyrin as a substrate. It can also produce glycyrrhetinic acid by oxidizing the 11th position of 11-deoxoglycyrrhetinic acid as a substrate.

[0072] The configuration of the plasmid region in the CYP88D6 expression vector is similar to that of the expression vector in the recombinant vector described in the third aspect. Alternatively, the recombinant vector described in Japanese Patent No. 5526323 may be used.

[0073] (2) CYP72A154 expression vector A "CYP72A154 expression vector" contains a gene and a fragment thereof (often referred to herein as a "CYP72A154 gene, etc.") that encodes a polypeptide having the activity of oxidizing the 30th position of oleanane-type triterpenoids, i.e., CYP72A154 and active fragments thereof (often referred to herein as "CYP72A154, etc."). Therefore, CYP72A154, etc. is expressed in the genetic recombinant organism by the CYP72A154 expression vector.

[0074] Specific examples of CYP72A154 include, but are not limited to, CYP72A154 derived from licorice (G. uralensis) consisting of the amino acid sequence set forth in SEQ ID NO: 9, CYP72A154 derived from 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. Further examples include polypeptides that have the activity of the second oxidation stage and consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in any of SEQ ID NOs: 9, 11, and 13, or consist of an amino acid sequence that has 80% or more identity to the amino acid sequence set forth in any of SEQ ID NOs: 9, 11, and 13.

[0075] Although not limited thereto, the genetic recombinant of the present invention can use mainly β-amyrin and 11-oxo-β-amyrin as substrates and oxidize their 30-position to produce 30-hydroxy-β-amyrin and 30-hydroxy-11-oxo-β-amyrin, respectively, by the catalytic activity of CYP72A154 expressed from a CYP72A154 expression vector. Also, using 30-hydroxy-11-oxo-β-amyrin as a substrate, it can further oxidize its 30-position to produce glycyrrhetinic acid.

[0076] The configuration of the plasmid region in the CYP72A154 expression vector is similar to that of the expression vector in the recombinant vector described in the third aspect. Alternatively, the recombinant vector described in Japanese Patent No. 5771846 may be used.

[0077] (3) UGT73P12 recombinant vector A "UGT73P12 recombinant vector" contains a gene and a fragment thereof (often referred to herein as a "UGT73P12 gene, etc.") that encodes a polypeptide having the activity of transferring glucuronic acid to the hydroxy group at the 2-position of glucuronic acid in an oleanane-type triterpenoid monoglucuronide, i.e., UGT73P12 or an active fragment thereof (often referred to herein as "UGT73P12, etc."). Therefore, UGT73P12, etc. is expressed in the genetic recombinant organism by the UGT73P12 expression vector.

[0078] A specific example of the UGT73P12 includes, but is not limited to, UGT73P12 derived from licorice (G. uralensis) and consisting of the amino acid sequence set forth in SEQ ID NO: 15. Other examples include polypeptides that have the second-stage glycosylation activity and consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that has 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0079] The configuration of the plasmid region in the UGT73P12 expression vector is similar to that of the expression vector in the recombinant vector described in the third aspect. Alternatively, the recombinant vector described in Japanese Patent No. 6344774 may be used.

[0080] (4) CSyGT expression vector The "CSyGT expression vector" corresponds to the CSyGT expression vector in the CSyGT recombinant vector described in the third embodiment, and therefore a detailed explanation thereof will be omitted here.

[0081] 6-2-2. Genetically modified organisms for the production of glycyrrhizin The "genetic recombinant for producing glycyrrhetinic acid" of the present invention refers to a transformant into which an expression vector containing at least the four enzyme genes has been introduced, or its progeny harboring such enzyme genes. Therefore, apart from the type of expression vector included, its basic structure may be the same as that of the transformant described in the fourth aspect and its progeny. However, since the present invention relates to a genetic recombinant capable of biosynthesizing glycyrrhizin from β-amyrin intracellularly, a preferred host is one capable of intracellularly biosynthesizing β-amyrin, the starting material in the biosynthetic system. β-Amyrin biosynthesis in the host may be based on an endogenous synthesis system or an exogenous synthesis system. Since β-amyrin, an oleanane-type triterpenoid, can be biosynthesized in many plants, when an endogenous synthesis system is used, the host of the present invention is preferably a plant. Plant species with high β-amyrin synthesis ability, vigorous fertility, and easy growth are preferred. Plants relatively closely related to licorice, i.e., legumes, are even more preferred. Examples include species belonging to the genus Glycyrrhiza, species belonging to the genus Glycine, and species belonging to the genus Lotus. On the other hand, when β-amyrin biosynthesis is based on an exogenous synthesis system, the host may be a biological species that is incapable of biosynthesizing β-amyrin itself. For example, by introducing an expression vector containing a β-amyrin synthase gene into yeast, the yeast transformant can be used as a host capable of intracellular biosynthesis of β-amyrin.

[0082] According to the present invention, even hosts that have not previously been able to biosynthesize glycyrrhizin can now biosynthesize glycyrrhizin as a metabolic product from β-amyrin as a starting material.

[0083] 7. Manufacturing method of glycyrrhizin Overview A seventh aspect of the present invention is a method for producing glycyrrhizin. The production method of the present invention is characterized in that glycyrrhizin is produced from β-amyrin using the genetic recombinant for producing glycyrrhizin of the sixth aspect as a biological production system. According to the production method of the present invention, it is possible to stably produce glycyrrhizin in large quantities without relying on extraction from licorice, which has heretofore been expensive.

[0084] 7-2. Method The production method of the present invention includes a culture step as an essential step and an "extraction step" as a selection step. (1)Culture process The "culturing step" in this embodiment may basically be the same as the culturing step described in the fifth embodiment. When the genetically modified organism is a plant, a method for culturing plants under known conditions may be applied. By this step, glycyrrhizin is produced in the genetically modified organism for producing glycyrrhizin of the sixth embodiment. (2) Extraction process The "extraction step" in this embodiment may basically be the same as the extraction step described in embodiment 5. When the genetically modified organism is a plant, the same method as that used to extract glycyrrhizin from licorice can be used.

[0085] The production method of the present invention makes it possible to stably and mass-produce glycyrrhizin from various genetic recombinants without relying on extraction from licorice. [Example]

[0086] The present invention will be specifically described in the following examples.

[0087] Example 1: Isolation of Glyma.06G324300, a gene similar to cellulose synthase from soybean Ripening seeds from greenhouse-grown soybean (Glycine max) cultivar "Williams 82" were collected. Total RNA was prepared using the RNA extraction reagent RNeasy Plant Mini Kit (QIAGEN) according to the attached protocol. 200 ng of the obtained total RNA was used to synthesize first-strand cDNA using the QuantiTech Reverse Transcription Kit (QIAGEN) according to the attached protocol. Using 1 μL of 5-fold diluted first-strand cDNA as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide deduced from Glyma.06G324300 were used as forward primers (SEQ ID NO: 17) and reverse primers (SEQ ID NO: 18), respectively. PCR was performed for 30 cycles using PrimeSTAR GXL DNA Polymerase (Takara Bio) at an annealing temperature of 55°C and a reaction temperature of 68°C. pDONR TM The forward primer had 12 artificial bases (AAAAAGCAGGCT) added to the 5' end, and the reverse primer had 12 artificial bases (AGAAAGCTGGGT) added to the 5' end, because these were necessary for the sequence-specific recombination reaction (GATEWAY attB × attP reaction) during cloning into pDONR221 (Thermo Fisher Technologies). The DNA fragment amplified from the seed-derived first-strand cDNA was cloned into pDONR221 by a sequence-specific recombination reaction (GATEWAY attB × attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies). TM The polynucleotide sequences of three independent clones obtained were determined, and the sequence obtained was SEQ ID NO:2, from which the polypeptide sequence deduced was SEQ ID NO:1.

[0088] Example 2: Search for genes homologous to Glyma.06G324300 from licorice We performed a gene homology search to identify Glyma.06G324300 homologous genes in licorice (Glycyrrhiza uralensis), a legume like soybean that is known to biosynthesize glycyrrhizin. Using the BLAST homology search function in the licorice genome database, Glycyrrhiza uralensis GDB (http: / / ngs-data-archive.psc.riken.jp / Gur-genome / index.pl), we identified a single nucleotide sequence, Glyur003152s00037491, encoding a protein with high amino acid identity to Glyma.06G324300. The polypeptide deduced from Glyur003152s00037491 showed 81% amino acid identity to Glyma.06G324300.

[0089] <Example 3: Isolation of a gene homologous to Glyma.06G324300 from licorice> Total RNA was prepared from licorice roots using PureLink Plant RNA Reagent (Thermo Fisher Scientific). One microgram of the resulting total RNA was used to synthesize first-strand cDNA using the SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μL of first-strand cDNA as a template, oligo DNAs corresponding to the N- and C-termini of the polypeptide deduced from Glyur003152s00037491 were used as the forward primer (SEQ ID NO: 19) and reverse primer (SEQ ID NO: 20), respectively. PCR was performed for 30 cycles using PrimeSTAR Max DNA Polymerase (Takara Bio) at an annealing temperature of 55°C and a reaction temperature of 72°C. pENTR TMThe forward primer had four artificial bases (cacc) added to the 5' end, which were necessary for cloning into the pENTR / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies). TM The resulting clones were cloned into the / D-TOPO entry vector, and the nucleotide sequences of the four independent clones were determined. The nucleotide sequence of the licorice Glyma.06G324300 homologous gene obtained in this manner is SEQ ID NO: 4, and the polypeptide sequence deduced from it is SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 was 82% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0090] Example 4: Search for genes homologous to Glyma.06G324300 from Lotus japonicus Using the same method as in Example 2, we searched for Glyma.06G324300 homologous genes as candidates for the orthologous gene of Glyma.06G324300 from Lotus japonicus. Using the BLAST homology search function in the Lotus japonicus genome information database, miyakogusa.jp (http: / / www.kazusa.or.jp / lotus / release1 / index.html), we found a single nucleotide sequence, Lj3g3v1981230, encoding a protein showing high amino acid identity to Glyma.06G324300. The polypeptide deduced from Lj3g3v1981230 showed 81.4% amino acid identity to Glyma.06G324300.

[0091] Example 5: Isolation of a gene homologous to Glyma.06G324300 from Lotus japonicus First-strand cDNA was synthesized using 1 μg of total RNA obtained from Lotus japonicus with 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 deduced from Lj3g3v1981230 were used as the forward primer (SEQ ID NO: 37) and reverse primer (SEQ ID NO: 38), respectively, and 30 cycles of PCR were performed using PrimeSTAR Max DNA Polymerase (Takara Bio) at an annealing temperature of 55°C and a reaction temperature of 72°C. TM The forward primer had four artificial bases (cacc) added to the 5' end, which were necessary for cloning into the pENTR / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies). TM The fragment was cloned into the / D-TOPO entry vector, and the polynucleotide sequences of two independent clones obtained were determined. The nucleotide sequence of the Glyma.06G324300 homologous gene of Lotus japonicus obtained in this manner is SEQ ID NO: 6, and the polypeptide sequence deduced from it is SEQ ID NO: 5. SEQ ID NO: 5 has 82% identity to the amino acid sequence shown in SEQ ID NO: 1.

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

[0093] The yeast (Saccharomyces cerevisiae) strain INVSc1 used in this study does not contain UDP-glucuronic acid, which serves as the donor substrate for the glycosyltransferase reaction predicted by the candidate gene product. Therefore, the UDP-glucose dehydrogenase (UGD) gene, which synthesizes UDP-glucuronic acid using UDP-glucose as a substrate, was introduced into a yeast expression destination vector. Specifically, using the cDNA of Arabidopsis thaliana UGD (AtUGD2) as a template, oligonucleotides corresponding to the N- and C-termini of the polypeptide were used as forward and reverse primers (SEQ ID NO: 21 and 22, respectively). PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) at an annealing temperature of 55°C and a reaction temperature of 72°C for 30 cycles. Because these primers are required for in-fusion cloning, a total of 19 bases (15 bases upstream of the cloning site in the destination vector and 4 bases (aaaa)) have been artificially added to the 5' end of the polynucleotide shown in SEQ ID NO: 23 (gggcggccgcactag).Furthermore, the reverse primer has 15 bases downstream of the cloning site in the destination vector added to the 3' end of the polynucleotide shown in SEQ ID NO: 24 (atccatcgatactag). The destination vector pESC-HIS-GW, created by inserting Gateway cassette A (Thermo Fisher Technologies) into the SrfI restriction enzyme site in MCS2 of the pESC-HIS (registered trademark) yeast expression vector (Agilent Technologies), was treated with SpeI restriction enzyme and mixed with a DNA fragment amplified from cDNA. The DNA fragment shown in SEQ ID NO: 25 was then inserted into MCS1 in pESC-HIS-GW using the In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio Inc.), yielding the destination vector pESC-HIS-AtUGD2-GW.

[0094] Example 7: Construction of yeast expression clones The plasmid (entry clone) containing the polynucleotide shown in SEQ ID NO: 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 SEQ ID NO: 2 was transferred to pESC-HIS by a sequence-specific recombination reaction (GATEWAY attL × attR reaction) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Technologies) to obtain the yeast expression vector pESC-HIS-AtUGD2-Glyma.06G324300 for the gene shown in SEQ ID NO: 2. In addition, the yeast expression vectors pESC-HIS-AtUGD2-Glyur003152s00037491 and pESC-HIS-AtUGD2-Lj3g3v1981230 for the genes shown in SEQ ID NO: 4 and 6 prepared in Examples 3 and 5 were obtained using the same method as above.

[0095] <Example 8: Introduction into glycyrrhetinic acid and soyasapogenol B-producing yeast strain> The yeast strain INVScI (Thermo Fisher Technologies) (MATa his3D1 leu2 trp1-289 ura3-52 MATAlpha his3D1 leu2 trp1-289 ura3-52) was co-transfected with the expression vector pYES3-BAS for the Lotus japonicus β-amyrin synthase (LjOSC1) gene, the co-expression vector pESC-CPR-CYP88D6 for the CYP88D6 gene and Lotus japonicus cytochrome P450 reductase (LjCPR1), and the expression vector pDEST52-CYP72A63 for the Medicago sativa orthologue of the licorice CYP72A154 gene. This resulted in a glycyrrhetinic acid-producing yeast strain (Figure 3(a)). At the same time, the β-amyrin synthase gene expression vector pYES3-BAS, the CYP93E3 gene and Lotus japonicus cytochrome P450 reductase (LjCPR1) co-expression vector pESC-CPR-CYP93E3, and the CYP72A566 gene expression vector pDEST52-CYP72A566 were introduced and co-expressed to obtain soyasapogenol B-producing yeast (Figure 3(b)). The pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 7 were introduced into these yeast strains, respectively. As a negative control, pESC-HIS-AtUGD2, which corresponds to an empty vector, was introduced into a glycyrrhetinic acid-producing yeast strain. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

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

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

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

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

[0100] The results are shown in Figures 4 and 5. Figure 4 shows the results of the enzyme activity of Glyma.06G324300 or its homologs when glycyrrhetinic acid and glucuronic acid were used as substrates. A single peak corresponding to glycyrrhetinic acid monoglucuronide was detected in (a) Sample A (black arrow). The retention time and mass spectrum of this peak closely matched those of glycyrrhetinic acid monoglucuronide. Similarly, peaks corresponding to glycyrrhetinic acid monoglucuronide were detected in (b) Sample B and (c) Sample C (black arrow). The retention time and mass spectrum of each peak closely matched those of glycyrrhetinic acid monoglucuronide. On the other hand, no peak corresponding to glycyrrhetinic acid monoglucuronide was detected in (d) Sample D, the negative control.

[0101] Figure 5 shows the results of the enzymatic activity of Glyma.06G324300 or its homologs when soyasapogenol B and glucuronic acid were used as substrates. A single peak corresponding to soyasapogenol B monoglucuronide was detected in sample E (a) (black arrow). Peaks corresponding to soyasapogenol B monoglucuronide were also detected in sample F (b) and sample G (c) (black arrow). The retention times and mass spectra of each peak were in good agreement with those of soyasapogenol B monoglucuronide. On the other hand, no peak corresponding to soyasapogenol B monoglucuronide was detected in sample H (d), the negative control.

[0102] <Example 11: Preparation of transformed yeast for substrate feeding assay> The yeast strain INVScI was transformed with pESC-HIS-AtUGD2-Glyma.06G324300, pESC-HIS-AtUGD2-Glyur003152s00037491, and pESC-HIS-AtUGD2-Lj3g3v1981230 obtained in Example 8. As a negative control, the same yeast strain INVScI was transformed with pESC-HIS-AtUGD2, which corresponds to the empty vector. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (Zymo Research) according to the attached protocol.

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

[0104] Example 13: Analysis of extracts from substrate feeding assay 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.

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

[0106] The results when glycyrrhetinic acid was used as the glycosyl acceptor substrate are shown in Figures 7 to 10. Figure 7 shows the results of the substrate feeding assay for sample I. A peak (white arrow) corresponding to the glycosyl acceptor substrate glycyrrhetinic acid was detected in (b), and a single peak (black arrow) thought to be the result of one molecule of glucuronic acid being added to glycyrrhetinic acid was detected in (c). The retention time and mass spectrum of this peak were in good agreement with those of glycyrrhetinic acid monoglucuronide.

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

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

[0109] Figure 11 shows the results of a substrate feeding assay using soyasapogenol B as the glycosyl acceptor substrate. Sample M (b) contained Glyma.06G324300 derived from soybean; sample N (c) contained Glyur003152s00037491 derived from licorice; and sample O (d) contained Lj3g3v1981230 derived from Miyakogusou. A peak corresponding to soyasapogenol B monoglucuronide, in which one molecule of glucuronic acid was added to soyasapogenol B, was detected in all samples (black arrow). The retention time and mass spectrum of this peak closely matched those of soyasapogenol B monoglucuronide. In contrast, no peak corresponding to soyasapogenol B monoglucuronide was detected in the negative control sample P (e).

[0110] The above results and the results from Example 10 demonstrated that the novel soybean-derived enzyme Glyma.06G324300 obtained in Example 1, the novel licorice-derived enzyme Glyur003152s00037491 obtained in Example 3, and the Lotus japonicus-derived enzyme Lj3g3v1981230 obtained in Example 5 possess glucuronyl transferase activity, converting glycyrrhetinic acid to glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the hydroxyl group at the 3-position of glycyrrhetinic acid. It was also demonstrated that the novel enzymes possess glucuronyl transferase activity, converting soyasapogenol B to soyasapogenol B monoglucuronide by transferring glucuronic acid to the hydroxyl group at the 3-position of soyasapogenol B. These results indicate that the novel enzymes obtained are glucuronyl transferases that transfer glucuronic acid to the hydroxyl group at the 3-position of oleanane-type triterpenoids.

[0111] Example 14: Isolation of a mutant lacking function of a gene homologous to Glyma.06G324300 in Lotus japonicus Based on Lotus japonicus gene and protein sequence information and the Lotus Base expression database (https: / / lotus.au.dk / ), we searched for mutant lines with LORE1 insertions at Lj3g3v1981230, yielding 19 hits. Two lines (30006020 and 30115796) were selected based on the LORE1 insertion site at Lj3g3v1981230 and the number of LORE1 insertions in other genes. Seeds were obtained from a distribution organization (Aarhus University, Denmark). Genomic DNA was extracted from the expanded cotyledons and PCR confirmed the LORE1 insertion at Lj3g3v1981230. PCR was performed using GoTaq® Colorless Master Mix (Promega) at an annealing temperature of 60°C and a reaction temperature of 72°C for 25 cycles. For PCR, a forward primer (30006020 is SEQ ID NO: 26, 30115796 is SEQ ID NO: 28), a reverse primer (30006020 is SEQ ID NO: 27, 30115796 is SEQ ID NO: 29), and a P2 primer (SEQ ID NO: 30) were used.

[0112] Example 15: Analysis of triterpenoid saponin composition in a mutant lacking function of the Glyma.06G324300 homologous gene of Lotus japonicus Whole plants of Lotus japonicus Glyma.06G324300 homologous gene loss-of-function mutant lines (product numbers 30006020 and 30115796) sown in Example 14 and aged one month were freeze-dried, and then 10 times the dry weight of the plants was added to 80% methanol. The plants were shaken at room temperature for 1 hour and centrifuged at 15,000 rpm for 5 minutes. The supernatant obtained from the centrifugation was clarified using a 0.45 μm pore size membrane filter (GL Chromatodisk 4P, GL Science), and 2 μL of each extract was subjected to LC-PDA / MS / MS analysis. An Ultimate 3000SD HPLC / LTQ orbitrap discovery MS (both Thermo Fisher Scientific) was used. The extract was applied to a reversed-phase column (C30, Develosil C30-UG-3, Nomura Chemical Co., Ltd.), and saponins were eluted with a linear gradient of acetonitrile containing 0.1% (v / v) formic acid (20-80% / 60 min) at a flow rate of 0.15 ml / min. The eluate was detected by UV absorption and mass spectrometry (parent ions in an orbitrap system and fragment ions in an ion trap system). The eluate was vaporized and positively ionized using electrospray ionization, and injected into the mass spectrometer. Soyasaponin Bb (m / z = 943.52) was used as the analytical standard, and each saponin molecule was annotated based on the fragment pattern obtained by MS / MS analysis.

[0113] As a result, as shown in (b) and (c) in Figures 12-1 and 12-2, the normally accumulated saponins (Bb, βg, etc.) in the homo mutant were below the detection limit, and abnormalities in the saponin composition were observed. These results demonstrate that the Lotus japonicus Glyma.06G324300 homologous gene (Lj3g3v1981230) actually functions in vivo in the saponin biosynthesis system.

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

[0115] Next, using the cloning vector containing the Lotus japonicus-derived Glyma.06G324300 homologous gene prepared in Example 5 as a template, PCR was carried out in the same manner as above using a forward primer (SEQ ID NO: 33) and a reverse primer (SEQ ID NO: 34) that amplify the region from the initiation codon to the termination codon of SEQ ID NO: 6, to obtain pDONR TM The polynucleotide sequences of the three independent clones obtained were determined and confirmed to be identical to SEQ ID NO: 6. The plasmid pDONR-Lj3g3v1981230 containing the polynucleotide was obtained as an entry clone. TMSince these are required for the sequence-specific recombination reaction (GATEWAY attB × attP reaction) during cloning into 221 (Thermo Fisher Technologies), the forward primer has the 12 bases (AAAAAGCAGGCT) shown in SEQ ID NO: 35 added to its 5' end, and the reverse primer has the 12 bases (AGAAAGCTGGGT) shown in SEQ ID NO: 36 added to its 5' end. The plasmid (entry clone) pDONR-Glyma.06g324300 containing the polynucleotide shown in SEQ ID NO: 2, the plasmid (entry clone) pDONR-Glyur003152s00037491 containing the polynucleotide shown in SEQ ID NO: 4, and the plasmid (entry clone) pDONR-Lj3g3v1981230 containing the polynucleotide shown in SEQ ID NO: 6, which were prepared in Example 1, were mixed with the destination vector pG35NGw, and the DNA fragment shown in SEQ ID NO: 6 was transferred into pCAMBIA-G35NGw by a sequence-specific recombination reaction (GATEWAY attL × attR reaction) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Technologies), to obtain the Lotus japonicus transformation vector pG35N-LjCSL containing the Lotus japonicus-derived Glyma.06G324300 homologous gene shown in SEQ ID NO: 6. In addition, using the same method as above, the soybean-derived Glyma.06G324300 gene shown in sequence number 2 and the licorice-derived Glyma.06G324300 homologous gene shown in sequence number 4 were transferred to obtain Lotus japonicus transformation vectors pG35N-GmCSL and pG35N-GuCSL, respectively.

[0116] Example 17: Rescue experiment by introducing soybean Glyma.06G324300 and Glyma.06G324300 homologous genes from licorice and Lotus japonicus into Lotus japonicus mutants The Glyma.06G324300 homologous gene was introduced into a Lotus japonicus Glyma.06G324300 homologous gene knockout mutant according to the method described in Diaz et al. (2005) "Induction of hairy roots for symbiotic gene expression studies." In Lotus japonicus Handbook, AJ Marquez, ed. (Dordrecht, The Netherlands: Springer), pp. 261-277. Seeds obtained from a homozygous mutant line of the Lotus japonicus Glyma.06G324300 homologous gene knockout mutant 30006020 obtained in Example 14 were sterilized for 20 minutes with hypochlorous acid (containing 0.02% Tween 20) at an effective chlorine concentration of 2% for 20 minutes, and then allowed to soak in sterile distilled water overnight. The seed coats of the imbibed seeds were removed, and the seeds were sown on 0.8% water agar medium. The seeds were protected from light with aluminum foil and cultured at 25°C for 4 days, followed by exposure to light for 1 day. The vector prepared in Example 16 was introduced into Agrobacterium (LBA1334), which was plated on the front side of L medium and cultured at 28°C for 1 day. The Agrobacterium cultured for 1 day was suspended in 10 mL of sterile water and placed in a round sterile Petri dish. A homozygous seedling of the Lotus japonicus Glyma.06G324300 homologous gene function deletion mutant 30006020 was immersed in the suspension, and the hypocotyl was cut with a razor blade. The cut seedlings were then placed on coculture medium, protected from light with aluminum foil, and cocultured at 21°C for 4 days. After co-cultivation, the plants were placed on HRE medium and grown at 23°C under a 16-hour light / 8-hour dark condition for 2 weeks. The plants that developed hairy roots were confirmed to exhibit GFP fluorescence under a fluorescent stereomicroscope.

[0117] Example 18: Analysis of triterpenoid saponin composition in Lotus japonicus hairy roots The plants with hairy roots obtained in Example 17 were transplanted into pots filled with vermiculite, supplemented with B&D hydroponic solution (Diaz et al., 2005), and grown for one month. Fully grown plants were freeze-dried and pulverized at 2500 rpm for 30 seconds using a Multi-Bead Shocker (Yasui Kikai Co., Ltd.). A volume of 80% methanol equivalent to 100 times the weight of the lyophilized material was added, and the mixture was shaken at room temperature for one hour. The mixture was then centrifuged at 15 krpm for five minutes to collect the supernatant. This supernatant was analyzed by LC-PDA / MS / MS using the method described in Example 15. As shown in Figure 13, saponin, which had disappeared in the mutant, was restored in the transformed hairy roots. This suggests that the Glyma.06G324300 homologous gene also catalyzes saponin synthesis in vivo.

[0118] Example 19: Search and isolation of genes homologous to Glyma.06G324300 from Astragalus A homology search was conducted to identify Glyma.06G324300 homologous genes in Astragalus sinicus, a legume like soybean. From a sequence dataset integrating RNA sequence data from the roots, stems, and leaves of Astragalus, a single nucleotide sequence, AsCSyGT, encoding a protein with high amino acid identity to Glyma.06G324300 was identified. First-strand cDNA was synthesized using 1 μg of total RNA from Astragalus stems using the SMART RACE cDNA amplification kit (Clontech) according to the attached protocol. Using 2 μL of first-strand cDNA as a template, oligo DNAs corresponding to the N-terminus and C-terminus of the polypeptide predicted from AsCSyGT were used as the forward primer (SEQ ID NO: 39) and reverse primer (SEQ ID NO: 40), respectively, and PCR was performed for 30 cycles at an annealing temperature of 55°C and a reaction temperature of 72°C using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). TMThe forward primer had four artificial bases (cacc) added to the 5' end, which were necessary for cloning into the pENTR / D-TOPO (registered trademark) entry vector (Thermo Fisher Technologies). TM The fragment was cloned into the / D-TOPO entry vector, and the polynucleotide sequences of two independent clones obtained were determined. The nucleotide sequence of the Glyma.06G324300 homologous gene from Astragalus solanacearum obtained in this manner is SEQ ID NO: 41, and the polypeptide sequence deduced from it is SEQ ID NO: 42. SEQ ID NO: 42 has 77% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0119] Example 20: Isolation of a gene homologous to Glyma.06G324300 from soybean Gene homology searches were performed in soybean to identify Glyma.06G324300 homologous genes as candidate paralogous genes of Glyma.06G324300. Using the BLAST homology search function in Soybase (https: / / soybase.org), a soybean genome information database, two nucleotide sequences, Glyma.04g255400 and Glyma.11g151800, encoding proteins showing high amino acid identity to Glyma.06G324300 were identified. Using the method described in Example 1, the two Glyma.06G324300 homologous genes, Glyma.04g255400 and Glyma.11g151800, were amplified and transformed into pDONR TM The oligonucleotides were cloned into pDONR221 (Thermo Fisher Technologies). Oligonucleotides corresponding to the N-terminus and C-terminus of the polypeptide deduced from Glyma.04g255400 were used as the forward primer (SEQ ID NO: 43) and reverse primer (SEQ ID NO: 44), respectively, and oligonucleotides corresponding to the N-terminus and C-terminus of the polypeptide deduced from Glyma.11g151800 were used as the forward primer (SEQ ID NO: 45) and reverse primer (SEQ ID NO: 46), respectively. TMThe forward primer had 12 artificial bases (AAAAAGCAGGCT) added to the 5' end, and the reverse primer had 12 artificial bases (AGAAAGCTGGGT) added to the 5' end, because these were necessary for the sequence-specific recombination reaction (GATEWAY attB × attP reaction) during cloning into pDONR221 (Thermo Fisher Technologies). The DNA fragment amplified from the seed-derived first-strand cDNA was cloned into pDONR221 by a sequence-specific recombination reaction (GATEWAY attB × attP reaction) using Gateway BP Clonase II Enzyme Mix (Thermo Fisher Technologies). TM The polynucleotide sequences of three independent clones obtained for each were determined. The sequences obtained are SEQ ID NO:47 and SEQ ID NO:49, and the polypeptide sequences deduced from them are SEQ ID NO:48 and SEQ ID NO:50. SEQ ID NO:48 and SEQ ID NO:50 have 93.9% and 71.1% identity, respectively, to the amino acid sequence shown in SEQ ID NO:1.

[0120] Example 21: Introduction of Glyma.06G324300 homologous genes from astragalus and soybean into glycyrrhetinic acid and soyasapogenol B-producing yeast strains Using the method described in Example 7, yeast expression clones of the Glyma.06G324300 homologous genes from the Chinese milk vetch obtained in Example 19 and the soybean obtained in Example 20, pESC-HIS-AsCSyGT, pESC-HIS-AtUGD2-Glyma04g255400, and pESC-HIS-AtUGD2-Glyma.11g151800, were constructed and introduced into glycyrrhetinic acid- and soyasapogenol B-producing yeast strains, respectively, using the method described in Example 8.

[0121] Example 22: In vivo enzyme assay using recombinant yeast into which Glyma.06G324300 homologous genes from Chinese milk vetch and soybean have been introduced Recombinant yeast was cultured and metabolites were extracted according to the method described in Example 9. As a result, a metabolite extract (Sample Q) derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 42 (AsCSyGT), a metabolite extract (Sample R) derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 48 (Glyma04g255400), and a metabolite extract (Sample S) derived from a glycyrrhetinic acid-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800) were obtained. Soyasapogenol B-producing yeast strains harboring 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 (Sample T) derived from a soyasapogenol B-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 42 (AsCSyGT), a metabolite extract (Sample U) derived from a soyasapogenol B-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 48 (Glyma04g255400), and a metabolite extract (Sample V) derived from a soyasapogenol B-producing yeast strain expressing the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800) were obtained.

[0122] Example 23: Analysis of metabolite extracts from yeasts transformed with Glyma.06G324300 homologous genes from Chinese milk vetch and soybean Samples for LC-MS analysis were prepared and analyzed by the method described in Example 10. The results are shown in Figures 14 and 15. In sample Q (a) shown in Figure 14, a single peak corresponding to glycyrrhetinic acid monoglucuronide was detected (black arrow). The retention time and mass spectrum of this peak matched well with those of glycyrrhetinic acid monoglucuronide. Similarly, peaks corresponding to glycyrrhetinic acid monoglucuronide were detected in sample R (b) and sample S (c) (black arrow). The retention time and mass spectrum of each peak matched well with those of glycyrrhetinic acid monoglucuronide. In sample T (a) shown in Figure 15, a single peak corresponding to soyasapogenol B monoglucuronide was detected (black arrow). Peaks corresponding to soyasapogenol B monoglucuronide were also detected (black arrow) in sample U (b) and sample V (c). The retention time and mass spectrum of each peak were in good agreement with those of soyasapogenol B monoglucuronide.

[0123] The above results demonstrate that AsCSyGT derived from astragalus obtained in Example 19 and Glyma04g255400 and Glyma11g151800 derived from soybean obtained in Example 20 possess primary glucuronid transfer activity, which converts glycyrrhetinic acid to glycyrrhetinic acid monoglucuronide by transferring glucuronic acid to the hydroxy group at position 3 of glycyrrhetinic acid. They also demonstrate primary glucuronid transfer activity, which converts soyasapogenol B to soyasapogenol B monoglucuronide by transferring glucuronic acid to the hydroxy group at position 3 of soyasapogenol B.

[0124] Example 24: Preparation of transformed yeast for substrate feeding assay of soybean Glyma.06G324300 homologous gene The yeast INVScI strain was transformed 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.

[0125] <Example 25: Substrate feeding assay using transformed yeast into which a soybean Glyma.06G324300 homologous gene has been introduced> The recombinant yeast obtained in Example 24 was cultured by the method described in Example 12. The resulting suspension of each yeast cell was divided into equal portions, and either ursolic acid, an ursane-type triterpenoid, or betulinic acid, a lupane-type triterpenoid, was added at a final concentration of 5 μM. The resulting yeast cells were then cultured again, and metabolites were extracted by the method described in Example 12. As a result, a feeding assay extract was obtained in which ursolic acid, an ursane-type triterpenoid, was added to transformed yeast expressing the polypeptide shown in SEQ ID NO: 48 (Glyma04g255400) or the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800), and betulinic acid, a lupane-type triterpenoid, was added.

[0126] Example 26: Analysis of extracts from a substrate feeding assay using transformed yeast carrying a soybean Glyma.06G324300 homologous gene The sample obtained in Example 25 was analyzed under the same conditions as in Example 10. MS was performed in SIM mode, with the m / z values ​​of the predicted reaction products (ursolic acid monoglucuronide = 631 (Figure 16, a) and betulinic acid monoglucuronide = 631 (Figure 17, a)) as parameters. Figure 16 shows the analysis results of a feeding assay extract (sample W) obtained by adding ursolic acid to a transformed yeast expressing the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800). A peak predicted to be ursolic acid monoglucuronide was detected in sample W (Figure 16b). On the other hand, no peak predicted to be ursolic acid monoglucuronide was detected in the negative control sample X (Figure 17c).

[0127] 17 shows the analysis results of a feeding assay extract (sample Y) obtained by adding betulinic acid to a transformed yeast expressing the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800). A peak estimated to be betulinic acid monoglucuronide was detected in sample Y ((b)). On the other hand, no peak estimated to be betulinic acid monoglucuronide was detected in the negative control sample Z ((c)).

[0128] From the above, the polypeptide shown in SEQ ID NO: 50 (Glyma.11g151800) is considered to be a glucuronyltransferase capable of transferring glucuronic acid to the 3-hydroxy group not only of oleanane-type triterpenoids but also of ursane-type triterpenoids such as ursolic acid and β-boswellic acid, and lupane-type triterpenoids such as betulinic acid.

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

Claims

1. A genetically modified organism capable of biosynthesizing β-amyrin and into which all of the polynucleotides encoding the polypeptides shown in (1) to (3) below have been introduced. (1) A compound having the activity of oxidizing the 11-position of an oleanane-type triterpenoid, which is a polypeptide comprising any of the amino acid sequences shown in (a) or (b); (a) the amino acid sequence set forth in SEQ ID NO: 7, or (b) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 7; (2) having the activity of oxidizing the 30-position of oleanane-type triterpenoids, and a polypeptide comprising any of the amino acid sequences shown in (a) or (b); (c) the amino acid sequence set forth in SEQ ID NO: 13, or (d) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13; (3) Transfer of glucuronic acid to the hydroxyl group at the 3-position of oleanane-type triterpenoids A polypeptide having an activity of transferring a polypeptide to a target gene, the polypeptide comprising either of the amino acid sequences shown in (e) or (f) below: Petite, (e) an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, and 5; or (f) a sequence having 90% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, and 5; The amino acid sequence having

2. 2. The genetic recombinant according to claim 1, further comprising a polynucleotide encoding the polypeptide shown in (4) below introduced thereinto: (4) A polypeptide having an activity of biosynthesizing UDP-glucuronic acid using UDP-glucose as a substrate.

3. 3. The genetic recombinant according to claim 2, wherein (4) is a polypeptide encoded by the base sequence shown in SEQ ID NO:

25.

4. The genetic recombinant according to any one of claims 1 to 3, wherein the host is a yeast.

5. A method for biosynthesizing glycyrrhetinic acid monoglucuronide from β-amyrin, the method comprising a step of culturing the genetic recombinant according to any one of claims 1 to 4.

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