WO2023090385A1 - Plant cells, plant tissue, plant body, and glycyrrhizin production method - Google Patents

Abstract

The present invention provides a technology for increasing glycyrrhizin content in a plant tissue or a plant body. The present invention pertains to: plant cells which have a modification in at least two target genes selected from the group consisting of genes encoding enzymes in a betulinic acid synthesis pathway, genes encoding enzymes in a soyasaponin synthesis pathway, and genes encoding enzymes in an oleanolic acid synthesis pathway, and in which the function and/or the expression of said target genes is deficient or reduced because of the modification; and a plant tissue or a plant body including the plant cells.

Description

Plant cell, plant tissue, plant body, and method for producing glycyrrhizin

The present disclosure relates to plant cells, plant tissues, plant bodies, methods for producing glycyrrhizin, and the like.

Glycyrrhizin is a component contained in plants of the genus Glycyrrhiza of the legume family. Glycyrrhizin, which is one of the important herbal medicines, is used in sweeteners, cosmetics and the like because of its properties. Glycyrrhizin is usually produced by extraction and purification from collected licorice. However, it takes a long time (generally three years or more) to harvest licorice, and the demand for glycyrrhizin is high. was

A plant tissue culture method is known as a method for producing useful substances derived from plants. It has been reported that the content of glycyrrhizin contained in the glycyrrhizin-like roots or stolones (delivering branches) is extremely low compared to the underground parts of wild Glycyrrhiza plants, and cannot be put to practical use for the production of glycyrrhizin.

An object of the present disclosure is to provide a technique for improving the glycyrrhizin content in plant tissues or plants.

As a result of intensive research in view of the above problems, the present inventors focused on the betulinic acid synthetic pathway, the soyasaponin synthetic pathway, and the oleanolic acid synthetic pathway among the synthetic pathways of various components that partially overlap with the glycyrrhizin synthetic pathway. Arrived. As a result of further intensive research, a gene encoding an enzyme on the betulinic acid synthesis pathway, a gene encoding an enzyme on the soyasaponin synthesis pathway, and a gene encoding an enzyme on the oleanolic acid synthesis pathway are selected. The present inventors have found that the above problems can be solved by modifying at least two or more types of target genes, which are modified to eliminate or reduce the function and/or expression of the target genes. Based on this knowledge, the inventor of the present invention has completed the invention of the present disclosure as a result of further research. That is, the present disclosure includes the following aspects.

Section 1. At least two or more target genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. A plant cell that has been modified such that the function and/or expression of the gene of interest is lost or reduced due to the modification.

Section 2. The target gene includes a gene encoding a triterpene 24-position carbon hydroxylase, a gene encoding a triterpene 22-position carbon hydroxylase, a gene encoding a triterpene 28-position carbon oxidase, and lupeol synthesis. Item 3. The plant cell according to Item 1 or 2, comprising at least two genes selected from the group consisting of enzyme-encoding genes.

Section 3. Item 1, wherein the target gene comprises at least two or more genes selected from the group consisting of genes encoding enzymes on the soyasaponin synthesis pathway.

Item 4. The plant cell according to Item 1 or 2, wherein the target gene comprises a gene encoding a triterpene 24-position carbon hydroxylase and a gene encoding a triterpene 22-position carbon hydroxylase.

Item 5. The target gene includes at least two genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and enzymes on the oleanolic acid synthesis pathway. Item 4. The plant cell of any one of items 1 to 3, comprising the encoding gene.

Item 6. The target gene includes a gene encoding lupeol synthase, a gene encoding a triterpene 24-position carbon hydroxylase, a gene encoding a triterpene 22-position carbon hydroxylase, and a triterpene 28-position carbon. Item 6. The plant cell according to any one of items 1 to 5, comprising a gene encoding an oxidase of

Item 7. Items 1 to 6, wherein a gene encoding at least one glycyrrhizin biosynthetic enzyme has been introduced.

Item 8. Any one of items 1 to 7, wherein the index value of the function and/or expression of the target gene is 10% or less relative to 100% of the index value of the function and/or expression when the target gene is not modified. plant cell according to .

Item 9. Item 8. The plant cell according to any one of Items 1 to 8, which is a plant cell of a leguminous plant.

Item 10. A plant tissue or plant body containing the plant cell according to any one of Items 1 to 9.

Item 11. Item 10, wherein the plant tissue or plant body according to Item 10 has a higher content of glycyrrhizin or at least one glycyrrhizin derivative than the plant tissue or plant body that does not contain plant cells.

Item 12. Item 10 or 11, which is a hairy root of Glycyrrhiza genus.

Item 13. A seed generated in the plant body according to any one of Items 10 to 12.

Item 14. At least two or more selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway in plant cells A method for producing a modified plant cell or a plant tissue or plant body containing the modified plant cell, comprising introducing a mutation that eliminates or reduces the function and/or expression of the target gene.

Item 15. A method for producing glycyrrhizin or a glycyrrhizin derivative, comprising extracting glycyrrhizin or at least one glycyrrhizin derivative from the plant tissue or plant body according to any one of items 10 to 12.

According to the present disclosure, it is possible to provide a technique for improving the glycyrrhizin content in plant tissues or plants.

2 shows the electrophoresis results of heteroduplex mobility analysis in Test Example 1. FIG. A to N are the results of using separate hairy root samples as templates for PCR. The upper row shows the PCR sample of the CYP93E3 gene, and the lower row shows the PCR sample of the CYP72A566 gene. FIG. 4 shows quantitative PCR results of CYP88D6 mRNA in hairy roots of Test Example 5. FIG. #21-26 each show separately generated hairy roots (CYP93E3/CYP72A566-double knockout + CYP88D6 overexpressing hairy roots). The others (wild-type) each show a separate wild-type licorice root hairy root. 2 shows the analysis results of triterpenoids by LC-MS in Test Example 6. FIG. Top row: wild-type hairy roots, second row from top: CYP93E3/ CYP72A566-double knockout hairy roots (Test Example 1) CYP716A179) (Test Example 2), and the bottom shows the analysis results of hairy roots with CYP93E3/CYP72A566-double knockout + CYP88D6 overexpression (Test Example 5). 2 shows the analysis results of triterpenoids by LC-MS in Test Example 6. FIG. The upper row shows the analysis results of CYP93E3 single knockout hairy roots (Test Example 4), the middle row shows the analysis results of CYP72A566 single knockout hairy roots (Test Example 1), and the lower row shows the analysis results of CYP716A179 single knockout hairy roots (Test Example 3). FIG. 1 shows a diagram of the triterpenoid biosynthetic pathway.

1. Definitions As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

"Identity" of amino acid sequences refers to the degree of matching of two or more comparable amino acid sequences to each other. Therefore, the higher the identity or similarity between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using the sequence analysis tool FASTA, using default parameters. or Algorithm BLAST by Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. ul SF "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTP based on such a BLAST algorithm has been developed. Specific methods of these analysis methods are known, and the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) can be referred to. "Identity" of base sequences is also defined according to the above.

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine correspond to conservative substitutions. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine, tryptophan; amino acid residues with β-branched side chains such as threonine, valine, isoleucine; aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine. Substitutions between amino acid residues are also conservative substitutions.

2. In one aspect of the present disclosure, plant cells are selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. A plant cell in which at least two or more selected target genes have been modified, and the function and/or expression of the target gene is lost or reduced due to the modification (herein, "plant cell of the present disclosure ). This will be explained below.

The plant cell is derived from a plant having at least one of the betulinic acid synthesis pathway, the soyasaponin synthesis pathway, and the oleanolic acid pathway, and is endogenously or exogenously introduced to biosyntheize glycyrrhizin. There are no particular restrictions as long as the plant can express the enzyme. Plants include a wide range of plants including, for example, the angiosperms magnolias, monocotyledons, eudicotyledons (roses I, roses II, chrysanthemums I, chrysanthemums II and outgroups thereof). can be done. More specific examples of plants include eggplants such as liverwort, tomatoes, green peppers, hot peppers, eggplants and tobacco; gourds such as cucumbers, pumpkins, melons and watermelons; vegetables such as cabbages, broccoli and Chinese cabbage; Raw and spicy vegetables such as celery, parsley, and lettuce; Green onions such as green onions, onions, and garlic; Other fruit vegetables such as strawberries and melons; Tap roots such as radishes, turnips, carrots, and burdock; Taro, cassava, and potatoes , potatoes, sweet potatoes, Chinese yams, etc.; grains, such as rice, corn, wheat, sorghum, barley, rye, seaweed, and buckwheat; Legumes; Soft vegetables such as asparagus, spinach, and Japanese honeysuckle; Flowers such as gentian, rose, bindweed, buttercup, bellflower, stock, carnation, and chrysanthemum; Oil crops such as rapeseed; Textile crops such as cotton and rush; Feed crops such as clover, dent corn, and alfalfa; Deciduous fruit trees such as myrtle, persimmon, apple, pear, grape, and peach; Citrus fruits such as oranges, lemons and grapefruits; Woody plants such as satsuki, azalea, oleander, osmanthus, birch, sycamore, sandalwood, sykunshi, yamaguruma, ilex, cedar, poplar and para rubber tree;

Among these plants, leguminous plants (e.g., licorice, soybean, Lotus japonicus, Astragalus membranaceus, fenugreek, Astragalus, Medicago, etc.), Araliaceae (e.g., Panax ginseng), Umbelliferae (e.g., Panax ginseng, etc.), Umbelliferae (e.g., radish) Centella asiatica, etc.), Cucurbitaceous plants (e.g., Jiaogulan, etc.), Ericaceous plants (e.g., Arctic japonicum, bearberry, etc.), Betulaceae plants (e.g., Betula oleracea, etc.), Gentianaceous plants (e.g., Safflower assembly, etc.), Rosaceae Plants (e.g., hawthorn, apple, Japanese quince, etc.), Myrtaceous plants (e.g., eucalyptus, cloves, etc.), Labiatae plants (e.g., lavender, lemon balm, marjoram, rosemary, sage, winter savory, musk grass, cat's mustache, carrots, etc.), Anacardiaceae plants (e.g., mangoes, etc.), Solanaceae plants (e.g., Ashwagandha, etc.), Apocynaceae plants (e.g., Apocynaceae, etc.), Oleaceae plants (e.g., olives, etc.), Euonymus plants (e.g., , Taiwan black crane, etc.), sycamore family plants (e.g., maple leaf sycamore, etc.), sycamore family plants (e.g., elderberry), sandalwood family plants (e.g., mistletoe, etc.), sykundiaceae plants (e.g., sykunshi, salmon, etc.), yamaguruma family plants (e.g. Apothecary japonicum etc.), Ilexaceae plants (e.g. Aohada etc.), Convolvulaceae plants (e.g. Gumbai bindweed etc.), Kakinokiaceae plants, Ranunculaceae plants (e.g. Azuma Tage etc.) and the like, more preferably include leguminous plants. Among leguminous plants, particularly preferred are plants of the genus Glycyrrhiza. Glycyrrhiza plants include, for example, Ural licorice, Spanish licorice, and Xinjiang licorice.

The plant cell is a plant having at least one of the betulinic acid synthetic pathway, the soyasaponin synthetic pathway, or the oleanolic acid pathway, and is endogenously or exogenously introduced to express the glycyrrhizin biosynthetic enzyme. As long as it is a suitable plant cell, it is not particularly limited, and can be cells of various plant tissues. Plant tissues include, for example, roots, stems, leaves, flowers, reproductive organs, and undifferentiated cells and tissues that differentiate into them. Glycyrrhizin biosynthetic enzymes include oxidase at position 11 or 30 of the triterpene skeleton, glucuronyltransferase 1, glucuronyltransferase 2, and the like (see FIG. 5). Examples of the 11-position oxidase include CYP88D6 (an enzyme having activity to convert β-amyrin into 11-oxo-β-amyrin). Examples of the 30-position oxidase include CYP72A154 (enzyme having activity to convert 11-oxo-β-amyrin into glycyrrhetinic acid), CYP72A63 (30-position oxidase derived from Lotus japonicus), and the like. Examples of glucuronyltransferase 1 include GuCSyGT (an enzyme that converts glycyrrhetinic acid into glycyrrhetinic acid monoglucuronide), spinach-derived CsyGT (an enzyme that converts medicagenic acid into yososside V), and the like. Examples of glucuronyltransferase 2 include UGT73P12 (enzyme having activity to convert glycyrrhetinic acid monoglucuronide into glycyrrhizin) and the like. In one embodiment, the plant cell is a cell into which a gene encoding at least one glycyrrhizin biosynthetic enzyme has been introduced.

A gene encoding an enzyme on the betulinic acid synthesis pathway (betulinic acid synthesis pathway gene) is not particularly limited as long as it is a gene encoding an enzyme on the pathway for synthesizing betulinic acid from 2,3-oxidosqualene. Enzymes on the betulinic acid synthesis pathway include, for example, lupeol synthase and triterpene 28-position oxidase.

Lupeol synthase is an enzyme that has the activity of converting 2,3-oxide squalene into lupeol on the betulinic acid synthesis pathway. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding lupeol synthase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding lupeol synthase (for example, identity analysis with these etc.) can be easily determined. An example of a gene encoding lupeol synthase is the LUS1 gene. Its amino acid sequence is shown in SEQ ID NO:1 and its coding base sequence is shown in SEQ ID NO:5.

　Triterpene 28-hydroxylase is an enzyme that has the activity of converting lupeol to betulinic acid on the betulinic acid synthesis pathway. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 28-hydroxylase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 28-hydroxylase (for example, It can be easily determined by identity analysis with these). An example of a gene encoding triterpene position 28 oxidase is the CYP716A179 gene. Its amino acid sequence is shown in SEQ ID NO:4 and its coding base sequence is shown in SEQ ID NO:8.

A gene encoding an enzyme on the soyasaponin synthesis pathway (soyasaponin synthesis pathway gene) is not particularly limited as long as it is a gene encoding an enzyme on the pathway for synthesizing soyasaponin from 2,3-oxidosqualene. Enzymes on the soyasaponin synthetic pathway include, for example, triterpene 24-hydroxylase, triterpene 22-hydroxylase, triterpene 21-hydroxylase, triterpene 3-glycosylase, triterpene 22-hydroxylase and the like.

Triterpene 24-hydroxylase is an enzyme that has the activity of converting β-amyrin to 24-OH-β-amyrin and the activity of converting sophoradiol to Soyasapogenol B on the soyasaponin synthesis pathway. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 24-hydroxylase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 24-hydroxylase (for example, It can be easily determined by identity analysis with these). An example of a gene encoding triterpene 24-hydroxylase is the CYP93E3 gene. Its amino acid sequence is shown in SEQ ID NO:2 and its coding base sequence is shown in SEQ ID NO:6.

Triterpene 22-hydroxylase is a gene for an enzyme that converts β-amyrin to sophoradiol and 24-OH-β-amyrin to Soyasapogenol B on the soyasaponin synthesis pathway. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 22-hydroxylase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 22-hydroxylase (for example, It can be easily determined by identity analysis with these). An example of a gene encoding triterpene 22-hydroxylase is the CYP72A566 gene. Its amino acid sequence is shown in SEQ ID NO:3 and its coding base sequence is shown in SEQ ID NO:7.

Triterpene 21-hydroxylase is an enzyme that converts β-amyrin to 21-hydroxy-β-amyrin, sophoradiol to cantoniensistriol, and soyasapogenol B to soyasapogenol A in the soyasaponin synthesis pathway. is the gene for In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 21-hydroxylase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 21-hydroxylase (for example, It can be easily determined by identity analysis with these). An example of a gene encoding soybean triterpene 21-hydroxylase is the CYP72A69 gene. Its amino acid sequence is shown in SEQ ID NO:42 and its coding base sequence is shown in SEQ ID NO:43.

Triterpene 3-glycosidase has the activity of adding the primary sugar to the 3-position of soyasapogenol A or soyasapogenol B on the soyasaponin synthetic pathway, the activity of adding the secondary sugar to the primary glycoside at the 3-position, or 3 It is an enzyme presumed to have the activity of adding a tertiary sugar to a secondary glycoside. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 3-glycosidase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 3-glycosidase (for example, It can be easily determined by identity analysis with these). An example is the GuCSyGT gene, which encodes a triterpene-3-glycosyltransferase. Its amino acid sequence is shown in SEQ ID NO:40 and its coding base sequence is shown in SEQ ID NO:41.

Triterpene 22-position glycosidase has the activity of adding the primary sugar to the 22nd position of soyasapogenol A and the activity of adding the secondary sugar to the primary glycoside at the 22nd position of soyasapogenol A on the soyasaponin synthesis pathway. is the estimated enzyme. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding the triterpene 22-position glycosidase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding the triterpene 22-position glycosidase (for example, It can be easily determined by identity analysis with these). An example of a gene encoding a triterpene 22-position glycosidase is the UGT73F4 gene. Its amino acid sequence is shown in SEQ ID NO:44 and its coding base sequence is shown in SEQ ID NO:45.

A gene encoding an enzyme on the oleanolic acid synthesis pathway (oleanolic acid synthesis pathway gene) is not particularly limited as long as it is a gene encoding an enzyme on the pathway for synthesizing oleanolic acid from 2,3-oxidosqualene. Enzymes on the oleanolic acid synthesis pathway include, for example, triterpene 28-oxidase.

　Triterpene 28-oxidase is a gene for an enzyme that converts β-amyrin to oleanolic acid on the oleanolic acid synthesis pathway. In various plants, the nucleotide sequence and amino acid sequence of the gene encoding triterpene 28 oxidase are known, or based on the nucleotide sequence and amino acid sequence of the known gene encoding triterpene 28 oxidase (for example, can be easily determined by identity analysis, etc.). An example of a gene encoding a triterpene position 28 oxidase is the CYP716A179 gene, as described above. Its amino acid sequence is shown in SEQ ID NO:4 and its coding base sequence is shown in SEQ ID NO:8.

The target gene for modification can be an ortholog or paralog of the gene indicated by the amino acid sequence or base sequence indicated by the above SEQ ID NO. For example, the target gene for modification is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and furthermore It may be a gene having an identity of preferably 98% or more, particularly preferably 99% or more, and having the above enzymatic activity.

The genes to be modified are at least two selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. more than seeds. In one embodiment, the target gene preferably includes a gene encoding a triterpene 24-position carbon hydroxylase, a gene encoding a triterpene 22-position carbon hydroxylase, and a triterpene 28-position carbon oxidase. It contains at least two or more genes selected from the group consisting of the encoding gene and the gene encoding lupeol synthase. In one embodiment, the target gene preferably contains at least two or more genes selected from the group consisting of genes encoding enzymes on the soyasaponin synthetic pathway, and particularly preferably encodes triterpene 24-hydroxylase. It contains a gene and a gene encoding triterpene 22-hydroxylase. In one embodiment, the target gene is preferably at least two or more genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and oleanol. A gene encoding an enzyme on the acid synthesis pathway, particularly preferably a gene encoding lupeol synthase, a gene encoding triterpene 24-hydroxylase, a gene encoding triterpene 22-hydroxylase, and a triterpene 28-position Contains genes encoding oxidases.

The target genes for modification include functionally normal mutants that can occur in nature. The gene to be modified may have base mutations such as substitutions, deletions, additions, and insertions as long as the activity of the encoding enzyme is not significantly impaired. The mutation is preferably a mutation that does not cause amino acid substitution or a mutation that causes conservative amino acid substitution in the protein translated from the mRNA.

The target gene for modification, for example, the amino acid sequence of the protein encoded by it is, for example, 95% or more, preferably 98% or more, more preferably 98% or more of the amino acid sequence of the protein encoded by the wild-type target gene of the homologous plant A gene with 99% or more identity. In addition, the target gene, for example, the amino acid sequence of the protein encoded by it is the same as the amino acid sequence of the protein encoded by the wild-type target gene of the same kind of plant, or one or more amino acid sequences relative to the amino acid sequence (eg, 2-10, preferably 2-5, more preferably 2-3, even more preferably 2) are genes whose amino acid sequences are substituted, deleted, added, or inserted.

In the plant cell of the present disclosure, the target gene is modified, and the function and/or expression of the target gene is lost or reduced due to the modification (the mutation introduced by the modification). Here, the "function" of the target gene indicates the enzymatic activity of the target gene described above. "Expression" of a gene of interest includes both expression of the mRNA of the gene of interest and expression of the protein of the gene of interest, preferably expression of the protein of the interest gene. "Deficient" indicates that the activity of the target gene protein and/or the expression level of the target gene is below the detection limit in samples obtained from the plant cells of the present disclosure. In addition, the term "decrease" refers to the activity of the target gene protein and/or the expression level of the target gene (index value of the function and/or expression of the target gene) in the sample obtained from the plant cells of the present disclosure. It is less than 100% (e.g., 70% or less , 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05 %, 0.02% or less, or 0.01% or less). The activity of the target gene protein and/or the expression level of the target gene can be measured according to known methods.

Mutations to be introduced into the target gene are not particularly limited as long as they are mutations that impair or reduce the function and/or expression of the target gene. Such mutations include, for example, gene disruption, mutations in protein coding regions, mutations in splicing regulatory regions, mutations in expression control regions (eg, promoters, activators, enhancers, etc.). Among these, mutations in protein coding regions are preferred. Plant cells of the present disclosure preferably have the mutation of the gene of interest on both chromosomes of the pair.

3. Plant tissue, plant body, seed In one aspect, the present disclosure includes plant tissue or plant body (herein, “plant tissue or plant body of the present disclosure”, “plant tissue or plant body of the present disclosure”, “plant cell of the present disclosure”, (also referred to as “plant tissue,” “plant body of the present disclosure”). Moreover, in one aspect, the present disclosure relates to seeds (in this specification, sometimes referred to as “seeds of the present disclosure”) generated in the plant body of the present disclosure. These will be described below.

"Plant body" means the entire plant, including all plant tissues (roots, stems, leaves, etc.). The plant tissue is not particularly limited as long as it is composed of part of the plant body, or undifferentiated tissue generated in part or the whole of the plant body, such as roots, stems, leaves, flowers, reproductive organs, and at least one selected from the group consisting of cells or tissues that differentiate into them. Seeds contain plant cells of the disclosure and can develop into plants of the disclosure. The seed can be obtained, for example, from a plant of the present disclosure in which the plant cells of the present disclosure have been integrated into germ cells.

From the viewpoint of glycyrrhizin production efficiency, the plant tissue of the present disclosure preferably contains at least one selected from the group consisting of roots and stems. From the viewpoint of glycyrrhizin production efficiency, the plant tissue of the present disclosure preferably contains underground parts of the plant (for example, roots (hairy roots, etc.), rhizomes, etc.).

The plant species of the plant tissue or plant body of the present disclosure are the same as the plants from which the plant cells of the present disclosure are derived.

The plant cells of the present disclosure have enhanced glycyrrhizin production efficiency due to lack or reduction in the function and/or expression of the target gene. Therefore, the plant tissue or plant body of the present disclosure containing this has enhanced glycyrrhizin production efficiency. Therefore, in one aspect, the plant tissue or plant body of the present disclosure (for example, by culturing or cultivating for a period in which glycyrrhizin is sufficiently accumulated) is transformed into a plant tissue or plant body that does not contain the plant cells of the present disclosure. The content of glycyrrhizin or at least one glycyrrhizin derivative is higher than that of The content of glycyrrhizin or at least one glycyrrhizin derivative in the plant tissue or plant body of the present disclosure is relative to the content of glycyrrhizin or at least one glycyrrhizin derivative in the plant tissue or plant body that does not contain plant cells of the present disclosure. , for example 1.5x, 2x, 5x, 10x, 20x, 50x, 100x or 150x.

Examples of glycyrrhizin derivatives include, but are not limited to, glycyrrhetinic acid monoglucuronide, licorice-saponin (LS)-A3, LS-B2, LS-C2, LS-D3, LS-E2, LS-F3, LS-G2, LS -H2, LS-J2, LS-K2, LS-L3, 18α-glycyrrhizin, apioglycyrrhizin, araboglycyrrhizin, periandrin I, periandrin II, periandrin III, periandrin IV and the like.

Four. A plant cell, a plant tissue or a plant body containing the plant cell of the present disclosure, a plant tissue or a plant body of the present disclosure, for example, a gene encoding an enzyme on the betulinic acid synthesis pathway, soyasaponin synthesis in the plant cell Introduce mutations that eliminate or reduce the function and/or expression of at least two or more target genes selected from the group consisting of genes encoding enzymes on the pathway and genes encoding enzymes on the oleanolic acid synthesis pathway. can be obtained by a method comprising From this point of view, in one aspect, the present disclosure provides, in plant cells, genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. A modified plant cell, or a plant tissue or plant body containing the modified plant cell, comprising introducing a mutation that eliminates or reduces the function and/or expression of at least two or more target genes selected from the group consisting of genes that It relates to a method (in this specification, it may be indicated as a “plant production method of the present disclosure”). This will be explained below.

Mutations in which the function and/or expression of the target gene are lost or reduced are the same as the explanation in "2. Plant cells".

The method of introducing mutations is not particularly limited, but from the viewpoint of production efficiency, a target-specific nuclease, an expression cassette of the nuclease, and an introduction containing at least one selected from the group consisting of mRNA of the nuclease is introduced into cells. There is a method of introduction.

The target-specific nuclease is not particularly limited as long as it can specifically cleave a specific site on genomic DNA and induce mutation. Target-specific nucleases include, for example, Cas proteins, TALEN proteins, ZFN proteins and the like.

The CRISPR/Cas system using Cas protein uses Cas protein, which is a nuclease (RGN; RNA-guided nuclease), and guide RNA. By introducing the system into cells, the guide RNA binds to the target site, and the DNA can be cleaved by the Cas protein called into the binding site.

The TALEN system using TALEN proteins uses artificial nucleases (TALENs) that contain DNA-binding domains of transcriptional activator-like (TAL) effectors in addition to DNA-cleaving domains (eg, FokI domains). By introducing the system into cells, TALENs bind to target sites via their DNA-binding domains and cleave DNA there. A DNA-binding domain that binds to a target site can be designed according to known schemes (e.g., Zhang F et al. (2011) Nature Biotechnology 29 (2); this paper is incorporated herein by reference). .

A ZFN system using ZFN proteins uses an artificial nuclease (ZFN) containing a nucleic acid cleavage domain conjugated to a DNA binding domain containing a zinc finger array. By introducing the system into cells, the ZFN binds to the target site via its DNA binding domain and cleaves the DNA there. A DNA-binding domain that binds to a target site can be designed according to known schemes.

Among the target-specific nucleases, the Cas protein is preferable from the viewpoint that the cleavage site can be determined more freely. Cas protein preferably includes Cas9 protein.

The target-specific nuclease expression cassette is not particularly limited as long as it is DNA capable of expressing the target-specific nuclease in the cells of the object of the plant production method of the present disclosure. A typical example of a target-specific nuclease expression cassette includes a DNA comprising a promoter and a target-specific nuclease coding sequence placed under the control of that promoter. In addition, the target-specific nuclease expression cassette alone or together with other sequences (eg, drug resistance gene, replication origin, etc.) may constitute a vector. The type of vector is not particularly limited.

When the target-specific nuclease is a Cas protein, the introduced material in the plant production method of the present disclosure further contains at least one selected from the group consisting of guide RNA expression cassettes and guide RNAs.

The guide RNA is not particularly limited as long as it is used in the CRISPR/Cas system. For example, the guide RNA can guide the Cas protein to the target site of the genomic DNA by binding to the target site of the genomic DNA and binding to the Cas protein. Various things can be used.

It is said that the 12 bases on the 3' side of the sequence that binds to the target sequence in the crRNA sequence are important for the binding of the guide RNA to the target site. Therefore, if the sequence that binds to the target sequence in the crRNA sequence is not completely identical to the target strand, the nucleotides that differ from the target strand are the 12 bases on the 3' side of the sequence that binds to the target sequence in the It is preferable to exist other than

In addition, the introduced material in the plant production method of the present disclosure can contain donor DNA. The use of donor DNA allows more precise introduction of desired mutations.

The introduction target is not particularly limited, and may be an undifferentiated plant tissue (e.g., callus), a part of a seed (e.g., hypocotyl, shoot apex, etc.), or a part of an adult ( For example, the stem apex, etc.) may be used.

The method of introduction is not particularly limited as long as it allows the substance to be introduced into the plant cells, and can be appropriately selected according to the type of substance to be introduced and the target of introduction. Examples of introduction methods include floral dip method, floral spray method, Agrobacterium method, particle gun method, infiltration method, toothpick inoculation method, suction injection method, leaf disk method, inflorescence infiltration method, and vacuum filtration method. , viral-mediated nucleic acid delivery, and the like. Among these, the Agrobacterium method is preferred from the viewpoint of convenience, safety, and the like.

A more specific example of the introduction method is shown below.

As a first specific example of the introduction method (Introduction example 1), a step of preparing a plasmid containing a promoter (e.g., T7 promoter, T3 promoter, 35S promoter, etc.) and a sequence containing an expression cassette downstream thereof (step a1 ), a step of obtaining plant virus genomic RNA from the plasmid obtained in step a1 by in vitro transcription (step b1), and inoculating a plant with the genomic RNA (active ingredient) obtained in step b1 (for example, rubbing inoculation method, particle gun inoculation method, etc.) (step c1). Alternatively, if the plasmid obtained in step a1 is a Ti plasmid containing a promoter capable of activating transcription in plant cells, such as the 35S promoter, instead of steps b1 and c1 above, for example, A step of introducing the plasmid obtained into Agrobacteria and culturing it (step b2), and inoculating the culture solution (culture solution containing the active ingredient) obtained in step b2 into plants (e.g., infiltration method, toothpick inoculation method , suction injection method, etc.) (step c2). Alternatively, instead of the above steps b1 and c1, for example, a method comprising a step (step c3) of inoculating a plant with the plasmid (active ingredient) obtained in step a1 (e.g., grinding inoculation method, particle gun inoculation method, etc.) It can be carried out. Alternatively, instead of step c2, for example, a method including a step (step c4) of performing a leaf disk method, an inflorescence infiltration method, a vacuum filtration method, or the like can be used. Desired proteins and peptides are produced from genomic RNA, plasmids, T-DNA, etc. introduced into plants by these methods.

As a second specific example of the introduction method (Introduction Example 2), a step of collecting the virus from a plant containing the plant virus (obtained, for example, in Introduction Example 1 above) (step d1), and in step d1 A method comprising the step of inoculating a plant with the collected virus (virus containing an active ingredient) (step e1). The collection in step d1 can be performed, for example, by recovering a virus fluid obtained by grinding a part of a plant containing a plant virus (eg, leaves, etc.). The inoculation in step e1 can be carried out by, for example, using an abrasive such as silicon carbide to scratch the site of the plant to be inoculated (eg, leaves) and contacting the site with the virus.

After the introduction, the plant cell, plant tissue, or plant body of the present disclosure can be obtained by growing the obtained plant or plant cell, or by growing the obtained plant through callus. . In addition, after the introduction, the introduced cells, tissues, etc. can be sorted with a drug, if necessary.

The method for selecting cells, tissues, etc. containing the desired mutation is shown below.

The target gene is amplified from the introduced cells, tissues, etc. by PCR, and the size of the amplified gene fragment is analyzed by heteroduplex mobility analysis to narrow down the cells, tissues, etc. into which the desired mutation has been introduced. be able to. It can also be narrowed down by performing an ELISA method using an anti-glycyrrhizin antibody and analyzing the glycyrrhizin content of cells, tissues, and the like. After narrowing down, it is possible to perform sequence analysis of the target gene and select cells, tissues, etc. into which the desired mutation has been introduced.

Five. Method for Producing Glycyrrhizin or a Glycyrrhizin Derivative In one aspect of the present disclosure, a method for producing Glycyrrhizin or a Glycyrrhizin derivative comprising extracting Glycyrrhizin or at least one type of Glycyrrhizin derivative from the plant tissue or body of the present disclosure (this specification In the book, it may be indicated as "the method for producing glycyrrhizin of the present disclosure".). This will be explained below.

The part to be extracted is not particularly limited. is mentioned. Among these, roots and rhizomes are preferred.

It is preferable to cut the plant tissue or plant body to be extracted as necessary.

The extraction solvent is not particularly limited. Examples of extraction solvents include water; alcohols such as methanol, ethanol, propanol and butanol; alkyl acetates such as methyl acetate, ethyl acetate, propyl acetate and butyl acetate; supercritical carbon dioxide and the like. Among these, water, alcohol (especially ethanol) and the like are preferred. The extraction solvent may be used singly or in combination of two or more.

The temperature of the solvent during extraction is not particularly limited, but is, for example, 0-100°C. The extraction time varies depending on the extraction method, solvent, temperature, etc., and is not particularly limited.

The obtained extract can be used as it is as glycyrrhizin or a glycyrrhizin derivative, or it can be used as glycyrrhizin or a glycyrrhizin derivative after being subjected to treatments such as dilution, concentration, drying and purification.

The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Reference example 1. Determination of target sequences for introducing mutations into target genes A gene encoding an enzyme on the betulinic acid synthesis pathway in Glycyrrhiza uralensis (LUS1 gene (amino acid sequence: SEQ ID NO: 1, coding base sequence: SEQ ID NO: 5) )), genes encoding enzymes in the soyasaponin synthesis pathway (CYP93E3 gene (amino acid sequence: SEQ ID NO: 2, coding nucleotide sequence: SEQ ID NO: 6) and CYP72A566 gene (amino acid sequence: SEQ ID NO: 3, coding nucleotide sequence: SEQ ID NO: 7 )), and genes encoding enzymes on the oleanolic acid synthesis pathway (CYP716A179 gene (amino acid sequence: SEQ ID NO: 4, coding base sequence: SEQ ID NO: 8)) were selected as target genes, and mutations were introduced into them. A target sequence was designed. Specifically, we use a web tool (CRISPR direct) to list candidate target sequences, and select two sequences for each gene that are close to the start codon and highly specific to the target gene. Selected. Selected target sequences (SEQ ID NOS: 9-16) are shown in Table 1.

Test example 1. Generation of CYP93E3/CYP72A566-double-knockout hairy roots and CYP72A566 single-knockout hairy roots Four gRNAs (two of each gene) that bind to target sequences and Arabidopsis codon usage bias to knockout the CYP93E3 and CYP72A566 genes A binary vector expressing Cas9 nuclease from S. pyogenes, codon-optimized for . Specifically, gRNA was cloned downstream of Arabidopsis thaliana-derived U6 promoter, and Cas9 nuclease was cloned downstream of cauliflower mosaic virus-derived 35S promoter into the T-DNA region of the vector. This vector was transformed into Agrobacterium Rhizogenes ATCC 15834 strain.

The hypocotyl of licorice was infected with Agrobacterium to induce hairy roots. After about 6 weeks, hairy roots were isolated and lysated according to the method described in the literature (Biotechniques 19:394-7, 1994).

Heteroduplex mobility analysis was performed to confirm the introduction of mutations into the target sequence of the target gene. Specifically, using the lysate as a template, a gene of about several 100 bp including the target sequence was amplified by PCR using target gene-specific primers. The PCR product was subjected to Multina (Shimadzu Corporation) and heteroduplex mobility analysis was performed. Those whose electrophoretic mobility of the PCR product was different from that of the wild type were selected as double knockout hairy root candidates (Fig. 1).

The PCR product of the double knockout candidate was cloned and sequence analysis was performed. Deletions/substitutions were confirmed in the target sequence of the target gene and double knockout hairy roots were identified. Hairy root F is a CYP93E3/ CYP72A566-double knockout, and hairy root M is a CYP72A566 single knockout (Tables 2 and 3: The first sequence (10 bp or more) in the table is SEQ ID NO: 17 in the sequence listing. ~25).

Test example 2. Generation of quadruple-knockout hairy roots (CYP93E3/ CYP72A566/LUS1/CYP716A179) To knockout the CYP93E3, CYP72A566, LUS1 and CYP716A179 genes, 8 gRNAs (2 for each gene) that bind to target sequences, and S A binary vector was constructed to co-express Cas9 nuclease from .pyogenes.

A lysate was prepared by inducing hairy roots in the same manner as in Test Example 1.

The hairy root lysate was subjected to competitive ELISA (Anal. Chem. 2001, 73, 5784-5790), glycyrrhizin in the lysate was quantified, and quadruple knockout candidate hairy roots were selected (Table 4, #1).

Using the selected hairy root lysate as a template, sequence analysis was performed in the same manner as in Test Example 1. Deletions/insertions within the target sequence of the target gene were confirmed and quadruple knockout hairy roots were identified (Table 5: Sequences first appearing in the table (>10 bp) are shown in SEQ ID NOs: 26-36 in the Sequence Listing). be done.).

Test example 3. Generation of CYP716A179 single-knockout hairy roots Four gRNAs (two for each gene) that bind to the target sequences of the LUS1 and CYP716A179 genes, and a binary vector that co-expresses Cas9 nuclease from S.pyogenes were constructed. Subsequent steps were carried out in the same manner as for the generation of CYP93E3/CYP72A566-double knockout hairy roots. As a result, only CYP716A179 single knockout was obtained (Table 6: The first sequence (10 bp or more) in the table is shown in SEQ ID NO: 37 in the Sequence Listing).

Test example 4. Generation of CYP93E3 Knockout Hairy Roots (Single Knockout) In addition to the two target sequences selected in Test Example 1, one target sequence was selected. A binary vector was constructed to co-express CYP93E3, three gRNAs that bind to gene target sequences, and the Cas9 nuclease from S. pyogenes. Subsequent steps were carried out in the same manner as for the generation of CYP93E3/CYP72A566-double knockout hairy roots. (Table 7: Sequences (10 bp or more) appearing first in the table are shown in SEQ ID NOs: 38-39 in the sequence listing).

Test example 5. Generation of CYP93E3/CYP72A566-double knockout + CYP88D6 overexpression hairy roots In order to generate hairy roots with knockout of CYP93E3 and CYP72A566 genes and overexpression of CYP88D6 gene, four types of gRNAs that bind to target sequences ( express Cas9 nuclease from S. pyogenes codon-optimized for Arabidopsis codon usage bias, and CYP88D6 from G. uralensis (amino acid sequence: SEQ ID NO: 46; coding sequence: SEQ ID NO: 47) A binary vector was generated. Specifically, gRNA was cloned downstream of Arabidopsis thaliana-derived U6 promoter, and Cas9 nuclease and CYP88D6 were cloned downstream of cauliflower mosaic virus-derived 35S promoter, respectively, in the T-DNA region of the vector. This vector was transformed into Agrobacterium rhizogenes ATCC 15834 strain.

A lysate was prepared by inducing hairy roots in the same manner as in Test Example 1.

The glycyrrhizin content of hairy roots was evaluated by the same method as Test Example 2, and candidate hairy roots with double knockout + CYP88D6 overexpression were selected.

In order to evaluate the expression level of CYP88D6, quantitative PCR was performed to select hairy roots with higher transcription levels of CYP88D6 compared to the wild type (Fig. 2).

Using the selected hairy root lysate as a template, sequence analysis was performed in the same manner as in Test Example 1. Deletion/insertion within the target sequence of the target gene was confirmed, and CYP93E3/ CYP72A566-double knockout + CYP88D6 overexpressing hairy roots were identified (Table 8: The first sequence (10 bp or more) in the table is the sequence listing. (SEQ ID NOS: 48-53).

Test example 6. Analysis of Triterpenoids by LC-MS Wild-type hairy roots and knockout hairy roots (Test Examples 1 to 5) were lyophilized, crushed with a multi-bead shocker (Yasui Kikai Co., Ltd.) and pulverized. Methanol was added to the hairy root powder, and triterpenoids were eluted by sonication. The filtered (0.2 um) solution was used as a triterpenoid solution and subjected to an LC-MS system, Aquity UPLC/MS system (Waters), to determine the triterpenoid composition.

The results are shown in Figures 3 and 4. Disruption of at least two genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. It was found that the glycyrrhizin content was increased by It was also found that the content of 11-deoxo-glycyrrhizin (licorice-saponin-B2), a kind of glycyrrhizin derivative, also increased.

Test example 7. Production of multiple knockout stolons Shoot tips are prepared from licorice stems or stolons. The method described in JP-A-2017-205104, consisting of a gene encoding an enzyme on the betulinic acid synthesis pathway, a gene encoding an enzyme on the soyasaponin synthesis pathway, and a gene encoding an enzyme on the oleanolic acid synthesis pathway A vector carrying gRNA and a Cas9 nuclease gene that bind to the target sequences of at least two or more target genes selected from the group, or a gRNA/Cas9 nuclease protein complex is introduced into the shoot apex. The shoot tips are transferred to an agar medium to form shoots. After selecting shoots containing foreign genes, stem parts containing axillary buds are differentiated into underground stems in a liquid medium by the method described in the literature (Plant biotechnology27, 59-66, 2010). By the same method as in Test Example 2, underground stems containing multiple knockout cells are identified.

Claims (15)

1. At least two or more target genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway. A plant cell that has been modified such that the function and/or expression of the gene of interest is lost or reduced due to the modification.
2. The target gene includes a gene encoding a triterpene 24-position carbon hydroxylase, a gene encoding a triterpene 22-position carbon hydroxylase, a gene encoding a triterpene 28-position carbon oxidase, and lupeol synthesis. 3. The plant cell according to claim 1, comprising at least two genes selected from the group consisting of enzyme-encoding genes.
3. 2. The plant cell according to claim 1, wherein said target gene comprises at least two or more genes selected from the group consisting of genes encoding enzymes on the soyasaponin synthesis pathway.
4. 3. The plant cell according to claim 1 or 2, wherein said gene of interest comprises a gene encoding a triterpene 24-position carbon hydroxylase and a gene encoding a triterpene 22-position carbon hydroxylase.
5. The target gene includes at least two genes selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and enzymes on the oleanolic acid synthesis pathway. A plant cell according to any one of claims 1 to 3, comprising an encoding gene.
6. The target gene includes a gene encoding lupeol synthase, a gene encoding a triterpene 24-position carbon hydroxylase, a gene encoding a triterpene 22-position carbon hydroxylase, and a triterpene 28-position carbon. The plant cell according to any one of claims 1 to 5, comprising a gene encoding an oxidase of
7. 7. The plant cell according to any one of claims 1 to 6, into which a gene encoding at least one glycyrrhizin biosynthetic enzyme has been introduced.
8. 8. Any one of claims 1 to 7, wherein the index value of the function and/or expression of the target gene is 10% or less relative to 100% of the index value of the function and/or expression when the target gene is not modified. The plant cell of claim 1.
9. The plant cell according to any one of claims 1 to 8, which is a plant cell of a leguminous plant.
10. A plant tissue or plant comprising the plant cell according to any one of claims 1 to 9.
11. 11. The plant tissue or plant according to claim 10, which has a higher content of glycyrrhizin or at least one glycyrrhizin derivative than the plant tissue or plant without the plant cells.
12. 12. The plant tissue according to claim 10 or 11, which is a hairy root of Glycyrrhiza genus.
13. A seed generated in the plant according to any one of claims 10 to 12.
14. At least two or more selected from the group consisting of genes encoding enzymes on the betulinic acid synthesis pathway, genes encoding enzymes on the soyasaponin synthesis pathway, and genes encoding enzymes on the oleanolic acid synthesis pathway in plant cells A method for producing a modified plant cell or a plant tissue or plant body containing the modified plant cell, comprising introducing a mutation that eliminates or reduces the function and/or expression of the target gene.
15. A method for producing glycyrrhizin or a glycyrrhizin derivative, comprising extracting glycyrrhizin or at least one glycyrrhizin derivative from the plant tissue or plant body according to any one of claims 10 to 12.

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