Steviol glycoside biosynthetic variant enzyme and use thereof

Abstract

The present invention provides a protein containing the amino acid sequence of SEQ ID NO: 2, a variant thereof, a polynucleotide encoding the protein, a transformant containing the polynucleotide, and a method for producing a steviol glycoside using the protein or a product containing the steviol glycoside.

Description

Technical Field

[0001] The present invention relates to a protein having an activity of transferring sugar to steviol glycoside, a polynucleotide encoding the protein, a transformant containing the polynucleotide, and a method for producing steviol glycoside or a product containing the protein or the transformant. Background Art

[0002] The leaves of Stevia rebaudiana in the Asteraceae family contain a secondary metabolite called steviol, which is a type of diterpenoid. Since its glycoside, steviol glycoside, is about 300 times sweeter than sugar, it is used as a low-calorie sweetener in the food industry. Obesity is becoming increasingly serious as a profound social problem internationally, and the demand for low-calorie sweeteners is also increasing from the perspective of improving health and reducing medical expenses. At present, artificially synthesized amino acid derivatives Aspartame and Acesulfame Potassium are used as artificial sweeteners, but naturally occurring low-calorie sweeteners such as steviol glycosides are safer, and it is expected that they will be easily understood by consumers (public acceptance).

[0003] Steviol glycosides of stevia are synthesized by adding sugars to steviol as a glycoside using various glycosyltransferases. Each steviol glycoside has its own unique taste characteristics, among which rebaudioside D (hereinafter, rebaudioside is sometimes referred to as "Reb") or RebM not only has high sweetness, but also has a sweet taste with less sweet aftertaste and bitterness, and is of high quality. However, such steviol glycosides with excellent taste characteristics are only contained in trace amounts in stevia, so attempts are being made to artificially synthesize them using glycosyltransferases. Some of the glycosyltransferases involved in the biosynthesis of steviol glycosides have been isolated, and the development of their variants is also underway (Patent Documents 1 to 3, Non-Patent Documents 1 to 2).

[0004] Patent Literature Patent document 1: WO2011 / 153378 Patent Document 2: WO2013 / 137487 Patent Document 3: WO2018 / 124141

[0005] Non-patent literature Non-patent document 1: Brandle and Telmer (2007) Phytochemistry 68, 1855-1863 Non-patent document 2: Richman et al (2005) Plant J. 41, 56-67 Summary of the invention

[0006] A glycotransferase that can synthesize desired steviol glycosides in high yield is sought.

[0007] The present invention provides a protein having high activity of adding a hexose to the 2nd position of a monomeric glucose residue at the 13th or 19th position of steviol glycoside.

[0008] In one embodiment, the present invention provides the following. [1] A protein selected from the group consisting of: (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, (b) a protein comprising an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residue at position 155 in the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, inserted and / or added, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I), and, (c) a protein comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2 and the same amino acid residue as the amino acid residue corresponding to the amino acid residue at position 155, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I); [Chemical 1] In the formula, R1 and R2 are each independently H, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C4~C 20 Alkyldiene, C6~C 18 Aryl, C6~C 20 Alkyl aryl, C6~C 20 Arylalkyl, C4~C 20 Cycloalkyl, C4~C 20 Cycloalkenyl, (C3~C 10 Cycloalkyl)C1~C 10 An alkyl group or a sugar residue, wherein at least one of R1 and R2 is a monomeric glucose residue. [2] The protein according to [1], wherein the amino acid residue at position 42 in the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding thereto is substituted with a phenylalanine residue. [3] The protein according to [1] or [2], wherein the hexose is glucose or rhamnose. [4] The protein according to any one of [1] to [3], wherein the compound represented by formula (I) is steviol monoside, rubusoside or stevioside. [5] A polynucleotide encoding the protein described in any one of [1] to [4]. [6] A vector, characterized in that it contains the polynucleotide described in [5]. [7] A non-human transformant, characterized in that it contains the polynucleotide described in [5]. [8] The transformant according to [7], characterized in that it contains at least one of UGT85C2, UGT76G1 and UGT74G1. [9] An extract, characterized in that it is an extract of the transformant described in [7] or [8].

[10] A food, medicine or industrial raw material, characterized in that it contains the extract described in [9].

[11] A method for producing the protein described in any one of [1] to [4], comprising culturing the non-human transformant described in [7] or [8].

[12] A method for producing steviol glycoside or a steviol glycoside composition, comprising culturing the non-human transformant according to [7] or [8].

[13] A method for producing a steviol glycoside or a steviol glycoside composition, comprising a step of reacting the protein according to any one of [1] to [4] with a UDP-sugar and the compound represented by the formula (I).

[14] The method according to

[13] , characterized in that the UDP-sugar is UDP-glucose or UDP-rhamnose.

[15] The method according to any one of

[12] to

[14] , wherein the steviol glycoside composition comprises at least one of RebE, steviol bioside, stevioside, and dulcoside A.

[0009] According to the present invention, a highly active glycosyltransferase that adds a hexose to the 2-position of a monomeric glucose residue at the 13th or 19th position of steviol glycoside and a method for producing steviol glycoside using the enzyme can be provided. In addition, by combining the protein of the present invention with other glycosyltransferases involved in the synthesis of steviol glycosides, steviol glycosides with good taste such as Reb D and Reb M can be produced in high yield. DETAILED DESCRIPTION

[0010] The present invention will be described in detail below. The following embodiments are for illustration of the present invention and are not intended to limit the present invention to the embodiments. The present invention can be implemented in various forms without departing from the gist of the present invention. In addition, all documents cited in this specification and other patent documents such as public gazettes and patent gazettes are incorporated into this specification as reference. In addition, this specification includes the contents described in the specification and drawings of the Japanese patent application (Japanese Patent Application No. 2022-110052) filed on July 7, 2022, which is the basis for the priority claim of this application.

[0011] 1. Glycotransferase In one aspect, the present invention provides a protein selected from the group consisting of the following (a) to (c) (hereinafter, sometimes referred to as "the protein of the present invention"). (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, (b) a protein comprising an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residue at position 155 of the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, inserted and / or added, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the following formula (I), and (c) A protein containing an amino acid sequence having a sequence identity of more than 90% with respect to the amino acid sequence of sequence number 2 and the same amino acid residue as the amino acid residue corresponding to the amino acid residue at position 155, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the following formula (I). [Chemistry 2] (wherein, R1 and R2 are each independent and represent H, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C4~C 20 Alkyl dienyl, C6~C 18 Aryl, C6~C 20 Alkyl aryl, C6~C 20 Arylalkyl, C4~C 20 Cycloalkyl, C4~C 20 Cycloalkenyl, (C3~C 10 Cycloalkyl)C1~C 10 Alkyl or sugar residue. Wherein, at least one of R1 and R2 is a monomeric glucose residue.

[0012] The amino acid residue corresponding to the amino acid residue at position 155 in the amino acid sequence of sequence number 2 refers to the amino acid residue existing at the other position when the amino acid residue at position 155 in the amino acid sequence of sequence number 2 is moved to another position by deletion, insertion, etc. of the amino acid residue in the protein (c) of the present invention. For example, when the protein (c) of the present invention is composed of an amino acid sequence in which the amino acid residue at position 30 is deleted in the amino acid sequence of sequence number 2, the "amino acid residue corresponding to the amino acid residue at position 155 in the amino acid sequence of sequence number 2" in the protein (c) is the amino acid residue at position 154. Which amino acid residue is the amino acid residue corresponding to the amino acid residue at position 155 in the amino acid sequence of sequence number 2 can be confirmed, for example, by comparing the amino acid sequence of sequence number 2 with the amino acid sequence of the protein (c) of the present invention.

[0013] In one embodiment, the protein of the present invention contains an amino acid sequence in which the amino acid residue at position 42 in the amino acid sequence of sequence number 2 or the amino acid residue corresponding thereto is substituted with a phenylalanine residue (e.g., sequence number 4). The amino acid residue corresponding to the amino acid residue at position 42 in the amino acid sequence of sequence number 2 refers to the amino acid residue present at the other position when the amino acid residue at position 42 in the amino acid sequence of sequence number 2 is moved to another position due to the deletion or insertion of the amino acid residue in the protein (b) or (c) of the present invention. For example, when the protein (b) of the present invention is composed of an amino acid sequence in which the amino acid residue at position 30 in the amino acid sequence of sequence number 2 is deleted, the "amino acid residue corresponding to the amino acid residue at position 42 in the amino acid sequence of sequence number 2" in the protein (b) refers to the amino acid residue at position 41. Which amino acid residue is the amino acid residue corresponding to the amino acid residue at position 42 in the amino acid sequence of sequence number 2 can be confirmed, for example, by comparing the amino acid sequence of sequence number 2 with the amino acid sequence of the protein (b) or (c) of the present invention.

[0014] In one embodiment, the protein of the present invention comprises a protein selected from the group consisting of: (a') a protein comprising the amino acid sequence of SEQ ID NO: 4, (b') a protein comprising an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residues at positions 42 and 155 of the amino acid sequence of SEQ ID NO: 4 are deleted, substituted, inserted and / or added, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I), and (c') contains an amino acid sequence having a sequence identity of more than 90% with respect to the amino acid sequence of sequence number 4, but the amino acid residues corresponding to the amino acid residues at positions 42 and 155 are respectively identical to the amino acid residues at positions 42 and 155 of sequence number 4, and a protein having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I).

[0015] The protein of the present invention may not exist naturally. The protein of the present invention may be artificially obtained by using, for example, the site-specific mutation introduction method described in "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997", "Nuc. Acids. Res., 10, 6487 (1982)", "Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)", "Gene, 34, 315 (1985)", "Nuc. Acids. Res., 13, 4431 (1985)", "Proc. Natl. Acad. Sci. USA, 82, 488 (1985)", etc.

[0016] In the present specification, examples of “a protein comprising an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residue at position 155 of the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, inserted and / or added, and which has an activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I)” include, for example, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to 41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31 , 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1 amino acid residue is deleted, substituted, inserted and / or added, and has the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I). The number of deletions, substitutions, insertions and / or additions of the above-mentioned amino acid residues is generally preferred to be as small as possible.

[0017] In addition, examples of the above-mentioned protein include a protein having a sequence identity of 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the amino acid sequence of SEQ ID NO: 2, an amino acid sequence identical to the amino acid residue corresponding to the amino acid residue at position 155, and having an activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound shown in formula (I). Generally, the larger the numerical value of the above-mentioned sequence identity, the more preferred.

[0018] Similarly, in the present specification, “a protein having an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residues at positions 42 and 155 of the amino acid sequence of SEQ ID NO: 4 are deleted, substituted, inserted and / or added, and having an activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I)” includes, for example, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to 41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, , 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1 amino acid residue is deleted, substituted, inserted and / or added, and has the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I). The number of deletions, substitutions, insertions and / or additions of the above-mentioned amino acid residues is generally preferred to be as small as possible.

[0019] In addition, examples of the above-mentioned protein include a protein having a sequence identity of 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to the amino acid sequence of SEQ ID NO: 4, an amino acid sequence identical to the amino acid residue corresponding to the amino acid residue at position 155, and having an activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound shown in formula (I). Generally, the larger the numerical value of the above-mentioned sequence identity, the more preferred.

[0020] In this manual, “C1 to C 20 The "alkyl" is preferably C1-C 10 The alkyl group is more preferably a C1 to C6 alkyl group. Examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and dodecanyl. In this manual, "C2 to C 20 The "alkenyl" is preferably C2-C 10 The alkenyl group is more preferably a C2 to C6 alkenyl group. Examples of the alkenyl group include, but are not limited to, vinyl, allyl, propenyl, isopropenyl, 2-methyl-1-propenyl, 2-methylallyl, and 2-butenyl. In this manual, "C2 to C 20 "Alkynyl" is preferably C2-C 10 The alkynyl group is more preferably a C2 to C6 alkynyl group. Examples of the alkynyl group include, but are not limited to, ethynyl, 2-propynyl, 2-butynyl, and the like. In this manual, "C4 to C 20 The "alkyl diene group" is preferably C4 to C 10 The alkyldienyl group is more preferably a C4 to C6 alkyldienyl group. Examples of the alkyldienyl group include, but are not limited to, 1,3-butadienyl and the like.

[0021] In this manual, "C6~C 18 The "aryl" is preferably C6-C 10 Aryl. Examples of the aryl group include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, indenyl, biphenyl, anthracenyl, and phenanthryl. In this manual, "C6~C 20 The "alkylaryl" is preferably C6-C 12 Alkylaryl. Examples of the alkylaryl group include, but are not limited to, o-tolyl, m-tolyl, p-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, o-cumyl, m-cumyl, p-cumyl, and mesityl. In this manual, "C6~C 20 The "arylalkyl" is preferably C6-C 12 Arylalkyl. Examples of the arylalkyl group include, but are not limited to, benzyl, phenethyl, diphenylmethyl, triphenylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, 2,2-diphenylethyl, 3-phenylpropyl, 4-phenylbutyl, and 5-phenylpentyl.

[0022] In this manual, "C4 to C 20 The cycloalkyl group is preferably C4 to C 10 Cycloalkyl. Examples of the cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In this manual, "C4 to C 20 The cycloalkenyl group is preferably C4 to C 10 Cycloalkenyl. Examples of the cycloalkenyl group include, but are not limited to, cyclopropenyl, cyclobutenyl, 2-cyclopenten-1-yl, 2-cyclohexen-1-yl, and 3-cyclohexen-1-yl. In this specification, as "(C3 to C 10 Cycloalkyl)C1~C 10 Examples of the "alkyl group" include, but are not limited to, methylcyclopropyl, ethylcyclopropyl, methylcyclobutyl, ethylcyclopentyl, and methylcyclohexyl.

[0023] In the present specification, the "sugar residue" is not particularly limited, and may be a sugar residue composed of one or more pentoses, hexoses (including deoxyhexoses), or a combination thereof. Examples of pentoses include ribose, arabinose, and lyxose, and examples of hexoses include allose, altrose, glucose, mannose, gulose, idose, galactose, talose, and rhamnose. The "sugar residue" is preferably a sugar residue composed of one or more hexose units, and more preferably a sugar residue of a glucose monomer (-Glc) or a glucose dimer (-Glc-Glc). In the sugar residue of a glucose dimer, the glucose residues are preferably bonded to each other in a β2,1 glycosidic bond.

[0024] The compound of formula (I) is preferably steviol monoglycoside, rubusoside or stevioside.

[0025] According to the protein of the present invention, the hexose added to the 2-position of the monomeric glucose residue at the 13-position or the 19-position of the compound represented by formula (I) is not particularly limited, and is preferably a hexose selected from glucose, rhamnose, mannose and galactose, and more preferably glucose or rhamnose. The hexose is most preferably glucose.

[0026] The activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I) can be verified, for example, by the following method: incubating in a buffer (for example, sodium phosphate buffer or potassium phosphate buffer) in a neutral region of pH 6.0 to 8.0 containing a test protein, 1 to 1000 μM (preferably 100 to 700 μM, most preferably 500 μM) of a UDP sugar (for example, UDP-glucose) and 1 to 500 μM (preferably 100 to 500 μM, most preferably 250 μM) of a substrate compound (compound of formula (I)) at a temperature of 20 to 40° C. for 10 minutes to 2 hours, purifying the reaction solution as needed and analyzing it by a known method such as HPLC analysis or LC-MS (Liquid Chromatography-Mass Spectrometry) analysis.

[0027] Alternatively, the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I) can be verified, for example, by the following method: a transformant (e.g., yeast) co-expressing the test protein and UGT85C2 is cultured in a medium containing glucose (e.g., 2 to 48 hours, preferably 3 to 24 hours, most preferably overnight), and then cultured in a medium containing steviol (e.g., 1 to 100 μg / mL, preferably 2 to 50 μg / mL, most preferably 10 μg / mL) for a predetermined time (e.g., 3 to 72 hours, preferably 6 to 48 hours, most preferably 2 nights) to obtain a culture supernatant, which is then analyzed by a known method such as HPLC analysis or LC-MS analysis.

[0028] As a result of the analysis, when a compound in which a hexose is attached to the 2-position of the monomeric glucose residue at the 13-position or 19-position of the compound represented by formula (I) (for example, steviol disaccharide, stevioside, dulcoside A, RebE, RebC, etc.) can be detected, the test protein can be said to have the activity of attaching a hexose to the 2-position of the monomeric glucose residue at the 13-position or 19-position of the compound represented by formula (I).

[0029] When the protein of the present invention is combined with UGT85C2 and steviol is used as a substrate, the activity of producing RebE is higher than that of a known protein (e.g., UGT91D-like3 (SEQ ID NO: 6)). The amount of RebE produced can be, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, 2.6 times or more, 2.8 times or more, 3 times or more, 3.5 times or more, or 4 times or more relative to the activity of UGT91D-like3. Furthermore, when the protein of the present invention is combined with UGT74G1, UGT76G1, and UGT85C2 and steviol is used as a substrate, the activity of producing RebD and RebM in addition to RebE is higher than that of known proteins (eg, UGT91D-like3). Relative to the activity of UGT91D-like3, the amount of RebD produced can be, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.8 times or more, 3 times or more, 3.2 times or more, 3.4 times or more, and the amount of RebM produced can be, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, 2.6 times or more, 2.8 times or more.

[0030] The deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues are deleted, substituted, inserted and / or added at any and one or more positions in the same sequence, and two or more of the deletion, substitution, insertion and addition may occur simultaneously.

[0031] Examples of amino acid residues that can be substituted for each other are shown below. Amino acid residues included in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, o-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine.

[0032] The protein of the present invention can be obtained by expressing a polynucleotide encoding the protein (refer to "polynucleotide of the present invention" described later) in a suitable host cell, or can be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethoxycarbonyl method) and the tBoc method (tert-butyloxycarbonyl method). In addition, chemical synthesis can be performed using peptide synthesizers such as Advanced Automation PeptideProtein Technologies, Perkin Elmer, Protein Technologies, PerSeptive, Applied Biosystems, and SHIMADZU.

[0033] 2. A method for producing steviol glycosides. By utilizing the activity of the protein of the present invention to add a hexose to the 2-position of the monomeric glucose residue at the 13-position or 19-position of the compound represented by formula (I), steviol glycosides can be produced easily and in large quantities.

[0034] Therefore, in another aspect, the present invention provides a method for producing steviol glycosides or steviol glycoside compositions, characterized in that it comprises a step of reacting the protein of the present invention with UDP-sugar and the compound represented by the formula (I) (hereinafter, sometimes referred to as "production method A of the present invention").

[0035] In this specification, the so-called "UDP-sugar" is a sugar of uridine diphosphate (UDP). Preferred examples of the sugar part in UDP-sugar include sugars composed of one or more hexoses. Examples of hexoses are as described above. UDP-sugar is preferably UDP-hexose, and more preferably selected from UDP-glucose, UDP-rhamnose, UDP-mannose and UDP-galactose. The UDP-sugar is most preferably UDP-glucose.

[0036] The production method A of the present invention includes a step (step 1) of reacting the protein of the present invention with a UDP sugar and a compound represented by formula (I). In step 1, a hexose is added to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I). The production method A of the present invention may further include a step (step 2) of purifying the steviol glycoside produced in step 1. In addition, the production method A of the present invention may further include a step (step 3) of further adding sugar to the steviol glycoside produced in step 1. Step 3, for example, may also include allowing other enzymes related to the biosynthesis of steviol glycosides such as UGT74G1, UGT76G1, and UGT85C2 to act on the steviol glycosides produced in step 1. The production method A of the present invention may further include a step (step 4) of producing the compound represented by formula (I) from steviol. Step 4, for example, may also include allowing other enzymes related to the biosynthesis of steviol glycosides such as UGT74G1, UGT76G1, and UGT85C2 to act on the steviol glycosides produced in step 1.

[0037] Examples of steviol glycosides produced by the production method A of the present invention include steviol disaccharide, stevioside, dulcoside A, RebE, RebC, etc., but are not limited to these. In addition, a steviol glycoside composition refers to a composition containing at least one steviol glycoside produced by the production method A of the present invention, for example, at least one of steviol disaccharide, stevioside, dulcoside A, RebE and RebC, preferably at least two. The steviol glycoside composition may also contain a compound used as a matrix, for example, it may also contain steviol, steviol monosaccharide, rubusoside, stevioside, etc.

[0038] The ratio of the steviol glycosides contained in the steviol glycoside composition, based on mass, may be, for example, 0.05-5.0, 0.10-4.5, 0.15-4.0, 0.20-3.5, 0.25-3.0, 0.30-2.5, 0.35-2.0, 0.40-1.5, 0.45-1.0, 0.47-0.8 or 0.48-0.6, particularly about 0.5, when combined with at least UGT74G1, UGT76G1 and UGT85C2, and may be, for example, 0.025-2.50, 0.050-2.25, 0.075-2.0, 0.080-2.0, 0.100-2.00, 0.110-2.00, 0.120-2.00, 0.130-2.00, 0.140-2.00, 0.150-2.00, 0.160-2.00, 0.170-2.00, 0.180-2.00, 0.190-2.00, 0.200-2.00, 0.200-2.00, 0.200-2.00, 0.200-3.00, 0. .10-1.75, 0.125-1.50, 0.150-1.25, 0.175-1.0, 0.20-0.75, 0.21-0.50, 0.22-0.40 or 0.23-0.30, in particular about 0.25, Reb D / Reb I may be, for example, 0.075-7.5, 0.150-6.75, 0.225-6.0, 0.30-5.25, 0.375-4.5, 0.450-3.75, 0.525-3.0, 0.60-2.25, 0.675-1.5, 0.70-1.0 or 0.725-0.8, in particular about 0.75, Reb D / stevioside may be, for example, 0.25-25.0, 0.5 0-22.5, 0.75-20.0, 1.0-17.5, 1.25-15.0, 1.50-12.5, 1.75-10.0, 2.0-7.5, 2.1-5.0, 2.2-4.0 or 2.3-3.00, especially about 2.5, RebM / RebA can be, for example, 0.05-5.0, 0.10-4.5, 0.15-4.0, 0.20-3.5, 0.25-3.0, 0.30-2.5, 0.35-2.0, 0.40-1.5, 0.45-1.0, 0.47-0.8 or 0.48-0.6, especially about 0.5, RebM / RebI can be, for example, 0.18-18, 0.28-16, 0. 38-14, 0.58-12, 0.78-10, 0.98-8, 1.18-6, 1.38-5, 1.58-4, 1.78-3 or 1.6-2, particularly about 1.8, RebM / stevioside may be, for example, 0.5-50, 1.0-45, 1.5-40, 2.0-35, 2.5-30, 3.0-25, 3.5-20, 4.0-15, 4.5-10, 4.7-8 or 4.8-6, particularly about 5, (RebD+RebM) / RebA may be, for example, 0.075-7.5, 0.150-6.75, 0.225-6.0, 0.30-5.25, 0.375-4.5, 0.450-3.75, 0.525-3.0, 0.60-2.25, 0.675-1.5, 0.70-1.0 or 0.725-0.8, especially about 0.75, (RebD+RebM) / RebI may be, for example, 0.25-25.0, 0.50-22.5, 0.75-20.0, 1.0-17.5, 1.25-15.0, 1.50-12.5, 1.75-10.0, 2.0-7.5, 2.1-5.0, 2.2-4.0 or 2.3-3.00, especially and (RebD+RebM) / stevioside may be, for example, 0.75-75.0, 1.50-67.5, 2.25-60.0, 3.0-52.5, 3.75-45.0, 4.5-37.5, 5.25-30.0, 6.0-22.5, 6.75-15.0, 7.0-10.0 or 7.25-8.0, particularly about 7.5. RebE / RebA may be, for example, about 0.005-0.50, 0.010-0.45, 0.01 5 to 0.40, 0.020 to 0.35, 0.025 to 0.30, 0.030 to 0.25, 0.035 to 0.20, 0.040 to 0.15, 0.045 to 0.10, 0.047 to 0.08 or 0.048 to 0.06, and in particular about 0.05, and RebE / RebI can be, for example, 0.025 to 2.50, 0.050 to 2.25, 0.075 to 2.0, 0.10 to 1.75, 0.125 to 1.50, 0.150 to 1.25 , 0.175-1.0, 0.20-0.75, 0.21-0.50, 0.22-0.40 or 0.23-0.30, especially about 0.25, RebE / stevioside can be, for example, 0.05-5.0, 0.10-4.5, 0.15-4.0, 0.20-3.5, 0.25-3.0, 0.30-2.5, 0.35-2.0, 0.40-1.5, 0.45-1.0, 0.47-0.8 or 0.48-0.6, especially about 0.5. .

[0039] The generated steviol glycosides are extracted with a suitable solvent (an aqueous solution such as water or an organic solvent such as alcohol, ether and acetone), and can be purified by a known method such as a gradient of ethyl acetate and other organic solvents: water, HPLC, gas chromatography, time-of-flight mass spectrometry (TOF-MS), ultra-performance liquid chromatography (UPLC), etc.

[0040] 3. Non-human transformants expressing the protein of the present invention Steviol glycosides can also be produced in cells of microorganisms (bacteria such as Escherichia coli or fungi such as yeast), plants, insects, mammals other than humans, etc. by using the protein of the present invention. In this case, the protein of the present invention can be expressed by introducing a polynucleotide encoding the protein of the present invention into a host cell from a microorganism, plant, insect, mammal other than humans, etc., and the protein of the present invention can be reacted with the UDP-sugar present in the above-mentioned cells and the compound represented by formula (I) to produce steviol glycosides.

[0041] Thus, the present invention provides a non-human transformant (hereinafter referred to as "the transformant of the present invention") containing a polynucleotide encoding a protein of the present invention (hereinafter sometimes referred to as "the polynucleotide of the present invention"). In one aspect, the polynucleotide of the present invention comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the base sequence of SEQ ID NO: 1, (b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, (c) a polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residue at position 155 in the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, inserted and / or added, and which has the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I), (d) a polynucleotide encoding a protein having an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2 and having the same amino acid residue as the amino acid residue corresponding to the amino acid residue at position 155, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I), and (e) a polynucleotide which hybridizes under highly stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotide of (a) or (b) above, and encodes a protein which contains an amino acid residue corresponding to the amino acid residue at position 155 of sequence number 2 and has the same amino acid sequence as the amino acid residue at position 155 of sequence number 2, and has the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I).

[0042] In one aspect, the polynucleotide of the present invention comprises a polynucleotide selected from the group consisting of: (a') a polynucleotide comprising the base sequence of SEQ ID NO: 3, (b') a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4, (c') a polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residues at positions 42 and 155 of the amino acid sequence of SEQ ID NO: 4 are deleted, substituted, inserted and / or added, and which has the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I), (d') a polynucleotide encoding a protein having an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 4 and having the same amino acid residues as those corresponding to the amino acid residues at positions 42 and 155, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I), and (e') hybridizes under highly stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of the polynucleotide of (a') or (b') above, and encodes an amino acid sequence containing amino acid residues corresponding to amino acid residues at positions 42 and 155 of sequence number 4, which are respectively identical to the amino acid residues at positions 42 and 155 of sequence number 4, and a polynucleotide having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by the above formula (I).

[0043] In this specification, the term "polynucleotide" refers to DNA or RNA. In this specification, the so-called "polynucleotide that hybridizes under highly stringent conditions" refers to, for example, a polynucleotide obtained by using, as a probe, all or a part of a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, a colony hybridization method, a plaque hybridization method, a Southern hybridization method, etc. As a hybridization method, for example, the method described in "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001" and "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997" can be used.

[0044] In this specification, the so-called "high stringency conditions" include, for example, (1) 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, 50°C, (2) 0.2×SSC, 0.1% SDS, 60°C, (3) 0.2×SSC, 0.1% SDS, 62°C, (4) 0.2×SSC, 0.1% SDS, 65°C, or (5) 0.1×SSC, 0.1% SDS, 65°C, but are not limited thereto. Among these conditions, the higher the temperature, the more efficient it is expected that DNA with a high degree of sequence identity can be obtained. However, as factors affecting the stringency of hybridization, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration need to be considered. If a person skilled in the art selects these factors appropriately, the same stringency can be achieved.

[0045] In addition, when a commercially available kit is used in hybridization, for example, Alkphos Direct Labelling and Detection System (GE Healthcare) can be used. At this time, according to the protocol attached to the kit, after incubation with the labeled probe for one night, the membrane is washed with a single wash buffer containing 0.1% (w / v) SDS at 55-60°C, and the hybridized DNA can be detected. Alternatively, when preparing the probe, when the probe is labeled with digoxigenin (DIG) using a commercially available reagent (for example, PCR Labeling Mix (Roche Diagnostics)), the DIG nucleic acid detection kit (Roche Diagnostics) can be used to detect hybridization.

[0046] Examples of polynucleotides that may hybridize other than the above include polynucleotides that have a sequence identity of 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with the polynucleotide of SEQ ID NO: 1 or 3 or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or 4 when calculated using default parameters using consistency search software such as FASTA and BLAST.

[0047] In addition, the sequence identity of the amino acid sequence or the base sequence can be determined using FASTA (Science 227 (4693): 1435-1441, (1985)) or the algorithm BLAST (Basic Local Alignment Search Tool) proposed by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). Programs called blastn, blastx, blastp, tblastn or tblastx based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When blastn is used to analyze the base sequence, for example, the parameters are set to score = 100 and wordlength = 12. In addition, when blastp is used to analyze the amino acid sequence, for example, the parameters are set to score = 50 and wordlength = 3. When using BLAST and GappedBLAST programs, the default parameters of each program are used. The polynucleotide of the present invention can be obtained by a known genetic engineering method or a known synthetic method.

[0048] The polynucleotide of the present invention is preferably introduced into a host in a state of being inserted into an appropriate expression vector. Suitable expression vectors typically contain the following substances. (i) a promoter that can be transcribed in the host cell, (ii) a polynucleotide of the present invention bound to the promoter, and (iii) An expression cassette contained as a component of a signal that is involved in the transcription termination and polyadenylation of RNA molecules and acts in the host cell. Examples of methods for producing expression vectors include methods using plasmids, phages, cosmids, and the like, but are not particularly limited.

[0049] The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. For example, depending on the type of host cell, an appropriate promoter sequence for expressing the polynucleotide of the present invention can be selected, and the promoter and the polynucleotide of the present invention can be combined into various plasmids to obtain a vector, which can be used as an expression vector.

[0050] The expression vector of the present invention, depending on the type of host to be introduced, contains an expression control region (e.g., a promoter, a terminator and / or a replication origin, etc.). As a promoter for bacteria, for example, trc promoter, tac promoter, lac promoter, etc. can be cited, as a promoter for yeast, for example, GAL1 promoter, GAL10 promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, PH05 promoter, etc. can be cited, as a promoter for mold, for example, amylase promoter, trpC promoter, etc. can be cited. In addition, as examples of plant promoters, 35S RNA promoter of cauliflower mosaic virus, rd29A gene promoter, rbcS promoter, and the enhancer sequence of the 35S RNA promoter of the above-mentioned cauliflower mosaic virus are attached to the mac-1 promoter of the 5' side of the mannopine synthase promoter sequence from Agrobacterium, etc. can be cited. As a promoter for animals, viral promoters (e.g., SV40 early promoter, SV40 late promoter, etc.) can be cited.

[0051] The expression vector preferably contains at least one selection marker. As the above-mentioned marker, auxotrophic markers (ura5, niaD, TRP1, URA3, HIS3, LEU2), drug resistance markers (hygromycin, bleomycin, G418), copper resistance gene (CUP1), cerulein resistance gene (mutated FAS2, PDR4) (Aritomi et al., Biosci Biotechnol Biochem. 2004; 68 (1): 206-14, Hussain et al., Gene, vol. 101, p. 149, 1991) and the like can be used.

[0052] The method for producing the transformant of the present invention is not particularly limited, and examples thereof include a transformation method in which an expression vector containing the polynucleotide of the present invention is introduced into a host.

[0053] The host cell used for transformation is not particularly limited, and various known cells can be used appropriately. For example, as examples of host cells, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe, etc.), plant cells, animal cells other than humans, etc. can be cited. The host cell can be a heterologous cell (a cell other than Stevia) or a homologous cell (a cell from Stevia). When the homologous cell is used as a transformant, the transformant of the present invention expresses the protein of the present invention introduced exogenously in addition to the endogenously expressed protein, and therefore promotes enzyme reactions, especially the biosynthesis of steviol glycosides, more than wild-type Stevia cells.

[0054] The host cell is preferably a host cell capable of producing the compound represented by formula (I). Here, the host cell is not limited to a cell capable of producing the compound represented by formula (I) in a natural state, and for example, may be a cell into which a known gene necessary for the production of the compound represented by formula (I) is introduced. Examples of genes encoding enzymes that contribute to the synthesis of the compound represented by formula (I) include, but are not limited to, known genes such as UGT85C2, UGT74G1, and UGT76G1 (Non-Patent Document 2).

[0055] When the host cell is a cell that cannot produce the compound represented by formula (I), steviol glycosides can be produced by adding the compound of formula (I) or a plant extract containing the compound as a substrate to a culture system of a transformant obtained by introducing the gene of the present invention into the host cell, even if a gene encoding an enzyme that contributes to the synthesis of the compound represented by formula (I) is not introduced.

[0056] Furthermore, by using a host cell into which a gene encoding a glycotransferase involved in the synthesis of steviol glycosides is introduced and expressing the polynucleotide of the present invention in the host cell, more highly glycosylated steviol glycosides (e.g., RebA, RebB, RebD, RebI, RebM, RebQ, etc.) can be produced. Examples of glycotransferases involved in the synthesis of steviol glycosidases include UGT74G1 (CDS: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8), UGT76G1 (CDS: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10), and UGT85C2 (CDS: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 12).

[0057] Suitable culture media and culture conditions for the host cells are well known in the art. In addition, the organisms to be transformed are not particularly limited, and examples thereof include various microorganisms or plants exemplified by the host cells above, or animals other than humans. In addition, for general molecular biology methods, reference can be made to "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)" and the like.

[0058] By culturing the transformant of the present invention, the transformant can produce the protein of the present invention. Thus, the present invention provides a method for producing the protein of the present invention (protein production method of the present invention), characterized in that the transformant of the present invention is used. The protein production method of the present invention includes a step of culturing the transformant of the present invention. Suitable culture media and conditions are well known in the art. In addition, the protein production method of the present invention may also contain a step of recovering the protein of the present invention after the step of culturing the transformant of the present invention. The recovery of the protein can be carried out by any known method. The present invention also provides a protein of the present invention produced by the protein production method of the present invention.

[0059] In addition, by culturing the transformant, the transformant can produce steviol glycosides. As mentioned above, by adding the compound of formula (I) or a plant extract containing the same compound as a substrate to the culture system of the transformant, the production of steviol glycosides can be promoted. By purifying the steviol glycosides accumulated in the transformant and / or secreted outside the transformant (e.g., in the culture), the target steviol glycoside can be obtained.

[0060] Thus, the present invention provides a method for producing a steviol glycoside or a steviol glycoside composition (method for producing steviol glycoside B of the present invention), characterized in that the transformant of the present invention is used. Suitable culture medium and conditions are well known in the art. In addition, the types and preparation methods of steviol glycosides are as described above for the method for producing steviol glycosides A of the present invention. The present invention further provides a steviol glycoside or a steviol glycoside composition produced by the method for producing steviol glycosides B of the present invention.

[0061] Any yeast can be used as a host cell for transformation, and specific examples thereof include yeasts of the genus Saccharomyces, but the host cell is not limited thereto. As a method for transforming yeast, a commonly used known method can be utilized. For example, it can be implemented by the method described in electroporation (Meth. Enzym., 194, p182 (1990)), spheroplast method (Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)), lithium acetate method (J. Bacteriology, 153, p163 (1983)), Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc., but is not limited thereto. Transformants can be obtained by selecting a strain group cultivated on a culture medium (e.g., a culture medium containing antibiotics or a culture medium lacking nutrients) having a selection pressure consistent with the selection marker used.

[0062] The transformant may be a plant transformant. The plant transformant is obtained by introducing an expression vector containing the polynucleotide of the present invention into a plant so that the polypeptide encoded by the polynucleotide is expressed. Alternatively, as long as the gene of the present invention is introduced in a manner that it can be inherited by offspring, a new plant body having the gene can be obtained by using the transformant of the present invention as a hybrid parent.

[0063] The expression vector used for transformation of the plant is not particularly limited as long as it is a vector that can express the polynucleotide of the present invention in the plant. Examples of the above-mentioned vector include a vector having a promoter that constitutively expresses the polynucleotide in plant cells or a vector having a promoter that is activated as an inducible promoter by external stimulation. Examples of promoters that constitutively express a polynucleotide in plant cells include 35S RNA promoter of cauliflower mosaic virus, rd29A gene promoter, rbcS promoter, mac-1 promoter, and the like. Examples of promoters that are inducible when activated by external stimuli include mouse mammary tumor virus (MMTV) promoter, tetracycline-responsive promoter, metallothionein promoter, and heat shock protein promoter.

[0064] The plant to be transformed in the present invention refers to any one of the whole plant, plant organs (e.g., leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g., epidermis, phloem, soft tissue, xylem, vascular bundle, palisade parenchyma, spongy tissue, etc.), or plant cultured cells, or plant cells in various forms (e.g., suspension cultured cells), protoplasts, leaf slices, callus, etc. The plant used for transformation is not particularly limited, and any one of the plants belonging to the monocotyledon class or the dicotyledon class may be used.

[0065] For gene introduction into plants, a transformation method known to those skilled in the art (eg, Agrobacterium method, gene gun method, PEG method, electroporation method, particle gun, etc.) is used. Cells or plant tissues into which the gene has been introduced are selected for resistance such as hygromycin resistance and then regenerated into plants according to conventional methods. Regeneration from transformed cells to plants can be carried out by methods known to those skilled in the art depending on the type of plant cells.

[0066] Whether the polynucleotide of the present invention has been introduced into a plant can be confirmed by detecting the polynucleotide of the present invention by PCR, Southern hybridization, Northern hybridization, etc., or by confirming the expression of a selection marker by a specific method (for example, culturing in a medium supplemented with a resistance agent or a medium lacking specific nutrients, etc.).

[0067] Once the polynucleotide of the present invention is obtained and incorporated into the transformed plant body of the genome, the offspring can be obtained by the sexual reproduction or asexual reproduction of the plant body. In addition, from the plant body or its offspring, or its asexual reproduction system, for example, seeds, fruits, cuttings, tubers, root tubers, plant strains, callus tissues, protoplasts, etc. are obtained, and based on these, the plant body can be mass-produced. Therefore, the present invention also includes the plant body into which the polynucleotide involved in the present invention is expressibly introduced, or the offspring of the plant having the same properties as the plant body, or the tissues from these.

[0068] In addition, the transformation method for various plants has been reported. There is no limitation on the transformant plant involved in the present invention. As a particularly preferred example, it is desirable to use a plant known for biosynthesis of various glycosides using steviol as aglycone, such as stevia or Rubus suavissimus.

[0069] Plants transformed with the polynucleotides of the present invention (hereinafter referred to as "plants of the present invention" or "plants of the present invention") can produce a large amount of steviol glycosides compared to their wild type when they have an appropriate substrate or when an appropriate substrate is added externally.

[0070] The plant of the present invention can be obtained simply by cultivating seeds, cuttings, bulbs, etc. of the plant of the present invention to obtain a complete plant body. Therefore, the plants of the present invention include whole plants, plant organs (such as leaves, petals, stems, roots, seeds, bulbs, etc.), plant tissues (such as epidermis, phloem, soft tissue, xylem, vascular bundles, palisade parenchyma, spongy tissue, etc.) or plant cultured cells, or various forms of plant cells (such as suspension cultured cells), protoplasts, leaf sections, callus tissue, etc.

[0071] The transformant of the present invention includes processed products of the transformant, such as dried products, freeze-dried products, pulverized products, etc. Non-limiting examples of processed products of the transformant include pulverized products of transformed cells (e.g. pulverized products of transformed yeast), dried leaves of transformed plants (e.g. dried leaves of transformed stevia plants), etc. In addition, the transformant of the present invention (including dried products, freeze-dried products, pulverized products, etc.) containing steviol glycosides can be used as raw materials for food, medicine, industrial raw materials, etc. When the transformant of the present invention contains steviol glycosides, the protein of the present invention is expressed, and a hexose is added to the 2nd position of the monomeric glucose residue at the 13th or 19th position of the steviol glycoside. Therefore, when the whole steviol glycosides contained in the transformant are extracted, the proportion of steviol glycosides with a hexose added to the 2nd position of the monomeric glucose residue at the 13th or 19th position of the steviol glycoside in the whole steviol glycosides is naturally increased compared with an organism (such as a wild-type stevia plant) that does not express the protein of the present invention. The change in the ratio affects the properties of the transformant, such as sensory properties such as sweetness.

[0072] The ratio of steviol glycosides contained in the transformant of the present invention containing steviol glycosides, based on the amount of production, in a transformant co-expressing at least UGT85C2, RebE / stevioside may be, for example, 0.05-5.0, 0.10-4.5, 0.15-4.0, 0.20-3.5, 0.25-3.0, 0.30-2.5, 0.35-2.0, 0.40-1.5, 0.45-1.0, 0.47-0.8 or 0.48-0.6, particularly about 0.5, and in a transformant co-expressing at least UGT74G1, UGT76G1 and UGT85C2, RebD / RebA may be, for example, 0.025-2.50, 0.050-2.50, 0.060-2.50, 0.080-2.50, 0.100-2.50, 0.200-3.50, 0.200-3.50, 0.200-3.50, 0.200-3.50, 0.200-3.50, 0.300-2.50, 0.350-2.0, 0.400-1.50, 0.450-1.00, 0.470-2.50, 0.500-2.50 .25, 0.075-2.0, 0.10-1.75, 0.125-1.50, 0.150-1.25, 0.175-1.0, 0.20-0.75, 0.21-0.50, 0.22-0.40 or 0.23-0.30, especially about 0.25, RebD / RebI, for example, 0.075-7.5, 0.150-6.75, 0.225-6.0, 0.30-5.25, 0.375-4.5, 0.450-3.75, 0.525-3.0, 0.60-2.25, 0.675-1.5, 0.70-1.0 or 0.725-0.8, especially about 0.75, RebD / stevioside, for example The ratio of RebM to RebA may be, for example, 0.25 to 25.0, 0.50 to 22.5, 0.75 to 20.0, 1.0 to 17.5, 1.25 to 15.0, 1.50 to 12.5, 1.75 to 10.0, 2.0 to 7.5, 2.1 to 5.0, 2.2 to 4.0, or 2.3 to 3.00, and may be particularly about 2.5. The ratio of RebM to RebA may be, for example, 0.05 to 5.0, 0.10 to 4.5, 0.15 to 4.0, 0.20 to 3.5, 0.25 to 3.0, 0.30 to 2.5, 0.35 to 2.0, 0.40 to 1.5, 0.45 to 1.0, 0.47 to 0.8, or 0.48 to 0.6, and may be particularly about 0.5. The ratio of RebM to RebI may be, for example, 0.1 8-18, 0.28-16, 0.38-14, 0.58-12, 0.78-10, 0.98-8, 1.18-6, 1.38-5, 1.58-4, 1.78-3 or 1.6-2, in particular about 1.8, RebM / stevioside can be, for example, 0.5-50, 1.0-45, 1.5-40, 2.0-35, 2.5-30, 3.0-25, 3.5-20, 4.0-15, 4.5-10, 4.7-8 or 4.8-6, in particular about 5, (RebD+RebM) / RebA can be, for example, 0.075-7.5, 0.150-6.75, 0.225-6.0, 0.30-5.25, 0.375-4.5, 0.450-3.75, 0.525-3.0, 0.60-2.25, 0.675-1.5, 0.70-1.0 or 0.725-0.8, especially about 0.75, (RebD+RebM) / RebI can be, for example, 0.25-25.0, 0.50-22.5, 0.75-20.0, 1.0-17.5, 1.25-15.0, 1.50-12.5, 1.75-10.0, 2.0-7.5, 2.1-5.0, 2.2-4.0 or 2.3 to 3.00, particularly about 2.5, (RebD + RebM) / stevioside may be, for example, 0.75 to 75.0, 1.50 to 67.5, 2.25 to 60.0, 3.0 to 52.5, 3.75 to 45.0, 4.5 to 37.5, 5.25 to 30.0, 6.0 to 22.5, 6.75 to 15.0, 7.0 to 10.0 or 7.25 to 8.0, particularly about 7.5, RebE / RebA may be, for example, about 0.005 to 0.50, 0.010 to 0. 45, 0.015-0.40, 0.020-0.35, 0.025-0.30, 0.030-0.25, 0.035-0.20, 0.040-0.15, 0.045-0.10, 0.047-0.08 or 0.048-0.06, especially about 0.05, RebE / RebI can be, for example, 0.025-2.50, 0.050-2.25, 0.075-2.0, 0.10-1.75, 0.125-1.50, 0.150- 1.25, 0.175-1.0, 0.20-0.75, 0.21-0.50, 0.22-0.40 or 0.23-0.30, especially about 0.25, RebE / stevioside can be, for example, 0.05-5.0, 0.10-4.5, 0.15-4.0, 0.20-3.5, 0.25-3.0, 0.30-2.5, 0.35-2.0, 0.40-1.5, 0.45-1.0, 0.47-0.8 or 0.48-0.6, especially about 0.5.

[0073] 4. Extracts of Transformants In addition, the present invention provides an extract of the above-mentioned transformant (hereinafter sometimes referred to as "the extract of the present invention"). The transformant of the present invention, when having a suitable substrate or when a suitable substrate is added from the outside, has a higher content of steviol glycosides than its wild type, so its extract can also contain steviol glycosides at a high concentration, especially steviol glycosides in which a hexose is added to the 2nd position of the monomeric glucose residue at the 13th or 19th position of the steviol glycoside.

[0074] The extract of the transformant of the present invention, even if the transformant is a cell derived from Stevia, has a higher ratio of steviol glycosides with hexose added to the 2-position of the monomeric glucose residue at the 13th or 19th position of steviol glycosides in the whole steviol glycosides than the extract of wild-type Stevia. In the transformant of the present invention, the introduced protein of the present invention is expressed, and a hexose is added to the 2-position of the monomeric glucose residue at the 13th or 19th position of steviol glycosides. Therefore, when the whole steviol glycosides are extracted, the ratio of steviol glycosides with hexose added to the 2-position of the monomeric glucose residue at the 13th or 19th position of steviol glycosides in the whole steviol glycosides is naturally increased compared with cells (such as wild-type Stevia cells) that do not express the protein. The change in the ratio affects the characteristics of the extract, such as sensory characteristics such as sweetness. The ratio of the steviol glycosides contained in the extract of the present invention is the same as that of the steviol glycoside composition of the present invention.

[0075] The extract of the transformant of the present invention can be obtained by crushing the transformant with glass beads, a homogenizer or an ultrasonic crusher, centrifuging the crushed product, and recovering the supernatant. In addition, when the transformant secretes steviol glycosides in vitro, the culture supernatant of the transformant is also included in the extract of the present invention. Furthermore, the above-mentioned method for extracting steviol glycosides can also be further subjected to an extraction step.

[0076] The extract of the transformant of the present invention can be used in conventional methods, for example, for the production of foods, pharmaceuticals, industrial raw materials, and the like.

[0077] In addition, the present invention provides a composition containing the steviol glycoside composition of the present invention, a transformant or an extract thereof (hereinafter, sometimes referred to as "the composition of the present invention"). The composition of the present invention may be in any shape, for example, it may be solid (for example, powder, granules), paste, gel or liquid. In addition, the composition of the present invention may be made into various forms. For example, the composition of the present invention may be made into the form of food, food additives, pharmaceuticals, quasi-drugs, cosmetics, etc., or may be used as a raw material for the manufacture of these products.

[0078] In the present invention, food is a general term for edible products, including foods specified in various countries and regions. The food in the present invention includes, for example, general foods in Japan (including functional foods, nutritional supplements, health supplements, natural foods and other health foods), health functional foods (specific health foods, functional labeled foods, nutritional functional foods, etc.), special purpose foods (specific health foods, patients' foods, pregnant women, lactation milk powder, infant formula milk, foods for people with dysphagia, etc.). The food in the present invention also includes foods for infants, formula milk for premature infants, foods for the elderly, foods for nursing, medical foods, etc.

[0079] The so-called nutritional supplement food refers to food that is fortified with specific nutrients. The so-called health food refers to food that is healthy or considered to be beneficial to health, including nutritional supplement food, natural food, diet food, etc. The so-called functional food refers to food used to supplement nutrients that realize the body's regulatory functions. The so-called food for infants refers to food provided to children up to about 6 years old. The so-called food for the elderly refers to food that has been processed in a way that is easier to digest and absorb than unprocessed food. The so-called infant formula refers to formula provided to children up to about 1 year old. The so-called formula for premature babies refers to formula provided to premature babies from birth to six months old.

[0080] The form of the food is not particularly limited and can be made into various forms. Examples of such forms include beverages, supplements, non-beverage foods, etc. The beverage can be any of an alcoholic beverage or a non-alcoholic beverage. Examples of non-alcoholic beverages include non-alcoholic beer, malt beverages, lactic acid bacteria beverages, fermented beverages, cocoa beverages, sports drinks, nutritional beverages, tea beverages (e.g., tea beverages such as green tea, oolong tea, brown rice tea, and black tea, barley tea, coix seed tea, sesame barley tea, buckwheat tea, cherry blossom tea, sweet tea, medicinal tea, and other tea beverages), refreshing drinks, coffee beverages (container coffee, liquid coffee, etc.), carbonated beverages (e.g., cola-flavored beverages, transparent carbonated beverages, ginger ale, fruit juice carbonated beverages, milk-containing carbonated beverages, or sugar-free carbonated beverages), functional beverages (e.g., sports drinks, energy drinks, health supplement beverages, and packaged gel drinks), fruit and vegetable beverages (e.g., 100% fruit juice beverages, fruit-containing beverages, low-juice refreshing beverages, fruit beverages containing fruit particles, or fruit pulp beverages), dairy beverages (e.g., milk, drinkable yogurt, lactic acid bacteria beverages, or milk-containing refreshing beverages), soy milk beverages (e.g., soy milk or soybean beverages), flavored water, and the like.

[0081] Alcoholic beverages refer to beverages containing alcohol raw materials, which may be carbonated shochu. Examples of alcohol raw materials include brewed liquor, distilled liquor, and mixed liquor. Examples of brewed liquor include wine and beer. Examples of distilled liquor include strong liquors (e.g., gin, vodka, rum, tequila, Newspirits (mixed liquors with less than 10% of whiskey-like original liquor) and raw material alcohol, etc.), liqueurs, whiskeys (e.g., whiskey, brandy, etc.), shochu, etc. Here, alcoholic beverages only need to contain detectable alcohol, for example, more than 1% by volume, more than 2% by volume, more than 3% by volume, more than 4% by volume, or more than 5% by volume of alcohol. Beverages include beverages of various calories. Preferably, the beverage is a low-calorie beverage (less than 20 kcal / 100 mL) or a calorie-free beverage (less than 5 kcal / 100 mL).

[0082] There is no limitation on non-beverage foods, and examples thereof include noodles (soba noodles, udon noodles, Chinese noodles, instant noodles, etc.), tofu, cakes and pastries (candy, chewing gum, chocolate, snacks, biscuits, cookies, gummy candies, cakes, wafers, pastry bread, chocolate, Japanese confectionery, etc.), bread, processed seafood or livestock foods (fish cakes, ham, sausages, etc.), dairy products (fermented milk, butter, cheese, yogurt, shortening, etc.), oils and fats and processed oil foods (salad oil, tempura oil, margarine, mayonnaise, shortening, fresh cream, dressings, low-fat margarine, etc.), seasonings (sauces, dressings, etc.), cooked or semi-cooked foods (stir-fried dishes, etc.), sterilized foods (curry, stews, rice bowls, porridge, mixed porridge, etc.), cold foods (ice cream, sorbet, shaved ice, etc.), powdered foods (powdered beverages, powdered soups, etc.), enteral liquid foods, etc. Preferred foods in the present invention include beverages, supplements, and the like.

[0083] The beverage can also be prepared as a containerized beverage in a state of being heat-sterilized and packed in a container. The container is not particularly limited, and examples thereof include PET bottles, aluminum cans, steel cans, paper boxes, cold cups, bottles, and the like. When heat sterilization is performed, the type thereof is not particularly limited, and for example, conventional methods such as UHT sterilization and autoclave sterilization can be used for implementation. The temperature of the heat sterilization process is not particularly limited, and for example, it is 65 to 130°C, preferably 85 to 120°C, for 10 to 40 minutes. Among them, as long as the same sterilization effect as the above conditions can be obtained, sterilization can also be performed at an appropriate temperature for a few seconds, for example, 5 to 30 seconds.

[0084] Food additives are products added to food ingredients during food manufacturing, and can be in the form of powder, granules, liquid, paste, etc. As pharmaceuticals, pharmaceuticals in various dosage forms can be cited. For example, as dosage forms suitable for oral administration, there are no limitations, and powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, chewables, syrups, etc. can be cited. In addition, as dosage forms suitable for parenteral administration, injections (such as solution injections, suspension injections, emulsion injections, injections prepared for use, etc.), ointments, pastes, aqueous solutions, wet compresses, patches, eye drops, nasal drops, inhalants, sprays, etc. can be cited. The quasi-drugs are not limited, and examples thereof include toothpaste, mouth fresheners, antiperspirants, cosmeceuticals, hair dyes, sanitary napkins, and the like. The cosmetics are not limited, and examples thereof include facial cleansers, makeup removers, lotions, beauty serums, facial masks, and sunscreens.

[0085] The composition of the present invention may also contain a combination of steviol glycosides in the same ratio as that of the steviol glycoside composition, transformant or extract thereof of the present invention. The composition of the present invention may also contain ingredients (additional ingredients) other than the steviol glycoside composition of the present invention, the transformant or its extract. The ingredients involved may vary according to the form, purpose, usage, etc. of the composition of the present invention, and non-limiting examples thereof include sweeteners, excipients, binders, disintegrants, coating agents, lubricants, colorants, flavoring agents, stabilizers, absorption promoters, pH modifiers, preservatives, antioxidants, flavors, vitamins, trace metal components, acid components, etc.

[0086] Examples of the sweetener include natural sweeteners, sugar alcohols, artificial sweeteners, high-intensity sweeteners, and low-calorie sweeteners. More specifically, examples include glucose, D-fructose, galactose, mannose, ribose, xylose, maltose, sucrose, lactose, arabinose, rare sugars (e.g., D-tagatose, D-sorbose, D-psicose (D-psicose), L-fructose, L-psicose (L-psicose), L-tagatose, L-sorbose, altrose, D-allose, talose, idose, gulose, etc.), peptide sweeteners (e.g., aspartame, neotame, alitame, etc.), sucrose derivatives (e.g., sucralose, etc.), synthetic sweeteners (e.g., acesulfame potassium, saccharin sodium, advantame, sweeteners, etc.), and tartrate. , sugar alcohols (e.g., erythritol, xylitol, sorbitol, maltitol, mannitol, etc.), monellin, curculin, brazzein sweet protein, thaumatin, mabinin, brazzein sweet protein, pentazine, hernandulcin, 4β-Hydroxyhernandulcin, miraculin, glycyrrhizin, praeruptoside A, steviol glycosides not contained in the steviol glycoside composition of the present invention, transformant or extract thereof, Siraitia grosvenorii (monk fruit) extract, mogrosides (e.g., MogV, MogIA1, MogIE1, MogIIA, MogIIA1, MogIIB, MogIIE, MogIII, MogIIIA1, MogIIIE, MobIV, MogIVA, simonoside I, etc.), Glycyrrhiza glabra (licorice) extract, Rubus suavissimus S.Lee (sweet tea) extract, Hydrangea macrophylla var.thunbergii (sweet tea) extract, Sclerochiton ilicifolius extract, Thaumataococcus daniellii Benth (West African arrowroot) extract, Dioscoreophyllum volkensii (Ceylon berry) extract, Curculigo latifolia (curculigo) extract, Richadella dulcifica (miracle fruit) extract, Pentadiplandra brazzeana (West African strawberry) extract, Capparis masaikai (caprine nut) extract, Lippia dulcis (sweet tongue grass) extract, neohesperidin dihydrochalcone, isomalt, high fructose syrup, fructose syrup, glucose-fructose syrup, oligosaccharides, honey, sugarcane juice (brown molasses), sugar (white sugar, sanwen sugar, brown sugar, wasanbon, etc.), maple syrup, molasses (treacle), maltose, etc.

[0087] In at least a specific exemplary embodiment of the composition of the present invention, the sweetener as an additional ingredient includes, for example, sugar alcohols such as erythritol, sorbitol, mannitol, xylitol, lactitol, isomalt, and maltitol. In other embodiments, the sweetener as an additional ingredient includes D-psicose, D-tagatose, a combination of erythritol and D-psicose, or a combination of erythritol, D-tagatose, and D-psicose.

[0088] When the composition of the present invention is made into food, it may also contain food additives as other ingredients. As food additives, for example, excipients (e.g., wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, alpha starch, casein, magnesium aluminate silicate, calcium silicate, etc.), binders (e.g., alpha starch, hydroxypropyl methylcellulose, polyvinyl polypyrrolidone, etc.), disintegrants (e.g., cellulose, hydroxypropyl cellulose, corn starch, etc.), fluidizers (e.g., light anhydrous silicic acid, etc.), oils and fats (e.g., vegetable oils such as soybean oil, sesame oil, olive oil, linseed oil, perilla oil, rapeseed oil, coconut oil, corn oil, or oils from animals and fish), nutrients (e.g., various minerals, various vitamins, amino acids), spices, sweeteners, flavoring agents, colorants, solvents (e.g., ethanol), salts, pH regulators, buffers, antioxidants, stabilizers, gelling agents, thickeners, lubricants, encapsulating agents, suspending agents, coating agents, preservatives, emulsifiers, etc.) may be cited. The food additives may be contained alone or in combination of two or more.

[0089] When the composition of the present invention is made into a pharmaceutical product or a quasi-pharmaceutical product, it may also contain pharmaceutically acceptable additives (e.g., surfactants, carriers, diluents, excipients, etc.) as other components. Pharmaceutically acceptable additives are well known in the pharmaceutical field, for example, as described in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), etc., which is incorporated herein by reference in its entirety.

[0090] The composition of the present invention can be prepared by mixing the steviol glycoside composition of the present invention, the transformant or its extract and the above-mentioned other components as needed, and processing them into a desired form. For example, when the composition of the present invention is in the form of a beverage, it can be prepared by adding the components contained in the beverage to a liquid such as water, and by a method including a mixing step. Thus, the present invention also relates to a method for preparing a composition (such as a food, a sweetener composition, a pharmaceutical product, etc.) containing steviol glycosides (hereinafter, sometimes referred to as "the method for preparing the composition of the present invention"), characterized in that it includes a step of combining the steviol glycoside composition of the present invention, the transformant or its extract and other components. The method for preparing the composition of the present invention may further include a step of preparing the steviol glycoside composition of the present invention, the transformant or its extract. The present invention further provides a composition (such as a food, a sweetener composition, a pharmaceutical product, etc.) containing steviol glycosides, characterized in that it is obtained by a method including a step of preparing the steviol glycoside composition of the present invention, the transformant or its extract, and a step of combining the steviol glycoside composition, the transformant or its extract obtained by the step with other components.

[0091] Composition of the present invention can be used in a manner suitable for various forms. For example, food can be taken in by non-oral nutritional routes such as nasal and enteral except oral intake. Food additives can be contained in the state in food and used by the same route as food. The route of administration of the adapted dosage form of pharmaceuticals is not limited, for example, can be administered orally, buccal, oral, intravenous, intramuscular, subcutaneous, intradermal, local, rectal, intra-articular, intra-articular package, intra-arterial, intra-portal vein, intraventricular, through mucosa, through skin, intranasal cavity, intraperitoneal, respiratory tract, tumor, brain, subarachnoid space, intraventricular, lung and intrauterine routes. The method of use of the adaptable form of quasi-pharmaceuticals and cosmetics can be used, for example, by applying to the skin, gargling, etc.

[0092] 5. Dead tissue or dead cells of transformants In addition, the present invention provides, from another aspect, a dead substance of tissue or cell of the above-mentioned transformant (hereinafter, sometimes referred to as "dead tissue or dead cell of the present invention"). The so-called "death" refers to a state in which the ability to reproduce, proliferate, regenerate and grow is lost, including the case of natural death not caused by human operation, and the case of death caused by human operation such as cutting, crushing, heating (including heating by gas such as air, heating by liquid such as water, heating by steam such as water vapor), freezing, drying, freeze drying, etc. The so-called dead tissue refers to a tissue in which all the cells contained are dead, and may also include dead transformants (transformants in which all the cells contained are dead). As specific examples of the dead tissue or dead cell of the present invention, for example, when the transformant is a plant transformant, dead substances of tissues or cells other than seeds can be listed. In a specific embodiment, the dead tissue or dead cell of the present invention also includes embryos, meristem cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices and callus dead substances. In a specific preferred embodiment, the dead tissue of the present invention is a dried leaf. When the transformant derived from the dead tissue or dead cell of the present invention contains steviol glycosides, the protein of the present invention is expressed and a hexose is added to the 2-position of the monomeric glucose residue at the 13th or 19th position of the steviol glycoside. Therefore, when the whole steviol glycosides contained in the dead tissue or dead cell are concentrated and extracted, the proportion of steviol glycosides with a hexose added to the 2-position of the monomeric glucose residue at the 13th or 19th position of the steviol glycoside in the whole steviol glycosides is naturally increased compared with an organism (e.g., a wild-type stevia plant) that does not express the protein of the present invention. The change in the ratio affects the properties of the dead tissue or dead cell, such as the sensory properties such as sweetness. Regarding the ratio of steviol glycosides contained in the dead tissue or dead cell of the transformant of the present invention containing steviol glycosides, the matters described in the above "3. Non-human transformants expressing the protein of the present invention" apply. The dead tissue or dead cell of the present invention itself can be used as a raw material for food, medicine, industrial raw material, etc. The present invention further provides an extract of dead tissue or dead cells of the present invention. The present invention further provides a composition containing the dead tissue or dead cells of the present invention or an extract thereof. Regarding the properties, morphology, etc. of these extracts and compositions, the matters described in the above "4. Extracts of transformants" apply. Example

[0093] Hereinafter, the present invention will be described in more detail using examples, but the scope of the present invention is not limited to these examples.

[0094] [Example 1] Construction of yeast expression system (1) Construction of yeast expression vector In order to evaluate the enzymatic activity of steviol glycosidase variants in a yeast expression system, the desired steviol glycosidase gene was inserted into the pESC vector (Agilent Technologies, Catalog #217455) to construct the following expression vector. In addition, UGT91D2 (V155T) represents a variant of UGT91D2 in which the valine residue at position 155 of UGT91D-like3 is substituted with a threonine residue, and UGT91D2 (Y42F / V155T) represents a variant of UGT91D2 in which the tyrosine residue at position 42 of UGT91D-like3 is substituted with a phenylalanine residue, and the valine residue at position 155 is substituted with a threonine residue. [Table 1] Table 1 Construction of expression vector name mark Inserting genes Insert sequence V1 pESc-Trp-91D2 TRP UGT91D-like3 Sequence Number 13 V2 pESc-Trp-91D2(V155T) TRP UGT91D2(V155T) Sequence Number 14 V3 pESc-Trp-91D2(V155T / Y42F) TRP UGT9ID2(Y42F / V155T) Sequence Number 15 V4 pESc-URA-85C2 URA UGT85C2 Sequence Number 16 V5 pESc-URA-74G1-85C2 URA UGT74G1+UGT85C2 Serial No. 16, 17 V6 pESc-His-74G1-76G1 HIS UGT74G1+UGT76G1 Serial No. 17, 18

[0095] (2) Yeast transformation The vector combination listed in Table 2 was introduced by the lithium acetate method using Saccharomyces cerevisiae YPH499 strain as a host, and the following transformants were obtained using a synthetic glucose agar medium lacking the amino acid corresponding to the auxotrophic marker gene. [Table 2] Table 2 Composition of transformants

[0096] [Example 2] Synthesis of steviol glycosides Using the transformants prepared in Example 1, steviol glycosides were synthesized from steviol. Specifically, each transformant (2 to 4 asexual propagation lines each) was cultured in 2 mL of glucose medium (synthetic glucose medium lacking the amino acid corresponding to the auxotrophic marker gene) at 30°C overnight, and the collected bacterial colony was cultured in 10 to 15 mL of galactose medium (synthetic galactose medium lacking the amino acid corresponding to the auxotrophic marker gene) supplemented with 10 μg / mL of steviol at 30°C for 2 nights. After the culture was completed, the culture solution was centrifuged to separate the supernatant and the bacterial cells. The culture supernatant was washed with acetonitrile, adsorbed to a Sep-Pak C186cc Vac Cartridge (Waters Corporation) equilibrated with water, washed with 20% acetonitrile, and then dissolved with 80% acetonitrile. After drying, the mixture was dissolved in 200 μL of 50% acetonitrile and the amount of steviol glycoside produced was analyzed by HPLC or LC-MS / MS. The HPLC conditions are shown below. Column: Shim-pack FC-ODS (4.6mm×150mm, 3μm, Shimadzu Corporation) Flow rate: 0.3mL / min Temperature: 40℃ Mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile Analysis by LC-MS / MS was performed using 1 mL of the sample solution in the MRM mode of LCMS8050 (Shimadzu Corporation).

[0097] The results based on transformants T1 (91D2 / 85C2) and T2 (91D2 (V155T) / 85C2) are shown in Table 3. The production amount of steviol glycosides was analyzed by HPLC. In addition, the values ​​in the table are relative values ​​(excluding the average value of asexual reproduction lines with extremely low production amounts) when the production amount of steviol bioglycosides based on transformant 1 is taken as 100%. [Table 3] Table 3 Steviol disodium glycoside production ability of transformants Steviol disodium glycoside production capacity (%) T1 100.0 T2 98.9

[0098] The results based on transformants T3 (91D2 / 85C2 / 74G1) and T4 (91D2 (V155T) / 85C2 / 74G1) are shown in Table 4. The amount of steviol glycoside produced was analyzed by LC-MS / MS, and the concentrations of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN and stevioside were quantified. The values ​​in the table are average values ​​excluding asexual propagation lines with extremely low production amounts, and ppm is based on mass. In addition, STV represents stevioside, and the values ​​in parentheses represent the ratio of T4 to T3. [Table 4] Table 4 RebE production ability of transformants

[0099] The results based on transformants T5 (91D2 / 85C2 / 74G1 / 76G1) and T6 (91D2 (V155T) / 85C2 / 74G1 / 76G1) are shown in Table 5. The production of steviol glycosides was analyzed by LC-MS / MS, and the concentrations of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebM, RebN and stevioside were quantified. The values ​​in the table are average values ​​excluding asexual propagation lines with extremely low production, and ppm is based on mass. In addition, STV represents the production of stevioside, and TSG (total steviol glycosides) represents the total production of the above-quantified steviol glycosides.

[0100] [Table 5] Table 5 Steviol glycoside production ability of transformants T5 T6 T6 / T5(%) RebA(ppm) 45.2 25.2 55.8 RebB(ppm) 8.85 6.48 73.2 RebD(ppm) 2.46 6.19 251.6 RebE(ppm) 0.63 1.35 214.3 RebG(ppm) 7.91 8.75 110.6 RebM(ppm) 6.64 12.8 192.8 STV(ppm) 6.13 2.53 41.2 RebD+RebM(ppm) 9.1 19.0 208.8 TSG(ppm) 77.8 63.2 81.3 RebD / RebA 0.05 0.25 451.3 RebD / STV 0.40 2.45 609.7 RebM / RebA 0.15 0.51 345.8 RebM / STV 1.08 5.06 467.1 RebE / RebA 0.01 0.05 374.5 RebE / STV 0.10 0.53 505.9 (RebD+RebM) / RebA 0.20 0.75 384.4 (RebD+RebM) / STV 1.48 7.51 519.2

[0101] The results based on transformants T5 (91D2 / 85C2 / 74G1 / 76G1), T6 (91D2 (V155T) / 85C2 / 74G1 / 76G1) and T7 (91D2 (Y43F / V155T) / 85C2 / 74G1 / 76G1) are shown in Table 6. The production amount of steviol glycosides was analyzed by LC-MS / MS, and the concentrations of RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN and stevioside were quantified. The values ​​in the table are the average values ​​of 4 asexual reproduction lines (the range of values ​​in parentheses is the minimum to maximum value), and ppm is based on mass. In addition, STV represents the production amount of stevioside, and TSG (total steviol glycosides) represents the total production amount of the above-mentioned steviol glycosides that were quantified. In addition, regarding Reb C, Reb F, Reb J, and Reb N, since the average value was less than 0.001 ppm, description thereof was omitted.

[0102] [Table 6] Table 6 Steviol glycoside production ability of transformants

[0103] The sequences mentioned in this specification are shown below. [Table 7]

[0104] [Table 8]

[0105] [Table 9]

[0106] [Table 10]

[0107] [Table 11]

[0108] [Table 12]

[0109] [Table 13]

[0110] [Table 14]

[0111] [Table 15]

Claims

1. A protein, characterized in that Select from the following: (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, (b) a protein comprising an amino acid sequence in which 1 to 48 amino acid residues other than the amino acid residue at position 155 in the amino acid sequence of SEQ ID NO: 2 are deleted, substituted, inserted and / or added, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I), and, (c) a protein comprising an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2 and the same amino acid residue as the amino acid residue corresponding to the amino acid residue at position 155, and having the activity of adding a hexose to the 2-position of the monomeric glucose residue at position 13 or 19 of the compound represented by formula (I); [Chemical 1] In the formula, R1 and R2 are each independently H, C1~C 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkynyl, C4~C 20 Alkyldiene, C6~C 18 Aryl, C6~C 20 Alkyl aryl, C6~C 20 Arylalkyl, C4~C 20 Cycloalkyl, C4~C 20 Cycloalkenyl, (C3~C 10 Cycloalkyl)C1~C 10 An alkyl group or a sugar residue, wherein at least one of R1 and R2 is a monomeric glucose residue.

2. The protein according to claim 1, characterized in that The amino acid residue at position 42 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding thereto is substituted with a phenylalanine residue.

3. The protein according to claim 1 or 2, characterized in that The hexose is glucose or rhamnose.

4. The protein according to any one of claims 1 to 3, characterized in that The compound represented by formula (I) is steviol monosaccharide, rubusoside or stevioside.

5. A polynucleotide, characterized in that Encodes the protein according to any one of claims 1 to 4.

6. A carrier, characterized in that Contains the polynucleotide according to claim 5.

7. A non-human transformant, characterized in that Contains the polynucleotide according to claim 5.

8. The transformant according to claim 7, characterized in that Contains at least one of UGT85C2, UGT76G1 and UGT74G1.

9. An extract, characterized in that The extract of the transformant according to claim 7 or 8.

10. A food, medicine or industrial raw material, characterized in that: Containing the extract according to claim 9.

11. A method for producing the protein according to any one of claims 1 to 4, characterized in that: The method comprises culturing the non-human transformant according to claim 7 or 8.

12. A method for producing steviol glycoside or a steviol glycoside composition, characterized in that: The method comprises culturing the non-human transformant according to claim 7 or 8.

13. A method for producing steviol glycoside or a steviol glycoside composition, characterized in that: The method comprises the step of reacting the protein according to any one of claims 1 to 4 with UDP-sugar and the compound represented by the formula (I).

14. The method according to claim 13, characterized in that The UDP-sugar is UDP-glucose or UDP-rhamnose.

15. The method according to any one of claims 12 to 14, characterized in that: The steviol glycoside composition comprises at least one of RebE, steviol bioside, stevioside, and dulcoside A.

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