Mutant glycosyltransferase and method for producing same, nucleic acid, vector, host cell, composition, and method for producing anthraquinone C glycoside

Abstract

Provided are: a mutant glycosyltransferase which has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and is capable of forming an anthraquinone C glycoside from an anthraquinone compound and a monosaccharide donor; a method for producing the mutant glycosyltransferase; a nucleic acid; a vector;

Description

TECHNICAL FIELD

[0001] The present application relates to a mutant glycosyltransferase, a method for producing the same, a nucleic acid, a vector, a host cell, a composition, and a method for producing an anthraquinone C glycoside. BACKGROUND

[0002] Glycosyltransferases are sugar transferases that catalyze a reaction in which a sugar residue is transferred from a sugar donor such as a sugar nucleotide to a sugar acceptor to form a glycosidic bond, and are applied to industrial processes for producing sugar glycosides using enzyme reactions.

[0003] Glycosides are classified into C glycosides, O glycosides, N glycosides, and the like, depending on the types of atoms to which the sugar portion and the non-sugar portion are bonded. Among them, C glycosides (for example, anthraquinone C glycosides) in which the sugar portion and the non-sugar portion are bonded by a carbon-carbon bond are more stable in an acidic environment than O glycosides in which the sugar portion and the non-sugar portion are bonded by an oxygen-carbon bond. In particular, anthraquinone C glycosides (for example, carmine pigments and the like), which are one of C glycosides, are useful compounds that are widely used in dyes, foods, cosmetics, and the like, and an effective production method is desired.

[0004] In Patent Literature 1, a method is described in which a mutant glycosyltransferase derived from Coccoidea is used to produce carmine pigments from kermesic acid or flavokermesic acid and glucose.

[0005] In Patent Literature 2, a host cell expressing a mutant glycosyltransferase for biosynthesizing carminic acid from polyketide building blocks is described.

[0006] In Non-Patent Literature 1, a mutant glycosyltransferase derived from Gentiana triflora having C glycosylation activity and a method for producing carminic acid from glucose using the same are described.

[0007] PRIOR ART

[0008] PATENT LITERATURE

[0009] Patent Literature 1: U.S. Patent No. 10100290

[0010] Patent Literature 2: U.S. Patent Application Publication No. 20210261992 Specification

[0011] NON-PATENT LITERATURE

[0012] Non-Patent Literature 1: Dongsoo Yang, Woo Dae Jang, Sang Yup Lee, Journal of American Chemical Society, 2021, 143, 14, 5364-5377. SUMMARY

[0013] PROBLEMS TO BE SOLVED BY THE INVENTION

[0014] However, in the conventional mutant glycosyltransferases, there is a tendency that the amount of the mutant glycosyltransferase produced from the host cell per unit volume (hereinafter, also referred to as "titer") is small due to the reasons that the protein expressed in the host cell is insolubilized and cannot be extracted, and the like. For example, it is described in Non-Patent Literature 1 that the expression of the mutant glycosyltransferase (DcUGT2) in Patent Literature 1 and Patent Literature 2 is difficult in a transformant in which Escherichia coli is used as a host cell. In Non-Patent Literature 1, it is described that, although the mutant glycosyltransferase (GtCGT) in which Escherichia coli is used as a host cell can be expressed, it is insolubilized from the extraction of the transformant, and thus the titer is small.

[0015] Therefore, in view of the above circumstances, the object of the present application is to provide a mutant glycosyltransferase having an excellent titer, a method for producing the same, a nucleic acid, a vector, a host cell, and a method for producing an anthraquinone C-glycoside.

[0016] MEANS FOR SOLVING THE PROBLEMS

[0017] The present application includes the following modes.

[0018] <1> A mutant glycosyltransferase having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NO: 1, and capable of forming an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor.

[0019] <2> The mutant glycosyltransferase according to <1>, which satisfies at least one condition selected from the group consisting of (1-A) to (6-A) below:

[0020] (1-A) the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, or an aliphatic amino acid residue,

[0021] (2-A) the amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue or an aliphatic amino acid residue,

[0022] (3-A) The amino acid residue corresponding to position 318 from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue,

[0023] (4-A) The amino acid residue corresponding to position 320 from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, an acidic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue,

[0024] (5-A) The amino acid residue corresponding to position 382 from the N terminus of SEQ ID NO: 1 is an aromatic amino acid residue,

[0025] (6-A) The amino acid residue corresponding to position 46 from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue.

[0026] (3-B) The amino acid residue corresponding to position 318 from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), a cysteine residue (C), an asparagine residue (N), a serine residue (S), an isoleucine residue (I), a leucine residue (L), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y),

[0027] (1-B) The amino acid residue corresponding to position 17 from the N terminus of SEQ ID NO: 1 is a histidine residue (H), a serine residue (S), or a glycine residue (G),

[0028] (2-B) The amino acid residue corresponding to position 20 from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), an asparagine residue (N), an isoleucine residue (I), or a valine residue (V),

[0029] (3-B) The amino acid residue corresponding to position 318 from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), a cysteine residue (C), an asparagine residue (N), a serine residue (S), an isoleucine residue (I), a leucine residue (L), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y),

[0030] (4-B) The amino acid residue corresponding to position 320 from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), an aspartic acid residue (D), a cysteine residue (C), a serine residue (S), a glycine residue (G), an isoleucine residue (I), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y),

[0031] (5-B) The amino acid residue corresponding to position 382 from the N terminus of SEQ ID NO: 1 is a tyrosine residue (Y),

[0032] (6-B) The amino acid residue corresponding to the 46th amino acid residue from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), a serine residue (S), a glycine residue (G), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y).

[0033] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0034] only the combination of (1-B) and (2-B);

[0035] only the combination of (3-B) and (4-B);

[0036] only the combination of (1-B), (2-B), and (5-B);

[0037] only the combination of (1-B), (2-B), (5-B), and (6-B);

[0038] only the combination of (1-B), (2-B), (3-B), (5-B), and (6-B);

[0039] only the combination of (1-B), (2-B), (4-B), (5-B), and (6-B); or

[0040] the combination of (1-B), (2-B), (3-B), (4-B), (5-B), and (6-B).

[0041] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0042] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0043] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0044] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0045] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0046] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0047] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0048] (3) The mutant glycosyltransferase according to <2>, which satisfies the following combination among the conditions of (1-B) to (6-B):

[0049] recovering the mutant glycosyltransferase from at least one of the host cell cultured and the culture medium.

[0050] <12> A composition comprising the mutant glycosyltransferase according to any one of <1> to <5>.

[0051] <13> A method for producing an anthraquinone C-glycoside, comprising the steps of:

[0052] contacting an anthraquinone compound and a monosaccharide donor with the mutant glycosyltransferase according to any one of <1> to <5> to form an anthraquinone C-glycoside.

[0053] Effects of the Invention

[0054] According to the present application, it is possible to provide a mutant glycosyltransferase having excellent potency, a method for producing the same, a nucleic acid, a vector, a host cell, and a method for producing an anthraquinone C-glycoside. DETAILED DESCRIPTION

[0055] Hereinafter, embodiments of the present application will be described. These descriptions and examples are intended to illustrate the embodiments, and not to limit the scope of the embodiments.

[0056] In the numerical ranges described in the present application, the upper limit value or the lower limit value described in one numerical range can be replaced with the upper limit value or the lower limit value of another numerical range described in stages. Also, in the numerical ranges described in the present application, the upper limit value or the lower limit value of the numerical range can be replaced with the value shown in the examples.

[0057] In the present application, each component can include a plurality of corresponding substances.

[0058] In the present application, the term "step" includes not only a single step, but also a case where the step cannot be clearly distinguished from other steps, as long as the intended purpose of the step can be achieved.

[0059] In the present application, the numerical range represented by "~" indicates a range including the values described before and after "~" as the minimum value and the maximum value, respectively.

[0060] Hereinafter, the glycosyltransferase activity for obtaining an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor will be simply referred to as "C-glycosylation activity".

[0061] In the present application, the description of the nucleotide sequence can be made focusing on the sequence on one of the strands even in the case where the nucleic acid strand of the nucleotide sequence forms a double strand, and in the other strand of the double strand, the description of the sequence should be applied by replacing the sequence with its complementary sequence.

[0062] In the present application, an amino acid residue is sometimes indicated by a single English letter. For example, a tryptophan residue is sometimes indicated by "W".

[0063] In the present application, even if a condition is indicated by a singular form, as long as no technical contradiction is caused, a plurality can not be excluded, unless specifically indicated.

[0064] Mutant glycosyltransferase

[0065] The mutant glycosyltransferase of the present application has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and is capable of forming an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor.

[0066] The mutant glycosyltransferase of the present application has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and is capable of forming an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor.

[0067] The mutant glycosyltransferase of the present application has 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and is capable of forming an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor.

[0068] The mutant glycosyltransferase of the present application can have 99.9% or less sequence identity with the amino acid sequence represented by SEQ ID NO: 75 (i.e., the amino acid sequence of the wild-type glycosyltransferase derived from Punica granatum).

[0069] In the present application, the mutant glycosyltransferase refers to a glycosyltransferase having one or more mutations with respect to the wild-type glycosyltransferase derived from Punica granatum.

[0070] The amino acid sequence represented by SEQ ID NO: 1 refers to a sequence in which the histidine residue (H) of the amino acid residue corresponding to the 20th position from the N terminus is changed to a valine residue (V) with respect to the amino acid sequence of the wild-type glycosyltransferase derived from Punica granatum represented by SEQ ID NO: 75.

[0071] In the amino acid sequence represented by SEQ ID NO: 1, it is considered that the valine residue (V) is superior in potency with respect to the wild-type glycosyltransferase derived from Punica granatum represented by SEQ ID NO: 75 by performing the mutation of replacing the histidine residue (H) of the above-described amino acid residue corresponding to the 20th position from the N terminus with the valine residue (V).

[0072] (Mutation position)

[0073] From the viewpoint of more excellent titer and C-glycosylation activity, the mutant glycosyltransferase of the present application preferably satisfies at least one condition selected from the group consisting of (1-A) to (6-A) below, more preferably satisfies 2 or more and 6 or less of the conditions, and further preferably satisfies 3 or more and 6 or less of the conditions.

[0074] (1-A) The amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, or an aliphatic amino acid residue,

[0075] (2-A) The amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue or an aliphatic amino acid residue,

[0076] (3-A) The amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue,

[0077] (4-A) The amino acid residue corresponding to the 320th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, an acidic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue,

[0078] (5-A) The amino acid residue corresponding to the 382nd position from the N terminus of SEQ ID NO: 1 is an aromatic amino acid residue,

[0079] (6-A) The amino acid residue corresponding to the 46th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue.

[0080] From the viewpoint of more excellent titer and C-glycosylation activity, the mutant glycosyltransferase of the present application preferably satisfies at least one condition selected from the group consisting of (1-A2) to (6-A2) below, more preferably satisfies 2 or more and 6 or less of the conditions, and further preferably satisfies 3 or more and 6 or less of the conditions.

[0081] (1-A2) The amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue,

[0082] (2-A2) The amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue,

[0083] (3-A2) The amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue or an aromatic amino acid residue,

[0084] (4-A2) The amino acid residue corresponding to the 320th amino acid residue from the N terminus of SEQ ID NO: 1 is a basic amino acid residue or an aliphatic amino acid residue,

[0085] (5-A2) The amino acid residue corresponding to the 382nd amino acid residue from the N terminus of SEQ ID NO: 1 is an aromatic amino acid residue,

[0086] (6-A2) The amino acid residue corresponding to the 46th amino acid residue from the N terminus of SEQ ID NO: 1 is an aromatic amino acid residue.

[0087] The basic amino acid residue refers to an amino acid residue composed of a basic amino acid.

[0088] The acidic amino acid residue refers to an amino acid residue composed of an acidic amino acid.

[0089] The aromatic amino acid residue refers to an amino acid residue composed of an aromatic amino acid. The aromatic amino acid refers to an amino acid having an aromatic group (e.g., phenyl group, etc.), and is not any of a basic amino acid and an acidic amino acid.

[0090] The polar uncharged amino acid residue refers to an amino acid residue composed of a polar uncharged amino acid. The polar uncharged amino acid refers to an amino acid having a polar group (e.g., thiol group, etc.) and not charged as a whole molecule in water (25°C), and is not any of an acidic amino acid, a basic amino acid, an aromatic amino acid.

[0091] The aliphatic amino acid residue refers to an amino acid residue composed of an aliphatic amino acid. The aliphatic amino acid refers to an aliphatic amino acid that is not any of a basic amino acid, an acidic amino acid, a polar uncharged amino acid, and an aromatic amino acid.

[0092] The 17th amino acid residue from the N terminus of SEQ ID NO: 1 refers to a glycine residue (G).

[0093] The 20th amino acid residue from the N terminus of SEQ ID NO: 1 refers to a valine residue (V).

[0094] The 318th amino acid residue from the N terminus of SEQ ID NO: 1 refers to a serine residue (S).

[0095] The 320th amino acid residue from the N terminus of SEQ ID NO: 1 refers to a tyrosine residue (Y).

[0096] The 382nd amino acid residue from the N terminus of SEQ ID NO: 1 refers to a tryptophan residue (W).

[0097] The 46th amino acid residue from the N terminus of SEQ ID NO: 1 refers to an isoleucine residue (I).

[0098] Examples of the basic amino acid residue (labeled as "basic" in the table), the acidic amino acid residue (labeled as "acidic" in the table), the polar uncharged amino acid residue (labeled as "polar uncharged" in the table), the aliphatic amino acid residue (labeled as "aliphatic" in the table), and the aromatic amino acid residue (labeled as "aromatic" in the table) are shown in the following table, but the respective amino acids are not limited thereto, and modified amino acid residues, abnormal amino acid residues, and the like are also included.

[0099] [Table 1]

[0100]

[0101] From the viewpoint of more excellent specific activity and C-glycosylation activity, the mutant glycosyltransferase of the present application preferably satisfies at least one condition selected from the group consisting of (1-B) to (6-B) below, more preferably satisfies two or more and six or fewer conditions, and further preferably satisfies three or more and six or fewer conditions:

[0102] (1-B) the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), a serine residue (S), or a glycine residue (G),

[0103] (2-B) the amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), an asparagine residue (N), an isoleucine residue (I), or a valine residue (V),

[0104] (3-B) the amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), a cysteine residue (C), an asparagine residue (N), a serine residue (S), an isoleucine residue (I), a leucine residue (L), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y),

[0105] (4-B) the amino acid residue corresponding to the 320th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), an aspartic acid residue (D), a cysteine residue (C), a serine residue (S), a glycine residue (G), an isoleucine residue (I), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y),

[0106] (5-B) the amino acid residue corresponding to the 382nd position from the N terminus of SEQ ID NO: 1 is a tyrosine residue (Y),

[0107] (6-B) the amino acid residue corresponding to the 46th position from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), a serine residue (S), a glycine residue (G), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y).

[0108] From the viewpoint of more excellent titer and C-glycosylation activity, the mutant glycosyltransferase of the present application preferably satisfies at least one condition selected from the group consisting of (1-B2) to (6-B2) below, more preferably 2 or more and 6 or fewer conditions, further preferably 3 or more and 6 or fewer conditions:

[0109] (1-B2) the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a serine residue (S),

[0110] (2-B2) the amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is an asparagine residue (N),

[0111] (3-B2) the amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is an arginine residue (R), or a phenylalanine residue (F),

[0112] (4-B2) the amino acid residue corresponding to the 320th position from the N terminus of SEQ ID NO: 1 is an arginine residue (R), or a valine residue (V),

[0113] (5-B2) the amino acid residue corresponding to the 382nd position from the N terminus of SEQ ID NO: 1 is a tyrosine residue (Y).

[0114] (6-B2) the amino acid residue corresponding to the 46th position from the N terminus of SEQ ID NO: 1 is a phenylalanine residue (F).

[0115] From the viewpoint of more excellent titer and C-glycosylation activity, the mutant glycosyltransferase of the present application preferably satisfies the following combinations among the conditions of (1-B) to (5-B) above:

[0116] only the combination of (1-B) and (2-B);

[0117] only the combination of (3-B) and (4-B);

[0118] only the combination of (1-B), (2-B), and (5-B);

[0119] only the combination of (1-B), (2-B), (5-B), and (6-B);

[0120] only the combination of (1-B), (2-B), (3-B), (5-B), and (6-B);

[0121] only the combination of (1-B), (2-B), (4-B), (5-B), and (6-B); or

[0122] the combination of (1-B), (2-B), (3-B), (4-B), (5-B), and (6-B).

[0123] More preferably, the following combinations are satisfied:

[0124] only the combination of (1-B) and (2-B);

[0125] only the combination of (1-B), (2-B) and (5-B);

[0126] only the combination of (1-B), (2-B), (5-B) and (6-B); or

[0127] the combination of (1-B), (2-B), (3-B), (4-B), (5-B) and (6-B),

[0128] Further preferably, the following combinations are satisfied:

[0129] only the combination of (1-B), (2-B) and (5-B);

[0130] only the combination of (1-B), (2-B), (5-B) and (6-B); or

[0131] the combination of (1-B), (2-B), (3-B), (4-B), (5-B) and (6-B).

[0132] In addition, in the above, for example, "only the combination of (1-B) and (2-B) is satisfied" means that, among (1-B) to (6-B), only (1-B) and (2-B) are satisfied, and (3-B), (4-B), (5-B) and (6-B) are not satisfied. In this case, there is no particular limitation on conditions other than (1-B) to (6-B). For example, there is no particular limitation on the amino acid residues other than the 17th, 20th, 46th, 318th, 320th and 382nd amino acid residues from the N terminus of SEQ ID NO: 1.

[0133] The mutant glycosyltransferase of the present application can also not satisfy the conditions represented by (1-A) to (6-A) and (1-B) to (6-B) above, as long as the sequence identity with the amino acid sequence represented by SEQ ID NO: 1 is within 80%.

[0134] The mutant glycosyltransferase of the present application can also include amino acid residue substitutions other than the conditions represented by (1-A) to (6-A) and (1-B) to (6-B) above, within the range in which the titer is obtained.

[0135] The total number of conditions selected from the group consisting of (1-A) to (6-A) and (1-B) to (6-B) above in the mutant glycosyltransferase is not particularly limited as long as it is 1 or more, and can be, for example, any one of 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1.

[0136] From the viewpoint of more excellent titer and C-glycosylation activity, the mutant glycosyltransferase preferably has one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 71, and more preferably has one amino acid sequence of SEQ ID NO: 59, SEQ ID NO: 70, and SEQ ID NO: 71.

[0137] In the mutant glycosyltransferase of the present application, the titer is preferably 1 (mg / L) or more, more preferably 3 (mg / L) or more, and further preferably 10 (mg / L) or more.

[0138] In the mutant glycosyltransferase of the present application, the C-glycosylation activity is preferably 1 (pkat / mg) or more, more preferably 10 (pkat / mg) or more, and further preferably 100 (pkat / mg) or more.

[0139] In the mutant glycosyltransferase of the present application, the C-glycosylation activity extracted from the culture solution is preferably 10 (pkat / L) or more, more preferably 100 (pkat / L) or more, and further preferably 1000 (pkat / L) or more.

[0140] The values of the titer, the C-glycosylation activity, and the C-glycosylation activity extracted from the culture solution are respectively as follows. Specifically, they are obtained by the methods described in the Examples described later.

[0141] (Titer)

[0142] Regarding the enzyme solution of the test subject, the enzyme amount (i.e., the amount of the mutant glycosyltransferase) is quantified from the concentration of the enzyme solution based on the absorbance at 280 nm determined by a spectrophotometer. Next, the enzyme diluent is electrophoretically developed with SDS-PAGE, the gel is dyed with CBB (Coomassie Brilliant Blue) staining solution, and the purity of the band containing the target protein (i.e., the band corresponding to the mutant glycosyltransferase) is quantified by image analysis. From the obtained enzyme amount and the culture solution amount, the titer is calculated according to the following formula.

[0143] Enzyme amount (mg) = enzyme solution concentration (mg / L) x enzyme solution amount (L) x (purity (%) / 100) Titer (mg / L) = enzyme amount (mg) / culture solution amount (L)

[0144] (C-glycosylation activity)

[0145] In 100 μL of a buffer (a mixed solution of 50 mM HEPES (pH 7.5), 1 mg / mL lysozyme, 0.5 mg / mL polymyxin B, 13 mM MgCl2, and 50 mM KCl), a 0.5 mM DMSO solution of an anthraquinone compound, a 5 mM aqueous monosaccharide donor solution, and 20 μg of the test enzyme were stirred thoroughly and reacted at 25°C. Subsequently, 100 μL of methanol was added to the reaction system to terminate the reaction, and the supernatant was obtained by centrifugation (4°C, 1800 g, 30 minutes).

[0146] The supernatant was measured using a high performance liquid chromatograph (HPLC) to quantitatively analyze the amount of anthraquinone C glycoside, the target product. The HPLC analysis conditions were as follows. The apparatus and column used were equivalent to those described below.

[0147] -HPLC analysis conditions-

[0148] Device: Nexera X2 (manufactured by Shimadzu Corporation)

[0149] Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.0×50mm

[0150] Mobile phase: A: water (0.1% formic acid), B: methanol (0.1% formic acid)

[0151] Time (min) / %B: 0 / 10, 0.4 / 10, 3.0 / 80, 4.0 / 100, 5.0 / 100, 5.1 / 10

[0152] Injection volume: 2 μL

[0153] Column temperature: 40°C

[0154] Flow rate: 0.4 mL / min

[0155] The C-glycosylation activity was calculated from the amount of anthraquinone C-glycoside produced, the reaction time, and the amount of enzyme used by HPLC according to the following formula.

[0156] C glycosylation activity (pkat / mg) = amount of C glycoside produced (pmol) / reaction time (s) / amount of enzyme used (mg)

[0157] (C glycosylation activity extracted from culture medium)

[0158] The enzyme activity extracted from the culture medium was calculated according to the following formula using the C-glycosylation activity and titer determined from the above evaluation.

[0159] Enzyme activity extracted from culture solution (pkat / L) = Anthraquinone C glycosylation activity (pkat / mg) x expression efficiency (mg / L)

[0160] The following illustrates the amino acid residue substitution of the mutant glycosyltransferase of the present application with respect to the amino acid sequence represented by SEQ ID NO: 1, but the present application is not limited thereto. In addition, in the table, for example, "G17H" described in the item of [effective mutation point] means that the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is substituted from a glycine residue (G) to a histidine residue (H).

[0161] [Table 2]

[0162] Effective mutation points 2 H19V 3 G17H 4 G17S 5 V20C 6 G17H, V20C 7 G17H, V20N 8 G17H, V20I 9 G17S, V20C 10 G17S, V20N 11 G17S, V20I 12 S318R 13 Y320H 14 Y320R 15 Y320V 16 S318H, Y320R 17 S318H, Y320V 18 S318R, Y320H 19 S318R, Y320R 20 S318R, Y320C

[0163] [Table 3]

[0164] Effective mutation points 21 S318R, Y320S 22 S318R, Y320I 23 S318R, Y320V 24 S318N, Y320V 25 S318G, Y320R 26 S318I, Y320R 27 S318I, Y320I 28 S318I, Y320V 29 S318L, Y320R 30 S318V, Y320R 31 S318V, Y320I 32 S318V, Y320L 33 S318V, Y320V 34 S318F, Y320R 35 S318F, Y320I 36 S318F, Y320V 37 S318Y, Y320I 38 S318Y, Y320V 39 W382Y 40 G17S, V20N, W382Y

[0165] [Table 4]

[0166] Effective mutation points 41 G17S, V20N, W382Y, I46C 42 G17S, V20N, W382Y, I46S 43 G17S, V20N, W382Y, I46G 44 G17S, V20N, W382Y, 146V 45 G17S, V20N, W382Y, I46F 46 G17S, V20N, W382Y, I46Y

[0167] [Table 5]

[0168] Effective mutation points 47 G17S, V20N, S318R, W382Y, I46F 48 G17S, V20N, S318V, W382Y, I46F 49 G17S, V20N, Y320R, W382Y, I46F 50 G17S, V20N, Y320, W382Y, I46F 51 G17S, V20N, S318H, Y320R, W382Y, I46F 52 G17S, V20N, S318H, Y320V, W382Y, I46F 53 G17S, V20N, S318R, Y320H, W382Y, I46F 54 G17S, V20N, S318R, Y320R, W382Y, I46F 55 G17S, V20N, S318R, Y320D, W382Y, I46F 56 G17S, V20N, S318R, Y320C, W382Y, I46F 57 G17S, V20N, S318R, Y320G, W382Y, I46F 58 G17S, V20N, S318R, Y320I, W382Y, I46F 59 G17S, V20N, S318R, Y320V, W382Y, I46F 60 G17S, V20N, S318R, Y320F, W382Y, I46F 61 G17S, V20N, S318C, Y320R, W382Y, I46F 62 G17S, V20N, S318G, Y320R, W382Y, I46F 63 G17S, V20N, S318I, Y320R, W382Y, I46F 64 G17S, V20N, S318I, Y320V, W382Y, I46F 65 G17S, V20N, S318L, Y320V, W382Y, I46F 66 G17S, V20N, S318V, Y320R, W382Y, I46F 67 G17S, V20N, S318V, Y320L, W382Y, I46F 68 G17S, V20N, S318V, Y320V, W382Y, I46F 69 G17S, V20N, S318V, Y320F, W382Y, I46F 70 G17S, V20N, S318F, Y320R, W382Y, 146F 71 G17S, V20N, S318F, Y320V, W382Y, I46F

[0169] The alignment of the sequences to each other can be performed by using a pair-wise alignment using BLAST (trademark) as a sequence homology search program. The parameters are set to the default parameters. In addition, the sequence homology is calculated based on the total length of the glycosyltransferase.

[0170] In the case of aligning the amino acid sequence represented by SEQ ID NO: 1 with the amino acid sequence of the comparison object, even if the amino acid residues corresponding to each other on the alignment are different from each other, the distance from the prescribed N terminus is sometimes different. For example, the valine residue (V) of the 20th amino acid residue from the N terminus in the amino acid sequence represented by SEQ ID NO: 1 means that it can be located at a position other than the 20th position from the N terminus on the amino acid sequence of the comparison object. In the present application, the "amino acid residue corresponding to the Xth position from the N terminus of SEQ ID NO: 1" means the amino acid residue corresponding to the Xth amino acid residue from the N terminus of SEQ ID NO: 1 on the alignment in the comparison object.

[0171] Specifically, for example, the valine of the 20th amino acid residue from the N terminus of SEQ ID NO: 1 can correspond to the 21st amino acid residue (not necessarily a valine residue) from the N terminus of the comparison object. In this case, the "amino acid residue corresponding to the 20th position from the N terminus" in the description of the present application means the 21st amino acid residue from the N terminus in the amino acid sequence of the comparison object.

[0172] The method for producing the mutant glycosyltransferase of the present application is not particularly limited, but for example, a vector containing a nucleic acid shown in a nucleotide sequence encoding the amino acid sequence of the mutant glycosyltransferase of the present application is prepared, and the mutant glycosyltransferase can be produced by the vector. Hereinafter, a detailed description will be given.

[0173] <Nucleic acid>

[0174] The nucleic acid of the present application is a nucleic acid encoding the mutant glycosyltransferase of the present application. More specifically, the nucleic acid of the present application has a nucleotide sequence encoding the amino acid sequence of the mutant glycosyltransferase of the present application.

[0175] As a method for synthesizing the nucleotide sequence encoding the amino acid sequence of the mutant glycosyltransferase of the present application, a method in which a mutation point is introduced into a nucleotide sequence encoding a wild-type glycosyltransferase derived from Punica granatum, a method in which a whole nucleotide sequence containing the mutation point is chemically synthesized, and the like can be given.

[0176] As a method for causing a mutation in a gene using a nucleotide sequence encoding a wild-type glycosyltransferase derived from Punica granatum as a mold, for example, a site-specific mutation method, a recombination PCR method, a method in which a specific portion of a nucleic acid is chemically synthesized, a method in which a gene is subjected to hydroxylamine treatment, a method in which a strain holding a gene is subjected to ultraviolet irradiation, a method in which a chemical agent such as nitroso guanidine and nitrous acid is subjected to treatment, a method in which a commercially available mutation introduction kit is used, and the like can be given.

[0177] The nucleic acid of the present application can be a deoxyribonucleic acid (DNA), or can be a ribonucleic acid (RNA).

[0178] < Vector >

[0179] The vector of the present application contains the nucleic acid of the present application.

[0180] As the vector of the present application, for example, a vector in which the mutant glycosyltransferase of the present application is introduced in a publicly known vector can be given. The vector of the present application can be any one of a viral vector, a bacteriophage vector, a plasmid vector, and the like.

[0181] The vector of the present application is preferably an expression vector.

[0182] The expression vector is only required to be an expression vector containing a nucleic acid shown in a nucleotide sequence encoding the amino acid sequence of the mutant glycosyltransferase of the present application. According to the expression vector of the present application, any host cell is transformed to obtain a transformant or a cell strain, and then the mutant glycosyltransferase of the present application can be produced by culturing the transformant and the cell strain.

[0183] The expression vector can include, in addition to the nucleotide sequence encoding the mutant glycosyltransferase of the present application, a nucleotide sequence constituting another region (hereinafter, also referred to simply as "another region") as needed.

[0184] As the other region, for example, a control region required for the transformant to produce the mutant glycosyltransferase, a region required for autonomous replication, and the like can be given.

[0185] As the control region required for the production of the mutant glycosyltransferase, a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, and the like can be given.

[0186] As examples of the promoter sequence, a trp promoter of a tryptophan operon derived from Escherichia coli, a lac promoter of a lactose operon, a PL promoter and a PR promoter derived from a bacteriophage λ, and the like can be given. Furthermore, a sequence artificially designed or altered, such as a tac promoter and a trc promoter, can also be used.

[0187] The sequence order on the expression vector of the control region is not particularly limited, but, for example, the promoter sequence and the ribosome binding sequence are preferably located upstream on the 5' end side than the gene encoding the mutant glycosyltransferase of the present application. Furthermore, the transcription termination sequence is preferably located downstream on the 3' end side than the gene encoding the mutant glycosyltransferase of the present application.

[0188] From the viewpoint of easily performing selection of the transformant, the expression vector can further include a nucleotide sequence encoding a selection gene that can be a selection marker.

[0189] The method of transforming the expression vector into a desired host cell and the method of producing the glycosyltransferase in the transformant are not particularly limited, and a publicly known general method and a host cell can be used.

[0190] <HOST CELL>

[0191] The host cell of the present application includes the expression vector of the present application.

[0192] The host cell according to the present application can produce a mutant glycosyltransferase having excellent potency.

[0193] The host cell of the present application can be exemplified by, for example, a method of constructing an expression vector including a nucleotide sequence encoding a mutant glycosyltransferase and, as needed, the above-described other region, and transforming the expression vector into a desired host cell.

[0194] As the host cell, prokaryotes (Escherichia coli, Bacillus subtilis, actinomycetes, and the like), yeasts, filamentous fungi, and the like can be given, and E. coli is preferred.

[0195] Escherichia coli is excellent in productivity of mass production because of its fast proliferation, low price and rich manufacturing history.

[0196] The host cell containing the expression vector (i.e., transformant) can be altered as necessary by introducing a silent mutation, etc., so that a codon of low frequency of use in the host cell becomes a codon of high frequency of use.

[0197] <Method for producing mutant glycosyltransferase>

[0198] The method for producing the mutant glycosyltransferase of the present application is a method for producing a mutant glycosyltransferase, comprising culturing the host cell of the present application described above in a culture medium; and recovering the mutant glycosyltransferase from at least one of the host cell cultured and the culture medium.

[0199] The culture conditions of the host cell of the present application, i.e., the host cell transformed by the expression vector (also referred to as transformant), can use publicly known conditions.

[0200] As the culture medium, any one of a synthetic culture medium or a natural culture medium can be used as long as it is a culture medium containing a carbon source, a nitrogen source, inorganic substances and other nutrients in appropriate amounts.

[0201] As the culture medium components, publicly known components for culture media can be used. For example, organic nutrient sources such as meat extract, yeast extract, malt extract, peptone, NZ amine and potato, carbon sources such as glucose, maltose, sucrose, starch and organic acids, nitrogen sources such as ammonium sulfate, urea and ammonium chloride, inorganic nutrient sources such as phosphate, magnesium, potassium and iron, and vitamins can be appropriately combined and used. Also, in order to perform selection of transformants based on drug selectivity, an antibiotic such as kanamycin can be used. In order to induce expression, an expression inducer such as an inducer for lac operon expression (isopropyl-β-thiogalactoside, etc.) can be used.

[0202] The pH of the culture medium can be appropriately selected, for example, in the range of pH 4 to pH 8.

[0203] As the culture method of the transformant, a conventional culture method such as shaking culture, aerated agitation culture, continuous culture, fed-batch culture, etc. of a liquid culture medium containing the transformant can be used.

[0204] The culture conditions are not particularly limited as long as they are conditions in which the transformant grows and the mutant glycosyltransferase can be produced, by appropriate selection of the type of transformant, culture medium and culture method.

[0205] The culture temperature is preferably 16°C to 45°C, more preferably 16°C to 40°C.

[0206] As for the cultivation period, for example, it is only necessary to cultivate in the range of 10 hours to 7 days (preferably, in the range of 15 hours to 24 hours) until the content of the protein having the target glycosyltransferase activity becomes maximum.

[0207] (Mutant glycosyltransferase recovery step)

[0208] The method for producing the mutant glycosyltransferase preferably includes a step of recovering the mutant glycosyltransferase from at least either one of the cultivated transformant and the culture medium after cultivation.

[0209] After the cultivation of the transformed host cell, the method for recovering the mutant glycosyltransferase can use a method conventionally used in the field.

[0210] For example, in the case where the mutant glycosyltransferase is secreted outside the cells of the transformant, a crude enzyme solution containing the mutant glycosyltransferase can be easily obtained by subjecting the culture of the transformant to centrifugal separation, filtration, or the like.

[0211] For example, in the case where the mutant glycosyltransferase is accumulated in the cells of the transformant, after the recovery by subjecting the cultivated transformant to centrifugal separation or the like, the recovered transformant is suspended in a buffer, and the cell membrane of the transformant is destroyed in the suspension by a known method such as lysozyme treatment, freeze-thawing, ultrasonic wave pulverization, whereby a crude enzyme solution containing the mutant glycosyltransferase can be recovered.

[0212] The crude enzyme solution containing the recovered mutant glycosyltransferase, in the case where separation and purification are required, can be further subjected to separation and purification, for example, by appropriately combining a membrane separation method based on ultrafiltration, salting-out, organic solvent precipitation, dialysis, or the like; a known chromatographic separation method such as ion exchange chromatography, gel filtration chromatography, or the like.

[0213] <Composition>

[0214] The composition of the present application is a composition containing the mutant glycosyltransferase of the present application. According to the composition of the present application, the titer is excellent.

[0215] The composition of the present application can further contain other materials other than the mutant glycosyltransferase. As the other materials, for example, there can be mentioned a pulverized product of the transformant (for example, cell membrane or the like), a buffer, a solvent, a culture solution containing a culture component, or the like, which are produced in the process of producing the mutant glycosyltransferase.

[0216] <Method for producing anthraquinone C glycoside>

[0217] The method for producing the anthraquinone C glycoside of the present application is a method for producing an anthraquinone C glycoside including a step of bringing an anthraquinone compound and a monosaccharide donor into contact with the mutant glycosyltransferase of the present application to form an anthraquinone C glycoside.

[0218] According to the method for producing an anthraquinone C glycoside of the present application, there is a tendency that an anthraquinone C glycoside can be efficiently produced.

[0219] The anthraquinone compound is not particularly limited as long as it has an anthraquinone skeleton and can function as a sugar acceptor in a sugar transfer reaction based on a glycosyltransferase, and a known anthraquinone compound can be used. As the anthraquinone compound, for example, kermesic acid, laccaic acid D, emodin, 2-methylanthraquinone, 1-chloroanthraquinone, 2-pentylanthraquinone, and the like can be mentioned.

[0220] The monosaccharide donor is not particularly limited as long as it can function as a donor of a monosaccharide in a sugar transfer reaction based on a glycosyltransferase, and a sugar nucleotide of a monosaccharide such as glucose, fructose, and galactose can be mentioned. As the sugar nucleotide, uridine diphosphate glucose and the like can be mentioned.

[0221] In particular, the mutant glycosyltransferase of the present application is excellent in a reaction in which an anthraquinone compound containing at least one of kermesic acid and laccaic acid D and a glucose donor (for example, uridine diphosphate glucose and the like) as a monosaccharide donor form an anthraquinone C glycoside containing a carmine pigment.

[0222] The method for bringing the anthraquinone compound and the monosaccharide donor into contact with the mutant glycosyltransferase of the present application is not particularly limited, but for example, a method in which the anthraquinone compound and the monosaccharide donor are mixed in a culture solution containing a transformant producing the mutant glycosyltransferase of the present application and a cell strain, a cell treatment, or an enzyme purification product can be mentioned.

[0223] Thus, the mutant glycosyltransferase of the present application is brought into contact with the anthraquinone compound and the monosaccharide donor, and catalyzes a reaction in which the anthraquinone C glycoside is formed from the anthraquinone compound and the monosaccharide donor.

[0224] The above-mentioned cell treatment refers to, for example, an extract and a pulverized product from a transformant.

[0225] The temperature at the time of the above-mentioned contact and reaction is, for example, preferably 10°C to 30°C, and more preferably 15°C to 30°C.

[0226] As one mode, a purified product of the mutant glycosyltransferase of the present application extracted and purified from a culture solution in which a transformant and a cell strain have been cultured can be mixed with a solvent containing at least the anthraquinone compound and the monosaccharide donor, and the two can be brought into contact and reacted, whereby the anthraquinone C glycoside is formed.

[0227] The pH of the aqueous solution at the time of the above-mentioned contact and reaction is preferably 6 to 8, and more preferably 7 to 8.

[0228] Details of the culture of the transformant are as described in the item of the method for producing a mutant glycosyltransferase of the present application.

[0229] Examples

[0230] The embodiments are further explained by the following examples, but the present application is not limited to the following examples in any way.

[0231] 1. Acquisition of wild-type and mutant glycosyltransferases

[0232] <Comparative Example 1: Acquisition of mutant glycosyltransferase (c1) for comparison>

[0233] The mutant glycosyltransferase (c1) was obtained by the method described in Non-Patent Literature 1.

[0234] <Comparative Example 2: Acquisition of mutant glycosyltransferase (c2) for comparison>

[0235] The mutant glycosyltransferase (c2) was obtained by the method described in Non-Patent Literature 1.

[0236] <Comparative Example 3: Acquisition of mutant glycosyltransferase (c3) for comparison>

[0237] The mutant glycosyltransferase (c3) described in Patent Literature 1 has a description that it is difficult to express based on Escherichia coli (i.e., low titer), and therefore was not acquired in the present application, and evaluation of the titer, C-glycosylation activity, and C-glycosylation activity extracted from the culture solution can not be performed.

[0238] <Reference Example 1: Acquisition of wild-type glycosyltransferase (r1) derived from Punica granatum>

[0239] The wild-type glycosyltransferase (r1) derived from Punica granatum was obtained by the method described in the literature (Nadia N. Ono, Xiaoqiong Qin, Alexander E. Wilson, Gang Li1, Li Tian, PLoS ONE, 11(5): e0156319).

[0240] The amino acid sequence of the mutant glycosyltransferase (c1) in Comparative Example 1 (SEQ ID NO: 72), the amino acid sequence of the mutant glycosyltransferase (c2) in Comparative Example 2 (SEQ ID NO: 73), the amino acid sequence of the mutant glycosyltransferase (c3) in Comparative Example 3 (SEQ ID NO: 74), and the amino acid sequence of the wild-type glycosyltransferase (r1) derived from Punica granatum in Reference Example 1 (SEQ ID NO: 75) are shown below, respectively.

[0241] SEQ ID NO: 72: MGSLTNNDNLHIFLVCFIGQGVVNPMLRLGKAFASKGLLVTLSAPEIVGTEIRKANNLNDDQPIKVGSGMIRFEFFDDGWESVNGSKPFDVWVYINHLDQTGRQKLPIMLKKHEETGTPVSCLILNPLVPWVADVADSLQIPCATLWVQSCASFSAYYHYHHGLVPFPTESEPEIDVQLPGMPLLKYDEVPDYLHPRTPYPFFGTNILGQFKNLSKNFCILMDTFYELEHEIIDNMCKLCPIKPIGPLFKIPKDPSSNGITGNFMKVDDCKEWLDSRPTSTVVYVSVGSVVYLKQEQVTEMAYGILNSEVSFLWVLRPPSKRIGTEPHVLPEEFWEKAGDRGKVVQWSPQEQVLAHPATVGFLTHCGWNSTQEAISSGVPVITFPQFGDQVTNAKFLVEEFKVGVRLGRGELENRIITRDEVERALREITSGPKAEEVKENALKWKKKAEETVAKGGYSERNLVGFIEEVARKTGTK

[0242] SEQ ID NO: 73: MGSLTNNDNLHIFLVCFIGQGVVNPMLRLGKAFASKGLLVTLSAPEIVGTEIRKANNLNDDQPIKVGSGMIRFEFFDDGWESVNGSKPFDVWQYINHLDQTGRQKLPIMLKKHEETGTPVSCLILNPLVPWVADVADSLQIPCATLWVQSCASFSAYYHYHHGLVPFPTESEPEIDVQLPGMPLLKYDEVPDFLHPRTPYPFFGTNILGQFKNLSKNFCILMDTFYELEHEIIDNMCKLCPIKPIGPLFKIPKDPSSNGITGNFMKVDDCKEWLDSRPTSTVVYVSVGSVVYLKQEQVTEMAYGILNSEVSFLWVLRPPSKRIGTEPHVLPEEFWEKAGDRGKVVQWSPQEQVLAHPATVGFLTHCGWNSTQEAISSGVPVITFPQFGDQVTNAKFLVEEFKVGVRLGRGELENRIITRDEVERALREITSGPKAEEVKENALKWKKKAEETVAKGGYSERNLVGFIEEVARKTGTK

[0243] SEQ ID NO: 74: MEFRLLILALFSVLMSTSNGAEILALFPIHGISNYNVAEALLKTLANRGHNVTVVTSFPQKKPVPNLYEIDVSGAKGLATNSIHFERLQTIIQDVKSNFKNMVRLSRTYCEIMFSDPRVLNIRDKKFDLVINAVFGSDCDAGFAWKSQAPLISILNARHTPWALHRMGNPSNPAYMPVIHSRFPVKMNFFQRMINTGWHLYFLYMYFYYGNGEDANKMARKFFGNDMPDINEMVFNTSLLFVNTHFSVDMPYPLVPNCIEIGGIHVKEPQPLPLEIQKFMDEAEHGVIFFTLGSMVRTSTFPNQTIQAFKEAFAELPQRVLWKFENENEDMPSNVLIRKWFPQNDIFGHKNIKAFISHGGNSGALEAVHFGVPIIGIPLFYDQYRNILSFVKEGVAVLLDVNDLTKDNILSSVRTVVNDKSYSERMKALSQLFRDRPMSPLDTAVYWTEYVIRHRGAHHLKTAGAFLHWYQYLLLDVITFLLVTFCAFCFIVKYICKALIHHYWSSSKSEKLKKN

[0244] Sequence number 75: MGSESSLVHVFLVSFPGQGHVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0245] 2. Obtaining of the mutant glycosyltransferase of the present application

[0246] Obtaining of the mutant glycosyltransferase (1) of the present application

[0247] The nucleic acid encoding the polypeptide of SEQ ID NO: 1 was codon-optimized and synthesized as a nucleic acid gene. Using a pET28a vector system into which the synthetic gene was cloned, competent cells of an E. coli strain BL21(DE3) were transformed, seeded on an LB agar medium containing 50 μg / mL of kanamycin, and incubated overnight at 37°C. The propagated colonies were inoculated in an LB liquid medium containing 50 μg / mL of kanamycin, and subjected to shaking culture overnight at 37°C, 200 rpm. A portion of the culture solution was inoculated in an LB liquid medium 0.3 L containing 50 μg / mL of kanamycin, and incubated at 37°C, 200 rpm. After the optical density (OD600) reached 0.6-0.8, isopropyl-β-thiogalactoside (IPTG) was added at a final concentration of 0.5 mM, and subjected to shaking culture at 16°C, 200 rpm for 18 hours. The supernatant was discarded by centrifugal separation (4°C, 3000 G, 20 minutes). The obtained cell pellet was suspended in 30 mL of a lysis buffer (1 x PBS, 10 mM imidazole solution (pH 7.4)), and subjected to treatment on ice using a homogenizer (BRANSON SONIFER Co.). The treated solution was subjected to centrifugal separation (4°C, 5000 G, 30 minutes), and the cell debris was discarded. After the obtained supernatant was subjected to His-tag purification, ultrafiltration, and replacement into a 50 mM HEPES buffer (pH 7.5), a purified enzyme solution was prepared, thereby obtaining a composition containing the mutant glycosyltransferase. The sequence of the above nucleic acid gene (SEQ ID NO: 76) is shown below.

[0248]

[0249] <Acquisition of mutant glycosyltransferases (2) to (71) of the present invention>

[0250] A gene was synthesized by codon optimization of a nucleic acid encoding a polypeptide of the sequence number shown in the table described later (in the table, marked as "enzyme sequence number"). Other than being the same specifications as the mutant glycosyltransferase (1), compositions containing the mutant glycosyltransferases (2) to (71) were obtained, respectively.

[0251] In the table described later, the single-letter notation of the capital English letter in the items [mutation 1G17] to [mutation 6I46] means the kind of the corresponding amino acid residue in the amino acid sequence represented by the sequence number 1. For example, the item [mutation 1G17] in Table 3 means a mutation related to the glycine residue (G) at the 17th amino acid residue from the N-terminal side in the amino acid sequence represented by the sequence number 1.

[0252] In the table described later, the blank in the items [mutation 1G17] to [mutation 6I46] means that the amino acid residue of each item is not mutated from the amino acid residue represented by the sequence number 1.

[0253] In the table described later, "H * " described in the item [mutation 2V20] of Reference Example 1 means that the 20th amino acid residue from the N-terminal side in the amino acid sequence of the wild-type glycosyltransferase derived from Punica granatum is a histidine residue (H).

[0254] In addition, the mutant glycosyltransferase (2) is a mutant glycosyltransferase that does not have mutations in G17, V20, S318, Y320, Y382, and I46 with respect to the sequence number 1, but as a result of having mutations in other amino acids, the amino acid sequence identity with respect to the sequence number 1 is 85%. With respect to the mutant glycosyltransferases (3) to (71), in addition to the mutations described in the table described later in G17, V20, S318, Y320, Y382, and I46 with respect to the sequence number 1, no mutations are present.

[0255] 3. Comparison of the titer and activity of glycosyltransferases

[0256] Using the obtained glycosyltransferases, the titer and enzyme activity were compared.

[0257] [evaluation of titer (a)]

[0258] The amount of enzyme (i.e., the amount of mutant glycosyltransferase) was quantified from the concentration of the purified enzyme solution based on the absorbance at 280 nm measured by a spectrophotometer, with respect to each example. Next, the enzyme diluent was electrophoretically developed by SDS-PAGE, the gel was stained by CBB (Coomassie Brilliant Blue) staining solution, and the purity of the band containing the target protein (i.e., the band corresponding to the mutant glycosyltransferase) was quantified by image analysis. The titer was calculated from the amount of enzyme and the amount of culture solution obtained according to the following formula and evaluated according to the following criteria. The results are shown in the table below.

[0259] Enzyme amount (mg) = enzyme solution concentration (mg / L) x enzyme solution amount (L) x (purity (%) / 100) Titer (mg / L) = enzyme amount (mg) / culture solution amount (L)

[0260] Evaluation Criteria

[0261] S: Titer is 10 (mg / L) or more

[0262] A: Titer is 3 (mg / L) or more and less than 10 (mg / L)

[0263] B: Titer is 1 (mg / L) or more and less than 3 (mg / L)

[0264] C: Titer is less than 1 (mg / L)

[0265] Evaluation of C-glycosylation activity (b)

[0266] Evaluation of C-glycosylation activity (b) in the case where betalamic acid was used as the substrate was performed with the specifications shown below.

[0267] In 100 μL of a buffer (a mixed solution of 50 mM HEPES (pH 7.5), 1 mg / mL lysozyme, 0.5 mg / mL polymyxin B, 13 mM MgCl2, and 50 mM KCl), 0.5 mM of a DMSO solution of betalamic acid, 5 mM of an aqueous solution of uridine diphosphate glucose (UDP-Glucose), and 20 μg of the purified enzyme of each example were stirred at 25°C for 3 hours and allowed to react. Thereafter, methanol 100 μL was added to the reaction system and the reaction was terminated, and centrifugal separation (4°C, 1800 G, 30 minutes) was performed to obtain a supernatant.

[0268] The supernatant was measured using a high-performance liquid chromatograph (HPLC) to quantitatively analyze the amount of anthraquinone C-glycoside as the target product. The analysis conditions of the HPLC were as follows.

[0269] HPLC Analysis Conditions

[0270] Apparatus: Nexera X2 (manufactured by Shimadzu Corporation)

[0271] Column: Waters ACQUITY UPLC BEH C18, 1.7 μm, 2.0 x 50 mm

[0272] Mobile phase: A: water (0.1% formic acid), B: methanol (0.1% formic acid)

[0273] Time (min) / %B: 0 / 10, 0.4 / 10, 3.0 / 80, 4.0 / 100, 5.0 / 100, 5.1 / 10

[0274] Injection amount: 2 μL

[0275] Column temperature: 40°C

[0276] Flow rate: 0.4 mL / min

[0277] Detection wavelength: kermesic acid 490 nm

[0278] Retention time: kermesic acid C glycoside 2.34 min

[0279] C glycosylation activity was calculated from the amount of C glycoside produced, the reaction time, and the amount of enzyme used by HPLC and evaluated using the following criteria. The results are shown in the table below.

[0280] C glycosylation activity (pkat / mg) = amount of C glycoside produced (pmol) / reaction time (s) / amount of enzyme used (mg)

[0281] Evaluation criteria

[0282] S: C glycosylation activity is 100 (pkat / mg) or more

[0283] A: C glycosylation activity is 10 (pkat / mg) or more and less than 100 (pkat / mg)

[0284] B: C glycosylation activity is 1 (pkat / mg) or more and less than 10 (pkat / mg)

[0285] C: C glycosylation activity is less than 1 (pkat / mg)

[0286] Evaluation of C glycosylation activity (a x b) extracted from culture solution

[0287] Enzyme activity extracted from culture solution was calculated from the C glycosylation activity and titer obtained from the above evaluation according to the following formula and evaluated using the following criteria. The results are shown in the table below.

[0288] Enzyme activity extracted from culture solution (pkat / L) = Anthraquinone C glycosylation activity (pkat / mg) x expression efficiency (mg / L)

[0289] - Evaluation Criteria -

[0290] S: Enzyme activity extracted from culture solution is 1000 (pkat / L) or more

[0291] A: Enzyme activity extracted from culture solution is 100 (pkat / L) or more and less than 1000 (pkat / L)

[0292] B: Enzyme activity extracted from culture solution is 10 (pkat / L) or more and less than 100 (pkat / L)

[0293] C: Enzyme activity extracted from culture solution is less than 10 (pkat / L)

[0294] [Table 6]

[0295]

[0296] [Table 7]

[0297]

[0298] [Table 8]

[0299]

[0300] [Table 9]

[0301]

[0302] As shown in the above tables, it was found that the potency of the mutant glycosyltransferases of the Examples was superior compared to the wild-type or mutant glycosyltransferases of the Comparative Examples. Furthermore, by introducing the mutations with respect to SEQ ID NO: 1, it was possible to obtain mutant glycosyltransferases having improved C-glycosylation activity. Among the mutant glycosyltransferases thus obtained, especially the mutant glycosyltransferases of the Examples having 90% or more sequence identity with respect to SEQ ID NO: 1, there was a tendency for the C-glycosylation activity extracted from the culture solution to be good.

[0303] 4. Evaluation of C-glycosylation activity with respect to laccaic acid D

[0304] Next, the C-glycosylation activity with respect to laccaic acid D as a substrate instead of kermesic acid was evaluated.

[0305] [Evaluation of C-glycosylation activity (b)]

[0306] The reaction procedure in the evaluation of C-glycosylation activity (b) in the case where laccaic acid D was used as a substrate was carried out in the same manner as in Example 1, except that the detection wavelength in HPLC analysis was set to the wavelength of laccaic acid D, 430 nm, and the retention time was set to the time of laccaic acid DC glycoside, 2.29 minutes. The results of each evaluation are shown in the table below.

[0307] In 100 μL of a buffer (a mixed solution of 50 mM HEPES (pH 7.5), 1 mg / mL lysozyme, 0.5 mg / mL polymyxin B, 13 mM MgCl2, and 50 mM KCl), 0.5 mM of a DMSO solution of laccaic acid D, 5 mM of an aqueous solution of uridine diphosphate glucose (UDP-Glucose), and 20 μg of each of the purified enzymes were stirred at 25°C for 24 hours and allowed to react. Thereafter, methanol 100 μL was added to the reaction system to terminate the reaction, and centrifugal separation (4°C, 1800 G, 30 minutes) was performed to obtain a supernatant.

[0308] Evaluation of C-glycosylation activity (a x b) extracted from culture solution

[0309] Evaluation of C-glycosylation activity (a x b) extracted from culture solution using each of the mutant glycosyltransferases was carried out in the same manner as in Example 1 described above. The results are shown in the table below.

[0310] In the table, the titer is the titer determined by the method described above.

[0311] [Table 10]

[0312]

[0313] As is apparent from the above table, each of the variants has excellent C-glycosylation activity also with respect to laccaic acid D.

[0314] 5. Study of C-glycosylation activity with respect to emodin

[0315] Next, C-glycosylation activity was evaluated in the case where emodin (6-methyl-l,3,8-trihydroxyanthraquinone) was used as a substrate instead of laccaic acid.

[0316] Evaluation of C-glycosylation activity (b)

[0317] The reaction procedure in the evaluation of C-glycosylation activity (b) in the case where emodin (6-methyl-l,3,8-trihydroxyanthraquinone) was used as a substrate was carried out in the same manner as in Example 1, except that the detection wavelength in HPLC analysis was set to the wavelength of emodin, 420 nm, and the retention time was set to the time of emodin C glycoside, 3.14 minutes. The results of each evaluation are shown in the table below.

[0318] In 100 μL of a buffer (a mixed solution of 50 mM HEPES (pH 7.5), 1 mg / mL lysozyme, 0.5 mg / mL polymyxin B, 13 mM MgCl2, and 50 mM KCl), 0.5 mM emodin DMSO solution, 5 mM uridine diphosphate glucose (UDP-Glucose) aqueous solution, and 20 μg of each purified enzyme were stirred at 25°C for 24 hours and allowed to react. Thereafter, methanol 100 μL was added to the reaction system and the reaction was terminated, and centrifugal separation (4°C, 1800 G, 30 minutes) was performed to obtain a supernatant.

[0319] Evaluation of C glycosylation activity (a x b) extracted from culture solution

[0320] Evaluation of C glycosylation activity (a x b) extracted from culture solution using each mutant glycosyltransferase was performed by the same method as in Example 1 described above. The results are shown in the table below.

[0321] In the table, the titer is the titer determined by the method described above.

[0322] [Table 11]

[0323]

[0324] As is apparent from the above table, each variant has excellent C glycosylation activity also with respect to emodin.

[0325] The amino acid sequence of each mutant glycosyltransferase is shown below.

[0326] SEQ ID NO: 1: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0327] SEQ ID NO: 2: MGSESLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNIGEEPSPI GDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKEVIPRMIKKNEEQNRPVSCLINNPFIPWV SDVAESLGLPSAMLWVQSCACFAAYYHYYHGLVPFPSESAMEIDVQLPCMPLLKHDEVPSFLYPTTP YPFLRRAIMGQYKNLDKPFCVLMDTFQELEHEIIEYMSKICPIKTVGPLFKNPKAPNANVRGDFMK ADDCISWLDSKPPASVVYVSFGSVVYLKQDQWDEIAFGLLNSGLNFLWVMKPPHKDSGYQLLTLPEG FLEKAGDKGKVVQWSPQEQVLAHPSVACFVTHCGWNSSMEALSSGMPVVAFPQWGDQVTDAKYLVDV FKVGVRMCRGEAENKLIMRDVVEKCLLEATVGPKAAEVKENALKWKAAAEAAVAEGGSSDRNIQAF VDEVKRRSIAIQSNKSEPKPVVQNAAVADHFGAKATTNGVAADLAGSNADGKVELVA

[0328] SEQ ID NO: 3: MGSESSLVHVFLVSFPHQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0329] SEQ ID NO: 4: MGSESSLVHVFLVSFPSQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0330] SEQ ID NO: 5: MGSESSLVHVFLVSFPGQGCVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0331] SEQ ID NO: 6: MGSESSLVHVFLVSFPHQGCVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0332] SEQ ID NO: 7: MGSESSLVHVFLVSFPHQGNVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0333] SEQ ID NO: 8: MGSESSLVHVFLVSFPHQGIVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0334] SEQ ID NO: 9: MGSESSLVHVFLVSFPSQGCVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0335] SEQ ID NO: 10: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0336] SEQ ID NO: 11: MGSESSLVHVFLVSFPSQGIVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0337] SEQ ID NO: 12: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0338] SEQ ID NO: 13: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGHTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0339] SEQ ID NO: 14: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0340] SEQ ID NO: 15: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0341] SEQ ID NO: 16: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDHGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0342] SEQ ID NO: 17: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDHGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0343] SEQ ID NO: 18: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGHTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0344] SEQ ID NO: 19: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0345] SEQ ID NO: 20: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGCTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0346] SEQ ID NO: 21: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGSTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0347] SEQ ID NO: 22: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0348] SEQ ID NO: 23: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0349] SEQ ID NO: 24: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDNGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0350] SEQ ID NO: 25: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDGGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0351] SEQ ID NO: 26: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDIGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0352] SEQ ID NO: 27: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDIGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0353] SEQ ID NO: 28: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDIGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0354] SEQ ID NO: 29: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDLGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0355] SEQ ID NO: 30: MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0356] SEQ ID NO: 31: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0357] SEQ ID NO: 32: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGLTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0358] SEQ ID NO: 33: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0359] SEQ ID NO: 34: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDFGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0360] SEQ ID NO: 35: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDFGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0361] SEQ ID NO: 36: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDFGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0362] SEQ ID NO: 37: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDYGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0363] SEQ ID NO: 38: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDYGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQWGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0364] SEQ ID NO: 39: "MGSESSLVHVFLVSFPGQGVVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0365] SEQ ID NO: 40: "MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESIGKQMRKASNISDQPAPVGDGFIRFEF FEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA"

[0366] SEQ ID NO: 41: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESCGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0367] SEQ ID NO: 42: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESSGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0368] SEQ ID NO: 43: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESGGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0369] SEQ ID NO: 44: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESVGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0370] SEQ ID NO: 45: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0371] SEQ ID NO: 46: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESYGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0372] SEQ ID NO: 47: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0373] SEQ ID NO: 48: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGYTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0374] SEQ ID NO: 49: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0375] SEQ ID NO: 50: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDSGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0376] SEQ ID NO: 51: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDHGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0377] SEQ ID NO: 52: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDHGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0378] SEQ ID NO: 53: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGHTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0379] Serial number 54: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0380] Serial number 55: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGDTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0381] SEQ ID NO: 56: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGCTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0382] SEQ ID NO: 57: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGGTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0383] SEQ ID NO: 58: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGITVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0384] SEQ ID NO: 59: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0385] SEQ ID NO: 60: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDRGFTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0386] SEQ ID NO: 61: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDCGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0387] SEQ ID NO: 62: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDGGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0388] SEQ ID NO: 63: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDIGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0389] SEQ ID NO: 64: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDIGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0390] SEQ ID NO: 65: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDLGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0391] SEQ ID NO: 66: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0392] SEQ ID NO: 67: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGLTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0393] SEQ ID NO: 68: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0394] Serial number 69: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDVGFTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0395] SEQ ID NO: 70: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLPSMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVVYLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDFGRTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEFKVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0396] SEQ ID NO: 71: MGSESSLVHVFLVSFPSQGNVNPLLRLGKRLASKGLLVTFTTPESFGKQMRKASNISDQPAPVGDG

[0397] FIRFEFFEDGWDEDEPRRQDLDQYLPQLEKVGKVLIPQMIQKNAEQGRPVSCLINNPFIPWVSDVAETLGLPSAMLWVQSCACFLAYYHYYHGLVPFPSENAMEIDVQLP SMPLLKHDEVPSFLYPTTPYPFLRRAILGQYKNLEKPFCILMDTFQELEHEIIEYTSKICPIKTVGPLFKNPKAPNTTVKGDFMKADDCIGWLDSKPASSVVYVSFGSVV YLKQDQWDEIAYGLLNSGVNFLWVMKPPHKDFGVTVLTLPEGFLEKAGDRGKVVQWSPQEQVLAHPATACFVTHCGWNSSMEALTSGMPVVAFPQYGDQVTDAKYLVDEF KVGVRMCRGEAEDKLITRDVVEQCLREATQGPKAAEMKKNALKWKAAAEASFVEGGSSDRNLQAFVDEVKRRSIEITASKPAVKAAPNGVVAAAESVVETKANGKVELAA

[0398] The invention of Japanese Patent Application No. 2023-042144, filed on March 16, 2023, is hereby incorporated by reference into this specification in its entirety. All documents, patent applications, and technical standards described in this specification are hereby incorporated by reference into this specification to the same extent as if each individual document, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A mutant glycosyltransferase having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and capable of forming an anthraquinone C-glycoside from an anthraquinone compound and a monosaccharide donor.

2. The mutant glycosyltransferase of claim 1, wherein, The mutant glycosyltransferase satisfies at least one condition selected from the group consisting of (1-A) to (6-A) below: (1-A) the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, or an aliphatic amino acid residue, (2-A) the amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue or an aliphatic amino acid residue, (3-A) the amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue, (4-A) the amino acid residue corresponding to the 320th position from the N terminus of SEQ ID NO: 1 is a basic amino acid residue, an acidic amino acid residue, a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue, (5-A) the amino acid residue corresponding to the 382nd position from the N terminus of SEQ ID NO: 1 is an aromatic amino acid residue, 3. The mutant glycosyltransferase of claim 2, wherein, (6-A) the amino acid residue corresponding to the 46th position from the N terminus of SEQ ID NO: 1 is a polar uncharged amino acid residue, an aliphatic amino acid residue, or an aromatic amino acid residue. The mutant glycosyltransferase satisfies at least one condition selected from the group consisting of (1-B) to (6-B) below: (1-B) the amino acid residue corresponding to the 17th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), a serine residue (S), or a glycine residue (G), (2-B) the amino acid residue corresponding to the 20th position from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), an asparagine residue (N), an isoleucine residue (I), or a valine residue (V), (3-B) the amino acid residue corresponding to the 318th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), a cysteine residue (C), an asparagine residue (N), a serine residue (S), an isoleucine residue (I), a leucine residue (L), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y), (4-B) the amino acid residue corresponding to the 320th position from the N terminus of SEQ ID NO: 1 is a histidine residue (H), an arginine residue (R), an aspartic acid residue (D), a cysteine residue (C), a serine residue (S), a glycine residue (G), an isoleucine residue (I), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y), (5-B) the amino acid residue corresponding to the 382nd position from the N terminus of SEQ ID NO: 1 is a tyrosine residue (Y), (6-B) the amino acid residue corresponding to the 46th position from the N terminus of SEQ ID NO: 1 is a cysteine residue (C), a serine residue (S), a glycine residue (G), a valine residue (V), a phenylalanine residue (F), or a tyrosine residue (Y).

4. The mutant glycosyltransferase of claim 3, wherein, The mutant glycosyltransferase satisfies the following combinations among the conditions of (1-B) to (6-B): only the combination of (1-B) and (2-B); only the combination of (3-B) and (4-B); only the combination of (1-B), (2-B), and (5-B); only the combination of (1-B), (2-B), (5-B), and (6-B); only the combination of (1-B), (2-B), (3-B), (5-B), and (6-B); only the combination of (1-B), (2-B), (4-B), (5-B), and (6-B); or the combination of (1-B), (2-B), (3-B), (4-B), (5-B), and (6-B).

5. The mutant glycosyltransferase of claim 1, wherein, The mutant glycosyltransferase has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

71.

6. A nucleic acid encoding the mutant glycosyltransferase according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid according to claim 6.

8. The vector of claim 7, wherein, The vector is an expression vector.

9. A host cell comprising the expression vector according to claim 8.

10. The host cell of claim 9, wherein, The host cell is Escherichia coli.

11. A method for producing a mutant glycosyltransferase, comprising the steps of: culturing the host cell according to claim 9 in a culture medium; and recovering the mutant glycosyltransferase from at least one of the cultured host cell and the culture medium.

12. A composition comprising the mutant glycosyltransferase according to any one of claims 1 to 5.

13. A method for producing an anthraquinone C-glycoside, comprising the steps of: a step of contacting an anthraquinone compound and a monosaccharide donor with the mutant glycosyltransferase according to any one of claims 1 to 5 to form an anthraquinone C-glycoside.

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