Mutant sucrose synthase, nucleic acid encoding mutant sucrose synthase, expression vector, transformant, method for producing mutant sucrose synthase, composition containing mutant sucrose synthase, method for producing uridine diphosphate-glucose, and method for producing sugar compound

Abstract

The mutant sucrose synthase satisfies the following (1) and / or (2) and has an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 1. (1) An amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than said amino acid residue and having improved enzyme activity compared to before substitution. (2) An amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than said amino acid residue and having improved enzyme activity compared to before substitution.

Description

Mutant sucrose synthase, nucleic acid encoding mutant sucrose synthase, expression vector, transformant, method for producing mutant sucrose synthase, composition containing mutant sucrose synthase, method for producing uridine diphosphate-glucose, method for producing sugar compounds

[0001] This disclosure relates to mutant sucrose synthase, nucleic acids encoding mutant sucrose synthase, expression vectors, transformants, methods for producing mutant sucrose synthase, compositions containing mutant sucrose synthase, methods for producing uridine diphosphate-glucose, and methods for producing sugar compounds.

[0002] Sucrose synthase is an enzyme that catalyzes the reaction of producing nucleotide diphosphate-glucose (such as uridine diphosphate-glucose) and fructose, and the reverse reaction, using sucrose and nucleotide diphosphates (NDPs) such as uridine diphosphate (UDP) as substrates. For example, plant-derived sucrose synthase has high specificity for uridine diphosphate among nucleotide diphosphates. Furthermore, nucleotide diphosphate-glucoses such as uridine diphosphate-glucose are used as substrates for glycosyltransferases and are raw materials for many carbohydrates and glycosides. For example, arbutin, known as a skin-whitening ingredient, is synthesized by glycosyltransferase using hydroquinone and uridine diphosphate-glucose as substrates.

[0003] Linda Bungaruang, Alexander Gutmann, Bernd Nidetzk y, Adv. Synth. Catal. 2013, 355, 2757 - 2763 discloses a system in which uridine diphosphate - glucose continuously supplied by sucrose synthase (GmSuSy) derived from soybeans is used for the C - glycosylation of phloretin by glycosyltransferase derived from rice. Also, Yehui Tao, Ping Sun, Ruxin Cai, Yan Li, Honghua Jia, Appl. Sci. 2022, 12, 3911 discloses a system for converting stevioside to rebaudioside A using sucrose synthase (AtSU S) derived from Arabidopsis thaliana and stevia glycosyltransferase UGT76G1.

[0004] By the way, if the enzyme activity of sucrose synthase is improved, it is possible to improve the productivity of substances in the system using sucrose synthase as described above. However, although various biological - derived sucrose synthases are known, no comparison of enzyme activities or examination of mutations in the enzyme - active site for improving enzyme activity has been carried out. Therefore, the present disclosure aims to identify mutations that improve the activity of sucrose synthase, and provides a mutant sucrose synthase with improved enzyme activity compared to before mutation, a nucleic acid encoding the mutant sucrose synthase, an expression vector, a transformant, a method for producing the mutant sucrose synthase, a composition containing the mutant sucrose synthase, a method for producing uridine diphosphate - glucose, and a method for producing a sugar compound.

[0005] As a result of intensive studies by the present inventors to achieve the above - described object, it has been found that by introducing a mutation into a predetermined amino acid residue in the amino acid sequence of sucrose synthase, the enzyme activity is significantly improved compared to before mutation, and the present disclosure has been completed. The present disclosure includes the following.

[0006] <1> A mutant sucrose synthase that satisfies the requirements of (1) and / or (2) below and has an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1. (1) The amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. (2) The amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. <2> The mutant sucrose synthase according to <1>, wherein the amino acid after substitution in (1) is an amino acid selected from the group consisting of tyrosine, serine, histidine, leucine, aspartic acid, and glycine. <3> The mutant sucrose synthase according to <1> or <2>, wherein the amino acid after substitution in (2) is an amino acid selected from the group consisting of serine, isoleucine, aspartic acid, glycine, and tyrosine. <4> A mutant sucrose synthase according to any one of <1> to <3>, wherein the amino acid residue corresponding to the 308th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is further replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. <5> A mutant sucrose synthase according to <4>, wherein the substituted amino acid is isoleucine or leucine. <6> A mutant sucrose synthase according to any one of <1> to <5>, wherein the amino acid residue corresponding to the 623rd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is further replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. <7> A mutant sucrose synthase according to <6>, wherein the substituted amino acid is an amino acid selected from the group consisting of leucine, histidine, arginine, valine, or tyrosine. <8> A nucleic acid encoding a mutant sucrose synthase according to any one of <1> to <7>. <9> An expression vector containing the nucleic acid according to <8>. A transformant containing the nucleic acid described in <10> and <8>.A method for producing mutant sucrose synthase, comprising culturing the transformant described in <11> and <10>, and recovering mutant sucrose synthase from the culture supernatant and / or culture. A composition comprising mutant sucrose synthase described in any one of <1> to <7>. A method for producing uridine diphosphate-glucose, comprising the steps of: carrying out an enzymatic reaction using mutant sucrose synthase described in any one of <1> to <7> to synthesize uridine diphosphate-glucose and fructose using sucrose and uridine diphosphate as substrates; and recovering uridine diphosphate-glucose, which is the reaction product of the enzymatic reaction. A method for producing a sugar compound, comprising the steps of: performing a first enzymatic reaction using a mutant sucrose synthase described in any one of <14>, <1> to <7> to synthesize uridine diphosphate-glucose and fructose using sucrose and uridine diphosphate as substrates; performing a second enzymatic reaction using a glycosyltransferase to synthesize a sugar compound using uridine diphosphate-glucose, the reaction product of the first enzymatic reaction, and a specific compound as substrates; and recovering the sugar compound, the reaction product of the second enzymatic reaction.

[0007] The mutant sucrose synthase of this disclosure can exhibit superior enzymatic activity compared to sucrose synthase having the amino acid sequence before mutation. In other words, this disclosure provides a mutant sucrose synthase with superior enzymatic activity, a nucleic acid encoding the mutant sucrose synthase, an expression vector containing the nucleic acid, a transformant containing the expression vector, a method for producing the mutant sucrose synthase, a composition containing the mutant sucrose synthase, a method for producing uridine diphosphate-glucose using the mutant sucrose synthase, and a method for producing sugar compounds using the mutant sucrose synthase.

[0008] This is a characteristic diagram showing the multiple alignments for the amino acid sequences of ZmSUS2 (SEQ ID NO: 1), DoSS1 (SEQ ID NO: 2), OsSUS2 (SEQ ID NO: 3), GmSuSy (SEQ ID NO: 4), StSUS4 (SEQ ID NO: 5), PsSUS2 (SEQ ID NO: 6), PsSUS3 (SEQ ID NO: 7), AtSUS1 (SEQ ID NO: 8), and AtSUS3 (SEQ ID NO: 9). This is a characteristic diagram showing the multiple alignments following Figure 1. This is a characteristic diagram showing the multiple alignments following Figure 2.

[0009] The embodiments of this disclosure are described below. The description is illustrative and does not limit the scope of this disclosure.

[0010] In the following embodiments, the components (including elemental steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values ​​and their ranges, and these do not limit the disclosure. For example, the disclosure allows for additions, omissions, substitutions, and changes to numbers, quantities, locations, ratios, materials, compositions, types, and sequences, etc., without departing from the intent of the disclosure.

[0011] In this disclosure, numerical ranges indicated using "~" include the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. If multiple types of the substance corresponding to each component exist in the composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified. The term "process" in this specification includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved.

[0012] In this disclosure, “gene” is not limited to protein-coding regions, but also includes, for example, expression regulatory regions, intron regions, and non-coding RNA (miRNA) of unknown function. In this disclosure, “DNA” and “RNA” are abbreviations for deoxyribonucleic acid and ribonucleic acid, respectively. In this disclosure, when “DNA” and “RNA” are mentioned, they mean molecules having multiple nucleotides in any form, including single-stranded, double-stranded, oligonucleotides, or polynucleotides. “Base sequence” and “nucleotide sequence” mean the order of nucleotides in single-stranded “DNA” and “RNA.”

[0013] [Mutant Sucrose Synthase] The mutant sucrose synthase of this disclosure is a mutant sucrose synthase that satisfies the following requirements (1) and / or (2) and has an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1. (1) The amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. (2) The amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution.

[0014] The mutant sucrose synthase of this disclosure is obtained by introducing a mutation that satisfies the requirements of (1) and / or (2) above into the pre-mutation sucrose synthase, and has higher enzyme activity compared to the pre-mutation sucrose synthase. Here, the enzyme activity of the mutant sucrose synthase and the pre-mutation sucrose synthase can be measured by the method described in the examples. That is, the sucrose synthase activity can be measured by adding the sucrose synthase to be evaluated to a reaction solution containing uridine diphosphate and sucrose as substrates, allowing the enzymatic reaction to proceed, and quantifying the uridine diphosphate-glucose as the reaction product. With respect to the mutant sucrose synthase of this disclosure, having high enzyme activity means that the sucrose synthase activity measured by the method described above is high in the enzyme solution obtained from the culture medium of cells that produce the mutant sucrose synthase.

[0015] Furthermore, the sucrose synthase into which the mutation is introduced may be wild-type sucrose synthase, or sucrose synthase having mutations other than those required in (1) and / or (2) above. Examples of sucrose synthases into which the mutation is introduced include wild-type sucrose synthases from various organisms, particularly wild-type sucrose synthases from plants. That is, the mutant sucrose synthase of this disclosure may be one in which a mutation requiring (1) and / or (2) above is introduced into a wild-type sucrose synthase and which has 70% or more identity with the amino acid sequence of SEQ ID NO: 1.

[0016] The sucrose synthase containing the amino acid sequence of SEQ ID NO: 1 is a wild-type sucrose synthase derived from maize (referred to as ZmSUS2). That is, the mutant sucrose synthase of this disclosure is one which has a mutation in the amino acid sequence of SEQ ID NO: 1 that satisfies the requirements of (1) and / or (2) above.

[0017] Furthermore, as a sucrose synthase to introduce mutations, we can mention a sucrose synthase having an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1. As an example of a sucrose synthase having an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1, we can mention one encoded by a homologous gene of ZmSUS2 derived from maize or a plant other than maize. Here, a homologous gene may be an ortholog, meaning a gene that is homologous between species, or a paralog, which is a multiple gene resulting from gene duplication. The value of identity between amino acid sequences can be calculated using the BLASTN or BLASTX program, which implement the BLAST algorithm (default setting). The value of identity is calculated by determining the number of amino acid residues that are perfectly identical when a pair of amino acid sequences are analyzed using pairwise alignment, and then calculating the percentage of the number of these amino acid residues among all the amino acid residues compared.

[0018] Furthermore, as a sucrose synthase to introduce mutations, we can mention sucrose synthases having an amino acid sequence that has more than 70% sequence similarity to the amino acid sequence of SEQ ID NO: 1. The sequence similarity value is calculated by determining the total number of amino acid residues that are perfectly identical when a pair of amino acid sequences are analyzed using pairwise alignment, and the total number of amino acid residues that are functionally similar in terms of physicochemical properties, and then calculating the percentage of this total number among all amino acid residues compared.

[0019] More specifically, sucrose synthases having an amino acid sequence that is 70% or more identical to the amino acid sequence of Sequence ID No. 1 include: sucrose synthase from Dendrobium officinale (hereinafter referred to as DoSS1), sucrose synthase from Oryza sativa (hereinafter referred to as OsSUS2), sucrose synthase from Glycine max (hereinafter referred to as GmSuSy), sucrose synthase from Solanum tuberosum (hereinafter referred to as StSUS4), and pea (Pisum) Examples include sucrose synthase 2 and sucrose synthase 3 derived from *Arabidopsis sativum* (hereinafter referred to as PsSUS2 and PsSUS3, respectively), and sucrose synthase 1 and sucrose synthase 3 derived from *Arabidopsis thaliana* (hereinafter referred to as AtSUS1 and AtSUS3, respectively).

[0020] DoSS1 has 82% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of DoSS1 is shown in SEQ ID NO: 2. OsSUS2 has 81% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of OsSUS2 is shown in SEQ ID NO: 3. GmSuSy has 77% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of GmSuSy is shown in SEQ ID NO: 4. StSUS4 has 75% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of StSUS4 is shown in SEQ ID NO: 5. PsSUS2 has 71% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of PsSUS2 is shown in SEQ ID NO: 6. PsSUS3 has 74% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of PsSUS3 is shown in SEQ ID NO: 7. AtSUS1 has 72% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of AtSUS1 is shown in SEQ ID NO: 8. AtSUS3 has 70% identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of AtSUS3 is shown in SEQ ID NO: 9.

[0021] Here, Figures 1 to 3 show the multiple alignments for the amino acid sequences of ZmSUS2 (SEQ ID NO: 1), DoSS1 (SEQ ID NO: 2), OsSUS2 (SEQ ID NO: 3), GmSuSy (SEQ ID NO: 4), StSUS4 (SEQ ID NO: 5), PsSUS2 (SEQ ID NO: 6), PsSUS3 (SEQ ID NO: 7), AtSUS1 (SEQ ID NO: 8), and AtSUS3 (SEQ ID NO: 9). The multiple alignments shown in Figures 1 to 3 are, from top to bottom, ZmSUS2 (the row labeled "Query" in Figures 1 to 3), DoSS1 (the row labeled "Query_182690" in Figures 1 to 3), OsSUS2 (the row labeled "Query_182691" in Figures 1 to 3), GmSuSy (the row labeled "Query_182686" in Figures 1 to 3), and StSUS4 (the row labeled "Query_182689" in Figures 1 to 3). PsSUS3 (row labeled "Query_182687" in Figures 1-3), AtSUS1 (row labeled "Query_182688" in Figures 1-3), PsSUS2 (row labeled "Query_182692" in Figures 1-3), and AtSUS3 (row labeled "Query_182693" in Figures 1-3) are shown.

[0022] Sucrose synthases containing any one of the amino acid sequences from SEQ ID NOs: 2 to 9, like sucrose synthases containing the amino acid sequence of SEQ ID NO: 1, will contain an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1, and will have improved activity compared to the pre-mutation version, by having a mutation that satisfies the requirements of (1) and / or (2) above.

[0023] [Mutation of Requirement (1)] Here, the amino acid residue to be substituted as defined in (1) above is the 348th amino acid residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1, but may be a different number in amino acid sequences other than SEQ ID NO: 1. In a sucrose synthase containing an amino acid sequence different from SEQ ID NO: 1, the amino acid residue to be substituted as defined in (1) above is referred to as the amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1. In a sucrose synthase containing an amino acid sequence different from SEQ ID NO: 1, the amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified by performing a pairwise alignment between the amino acid sequence different from SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 1. That is, by pairwise alignment, the amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified in an amino acid sequence different from SEQ ID NO: 1.

[0024] Specifically, based on the multiple alignments shown in Figures 1 to 3, the positions of the amino acid residues corresponding to the 348th position from the N-terminus in ZmSUS2 can be identified for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1, and AtSUS3. These positions are enclosed in a box labeled "(1)" in Figure 2.

[0025] More specifically, in DoSS1 containing the amino acid sequence of SEQ ID NO: 2, the amino acid residue to be substituted as defined in (1) above is located at position 345; in OsSUS2 containing the amino acid sequence of SEQ ID NO: 3, the amino acid residue to be substituted as defined in (1) above is located at position 340; in GmSuSy containing the amino acid sequence of SEQ ID NO: 4, the amino acid residue to be substituted as defined in (1) above is located at position 343; and in StSUS4 containing the amino acid sequence of SEQ ID NO: 5, the amino acid residue to be substituted as defined in (1) above is located at position 343. In PsSUS2 containing the amino acid sequence of SEQ ID NO: 6, the amino acid residue to be substituted as defined in (1) above is located at position 346; in PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, the amino acid residue to be substituted as defined in (1) above is located at position 341; in AtSUS1 containing the amino acid sequence of SEQ ID NO: 8, the amino acid residue to be substituted as defined in (1) above is located at position 345; and in AtSUS3 containing the amino acid sequence of SEQ ID NO: 9, the amino acid residue to be substituted as defined in (1) above is located at position 345.

[0026] Furthermore, in sucrose synthase (ZmSUS2) containing the amino acid sequence of SEQ ID NO: 1, the amino acid residue to be substituted as defined in (1) above is threonine, but in amino acid sequences different from SEQ ID NO: 1, it may be a different amino acid residue. In sucrose synthase containing an amino acid sequence different from SEQ ID NO: 1, the amino acid residue corresponding to the 348th threonine from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified by performing a pairwise alignment between the amino acid sequence different from SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 1. That is, by pairwise alignment, the amino acid residue corresponding to the 348th threonine from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified in an amino acid sequence different from SEQ ID NO: 1.

[0027] Specifically, based on the multiple alignments shown in Figures 1 to 3, the type of amino acid residue corresponding to the 348th position from the N-terminus in ZmSUS2 can be identified for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1, and AtSUS3 (the box labeled "(1)" in Figure 2).

[0028] More specifically, in DoSS1 containing the amino acid sequence of SEQ ID NO: 2, the amino acid residue to be substituted as defined in (1) above is valine; in OsSUS2 containing the amino acid sequence of SEQ ID NO: 3, the amino acid residue to be substituted as defined in (1) above is valine; in GmSuSy containing the amino acid sequence of SEQ ID NO: 4, the amino acid residue to be substituted as defined in (1) above is valine; in StSUS4 containing the amino acid sequence of SEQ ID NO: 5, the amino acid residue to be substituted as defined in (1) above is valine; in PsSUS2 containing the amino acid sequence of SEQ ID NO: 6, the amino acid residue to be substituted as defined in (1) above is lysine; in PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, the amino acid residue to be substituted as defined in (1) above is valine; in AtSUS1 containing the amino acid sequence of SEQ ID NO: 8, the amino acid residue to be substituted as defined in (1) above is valine; and in AtSUS3 containing the amino acid sequence of SEQ ID NO: 9, the amino acid residue to be substituted as defined in (1) above is lysine.

[0029] Furthermore, as specified in (1) above, the substituted amino acid residue is a different amino acid from the original amino acid, and the enzyme activity is improved compared to the original amino acid. The enzyme activity of sucrose synthase before and after the substitution mutation can be measured and quantitatively compared according to the method described above. Conventional known genetic engineering techniques can be used as appropriate for substituting a specific amino acid residue with another amino acid. In short, the base sequence of the wild-type gene encoding the protein to be mutated can be identified, and a mutation can be introduced to encode the substituted protein using a site-directed mutagenesis kit or the like. The mutated gene can then be recovered, for example, incorporated into an expression vector, according to standard procedures. Mutations in genes can be introduced using known methods such as the Kunkel method or the Gapped duplex method, or similar methods. For example, mutations can be introduced using site-directed mutagenesis kits (e.g., Mutant-K (TAKARA Bio) or Mutant-G (TAKARA Bio)), or using TAKARA Bio's LA PCR in vitro Mutagenesis series kits.

[0030] In particular, in (1) above, the substituted amino acid residue is preferably an amino acid selected from the group consisting of tyrosine, serine, histidine, leucine, aspartic acid, and glycine. By making the amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 an amino acid selected from the group consisting of tyrosine, serine, histidine, leucine, aspartic acid, and glycine, a mutant sucrose synthase with excellent activity can be obtained.

[0031] [Mutation in Requirement (2)] The amino acid residue to be substituted as specified in (2) above is the 618th amino acid residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1, but it may be a different number in amino acid sequences different from SEQ ID NO: 1. In sucrose synthase containing an amino acid sequence different from SEQ ID NO: 1, the amino acid residue to be substituted as specified in (2) above can be identified in the same way as in the case of (1) above.

[0032] Specifically, based on the multiple alignments shown in Figures 1 to 3, the positions of the amino acid residues corresponding to the 618th position from the N-terminus in ZmSUS2 can be identified for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1, and AtSUS3. These positions are enclosed in a box labeled "(2)" in Figure 3.

[0033] More specifically, in DoSS1 containing the amino acid sequence of SEQ ID NO: 2, the amino acid residue to be substituted as defined in (2) above is located at position 615; in OsSUS2 containing the amino acid sequence of SEQ ID NO: 3, the amino acid residue to be substituted as defined in (2) above is located at position 610; in GmSuSy containing the amino acid sequence of SEQ ID NO: 4, the amino acid residue to be substituted as defined in (2) above is located at position 613; and in StSUS4 containing the amino acid sequence of SEQ ID NO: 5, the amino acid residue to be substituted as defined in (2) above is located at position 613. In PsSUS2 containing the amino acid sequence of SEQ ID NO: 6, the amino acid residue to be substituted as defined in (2) above is located at position 617; in PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, the amino acid residue to be substituted as defined in (2) above is located at position 611; in AtSUS1 containing the amino acid sequence of SEQ ID NO: 8, the amino acid residue to be substituted as defined in (2) above is located at position 615; and in AtSUS3 containing the amino acid sequence of SEQ ID NO: 9, the amino acid residue to be substituted as defined in (2) above is located at position 616.

[0034] Furthermore, in sucrose synthase (ZmSUS2) containing the amino acid sequence of SEQ ID NO: 1, the amino acid residue to be substituted as defined in (2) above is asparagine, but in amino acid sequences different from SEQ ID NO: 1, it may be a different amino acid residue. In sucrose synthase containing an amino acid sequence different from SEQ ID NO: 1, the amino acid residue corresponding to the 618th asparagine from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified by performing a pairwise alignment between the amino acid sequence different from SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 1. That is, by pairwise alignment, the amino acid residue corresponding to the 618th asparagine from the N-terminus in the amino acid sequence of SEQ ID NO: 1 can be identified in an amino acid sequence different from SEQ ID NO: 1.

[0035] Specifically, based on the multiple alignments shown in Figures 1 to 3, the type of amino acid residue corresponding to the 618th position from the N-terminus in ZmSUS2 can be identified for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1, and AtSUS3 (the box labeled "(2)" in Figure 3).

[0036] More specifically, in DoSS1 containing the amino acid sequence of SEQ ID NO: 2, the amino acid residue to be substituted as specified in (2) above is lysine; in OsSUS2 containing the amino acid sequence of SEQ ID NO: 3, the amino acid residue to be substituted as specified in (2) above is asparagine; in GmSuSy containing the amino acid sequence of SEQ ID NO: 4, the amino acid residue to be substituted as specified in (2) above is lysine; in StSUS4 containing the amino acid sequence of SEQ ID NO: 5, the amino acid residue to be substituted as specified in (2) above is lysine; in PsSUS2 containing the amino acid sequence of SEQ ID NO: 6, the amino acid residue to be substituted as specified in (2) above is lysine; in PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, the amino acid residue to be substituted as specified in (2) above is lysine; in AtSUS1 containing the amino acid sequence of SEQ ID NO: 8, the amino acid residue to be substituted as specified in (2) above is lysine; and in AtSUS3 containing the amino acid sequence of SEQ ID NO: 9, the amino acid residue to be substituted as specified in (2) above is asparagine.

[0037] Furthermore, as specified in (2) above, the substituted amino acid residue is a different amino acid from the original amino acid, and the enzyme activity is improved compared to the original amino acid. The enzyme activity of sucrose synthase before and after the substitution mutation can be measured and quantitatively compared according to the method described above. Conventional known genetic engineering techniques can be used as appropriate for the method of substituting a specific amino acid residue with another amino acid. In short, the base sequence of the wild-type gene encoding the protein to be mutated can be identified, and a mutation can be introduced to encode the substituted protein using a site-directed mutagenesis kit or the like. The mutated gene can then be recovered, for example, incorporated into an expression vector, according to standard procedures. Mutations in genes can be introduced using known methods such as the Kunkel method or the Gapped duplex method, or similar methods. For example, mutations can be introduced using site-directed mutagenesis kits (e.g., Mutant-K (TAKARA Bio) or Mutant-G (TAKARA Bio)), or using TAKARA Bio's LA PCR in vitro Mutagenesis series kits.

[0038] In particular, in (2) above, the substituted amino acid residue is preferably an amino acid selected from the group consisting of serine, isoleucine, aspartic acid, glycine, and tyrosine. By making the amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 an amino acid selected from the group consisting of serine, isoleucine, aspartic acid, glycine, and tyrosine, a mutant sucrose synthase with excellent activity can be obtained.

[0039] [Other Mutations 1] The mutant sucrose synthase of this disclosure preferably has the following mutations in addition to the mutations specified in (1) and / or (2) above. For example, a mutation can be made in which the amino acid residue corresponding to the 308th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. Further improvement in activity can be achieved by mutating the amino acid residue corresponding to the 308th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 in addition to the mutations specified in (1) and / or (2) above.

[0040] Specifically, based on the multiple alignments shown in Figures 1 to 3, the positions of the amino acid residues corresponding to the 308th position from the N-terminus in ZmSUS2 can be identified for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1, and AtSUS3. These positions are enclosed in a box labeled "(3)" in Figure 2.

[0041] In ZmSUS2, which contains the amino acid sequence of SEQ ID NO: 1, the 308th amino acid residue from the N-terminus is valine. In DoSS1, which contains the amino acid sequence of SEQ ID NO: 2, the amino acid residue corresponding to position 308 is valine. In OsSUS2, which contains the amino acid sequence of SEQ ID NO: 3, the amino acid residue corresponding to position 308 is valine. In GmSuSy, which contains the amino acid sequence of SEQ ID NO: 4, the amino acid residue corresponding to position 308 is valine. In StSUS4, which contains the amino acid sequence of SEQ ID NO: 5, the amino acid residue corresponding to position 308 is valine. In PsSUS2, which contains the amino acid sequence of SEQ ID NO: 6, the amino acid residue corresponding to position 308 is valine. In PsSUS3, which contains the amino acid sequence of SEQ ID NO: 7, the amino acid residue corresponding to position 308 is isoleucine. In AtSUS1, which contains the amino acid sequence of SEQ ID NO: 8, the amino acid residue corresponding to position 308 is valine. In AtSUS3, which contains the amino acid sequence of Sequence ID No. 9, the amino acid residue corresponding to position 308 is valine.

[0042] Also, in this case as well, the enzyme activities of sucrose synthase before and after the substitution mutation can be measured according to the method described above and quantitatively compared. Also, the method of substituting a specific amino acid residue with another amino acid is the same as described above.

[0043] In particular, the amino acid residue corresponding to the 308th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 preferably mutates to isoleucine or leucine. However, in PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, since the amino acid residue corresponding to the 308th position is isoleucine, it is preferable to mutate the isoleucine to leucine.

[0044] [Other Mutation 2] The mutant sucrose synthase of the present disclosure preferably has the following mutations in addition to the mutations defined in (1) and / or (2) above. For example, a mutation can be mentioned in which the amino acid residue corresponding to the 623rd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than the amino acid residue and the enzyme activity is improved compared to before the substitution. In addition to the mutations defined in (1) and / or (2) above, by mutating the amino acid residue corresponding to the 623rd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1, further improvement in activity can be achieved.

[0045] Specifically, based on the multiple alignment shown in FIGS. 1 to 3, for DoSS1, OsSUS2, GmSuSy, StSUS4, PsSUS2, PsSUS3, AtSUS1 and AtSUS3, the position of the amino acid residue corresponding to the 623rd position from the N-terminus in ZmSUS2 can be specified. This position is surrounded by a frame marked with "(4)" in FIG. 3.

[0046] In ZmSUS2, which contains the amino acid sequence of SEQ ID NO: 1, the 623rd amino acid residue from the N-terminus is lysine. In DoSS1, which contains the amino acid sequence of SEQ ID NO: 2, the amino acid residue corresponding to the 623rd position is leucine. In OsSUS2, which contains the amino acid sequence of SEQ ID NO: 3, the amino acid residue corresponding to the 623rd position is arginine. In GmSuSy, which contains the amino acid sequence of SEQ ID NO: 4, the amino acid residue corresponding to the 623rd position is leucine. In StSUS4, which contains the amino acid sequence of SEQ ID NO: 5, the amino acid residue corresponding to the 623rd position is leucine. In PsSUS2, which contains the amino acid sequence of SEQ ID NO: 6, the amino acid residue corresponding to the 623rd position is arginine. In PsSUS3, which contains the amino acid sequence of SEQ ID NO: 7, the amino acid residue corresponding to the 623rd position is leucine. In AtSUS1, which contains the amino acid sequence of SEQ ID NO: 8, the amino acid residue corresponding to the 623rd position is asparagine. In AtSUS3, which contains the amino acid sequence of SEQ ID NO: 9, the amino acid residue corresponding to position 623 is arginine.

[0047] In this case as well, the enzyme activity of sucrose synthase before and after the substitution mutation can be measured and quantitatively compared according to the method described above. The method for substituting a specific amino acid residue with another amino acid is also the same as described above.

[0048] In particular, the amino acid residue corresponding to the 623rd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 preferably mutates to leucine, histidine, arginine, valine or tyrosine. However, in DoSS1 containing the amino acid sequence of SEQ ID NO: 2, GmSuSy containing the amino acid sequence of SEQ ID NO: 4, StSUS4 containing the amino acid sequence of SEQ ID NO: 5, and PsSUS3 containing the amino acid sequence of SEQ ID NO: 7, since the amino acid residue corresponding to the 623rd position is leucine, it is preferable to mutate the leucine to histidine, arginine, valine or tyrosine. Further, in OsSUS2 containing the amino acid sequence of SEQ ID NO: 3, PsSUS2 containing the amino acid sequence of SEQ ID NO: 6, and AtSUS3 containing the amino acid sequence of SEQ ID NO: 9, since the amino acid residue corresponding to the 623rd position is arginine, it is preferable to mutate the arginine to leucine, histidine, valine or tyrosine.

[0049] [Nucleic acid encoding mutant sucrose synthase] The nucleic acid encoding the mutant sucrose synthase of the present disclosure encodes the mutant sucrose synthase described above. Nucleic acid means a nucleic acid including naturally occurring nucleic acids such as DNA and RNA, artificial nucleic acids such as nucleic acid molecules obtained by chemically modifying PNA (peptide nucleic acid), bases, sugars, phosphodiester moieties, etc. Further, the nucleic acid encoding the mutant sucrose synthase means that it includes both a region including an expression control region and a coding region, and a region consisting only of a coding region.

[0050] The base sequence of the nucleic acid encoding the mutant sucrose synthase of the present disclosure can be appropriately designed based on the amino acid sequence of the mutant sucrose synthase described above. At this time, the codons suitable for the host cell to be used are considered in the base sequence to be designed, and the degeneracy of the codons is also considered. The base sequence of the nucleic acid encoding the mutant sucrose synthase can be designed, for example, from the base sequence of the nucleic acid encoding the wild-type sucrose synthase.

[0051] As an example, Sequence ID No. 10 shows an example of a nucleotide sequence design that encodes ZmSUS2, including the amino acid sequence of Sequence ID No. 1. The nucleotide sequence of the nucleic acid encoding mutant sucrose synthase can be designed by changing the codons that encode the amino acids to be substituted in the nucleotide sequence of Sequence ID No. 10. Specifically, the codon encoding the 348th amino acid in the amino acid sequence of Sequence ID No. 1 corresponds to positions 1042 to 1044 in the nucleotide sequence of Sequence ID No. 1. The codon encoding the 618th amino acid in the amino acid sequence of Sequence ID No. 1 corresponds to positions 1852 to 1854 in the nucleotide sequence of Sequence ID No. 1. The codon encoding the 308th amino acid in the amino acid sequence of Sequence ID No. 1 corresponds to positions 922 to 924 in the nucleotide sequence of Sequence ID No. 1. The codon encoding the 623rd amino acid in the amino acid sequence of Sequence ID No. 1 corresponds to positions 1867 to 1869 in the nucleotide sequence of Sequence ID No. 1. Then, by designing these codons to encode the mutated amino acids, the nucleotide sequence of the nucleic acid encoding mutant sucrose synthase can be determined.

[0052] [Expression Vector] The expression vector of this disclosure contains a nucleic acid encoding the mutant sucrose synthase of this disclosure. Preferably, the expression vector of this disclosure is capable of autonomous replication in host cells as described later. Preferably, the expression vector contains a promoter, a ribosome binding sequence, and a transcription termination sequence for expressing the nucleic acid encoding the mutant sucrose synthase. The expression vector may also contain a gene that controls promoter activity. Any promoter that can be expressed in a host such as E. coli may be used. For example, E. coli-derived promoters such as the trp promoter, lac promoter, PL promoter, and PR promoter, or phage-derived promoters such as the T7 promoter can be used. Furthermore, artificially designed and modified promoters such as the tac promoter may also be used.

[0053] Furthermore, to construct expression vectors, vectors such as M13 phage or its variants, λ phage or its variants, pBR322 or its variants (pB325, pAT153, pUC8, etc.) can be used as E. coli host vectors; pYepSec1, pMFa, pYES2, etc. can be used as yeast host vectors; pAc, pVL, etc. can be used as insect cell host vectors; and pCDM8, pMT2PC, etc. can be used as mammalian cell host vectors.

[0054] An expression vector can be constructed by inserting the nucleic acid of this disclosure into the vector described above. In this case, the insertion of a selected marker gene, the insertion of a promoter, etc., can be performed by referring to standard recombinant DNA techniques (e.g., Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

[0055] [Transformed organisms] The transformed organisms of the present disclosure include a nucleic acid encoding the mutant sucrose synthase of the present disclosure as described above. That is, the transformed organisms of the present disclosure are obtained by introducing the nucleic acid encoding the mutant sucrose synthase of the present disclosure into a suitable host cell. An example of a method for introducing the nucleic acid encoding the mutant sucrose synthase of the present disclosure into a host cell is to introduce the expression vector described above into the host cell.

[0056] Here, as host cells, microorganisms capable of expressing nucleic acids encoding mutant sucrose synthase can be used. Examples of usable microorganisms include bacteria belonging to the Escherichia genus, such as Escherichia coli; Corynebacterium glutamicum, such as Corynebacterium glutamicum; Bacillus, such as Bacillus subtilis; Pseudomonas, such as Pseudomonas putida; and Rhizobium, such as Rhizobium meliloti. Furthermore, examples of usable microorganisms include yeasts of the genus Candida such as Candida Shehatae, yeasts of the genus Pichia such as Pichia stipitis, yeasts of the genus Pachysolen such as Pachysolen tannophilus, yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe, with Saccharomyces cerevisiae being particularly preferred.

[0057] The method for introducing an expression vector is not particularly limited as long as it involves introducing DNA into the host cell. Examples include methods using calcium ions [Cohen, S. N., et al.: Proc. Natl. Acad. Sci., USA, 69:2110-2114 (1972)] and electroporation.

[0058] [Method for Producing Mutant Sucrose Synthase] The method for producing mutant sucrose synthase according to this disclosure is a method of culturing the transformant described above and recovering mutant sucrose synthase from the culture supernatant and / or culture. The step of culturing the transformant can be carried out by inoculating the transformant into a suitable medium and preparing the culture conditions in accordance with conventional methods. The culture conditions should be selected taking into account the nutritional and physiological properties of the host cell. The medium used should preferably contain nutrients that the host cell can utilize and that enable efficient protein expression in the transformant. Therefore, it is preferable that the medium contains a carbon source, a nitrogen source and other essential nutrients necessary for the growth of the host cell, and it does not matter whether it is a natural medium or a synthetic medium.

[0059] For example, the culture medium can use saccharified saccharified liquid, molasses, dextran, starch, etc. as a carbon source, and ammonium salts, nitrates, amino acids, peptones, casein, etc. as a nitrogen source. Other nutrients that may be included as desired include inorganic salts, vitamins, antibiotics, etc. When the host cell is E. coli, LB medium, M9 medium, etc., can be suitably used. There are no particular restrictions on the culture form, but liquid medium is suitably used from the viewpoint of large-scale culture.

[0060] In the recovery process, mutant sucrose synthase is recovered from the culture of the transformants obtained in the culture process described above. The recovered mutant sucrose synthase may be crude enzyme solution or isolated and purified. Specifically, the culture of the transformants is recovered by means of centrifugation, filtration, etc., and then lysed or disrupted by enzymatic disruption methods such as lysozyme treatment, or physical disruption methods such as sonication, freeze-thaw cycles, or osmotic shock. Subsequently, the culture supernatant obtained by centrifugation can be obtained as crude enzyme solution. Furthermore, the solubilized fraction of the obtained crude enzyme solution can be collected by means of filtration, etc., and the mutant sucrose synthase can be isolated and purified by known protein purification methods, such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, hydrophobicity, anion chromatography, cation chromatography, hydroxyapatite chromatography, etc., either alone or in appropriate combinations.

[0061] [Composition] The composition of the present disclosure contains the mutant sucrose synthase described above. The composition of the present disclosure is not particularly limited, but can be in a form used for the production of substances using mutant sucrose synthase. In this case, the composition of the present disclosure may be a mixture of mutant sucrose synthase and a buffer solution with a pH suitable for mutant sucrose synthase. In addition, the mutant sucrose synthase in the composition of the present disclosure may be isolated and purified, or it may be a crude enzyme solution. Furthermore, the composition of the present disclosure may contain some of the substrates used in the enzymatic reaction by mutant sucrose synthase. For example, mutant sucrose synthase uses uridine diphosphate and sucrose as substrates to produce uridine diphosphate-glucose and fructose through an enzymatic reaction. Therefore, the composition of the present disclosure may be a mixture of mutant sucrose synthase and either uridine diphosphate or sucrose.

[0062] Furthermore, the compositions of this disclosure may also include mutant sucrose synthase and other enzymes that utilize the reaction product of the mutant sucrose synthase as a substrate. In this case, the other enzyme may be a glycosyltransferase that uses uridine diphosphate-glucose, which is the reaction product of mutant sucrose synthase, as a substrate. That is, an example of a composition of this disclosure is a composition that includes mutant sucrose synthase and a glycosyltransferase. Other examples include uridine diphosphate-glucose dehydrogenase, uridine diphosphate-glucose pyrophosphorylase, uridine diphosphate-glucose pyrophosphatase, uridine diphosphate-glucose-hexose-1-phosphate uridylyltransferase, and uridine diphosphate-galactose-4-epimerase.

[0063] [Method for Producing Uridine Pyrophosphate-Glucose] Uridine pyrophosphate-glucose can be produced by utilizing the mutant sucrose synthase of the present disclosure. The mutant sucrose synthase of the present disclosure has particularly excellent enzymatic activity, and therefore, by using it in the method for producing uridine pyrophosphate-glucose, the productivity of uridine pyrophosphate-glucose can be improved. The method for producing uridine pyrophosphate-glucose of the present disclosure comprises the steps of carrying out an enzymatic reaction using the mutant sucrose synthase of the present disclosure described above, using sucrose and uridine pyrophosphate as substrates to synthesize uridine pyrophosphate-glucose and fructose, and recovering the uridine pyrophosphate-glucose, which is the reaction product of the enzymatic reaction.

[0064] [Method for Producing Sugar Compounds] Sugar compounds can be produced using the mutant sucrose synthase of this disclosure and the uridine diphosphate-glucose produced by the mutant sucrose synthase. Glycosyltransferase can be used for the synthesis of sugar compounds using uridine diphosphate-glucose. Specifically, the method for producing sugar compounds of this disclosure includes the steps of: a first enzymatic reaction in which uridine diphosphate-glucose and fructose are synthesized using sucrose and uridine diphosphate as substrates with the mutant sucrose synthase of this disclosure described above; a second enzymatic reaction in which a sugar compound is synthesized using uridine diphosphate-glucose, the reaction product of the first enzymatic reaction, and a specific compound as substrates with a glycosyltransferase; and a step of recovering the sugar compound, which is the reaction product of the second enzymatic reaction.

[0065] The method for producing the sugar compound described herein is not particularly limited, but can be applied to systems such as a system for producing arbutin as a sugar compound using hydroquinone as a specific compound, a system for producing vanillin glucoside as a sugar compound using vanillin as a specific compound, a system for producing menthol glucoside as a sugar compound using menthol as a specific compound, and a system for producing rebaudioside A using steviol glycosides. Furthermore, specific examples of glycosyltransferases used in this production method include UGT72B1 derived from Arabidopsis thaliana, UGT72B11 derived from Pilosella officinarum, UGT76G1 derived from Stevia rebaudiana, and yjiC derived from Bacillus subtilis.

[0066] The present disclosure will be described in more detail below with reference to examples, but the technical scope of the present disclosure is not limited to the following examples.

[0067] 1. Obtaining Wild-Type Sucrose Synthase <Comparative Example 1: Obtaining Soybean-Derived Wild-Type Sucrose Synthase (GmSuSy)> As Comparative Example 1, the codons of soybean-derived wild-type sucrose synthase were optimized, and a synthetic gene encoding soybean-derived wild-type sucrose synthase with the optimized codons was obtained. Using the pET28a vector system cloned from this synthetic gene, competent cells of Escherichia coli strain BL21 (DE3) were transformed, seeded on LB agar medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C. The proliferated colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight with shaking at 37°C and 200 rpm. A portion of the culture solution was inoculated into 0.3 L of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm. After the optical density at 600 nm (OD600) reached 0.6–0.8, isopropyl-β-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, and the mixture was incubated with shaking at 25°C and 200 rpm for 18 hours. The supernatant was discarded after centrifugation (4°C, 3000 G, 20 min).

[0068] The obtained cell pellet was suspended in 10 mL of lysis buffer (20 mM HEPES buffer (pH 7.5), 300 mM sodium chloride solution) and processed on ice using a homogenizer (BRANSON SONIFER). The processed solution was centrifuged (4°C, 3000 G, 30 min) and the cell debris was discarded. The obtained supernatant was purified with a His tag, ultrafiltered, and replaced with 50 mM HEPES buffer (pH 7.5) to obtain a purified enzyme solution containing wild-type sucrose synthase derived from soybeans. The amino acid sequence of wild-type sucrose synthase derived from soybeans (GmSuSy) is shown in Sequence ID No. 4.

[0069] <Comparative Example 2: Obtaining Wild-Type Sucrose Synthase (AtSUS1) Derived from Arabidopsis thaliana> As Comparative Example 2, a composition containing wild-type sucrose synthase (AtSUS1) derived from Arabidopsis thaliana was obtained in accordance with the method described in Comparative Example 1. The amino acid sequence of wild-type sucrose synthase (AtSUS1) derived from Arabidopsis thaliana is shown in Sequence ID No. 4.

[0070] <Comparative Example 3: Obtaining Wild-Type Sucrose Synthase (ZmSUS2) Derived from Maize> As Comparative Example 3, a composition containing wild-type sucrose synthase (ZmSUS2) derived from maize was obtained in accordance with the method described in Comparative Example 1. The amino acid sequence of the wild-type sucrose synthase derived from maize is shown in SEQ ID NO: 1.

[0071] 2. Obtaining Mutant Sucrose Synthase <Examples 1-19: Obtaining Mutant Sucrose Synthase with Mutations 1-4> The nucleic acid encoding ZmSUS2 shown in Sequence ID No. 1 was optimized for codons, and the 308th, 348th, 618th, and 623rd positions from the N-terminus were used as mutation sites. Site-saturated mutations were introduced by PCR. Competent cells of Escherichia coli strain BL21 (DE3) were transformed using the pET28a vector system, which contained the synthesized gene into which the mutations were introduced. The cells were seeded on LB agar medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. The resulting colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight with shaking at 37°C and 1050 rpm. A portion of the culture medium was inoculated into 800 μL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 1050 rpm. After the optical density at 600 nm (OD600) reached 0.6–0.8, isopropyl-β-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, and the culture was incubated with shaking at 25°C and 1050 rpm for 18 hours. The supernatant was discarded after centrifugation (4°C, 1800 G, 30 min).

[0072] The obtained cell pellet was suspended in 250 μL of lysis 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) and shaken at 25°C and 1050 rpm for 2 hours. The treated solution was centrifuged (4°C, 1800 G, 30 min), and the supernatant was used as a crude enzyme solution to obtain a composition containing mutant sucrose synthase.

[0073] 3. Comparison of Sucrose Synthase Activity [Evaluation of Activity Extracted from Culture Medium] The activity extractable from the culture medium when uridine diphosphate was used as a substrate was evaluated according to the specifications shown below. First, 20 μL of the obtained crude enzyme solution, a 2 mM aqueous solution of uridine diphosphate, and a 100 mM sucrose solution were reacted in 20 mM HEPES buffer (pH 6.5) by stirring at 40°C for 30 minutes. Then, 150 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 min).

[0074] The supernatant was analyzed using high-performance liquid chromatography (HPLC) to quantitatively determine the amount of the target product, uridine diphosphate glucose. The HPLC analysis conditions were as follows: -HPLC Analysis Conditions- Instrument: Nexera X2 (Shimadzu Corporation) Column: Imtakt Scherzo SS-C18, 3 μm, 2.0 × 50 mm Mobile phase: A: 5 mM phosphate buffer (pH 6.8) / acetonitrile = 95 / 5, B: 50 mM phosphate buffer (pH 6.8) / acetonitrile = 50 / 50 Time (min) / %B: 0 / 0, 1.0 / 0, 4.0 / 60, 4.1 / 100, 6.5 / 100, 6.6 / 0, 9.0 / 0 Injection volume: 1 μL Column temperature: 40°C Flow rate: 0.5 mL / min Detection wavelength: 280 nm Retention time: Uridine diphosphate glucose 0.77 min, Uridine diphosphate 3.03 min

[0075] The amount of uridine diphosphate glucose produced was quantified by HPLC, and the activity ratio of the mutant sucrose synthase was calculated, with the activity of wild-type sucrose synthase derived from maize (ZmSUS2: Comparative Example 3) that could be isolated from the culture medium set to 1.0. Table 1 summarizes the mutant sucrose synthases whose activity exceeded 1.0 when isolated from the culture medium in this experiment.

[0076]

[0077] In Table 1, the items [Mutation 1 T348], [Mutation 2 N618], [Mutation 3 V308] to [Mutation 4 K623] refer to the 308th, 348th, 618th, and 623rd amino acid residues from the N-terminus, respectively, which were introduced as mutation sites. The capital letter to the left of the number represents the single-letter designation of the wild-type amino acid. For example, the item [Mutation 1 T348] in Table 1 refers to a mutation in the threonine residue (T), which is the 348th amino acid residue from the N-terminus in the amino acid sequence represented by Sequence ID No. 1. The capital letters to the right of Examples 1 to 19 in Table 1 represent the substituted amino acid (single-letter designation). In Table 1, blank spaces indicate that the amino acid residue for each item has not mutated from the amino acid residue represented by Sequence ID No. 1.

[0078] As shown in Table 1, mutant sucrose synthases with improved activity were obtained by introducing mutations at positions 308, 348, 618, and 623 from the N-terminus of ZmSUS2. In particular, mutant sucrose synthases in which the valine at position 308 from the N-terminus of ZmSUS2 was replaced with isoleucine or leucine showed excellent activity. Furthermore, mutant sucrose synthases in which the threonine at position 348 from the N-terminus of ZmSUS2 was replaced with an amino acid selected from the group consisting of tyrosine, serine, histidine, leucine, aspartic acid, and glycine showed excellent activity. In addition, mutant sucrose synthases in which the asparagine at position 618 from the N-terminus of ZmSUS2 was replaced with an amino acid selected from the group consisting of serine, isoleucine, aspartic acid, glycine, and tyrosine showed excellent activity. Furthermore, it was found that mutant sucrose synthase in which the 623rd lysine position from the N-terminus of ZmSUS2 was replaced with an amino acid selected from the group consisting of leucine, histidine, arginine, valine, or tyrosine exhibited superior activity.

[0079] [Evaluation of Titer] For the mutant sucrose synthases of Examples 1 to 7, purified enzyme solutions were prepared according to the method described in Comparative Example 1. Then, for the purified enzyme solutions of Comparative Examples 1 to 3 and Examples 1 to 7, the amount of enzyme (amount of wild-type sucrose synthase or mutant sucrose synthase) was quantified from the concentration of the purified enzyme solution based on the absorbance at 280 nm measured by a spectrophotometer. Subsequently, the enzyme dilution was developed by SDS-PAGE electrophoresis, and after CBB staining with CBB (Coomassie Brilliant Blue) staining solution, the purity of the band containing the target protein (band corresponding to wild-type sucrose synthase or mutant sucrose synthase) was quantified by image analysis. From the obtained amount of enzyme and culture medium volume, the titer was calculated based on the following formula, and the results of evaluation according to the following criteria are shown in Table 2.

[0080] Enzyme amount (mg) = Enzyme solution concentration (mg / L) × Enzyme solution volume (L) × (Purity (%) / 100) Potency (mg / L) = Enzyme amount (mg) / Culture medium volume (L)

[0081] -Evaluation Criteria- S: Potency of 15 mg / L or higher A: Potency of 10 mg / L or higher but less than 15 mg / L B: Potency of 5 mg / L or higher but less than 10 mg / L C: Potency of less than 5 mg / L

[0082]

[0083] In addition, the right-hand columns for Comparative Examples 1 and 2 in Table 2 indicate the types of amino acids corresponding to positions 348, 618, 308, and 623 in wild-type sucrose synthase derived from maize (ZmSUS2). As shown in Table 2, wild-type ZmSUS2 and the mutant sucrose synthases of Examples 1 to 7 were found to have superior titers compared to GmSuSy in Comparative Example 1 and AtSUS1 in Comparative Example 2.

[0084] The disclosure of Japanese Patent Application No. 2024-164193, filed on September 20, 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A mutant sucrose synthase that satisfies the following requirements (1) and / or (2), and has an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO:

1. (1) The amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution. (2) The amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution.

2. The mutant sucrose synthase according to claim 1, wherein the substituted amino acid in (1) above is an amino acid selected from the group consisting of tyrosine, serine, histidine, leucine, aspartic acid, and glycine.

3. The mutant sucrose synthase according to claim 1, wherein the substituted amino acid in (2) above is an amino acid selected from the group consisting of serine, isoleucine, aspartic acid, glycine, and tyrosine.

4. The mutant sucrose synthase according to claim 1, wherein the amino acid residue corresponding to the 308th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution.

5. The mutant sucrose synthase according to claim 4, wherein the substituted amino acid is isoleucine or leucine.

6. The mutant sucrose synthase according to claim 1, wherein the amino acid residue corresponding to the 623rd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the said amino acid residue that improves enzyme activity compared to before the substitution.

7. The mutant sucrose synthase according to claim 6, wherein the substituted amino acid is an amino acid selected from the group consisting of leucine, histidine, arginine, valine, or tyrosine.

8. A nucleic acid encoding the mutant sucrose synthase described in claim 1.

9. An expression vector comprising the nucleic acid described in claim 8.

10. A transformant comprising the nucleic acid described in claim 8.

11. A method for producing mutant sucrose synthase, comprising culturing the transformant described in claim 10 and recovering mutant sucrose synthase from the culture supernatant and / or culture.

12. A composition comprising the mutant sucrose synthase described in claim 1.

13. A method for producing uridine diphosphate-glucose, comprising the steps of: carrying out an enzymatic reaction using the mutant sucrose synthase described in claim 1 to synthesize uridine diphosphate-glucose and fructose using sucrose and uridine diphosphate as substrates; and recovering the uridine diphosphate-glucose, which is the reaction product of the enzymatic reaction.

14. A method for producing a sugar compound, comprising: a first enzymatic reaction in which sucrose and uridine diphosphate are used as substrates to synthesize uridine diphosphate-glucose and fructose using the mutant sucrose synthase described in claim 1; a second enzymatic reaction in which a sugar compound is synthesized using uridine diphosphate-glucose, which is the reaction product of the first enzymatic reaction, and a specific compound as substrates using a glycosyltransferase; and a step of recovering the sugar compound, which is the reaction product of the second enzymatic reaction.

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