Polypeptide aggregation capacity evaluation method and vector used in the method

Abstract

To provide a polypeptide aggregation capacity evaluation method and a vector used in the method.SOLUTION: A polypeptide aggregation capacity evaluation method comprises measuring an amount of deoxyviolacein and / or violacein in a cell that contains a vector in which nucleotide sequences encoding a vioC protein and a vioE protein, to which a nucleotide sequence encoding a polypeptide to be evaluated is added, are included in the same vector or in different vectors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for evaluating the aggregation ability of a polypeptide and a vector used in the method, etc. Specifically, the present disclosure relates to a method for evaluating the aggregation ability of a polypeptide, etc., which comprises measuring the amount of deoxyviolacein and / or violacein in cells containing vectors containing nucleotide sequences encoding vioC protein and vioE protein, to which nucleotide sequences encoding a polypeptide to be evaluated have been added, either in the same or different vectors. [Background technology]

[0002] It is known that in yeast, glycolytic enzymes and the like form protein aggregates called G-bodies (glycolytic bodies) under hypoxic conditions (Non-Patent Document 1). For example, it has been reported that the N-terminal amino acid sequence (scENO(1-30)) of the glycolytic enzyme enolase (Eno2p) contributes to the formation of G-bodies under hypoxic conditions (Non-Patent Document 2). It has also been reported that the amino acid sequence of yeast pyruvate kinase (Cdc19p) (e.g., the amino acid sequence from positions 217 to 243; SC3) has the ability to self-assemble (Patent Document 1, Non-Patent Document 3). The amino acid sequences involved in the assembly of these metabolic enzymes are called "MetaFos-tags" (META (metabolic enzymes transiently assembling) body-forming peptide sequence tags)

[0003] The formation of metabolic enzyme aggregates is said to have the following effects: it can improve metabolic efficiency by suppressing the amount of intermediates present; it can prevent reactant intermediates from being used in competing reactions; it can protect unstable intermediates; it can shorten the apparent reaction time by reducing the waiting time between successive enzymatic reactions (Non-Patent Document 4). For example, there have been reports using a polypeptide called FUS (Fused in Sarcoma), a human-derived RNA-binding protein known to have the property of forming aggregates within cells (Non-Patent Document 5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-23225 [Non-patent literature]

[0005] [Non-Patent Document 1] Cell Rep. 2017 Jul 25;20(4):895-908. [Non-patent document 2] Eukaryot Cell. 2013 Aug;12(8):1106-19. [Non-patent document 3] PLoS One.2023 Apr13;18(4):e0283002. [Non-patent document 4] Microorganisms. 2022 Jan 21;10(2):232. [Non-Patent Document 5] Nat Chem Biol. 2019 Jun;15(6):589-597. Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present disclosure is to provide a method for evaluating the aggregation ability of a polypeptide and a vector to be used in the method. [Means for solving the problem]

[0007] The present inventors discovered that the aggregation-forming ability of a polypeptide to be evaluated can be evaluated by adding the polypeptide to be evaluated to proteins vioC and vioE, which are involved in the biosynthesis of violacein, and have made further improvements.

[0008] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. A method for evaluating the aggregation ability of a polypeptide, comprising measuring the amount of deoxyviolacein and / or violacein in cells containing vectors containing base sequences encoding the vioC protein and the vioE protein, to which a base sequence encoding the polypeptide to be evaluated has been added, either in the same or different vectors. Section 2. Item 10. The method according to Item 1, wherein the cell contains a vector containing a nucleotide sequence encoding the vioA protein and / or the vioB protein in the same or a different vector as the vector. Section 3. The cells A vector comprising a base sequence encoding the vioC protein and the vioE protein to which a base sequence encoding a polypeptide to be evaluated has been added; and Item 1. The method according to Item 1, wherein the vector comprises a nucleotide sequence encoding the vioA protein and the vioB protein. Section 4. Item 4. The method according to any one of Items 1 to 3, wherein the nucleotide sequence encoding the polypeptide to be evaluated is added to the 5'-end side of the nucleotide sequence encoding the vioC protein and the vioE protein. Section 5. Item 4. The method according to any one of Items 1 to 3, wherein the vector is a plasmid vector. Section 6. Item 4. The method according to any one of Items 1 to 3, wherein the vector further comprises a nucleotide sequence encoding a vioD protein. Section 7. A vector for use in the method according to any one of Items 1 to 3. Section 8. A method for producing a useful substance, comprising culturing cells containing, on the same or different vectors, vectors containing a base sequence encoding two or more enzymes to which a base sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 13; or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 13 and which has the ability to form aggregates has been added. [Effects of the Invention]

[0009] A method for evaluating the aggregation ability of a polypeptide is provided, as well as a vector for use in the method. [Brief explanation of the drawings]

[0010] [Figure 1] A schematic diagram of the violacein biosynthetic pathway is shown. [Figure 2] The structure of the plasmid used in Experimental Example 1 is shown below. [Figure 3] The results of microscopic observation are shown (scale bar = 10 μm). [Figure 4] The results of measuring the amounts of violacein and deoxyviolacein produced by HPLC analysis are shown (n=3, bar=mean±SD, t-test, *P<0.05, NS: not significant). [Figure 5] The results show the amount of deoxyviolacein produced when the amount of violacein produced is set to 1 (n=3, bar=mean±SD, t-test, *P<0.05). [Figure 6] The structure of the plasmid used in Experimental Example 2 is shown below. [Figure 7] The results of microscopic observation are shown (scale bar = 10 μm). [Figure 8]The results of measuring the amount of deoxyviolacein produced by HPLC analysis are shown (n=3, bar=mean±SD, Tukey test, NS: no significant difference, P<0.05 is indicated by different letters). [Figure 9] The results show the amount of deoxyviolacein produced, with the amount of violacein produced being set at 1 (n=3, bar=mean±SD, Tukey-test, P<0.05 is indicated by different letters). DETAILED DESCRIPTION OF THE INVENTION

[0011] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a method for evaluating the aggregation ability of a polypeptide, the method comprising measuring the amount of deoxyviolacein and / or violacein in cells containing vectors containing nucleotide sequences encoding the vioC protein and the vioE protein, to which nucleotide sequences encoding the polypeptides to be evaluated have been added, either in the same or different vectors. This method may also be referred to as the "evaluation method of the present disclosure."

[0012] The biosynthetic pathway of the anticancer pigment violacein is shown in Figure 1. The vioA protein is an enzyme that metabolizes L-tryptophan to indole-3-pyruvic acid imine (IPA-imine). The vioB protein is an enzyme that dimerizes IPA-imine (to produce an IPA-imine dimer). The vioE protein is an enzyme that metabolizes IPA-imine dimer to protodeoxyviolaceic acid. The vioD protein is an enzyme that metabolizes protodeoxyviolaceic acid to protoviolaceic acid. The vioC protein is an enzyme that metabolizes protodeoxyviolaceic acid to deoxyviolacein or protoviolaceic acid to violacein. In this specification, the vioA protein, vioB protein, vioC protein, vioD protein, and vioE protein may be collectively referred to as "vio proteins."

[0013] The vio protein used in the present disclosure may be wild-type or may have a mutation as long as the protein functions. Whether the protein has a function can be confirmed by known methods. For example, if the expected enzyme reaction product is produced when the protein is contacted with a substrate, the protein can be determined to have a function. Examples of such mutations include conservative substitutions.

[0014] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with an amino acid having a side chain with similar properties to that of the amino acid. Specific examples of conservative substitution include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine; etc.

[0015] The vio protein can be, for example, a vio protein derived from a microorganism that biosynthesizes violacein. Examples of microorganisms that biosynthesize violacein include the genera Chromobacterium, Duganella, Pseudoalteromonas, Janthinobacterium, Iodobacter, Rugamonas, and Massilia. Among these, the genus Pseudoalteromonas is preferred.

[0016] SEQ ID NO: 3 is the amino acid sequence constituting the vioA protein derived from Pseudoalteromonas luteoviolacea. For example, the vioA protein used in the present disclosure may be a polypeptide having the amino acid sequence of SEQ ID NO: 3 in which one or more amino acids have been deleted, substituted, or added. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence of SEQ ID NO: 3 may be, for example, 65, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0017] Techniques for introducing mutations such as deletion, substitution, or addition of amino acids into a specific amino acid sequence are known in the art and can be performed using any method, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, or random mutagenesis.

[0018] The vioA protein used in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence set forth in SEQ ID NO: 3. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0019] Amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / using default parameters.

[0020] SEQ ID NO: 4 is a nucleotide sequence encoding the vioA protein derived from Pseudoalteromonas luteoviolacea. For example, the nucleotide sequence encoding the vioA protein used in the present disclosure is exemplified by a nucleotide sequence showing 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 4. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0021] Nucleotide sequence identity can be calculated using commercially available analytical tools or those available via telecommunications lines (Internet), such as software such as FASTA, BLAST, PSI-BLAST, and SSEARCH. Specifically, the main initial conditions generally used for BLAST searches are as follows: In Advanced BLAST 2.1, the blastn program is used with various parameters set to default values ​​to perform a search, and the nucleotide sequence identity value (%) can be calculated.

[0022] SEQ ID NO: 5 is the amino acid sequence constituting the vioB protein derived from Pseudoalteromonas luteoviolacea. For example, the vioB protein used in the present disclosure may be a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 5. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence of SEQ ID NO: 5 may be, for example, 150, 100, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0023] The vioB protein used in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence set forth in SEQ ID NO: 5. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0024] SEQ ID NO: 6 is a nucleotide sequence encoding the vioB protein derived from Pseudoalteromonas luteoviolacea. For example, the nucleotide sequence encoding the vioB protein used in the present disclosure is exemplified by a nucleotide sequence showing 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 6. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0025] SEQ ID NO: 7 is the amino acid sequence constituting the vioC protein derived from Pseudoalteromonas luteoviolacea. For example, the vioC protein used in the present disclosure may be a polypeptide having the amino acid sequence of SEQ ID NO: 7 in which one or more amino acids have been deleted, substituted, or added. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence of SEQ ID NO: 7 may be, for example, 65, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0026] The vioC protein used in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence set forth in SEQ ID NO: 7. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0027] SEQ ID NO: 8 is a nucleotide sequence encoding the vioC protein derived from Pseudoalteromonas luteoviolacea. For example, an example of a nucleotide sequence encoding the vioC protein used in the present disclosure is a nucleotide sequence that shows 85% or more identity to the nucleotide sequence set forth in SEQ ID NO: 8. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0028] SEQ ID NO: 9 is the amino acid sequence constituting the vioD protein derived from Pseudoalteromonas luteoviolacea. For example, the vioD protein used in the present disclosure may be a polypeptide having the amino acid sequence of SEQ ID NO: 9 in which one or more amino acids have been deleted, substituted, or added. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence of SEQ ID NO: 9 may be, for example, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0029] The vioD protein used in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence set forth in SEQ ID NO: 9. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0030] SEQ ID NO: 10 is a nucleotide sequence encoding the vioD protein derived from Pseudoalteromonas luteoviolacea. For example, an example of a nucleotide sequence encoding the vioD protein used in the present disclosure is a nucleotide sequence that shows 85% or more identity to the nucleotide sequence set forth in SEQ ID NO: 10. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0031] SEQ ID NO: 11 is the amino acid sequence constituting the vioE protein derived from Pseudoalteromonas luteoviolacea. For example, the vioE protein used in the present disclosure may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 in which one or more amino acids have been deleted, substituted, or added. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence set forth in SEQ ID NO: 11 may be, for example, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0032] The vioE protein used in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence set forth in SEQ ID NO: 11. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0033] SEQ ID NO: 12 is a nucleotide sequence encoding the vioE protein derived from Pseudoalteromonas luteoviolacea. For example, the nucleotide sequence encoding the vioE protein used in the present disclosure is exemplified by a nucleotide sequence showing 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 12. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0034] In the vectors used in the evaluation method of the present disclosure, the nucleotide sequences encoding the vioC protein and the vioE protein may be contained in the same vector or different vectors, preferably the same vector.

[0035] Preferably, the vector used in the evaluation method of the present disclosure further comprises a nucleotide sequence encoding the vioA protein. The nucleotide sequence encoding the vioA protein may be contained in the same vector as the vector comprising the nucleotide sequences encoding the vioC protein and / or the vioE protein, or in a different vector. In particular, it is preferable that the nucleotide sequence encoding the vioA protein be contained in a vector different from the vector comprising the nucleotide sequences encoding the vioC protein and the vioE protein.

[0036] Preferably, the vector used in the evaluation method of the present disclosure further contains a nucleotide sequence encoding the vioB protein. The nucleotide sequence encoding the vioB protein may be contained in the same vector as the vector containing the nucleotide sequences encoding the vioC protein and / or the vioE protein, or may be contained in a different vector. In particular, it is preferable that the nucleotide sequence encoding the vioB protein be contained in a vector different from the vector containing the nucleotide sequences encoding the vioC protein and the vioE protein.

[0037] The nucleotide sequence encoding the vioB protein may be contained in the same vector as the vector containing the nucleotide sequence encoding the vioA protein, or in a different vector, and is preferably contained in the same vector as the vector containing the nucleotide sequence encoding the vioA protein.

[0038] In other words, the evaluation method of the present disclosure is A vector comprising a base sequence encoding the vioC protein and the vioE protein to which a base sequence encoding a polypeptide to be evaluated has been added; and It is preferable to use a vector containing a nucleotide sequence encoding the vioA protein and the vioB protein.

[0039] The vector used in the evaluation method of the present disclosure may further contain a nucleotide sequence encoding the vioD protein. The nucleotide sequence encoding the vioD protein may be contained in the same vector as a vector containing nucleotide sequences encoding other vio proteins (vioA protein, vioB protein, vioC protein, vioE protein), or may be contained in a different vector. In particular, it is preferable that the nucleotide sequence encoding the vioD protein be contained in the same vector as a vector containing nucleotide sequences encoding the vioC protein and vioE protein. In other words, the evaluation method of the present disclosure is A vector comprising a base sequence encoding the vioC protein and the vioE protein to which a base sequence encoding a polypeptide to be evaluated has been added, and a base sequence encoding the vioD protein; and It is preferable to use a vector containing a nucleotide sequence encoding the vioA protein and the vioB protein.

[0040] In the vector used in the evaluation method of the present disclosure, a nucleotide sequence encoding a polypeptide to be evaluated is added to the nucleotide sequences encoding the vioC protein and the vioE protein. The polypeptide to be evaluated may be used singly or in combination of two or more.

[0041] The polypeptide to be evaluated is preferably a polypeptide capable of forming an aggregate.

[0042] The number of amino acids constituting the polypeptide to be evaluated is not particularly limited, and may be, for example, about 3 to 300, or about 10 to 300. For example, the number of amino acids constituting FUS(1-215) used in the Examples described below is 215, the number of amino acids constituting SC3 is 28, and the number of amino acids constituting scENO(1-30) is 30.

[0043] The polypeptides to be evaluated that are added to the vioC protein and the vioE protein may be the same or different. It is preferable that they are the same. Addition of the same polypeptide is believed to enable the vioC protein and the vioE protein to form an aggregate. Even when different polypeptides are added, they are believed to be able to form an aggregate as long as the localization of the polypeptides is the same.

[0044] The nucleotide sequence encoding the polypeptide to be evaluated may be added to the 5'-end or the 3'-end, with the 5'-end being preferred.

[0045] The nucleotide sequence encoding the polypeptide to be evaluated may be added directly to the nucleotide sequence encoding the vio protein, or may be added indirectly via a linker or the like.

[0046] When the vector used in the evaluation method of the present disclosure contains at least one protein selected from the group consisting of the vioA protein, the vioB protein, and the vioD protein, for example, a nucleotide sequence encoding a polypeptide to be evaluated may be added to the nucleotide sequence encoding the protein. Specifically, the nucleotide sequence encoding the polypeptide to be evaluated may be added to the nucleotide sequence encoding the vioA protein, the nucleotide sequence encoding the polypeptide to be evaluated may be added to the nucleotide sequence encoding the vioB protein, or the nucleotide sequence encoding the polypeptide to be evaluated may be added to the nucleotide sequence encoding the vioD protein.

[0047] When a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioA protein, the vioB protein, or the vioD protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioC protein and / or the vioE protein. In other words, when a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioA protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioC protein and / or the vioE protein. When a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioB protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioC protein and / or the vioE protein. When a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioD protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioC protein and / or the vioE protein. Furthermore, when a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioA protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioB protein and / or the vioD protein. When a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioB protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioA protein and / or the vioD protein. When a nucleotide sequence encoding a polypeptide to be evaluated is added to a nucleotide sequence encoding the vioD protein, the polypeptide may be the same as or different from, for example, the polypeptide to be evaluated added to the vioA protein and / or the vioB protein. It is believed that by adding the same polypeptide, the vio protein to which the polypeptide has been added can form an aggregate. Furthermore, even when different polypeptides are added, it is believed that aggregates can be formed as long as the polypeptides are localized in the same way. It is also believed that by using polypeptides with different localizations, two or more different aggregates can be simultaneously formed. For example, the polypeptides to be evaluated added to the vioA protein and the vioB protein are different polypeptides that show the same localization, and the polypeptides to be evaluated added to the vioC protein and the vioE protein, and / or the vioD protein are different polypeptides that show the same localization.

[0048] The vector used in the evaluation method of the present disclosure may contain any other base sequence in addition to the base sequences described above, such as a promoter (e.g., the CUP1 promoter (a copper ion-responsive promoter) or other inducible and / or constitutive promoters), an enhancer, a terminator, a polyadenylation signal, a selection marker, a replication origin, a base sequence encoding a fluorescent protein, a base sequence encoding an amino acid sequence that interacts with a fluorescent probe, or a base sequence encoding an amino acid sequence used to confirm the localization of a protein.

[0049] Preferably, the vector used in the evaluation method of the present disclosure does not contain a base sequence encoding an optogenetic clustering system-associated protein. As used herein, the term "optogenetic clustering system-associated protein" refers to a protein that undergoes clustering in response to light intensity, such as CRY2 (cryptochrome 2), which dimerizes upon blue light stimulation; PixD and PixE, which form a complex in the dark and dissociate into a PixE monomer and a PixD dimer upon blue light stimulation; or mutants thereof that function as optogenetic clustering system-associated proteins.

[0050] The vector is not particularly limited and can be appropriately selected depending on the type of host cell into which the vector is to be introduced. Examples include plasmid vectors and viral vectors. Among these, plasmid vectors are preferred.

[0051] The cells are not particularly limited as long as they can produce deoxyviolacein or violacein by introducing the above-mentioned vector, and may be either prokaryotic or eukaryotic. Examples include prokaryotic cells such as Escherichia bacteria (e.g., Escherichia coli), actinomycetes (e.g., Streptomyces), Bacillus bacteria (e.g., Bacillus subtilis), Streptococcus, and Staphylococcus; yeast cells such as Saccharomyces, Piscia, and Kluyveromyces; fungal cells such as Aspergillus, Penicillium, Talaromyces, Trichoderma, Hypocrea, and Acremonium; insect cells; plant cells; and animal cells. Among these, yeast cells are preferred, with Saccharomyces cells being more preferred.

[0052] The method for constructing a vector and the method for introducing a vector into a cell can be any method known in the art under any conditions, depending on the type of vector and the type of cell.

[0053] The evaluation method of the present disclosure preferably includes a step of culturing the above-described cells, which allows the above-described cells to produce deoxyviolacein and / or violacein.

[0054] The culture conditions are not particularly limited as long as they allow the production of deoxyviolacein and / or violacein, and can be appropriately selected depending on the type of cells used, etc.

[0055] Since violacein is synthesized from tryptophan, the medium used for culture may contain, for example, a compound that enables the cells used in the evaluation method of the present disclosure to synthesize tryptophan. Examples of such compounds include a carbon source such as glucose, a nitrogen source such as amino acids, and a phosphate such as dipotassium hydrogen phosphate. More specifically, when yeast is used, for example, an SC+HM medium or the like can be used. Furthermore, depending on the type of vio protein introduced into the vector used in the evaluation method of the present disclosure, for example, tryptophan, IPA-imine, IPA-imine dimer, etc. may be added to the medium. For example, when the vector contains a nucleotide sequence encoding the vioC protein and the vioE protein, for example, IPA-imine dimer may be added to the medium. For example, when the vector contains a nucleotide sequence encoding the vioB protein, the vioC protein, and the vioE protein, IPA-imine may be added to the medium. For example, when the vector contains a nucleotide sequence encoding the vioA protein, the vioB protein, the vioC protein, and the vioE protein, tryptophan may be added to the medium. The concentrations of various compounds added to the medium can be appropriately set depending on the type of compound.

[0056] The culture temperature can be appropriately selected depending on the type of cells used. When yeast is used, the culture temperature can be, for example, about 4 to 50°C, or may be about 13 to 45°C. The culture time can be appropriately selected depending on the type of cells used. When yeast is used, the culture time can be, for example, 1 hour or more, and may be about 1 to 168 hours, or about 12 to 168 hours. The pH during culture can be appropriately selected depending on the type of cells used. When yeast is used, the pH can be, for example, about 2 to 8, or may be about 4 to 8. In the culturing step, shaking, stirring, etc. can be carried out as necessary.

[0057] When the cells used in the evaluation method of the present disclosure contain a vector containing a base sequence encoding the vioD protein, the cells can produce deoxyviolacein and violacein. When the cells used in the evaluation method of the present disclosure do not contain a vector containing a base sequence encoding the vioD protein, the cells do not produce violacein but can produce deoxyviolacein.

[0058] The evaluation method of the present disclosure preferably includes a step of extracting deoxyviolacein and / or violacein from the cultured cells. The extraction method is not particularly limited, and any method known in the art can be used under any conditions. For example, as shown in the Examples below, a method in which the pigment is extracted using an organic solvent such as methanol at a high temperature (e.g., 60 to 100°C, more preferably 80 to 100°C) for about 5 to 30 minutes can be used. In the extraction step, shaking, stirring, etc. may be carried out as necessary.

[0059] The evaluation method of the present disclosure preferably includes a step of measuring the amount of deoxyviolacein and / or violacein. The measurement method is not particularly limited and may be, for example, HPLC analysis. Examples of HPLC analysis methods include the method described in the Examples (gradient elution using different mobile phases: MilliQ water (solvent A) and acetonitrile (solvent B)). More specifically, the method may involve starting with 5% solvent B, linearly increasing solvent B from 5 to 95% after 0 to 10 minutes, maintaining solvent B at 95% after 10 to 13 minutes, linearly decreasing solvent B from 95 to 5% after 13 to 13.5 minutes, and maintaining solvent B at 5% after 13.5 to 23.5 minutes, with an injection volume of 10 μL, a flow rate of 0.9 mL / min, a column temperature of 40°C, and a detection wavelength of 565 nm. In this method, the retention time of violacein is 9.983 to 10.008 seconds, and the retention time of deoxyviolacein is 10.904 to 10.945 seconds.

[0060] Furthermore, for example, the amount of deoxyviolacein relative to the amount of violacein may be calculated from the measured amounts of deoxyviolacein and violacein.

[0061] According to the evaluation method of the present disclosure, the ability to form aggregates can be evaluated based on the amount of deoxyviolacein, the amount of violacein, and / or the amount of deoxyviolacein relative to the amount of violacein. Furthermore, since the evaluation method of the present disclosure evaluates the ability to form aggregates based on the amount of deoxyviolacein, the amount of violacein, and / or the amount of deoxyviolacein relative to the amount of violacein, it is also possible to evaluate the effect of aggregate formation on substance production in, for example, any metabolic pathway (more specifically, any metabolic pathway involving two or more enzymatic reactions, any metabolic pathway having a branch point, etc.). In other words, it is presumed that the evaluation method of the present disclosure can evaluate whether substance production is actually efficient through aggregate formation in any metabolic pathway (more specifically, any metabolic pathway involving two or more enzymatic reactions, any metabolic pathway having a branch point, etc.) by evaluating the ability to form aggregates.

[0062] In the evaluation method of the present disclosure, when the same polypeptide to be evaluated is added to the vioC protein and the vioE protein, compared to when the polypeptide to be evaluated is not used, if the amount of deoxyviolacein produced increases, the amount of violacein produced decreases, and / or the amount of deoxyviolacein produced relative to the amount of violacein produced increases, the polypeptide to be evaluated can be evaluated as having the ability to form aggregates. Furthermore, it is presumed that the polypeptide to be evaluated can be evaluated as promoting substance production through aggregate formation. In the evaluation method of the present disclosure, when a different polypeptide to be evaluated is added to the vioC protein and the vioE protein, compared to when the polypeptide to be evaluated is not used, if the amount of deoxyviolacein produced increases, the amount of violacein produced decreases, and / or the amount of deoxyviolacein produced relative to the amount of violacein produced increases, the polypeptide to be evaluated can be evaluated as having the ability to form aggregates. Furthermore, it is presumed that the combination of polypeptides to be evaluated can be evaluated as promoting substance production through aggregate formation. In the evaluation method of the present disclosure, when the same polypeptide to be evaluated II is added to the vioC protein and the vioE protein, compared to when the same polypeptide to be evaluated I is added, if the amount of deoxyviolacein produced increases, the amount of violacein produced decreases, and / or the amount of deoxyviolacein produced relative to the amount of violacein produced increases, the polypeptide to be evaluated II can be evaluated as having a higher ability to form aggregates than the polypeptide to be evaluated I. Furthermore, it is presumed that the polypeptide to be evaluated II can be evaluated as more capable of promoting substance production through aggregate formation than the polypeptide to be evaluated I. In the evaluation method of the present disclosure, when a target polypeptide different from the target polypeptides added to the vioC and vioE proteins is added to the vioD protein, compared to when the target polypeptide is not used, if the amount of deoxyviolacein produced increases, the amount of violacein produced decreases, and / or the amount of deoxyviolacein produced relative to the amount of violacein produced, it can be evaluated that the target polypeptide added to the vioD protein and the target polypeptides added to the vioC and vioE proteins do not have the ability to form aggregates. Furthermore, it is presumed that the target polypeptide added to the vioD protein and the target polypeptides added to the vioC and vioE proteins can be evaluated as suppressing substance production by not forming aggregates. In the evaluation method of the present disclosure, when the same polypeptide to be evaluated as that added to the vioC and vioE proteins is added to the vioA and vioB proteins or the vioA, vioB, and vioD proteins in the evaluation method is added to the vioA and vioB proteins or the vioA, vioB, and vioD proteins in the evaluation method, compared to when the same polypeptide to be evaluated as that added to the vioC and vioE proteins is added, the polypeptide to be evaluated can be evaluated as having the ability to form aggregates. Furthermore, it is presumed that the polypeptide to be evaluated can be evaluated as promoting substance production through aggregate formation. In the evaluation method of the present disclosure, when a target polypeptide different from the target polypeptide added to the vioC and vioE proteins is added to the vioA and vioB proteins or the vioA, vioB, and vioD proteins, compared to when the target polypeptide is not added to the vioA and vioB proteins or the vioA, vioB, and vioD proteins, and the amount of deoxyviolacein and / or violacein produced is increased, the target polypeptide can be evaluated as having the ability to form aggregates. Furthermore, it is presumed that the target polypeptide can be evaluated as promoting substance production through aggregate formation. In the evaluation method of the present disclosure, when a target polypeptide II to be evaluated that is the same as the target polypeptide I to be evaluated that is added to the vioC and vioE proteins is added to the vioA and vioB proteins, or the vioA, vioB, and vioD proteins, compared to a target polypeptide I to be evaluated that is the same as the target polypeptide I to be evaluated that is added to the vioC and vioE proteins, and the amount of deoxyviolacein produced and / or the amount of violacein produced is increased, the target polypeptide II to be evaluated can be evaluated as having a higher ability to form aggregates than the target polypeptide I to be evaluated. Furthermore, it is presumed that the target polypeptide II to be evaluated can be evaluated as having a higher ability to form aggregates than the target polypeptide I to be evaluated as having a higher ability to promote substance production through aggregate formation.

[0063] Furthermore, by evaluating the aggregation ability of polypeptides using the evaluation method of the present disclosure, polypeptides or combinations thereof that exhibit desired selectivity in violacein biosynthesis (deoxyviolacein amount, violacein amount, and / or deoxyviolacein amount relative to violacein amount) can be selected from the target polypeptides added to the vioC protein and vioE protein. For example, by applying the combination to any other metabolic pathway (more specifically, any metabolic pathway involving two or more enzymatic reactions, any metabolic pathway with a branching point, etc.), it is believed that substances exhibiting similar selectivity can be produced in the metabolic pathway.

[0064] According to the evaluation method of the present disclosure, the target polypeptide can be easily evaluated by introducing the above-mentioned vector.

[0065] The present disclosure also encompasses vectors to be used in the above-described evaluation methods of the present disclosure.

[0066] The present disclosure also encompasses a method for producing a useful substance, comprising culturing cells containing, in the same or different vectors, vectors containing nucleotide sequences encoding two or more enzymes to which a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 13 and which has the ability to form aggregates has been added. In this specification, this production method may be referred to as the "production method of the present disclosure."

[0067] SEQ ID NO: 13 is an amino acid sequence in which an initiation codon has been added to the N-terminus of the amino acid sequence (SC3) of yeast pyruvate kinase (Cdc19p) from positions 217 to 243. As shown in the Examples below, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 has the ability to form aggregates and can efficiently produce substances through the formation of aggregates.

[0068] In the amino acid sequence shown in SEQ ID NO: 13, the upper limit of the number of deleted, substituted, or added amino acids may be, for example, 5, 4, 3, or 2. Specifically, it may be 1 to 5, or 1 to 4. The ability to form an aggregate can be evaluated by the method described above.

[0069] A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 13 may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity to the amino acid sequence shown in SEQ ID NO: 13. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0070] SEQ ID NO: 14 is a nucleotide sequence encoding an amino acid sequence in which an initiation codon has been added to the N-terminus of the amino acid sequence (SC3) from positions 217 to 243 of yeast pyruvate kinase (Cdc19p). For example, an example of a nucleotide sequence encoding a polypeptide capable of forming aggregates, which has an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 13, is a nucleotide sequence that shows 85% or more identity to the nucleotide sequence of SEQ ID NO: 14. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0071] The useful substance is not particularly limited, and is preferably one that is synthesized through two or more enzymatic reactions, such as deoxyviolacein.

[0072] The enzyme is not particularly limited as long as it can produce the desired useful substance. When producing deoxyviolacein, examples include the vioA protein, vioB protein, vioC protein, vioD protein, and vioE protein.

[0073] The number of optional enzymes is not particularly limited, and may be, for example, 2 to 7 or 3 to 5.

[0074] For the manufacturing method of the present disclosure, the description of the "Evaluation method of the present disclosure" can be cited. The medium used for cell culture can contain compounds as needed depending on the type of useful substance to be produced.

[0075] According to the production method of the present disclosure, the amount of a useful substance produced is preferably 1.5 times or more greater than when a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 15 is added to a nucleotide sequence encoding an arbitrary enzyme. In other words, the production method of the present disclosure is a production method for a useful substance, comprising culturing cells containing, in the same or different vectors, vectors containing nucleotide sequences encoding two or more arbitrary enzymes to which a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 or a polypeptide capable of forming aggregates, the polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 13, can be said to be a production method in which the amount of a useful substance produced is 1.5 times or more greater than when a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 15 is added to a nucleotide sequence encoding an arbitrary enzyme.

[0076] SEQ ID NO: 1 is the amino acid sequence of positions 1 to 215 of FUS (Fused in Sarcoma), a human-derived RNA-binding protein. SEQ ID NO: 15 is the amino acid sequence of positions 1 to 30 (scENO) of yeast glycolytic enzyme enolase (Eno2p).

[0077] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0078] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0079] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0080] Experimental Example 1: Enzyme assembly experiment using the assembly domain of FUS The plasmids used in Experimental Example 1 are shown in Table 1 and Figure 2. The amino acid and nucleotide sequences of FUS(1-215) are shown in SEQ ID NOs: 1 and 2, and the amino acid and nucleotide sequences of vioA to E proteins are shown in SEQ ID NOs: 3 to 12.

[0081] [Table 1]

[0082] yeast culture Saccharomyces cerevisiae BY4741 was transformed with p426-VioA-VioB and either one of two plasmids, pLEU-FUSN_VioE_FR-VioD-FUSN_VioC_EGFP, pLEUFUSN_VioE_FR-VioD-VioC_EGFP, pLEU-VioE_FR-VioD-FUSN_VioC_EGFP, or pLEU-VioE_FR-VioD-VioC_EGFP. The transformed colonies were cultured in SC+HM liquid medium (0.67% (w / v) Yeast nitrogen base without amino acid residues). The bacteria were inoculated into 5 mL (2 tubes = 10 mL) of a 10 mL tube containing 200 kJ / mL of 100 kJ / mL ... 600The pre-preculture solution was collected so that the β-amylindrical ratio was 0.1 and centrifuged at 4°C and 5,000 rpm for 5 minutes. The bacterial cells were then collected and suspended in 100 mL of fresh SC+HM liquid medium (2 tubes = 200 mL), followed by shaking culture at 30°C and 100 rpm for 24 hours (preculture). The culture was then divided into samples for microscopic observation and samples for HPLC analysis. In the microscopic observation samples, the initial OD of this culture was 600 The pre-culture solution was collected and centrifuged at 4°C and 5,000 rpm for 5 minutes so that the ratio of Cu to Cu was 0.5. The cells were then collected and suspended in 5 mL of fresh SC+HM liquid medium. 2+ A 10 mM CuSO4 solution was added to the mixture so that the final concentration was 100 μM, and the mixture was cultured at 30°C and 300 rpm with shaking for 24 hours (main culture). In the HPLC analysis sample, the initial OD of this culture 600 The pre-culture solution was collected and centrifuged at 4°C and 5,000 rpm for 5 minutes so that the ratio of Cu to HM was 0.5. The cells were then collected and suspended in 1 L of fresh SC+HM liquid medium. 2+ A 10 mM CuSO4 solution was added to the mixture so that the final concentration was 100 μM, and the mixture was cultured at 30°C and 100 rpm with shaking for 96 hours (main culture).

[0083] Cell fixation for microscopic observation samples 1 mL of each culture medium was collected and centrifuged at 5,000 rpm at 4°C for 5 minutes. The collected cells were suspended in 500 μL of 1x PBS buffer and centrifuged again at 5,000 rpm at 4°C for 5 minutes. The collected cells were suspended in 100 μL of 4% paraformaldehyde phosphate buffer. The suspension was left to stand overnight at 4°C and used as a sample for microscopic observation.

[0084] Microscopic observation The sample for microscopic observation was placed on a glass slide and covered with a cover glass. Images were then taken using the HS all-in-one fluorescence microscope BZ-9000 in bright field, red fluorescence, and green fluorescence fields. The captured images were then adjusted for brightness and contrast and overlaid using the BZ-II analysis application.

[0085] pigment extraction The entire culture medium was collected and centrifuged at 5,000 rpm at 4°C for 5 minutes. The collected cells were suspended in 100 mL of 1x PBS buffer, transferred to a 50 mL Falcon tube, and centrifuged at 5,000 rpm at 4°C for 5 minutes. Similarly, the collected cells were suspended again in 30 mL of 1x PBS buffer, transferred to a 15 mL Falcon tube, and centrifuged at 5,000 rpm at 4°C for 5 minutes. Five mL of methanol was added to the collected cells and vortexed. The mixture was then incubated at 95°C for 15 minutes. During this incubation, vortexing was performed three times for 3 seconds. After incubation, the mixture was centrifuged at 5,000 rpm at 4°C for 5 minutes, and the supernatant was collected in an Eppendorf tube. The mixture was again centrifuged at 5,000 rpm at 4°C for 5 minutes, and the supernatant was collected. The supernatant was concentrated at 50°C and 1,700 rpm in the "IR & HEAT" mode of an infrared vacuum concentration centrifuge, and the final sample suspended in 250 μL of methanol was used as the HPLC analysis sample.

[0086] HPLC analysis Violacein and deoxyviolacein were quantitatively analyzed using a HPLC system equipped with a COSMOSIL Packed Column 5C18-AR-II, φ4.6 × 150 mm. The compounds were separated by gradient elution using different mobile phases: MilliQ water (solvent A) and acetonitrile (solvent B). Starting at 5% solvent B, the solvent B was linearly increased from 5 to 95% over 0–10 min, held at 95% over 10–13 min, linearly decreased from 95 to 5% over 13–13.5 min, and held at 5% over 13.5–23.5 min. The injection volume was 10 μL, the flow rate was 0.9 mL / min, the column temperature was 40 °C, and the detection wavelength was 565 nm. The retention times were 9.983 to 10.008 seconds for violacein and 10.904 to 10.945 seconds for deoxyviolacein.

[0087] The results of the microscopic observation are shown in Figure 3. FusionRed indicates the fluorescence of the VioE enzyme, and EGFP indicates the fluorescence of the VioC enzyme. As shown in Figure 3, in yeast transformed with the plasmid pLEUFUSN_VioE_FR-VioD-FUSN_VioC_EGFP, in which both VioE and VioC were FUSN-conjugated, both Fusion Red and EGFP formed aggregates, confirming their colocalization. Furthermore, in yeast transformed with the plasmid pLEUFUSN_VioE_FR-VioD-FUSN_VioC_EGFP, in which only VioE was FUSN-conjugated, only Fusion Red formed aggregates. In yeast transformed with the plasmid pLEUFUSN_VioE_FR-VioD-FUSN_VioC_EGFP, in which only VioC was FUSN-conjugated, only EGFP formed aggregates. No aggregate formation was observed in yeast transformed with the FUSN-free plasmid pLEU-VioE_FR-VioD-VioC_EGFP (VioE+VioC).

[0088] The results of HPLC analysis of the amounts of violacein and deoxyviolacein produced are shown in Figure 4. The HPLC analysis results were calculated from the peak areas. The amount of deoxyviolacein produced, with the amount of violacein defined as 1, is shown in Figure 5. As shown in Figure 4, the amount of violacein produced by yeast transformed with the FUSN-containing plasmid pLEU-FUSN_VioE_FR-VioD-FUSN_VioC_EGFP (FUSNVioE+FUSN-VioC) tended to be significantly lower than that produced by yeast transformed with the non-FUSN-containing plasmid pLEU-VioE_FR-VioDVioC_EGFP (VioE+VioC). Furthermore, as shown in FIG. 5, when the amount of violacein produced by the FUSN-introduced yeast (FUSN-VioE+FUSN-VioC) was set to 1, the amount of deoxyviolacein produced was significantly higher than that of the FUSN-unintroduced yeast (VioE+VioC).

[0089] Experimental example 2: Enzyme assembly formation experiment using Metafos-tag The plasmids used in Experimental Example 2 are shown in Table 2 and Figure 6. The amino acid sequences and nucleotide sequences of SC3 and scENO(1-30) are shown in SEQ ID NOs: 13-16.

[0090] [Table 2]

[0091] The results of microscopic observation performed in the same manner as in Experimental Example 1 are shown in FIG. As shown in Figure 7, in yeast transformed with plasmids carrying FUSN, SC3, and scENO(1-30) for both VioE and VioC (FUS, SC3, ENO), aggregate formation was observed for both FusionRed and EGFP, confirming their colocalization. However, no aggregate formation was observed in yeast transformed with a plasmid lacking the aggregation tag (CONT1).

[0092] The amount of deoxyviolacein produced was measured by HPLC analysis using the same method as in Experimental Example 1. The results are shown in Figure 8. The amount of deoxyviolacein produced, when the amount of violacein produced was set to 1, is also shown in Figure 9. As shown in Figure 8, the yeast transformed with the plasmid pLEUSC3_VioE_FR-VioD-SC3_VioC_EGFP, in which both VioE and VioC were added with SC3 (SC3), produced significantly higher amounts of deoxyviolacein than the strain without the aggregation tag (CONT1). As shown in Figure 9, the amount of deoxyviolacein produced by SC3, when the amount of violacein produced was set to 1, was significantly higher than that of CONT1. These results demonstrate that SC3 has the best aggregation ability compared to FUS and scENO, and therefore exhibits efficient deoxyviolacein production.

Claims

1. A method for evaluating the aggregation ability of a polypeptide, comprising measuring the amount of deoxyviolacein and / or violacein in cells containing vectors containing base sequences encoding the vioC protein and the vioE protein, to which a base sequence encoding the polypeptide to be evaluated has been added, the vector being the same or different.

2. The method of claim 1 , wherein the cell contains a vector containing a nucleotide sequence encoding the vioA protein and / or the vioB protein, either in the same vector or in a different vector.

3. The cells A vector comprising a base sequence encoding the vioC protein and the vioE protein to which a base sequence encoding a polypeptide to be evaluated has been added; and The method of claim 1 , comprising a vector containing a nucleotide sequence encoding the vioA protein and the vioB protein.

4. The method according to any one of claims 1 to 3, wherein the nucleotide sequence encoding the polypeptide to be evaluated is added to the 5'-end side of the nucleotide sequence encoding the vioC protein and the vioE protein.

5. The method according to any one of claims 1 to 3, wherein the vector is a plasmid vector.

6. The method according to any one of claims 1 to 3, wherein the vector further comprises a base sequence encoding a vioD protein.

7. A vector for use in the method according to any one of claims 1 to 3.

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