KR102342635B1 - Polynucleotides to enhance expression of a desired polypeptide and their uses - Google Patents

Abstract

The present application relates to polynucleotides for enhancing the expression of a target protein and uses thereof.

Description

Polynucleotides to enhance expression of a target protein and their uses {Polynucleotides to enhance expression of a desired polypeptide and their uses}

The present application relates to polynucleotides for enhancing the expression of a target protein and uses thereof.

Initiation efficiency is the most important determinant of translation efficiency. In bacteria, the 30S ribosomal subunit, aided by initiation factors 1, 2, 3 and fMet-tRNA ^fMet , recognizes the translation initiation region (TIR) of mRNA. TIR recognition is followed by binding of the 50S ribosomal subunit and release of initiation factors. In this process, the initiation efficiency (rate) may be limited depending on the binding of the 30S ribosome subunit to the TIR.

The following sequence elements of TIR contribute to its efficiency. (a) an initiation codon, most commonly AUG, but sometimes GUG, and very rarely UUG, AUU or CUG; (b) Shine-Dalgarno (SD) sequence; (c) Regions upstream of the SD sequence and downstream of the initiation codon containing A/U-rich sequences, also referred to as enhancers of translation. In addition, it is known that the spacing between these sequence elements is often important for efficiency, for example, the distance between the SD sequence and the starting triplet significantly affects the conversion efficiency (BMC Mol Biol. 2007 Oct 31;8:100). .).

Under this background, as a result of intensive research efforts to increase the expression of 4-epimerase by introducing a mutation in TIR that affects expression efficiency, the present applicants discovered a polynucleotide that enhances expression and completed the present application.

One object of the present application is to include i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine in the base sequence of SEQ ID NO: 1, To provide a polynucleotide for expression.

Another object of the present application is to provide a polynucleotide comprising a polynucleotide for the expression and encoding a protein of interest.

Another object of the present application is to provide a vector including a polynucleotide for the expression.

Another object of the present application is a polynucleotide for the expression; a polynucleotide comprising a polynucleotide for the expression and encoding a protein of interest; Or a vector comprising a polynucleotide for the expression; to provide a microorganism expressing a target protein comprising the.

Another object of the present application is to provide a method for producing a target protein, comprising the step of culturing the microorganism in a medium.

Another object of the present application is to provide a method for producing a target product, comprising the steps of culturing the microorganism in a medium to produce a target protein and contacting the produced target protein with a substrate.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below. In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.

In one aspect of the present application for achieving the above object, in the nucleotide sequence of SEQ ID NO: 1, i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine It is to provide a polynucleotide for expression, including the substitution of.

In the present application, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain shape.

In the present application, the term "polynucleotide for expression" refers to a polynucleotide (ie, RNA or a DNA sequence encoding the mRNA) corresponding to a translation initiation region (TIR) of an mRNA. TIR is a target Refers to the nucleic acid region that provides the efficiency of translation initiation of a gene.Generally, the TIR in a specific translation unit comprises ribosome binding site (RBS) and 5' and 3' sequence to RBS.RBS is at least Shine -Defined to contain Shine-Dalgarno region and initiation codon (AUG).Thus, TIR also includes at least part of the nucleic acid sequence to be translated.Meanwhile, in this application, TIR refers to the region within mRNA. It is not limited to, and can be used to refer to the DNA encoding the mRNA.The polynucleotide according to the present application increases the translation efficiency as the translation initiation activity increases, so that the expression of the target protein encoded by the polynucleotide is reduced. Compared with wild-type or unmodified polynucleotide before substitution, the polynucleotide of the present application is "polynucleotide with increased expression compared to wild-type or unmutated polynucleotide" or "polynucleotide that increases translation initiation activity" nucleotides”.

In the present application, the term “start codon” may be a sequence expressed as AUG in RNA and ATG in DNA. However, the present invention is not limited thereto, and those skilled in the art may appropriately select and use codons such as UUG and GUG according to the microorganism within the range capable of increasing the expression of the target protein.

The polynucleotide for expression may have an increased translation initiation activity compared to the TIR or wild-type TIR of SEQ ID NO: 1 before substitution, thereby increasing translation efficiency, but is not limited thereto. On the other hand, the polynucleotide for expression of the present application includes one or more substitutions in the polynucleotide of SEQ ID NO: 1, and may also be referred to as a 'polynucleotide variant'.

In the present application, the term "polynucleotide variant" refers to a polynucleotide in which any one or more nucleotides constituting the polynucleotide are substituted with other nucleotides. The polynucleotide variant may be used interchangeably with terms such as variant polynucleotide and variant polynucleotide.

In the present application, the term “fructose-4-epimerase” refers to an enzyme having fructose-4-epimerization activity that epimerizes the 4th carbon position of fructose to convert fructose into tagatose. For the purpose of the present application, if tagatose can be produced using fructose as a substrate, it may be included without limitation, and may be used in combination with 'D-fructose C4-epimerase'. As an example, tagatose-bisphosphate aldolase or tagatose-bisphosphate class II accessory protein with EC 4.1.2.40 in KEGG (Kyoto Encyclopedia of Genes and Genomes), a known database II accessory protein) may be included as a fructose-4-epimerase enzyme if it has an activity to convert fructose into tagatose as a substrate. The tagatose-diphosphate aldolase was previously prepared with glycerone phosphate and D-tagatose 1,6-bisphosphate as a substrate as shown in [Scheme 1] below. It is known as an enzyme that produces D-glyceraldehyde 3-phosphate.

[Scheme 1]

D-tagatose 1,6-diphosphate <=> glycerone phosphate + D-glyceraldehyde 3-phosphate

For example, if tagatose 6-phosphate kinase (EC 2.7.1.144) has an activity to convert fructose into tagatose as a substrate, it may be included as a fructose-4-epimerase. The tagatose-6-phosphate kinase was previously prepared by using ATP and D-tagatose 6-phosphate as substrates as shown in [Scheme 2] below to produce ADP and D-tagatose 1,6- It is known as an enzyme that produces D-tagatose 1,6-bisphosphate.

[Scheme 2]

ATP + D-tagatose 6-phosphate <=> ADP + D-tagatose 1,6-diphosphate

The activity of the fructose-4-epimerase is 0.01% or more, specifically 0.1% of the conversion rate from fructose, a substrate, to tagatose (conversion rate = tagatose weight / initial fructose weight *100)

or more, more specifically, it may be 0.3% or more. More specifically, the conversion may be in the range of 0.01% to 100%, and in the range of 0.1% to 50%.

Fructose-4-epimerase of the present application may be an enzyme derived from a microorganism or mutant, e.g., a course moto O Elia (Kosmotoga olearia), sseomeon air load Riggs decene sheath (Thermanaerothrix daxensis), also written mousse Pro Fundi ( Rhodothermus profundi ), Rhodothermus marinus ( Rhodothermus marinus ), Limnochorda pilosa ( Limnochorda pilosa ), Caldithrix abyssi , Caldilinea aerophila ) Sulfuricus ( Thermoanaerobacter thermohydrosulfuricus ), Ecidobacteriales bacterium ( Acidobacteriales bacterium ), Caldicellulosiruptor kronotskyensis , Thermoanaerobacterium thermosaccharolyticum or Thermoanaerobacterium thermosaccharolyticum Monas sp. H103 (Pseudoalteromonas sp. H103), but may be derived from an enzyme or a variant, not limited to this, the particular course moto O Elia, but may be derived from an enzyme or a variant, not limited to this. The Cosmotoga Oelia-derived enzyme may include SEQ ID NO: 7.

In the present application, SEQ ID NO: 1 refers to a sequence corresponding to TIR in the nucleotide sequence encoding fructose-4-epimerase. SEQ ID NO: 1 has been described as a representative DNA sequence encoding the TIR region of mRNA, but the mRNA sequence encoded by SEQ ID NO: 1, that is, an RNA sequence in which thymine (T) of SEQ ID NO: 1 is uracil (U). it is self-evident that Hereinafter, a variant of SEQ ID NO: 1, that is, a sequence in which some bases are substituted is also the same.

SEQ ID NO: 1 may include a start codon, and specifically, SEQ ID NO: 1 may include polynucleotides 1 to 45 based on A of ATG, which is a start codon.

The sequence of SEQ ID NO: 1 can be obtained from a known database, GenBank of NCBI or KEGG (Kyoto Encyclopedia of Genes and Genomes). As an example, Kosmotoga olearia ( Kosmotoga olearia ) may be derived, but is not limited thereto. In addition, a sequence having the same activity as the base sequence may be included without limitation. In addition, it may include the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having 70% or more homology or identity therewith, but is not limited thereto. Specifically, the base sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 1 and SEQ ID NO: 1 It may include a nucleotide sequence. In addition, as long as it is a nucleotide sequence having such homology or identity and exhibiting efficacy corresponding to the polynucleotide, it is apparent that a polynucleotide having a nucleotide sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application.

That is, even if it is described as a 'polynucleotide having a nucleotide sequence described in a specific SEQ ID NO:' in the present application, if it has the same or corresponding activity as a polynucleotide consisting of the nucleotide sequence of the SEQ ID NO: some sequences are deleted, It is apparent that a polynucleotide having a modified, substituted, conservatively substituted or added base sequence may also be used in the present application. For example, if it has the same or corresponding activity as the polynucleotide, adding a sequence that does not change the function of the polynucleotide before or after the base sequence, naturally occurring mutation, or silent mutation or conservation thereof It is obvious that the substitution is not excluded, and it is within the scope of the present application even if it has such sequence additions or mutations.

In the nucleotide sequence of SEQ ID NO: 1, which is a sequence corresponding to TIR in the nucleotide sequence encoding fructose-4-epimerase in the nucleotide sequence encoding the fructose-4-epimerase, the polynucleotide for the expression, that is, the polynucleotide variant, is based on A of ATG, the initiation codon in TIR. to any one or more of the nucleotides at positions 1 to 45 may be mutated, but is not limited thereto. Specifically, the nucleotide mutation may be a mutation in which any one or more nucleotides selected from the group consisting of the 24th, 27th, and 33rd positions based on A of ATG, the initiation codon in TIR, are substituted with other nucleotides, and more specifically , all of the 24th, 27th, and 33rd positions may be substituted with different nucleotides. In addition, any one or more nucleotides selected from the group consisting of the 6th, 12th, 15th, 18th, 30th, 34th, 36th, 39th, 42nd and 45th nucleotides are substituted with other nucleotides, It may be a mutated one, but is not limited thereto.

The nucleotide mutation is not particularly limited as long as it is made within the range in which only the codon is changed without altering the sequence of the amino acid encoding the fructose-4-epimerase, but specifically, in the nucleotide sequence of SEQ ID NO: 1 i ) the substitution of thymine (T) for cytosine (C) at the 24th base, ii) the substitution of thymine (T) at the 27th base for cytosine (C), or iii) the substitution of cytosine for adenine (A) at the 33rd base It may include any one or more substitutions selected from the group consisting of substitution with (C), and may include all substitutions of i) to iii). In addition, any one selected from the group consisting of the 6th, 12th, 15th, 18th, 30th, 34th, 36th, 39th, 42th and 45th in the nucleotide sequence of SEQ ID NO: 1 In the case of further including the above substitutions, the substitution is that the 6th base adenine (A) is guanine (G), the 12th base thymine (T) is cytosine (C), and the 15th base thymine (T) is guanine (G) ), the 18th base thymine (T) to guanine (G), the 30th base thymine (T) to guanine (G), the 34th base thymine (T) to cytosine (C), and the 36th guanine (G) ) is thymine (T), the 39th base adenine (A) is replaced with guanine (G), the 42nd base adenine (A) is replaced with guanine (G), and the 45th guanine (G) is replaced with adenine (A) Any one or more of these may be selected, but the present invention is not limited thereto.

The polynucleotide for the expression may be the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

In addition, the polynucleotide for expression is hybridized under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or a part of the nucleotide sequence constituting the polynucleotide for expression. Thus, any polynucleotide sequence having translation initiation activity may be included without limitation. The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are described in, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology. , John Wiley & Sons, Inc., New York). For example, between genes with high homology or identity, 70% or more, 80% or more, specifically 85% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more , More specifically, the conditions in which genes having 99% or more homology or identity are hybridized and genes having lower homology or identity do not hybridize, or the washing conditions of normal Southern hybridization are 60°C, 1×. SSC, 0.1% SDS, specifically at a salt concentration and temperature equivalent to 60° C., 0.1×SSC, 0.1% SDS, more specifically 68° C., 0.1×SSC, 0.1% SDS, once, specifically 2 Conditions for washing three to three times can be enumerated.

Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also encompass substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the overall sequence.

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art.

As used herein, the term "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related, and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the entire or full-length sequence under moderate or high stringent conditions. or more than 90% hybrid. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: using default parameters as in 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing the sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the term “homology” or “identity” refers to a relevance between sequences.

In the present application, the term “untranslated region (UTR)” refers to an mRNA region that is not translated into a protein. If the UTR is found on the 5' side, it is named 5' UTR (5' leader), and if it is found on the 3' side, it is named 3' UTR (3' trailer). The 5'UTR is upstream from the coding sequence, and there is a sequence within the 5'UTR that can be recognized by the ribosome to bind the ribosome and initiate translation. The 3' UTR is found immediately after the translation stop codon and plays an important role in translation termination as well as transcription termination gene expression. The untranslated mRNA region is known to be involved in many regulatory aspects of gene expression, such as expression level. The 5' UTR of prokaryotes consists of the Shine-Dalgarno sequence (5'-AGGAGGU-3'). This sequence is found 3 to 10 base pairs upstream from the start codon. The eukaryotic 5' UTR contains the Kozak consensus sequence (ACCAUGG). The 5'-UTR portion is largely divided into two types. Most eukaryotic mRNA has an RNA, a promoter part with 7-methyl-guanosine (cap) at the 5' end, and for protein synthesis, the protein synthesis initiation complex recognizes the cap and serves as the start codon. Proceed to AUG to start protein synthesis. However, some RNAs have an IRES (Internal Ribosome Entry Site) sequence instead of a cap, so they synthesize proteins. IRES is possessed by many viruses as well as some eukaryotic mRNAs. Based on this, the method of mRNA protein synthesis can be largely divided into cap-dependent transcriptional reaction and IRES-dependent transcriptional reaction.

In the present application, UTR may mean 5' UTR, and 5' UTR may be represented by an mRNA or DNA sequence.

The 5' UTR may be included without limitation as long as it has translation initiation activity in combination with a polynucleotide variant or has an increased expression level compared to a wild-type or unmutated polynucleotide, but specifically any of SEQ ID NO: 5 or SEQ ID NO: 6 It may include one or more.

Another aspect of the present application is to provide a polynucleotide comprising a polynucleotide for the expression and encoding a target protein.

The polynucleotide for the expression is as described above.

As the polynucleotide encoding the target protein includes a polynucleotide for the expression, that is, a polynucleotide variant, the amino acid sequence is not changed and the expression of the encoded target protein or target polypeptide includes a wild-type TIR before mutation. It can be increased compared to the gene that The polynucleotide encoding the target protein is located in the coding region within a range that does not change the amino acid sequence of the protein due to codon degeneracy, or considering codons preferred in the organism to express the polynucleotide. Various modifications may be made. The polynucleotide sequence is the same as described above.

The gene encoding the target protein may be a gene encoding a polypeptide as a specific target nucleic acid to be transcribed. The gene of interest may be any gene desired by those skilled in the art, and may be a gene derived from a foreign species or another strain other than the host strain, or a modified form of the gene, but is not limited thereto. The target protein may be a polypeptide whose expression level is to be increased. The polypeptide includes a fluorescent protein and a polypeptide involved in the production of a target substance, and may be an enzyme or a membrane protein (transporter). In addition, the target substance may be a saccharide, an amino acid and/or a nucleic acid, but is not limited thereto. The saccharide may specifically be tagatose or allulose, but is not limited thereto. For example, the tagatose-producing enzyme may be a fructose-4-epimerase or an arabinose isomerase, but is not limited thereto. For example, the allulose-producing enzyme may be a fructose-3-epimerase, but is not limited thereto. The amino acid producing enzyme may include, without limitation, not only amino acids but also those involved in the synthesis of their precursors, for example, acetohydroxy acid synthase, pyruvic acid dehydrogenase, amino acid aminotransferase, aspartokinase, acetyl homo It may be serine sulfidylase, cystathionine gamma synthase, but is not limited thereto. The nucleic acid may specifically be IMP, XMP, or GMP, but is not limited thereto, and the nucleic acid producing enzyme may also include an enzyme involved in the synthesis of its precursor without limitation. For example biotin carboxylase, glyceraldehyde-3-phosphate dehydrogenase, lipoamide dehydrogenase, fumaase, 5'-nucleotidase, xanthyl acid aminase, or proline dehydrogenase may be, but is not limited thereto. In addition, the membrane protein includes those involved in the discharge or inflow of a target substance. However, the types of the above-described proteins are merely examples and are not limited thereto. Meanwhile, the above-mentioned target protein includes not only the wild type but also mutants thereof. For example, the target protein may be fructose-4-epimerase, including a polynucleotide for the expression, and fructose-4-epimerization encoded from a polynucleotide encoding fructose-4-epimerase. The expression of the enzyme can be enhanced compared to the enzyme in which the polynucleotide for expression is encoded from a polynucleotide containing a wild-type TIR before mutation or.

Another aspect of the present application is to provide a vector comprising a polynucleotide for the expression.

The polynucleotide for the expression is as described above.

In the present application, the term "vector" refers to a DNA preparation containing the base sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

The vector may be in the form of an operably linked polynucleotide for expression of the present application.

In addition, the vector includes a polynucleotide for the expression, and may include a polynucleotide encoding a target protein, and may be in a form in which the polynucleotide encoding the target protein is operably linked.

In the present application, the term "operably linked" generally refers to a nucleotide expression control sequence and a nucleotide sequence encoding a target protein are operably linked to perform a function, thereby affecting the expression of the coding nucleotide sequence. The operable linkage with the vector can be prepared using a genetic recombination technique known in the art, and site-specific DNA cutting and ligation can be made using a cutting and ligation enzyme in the art.

The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as a phage vector or a cosmid vector, and pHCP (Korea Patent Publication No. 10- 2018-0092110), pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

For example, a polynucleotide encoding a target protein may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule is inserted, and to confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or expression of a surface polypeptide. markers may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.

In another aspect of the present application, in the base sequence of SEQ ID NO: 1, i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine. , polynucleotides for expression; a polynucleotide comprising a polynucleotide for the expression and encoding a protein of interest; Or a vector comprising a polynucleotide for the expression; to provide a microorganism expressing a target protein comprising the.

The polynucleotide for the expression, the polynucleotide encoding the target protein, and the vector are the same as described above.

As used herein, the term “a microorganism expressing a target protein” refers to a microorganism having a naturally weak target protein-producing ability or a microorganism to which a target protein-producing ability is imparted to a parent strain lacking the target protein-producing ability.

The microorganism may be a recombinant microorganism produced by using a method of introducing a vector including a polynucleotide for expression into a host cell. The method for transforming the vector may include any method of introducing a polynucleotide into a cell, and may be performed by selecting an appropriate standard technique as known in the art. For example, electroporation (electroporation), calcium phosphate co-precipitation (calcium phosphate co-precipitation), retroviral infection (retroviral infection), microinjection (microinjection), DEAE-dextran (DEAEdextran), cationic liposome (cationic) liposome) method and heat shock method, but is not limited thereto.

In the present application, the term "transformation" means introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell.

The transformed gene may include both a form inserted into a chromosome of a host cell and a form located extrachromosomally, as long as it can be expressed in the host cell. In addition, the gene includes DNA and RNA as polynucleotides, and can be used without limitation as long as it can be introduced into a host cell and expressed. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a recombinant vector capable of self-replication. In addition, the gene may be introduced into a host cell by itself or in the form of a polynucleotide construct, and may be operably linked to a sequence necessary for expression in the host cell.

In the present application, the term polynucleotide or polypeptide/protein “to be expressed” refers to a state in which a target polynucleotide or polypeptide is introduced into or modified to be expressed in a microorganism, wherein the polynucleotide or polypeptide is expressed in the microorganism. In the case of an existing protein, it refers to a state in which its activity is enhanced compared to before endogenous or modified.

Specifically, "introduction of a polypeptide (or protein)" refers to exhibiting improved activity compared to the intrinsic activity or activity prior to modification of the target protein, or exhibiting the activity of a specific polypeptide that the microorganism did not originally have. For example, a polynucleotide encoding a specific polypeptide may be introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific polypeptide may be introduced into a microorganism to exhibit its activity. In addition, "enhancement of activity" means that the activity is improved compared to the intrinsic activity or activity before modification of a specific polypeptide possessed by the microorganism. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain before the transformation when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors.

Specifically, the enhancement of the activity of the present application is a method of linking and expressing a polynucleotide for expression of the present application to the top of a gene encoding a target protein, a polynucleotide for expression of the present application and/or a gene encoding a target protein A method for increasing the intracellular copy number of a gene encoding a target protein by introducing a vector capable of replicating and functioning independently of a host to an operably linked method, a polynucleotide for expression of the present application and/or From the group consisting of a method of replacing the expression control sequence with a sequence with strong modification or activity to increase the expression of the gene encoding the target protein, and a method of additionally introducing a mutation into the gene encoding the target protein to enhance the activity of the target protein It may be made by any one or more methods selected, but is not limited thereto.

The increase in the intracellular copy number of the gene, but is not particularly limited thereto, may be performed by introducing the vector into a host cell or insertion into a chromosome in the host cell. Specifically, a vector capable of replicating and functioning independently of a host, to which a gene encoding a polynucleotide and/or a target protein for expression of the present application is operably linked, may be introduced into a host cell. Alternatively, a gene encoding a polynucleotide and/or a target protein for expression of the present application may be inserted into a chromosome in a host cell to which a gene encoding a polynucleotide and/or a target protein is operably linked. The vector may be introduced into the chromosome of the host cell. Insertion of a gene encoding a polynucleotide for expression and/or a target protein into a chromosome may be accomplished by any method known in the art, for example, by homologous recombination.

Next, modifying the expression control sequence to increase the expression of the gene encoding the polynucleotide and/or the target protein of the present application is not particularly limited thereto, but the nucleic acid sequence is deleted to further enhance the activity of the expression control sequence. , insertion, non-conservative or conservative substitution, or a combination thereof by inducing a mutation in the sequence, or by replacing the nucleic acid sequence with a stronger activity. The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence for regulating the termination of transcription and translation, and the like.

A strong promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter, but is not limited thereto. Examples of known strong promoters include cj1 to cj7 promoter (Korean Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter , SPL13 (sm3) promoter (Korean Patent No. 10-1783170), O2 promoter (Korean Patent No. 10-1632642), tkt promoter and yccA promoter, but are not limited thereto.

In addition, the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but a mutation in the expression control sequence by deleting, inserting, non-conservative or conservative substitution of a nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing or replacing the polynucleotide sequence improved to have stronger activity.

The introduction and enhancement of the activity of the target protein may be one in which the expression, activity or concentration of the corresponding polypeptide is increased based on the expression, activity or concentration of the polypeptide in a wild-type or unmodified microbial strain, but is not limited thereto it is not

In the present application, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in a microorganism, it is a native strain itself, or a microorganism that does not contain a gene encoding the polynucleotide and the target protein , or refers to a microorganism that has not been transformed with a vector including a gene encoding the polynucleotide and the target protein.

The recombinant microorganism may include any of prokaryotic microorganisms and eukaryotic microorganisms as long as the microorganisms capable of producing the target protein, including the polynucleotide for expression of the present application, the polynucleotide encoding the target protein, or a vector including the same. For example , Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, and Brevibacterium genus A microbial strain belonging to may be included, specifically, Corynebacterium ( Corynebacterium ) It may be a genus, for example, Corynebacterium ( Corynebacterium ) The genus is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium Ammonia Genes ( Corynebacterium ammoniagenes ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium caluna ( Corynebacterium callunae ), Corynebacterium stationis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Coryne ) , Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans Cxorynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ) or Corynebacterium flavescens ( Corynebacterium flavescens ), etc. may be , More specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ) It may be, but is not limited thereto.

For the purposes of the present application, the recombinant microorganism may be a microorganism in which the production of the target protein is increased compared to that of a wild-type or unmodified microorganism strain.

In another aspect of the present application, in the base sequence of SEQ ID NO: 1, i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine. , polynucleotides for expression; a polynucleotide comprising a polynucleotide for the expression and encoding a protein of interest; Or a vector comprising a polynucleotide for the expression; to provide a method for producing a target protein, comprising the step of culturing a microorganism expressing the target protein comprising a medium.

The polynucleotide for the expression, the polynucleotide encoding the target protein, and the vector are the same as described above.

In the present application, the term "cultivation" means growing the microorganism in an appropriately controlled environmental condition. The culture process of the present application may be made according to an appropriate medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be batch, continuous, and fed-batch, but is not limited thereto.

In the present application, the term "medium" refers to a material mixed with nutrients required for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganisms of the present application may be used without any particular limitation as long as they are conventional media used for culturing microorganisms. It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids and/or vitamins.

As the carbon source in the present application, carbohydrates such as glucose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium monobasic phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.

In the present application, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner during culturing of the microorganism to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without injection of gas or without gas to maintain anaerobic and microaerobic conditions. it is not

The temperature of the medium may be 20 °C to 50 °C, specifically 30 °C to 37 °C, but is not limited thereto. The incubation period may be continued until a desired production amount of a useful substance is obtained, and specifically, it may be 10 hours to 100 hours, but is not limited thereto.

The target protein produced by the culture may be discharged into the medium or may remain in the cell without being discharged.

The method for producing the target protein may include recovering the target protein from the cultured medium or microorganism.

The method for recovering the target protein produced in the culturing step of the present application may be to collect the target protein from the culture medium using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. may be used, and a target protein may be recovered from a medium or microorganism using a suitable method known in the art.

In addition, the recovery step may include a purification process, and may be performed using a suitable method known in the art. Accordingly, the recovered target protein may be a purified form or a microbial fermentation broth containing the target protein (Introduction to Biotechnology and Genetic Engineering, A. J. Nair., 2008).

In another aspect of the present application, in the base sequence of SEQ ID NO: 1, i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine. , polynucleotides for expression; a polynucleotide comprising a polynucleotide for the expression and encoding a protein of interest; Or a vector comprising a polynucleotide for the expression; culturing a microorganism expressing the target protein comprising the step of culturing the target protein in a medium to produce the target protein and contacting the produced target protein with a substrate, the target product production to provide a way

The method for producing the target product may further include recovering the target product.

The method of recovering the target product of the present application may be to collect the target product using a suitable method known in the art according to a method of contacting the target protein with a substrate. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. may be used, and a desired product may be recovered using a suitable method known in the art.

In addition, the recovery step may include a purification process, and may be performed using a suitable method known in the art.

Another aspect of the present application includes i) substitution of the 24th base with thymine, ii) the substitution of the 27th base with cytosine, and iii) the substitution of the 33rd base with cytosine in the base sequence of SEQ ID NO: 1 It is to provide a method for increasing the expression level of the target protein.

Including the polynucleotide for expression of the present application, the polynucleotide encoding the target protein has a significantly higher expression level of the target protein than before mutation or when wild-type TIR is included. Economical is high.

1 is a schematic diagram of a pHCP plasmid.
Figure 2 is a diagram showing the 5' UTR and TIR portion changed in the vector.
3 is a diagram showing fluorescence values according to TIR library expression.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Preparation of Vector

For the plasmid, a pHCP plasmid in which the 21st position of orfA2 (parB) was mutated to adenine (A) (Korean Publication No. 10-2018-0092110, FIG. 1) was used for the plasmid. In the present application, an attempt was made to obtain a plasmid with a higher copy number than the existing pHCP, and for this purpose, the sequence of 98bp, which is the ctRNA part of pHCP, was randomly changed and screened. As a result, the 22nd nucleotide of the ctRNA part was finally substituted with C (cytosine). A polynucleotide in which the 79th nucleotide was substituted with C (cytosine) was identified, and a high copy number plasmid containing it was designated as a pHCP7 vector (SEQ ID NO: 4).

The current expression level was predicted using the existing 5' UTR designer tool to increase the expression level by introducing a mutation in the 5' UTR (Untranslated region) part, which is known to affect the expression level. The conventionally constructed expression system is a system in which a new TN (KNF4E) tagatose convertase is expressed in a pHCP7 vector (Application No. 10-2019-0082850). The expression level of the existing expression system predicted using the UTR designer tool (Seo, Sang Woo, et al. "Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency." Metabolic engineering 15 (2013): 67-74.) was 42,337 were predicted. Based on the sequence of the existing expression system, the 5' UTR library sequence was predicted by diversifying the expression amount to be constructed through the 5' UTR library in the range of 1,002 to 1,000,000 (Fig. 2). did For the 5' UTR sequence, the 5' UTR sequence of the existing expression system (SEQ ID NO: 5) and the library 5' UTR sequence (SEQ ID NO: 6) were used. The library sequence introduced to improve the 5' UTR region for effectively increasing the expression level may have a variety of ^{sequences of 2.56 x 10 2 .} The PCR products with various TIR part pHCP 7 vector was modified by inserting a Kpn I, Nde I restriction site in order to build a library including the same. This was constructed with a size of ^{5.5 x 10 3} larger than the range of diversity to be constructed in E. coli for genetic manipulation.

Example 2: TIR library construction

Except for the 5' UTR part, The TIR (translation initiation region) part, which is known to affect the expression level, is the target protein TN ( A degenerated primer was prepared for the 14 amino acid sequence of the front part of KNF4E) (fructose-4-epimerase). In order to effectively confirm the difference in expression level due to the altered sequence, a library was constructed by fusion of sfGFP, a fluorescent protein, after 33 amino acid sequences in the front part of TN (KNF4E). Except for ATG, which is the start codon of TN (KNF4E), the TIR part was changed by changing only the codon using the primer for the 14 amino acid sequence that is the TIR part. This is 1.47 x 10⁵Variations in size were included. The altered TIR portion in the vector is shown in FIG. 2 . The TN(KNF4E)-sfGFP gene containing a library with an altered TIR region was amplified using PCR, whichNdeI,NotAfter cleavage with the I restriction enzyme site, it was additionally introduced into the vector of Example 1, so that the difference in the expression amount of sfGFP, a fluorescent protein, could be confirmed as a fluorescence value. The library of TIR fractions was 1.8 x 10 in E. coli.⁵ It was constructed to size, and it was recovered and transformed into a Corynebacterium glutamicum strain.

Example 3: Comparison of fluorescence expression level of TIR library

In the library transferred to the Corynebacterium glutamicum strain, the intensity of the fluorescent protein expressed was compared using a fluorescence-activated cell sorting (FACS) to compare the expression level. Based on the existing pHCP 7_H4 promoter_T7 RBS_TN(KNF4E)sfGFP expression system, clones with increased fluorescent protein expression were identified.

Specifically, as a result of confirming the fluorescence value of each library, as shown in FIG. 3 , compared to the pHCP 7_H4 promoter_T7 RBS_TN(KNF4E)sfGFP having an average value of about 2,154, L09 (SEQ ID NO: 2) was 3,555, L16 (SEQ ID NO: 3) ) was confirmed to have a high fluorescence value of 2,496, and it was confirmed that a change in the sequence of this part affects the expression level. L09 and L16 confirmed that the UTR sequence is SEQ ID NO: 5.

The strains transformed with the L09 and L16 libraries showing the high fluorescence values were named Corynebacterium glutamicum CF01-0012 and Corynebacterium glutamicum CF01-0013, respectively, and deposited at the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, as of October 18, 2019. and were given accession numbers KCCM12608P and KCCM12609P.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Korea Microorganism Conservation Center (Overseas) KCCM12608P 20191018 Korea Microorganism Conservation Center (Overseas) KCCM12609P 20191018

<110> CJ CheilJedang Corporation <120> Polynucleotides to enhance expression of a desired polypeptide and their uses <130> KPA191001-KR <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 45 <212> DNA <213> Unknown <220> <223> TIR of Kosmotoga olearia fructose 4-epimerase wild type <400> 1 atgaaaaaac atcctcttca ggacattgtt tcattgcaaa aacag 45 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> L09 TIR <400> 2 atgaaaaaac accctcttca ggatatcgtt tcccttcaaa agcaa 45 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> L16 TIR <400> 3 atgaagaaac acccgctgca ggatatcgtg tccctgcaga aacag 45 <210> 4 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> pHCP7 <400> 4 gctgcacgaa tacctgaaaa acgttgaacg ccccgtgagc ggtaactcac agggcgtcgg 60 ctaaccccca gtccaaacct gggagaaagc gctcaaaa 98 <210> 5 <211> 20 <212> DNA <213> Unknown <220> <223> UTR wild type <400> 5 aactttaaga aggagatata 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> UTR library <220> <221> misc_feature <222> (2) <223> R is G or A. <220> <221> misc_feature <222> (7) <223> R is G or A. <220> <221> misc_feature <222> (10) <223> R is G or A. <220> <221> misc_feature <222> (12) <223> R is G or A. <220> <221> misc_feature <222> (15) <223> S is G or C. <220> <221> misc_feature <222> (6) <223> K is G or T/U. <220> <221> misc_feature <222> (19) <223> K is G or T/U. <220> <221> misc_feature <222> (20) <223> W is A or T/U. <400> 6 arcttkragr argasatakw 20 <210> 7 <211> 1308 <212> DNA <213> Unknown <220> <223> Kosmotoga olearia fructose 4-epimerase <400> 7 atgaaaaaac atcctcttca ggacattgtt tcattgcaaa aacagggaat acccaaaggg 60 gttttctctg tatgtagtgc caatagattt gttattgaaa ccactctgga atatgcgaag 120 atgaaaggga caacggttct tatagaggcc acctgcaatc aggtaaacca gttcggtggc 180 tacaccggta tgactcctgc tgatttcaga gaaatggttt tttctatcgc tgaggatatt 240 ggacttccca aaaataaaat catccttggt ggcgaccatc ttggcccaaa tccctggaag 300 ggtcagccgt cagatcaggc tatgcgtaac gccattgaaa tgattcgaga atacgctaaa 360 gctgggttta ccaagcttca tctggatgcc agcatgcgtc ttgcagacga tccggggaac 420 gaaaacgagc cgctgaaccc ggaagttata gcggaaagaa cagctcttct ctgtcttgaa 480 gccgagaggg cttttaaaga atccgccggt tctctccggc ctgtttacgt tattggtacg 540 gatgttccgc caccgggtgg agcgcaaaac gaaggtaaat cgattcatgt aaccagtgtt 600 caggattttg agcgtaccgt tgagttgacc aaaaaggcat ttttcgacca tggtttgtat 660 gaagcctggg gaagggtgat tgcggttgtt gtgcaaccgg gagtagaatt cgggaatgaa 720 catatattcg aatatgatag aaatcgagcg agagaactta ctgaggcgat aaaaaagcat 780 ccaaatatag tttttgaagg tcactcgaca gattatcaaa cggcaaaagc attgaaagaa 840 atggtagaag acggtgtagc catactcaag gttgggccag ctctaacatt tgcgctcaga 900 gaggcttttt ttgcgttgag cagcattgaa aaagagttat tttatgatac acccgggctt 960 tgttcaaact ttgttgaagt tgtcgagaga gcgatgcttg acaatccaaa acattgggaa 1020 aaatattacc agggagaaga gagagaaaat agattagccc gtaaatacag ctttctcgat 1080 cgcttgaggt attactggaa tcttcctgag gttagaacag cggtgaataa gctgataacc 1140 aaccttgaaa caaaagaaat cccgttaacg cttataagcc agttcatgcc gatgcagtac 1200 caaaaaatca gaaacggttt gctaagaaag gatccaataa gccttataaa agatcgaatt 1260 acccttgttc ttgatgacta ctatttcgca actcaccctg aatgttga 1308

Claims (13)

TIR (translation initiation region) comprising the nucleotide sequence of SEQ ID NO: 2 or 3.
delete delete The TIR of claim 1 ; and a UTR of SEQ ID NO: 5 on top of the TIR.
A polynucleotide comprising the TIR of claim 1 and encoding a protein of interest.
The polynucleotide of claim 5, wherein the target protein is fructose-4-epimerase.
A vector comprising the TIR of claim 1.
TIR comprising the nucleotide sequence of SEQ ID NO: 2 or 3; a polynucleotide comprising the TIR and encoding a protein of interest; Or a vector comprising the TIR; Corynebacterium genus microorganism expressing a target protein comprising a.
The microorganism according to claim 8, wherein the microorganism is Corynebacterium glutamicum.
TIR comprising the nucleotide sequence of SEQ ID NO: 2 or 3; a polynucleotide comprising the TIR and encoding a protein of interest; Or a vector comprising the TIR; comprising the step of culturing a microorganism expressing the target protein comprising the in a medium, the target protein production method.
11. The method of claim 10, further comprising the step of recovering the target protein from the cultured microorganism or culture, the target protein production method.
TIR comprising the nucleotide sequence of SEQ ID NO: 2 or 3; a polynucleotide comprising the TIR and encoding a protein of interest; Or a vector comprising the TIR; culturing a microorganism expressing the target protein comprising the step of culturing the target protein in a medium, and the step of contacting the produced target protein with a substrate, a target product production method.
13. The method of claim 12, further comprising the step of recovering the target product.

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