KR102198826B1 - Novel vector having enhanced copy number and use thereof - Google Patents

Abstract

The present application relates to a vector having an increased copy number and a method for producing a target substance using the same. A plasmid of the present application has a high copy number, and thus can be used as a vector for high-efficiency production of a target protein.

Description

BACKGROUND OF THE INVENTION [0002] A vector having an increased copy number and its use

The present application relates to a vector having an increased copy number and a method for producing a target substance using the same.

Microorganisms in Corynebacterium are strains that are applied in various industrial fields. It is a strain mainly used for the production of amino acids and nucleic acids, and since it is generally considered safe, it is also used to produce various metabolites and food additives. It is not only used for the production of these various metabolites and food additives, but is also widely used to produce recombinant proteins.

An important objective in a process using microorganisms of the genus Corynebacterium is to obtain as much product as possible with excellent quality at an effective cost. To this end, the amount of the gene encoding the target product, the recombinant protein, becomes a very important factor, and a highly efficient gene expression system is required to induce overexpression of the target gene in microorganisms in Corynebacterium.

In recent years, as plasmids have become major in the field of gene therapy, studies to increase the number of genes possessed by microorganisms are generally conducted as a method to increase the expression level of specific genes. Accordingly, efforts are being made to develop a plasmid with a high copy number, and by inserting a desired gene into a plasmid with a high copy number and transforming the recombinant plasmid into a microorganism again, the effect of increasing the gene by the number of copies of the plasmid per microorganism is expected. I can.

The plasmid used in Corynebacterium glutamicum requires an origin of replication in E. coli required for easy construction of a recombinant protein and a replication origin in Corynebacterium glutamicum for use in an expression host. The pCES208 plasmid (Korean Publication 10-2018-0092110) contains the pCG1 origin of replication isolated from Corynebacterium glutamicum and has been used to produce recombinant proteins. It is known that the open reading frame (ORF) and ctRNA (antisense RNA) influence the regulation of replication.

Under this technical background, the inventors of the present application completed this application by introducing mutations in the copy number control site of the plasmid in order to use the plasmid with higher efficiency, and confirming that the target protein can be highly expressed by increasing the copy number. I did.

The object of the present application is to provide a polynucleotide sequence encoding ctRNA, and a vector including the same.

Another object of the present application is to provide a composition for gene expression comprising the polynucleotide.

Another object of the present application is to provide a microorganism containing the vector.

Another object of the present application is to provide a method for increasing the expression of a protein of interest, comprising introducing the vector into a microorganism.

Another object of the present application is to provide a method of producing a target protein comprising culturing the microorganism.

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 various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.

Further, those of ordinary skill in the art may recognize or ascertain a number of equivalents to certain aspects of the present application described in this application using only routine experimentation. Also, such equivalents are intended to be included in this application.

One aspect of the present application provides a polynucleotide sequence encoding ctRNA, and a vector including the same.

In the present application, the term "polynucleotide" is a polymer of nucleotides in which nucleotide units are connected in a long chain by covalent bonds, and is a DNA strand having a predetermined length or more.

For the purposes of the present application, the polynucleotide may be a region encoding ctRNA in a vector.

In the present application, the term "ctRNA" is an abbreviation of counter-transcribed RNA, and is a sequence that controls replication. Specifically, it may be a sequence involved in controlling the copy number of the vector. The ctRNA can inhibit transcription by complementarily binding to the mRNA sequence transcribed in the vector, and thus may be referred to as "antisense RNA". Specifically, the ctRNA may be involved in the regulation of the number of copies by complementary binding to the mRNA encoding the replication initiation protein.

The polynucleotide sequence of the present application is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with SEQ ID NO: 1 and/or SEQ ID NO: 1 One or more nucleotides in a polynucleotide sequence having homology or identity may be substituted with other nucleotides, but are not limited thereto.

The substitution may include substitution of the 22nd and/or 79th nucleotide in the polynucleotide sequence of SEQ ID NO: 1 with another nucleotide. The other nucleotide is not limited as long as it is different from the nucleotide before the mutation.

Specifically, the polynucleotide sequence may be a polynucleotide sequence encoding ctRNA, in which the 22nd nucleotide and/or the 79th nucleotide in the polynucleotide sequence of SEQ ID NO: 1 is substituted with another nucleotide. The 22nd nucleotide may be substituted with C (cytosine). The 79th nucleotide may be substituted with T (thymine). However, the present invention is not limited thereto, and is not limited and is included in the scope of the present application as long as the number of copies of the vector can be increased by being included in the vector. That is, it is obvious that those that are identical to or have 80%, 85%, 90%, 95% or more homology to the polynucleotide sequence described above are included in the scope of the present application as long as the copy number of the vector can be increased.

The polynucleotide sequence may include a polynucleotide sequence of SEQ ID NO: 2 or 3, or a polynucleotide sequence having at least 80%, 85%, 90%, or 95% homology with each sequence, but is not limited thereto. Does not.

Even if it is described as'polynucleotide described with a specific sequence number' in the present application, if it has the same or corresponding activity as the polynucleotide containing the sequence or the polynucleotide sequence of the sequence number, some sequences are deleted, modified, It is obvious that polynucleotides having substituted or added nucleotide sequences can also be used in the present application. For example, if it has the same or corresponding activity as the polynucleotide, it does not exclude the addition of meaningless sequences before and after the nucleotide sequence of the corresponding sequence number, or mutations that may occur naturally, or silent mutations thereof. It is obvious that, even when having such a sequence addition or mutation, it falls within the scope of the present application.

Homology and identity refer to the degree to which two given base sequences are related and can be expressed as a percentage.

The terms homology and identity can often be used interchangeably.

The sequence homology or identity of conserved polynucleotides is determined by standard alignment algorithms, and the default gap penalty established by the program used can be used together. Substantially, homologous or identical sequences are generally in moderate or high stringent conditions along at least about 50%, 60%, 70%, 80% or 90% of the sequence or full-length. (stringent conditions) can be hybridized. Polynucleotides containing degenerate codons instead of codons in hybridizing polynucleotides are also contemplated.

Whether any two polynucleotide sequences have homology, similarity or identity is determined, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: Can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Alternatively, as performed in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, 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, homology, similarity or identity can be determined using BLAST, or ClustalW of the National Center for Biotechnology Information.

Homology, similarity or identity of polynucleotides can be found in, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2:482, for example, Needleman et al. (1970), J Mol Biol. 48: 443 can be determined by comparing sequence information using a GAP computer program. In summary, the GAP program defines the total number of symbols in the shorter of two sequences, divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). The default parameters for the GAP program are (1) a monolithic comparison matrix (contains 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. As disclosed by 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 the relevance between sequences.

In addition, the polynucleotide of the present application has various modifications to the coding region within a range that does not change the polynucleotide sequence due to the degeneracy of the codon or in consideration of the preferred codon in the organism to express the polynucleotide. This can be done. In addition, a probe that can be prepared from a known gene sequence, for example, hybridizes under stringent conditions with a complementary sequence for all or part of the nucleotide sequence, and at least one nucleotide in the polynucleotide sequence of SEQ ID NO: 1 is converted to another nucleotide. Any sequence that is substituted and encodes ctRNA may be included without limitation. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition , Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989). For example, between genes with high homology or identity, 40% or more, specifically 70% or more, 80% or more, 85% or more, 90% or more, more specifically 95% or more, More specifically, genes having a homology or identity of 97% or more, in particular, 99% or more, are hybridized, and genes with lower homology or identity are not hybridized, or in general Southern hybridization (southern hybridization). hybridization) at a salt concentration and temperature equivalent to 60°C, 1XSSC, 0.1% SDS, specifically 60°C, 0.1XSSC, 0.1% SDS, and more specifically 68°C, 0.1XSSC, 0.1% SDS, The conditions for washing once, specifically two to three times, can be enumerated.

Hybridization requires that two nucleic acids have a complementary sequence, although a mismatch between bases is possible depending on the stringency of the 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. Thus, the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire 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 a person skilled in the art according to the purpose.

The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art.

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

The composition for gene expression refers to a composition capable of expressing a target gene including the sequence of the present application. For example, the composition for gene expression may include the polynucleotide, and may include, without limitation, a composition for expressing a target gene. Alternatively, a configuration that allows the polynucleotide to function as a copy number regulating factor may also be included, but is not limited thereto. In the composition for gene expression of the present application, the polynucleotide may be in a form contained in a vector to express a target gene operably linked in the introduced host cell.

In the present application, the term "vector" refers to a DNA preparation containing a base sequence encoding the target polynucleotide operably linked to a suitable control sequence so that the target polynucleotide can be expressed in a suitable host. The regulatory sequence includes a promoter capable of initiating transcription, any 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. Sequences encoding ctRNA for purposes may also be included. Vectors can be transformed into a suitable host and then replicated or function independently of the host genome, and can be integrated into the genome itself.

The vector used in the present application is not particularly limited as long as it can be replicated in the host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages, and specifically, may be plasmid vectors.

In the present application, the term "plasmid" refers to a cyclic non-chromosomal element present in bacteria. For the preparation of plasmids, delivery and binding of plasmid DNA, transformation, etc., those of ordinary skill in the art may use known methods, and the known methods are described in the literature [eg, Sambrook, J. etal.,'Molecular Cloning: A Laboratory Manual, Second Edition', Cold Spring Harbor Laboratory Press (1989)].

As the plasmid vector, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used, specifically pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC A vector or the like may be used, but is not limited thereto.

The target polynucleotide linked to the vector may be a gene encoding a protein of interest, and may be a gene encoding a polypeptide with a specific target nucleic acid to be transcribed. The target gene may be any gene desired by a person skilled in the art, and may be a gene derived from a foreign species or other strain other than a host strain, or a gene in a modified form, but is not limited thereto. The target protein may be a protein for which the expression level is to be increased. The target protein includes a fluorescent protein and a protein involved in the production of the target material, and may be an enzyme or a membrane protein (transporter). The target substance may be a saccharide, an amino acid and/or a nucleic acid, but is not limited thereto.

The saccharide may be specifically tagatose or allulose, but is not limited thereto. For example, the tagatose-producing enzyme may be a fructose-4-epimerase or 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 its precursors, for example acetohydroxy acid synthase, pyruvate dehydrogenase, amino acid aminotransferase, aspartokinase, acetylhomo Serine sulfhydrylase, cystathionine gamma synthase may be, but is not limited thereto. The nucleic acid may be specifically IMP, XMP, or GMP, but is not limited thereto, and the nucleic acid-producing enzyme may also include, without limitation, enzymes involved in its precursor synthesis. For example, biotin carboxylase, glyceraldehyde-3-phosphate dehydrogenase, lipoamide dehydrogenase, fumarase, 5'-nucleotidase, xanthylate 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 the target substance. However, the types of proteins described above are only examples and are not limited thereto. Meanwhile, the above-described target protein includes not only the wild type but also its mutant.

The vector may contain a selectable marker for selecting a host cell containing the vector. The selectable marker is for selecting cells transformed with a vector, and markers conferring a selectable phenotype such as drug resistance, auxotrophic resistance, resistance to cytotoxic agents, or expression of surface proteins may be used. Since only cells expressing a selectable marker survive in an environment treated with a selective agent, transformed cells can be selected.

In the present application, the vector may be a shuttle vector. In the present application, the term "shuttle vector" means a vector that can be maintained in a plurality of strains. The shuttle vector may be a shuttle vector between microorganisms of the genus Corybacterium and microorganisms of genus Escherichia, specifically, may be a shuttle vector between Corynebacterium glutamicum and Escherichia coli , but is limited thereto. It doesn't work.

The Escherichia coli-Corynebacterium glutamicum shuttle vector is capable of replication and/or maintenance in both Escherichia coli and Corynebacterium glutamicum, and includes both the origin of replication of Escherichia coli and the origin of Corynebacterium glutamicum. can do. Further, for example, after cloning a foreign gene or a gene causing a mutation in E. coli into a shuttle vector, the shuttle vector may be introduced into Corynebacterium glutamicum to induce a desired trait, but is not limited thereto.

In the present application, the vector may include parB and/or repA at a site upstream or downstream of the polynucleotide of the present application. parB is a sequence encoding a protein that binds to centromere, and is involved in the regulation of the number of copies of the plasmid. repA refers to a sequence encoding a protein that codes for a replication start site of a plasmid, and each sequence can be appropriately selected and used from a known database. parB and repA may also include mutated sequences for replication and/or copy number control, but are not limited thereto, and naturally mutated sequences are also included.

In addition, the vector may include genes involved in replication and/or copy number control, such as an origin of replication and a promoter, and may include a restriction enzyme site, but is not limited thereto.

In this application, "copy number" means the average number of plasmids present in a cell. The number of copies can be determined by adjusting the copy start origin or the open reading frame of the copy control site. For example, it is possible to control the expression level of an open reading frame essential for replication, or by inducing a mutation in the open reading frame. The copy number of the vector of the present application includes the polynucleotide of the present application, so that the polynucleotide of the present application may not be included or the copy number may be maintained higher than that of the unmodified or natural vector, but is not limited thereto.

Another aspect of the present application provides a microorganism comprising the vector.

Specifically, the microorganism may be a microorganism transformed with a vector including the polynucleotide and the gene encoding the target protein, or may be a microorganism including the polynucleotide and the gene encoding the target protein, but is not limited thereto. .

In the present application, the term "transformation" refers to 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. Transformed polynucleotides may include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the protein of interest. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" in the above means that a promoter sequence for initiating and mediating transcription of the polynucleotide of the present application and the gene sequence are functionally linked. The method of transforming the vector may include any method of introducing a polynucleotide into a cell, and as known in the art, a suitable standard technique may be selected and performed. For example, electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposomes (cationic) liposome) method, thermal shock method, protoplasm fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fiber, agrobacteria mediated transformation, PEG, dextran sulfate, lipofectamine and drying/ Inhibitory mediated transformation and the like may be included, but are not limited thereto.

The microorganism is not limited to any one capable of producing a protein of interest, and may be, for example, Corynebacterium sp., or Escherichia sp., and specifically Corynebacterium. Bacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium flavu m ( Brevibacterium flavu m), Corynebacterium It may be thermoaminogenes ( Corynebacterium thermoaminogenes ), Corynebacterium episodes ( Corynebacterium efficiens ), Corynebacterium stationis ( Corynebacterium stationis ) or Escherichia coli . It is not limited thereto.

Another aspect of the present application provides a method of increasing the expression of a target protein comprising introducing the vector of the present application into a microorganism.

Since the vector including the polynucleotide of the present application has an increased copy number, it can be used to increase the expression of a target protein in a microorganism. The target protein is as described above.

Another aspect of the present application provides a method for producing a target substance, comprising culturing a microorganism containing the vector of the present application.

The target material is as described above. When the target substance is a substance other than a protein, the production method may be a method of producing the target substance by including a gene encoding a protein involved in the production of the target substance in the vector, but is not limited thereto. In the above method, the step of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited thereto, but a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) is used to provide an appropriate pH (eg, pH 5 to 9, specifically Is capable of adjusting pH 6 to 8, most specifically pH 6.8), and maintaining aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and may be cultured for about 10 to 160 hours, but is not limited thereto.

In addition, the culture medium used is a carbon source such as sugar and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g., soybean oil). , Sunflower seed oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid and linoleic acid), alcohols (such as glycerol and ethanol) and organic acids (such as acetic acid), etc. It can be used, but is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) or the like may be used individually or in combination, but is not limited thereto. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a sodium-containing salt corresponding thereto may be used individually or in combination as a source of phosphorus, but are not limited thereto. In addition, the medium may contain essential growth-promoting substances such as other metal salts (eg, magnesium sulfate or iron sulfate), amino acids and vitamins.

Since the plasmid of the present application has a high copy number, it can be used as a vector for highly efficient production of a target protein, and is very useful for industrial applications.

1 is a schematic diagram of a pHCP plasmid expressing a fluorescent protein in order to use the FACS screening method of the present application.
2 shows a process of screening a plasmid having an increased copy number through a fluorescent flow cytometer for the present application. Empty indicates the value of the pHCP plasmid itself that does not express a fluorescent protein (Negative control), and WT indicates a cell expressing a fluorescent protein by combining a part of the D-tagatose converting enzyme and a fluorescent protein in pHCP. 6 ^th represents cells analyzed after 6 screening processes. The X-axis represents the relative amount of fluorescence of the cell, and the mean value represents the average value of fluorescence.
Figure 3 shows the amount of the plasmid obtained at the cell concentration as shown in the photograph of agarose electrophoresis after harvesting the plasmids obtained through screening in the present application, treatment with restriction enzymes. pCES208 is a plasmid that is the parent of pHCP and has a low copy number, and pHCP is a plasmid with a higher copy number than pCES208. As a control, a plasmid expressing H4_sfGFP in pCES208 and pHCP plasmid was used. In order to confirm the number of copies of the pHCP 7 and pHCP 51 plasmids discovered in the present application, an experimental group in which a gene in which the 99bp front part of D-tagatose converting enzyme (TN) and sfGFP are combined was expressed using the H4 promoter was used. The obtained plasmid systems were cut with one restriction enzyme site called Nco I and placed on an agarose gel to check the size and concentration. As a result, darker bands than the pHCP used as the reference were found in the plasmids pHCP51 and pHCP7 discovered in the present application. In addition, it can be confirmed that the plasmid amount obtained by harvesting the cells with an OD of 600 nm 4 was obtained at a concentration higher than that of pHCP.
FIG. 4 shows the stability of the plasmid when subcultured for 5 days using a system in which a part of D-tagatose converting enzyme and a fluorescent protein are expressed in combination in the high copy number plasmids discovered in the present application. It is a schematic diagram of comparison by value.
Figure 5 is a plasmid used to confirm the number of copies of the plasmid of interest in the present application, the system expressing the D-tagatose converting enzyme, the pHCP plasmid, and the plasmids (pHCP 7, pHCP 51) confirmed in the invention It is a schematic diagram showing the number of copies.
FIG. 6 is a schematic diagram showing a protein expressed by expressing D-tagatose converting enzyme in the plasmids discovered in the present application, and analyzed and quantified by SDS-PAGE. The arrow part is the target D-tagatose converting enzyme, the total protein is indicated as Total, and the water-soluble protein is indicated as Soluble. C is a cell having a plasmid system without an expression protein, H is using pHCP, and pHCP 7, pHCP 51 is using the plasmid system discovered in this application.
7 is a schematic diagram showing the conversion rate of tagatose after expressing D-tagatose converting enzyme in Corynebacterium glutamicum using the plasmid discovered in the present application. Each% represents the amount converted to tagatose from the substrate used.

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

Example 1: Random modification of ctRNA portion using Error prone PCR

In order to screen a plasmid having a high copy number, the ctRNA portion of the plasmid was randomly modified using the error prone PCR method. Error prone PCR was modified to have an error rate of about 1% by using dATP, dCTP, dGTP, dTTP and MgCl ₂ at different concentrations. As the plasmid, a pHCP plasmid (Korean Publication 10-2018-0092110) in which the 21st position of orfA2 (parB) was mutated to adenine (A) was used for high copy water. 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 portion of pHCP, was randomly changed. The randomly modified portion contains the ctRNA portion of pHCP (Fig. 1), and the randomly modified ctRNA portion PCR product was originally prepared using the Nco I, Eco RI restriction enzyme sites in front of and behind the PCR product containing the ctRNA altered sequence. By replacing the same restriction enzyme site of the pHCP plasmid, a plasmid library of various copies was constructed. The plasmid library was constructed with a size of 1.4 x 10 ⁷ in E. coli, and the resulting plasmids were recovered and constructed with a 2 x 10 ⁴ size library in the target Corynebacterium glutamicum host and used for screening.

Example 2: Screening of cells expressing fluorescent signal protein by fluorescent flow cytometry

The library constructed to obtain a plasmid with a high copy number was transformed into Corynebacterium glutamicum and cultured at 30°C for 24 hours using Brain Heart Infusion (BHI) medium, and then passaged once under the same conditions. After proceeding, analysis and screening were performed using FACS. Since the constructed library is used as a reporter protein by combining D-tagatose converting enzyme and fluorescent protein (GFP) of Korean Patent Registration No. 10-1550796, cells with the fluorescence intensity of the top 1% are classified based on the fluorescence signal using this. Obtained by. The obtained cells were inoculated into a new BHI medium and cultured at 30° C. for 24 hours, followed by one passage and then the screening process was repeated. As a result of the screening for a total of 6 times, cell groups having a fluorescence intensity increased by about 1.8 times compared to that of the pHCP plasmid were obtained (FIG. 2). This was plated on BHI solid medium to separate into single cells, and plasmids obtained from single cells were analyzed.

Example 3: Analysis of the discovered plasmid

Plasmids obtained from single cells were confirmed whether there was a change in the ctRNA portion through sequence analysis. As a result of sequence analysis, it was confirmed that it had the ctRNA sequence of SEQ ID NO: 2 or 3. That is, a plasmid having a sequence in which the sequence of the pHCP plasmid and the ctRNA part was changed by 1 bp was identified, and PCR was performed to confirm that there was no change in the sequence of the cloned part. Finally, a polynucleotide in which the 22nd nucleotide of the ctRNA part is substituted with C (cytosine), a polynucleotide in which the 79th nucleotide is substituted with C (cytosine) and a polynucleotide in which the 22nd nucleotide of the ctRNA part is substituted with T (thymine), Polynucleotides in which the 79th nucleotide was substituted with T (thymine) were identified, and these were designated as pHCP7 and pHCP51 plasmids, respectively. In order to confirm the amount of plasmid in the same cell concentration, a plasmid having a complex gene of D-tagatose converting enzyme and sfGFP was obtained in the presence of the H4 promoter. After treatment with restriction enzymes, a nanodrop was used to confirm the concentration using electrophoresis. The concentration was measured. As for the amount of plasmid compared to the cell concentration, pHCP7 and pHCP51 discovered in the present application were more than pHCP, which could be clearly confirmed in the agarose electrophoresis picture (Fig. 3).

Example 4: Confirmation of fluorescence protein expression and stability of the discovered plasmid

The stability of the expression of the fluorescent protein of the plasmid was confirmed by performing subculture for 5 days based on the fluorescent signal when the discovered fluorescent proteins of pHCP7 and pHCP51 and the reference pHCP were expressed. Corynebacterium strains having each plasmid were inoculated in BHI medium and cultured at 30° C. for 24 hours, and then, the fluorescent signal was measured using the strain that passed through one passaging process as Day1. Day1 was subcultured again and cultured at 30°C for 24 hours, and then Day2 was designated as Day2, and the fluorescence signal was also measured. In the same way, the fluorescence intensity of each day was measured through passage culture for a total of 5 days (FIG. 4). The fluorescence intensity was compared with the Absorbance unit (A.U) using TECAN Infinite M200Pro (Tecan Group Ltd, Mδnnedorf, Switzerland). A.U is a value obtained by dividing the fluorescence signal of 495nm Excitation and 515nm Emission value by the absorbance measured at 595nm. As shown in Fig. 4, as a negative control, it is a blank plasmid that does not express a fluorescent protein. Fluorescent protein expression using the pHCP plasmid was expressed as pHCP_TNsfGFP, and pHCP7 and pHCP51 were expressed as pHCP7_TNsfGFP and pHCP51_TNsfGFP, respectively. As a result of measuring the fluorescence intensity for 5 days, it was confirmed that the fluorescence values in the discovered pHCP7 and pHCP51 were higher than that in pHCP, and the fluorescence intensity that the fluorescence protein was stably expressed without decreasing the fluorescence value even in the passage for 5 days It could be confirmed through.

Example 5: Confirmation of the number of copies of the discovered plasmid

To confirm the number of copies of the discovered pHCP7 and pHCP51 plasmids, the reporter protein gene D-tagatose converting enzyme used for screening and the complex gene of fluorescent protein were removed, and the desired D-tagatose converting enzyme was inserted. It was used to confirm the number of copies (Fig. 5a). In order to confirm the number of copies of the plasmid, a quantitative method compared to the genome gene was performed using real-time PCR. The gene site used to confirm the number of copies of the plasmid used the p15A ori part of E. coli as the amplification site, and the SigB gene part of the genome gene of Corynebacterium was used as the amplification site. Compared to the number of copies of pHCP, it was confirmed that the discovered pHCP 7, pHCP 51 had 2.3 times and 3 times the number of copies, respectively (Fig. 5b). It was confirmed that the plasmid provided in the present application can be used as an improved plasmid having a greater number of copies than the existing pHCP plasmid.

The accession to the Corynebacterium glutamicum were Corynebacterium glutamicum _pHCP7, Corynebacterium glutamicum _pHCP51 as naming and the depositary institution Korea Culture Center of Microorganisms under the Budapest Treaty in June 2019, 04 dates including the pHCP7 and pHCP51, accession number KCCM12542P, Received KCCM12543P.

Example 6: Production of D-tagatose converting enzyme from the discovered plasmid

In order to confirm that the plasmid with improved copy number found in the present application can directly increase the amount of protein expression, the expression of the target product, D-tagatose converting enzyme, was confirmed. To confirm the expression of the system in which the D-tagatose converting enzyme was inserted into each plasmid in Corynebacterium, each of the cells was inoculated in BHI medium and cultured at 30°C for 24 hours, followed by one passaging process under the same conditions. After harvesting with an OD of _{600 nm} 4, cell disruption was performed using an ultrasonic disruptor. Each of the cells was cultured for the same time and subjected to ultrasonic disruption, and protein expression analysis was performed under the same conditions. In order to confirm the protein expression, the expression of the 56.6kDa D-tagatose converting enzyme was confirmed through the expression protein analysis process using SDS-PAGE. Protein analysis using Western blot was also carried out, which was confirmed using an anti-histidine antibody using 6 histidine tags attached to D-tagatose converting enzyme, and as only a band of the expected protein size was identified, the desired D-tag It was possible to confirm the expression and amount of the toss converting enzyme. Using the SDS-PAGE result, the expression amount of D-tagatose converting enzyme relative to the total protein produced was quantitatively analyzed using the Gel Analyzer program. In FIG. 6, C is a vector without an expression gene, H is a D-tagatose converting enzyme using pHCP, and 51 and 7 are expression levels in an expression system using pHCP 51 and pHCP 7, respectively. As a result, it was confirmed that the expression level of those using pHCP51 increased by 3.8% compared to pHCP, and that of the expression system using pHCP7 increased the expression level of 5.3% compared to pHCP (Fig. 6). It was confirmed that it is more useful because it showed an increase in the amount of expression in protein expression than in pHCP.

Example 7: Tagatose conversion using the discovered plasmid-based whole cell

In order to show that the system using the plasmids discovered in this application is more useful, the D-tagatose converting enzyme expression system was applied to the plasmids discovered in Corynebacterium, and Tagatose conversion reaction was performed using the production strain itself. To confirm the increase in toss production. For conversion of tagatose, Corynebacterium is inoculated in BHI medium, cultured at 30° C. for 24 hours, and then subcultured in a 250 mL flask at the same temperature and time conditions as in the same medium. The cultured Corynebacterium was centrifuged 45 mL at 3500 rpm for 10 minutes to obtain only the strain, and then the strain was added at 20% weight of the total reaction to react. In the tagatose conversion reaction used in the present application, 20% of fructose was used as a substrate, and the reaction conditions were 50mM Tris-HCl (pH8.0) including 1mM NiSO ₄ . For the reaction, the strain and the substrate were separately pretreated at 60° C. for 30 minutes, mixed, and then reacted at 60° C. for 2 hours, followed by centrifugation at 13000 rpm for 10 minutes, and the supernatant was recovered and analyzed by HPLC. In HPLC, tagatose produced by the RID detector and fructose as a substrate were quantified using an HPX 87H column using a 5mM H ₂ SO ₄ solvent. The pHCP (negative control) is a plasmid in which D-tagatose converting enzyme is not inserted, and the other is the conversion result using a vector expressing D-tagatose converting enzyme (TN full) at pHCP, pHCP51, and pHCP7. Compared to the use of the pHCP plasmid, it was confirmed that the tagatose conversion rate of the system expressed by using the pHCP 51 discovered in the present application increased by 0.9%, and that with the pHCP7 increased by 1.6%. Through this, it was confirmed that the plasmid system discovered in the present application has a high copy number, and shows the result of increasing the conversion activity of the strain itself as well as affecting the expression level of the desired protein.

From the above description, those skilled in the art to which the present application belongs will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

Korea Microorganism Conservation Center (overseas) KCCM12542P 20190604 Korea Microorganism Conservation Center (overseas) KCCM12543P 20190604

<110> CJ CheilJedang Corporation Korea Advanced Institute of Science and Technology <120> Novel vector having enhanced copy number and use thereof <130> KPA190630-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> pHCP WT <400> 1 gctgcacgaa tacctgaaaa atgttgaacg ccccgtgagc ggtaactcac agggcgtcgg 60 ctaaccccca gtccaaacct gggagaaagc gctcaaaa 98 <210> 2 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> pHCP7 <400> 2 gctgcacgaa tacctgaaaa acgttgaacg ccccgtgagc ggtaactcac agggcgtcgg 60 ctaaccccca gtccaaacct gggagaaagc gctcaaaa 98 <210> 3 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> pHCP51 <400> 3 gctgcacgaa tacctgaaaa atgttgaacg ccccgtgagc ggtaactcac agggcgtcgg 60 ctaaccccca gtccaaactt gggagaaagc gctcaaaa 98

Claims (11)

I) 22nd nucleotide in the polynucleotide sequence of SEQ ID NO: 1 is substituted with C (cytosine); ii) nucleotide 79 is substituted with T (thymine); Or iii) a polynucleotide encoding a ctRNA comprising a substitution combination of i) and ii).
The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
A composition for gene expression comprising the polynucleotide of claim 1.
A vector comprising a gene encoding the polynucleotide of claim 1 and a protein of interest.
The vector of claim 4, wherein the protein of interest is an enzyme that produces at least one of saccharides, amino acids, and nucleic acids.
The vector of claim 4, wherein the vector further comprises parB and repA.
The vector of claim 6, wherein the parB is located upstream of the polynucleotide encoding the ctRNA, and the repA is located downstream of the polynucleotide encoding the ctRNA.
The polynucleotide of claim 1; Or, a microorganism comprising a vector containing the same.
The microorganism according to claim 8, wherein the microorganism is a microorganism of the genus Corynebacterium or genus Escherichia.
A method for producing a target substance comprising the step of culturing the microorganism of claim 8 or 9.
The method of claim 10, wherein the target substance is any one or more of saccharides, amino acids, and nucleic acids.

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