KR20220049827A - A recombinant Corynebacterium having enhanced secreted production efficiency of target protein - Google Patents

Abstract

The present invention relates to a recombinant Corynebacterium in which a function of the secretion signal peptide of cg2052 or the secretion signal peptide of cg1514, which is endogenously present in microorganisms of the genus Corynebacterium, is inhibited, thereby improving the ability to secrete and produce a target protein. The recombinant microorganism of the genus Corynebacterium according to the present invention can remove genes that induce the secretion of existing impurities and secrete the target protein with high purity and efficiency, so that the target protein can be produced very easily without cell disruption, so that it is advantages for industrial applications.

Description

Recombinant Corynebacterium having enhanced secreted production efficiency of target protein {A recombinant Corynebacterium having enhanced secreted production efficiency of target protein}

The present invention relates to a recombinant Corynebacterium having improved secretion production ability of a target protein, and more particularly, the function of the secretion signal peptide of cg2052 or the secretion signal peptide of cg1514 inherently present in microorganisms of the genus Corynebacterium is inhibited. It relates to a recombinant microorganism of the genus Corynebacterium having an improved extracellular secretion and production ability of the target protein into which a gene encoding the target protein is introduced.

Microorganisms of the genus Corynebacterium are mainly used for the production of amino acids and nucleic acids in the industrial field, and their safety has been recognized and is used to produce various metabolites and food additives. Microorganisms of the genus Corynebacterium are known to not only have a secretory system internally, but also do not produce proteases, so they can efficiently produce industrially useful recombinant proteins. That is, in general, when a recombinant protein is produced using another microorganism, a cell disruption process is required to recover the produced recombinant protein, whereas when the recombinant protein is produced using a microorganism of the genus Corynebacterium, Corynebacterium. Since a separate crushing process is not required by the intrinsic secretion system of the genus microorganism, the recombinant protein produced by the industrial process using the microorganism of the genus Corynebacterium can be easily recovered, enabling economical and effective recombinant protein production.

Although the secretion system of the existing host is used as a method for producing a specific recombinant protein (Yim, S. S. et al., (2016). Biotechnology and bioengineering, 113(1), 163-172.), the target recombinant protein Research to improve strains for inducing efficient secretion production of In this study, secretory signal codons used for secretory production are optimized to increase secretory capacity (Korean Patent Registration No. 1009197040000), or the number of copies of a plasmid system for secretory production of recombinant proteins is increased (Choi, J. W. et al. , (2018). Applied microbiology and biotechnology, 102(2), 873-883.), a method using an increase in expression amount by codon optimization of a recombinant protein (Wang, X. et al., (2010). Protein expression and purification, 72(1), 101-106.) and the like.

However, the above methods focus on the expression of the recombinant protein, and studies on how to effectively secrete the expressed recombinant protein into extracellular cells are insufficient.

In the present invention, intrinsic secretion of microorganisms of the genus Corynebacterium by paying attention to the problem that a separate process of cell disruption is eventually required to produce the recombinant protein when the expression level of the recombinant protein in the cell is increased but it is not effectively secreted out of the cell How to improve the system to improve the secretion of the target recombinant protein and to reduce the secretion impurities. By confirming that it can be increased, the present invention was completed.

Korean Patent Registration 10-09197040000

Yim, S. S. et al., (2016). Biotechnology and bioengineering, 113(1), 163-172 Choi, J. W. et al., (2018). Applied microbiology and biotechnology, 102(2), 873-883 Wang, X. et al., (2010). Protein expression and purification, 72(1), 101-106

The present invention is to provide a novel recombinant microorganism of the genus Corynebacterium having improved extracellular secretion ability of a target protein.

In order to achieve the above object, the present invention provides a secretory signal peptide of cg2052 represented by the amino acid sequence of SEQ ID NO: 1 and/or cg1514 represented by the amino acid sequence of SEQ ID NO: 2 that are inherently present in microorganisms of the genus Corynebacterium. The function of the signal peptide is inhibited,

Provided is a recombinant microorganism of the genus Corynebacterium having an improved extracellular secretion and production ability of the target protein into which a gene encoding a target protein is introduced.

In the present invention, the target protein may be characterized in that it is introduced by being operably linked to a secretion signal peptide that induces secretion of the target protein in a microorganism of the genus Corynebacterium.

In the present invention, the secretion signal peptide inducing secretion of the target protein is a Cg2052 secretion signal peptide, a Cg1514 secretion signal peptide, a CgR0949 secretion signal peptide, a TorA secretion signal peptide, a CspA secretion signal peptide, a Prorin B (PorB) secretion signal peptide, It may be characterized in that it is selected from the group consisting of CgR_2070 secretion signal peptide and AprE secretion signal peptide.

In the present invention, the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide or a TorA secretion signal peptide, and the recombinant microorganism is a gene encoding one or more proteins selected from the group consisting of TatA, TatB and TatC. It may be characterized in that it is additionally introduced or amplified.

In the present invention, the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide, and the recombinant microorganism may be characterized in that genes encoding TatA and TatC proteins are additionally introduced or amplified.

The present invention also relates to a method for secretion and production of a target protein comprising the steps of:

(a) culturing the recombinant microorganism of the genus Corynebacterium to express, secrete, and produce the target gene: and

(b) recovering the secreted/produced target protein.

The recombinant microorganism of the genus Corynebacterium according to the present invention can secrete the target protein with high efficiency and purity by removing the gene that induces the secretion of the existing impurities, so that the target protein can be produced very easily without cell disruption, so industrial application advantageous to

1 is a schematic diagram of a basic plasmid for use in a system for secretion production. This is a plasmid pHCP_CgR in which the CgR0949 signal secretion peptide for secretion production is inserted into the pHCP vector.
2 is a schematic diagram of the pHCP_CgR_DagA plasmid.
3 is a schematic diagram of the pHCP_CgR_DagA_TatAC plasmid.
4 is a schematic diagram of the pHCP_CgR_DagA_TatACB plasmid.
5 is a result of analyzing the extracellular secretion production of beta-agarase (DagA) after flask culture of recombinant Corynebacterium glutamicum. Figure 5a is a result of SDS-PAGE, Figure 5b is a result of Western blotting. Lane M is a protein marker, Lane 1 is pHCP, Lane 2 is pHCP-CgR-DagA, Lane 3 is pHCP-CgR-DagA-TatAC, Lane 4 is pHCP-CgR-DagA-TatACB. This secretory expression system was confirmed in WT C. glutamicum ATCC13032.
6 is a result of high-concentration fed-batch culture by transforming pHCP_CgR_DagA_TatAC into WT C. glutamicum ATCC13032. Figure 6a shows the results of fed-batch culture by time, ● indicates the growth curve, ■ indicates the amount of glucose, ▲ indicates the amount of secreted DagA. 6b is an SDS-PAGE result of the secreted protein over time. ◀ indicates secreted DagA, and ◁ indicates unknown protein.
FIG. 7 is a result of FPLC performed to isolate the unknown protein of FIG. 6 . 7A is a FPLC result measured at UV280. Figure 7b is a SDS-PAGE result of the protein recovered from each fraction number of Figure 7a. M lane is a protein marker.
8 is an N-terminal amino acid analysis result of the 13 fraction obtained in FIG. 7 . Five amino acids were analyzed using this analysis method, and the result was obtained as a protein with the N-terminal of EEVSG, confirming that it is the Cg2052 gene of Corynebacterium.
9 is a fed-batch culture result of DagA using C. glutamicum SP001 in which the secretion signal peptide of the Cg2052 gene has been removed. In Figure 9, ● indicates a growth curve, ■ indicates the amount of glucose, ▲ indicates the amount of secreted protein. 9b is an SDS-PAGE result of the secreted protein over time. ◀ indicates secreted DagA, and ◁ indicates the second unknown protein.
FIG. 10 is a result of performing MALDI-TOF-MS to confirm the second unknown protein of FIG. 9 .
11 schematically illustrates the secretion expression system of the Corynebacterium SP002 strain in which the Cg1514 gene and the Cg2052 gene are removed.
12 is a fed-batch culture result of DagA using C. glutamicum SP002 in which the secretion signal peptide of the Cg1514 gene has been removed from C. glutamicum SP001. In Figure 12a, ● indicates a growth curve, ■ indicates the amount of glucose, ▲ indicates the amount of secreted protein. 12b is an SDS-PAGE result of the secreted protein over time. ◀ indicates secreted DagA.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

Since the microorganisms of the genus Corynebacterium inherently have a protein secretion system, they are widely used for industrially useful recombinant protein production. However, when the microorganism of the genus Corynebacterium is further improved, the purity and production of the recombinant protein can be further improved. In the present invention, it was confirmed that the secretion of the target protein can be significantly increased by strengthening some secretion signal genes and weakening some secretion signal genes in the microorganisms of the genus Corynebacterium.

Accordingly, in one aspect, the present invention provides a secretion signal peptide of cg2052 represented by the amino acid sequence of SEQ ID NO: 1 and/or a secretion signal peptide of cg1514 represented by the amino acid sequence of SEQ ID NO: 2 that is inherently present in a microorganism of the genus Corynebacterium It relates to a recombinant microorganism of the genus Corynebacterium in which the function of the target protein is suppressed and the gene encoding the target protein is introduced, and the extracellular secretion and production ability of the target protein is improved.

The Cg2052 and Cg1514 secretion signal peptides act competitively for the target protein to be secreted out of the cell through the recombinant microorganism of the genus Corynebacterium, respectively. When the function of the Cg2052 and/or Cg1514 secretion signal peptide is inhibited, There is an effect that the secretion effect is improved and the purity of the secreted target protein is improved.

In the present invention, the target protein may be characterized in that it is introduced by being operably linked to a secretion signal peptide that induces secretion of the target protein in a microorganism of the genus Corynebacterium.

In the present invention, the secretion signal peptide inducing secretion of the target protein is the Cg2052 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 1, the Cg1514 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 CgR0949 secretion signal peptide represented by, TorA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 4, CspA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 5, Prorin B (PorB) represented by the amino acid sequence of SEQ ID NO: 6 It may be characterized in that it is selected from the group consisting of a secretion signal peptide, a CgR_2070 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 7, and an AprE secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 8, but is not limited thereto. That is, it will be apparent to those skilled in the art that a secretion signal peptide capable of inducing secretion of a target protein in a microorganism of the genus Corynebacterium can be used without limitation.

In the present invention, the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 3 or a TorA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 4, and the recombinant microorganism is TatA , TatB and TatC may be characterized in that the gene encoding any one or more proteins selected from the group are additionally introduced or amplified.

Preferably, the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 3, and the recombinant microorganism has TatA and TatC proteins encoding genes are additionally introduced or amplified. can be characterized.

In the present invention, TatA, TatB, and TatC proteins can improve the extracellular secretion ability of the CgR0949 secretion signal peptide or the TorA secretion signal peptide.

In the present invention, that the gene is additionally introduced means not only when the gene is independently introduced into the host microorganism genome in the form of a plasmid or vector, but also when the gene is introduced into the host microorganism and integrated into the genome itself. includes

In one aspect, in the present invention, a recombinant vector into which a gene encoding a secretion signal peptide and a gene encoding a target protein are introduced may be used. In this case, the gene encoding the secretion signal peptide and the gene encoding the target protein are preferably introduced into one recombinant vector. The target protein is operably linked to the secretion signal peptide and expressed, thereby promoting extracellular secretion of the target protein.

In addition, in one aspect, in the present invention, a recombinant vector into which a gene encoding any one or more proteins selected from the group consisting of TatA, Tat B and TatC is introduced may be used. In this case, the gene encoding TatA, the gene encoding TatB, and the gene encoding TatC may each be introduced into a host microorganism using separate recombinant vectors, and two or more genes are inserted into one vector, That is, it can be introduced into a host microorganism using one or two recombinant vectors.

The vector that can be used for the introduction of the gene in the present invention may be any one or more selected from the group consisting of pHCP, pTac15K, pBBR1MCS, pEKEx1, pXMJ19 and pCES208, but is not limited thereto.

In another aspect, the present invention relates to a method for secretion and production of a target protein comprising the following steps:

(a) culturing the recombinant microorganism of the genus Corynebacterium to express, secrete, and produce the target gene: and

(b) recovering the secreted/produced target protein.

In the present invention, the Corynebacterium genus microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium acetoglutamicum ( Corynebacterium acetoglutamicum ), Corynebacterium acetoacidophilum ( Corynebacterium acetoacidophilum ) , Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenes ) and Corynebacterium ammoniagenes ) It may be selected from the group consisting of, but is not limited thereto.

In the present invention, when using Corynebacterium microorganisms of the other genus, Although the names referring to the Cg2052 and Cg1514 secretion signal peptides are different, the secretion signal peptides corresponding to the Cg2052 and Cg1514 secretion signal peptides will be apparent to those skilled in the art when judging based on the homology of the amino acid sequence. Accordingly, it will be apparent to those skilled in the art that a recombinant microorganism of the genus Corynebacterium in which the function of the secretion signal peptide corresponding to these Cg2052 and Cg1514 secretion signal peptides is suppressed falls within the scope of the present invention.

In the present invention, The secretion signal peptide of cg2052 represented by the amino acid sequence of SEQ ID NO: 1 and the secretion signal peptide of cg1514 represented by the amino acid sequence of SEQ ID NO: 2, which are inherently present in microorganisms of the genus Corynebacterium, are other than Corynebacterium glutamicum. In the microorganisms of the genus Corynebacterium, respectively, for the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2, 80% or more, preferably 90% or more, such as 95% or more, 96% or more, 97% or more , the effect of the present invention can be achieved by inhibiting the function of the secretion signal peptide corresponding to these secretion signal peptides having sequence homology of 98% or more, or 99% or more.

In the present invention, that the gene is introduced or amplified means that, when the peptide or protein produced by the gene is not present in the host microorganism, it is artificially expressed in the host microorganism to have the activity or function of the peptide or protein, It includes those modified to enhance the activity or function of the peptide or protein produced by the gene compared to the intrinsic activity or function.

In the present invention, "modified to enhance intrinsic activity or function" means that a gene exhibiting activity or function is introduced or the copy number of the gene is increased (eg, expression using a plasmid into which the gene is introduced), the gene The activity or function of the microorganism after the manipulation is increased compared to the activity of the microorganism before the manipulation, such as deletion of an expression inhibitory regulatory factor or modification of the expression control sequence, for example, use of an improved promoter means a state of being

In the present invention, "enhancement of peptide function" means that the function of the peptide itself is newly introduced or increased to induce an effect beyond the original function, as well as increase of intrinsic gene function, intrinsic gene amplification from internal or external factors, the Deletion of inhibitory regulatory factors of gene expression, increase in gene copy number, introduction of genes from outside, modification of expression control sequences, in particular, include those whose functions are increased by increasing the function of peptides by replacement or modification of promoters and mutations in genes do.

In the present invention, "inhibition of peptide function" means that all or part of the gene encoding the peptide is deleted, or the function of the gene is suppressed so that the peptide or protein produced by the gene is removed from the host microorganism as well as , including those modified so that the peptide or protein produced by the gene is weakened compared to its intrinsic activity or function.

In the present invention, the inhibition of the secretion signal peptide function is all or part of the secretion signal peptide is deleted or its function is suppressed, as well as all of the intrinsic genes encoding the peptide secretion induced by the secretion signal peptide. or a case in which the secretion function of the secretion signal peptide does not function properly because a part is deleted or its function is inhibited.

In the present invention, "modified to be weakened compared to intrinsic activity or function" means deletion of a gene exhibiting activity or function or inactivation of a gene (eg, substitution with a mutant gene), attenuation of gene expression (eg, , substitution with a weak promoter, introduction of siRNA, gRNA, sRNA, etc., substitution of the start codon from ATG to GTG, etc.), inhibition of the function of the peptide expressed by the gene (e.g., a non-competitive repressor or a competitive repressor) It refers to a state in which the function of the microorganism after the operation is reduced compared to the function of the microorganism before the operation such as addition) is performed.

The term "intrinsic activity or function" used in the present invention means the activity or function possessed by enzymes, peptides, proteins, etc. that the original microorganism has in an unmodified state, and "modified to be enhanced compared to the intrinsic activity or function" “Being means that the activity or function is newly introduced or improved compared to the activity or function of the peptide in the state before modification, and the meaning of “modified to be weakened compared to the intrinsic activity or function” means that the activity or function is in the state before modification. It means that the activity or function disappears or is further reduced when compared to the activity or function of the peptide or the like.

In the present invention, the term "deletion" is a concept encompassing preventing the corresponding gene from being expressed through a method of mutating, substituting, or deleting a part or all of the base of the gene, or preventing activity or function from being expressed even if it is expressed.

In the present invention, "overexpression" refers to expression at a level higher than the level at which the corresponding gene is expressed in cells under normal conditions, and the promoter of a gene existing on the genome is replaced with a strong promoter, or the gene is cloned into an expression vector. This is a concept that includes increasing the expression level through a method of transforming cells into cells.

As used herein, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. A vector can be a plasmid, a phage particle or simply a potential genomic insert. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since a plasmid is currently the most commonly used form of a vector, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows efficient replication to include several to hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector are selected. It has a structure including an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method. After ligation, the vector must be transformed into an appropriate host cell. Transformation can be readily accomplished using the calcium chloride method or electroporation (Neumann, et al., EMBO J., 1:841, 1982) and the like.

The promoter of the vector may be constitutive or inducible, and may be further modified for the effect of the present invention. In addition, the expression vector contains a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication (Ori). Vectors may be self-replicating or integrated into host genomic DNA. Preferably, the gene transferred by being inserted into the vector is irreversibly fused into the genome of the host cell so that the gene expression in the cell is stably maintained for a long period of time.

A base sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. It can be a gene and regulatory sequence(s) linked in such a way as to enable gene expression when an appropriate molecule (eg, a transcriptional activation protein) is bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a preprotein that participates in secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, in contact and in reading frame. However, the enhancer does not need to be in contact. Linking of these sequences is accomplished by ligation (ligation) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and/or translational expression control sequences to function in the selected expression host. Preferably, the expression control sequence and/or the gene is contained in a single recombinant vector that contains both a bacterial selection marker and a replication origin. When the host cell is a eukaryotic cell, the recombinant vector must further contain an expression marker useful in the eukaryotic expression host.

A host cell transformed with the above-described recombinant vector constitutes another aspect of the present invention. As used herein, the term “transformation” refers to the introduction of DNA into a host such that the DNA becomes capable of replication either as an extrachromosomal factor or by chromosomal integrity.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function equally against the same expression system. However, a person skilled in the art can make an appropriate selection among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The copy number of the vector, its ability to control the copy number and the expression of other proteins encoded by the vector, for example antibiotic markers, must also be considered.

In addition, the gene introduced in the present invention can be characterized in that it is introduced into the genome of the host cell and exists as a chromosomal factor. For those skilled in the art to which the present invention pertains, it will be apparent that even when the gene is inserted into the genome chromosome of the host cell, it will have the same effect as when the recombinant vector is introduced into the host cell as described above.

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 invention is not limited to these Examples and Experimental Examples.

Example 1: Secretory Production of Model Proteins for Confirmation of Secretion Production

Using the secretion system inherent in the Corynebacterium sp. strain, β (DagA) was selected as a model protein to compare protein secretion and production capacity, and the experiment was conducted. First, in order to enhance secretion production, pXD5 (S.S. Yim, et al., Biotechnology Journal 12(11) (2017) 1700040.) from CgR0949-F, CgR0949-R primers (SEQ ID NOs: 16, 17) using the secretion signal The peptide CgR0949 (SEQ ID NO: 3) was amplified by PCR. PCR was performed using a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) instrument, and using PrimeSTAR HS polymerase (Takara Bio, Inc., Shiga, Japan), the gene was amplified from pXD5 using the primers described. .

The PCR reaction conditions were 98 °C 5 min 1 Cycle, 98 °C 5 min-55 °C-20 sec-72 °C-30 sec 25 Cycles, 72 °C-5 min 1 Cycle. The PCR product was transformed into a pHCP vector (Korean Publication No. 10-2018-0092110, Choi, JW, Yim, SS, & Jeong, KJ (2018). Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum. Applied microbiology and biotechnology , 102 (2), 873-883.) was inserted using Bam HI, Xba I restriction enzyme, which was named pHCP_CgR [Fig. 1]. First, βagarase (X05811.1) is a gene derived from Streptomyces coelicolor A3, pUWL201PW-DagA (U. Temuujin et. al., Applied microbiology and biotechnology 92(4) (2011) 749-759.) DagA_F, DagA_R primer ( After PCR amplification using SEQ ID NOs: 18 and 19), it was inserted into the pHCP_CgR vector using Sfi I restriction enzyme. This was named pHCP-CgR-DagA [Fig. 2]. For an efficient secretion system using the CgR0949 secretion signal peptide, a system for co-expressing TatAC and a system for co-expressing TatABC were additionally constructed. TatAC (SEQ ID NO: 9) was amplified by PCR using TatAC-F and TatAC-R primers (SEQ ID NO: 20, 21) in C. glutamicum ATCC13032 (ATCC, USA). PCR was performed using a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) instrument, and using PrimeSTAR HS polymerase (Takara Bio, Inc., Shiga, Japan), the gene was amplified from pXD5 using the primers described. . The PCR reaction conditions were 98 °C 5 min 1 Cycle, 98 °C 5 min-55 °C-20 sec-72 °C-1 min 30 sec 25 Cycle, 72 °C-5 min 1 Cycle. The PCR product was inserted into the site of the Not I restriction enzyme of pHCP-CgR-DagA to prepare pHCP-CgR-DagA-TatAC [Fig. 3]. Also TatB gene (SEQ ID NO: 14) was amplified using TatB-F, TatB-R primers (SEQ ID NOs: 22, 23), and inserted into the Bgl II and Spe I sites of pHCP-CgR-DagA-TatAC, which was named pHCP-CgR-DagA-TatACB [Fig. 4].

Example 2: C. glutamicum Flask culture on ATCC13032

Secretion production was carried out using the expression system constructed in the present invention.

After transformation into Corynebacterium glutamicum ATCC13032 with pHCP_CgR, pHCP-CgR-DagA, pHHCP-CgR-DagA-TatAC, pHCP-CgR-DagA-TatABC (Van der Rest, ME, Lange, C., & Molenaar, D. (1999). A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. See Applied Microbiology and Biotechnology , 52 (4), 541-545). ) was inoculated into the medium and incubated at 30 ℃ 200rpm for 17 hours. Thereafter, the cultured strain was transferred to 50 mL of fresh BHI medium in a 250 mL flask by 1/100 of a volume and cultured under the same conditions. After culturing for 24 hours, the Corynebacterium glutamicum culture medium was centrifuged at 13000 rpm for 10 minutes, the supernatant was taken, the supernatant and acetone were mixed in equal amounts and stored at -20°C for 1 hour, 13000rpm, after centrifugation for 10 minutes , the protein was obtained through an acetone precipitation method to recover the precipitated protein. SDS-PAGE was performed with the obtained protein, and the amount of β produced in each strain was compared. As shown in FIG. 5, it was confirmed that secretory production was only produced in pHCP-CgR-DagA-TatAC, pHCP-CgR-DagA-TatABC, and among them, it was confirmed that the strain including pHCP-CgR-DagA-TatAC produced more β, It was used for high-concentration fed-batch culture.

Example 3: C. glutamicum High-concentration fed-batch culture using ATCC13032

2L high-concentration fed-batch culture was performed using the secretory expression pHCP-CgR-DagA-TatAC system confirmed in Example 1. The cultures grown overnight in 10 mL of BHI media were transferred to a 50 mL flask culture containing 15 g/L yeast extract, 7 g/L casamino acid, and 2 g/L urea in minimal media and pre-cultured at 30 °C. After overnight incubation, this was inoculated into a fermenter, and 13 N ammonia was added to maintain pH 7.0 while monitoring the temperature at 30° C. and pH. The amount of dissolved oxygen was maintained at 30% ( v/v ) by adding rpm and oxygen. Glucose was analyzed using the YSI 2300 STAT Plus instrument, and the growth rate was analyzed by analyzing the optical density at OD _600nm (FIG. 6a). Proteins were analyzed by SDS-PAGE (Fig. 6b). As a result, it was confirmed that the protein, DagA (36 kDa), was secreted at 1.5 g/L, and through ImageJ, an SDS-PAGE image analysis program, it was confirmed that DagA corresponds to about 54% of the total secreted protein. However, a protein corresponding to about 45% of the size of about 27 kDa smaller than that of the DagA protein was identified.

Example 4: Implementation of FPLC analysis for identification of unknown proteins

In order to confirm the unknown protein observed in Example 2, only the supernatant of the fermented product was recovered and purified using FPLC. As shown in Figure 7, samples corresponding to each fraction number were taken, and as a result of SDS-PAGE analysis, it was possible to purify a protein of the same size as the unknown protein in fraction 13, and N-terminal amino acid analysis was performed using this. Five amino acid sequences were analyzed through As a result, the amino acid sequence of EEVSG was confirmed as shown in FIG. 8, and it was confirmed that it was a part of the Cg2052 gene (CAF20211.1) of C. glutamicum ATCC13032. It was decided to remove the Cg2052 gene from C. glutamicum ATCC13032 for the production of high-purity target protein secretion. After analyzing the secretion signal sequence of the Cg2052 protein through the SignalP-5.0 program, in order to remove only the secretion signal sequence (SEQ ID NO: 1), 500 bp in front of the secretion signal sequence Cg2052A-F, Cg2052A-R (SEQ ID NO: 24, 25) amplified with, and 500 bp after the secretion signal sequence is amplified through Cg2052B-F, Cg2052B-R (SEQ ID NOs: 26, 27) and linked again by overlapping PCR method to pK19mobSacB (Life Science Market) Sal I and BamH Recombinant pK19-Cg2052ss was prepared using restriction enzyme I. This vector was prepared to be at least 1 ug/ul or more, and the primary recombination was induced by electroporation in the Corynebacterium glutamicum ATCC 13032 strain. At this time, the electroporated strain was smeared on an LB agar plate containing 20 ug/ul of kanamycin to isolate colonies, and then it was confirmed through PCR and sequencing whether it was properly inserted into the induced position on the genome. The isolated strain was cultured overnight on LB agar liquid medium containing 10% sucrose to induce secondary recombination again, and then spread on a sucrose agar plate of the same concentration to isolate colonies. After confirming whether the antibiotic kanamycin resistance among the finally isolated colonies was confirmed, it was confirmed through nucleotide sequence analysis whether the Cg2052 gene was deleted among the non-antibiotic-resistant strains. The strain prepared in this way was named C. glutamicum SP001 strain.

Example 5: C. glutamicum High-concentration fed-batch culture using SP001 strain

After transformation into the C. glutamicum SP001 strain using the same pHCP-CgR-DagA-TatAC, 2L high-concentration fed-batch culture was performed. The method was performed in the same manner as in the fed-batch culture of Example 2, and the growth rate, glucose consumption, and secreted expression protein amount were measured as shown in FIG. 9a. However, as shown in FIG. 9b , it was confirmed that, in addition to the protein DagA (36 kDa), a second unknown protein with a size of 27 kDa was secreted in an amount accounting for about 33% of the total secreted protein.

Example 6: C. glutamicum Construction of SP002 strain

As a result of performing an analysis using MALDI-TOF-MS to identify the second unknown protein identified in Example 4, a result as shown in FIG. 10 was obtained, which is the Cg1514 protein (CAF21351.1 of C. glutmaicum ATCC13032) ) could be confirmed. It was confirmed that a high amount of Cg1514 protein was secreted in addition to the target protein in fed-batch culture using the C. glutamicum SP001 strain. It was decided to remove the gene. After analyzing the secretion signal sequence (SEQ ID NO: 2) of the Cg1514 protein through the SignalP-5.0 program, in order to remove only the secretion signal sequence, 500 bp in front of the secretion signal sequence Cg1514A-F, Cg1514A-R (SEQ ID NO: 28, 29) After amplification of the secretory signal sequence, 500 bp after the secretion signal sequence is amplified through Cg1514B-F, Cg1514B-R (SEQ ID NOs: 30, 31) and linked again by overlapping PCR method using Sal I and BamHI restriction enzymes for pK19mobSacB to prepare pK19-Cg1514ss for recombination. After this process, C. glutamicum SP002 in which the Cg1514 gene was further removed from the C. glutamicum SP001 strain was prepared in the same manner as in Example 3.

Example 7: High-concentration fed-batch culture using C. glutamicum SP002 strain

Finally, the secretory production of DagA, a model protein, was confirmed through high-concentration fed-batch culture using the constructed strain SP002. Fed-batch culture was performed in the same manner as described in Example 3, and protein analysis was analyzed using SDS-PAGE without a concentration process. As shown in FIG. 12, the concentration of secreted DagA was 2.0 g/L, and the purity was confirmed to be 93%. This is a result confirming that the improved SP002 strain is a strain suitable for secretion capable of high-concentration secretion by enabling high-purity secretion in secretion production.

Example 8: Sequence information

Cg2052 secretion signal amino acid sequence (SEQ ID NO: 1)

MLRKTVTGGIVALIATATLMNSVSSA

Cg1514 secretion signal amino acid sequence (SEQ ID NO: 2)

MLNRVSRIAGASAITLCIGLTTILSPTSTAQS

CgR0949 secretion signal amino acid sequence (SEQ ID NO: 3)

MQINRRGFLKATAGLATIGAASMFMPKANALGA

TorA secretion signal peptide amino acid sequence (from Escherichia coli str. K-12, AYG19906.1) (SEQ ID NO: 4)

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA

CspA secretion signal peptide amino acid sequence (from Corynebacterium ammoniagenes , BAB62413.1) (SEQ ID NO: 5)

MKRMKSLAAALTVAGAMLAAPVATA

PorB secretion signal peptide amino acid sequence (from Corynebacterium glutamicum ATCC13032, DAA01140.1) (SEQ ID NO: 6)

MKLSHRIAAMAATAGITVAAFAAPASA

CgR_2070 secretion signal peptide amino acid sequence (from Corynebacterium glutamicum R, BAF55069.1) (SEQ ID NO: 7)

MGKHRRNNSNATRKAVAASAVALGATAAIASPAQA

AprE secretion signal peptide amino acid sequence (from Bacillus subtilis , WP_014479360.1) (SEQ ID NO: 8)

MRSKKLWISLLFALTLIFTMAFSNMSAQA

SEQ ID NO: 9 -TatAC

ATGTCCATTGTTGAGCACATCAAAGAGTTTCGACGCCGACTTCTTATCGCTCTGGCGGGCATCCTCGTGGGCACCATTATCGGCTTTATTTGGTACGATTTCTCATTTTGGCAGATCCCCACTTTGGGCGAGCTGCTGAGGGATCCGTACTGTTCTCTGCCTGCTGAATCCCGCTGGGCCATGAGCGACTCAGAGGAATGTCGACTGCTCGCAACCGGCCCGTTTGATCCATTCATGCTTCGCCTTAAAGTAGCGGCGTTGGTGGGTATGGTTCTTGGCTCACCCGTGTGGCTGAGCCAGCTGTGGGGCTTTATCACCCCAGGTTTGATGAAGAATGAGCGCCGTTACACCGCAATCTTCGTCACGATTGCTGTTGTGCTGTTTGTCGGCGGTGCTGTTCTTGCGTACTTCGTCGTTGCATATGGTTTGGAGTTCCTCCTTACCATTGGTGGAGACACCCAGGCAGCGGCCCTGACTGGTGATAAGTACTTCGGATTCTTGCTCGCGTTGTTGGCGATTTTCGGCGTGAGCTTCGAAGTTCCACTGGTGATCGGCATGCTCAACATTGTGGGTATCTTGCCTTACGATGCCATTAAAGATAAGCGACGCATGATCATCATGATTTTGTTCGTGTTCGCTGCTTTCATGACACCCGGCCAGGATCCTTTCACCATGTTGGTGTTGGCGCTTTCACTCACCGTTCTGGTAGAGCTTGCCCTGCAGTTCTGTCGTTTCAACGACAAACGCCGGGACAAGAAGCGCCCAGAATGGCTTGATGGCGATGACCTCTCTGCATCACCACTGGATACTTCTGCTGGTGGAGAAGATGCTCCAAGCCCAGTCGAAACCCCAGAGGCGGTGGAGCCTTCGCGGATGCTGAACCCAAGTGGGGAGGCGTCGATAAGCTATAAACCCGGGCGCGCCGACTTCGGTGACGTGCTCTAA

SEQ ID NO: 10-TatA (nucleotide sequence)

ATGTCCCTCGGACCATGGGAAATTGGAATCATTGTCCTGCTGATCATCGTGCTGTTCGGCGCGAAGAAGCTGCCTGATGCAGCTCGTTCCATCGGCCGTTCCATGCGCATCTTCAAGTCTGAAGTCAAAGAAATGAACAAGGACGGCGATACCCCAGAACAACAGCAGCAGCCTCAGCAGCAGATTGCGCCCAACCAGATCGAGGCTCCTCAGCCAAACTTTGAGCAGCACTACCAGGGACAGCAGGTTCAGCAGCCTCAGAACCCTCAGACCCCTGACTACCGTCAGAACTACGAGGATCCAAACCGCACCTCTTAA

SEQ ID NO: 11-TatA (amino acid sequence)

MSLGPWEIGIIVLLIIVLFGAKKLPDAARSIGRSMRIFKSEVKEMNKDGDTPEQQQQPQQQIAPNQIEAPQPNFEQHYQGQQVQQPQNPQTPDYRQNYEDPNRTS*

SEQ ID NO:12-TatC (nucleotide sequence)

ATGTCCATTGTTGAGCACATCAAAGAGTTTCGACGCCGACTTCTTATCGCTCTGGCGGGCATCCTCGTGGGCACCATTATCGGCTTTATTTGGTACGATTTCTCATTTTGGCAGATCCCCACTTTGGGCGAGCTGCTGAGGGATCCGTACTGTTCTCTGCCTGCTGAATCCCGCTGGGCCATGAGCGACTCAGAGGAATGTCGACTGCTCGCAACCGGCCCGTTTGATCCATTCATGCTTCGCCTTAAAGTAGCGGCGTTGGTGGGTATGGTTCTTGGCTCACCCGTGTGGCTGAGCCAGCTGTGGGGCTTTATCACCCCAGGTTTGATGAAGAATGAGCGCCGTTACACCGCAATCTTCGTCACGATTGCTGTTGTGCTGTTTGTCGGCGGTGCTGTTCTTGCGTACTTCGTCGTTGCATATGGTTTGGAGTTCCTCCTTACCATTGGTGGAGACACCCAGGCAGCGGCCCTGACTGGTGATAAGTACTTCGGATTCTTGCTCGCGTTGTTGGCGATTTTCGGCGTGAGCTTCGAAGTTCCACTGGTGATCGGCATGCTCAACATTGTGGGTATCTTGCCTTACGATGCCATTAAAGATAAGCGACGCATGATCATCATGATTTTGTTCGTGTTCGCTGCTTTCATGACACCCGGCCAGGATCCTTTCACCATGTTGGTGTTGGCGCTTTCACTCACCGTTCTGGTAGAGCTTGCCCTGCAGTTCTGTCGTTTCAACGACAAACGCCGGGACAAGAAGCGCCCAGAATGGCTTGATGGCGATGACCTCTCTGCATCACCACTGGATACTTCTGCTGGTGGAGAAGATGCTCCAAGCCCAGTCGAAACCCCAGAGGCGGTGGAGCCTTCGCGGATGCTGAACCCAAGTGGGGAGGCGTCGATAAGCTATAAACCCGGGCGCGCCGACTTCGGTGACGTGCTCTAA

SEQ ID NO: 13 -TatC (amino acid sequence)

MSIVEHIKEFRRRLLIALAGILVGTIIGFIWYDFSFWQIPTLGELLRDPYCSLPAESRWAMSDSEECRLLATGPFDPFMLRLKVAALVGMVLGSPVWLSQLWGFITPGLMKNERRYTAIFVTIAVVLFVGGAVLAYFVVAYGLEFLLTIGGDTQAAALTGDKYFGFLLALLAIFGVSFEVPLVIGMLNIVGILPYDAIKDKRRMIIMILFVFAAFMTPGQDPFTMLVLALSLTVLVELALQFCRFNDKRRDKKRPEWLDGDDLSASPLDTSAGGEDAPSPVETPEAVEPSRMLNPSGEASISYKPGRADFGDVL *

SEQ ID NO: 14-TatB (nucleotide sequence)

ATGTTTTCTAGCGTGGGTTGGGGAGAGATCTTCCTCTTAGTCGTTGTGGGCCTTGTTGTCATCGGCCCGGAACGGTTGCCTCGTTTGATCCAGGACGCACGCGCTGCGCTGCTCGCTGCACGTACCGCTATCGACAATGCAAAGCAGTCGTTGGACAGTGATTTTGGTTCGGAATTTGATGAAATCCGAAAGCCACTAACCCAGGTTGCACAGTACAGCCGGATGAGCCCCAAGACGGCCATCACTAAGGCGTTATTTGATAATGATTCCTCGTTCCTGGATGACTTTGATCCAAAGAAGATCATGGCCGAAGGAACAGAAGGCGAAGCTCAGCGCAACAAGCAGGCAGCTGACAACAATGCGAATGTGGTGGAACGTCCAGCTGATGGTTCCACCGCACGCCCAACGCAAAACGATCCAAAAGACGGCCCGAATTACTCAGGTGGCGTCTCTTGGACCGATATTATTTAG

SEQ ID NO: 15-TatB (amino acid sequence)

MFSSVGWGEIFLLVVVGLVVIGPERLPRLIQDARAALLAARTAIDNAKQSLDSDFGSEFDEIRKPLTQVAQYSRMSPKTAITKALFDNDSSFLDDFDPKKIMAEGTEGEAQRNKQAADNNANVVERPADGSTARPTQNDPKDGPNYSGGVSWTDII*

primer sequence Primer Sequences (5′ to 3′) SEQ ID NO: CgR0949-F gcatgggatccatgcaaataaaccgccgaggcttcttaaaagc 16 CgR0949-R tgggcctctagatgctccaagggcgttggcctttggc 17 DagA_F CTAGAGGCCCAGCCGGCCAAGCAGACCTCGAATGGGAAC 18 DagA_R ggccgcggcccccgaggccctatttgtcatcgtcatctttataatccacggcctgatacgtcctgacccagcggtagta 19 TatAC-F ggggccgcggccgcaagaaggagatataatgtccctcggaccatgg 20 TatAC-R aggcagcggccgcaactagtgtgtcagatctttagagcacgtcaccgaagtcggcgcgcccgg 21 TatB-F ctctaaagatcttttttaatccttatttacattttctgaaagaccggtc 22 TatB-R cggccgcaactagtctaaataatatcggtccaagagacgccacctgagtaat 23 Cg2052A-F TGACAGTCGACAGGTCACGACACAGAGT 24 Cg2052A-R CAACAGTGTTTCAAACTAAAAAACGACGGGATTCCTTTCTGTTTTGC 25 Cg2052B-F GCAAAACAGAAAGGAATCCCGTCGTTTTTTAGTTTGAAACACTGTTG 26 Cg2052B-R TGTCAGGATCCAAAACTTCTTCGAGATCACTCTTTTG 27 Cg1514A-F TAGACGTCGACCATCAACAACGCAAAATACTGGAT 28 Cg1514A-R AGGGGTGATCTGTTCGAGCAAAAAGAGCTCCTGATCATGTA 29 Cg1514B-F TACATGATCAGGAGCTCTTTTTGCTCGAACAGATCACCCCT 30 Cg1514B-R TGTCAGGTACCATTGTCTCGTGGTTTGTTGC 31

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Advanced Institute of Science and Technology <120> A recombinant Corynebacterium having enhanced secreted production efficiency of target protein <130> P20-B251 <160> 31 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> PRT <213> Corynebacterium glutamicum <400> 1 Met Leu Arg Lys Thr Val Thr Gly Gly Ile Val Ala Leu Ile Ala Thr 1 5 10 15 Ala Thr Leu Met Asn Ser Val Ser Ser Ala 20 25 <210> 2 <211> 32 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Leu Asn Arg Val Ser Arg Ile Ala Gly Ala Ser Ala Ile Thr Leu 1 5 10 15 Cys Ile Gly Leu Thr Thr Ile Leu Ser Pro Thr Ser Thr Ala Gln Ser 20 25 30 <210> 3 <211> 33 <212> PRT <213> Corynebacterium glutamicum <400> 3 Met Gln Ile Asn Arg Arg Gly Phe Leu Lys Ala Thr Ala Gly Leu Ala 1 5 10 15 Thr Ile Gly Ala Ala Ser Met Phe Met Pro Lys Ala Asn Ala Leu Gly 20 25 30 Ala <210> 4 <211> 39 <212> PRT <213> Escherichia coli <400> 4 Met Asn Asn Asn Asp Leu Phe Gln Ala Ser Arg Arg Arg Phe Leu Ala 1 5 10 15 Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu 20 25 30 Thr Pro Arg Arg Ala Thr Ala 35 <210> 5 <211> 25 <212> PRT <213> Corynebacterium ammoniagenes <400> 5 Met Lys Arg Met Lys Ser Leu Ala Ala Ala Leu Thr Val Ala Gly Ala 1 5 10 15 Met Leu Ala Ala Pro Val Ala Thr Ala 20 25 <210> 6 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Corynebacterium glutamicum <400> 6 Met Lys Leu Ser His Arg Ile Ala Ala Met Ala Ala Thr Ala Gly Ile 1 5 10 15 Thr Val Ala Ala Phe Ala Ala Pro Ala Ser Ala 20 25 <210> 7 <211> 35 <212> PRT <213> Corynebacterium glutamicum <400> 7 Met Gly Lys His Arg Arg Asn Asn Ser Asn Ala Thr Arg Lys Ala Val 1 5 10 15 Ala Ala Ser Ala Val Ala Leu Gly Ala Thr Ala Ala Ile Ala Ser Pro 20 25 30 Ala Gln Ala 35 <210> 8 <211> 29 <212> PRT <213> Bacillus subtilis <400> 8 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala 20 25 <210> 9 <211> 945 <212> DNA <213> Corynebacterium glutamicum <400> 9 atgtccattg ttgagcacat caaagagttt cgacgccgac ttcttatcgc tctggcgggc 60 atcctcgtgg gcaccattat cggctttatt tggtacgatt tctcattttg gcagatcccc 120 actttgggcg agctgctgag ggatccgtac tgttctctgc ctgctgaatc ccgctgggcc 180 atgagcgact cagaggaatg tcgactgctc gcaaccggcc cgtttgatcc attcatgctt 240 cgccttaaag tagcggcgtt ggtgggtatg gttcttggct cacccgtgtg gctgagccag 300 ctgtggggct ttatcacccc aggtttgatg aagaatgagc gccgttacac cgcaatcttc 360 gtcacgattg ctgttgtgct gtttgtcggc ggtgctgttc ttgcgtactt cgtcgttgca 420 tatggtttgg agttcctcct taccattggt ggagacaccc aggcagcggc cctgactggt 480 gataagtact tcggattctt gctcgcgttg ttggcgattt tcggcgtgag cttcgaagtt 540 ccactggtga tcggcatgct caacattgtg ggtatcttgc cttacgatgc cattaaagat 600 aagcgacgca tgatcatcat gattttgttc gtgttcgctg ctttcatgac acccggccag 660 gatcctttca ccatgttggt gttggcgctt tcactcaccg ttctggtaga gcttgccctg 720 cagttctgtc gtttcaacga caaacgccgg gacaagaagc gcccagaatg gcttgatggc 780 gatgacctct ctgcatcacc actggatact tctgctggtg gagaagatgc tccaagccca 840 gtcgaaaccc cagaggcggt ggagccttcg cggatgctga acccaagtgg ggaggcgtcg 900 ataagctata aacccgggcg cgccgacttc ggtgacgtgc tctaa 945 <210> 10 <211> 318 <212> DNA <213> Corynebacterium glutamicum <400> 10 atgtccctcg gaccatggga aattggaatc attgtcctgc tgatcatcgt gctgttcggc 60 gcgaagaagc tgcctgatgc agctcgttcc atcggccgtt ccatgcgcat cttcaagtct 120 gaagtcaaag aaatgaacaa ggacggcgat accccagaac aacagcagca gcctcagcag 180 cagattgcgc ccaaccagat cgaggctcct cagccaaact ttgagcagca ctaccaggga 240 cagcaggttc agcagcctca gaaccctcag acccctgact accgtcagaa ctacgaggat 300 ccaaaccgca cctcttaa 318 <210> 11 <211> 105 <212> PRT <213> Corynebacterium glutamicum <400> 11 Met Ser Leu Gly Pro Trp Glu Ile Gly Ile Ile Val Leu Leu Ile Ile 1 5 10 15 Val Leu Phe Gly Ala Lys Lys Leu Pro Asp Ala Ala Arg Ser Ile Gly 20 25 30 Arg Ser Met Arg Ile Phe Lys Ser Glu Val Lys Glu Met Asn Lys Asp 35 40 45 Gly Asp Thr Pro Glu Gln Gln Gln Gln Pro Gln Gln Gln Ile Ala Pro 50 55 60 Asn Gln Ile Glu Ala Pro Gln Pro Asn Phe Glu Gln His Tyr Gln Gly 65 70 75 80 Gln Gln Val Gln Gln Pro Gln Asn Pro Gln Thr Pro Asp Tyr Arg Gln 85 90 95 Asn Tyr Glu Asp Pro Asn Arg Thr Ser 100 105 <210> 12 <211> 945 <212> DNA <213> Corynebacterium glutamicum <400> 12 atgtccattg ttgagcacat caaagagttt cgacgccgac ttcttatcgc tctggcgggc 60 atcctcgtgg gcaccattat cggctttatt tggtacgatt tctcattttg gcagatcccc 120 actttgggcg agctgctgag ggatccgtac tgttctctgc ctgctgaatc ccgctgggcc 180 atgagcgact cagaggaatg tcgactgctc gcaaccggcc cgtttgatcc attcatgctt 240 cgccttaaag tagcggcgtt ggtgggtatg gttcttggct cacccgtgtg gctgagccag 300 ctgtggggct ttatcacccc aggtttgatg aagaatgagc gccgttacac cgcaatcttc 360 gtcacgattg ctgttgtgct gtttgtcggc ggtgctgttc ttgcgtactt cgtcgttgca 420 tatggtttgg agttcctcct taccattggt ggagacaccc aggcagcggc cctgactggt 480 gataagtact tcggattctt gctcgcgttg ttggcgattt tcggcgtgag cttcgaagtt 540 ccactggtga tcggcatgct caacattgtg ggtatcttgc cttacgatgc cattaaagat 600 aagcgacgca tgatcatcat gattttgttc gtgttcgctg ctttcatgac acccggccag 660 gatcctttca ccatgttggt gttggcgctt tcactcaccg ttctggtaga gcttgccctg 720 cagttctgtc gtttcaacga caaacgccgg gacaagaagc gcccagaatg gcttgatggc 780 gatgacctct ctgcatcacc actggatact tctgctggtg gagaagatgc tccaagccca 840 gtcgaaaccc cagaggcggt ggagccttcg cggatgctga acccaagtgg ggaggcgtcg 900 ataagctata aacccgggcg cgccgacttc ggtgacgtgc tctaa 945 <210> 13 <211> 314 <212> PRT <213> Corynebacterium glutamicum <400> 13 Met Ser Ile Val Glu His Ile Lys Glu Phe Arg Arg Arg Arg Leu Leu Ile 1 5 10 15 Ala Leu Ala Gly Ile Leu Val Gly Thr Ile Ile Gly Phe Ile Trp Tyr 20 25 30 Asp Phe Ser Phe Trp Gln Ile Pro Thr Leu Gly Glu Leu Leu Arg Asp 35 40 45 Pro Tyr Cys Ser Leu Pro Ala Glu Ser Arg Trp Ala Met Ser Asp Ser 50 55 60 Glu Glu Cys Arg Leu Leu Ala Thr Gly Pro Phe Asp Pro Phe Met Leu 65 70 75 80 Arg Leu Lys Val Ala Ala Leu Val Gly Met Val Leu Gly Ser Pro Val 85 90 95 Trp Leu Ser Gln Leu Trp Gly Phe Ile Thr Pro Gly Leu Met Lys Asn 100 105 110 Glu Arg Arg Tyr Thr Ala Ile Phe Val Thr Ile Ala Val Val Leu Phe 115 120 125 Val Gly Gly Ala Val Leu Ala Tyr Phe Val Val Ala Tyr Gly Leu Glu 130 135 140 Phe Leu Leu Thr Ile Gly Gly Asp Thr Gln Ala Ala Ala Leu Thr Gly 145 150 155 160 Asp Lys Tyr Phe Gly Phe Leu Leu Ala Leu Leu Ala Ile Phe Gly Val 165 170 175 Ser Phe Glu Val Pro Leu Val Ile Gly Met Leu Asn Ile Val Gly Ile 180 185 190 Leu Pro Tyr Asp Ala Ile Lys Asp Lys Arg Arg Met Ile Ile Met Ile 195 200 205 Leu Phe Val Phe Ala Ala Phe Met Thr Pro Gly Gln Asp Pro Phe Thr 210 215 220 Met Leu Val Leu Ala Leu Ser Leu Thr Val Leu Val Glu Leu Ala Leu 225 230 235 240 Gln Phe Cys Arg Phe Asn Asp Lys Arg Arg Asp Lys Lys Arg Pro Glu 245 250 255 Trp Leu Asp Gly Asp Asp Leu Ser Ala Ser Pro Leu Asp Thr Ser Ala 260 265 270 Gly Gly Glu Asp Ala Pro Ser Pro Val Glu Thr Pro Glu Ala Val Glu 275 280 285 Pro Ser Arg Met Leu Asn Pro Ser Gly Glu Ala Ser Ile Ser Tyr Lys 290 295 300 Pro Gly Arg Ala Asp Phe Gly Asp Val Leu 305 310 <210> 14 <211> 471 <212> DNA <213> Corynebacterium glutamicum <400> 14 atgttttcta gcgtgggttg gggagagatc ttcctcttag tcgttgtggg ccttgttgtc 60 atcggcccgg aacggttgcc tcgtttgatc caggacgcac gcgctgcgct gctcgctgca 120 cgtaccgcta tcgacaatgc aaagcagtcg ttggacagtg attttggttc ggaatttgat 180 gaaatccgaa agccactaac ccaggttgca cagtacagcc ggatgagccc caagacggcc 240 atcactaagg cgttatttga taatgattcc tcgttcctgg atgactttga tccaaagaag 300 atcatggccg aaggaacaga aggcgaagct cagcgcaaca agcaggcagc tgacaacaat 360 gcgaatgtgg tggaacgtcc agctgatggt tccaccgcac gcccaacgca aaacgatcca 420 aaagacggcc cgaattactc aggtggcgtc tcttggaccg atattattta g 471 <210> 15 <211> 156 <212> PRT <213> Corynebacterium glutamicum <400> 15 Met Phe Ser Ser Val Gly Trp Gly Glu Ile Phe Leu Leu Val Val Val 1 5 10 15 Gly Leu Val Val Ile Gly Pro Glu Arg Leu Pro Arg Leu Ile Gln Asp 20 25 30 Ala Arg Ala Ala Leu Leu Ala Ala Arg Thr Ala Ile Asp Asn Ala Lys 35 40 45 Gln Ser Leu Asp Ser Asp Phe Gly Ser Glu Phe Asp Glu Ile Arg Lys 50 55 60 Pro Leu Thr Gln Val Ala Gln Tyr Ser Arg Met Ser Pro Lys Thr Ala 65 70 75 80 Ile Thr Lys Ala Leu Phe Asp Asn Asp Ser Ser Phe Leu Asp Asp Phe 85 90 95 Asp Pro Lys Lys Ile Met Ala Glu Gly Thr Glu Gly Glu Ala Gln Arg 100 105 110 Asn Lys Gln Ala Ala Asp Asn Asn Ala Asn Val Val Glu Arg Pro Ala 115 120 125 Asp Gly Ser Thr Ala Arg Pro Thr Gln Asn Asp Pro Lys Asp Gly Pro 130 135 140 Asn Tyr Ser Gly Gly Val Ser Trp Thr Asp Ile Ile 145 150 155 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> CgR0949-F <400> 16 gcatgggatc catgcaaata aaccgccgag gcttcttaaa agc 43 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CgR0949-R <400> 17 tgggcctcta gatgctccaa gggcgttggc ctttggc 37 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DagA_F <400> 18 ctagaggccc agccggccaa gcagacctcg aatgggaac 39 <210> 19 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> DagA_R <400> 19 ggccgcggcc cccgaggccc tatttgtcat cgtcatcttt ataatccacg gcctgatacg 60 tcctgaccca gcggtagta 79 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> TatAC-F <400> 20 ggggccgcgg ccgcaagaag gagatataat gtccctcgga ccatgg 46 <210> 21 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> TatAC-R <400> 21 aggcagcggc cgcaactagt gtgtcagatc tttagagcac gtcaccgaag tcggcgcgcc 60 cgg 63 <210> 22 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> TatB-F <400> 22 ctctaaagat cttttttaat ccttatttac attttctgaa agaccggtc 49 <210> 23 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> TatB-R <400> 23 cggccgcaac tagtctaaat aatatcggtc caagagacgc cacctgagta at 52 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cg2052A-F <400> 24 tgacagtcga caggtcacga cacagagt 28 <210> 25 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Cg2052A-R <400> 25 caacagtgtt tcaaactaaa aaacgagggg attcctttct gttttgc 47 <210> 26 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Cg2052B-F <400> 26 gcaaaacaga aaggaatccc gtcgtttttt agtttgaaac actgttg 47 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Cg2052B-R <400> 27 tgtcaggatc caaaacttct tcgagatcac tcttttg 37 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Cg1514A-F <400> 28 tagacgtcga ccatcaacaa cgcaaaatac tggat 35 <210> 29 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Cg1514A-R <400> 29 aggggtgatc tgttcgagca aaaagagctc ctgatcatgt a 41 <210> 30 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Cg1514B-F <400> 30 tacatgatca ggagctcttt ttgctcgaac agatcacccc t 41 <210> 31 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Cg1514B-R <400> 31 tgtcaggtac cattgtctcg tggtttgttg c 31

Claims (6)

The function of the secretion signal peptide of cg2052 represented by the amino acid sequence of SEQ ID NO: 1 and/or the secretion signal peptide of cg1514 represented by the amino acid sequence of SEQ ID NO: 2, which is inherently present in microorganisms of the genus Corynebacterium, is inhibited,
A recombinant microorganism of the genus Corynebacterium having an improved extracellular secretion and production ability of the target protein into which a gene encoding the target protein has been introduced.
The recombinant microorganism of the genus Corynebacterium according to claim 1, wherein the target protein is operably linked to a secretion signal peptide that induces secretion of the target protein in the microorganism of the genus Corynebacterium.
The method according to claim 2, wherein the secretion signal peptide inducing secretion of the target protein is a Cg2052 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 1, a Cg1514 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 3 CgR0949 secretion signal peptide represented by the sequence, TorA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 4, CspA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 5, Prorin B (PorB) represented by the amino acid sequence of SEQ ID NO: 6 ) Secretion signal peptide, CgR_2070 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 7, and AprE secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 8 Recombinant microorganism of the genus Corynebacterium, characterized in that it is selected from the group consisting of.
The method according to claim 3, wherein the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 3 or a TorA secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 4, and the recombinant microorganism is A recombinant microorganism of the genus Corynebacterium, characterized in that a gene encoding any one or more proteins selected from the group consisting of TatA, TatB and TatC is additionally introduced or amplified.
The method according to claim 4, wherein the secretion signal peptide inducing secretion of the target protein is a CgR0949 secretion signal peptide represented by the amino acid sequence of SEQ ID NO: 3, and the recombinant microorganism has TatA and TatC proteins encoding genes are additionally introduced or Recombinant microorganism of the genus Corynebacterium, characterized in that amplified.
A method for secretion and production of a target protein comprising the steps of:
(a) culturing the recombinant microorganism of the genus Corynebacterium according to any one of claims 1 to 5 to express, secrete, and produce the target gene: and
(b) recovering the secreted/produced target protein.

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