# Patent

## Title
Shuttle vector for Corynebacterium or E. coli

## Abstract

Disclosed is a shuttle vector that can be used for Corynebacterium and E. coli, containing: a repressor selected from a group consisting of a lacI repressor and a tetR repressor; a promoter selected from a group consisting of a trc promoter, a tetA promoter and a LacUV5 promoter; a replication origin pBL1 derived from Corynebacterium glutamicum; and a replication origin ColE1 of E. coli. A host cell transformed with the shuttle vector can effectively produce industrially useful substances. Also, the shuttle vector may be used to easily insert various combinations of target genes and, as a result, a variety of vectors can be prepared effectively.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to Korean Patent Application No. 10-2014-0146255, filed on Oct. 27, 2014, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference.
BACKGROUND
1. Field
The present disclosure relates to a shuttle vector that can be used for both Corynebacterium and E. coli, a host cell transformed with the shuttle vector, a method for producing a substance using the transformed host cell and a multi-shuttle vector using the shuttle vector.
[Description about National Research and Development Support]
This study was supported by Ministry of Science, ICT and Future Planning, Republic of Korea (Cooperation research project: Creative Amalgamation Research, Project No. CAP-11-1-KIST) under the superintendence of the Fundamental technology research society.
2. Description of the Related Art
At present, the newest metabolic engineering techniques such as next-generation genome sequencing and fast DNA synthesis are used to produce useful bioproducts. Bioproduct production using E. coli and yeast, which are the most widely employed industrially at present, has been being developed consistently. For production of foreign metabolites, reconstruction of heterologous genes into cells is an essential process. Also, an optimizing process is necessary to maximize the production of target substances. A usual method of introducing a heterologous metabolic pathway for production of a target substance is to insert a specific gene into a plasmid or a genome. More recently, sequence- and ligase-independent cloning (SLIC), Gibson DNA assembly, circular polymerase extension cloning (CPEC), etc. are used as advanced cloning methods.
SUMMARY
In an aspect, the present disclosure is directed to providing a shuttle vector that can operate in both Corynebacterium and E. coli. 
In another aspect, the present disclosure is directed to providing a vector that can express two or more proteins at the same time, with two or more target genes inserted therein.
In another aspect, the present disclosure is directed to providing a vector that can operate effectively in industrially useful Corynebacterium glutamicum. 
In another aspect, the present disclosure is directed to providing an expression vector that can effectively produce industrially useful substances and a host cell transformed with the vector.
In another aspect, the present disclosure is directed to providing a method for effectively producing industrially useful substances.
In another aspect, the present disclosure is directed to providing a method for preparing a multi-shuttle vector that can express two or more target proteins which can invoke a mechanism for producing useful substances or which themselves are industrially useful.
In another aspect, the present disclosure is directed to providing a vector that can sustain strong expression even when the host cell is in a stationary phase.
In an aspect, the present disclosure provides a shuttle vector for Corynebacterium and E. coli, containing: a repressor selected from a group consisting of a lacI repressor and a tetR repressor; a promoter selected from a group consisting of a trc promoter, a tetA promoter and a LacUV5 promoter; a replication origin pBL1 derived from Corynebacterium glutamicum; and a replication origin ColE1 of E. coli. 
In an aspect, the present disclosure provides a host cell transformed with the shuttle vector.
In an aspect, the present disclosure provides a method for producing a substance, including culturing the transformed host cell.
In an aspect, the present disclosure provides a method for preparing a multi-shuttle vector containing two or more target genes, including inserting a target gene into the shuttle vector according to an exemplary embodiment of the present disclosure, wherein the shuttle vector already contains one or more target gene (i.e., “pre-existing target gene”) before the insertion of the target gene, and the step of inserting includes forming complementary binding between the BglII site located upstream of the pre-existing target gene of the shuttle vector or the target gene to be inserted into the shuttle vector and the BamHI site located downstream of the pre-existing target gene or another target gene to be inserted into the shuttle vector.
The vector disclosed in the present disclosure may be used to produce various chemicals and amino acids. Specifically, it can express two or more target proteins which can invoke a mechanism for producing useful substances or which themselves may be industrially useful. In another aspect, the vector provided by the present disclosure can sustain strong expression even when the host cell is in a stationary phase. In addition, time and cost can be saved greatly and the level of gene expression can be effectively controlled owing to simple gene assembly.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically illustrates BglBrick cloning.
FIG. 2 schematically illustrates a procedure of obtaining a vector wherein target genes A-B and target genes B-A are inserted through BglBrick cloning.
FIG. 3 schematically illustrates development of a pBbEB1c-RFP vector as one of CoryneBrick vectors using the CPEC method.
FIG. 4 illustrates assembly of pBbEB2c-RFP and pBbEB5c-RFP vectors as CoryneBrick vectors through restriction enzyme treatment and ligation.
FIG. 5 shows a result of measuring RFP-specific fluorescence and growth of C. glutamicum strains transformed with three CoryneBrick vectors (a. pBbEB1c-RFP, b. pBbEB2c-RFP, c. pBbEB5c-RFP) (IPTG or aTc induction; no induction).
FIG. 6 illustrates a metabolic pathway of xylose.
FIG. 7 shows growth of Corynebacterium glutamicum in which the target genes xylA and xylB are introduced and xylose concentrations in a medium containing 1% xylose.
FIG. 8 shows a map of a pBbE1c-RFP vector as a template for constructing a vector according to an exemplary embodiment of the present disclosure.
FIG. 9a and FIG. 9b show the sequence of a pBbEB1c-RFP vector according to an exemplary embodiment of the present disclosure. The underlined portions indicate lacI (76-1167), pTrc (1224-1463) and pBL1 (2477-5031), in that order.
FIG. 10a and FIG. 10b show the sequence of a pBbEB2c-RFP vector according to an exemplary embodiment of the present disclosure. The underlined portions indicate tetR (21-627), tetA (651-712) and pBL1 (1885-4280), in that order.
FIG. 11a and FIG. 11b show the sequence of a pBbEB5c-RFP vector according to an exemplary embodiment of the present disclosure. The underlined portions indicate lacI (49-1140), LacUV5 (1232-1585) and pBL1 (2599-5153), in that order.



DETAILED DESCRIPTION
In the present disclosure, a “multi-shuttle vector” refers to a shuttle vector which contains two or more target genes or two or more kinds of target genes desired to be overexpressed and can express two or more target proteins or two or more kinds of target proteins at the same time upon transformation. The expressed two or more proteins may be used as they are or the two or more proteins may invoke a mechanism for producing desired useful substances.
Genes within all the vectors disclosed in the present disclosure are operably linked with each other. The term “operable” means that the target gene can be expressed normally.
In an aspect, the present disclosure provides a shuttle vector for Corynebacterium and E. coli, containing: a repressor selected from a group consisting of a lacI repressor and a tetR repressor; a promoter selected from a group consisting of a trc promoter, a tetA promoter and a LacUV5 promoter; a replication origin pBL1 derived from Corynebacterium glutamicum; and a replication origin ColE1 of E. coli. 
Since the vector can be used both for Corynebacterium and E. coli, it is possible to easily recombine a gene in E. coli and to obtain a desired protein in a Corynebacterium strain through transformation.
For example, a template vector of the shuttle vector for Corynebacterium and E. coli may be pBbE1c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). Also, the repressor and the promoter may be ones derived, for example, from pBbA2k-RFP and/or pBbE5c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). Also, the replication origin pBL1 derived from Corynebacterium glutamicum may be one derived, for example, from pXMJ19 (Jakoby M et al., (1999) “Construction and application of new Corynebacterium glutamicum vectors.” Biotechnology Techniques 13, 437-441).
In an aspect, the vector may further contain a chloramphenicol-resistant reporter gene.
In another aspect, the vector may further contain a red fluorescent protein (RFP) gene. Since the red fluorescent protein (RFP) gene emits fluorescence when expressed in a host cell, it informs that the vector operates normally and the target gene has been expressed normally. Another target gene may be inserted at the site of the red fluorescent protein (RFP) gene. The vector may contain a BglII site and a BamHI site as restriction enzyme sites on both sides of the red fluorescent protein (RFP) gene.
For example, the chloramphenicol-resistant reporter gene and the red fluorescent protein (RFP) gene may be ones derived from the template vector pBbE1c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12).
In another aspect, the vector may further contain a BglII site and a BamHI site as restriction enzyme sites. In another aspect, the vector may further contain an EcoRI, a BglII site, a BamHI site and an XhoII site as restriction enzyme sites.
In another aspect, the shuttle vector for Corynebacterium and E. coli may further contain a target gene encoding a target protein desired to be overexpressed.
In another aspect, the shuttle vector for Corynebacterium and E. coli may contain two or more target genes.
In an aspect, the two or more target genes may be ones derived from different vectors. The different vectors may have a BglII site and a BamHI site on both sides of the target gene and the two target genes may be contained in one vector through complementary binding between the BglII site of one vector and the BamHI site of another vector.
BglBrick cloning is a cloning method which does not require a PCR amplification process. For example, target genes A and B respectively contained in two vectors can be simply cloned into one vector. Referring to FIG. 1, each vector has EcoRI site, BglII, BamHI and XhoI sites as restriction enzyme sites, which allows binding of partB immediately downstream of the target gene partA. It is possible because, upon enzyme treatment, the BamHI and the BglII sites can bind again with each other because their DNA strands are complementary to each other. In this manner, various genes can be simply cloned into one vector.
In an aspect, the BglII site and the BamHI site may be located on both sides of the target gene. In another aspect, the order of the target gene and the restriction enzyme sites may be: EcoRI->BglII site->target gene->BamHI site->XhoII site. For example, let's call two vectors arranged in this order and having different target genes, i.e. a target gene A and a target gene B, a vector A and a vector B, respectively. By treating the vector A with restriction enzymes EcoRI and BamHI and treating the vector B with restriction enzymes EcoRI and BglII and then ligating the cut BglII site and the cut BamHI site through complementary binding, a shuttle vector in which the target genes A-B are inserted can be obtained (FIG. 2). Conversely, by treating the vector A with BglII and XhoI, and treating the vector B with BamHI and XhoI and then ligating, the target genes can be inserted in the order of B-A (FIG. 2).
In an aspect, the pBbEB1c-RFP shuttle vector may be a vector wherein a replication origin pBL1 derived from Corynebacterium glutamicum is inserted into a vector of SEQ ID NO 4 and the replication origin pBL1 may be located upstream of a replication origin ColE1 of E. coli. 
For example, the shuttle vector according to the present disclosure may be a shuttle vector prepared newly using the BglBrick vectors published in the literature such as pBbE1c-RFP, pBbA2k-RFP and pBbE5c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and pXMJ19 (Jakoby M et al., (1999) “Construction and application of new Corynebacterium glutamicum vectors.” Biotechnology Techniques 13, 437-441). The shuttle vector according to the present disclosure will be called a CoryneBrick vector. In an aspect, the CoryneBrick vector may be prepared by a CPEC cloning method or a traditional restriction-ligation cloning method. First, a pBbE1c-RFP vector may be prepared by inserting the Corynebacterium glutamicum replication origin pBL1 of a pXMJ19 vector into a specific portion using the CPEC method. For example, the vector may be a vector having a sequence of SEQ ID NO 1.
In SEQ ID NO 1, lacI is 76-1167, pTrc is 1224-1463 and pBL1 is 2477-5031.
In another aspect, the shuttle vector for Corynebacterium and E. coli may be a pBbEB2c-RFP vector containing a tetR repressor and a tetA promoter. In an aspect, the vector may be one prepared by removing a lacI repressor and a trc promoter from a vector of SEQ ID NO 1 using a restriction enzyme and inserting a tetR repressor and a tetA promoter. For example, the pBbEB2c-RFP vector may have a sequence of SEQ ID NO 2.
In SEQ ID NO 2, tetR is 21-627, tetA is 651-712 and pBL1 is 1885-4280.
In another aspect, the shuttle vector for Corynebacterium and E. coli may be a pBbEB5c-RFP vector containing a lacI repressor and a LacUV5 promoter. In an aspect, the vector may be one prepared by removing a lacI repressor and a trc promoter from a vector of SEQ ID NO 1 using a restriction enzyme, and inserting a lacI repressor and a LacUV5 promoter. For example, the pBbEB5c-RFP vector may have a sequence of SEQ ID NO 3.
In SEQ ID NO 3, lacI is 49-1140, LacUV5 is 1232-1585 and pBL1 is 2599-5153.
In an aspect, the present disclosure provides a host cell transformed with any of the above-described shuttle vectors. The host cell may be a Corynebacterium or E. coli cell. In an exemplary embodiment of the present disclosure, a Corynebacterium glutamicum strain may be used as a host cell to be transformed with the shuttle vector. Corynebacterium glutamicum is a bacterial strain producing various amino acids and nucleotides and is the most widely industrially used strain at present. The wild type is known to be capable of using glucose and sucrose as carbon sources, but not xylose and cellobiose or starch. A variety of amino acids and chemicals can be produced using the carbon sources that could not be used by Corynebacterium glutamicum, using a metabolic engineering technique.
In an aspect, the present disclosure provides a method for producing a substance, including culturing the transformed host cell described above. The method may produce two or more proteins at the same time. The produced two or more proteins may invoke a mechanism necessary for producing useful substances or the proteins themselves may be industrially useful. In an aspect, the substance may be an amino acid.
In an aspect, the present disclosure provides a method for preparing a multi-shuttle vector containing two or more target genes. For example, the method may include inserting a target gene into the shuttle vector according to an exemplary embodiment of the present disclosure, wherein the shuttle vector already contains one or more target gene before the insertion of the target gene, and the step of inserting includes forming complementary binding between the BglII site located upstream of the pre-existing target gene or the target gene to be inserted into the shuttle vector; and the BamHI site located downstream of the pre-existing target gene or another target gene to be inserted into the shuttle vector.
In an aspect, the complementary binding may be formed between the BglII site located upstream of the pre-existing target gene and the BamHI site located downstream of another target gene to be inserted into the shuttle vector, and, as a result of the complementary binding, the inserted target gene may be inserted upstream of the pre-existing target gene. For example, a procedure of inserting a target gene A to a shuttle vector already having a target gene B such that the genes are in the order of A-B is shown in the left side of FIG. 2. The target gene A to be inserted is designed such that it has EcoRI and BglII restriction enzyme sites at the upstream side, and a BamHI restriction enzyme site at the downstream side. This may be accomplished by using a vector having the target gene A, and having EcoRI and BglII restriction enzyme sites upstream thereof, and BamHI and XhoI restriction enzyme sites downstream thereof. By treating the vector with restriction enzymes EcoRI and BamHI, the target gene A having EcoRI and BglII restriction enzyme sites at the upstream side and a BamHI restriction enzyme site at the downstream side may be obtained. Meanwhile, a vector having the target gene B and having EcoRI and BglII restriction enzyme sites upstream of B, and BamHI and XhoI restriction enzyme sites downstream thereof is treated with restriction enzymes EcoRI and BglII. When the restriction enzyme site-containing and target A- and target B-containing vectors are mixed, complementary binding is formed between the BglII site located upstream of the target gene B of the vector and the BamHI site located downstream of the target gene A to be inserted into the shuttle vector and, as a result, a vector wherein the target genes are inserted in the order of A-B can be obtained.
In another aspect, the complementary binding may be formed between the BglII site located upstream of another target gene to be inserted into the shuttle vector and the BamHI site located downstream of the target gene of the shuttle vector, and, as a result of the complementary binding, the target gene may be inserted downstream of the pre-existing target gene. For example, a procedure of inserting a target gene A to a shuttle vector already having a target gene B such that the genes are in the order of B-A is shown in the right side of FIG. 2. The target gene A to be inserted is designed such that it has a BglII restriction enzyme site at the upstream side and BamHI and XhoI restriction enzyme sites at the downstream side. This may be accomplished by using a vector having the target gene A and having EcoRI and BglII restriction enzyme sites upstream of the A, and BamHI and XhoI restriction enzyme sites downstream of the A. By treating the vector with restriction enzymes BglII and XhoI, the target gene A having EcoRI and BglII restriction enzyme sites at the upstream side, and BamHI and XhoI restriction enzyme sites at the downstream side may be obtained. Meanwhile, a vector having the target gene B, and having EcoRI and BglII restriction enzyme sites upstream of B and BamHI and XhoI restriction enzyme sites downstream thereof is treated with restriction enzymes BamHI and XhoI. When the restriction enzyme site-containing and target A- and target B-containing vectors are mixed, complementary binding is formed between the BamHI site located downstream of the target gene B of the vector and the BglII site located upstream of the target gene A to be inserted into the shuttle vector and, as a result, a vector wherein the target genes are inserted in the order of B-A can be obtained.
In another aspect, the method may further include, before the step of inserting, preparing a restriction enzyme site-comprising target gene wherein the BglII site is located upstream of the target gene to be inserted and the BamHI site is located downstream of the target gene to be inserted. The restriction enzyme site-containing target gene may be prepared according to any method known in the art. For example, as described above, a vector having the target gene to be inserted and having EcoRI and BglII restriction enzyme sites upstream of the target and BamHI and XhoI restriction enzyme sites downstream thereof may be used.
Example 1
Construction of pBbEB1c-RFP as Novel CoryneBrick Vector
A BglBrick plasmid pBbE1c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and a pXMJ19 vector having a C. glutamicum replication origin pBL1 (Jakoby M et al., (1999) “Construction and application of new Corynebacterium glutamicum vectors.” Biotechnology Techniques 13, 437-441) were used as DNA templates for construction of a CoryneBrick vector pBbEB1c-RFP. The C. glutamicum replication origin pBL1 of the pXMJ19 was assembled into the pBbE1c-RFP using the CPEC method (Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441). The resulting vector was transformed into E. coli HIT-DH5α (Cat# RH617-J80, RBC Bioscience) and then extracted with mini-prep. Since the assembled vector had the possibility of mutation because it was prepared through PCR, the entire sequence was identified by plasmid sequencing. Thus obtained CoryneBrick vector was named as pBbEB1c-RFP (FIG. 3).
Example 2
Construction of pBbEB2c-RFP and pBbEB5c-RFP Using pBbEB1c-RFP CoryneBrick Vector
The pBbEB1c-RFP prepared in Example 1 and the previously known pBbA2k-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and pBbE5c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) were used to prepare CoryneBrick vectors pBbEB2c-RFP and pBbEB5c-RFP. Because only the promoter and repressor portions of pBbA2k-RFP and pBbE5c-RFP were necessary, instead of using the CPEC method as in Example 1, the promoter and repressor sites of the pBbEB1c-RFP prepared in Example 1 were removed with restriction enzymes and the promoter and repressor portions of pBbA2k-RFP and pBbE5c-RFP were inserted there. The used restriction enzymes were AatII and EcoRI. The promoter and repressor portions of pBbA2k-RFP and pBbE5c-RFP and the promoter and repressor portions of pBbEB1c-RFP were cut using the restriction enzymes and then the promoter and repressor portions of pBbA2k-RFP and pBbE5c-RFP were inserted into the restriction enzyme-treated pBbEB1c-RFP to prepare pBbEB2c-RFP and pBbEB5c-RFP, respectively (FIG. 4).
Example 3
Testing of CoryneBrick Vector for Gene Expression in C. glutamicum 
It was tested whether the three CoryneBrick vectors prepared in Examples 1 and 2 are suitable for gene expression in C. glutamicum. The expression kinetics of the red fluorescent protein (RFP) inserted in each vector was analyzed. For the analysis, excitation and emission at wavelengths of 535 nm and 620 nm, respectively, were measured using an automatic microplate reader (Tecan Infinite M200 pro, Tecan Group Ltd., Switzerland). Specific fluorescence (fluorescence intensity/cell density) during the growth of the strain was measured using the instrument. After transformation of pBbEB1c-RFP, pBbEB2c-RFP and pBbEB5c-RFP into the Corynebacterium glutamicum strain (ATCC 13032), the transformed cells were cultured overnight in BHIS (brain-heart infusion, supplemented) medium at 30° C. The cultured cells were cultured in fresh BHIS medium for 4 hours using 1 mM IPTG (pBbEB1c-RFP, pBbEB5c-RFP) or 100 nM aTc (pBbEB2c-RFP) as an inducer with a start OD of 0.1. 200 μL of the culture was transferred onto a 96-well plate and fluorescence intensity and optical density were measured at 30° C. and 200 rpm for 50 hours. The result is shown in FIG. 5.
FIG. 5 shows the RFP-specific fluorescence and growth of the C. glutamicum strain transformed with the three CoryneBrick vectors (a. pBbEB1c-RFP, b. pBbEB2c-RFP, c. pBbEB5c-RFP) (red: IPTG or aTc induction, black: no induction). As seen from a and c in FIG. 5, the pBbEB1c-RFP and the pBbEB5c-RFP induced with 1 mM IPTG exhibited 5-fold and 2.5-fold increased specific fluorescence in 48 hours as compared to the non-induced control. The cells not induced with IPTG maintained a constant value. From this result, it was confirmed that the pBbEB1c-RFP having the trc promoter exhibits about 2-fold stronger RFP gene expression as compared to the pBbEB5c-RFP having the lacUV5 promoter. Similarly to the pBbEB5c-RFP, the pBbEB2c-RFP having the tetA promoter and the tetR repressor exhibited about 2.5-fold stronger specific fluorescence when induced with 100 nm aTc as compared to the non-induced cells.
From this result, it was confirmed that the pBbEB1c having the trc promoter exhibits about 2-fold stronger gene expression as compared to the pBbEB5c having the lacUV5 promoter or the pBbEB2c having the tetA promoter.
Example 4
Culturing of C. glutamicum Strain Wherein pBbEB1c-xylA, pBbEB1c-xylA-xylB and pBbEB1c-xylB-xylA are Introduced in Medium Containing 1% Xylose
After the operation of the CoryneBrick vectors in C. glutamicum was confirmed, codons were optimized using the Gene Designer 2.0 software (DNA2.0, MenloPark, CA, USA). E. coli xylA (xylose isomerase) and xylB (xylulokinase) genes prepared by the BglBrick method were inserted into the pBbEB1c-RFP vector and it was investigated whether xylose was consumed. It was because, although the wild-type C. glutamicum strain is presumed to have the xylB gene for metabolizing xylose, it cannot use xylose as a carbon source because it lacks the xylA gene. Since it is already known that C. glutamicum to which xylA and xylB have been introduced can consume xylose, if the Corynebacterium glutamicum could consume xylose, it directly confirms that the CoryneBrick vector constructed according to the present disclosure operates normally.
The C. glutamicum strain was transformed with codon-optimized E. coli xylA and xylB using the CoryneBrick vector. The codon-optimized E. coli xylA and xylB genes were acquired from GenScript, and EcoRI and BglII sites were attached upstream of the gene and a BamHI site was attached downstream of the gene for use in BglBrick cloning. First, the xylA gene was inserted into the pBbEB1c vector using restriction enzymes EcoRI and BamHI. The resulting pBbEB1c-xylA was transformed into Corynebacterium glutamicum (CgEcXylA) and cultured for 56 hours in CGXII medium+1% xylose medium at 30° C. and 200 rpm, with a start OD of 1. The specific growth rate was 0.09/h and the final OD was about 15.7.
Then, the codon-optimized xylB gene was inserted upstream or downstream of xylA by the BglBrick method. After transformation, Corynebacterium glutamicum was cultured in the presence of 1% xylose. To prepare pBbEB1c-xylA-xylB, the pBbEB1c-xylA was treated with BamHI and XhoI restriction enzymes and the xylB gene was ligated after treating with BglII and XhoI restriction enzymes. To prepare pBbEB1c-xylB-xylA, the pBbEB1c-xylA was treated with EcoRI and BglII restriction enzymes and xylB was ligated after treating with EcoRI and BamHI restriction enzymes. After the culturing, the Corynebacterium glutamicum transformed with the pBbEB1c-xylA-xylB (CgEcXylAB) showed a specific growth rate of 0.11/h, which is slightly faster than that for CgEcXylA, and a final OD, which is similar to that for CgEcXylA. The Corynebacterium glutamicum transformed with the pBbEB1c-xylB-xylA (CgEcXylBA) showed no significant difference in specific growth rate and final OD as compared to CgEcXylA (see FIG. 6).
Xylose consumption of each strain was analyzed by HPLC after sampling the culture every hour and centrifuging the sample at 14000 rpm for 10 minutes. The result is shown in FIG. 6.
SEQ ID NO 4: pBbE1c-RFP (4051 bps)









gacgtcgacaccatcgaatggtgcaaaacctttcg



 



cggtatggcatgatagcgcccggaagagagtcaat



 



tcagggtggtgaatgtgaaaccagtaacgttatac



 



gatgtcgcagagtatgccggtgtctcttatcagac



 



cgtttcccgcgtggtgaaccaggccagccacgttt



 



ctgcgaaaacgcgggaaaaagtggaagcggcgatg



 



gcggagctgaattacattcccaaccgcgtggcaca



 



acaactggcgggcaaacagtcgttgctgattggcg



 



ttgccacctccagtctggccctgcacgcgccgtcg



 



caaattgtcgcggcgattaaatctcgcgccgatca



 



actgggtgccagcgtggtggtgtcgatggtagaac



 



gaagcggcgtcgaagcctgtaaagcggcggtgcac



 



aatcttctcgcgcaacgcgtcagtgggctgatcat



 



taactatccgctggatgaccaggatgccattgctg



 



tggaagctgcctgcactaatgttccggcgttattt



 



cttgatgtctctgaccagacacccatcaacagtat



 



tattttctcccatgaagacggtacgcgactgggcg



 



tggagcatctggtcgcattgggtcaccagcaaatc



 



gcgctgttagcgggcccattaagttctgtctcggc



 



gcgtctgcgtctggctggctggcataaatatctca



 



ctcgcaatcaaattcagccgatagcggaacgggaa



 



ggcgactggagtgccatgtccggttttcaacaaac



 



catgcaaatgctgaatgagggcatcgttcccactg



 



cgatgctggttgccaacgatcagatggcgctgggc



 



gcaatgcgcgccattaccgagtccgggctgcgcgt



 



tggtgcggatatctcggtagtgggatacgacgata



 



ccgaagacagctcatgttatatcccgccgttaacc



 



accatcaaacaggattttcgcctgctggggcaaac



 



cagcgtggaccgcttgctgcaactctctcagggcc



 



aggcggtgaagggcaatcagctgttgcccgtctca



 



ctggtgaaaagaaaaaccaccctggcgcccaatac



 



gcaaaccgcctctccccgcgcgttggccgattcat



 



taatgcagctggcacgacaggtttcccgactggaa



 



agcgggcagtgagcgcaacgcaattaatgtaagtt



 



agcgcgaattgatctggtttgacagcttatcatcg



 



actgcacggtgcaccaatgcttctggcgtcaggca



 



gccatcggaagctgtggtatggctgtgcaggtcgt



 



aaatcactgcataattcgtgtcgctcaaggcgcac



 



tcccgttctggataatgttttttgcgccgacatca



 



taacggttctggcaaatattctgaaatgagctgtt



 



gacaattaatcatccggctcgtataatgtgtggaa



 



ttgtgagcggataacaatttcagaattcaaaagat



 



cttttaagaaggagatatacatatggcgagtagcg



 



aagacgttatcaaagagttcatgcgtttcaaagtt



 



cgtatggaaggttccgttaacggtcacgagttcga



 



aatcgaaggtgaaggtgaaggtcgtccgtacgaag



 



gtacccagaccgctaaactgaaagttaccaaaggt



 



ggtccgctgccgttcgcttgggacatcctgtcccc



 



gcagttccagtacggttccaaagcttacgttaaac



 



acccggctgacatcccggactacctgaaactgtcc



 



ttcccggaaggtttcaaatgggaacgtgttatgaa



 



cttcgaagacggtggtgttgttaccgttacccagg



 



actcctccctgcaagacggtgagttcatctacaaa



 



gttaaactgcgtggtaccaacttcccgtccgacgg



 



tccggttatgcagaaaaaaaccatgggttgggaag



 



cttccaccgaacgtatgtacccggaagacggtgct



 



ctgaaaggtgaaatcaaaatgcgtctgaaactgaa



 



agacggtggtcactacgacgctgaagttaaaacca



 



cctacatggctaaaaaaccggttcagctgccgggt



 



gcttacaaaaccgacatcaaactggacatcacctc



 



ccacaacgaagactacaccatcgttgaacagtacg



 



aacgtgctgaaggtcgtcactccaccggtgcttaa



 



ggatccaaactcgagtaaggatctccaggcatcaa



 



ataaaacgaaaggctcagtcgaaagactgggcctt



 



tcgttttatctgttgtttgtcggtgaacgctctct



 



actagagtcacactggctcaccttcgggtgggcct



 



ttctgcgtttatacctagggcgttcggctgcggcg



 



agcggtatcagctcactcaaaggcggtaatacggt



 



tatccacagaatcaggggataacgcaggaaagaac



 



atgtgagcaaaaggccagcaaaaggccaggaaccg



 



taaaaaggccgcgttgctggcgtttttccataggc



 



tccgcccccctgacgagcatcacaaaaatcgacgc



 



tcaagtcagaggtggcgaaacccgacaggactata



 



aagataccaggcgtttccccctggaagctccctcg



 



tgcgctctcctgttccgaccctgccgcttaccgga



 



tacctgtccgcctttctcccttcgggaagcgtggc



 



gctttctcatagctcacgctgtaggtatctcagtt



 



cggtgtaggtcgttcgctccaagctgggctgtgtg



 



cacgaaccccccgttcagcccgaccgctgcgcctt



 



atccggtaactatcgtcttgagtccaacccggtaa



 



gacacgacttatcgccactggcagcagccactggt



 



aacaggattagcagagcgaggtatgtaggcggtgc



 



tacagagttcttgaagtggtggcctaactacggct



 



acactagaaggacagtatttggtatctgcgctctg



 



ctgaagccagttaccttcggaaaaagagttggtag



 



ctcttgatccggcaaacaaaccaccgctggtagcg



 



gtggtttttttgtttgcaagcagcagattacgcgc



 



agaaaaaaaggatctcaagaagatcctttgatctt



 



ttctacggggtctgacgctcagtggaacgaaaact



 



cacgttaagggattttggtcatgactagtgcttgg



 



attctcaccaataaaaaacgcccggcggcaaccga



 



gcgttctgaacaaatccagatggagttctgaggtc



 



attactggatctatcaacaggagtccaagcgagct



 



cgatatcaaattacgccccgccctgccactcatcg



 



cagtactgttgtaattcattaagcattctgccgac



 



atggaagccatcacaaacggcatgatgaacctgaa



 



tcgccagcggcatcagcaccttgtcgccttgcgta



 



taatatttgcccatggtgaaaacgggggcgaagaa



 



gttgtccatattggccacgtttaaatcaaaactgg



 



tgaaactcacccagggattggctgagacgaaaaac



 



atattctcaataaaccctttagggaaataggccag



 



gttttcaccgtaacacgccacatcttgcgaatata



 



tgtgtagaaactgccggaaatcgtcgtggtattca



 



ctccagagcgatgaaaacgtttcagtttgctcatg



 



gaaaacggtgtaacaagggtgaacactatcccata



 



tcaccagctcaccgtctttcattgccatacgaaat



 



tccggatgagcattcatcaggcgggcaagaatgtg



 



aataaaggccggataaaacttgtgcttatttttct



 



ttacggtctttaaaaaggccgtaatatccagctga



 



acggtctggttataggtacattgagcaactgactg



 



aaatgcctcaaaatgttctttacgatgccattggg



 



atatatcaacggtggtatatccagtgatttttttc



 



tccattttagcttccttagctcctgaaaatctcga



 



taactcaaaaaatacgcccggtagtgatcttattt



 



cattatggtgaaagttggaacctcttacgtgccga



 



tcaacgtctcattttcgccagatatc

## Claims

1. A shuttle vector for Corynebacterium glutamicum and E. coli, comprising:
a repressor selected from a group consisting of a lacI repressor and a tetR repressor;
a promoter selected from a group consisting of a trc promoter, a tetA promoter and a LacUV5 promoter; a replication origin pBL1 derived from Corynebacterium glutamicum; and a replication origin ColE1 of E. coli, 
wherein the vector is
a pBbEB1c vector comprising a replication origin pBL1, a replication origin ColE1, a lacI repressor and a trc promoter;
a pBbEB2c vector comprising a replication origin pBL1, a replication origin ColE1, a tetR repressor and a tetA promoter; or
a pBbEB5c vector comprising a replication origin pBL1, a replication origin ColE1, a lacI repressor and a LacUV5 promoter.

2. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector further comprises a chloramphenicol-resistant reporter gene.

3. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector further comprises a red fluorescent protein (RFP) gene.

4. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector further comprises a BglII site and a BamHI site as restriction enzyme sites.

5. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 4, wherein the shuttle vector for Corynebacterium glutamicum and E. coli further comprises a target gene encoding a target protein desired to be overexpressed.

6. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 5, wherein the BglII site and the BamHI site are located on both sides of the target gene.

7. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 6, wherein the shuttle vector for Corynebacterium glutamicum and E. coli comprises two or more target genes.

8. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 7, wherein the two or more target genes are comprised in one vector through complementary binding between the BglII site located upstream of one target gene and the BamHI site located downstream of another target gene.

9. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector is a pBbEB1c-RFP vector.

10. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the shuttle vector is a vector wherein a replication origin pBL1 derived from Corynebacterium glutamicum is inserted into a vector of SEQ ID NO 4 and the replication origin pBL1 is located upstream of a replication origin ColE1 of E. coli provided that an RFP sequence in the pBbEB1c-RFP vector may be deleted or replaced with a different sequence.

11. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector has a sequence of SEQ ID NO 1 provided that an RFP sequence in the pBbEB1c-RFP vector may be deleted or replaced with a different sequence.

12. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector is a pBbEB2c-RFP vector.

13. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector is one prepared by removing a lacI repressor and a trc promoter from a vector of SEQ ID NO 1 using a restriction enzyme and inserting a tetR repressor and a tetA promoter provided that an RFP sequence in the pBbEB2c-RFP vector may be deleted or replaced with a different sequence.

14. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector has a sequence of SEQ ID NO 2 provided that an RFP sequence in the pBbEB2c-RFP vector may be deleted or replaced with a different sequence.

15. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector is a pBbEB5c-RFP vector.

16. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector is one prepared by removing a lacI repressor and a trc promoter from a vector of SEQ ID NO 1 using a restriction enzyme and inserting a lacI repressor and a LacUV5 promoter provided that an RFP sequence in the pBbEB5c-RFP vector may be deleted or replaced with a different sequence.

17. The shuttle vector for Corynebacterium glutamicum and E. coli according to claim 1, wherein the vector has a sequence of SEQ ID NO 3 provided that an RFP sequence in the pBbEB5c-RFP vector may be deleted or replaced with a different sequence.

18. A host cell transformed with the shuttle vector according to claim 1.

19. The host cell according to claim 18, wherein the host cell is a Corynebacterium glutamicum or E. coli.

20. The host cell according to claim 19, wherein the host cell is a Corynebacterium glutamicum.