Patent Publication Number: US-2016230179-A1

Title: Shuttle plasmid replicable in both clostridia and escherichia coli

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of Korean Patent Application No. 10-2015-0020510, filed on Feb. 10, 2015, which is hereby incorporated by reference in its entirety into this application. 
     BACKGROUND 
     1. Technical Field 
     The present invention relates to a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli.    
     2. Description of the Related Art 
     Microorganisms of the genus  Clostridium  are Gram-positive bacteria. They are obligate anaerobes capable of producing spores and have a variety of activity as biocatalysts. Recently, as the full genomes of some microorganisms of the genus  Clostridium  have recently been sequenced, it is expected that studies into  Clostridium  will contribute to the production of biofuels from renewable biomass and development of methods for interpreting and preventing pathogenic mechanisms of some microorganisms of the genus  Clostridium.  Although  Clostridium  is important industrially and medicinally, research of  Clostridium  has been extremely limited due to absence of useful genetic engineering techniques. Accordingly, there is a need for development of effective host/plasmid systems useful for developing  Clostridium  strains having an effectively high metabolic activity. 
     On the other hand, in order to replicate a plasmid in  acetobutylicum  and/or  Escherichia coli,  shuttle plasmids such as pMTL500E (Minton N P, Oultram J D. 1988. Host: vector systems for gene cloning in  Clostridium.  Microbiological Sciences 5: 310-315), pIM1 (Mermelstein L D, Papoutsakis E T. 1993. In vivo methylation in  Escherichia coli  by the  Bacillus subtilis  phage phi 3TI methyltransferase to protect plasmids from restriction upon transformation of  Clostridium acetobutylicum  ATCC 824. Appl Environ Microbiol 59:1077-1081.), pJIR418 (Sloan J, Warner T A, Scott P T, Bannam T L, Berryman D I, Rood J I. 1992. Construction of a sequenced  Clostridium perfringens - Escherichia coli  shuttle plasmid. Plasmid 27:207-219.), and pCB 102 (Fox M E, Lemmon M J, Mauchline M L, Davis T O, Giaccia A J, Minton N P, Brown J M. 1996. Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene therapy 3: 173-178.), and the like have been developed. The Clostridial replication origins (origins of replication) are derived from pAM β1, pIMP13, pIP404, and pCB102, respectively. However, no plasmid is used industrially since their segregational stability is very low under culture conditions with no antibiotic present. Namely, since use of antibiotics may deteriorate economic feasibility of microorganisms in terms of industrial application or can cause problems related to environmental stability, in order for plasmids to be used industrially, segregational stability of the plasmids in a medium containing no antibiotics should be guaranteed. The shuttle plasmids such as pMTL500E, pIM1, pJIR418, pCB102, and the like do not possess the requisite segregational stability. 
     The present inventors have endeavored to find a novel plasmid having excellent segregational stability in a culture medium containing no antibiotics and capable of replication in  Clostridium acetobutylicum,  and constructed plasmids including specific replication origins and regions encoding replication proteins, and the like which are found to be replicable in both  Clostridium  and  Escherichia coli  and have high segregational stability. The present invention is based on this finding. 
     BRIEF SUMMARY 
     It is an aspect of the present invention to provide a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli.    
     In accordance with one aspect of the present invention, there is provided a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli,  including: a base sequence of a first replication origin replicable in  Escherichia coli;  and a base sequence of a second replication origin derived from pUB110 plasmid. 
     The shuttle plasmid may be replicable in both  Clostridium  and  Escherichia coli.    
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and other aspects, features, and advantages of the present invention will become apparent from the detailed description of the following embodiments in conjunction with the accompanying drawings, in which: 
         FIG. 1  shows a genetic map of cryptic plasmid pUB110 derived from  Staphylococus aureus;    
         FIG. 2  shows a process for constructing a shuttle plasmid pLK1-MCS from pUB110 cryptic plasmid and pMTL500E plasmid including a base sequence of a replication origin of pUC19 plasmid; 
         FIG. 3  shows segregational stability of a shuttle plasmid pLK1-MCS of the present invention; and 
         FIG. 4  shows a process for constructing a recombinant plasmid in which a recombinant gene is cloned so that acetone is converted into isopropanol by using the shuttle plasmid of the present invention. 
     
    
    
     EXPLANATION OF SEQ ID NO 
     SEQ ID NO: 1 and SEQ ID NO: 2 are base sequences of two primers used in the amplification of a region encoding a replication protein and a replication origin of pUB110 plasmid. 
     SEQ ID NO: 3 is a base sequence of a replication origin of pUB110 plasmid. 
     SEQ ID NO: 4 is a base sequence of a region encoding a replication protein of pUB110 plasmid. 
     SEQ ID NO: 5 is an amino acid sequence of a replication protein (RepA) of pUB110 plasmid. 
     SEQ ID NO: 6 is a base sequence of a DNA fragment including a multiple cloning site used in the construction of a shuttle plasmid pLK1-MCS of the present invention. 
     SEQ ID NO: 7 is a base sequence of an erythromycin resistance gene. 
     SEQ ID NO: 8 is a base sequence of an ampicillin resistance gene. 
     SEQ ID NO: 9 is a base sequence of a replication origin of pUC19 plasmid. 
     SEQ ID NO: 10 is a base sequence of a shuttle plasmid pLK1-MCS of the present invention. 
     SEQ ID NO: 11 and SEQ ID NO: 12 are base sequences of primers used in the amplification of a region encoding a secondary alcohol dehydrogenase through PCR reaction. 
     DETAILED DESCRIPTION 
     The present invention relates to a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli  including: 
     a base sequence of a first replication origin replicable in  Escherichia coli;  and 
     a base sequence of a second replication origin derived from pUB110 plasmid. 
     In addition, the present invention relates to a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli  including: 
     a base sequence of pMTL500E plasmid including a replication origin of pUC19 plasmid (pUC origin) and ampicillin and erythromycin antibiotic resistance genes; and 
     a base sequence of a base sequence of a replication protein and a replication origin derived from pUB110 plasmid (pUB110 origin). 
     Further, the present invention relates to a method for constructing a transformed microorganism including: 
     preparing a shuttle plasmid of the present invention; and 
     introducing the shuttle plasmid into a microorganism. 
     Furthermore, the present invention relates to a transformed microorganism including a shuttle plasmid of the present invention. 
     Furthermore, the present invention relates to a method for producing a culture including: 
     culturing a transformed microorganism including a shuttle plasmid of the present invention; and 
     harvesting the culture. 
     Hereinafter, embodiments of the present invention will be described in detail. 
     Base Sequence of a First Replication Origin Replicable in  Escherichia coli    
     The shuttle plasmid of the present invention includes a base sequence of a first replication origin replicable in  Escherichia coli.  The base sequence of the first replication origin may be preferably a base sequence of a replication origin derived from pUC19 plasmid. Namely, the base sequence of the first replication origin may be derived from pUC (pUC origin). In addition, the base sequence of a replication origin derived from pUC19 plasmid may be derived from pMTL500E plasmid. 
     Base Sequence of a Second Replication Origin Derived from pUB110 Plasmid 
     The shuttle plasmid of the present invention includes a base sequence of a second replication origin derived from pUB110 plasmid. pUB110 plasmid is a cryptic plasmid derived from  Staphylococcus aureus,  and the genetic map thereof is depicted in  FIG. 1 . The base sequence of a replication origin derived from pUB110 plasmid may be a base sequence of SEQ ID NO: 3. In addition, the base sequence may have 70% or more, preferably 80% or more, more preferably 90% or more homology with SEQ ID NO: 3 and maintain functionality of the replication origin. 
     Base Sequence Encoding a Replication Protein Derived from pUB110 Plasmid 
     The shuttle plasmid of the present invention may include a base sequence encoding a replication protein and derived from pUB110 plasmid. The base sequence encoding the replication protein may be a base sequence of SEQ ID NO: 4. The base sequence may have 70% or more, preferably 80% or more, more preferably 90% or more homology with SEQ ID NO: 4 and encode a protein maintaining the functionality of the replication protein. On the other hand, the amino acid sequence of the replication protein may be an amino acid sequence of SEQ ID NO: 5. The amino acid sequence may have 70% or more, preferably 80% or more, more preferably 90% or more homology with SEQ ID NO: 5 and maintain the functionality of the replication protein. 
     First Antibiotic Resistance Gene 
     The shuttle plasmid of the present invention may include a first antibiotic resistance gene capable of being expressed in  Escherichia coli  and serving as a selective marker in  Escherichia coli.  The first antibiotic resistance gene is expressed in  Escherichia coli  and serves as a selective marker. Preferably, the first antibiotic resistance gene is an ampicillin antibiotic resistance gene. 
     Second Antibiotic Resistance Gene 
     The shuttle plasmid of the present invention may include a second antibiotic resistance gene capable of being expressed in  Clostridium  and serving as a selective marker in  Clostridium.  The second antibiotic resistance gene is expressed in  Clostridium,  preferably  Clostridium acetobutylicum  and serves as a selective marker. Preferably, the second antibiotic resistance gene is an erythromycin antibiotic resistance gene. 
     However, it is not intended that the first antibiotic resistance gene is limited to the ampicillin antibiotic resistance gene, or the second antibiotic resistance gene is limited to erythromycin antibiotic resistance gene. For example, the first antibiotic resistance gene and the second antibiotic resistance gene may be identical. The first antibiotic resistance gene may be any antibiotic resistance gene so long as such gene can serve as a selective marker in  Escherichia coli.  The second antibiotic resistance gene may be any antibiotic resistance gene so long as such gene can serve as a selective marker in  Clostridium.    
     Shuttle Plasmid of the Present Invention 
     The shuttle plasmid of the present invention is a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli  including: 
     a base sequence of a first replication origin replicable in  Escherichia coli;  and 
     a base sequence of a second replication origin derived from pUB110 plasmid. 
     Further, the shuttle plasmid of the present invention may be a shuttle plasmid replicable in both  Clostridium  and  Escherichia coli  including: 
     a base sequence of a first replication origin replicable in  Escherichia coli;    
     a base sequence of a second replication origin derived from pUB110 plasmid; 
     a base sequence encoding a replication protein derived from pUB110 plasmid; 
     a first antibiotic resistance gene expressed in  Escherichia coli;  and 
     a second antibiotic resistance gene expressed in  Clostridium.    
     The shuttle plasmid of the present invention may include a base sequence of SEQ ID NO: 7, or a base sequence having 80% or more, preferably 90% or more, more preferably 95% or more homology with SEQ ID NO: 7 so long as the base sequence maintains replication capability in both  Escherichia coli  and  Clostridium,  and expression and selection marker capability of antibiotic resistance genes in both  Escherichia coli  and  Clostridium.  Further, the shuttle plasmid of the present invention may be the pLK1-MCS. In addition, the shuttle plasmid of the present invention can be easily constructed using pUB110 cryptic plasmid and pMTL500E plasmid, wherein pMTL500E plasmid includes a replication origin of pUC19 plasmid, the first antibiotic resistance gene and the second antibiotic resistance gene ( FIG. 2 ). 
     The shuttle plasmid of the present invention is a shuttle vector replicable in both  Clostridium  and  Escherichia coli.  Particularly, the shuttle plasmid has high segregational stability in  Clostridium.  Therefore, use of the shuttle plasmid of the present invention may allow the shuttle plasmid and a target gene to be recombined, which functions as an operably linked recombinant shuttle plasmid. Further, the shuttle plasmid of the present invention is capable of replication in both  Clostridium  and  Escherichia coli  under an environment with no antibiotics and under an environment with antibiotics. Specifically, the shuttle plasmid of the present invention is capable of replication in both  Clostridium  and  Escherichia coli  under the environment with no antibiotics, and has high segregational stability, thereby being industrially applicable. 
     Further, the shuttle plasmid of the present invention has high segregational stability in  Clostridium  strains, and is sufficiently replicable in a culture medium containing no antibiotics, thereby ensuring stability in a fermentation process or a biotransformation process. 
       Clostridium  may include  Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butylicum, Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum  or  Clostridium tyrobutyricum.  Preferably,  Clostridium  is  Clostridium acetobutylicum.    
     Method for Constructing Transformed Microorganisms 
     The present invention provides a method for constructing a transformed microorganism including: preparing a shuttle plasmid of the present invention; and introducing the shuttle plasmid into a microorganism. 
     After preparing the shuttle plasmid, a foreign gene is cloned to the shuttle plasmid, which is introduced into a microorganism. 
     Furthermore, the present invention provides a method for producing a transformed microorganism including: preparing a shuttle plasmid of the present invention; cloning a foreign gene into the shuttle plasmid; and introducing the shuttle plasmid into which the foreign gene is cloned into a microorganism. 
     In addition, the present invention provides a transformed microorganism including the shuttle plasmid of the present invention. The microorganism may be  Escherichia coli  or  Clostridium.    
     Method for Producing Culture Products 
     The present invention provides a method for producing a culture including: culturing a transformed microorganism including the shuttle plasmid of the present invention; and harvesting the culture. 
     Further, the present invention provides a method for producing a fermented product including: culturing a transformed microorganism including the shuttle plasmid of the present invention; collecting the culture; and harvesting a fermented product produced by the transformed microorganism from the culture. 
     Further, the present invention provides a method for producing a fermented product including: preparing a shuttle plasmid of the present invention; cloning a foreign gene into the shuttle plasmid; and introducing the shuttle plasmid into a microorganism to construct a transformed microorganism; culturing the transformed microorganism; collecting the culture; and harvesting a fermented product from the culture. 
     The microorganism may be  Escherichia coli  or  Clostridium.  The culture contains fermented products produced by the transformed microorganism. The fermented products may be fermented products originally produced by the microorganism or fermented products produced by the foreign gene. For example, the fermented products may be alcohols, organic acids, ketones, and the like, preferably, alcohols having carbon number of 7 or less, polyhydric alcohols, and the like. For example, the fermented products may be butanol, isopropanol, ethanol, 1,3-propanol, 2,3-butandiol, propionic acid, acetone, and the like, without being limited thereto. 
     The above and other aspects, features, and advantages of the present invention will become apparent from the detailed description of the following embodiments in conjunction with the accompanying drawings. However, it should be understood that the present invention is not limited to the following embodiments and may be embodied in different ways, and that the embodiments are provided for complete disclosure and thorough understanding of the invention by those skilled in the art. The scope of the invention should be defined only by the accompanying claims and equivalents thereof. 
     Materials and Methods 
     pUB110 plasmid and  Clostridium acetobutylicum  ATCC 824 were purchased from the US strain depository authority, American Type Culture Collection (ATCC). 
     EXPERIMENTAL EXAMPLE 1 
     Construction of pLK1-MCS Shuttle Plasmid 
     A region encoding a replication protein and a replication origin of pUB110 plasmid were amplified and obtained using a primer having a base sequence of SEQ ID NO: 1 and a primer having a base sequence of SEQ ID NO: 2 and using pUB110 cryptic plasmid as a template. The base sequence of the replication origin of pUB110 plasmid is the base sequence of SEQ ID NO: 3; the base sequence encoding a replication protein (RepA) is the base sequence of SEQ ID NO: 4; and the amino acid sequence of a replication protein is the amino acid of SEQ ID NO: 5. 
     100 μl of PCR reaction mixture was prepared by combining 250 μM dNTP, 20 pmol of each primer, 1.5 mM MgCl2, 10 μl of 10× buffer, 100 ng of DNA template, and 1 unit of pfu polymerase. In PCR reaction, the reaction repeated 30 cycles consisting of initial denaturing at 95° C. for 5 minutes, followed by denaturing at 95° C. for one minute, annealing at 58° C. for one minute and then polymerizing at 72° C. for two minutes. PCR reaction in the following Examples was performed in the same manner as above. The amplified DNA fragment was purified on a 1% agarose gel, and then digested with SacI/BglII restriction enzymes to isolate a DNA fragment. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
             
            
               
                 SEQ ID NO: 1 
                 ATAGAGCTCACGAAGTCGAGATCAGGGAATGA 
               
               
                   
                 G 
               
               
                   
               
               
                 SEQ ID NO: 2 
                 GCGAGATCTCTCGTCTTCCTAAGCATCCTTCA 
               
               
                   
                 ATCC 
               
               
                   
               
               
                 SEQ ID NO: 3 
                 CTTGTTCTTTCTTATCTTGATACATATAGAAA 
               
               
                   
                 TAACGTCATTTTTATTTTAGTTGCTGAAAGGT 
               
               
                   
                 GCGTTGAAGTGTTGGTATGTATGTGTTTTAAA 
               
               
                   
                 GTATTGAAAACCCTTAAAATTGGTTGCACAGA 
               
               
                   
                 AAAACCCCATCTGTTAAAGTTATAAGTGACTA 
               
               
                   
                 AACAAATAACTAAATAGA 
               
               
                   
               
               
                 SEQ ID NO: 4 
                 ATGGGGCTTTCTTTTAATATTATGTGTCCTAA 
               
               
                   
                 TAGTAGCATTTATTCAGATGAAAAATCAAGGG 
               
               
                   
                 TTTTAGTGGACAAGACAAAAAGTGGAAAAGTG 
               
               
                   
                 AGACCATGGAGAGAAAAGAAAATCGCTAATGT 
               
               
                   
                 TGATTACTTTGAACTTCTGCATATTCTTGAAT 
               
               
                   
                 TTAAAAAGGCTGAAAGAGTAAAAGATTGTGCT 
               
               
                   
                 GAAATATTAGAGTATAAACAAAATCGTGAAAC 
               
               
                   
                 AGGCGAAAGAAAGTTGTATCGAGTGTGGTTTT 
               
               
                   
                 GTAAATCCAGGCTTTGTCCAATGTGCAACTGG 
               
               
                   
                 AGGAGAGCAATGAAACATGGCATTCAGTCACA 
               
               
                   
                 AAAGGTTGTTGCTGAAGTTATTAAACAAAAGC 
               
               
                   
                 CAACAGTTCGTTGGTTGTTTCTCACATTAACA 
               
               
                   
                 GTTAAAAATGTTTATGATGGCGAAGAATTAAA 
               
               
                   
                 TAAGAGTTTGTCAGATATGGCTCAAGGATTTC 
               
               
                   
                 GCCGAATGATGCAATATAAAAAAATTAATAAA 
               
               
                   
                 AATCTTGTTGGTTTTATGCTGCAACGGAAGTG 
               
               
                   
                 ACAATAAATAATAAAGATAATTCTTATAATCA 
               
               
                   
                 GCACATGCATGTATTGGTATGTGTGGAACCAA 
               
               
                   
                 CTTATTTTAAGAATACAGAAAACTACGTGAAT 
               
               
                   
                 CAAAAACAATGGATTCAATTTTGGAAAAAGGC 
               
               
                   
                 AATGAAATTAGACTATGATCCAAATGTAAAAG 
               
               
                   
                 TTCAAATGATTCGACCGAAAAATAAATATAAA 
               
               
                   
                 TCGGATATACAATCGGCAATTGACGAAACTGC 
               
               
                   
                 AAAATATCCTGTAAAGGATACGGATTTTATGA 
               
               
                   
                 CCGATGATGAAGAAAAGAATTTGAAACGTTTG 
               
               
                   
                 TCTGATTTGGAGCAAGGTTTACACCGTAAAAG 
               
               
                   
                 GTTAATCTCCTATGGTGGTTTGTTAAAAGAAA 
               
               
                   
                 TACATAAAAAATTAAACCTTGATGACACAGAA 
               
               
                   
                 GAAGGCGATTTGATTCATACAGATGATGACGA 
               
               
                   
                 AAAAGCCGATGAAGATGGATTTTCTATTATTG 
               
               
                   
                 CAATGTGGAATTGGGAACGGAAAAATTATTTT 
               
               
                   
                 ATTAAAGAGTAG 
               
               
                   
               
               
                 SEQ ID NO: 5 
                 MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKV 
               
               
                   
                 RPWREKKIANVDYFELLHILEFKKAERVKDCA 
               
               
                   
                 ETLEYKQNRETGERKLYRVWFCKSRLCPMCNW 
               
               
                   
                 RRAMKHGIQSQKVVAEVIKQKPTVRWLFLTLT 
               
               
                   
                 VKNVYDGEELNKSLSDMAQGFRRNIMQYKKIN 
               
               
                   
                 KNLVGFMRATEVTINNKDNSYNQHMHVLVCVE 
               
               
                   
                 PTYFKNTENYVNQKQWIQFWKKAMKLDYDPNV 
               
               
                   
                 KVQMIRPKNKYKSDIQSAIDETAKYPVKDTDF 
               
               
                   
                 MTDDEEKNLKRLSDLEEGLHRKRLISYGGLLK 
               
               
                   
                 EIHKKLNLDDTEEGDLIHTDDDEKADEDGFSI 
               
               
                   
                 IAMWNWERKNYFIKE 
               
               
                   
               
            
           
         
       
     
     On the other hand, as depicted in  FIG. 2 , pMTL500E plasmid was cleaved with SacI/BamHI restriction enzymes and the resulting DNA fragment containing an ampicillin resistance gene, an erythromycin resistance gene and a replication origin region of pUC19 plasmid (pUC origin) were purified in a 1% agarose gel, and then, the digested fragment ligated with DNA fragments (SEQ ID NOs: 3 and 4) comprising a replication origin of pUB110 and a replication protein, which was amplified by PCR reaction as mentioned above, digested with SacI/BglII restriction enzymes to construct pLK1-temp. The constructed pLK1-temp was digested with PvuII/SacI restriction enzymes, cloned together with a DNA fragment excised from DNA (SEQ ID NO: 6) by digestion of SmaI/SacI, which include a promoter and a multiple cloning site synthesized by Bioneer Corp. to construct a final pLK1-MCS (SEQ ID NO: 10,  FIG. 2 ). The pLK1-MCS includes a base sequence of the erythromycin resistance gene (SEQ ID NO: 7), a base sequence of the ampicillin resistance gene (SEQ ID NO: 8) and a base sequence of the replication origin of pUC19 plasmid (SEQ ID NO: 9). 
     The pLK1-MCS (DNA plasmid) was deposited with accession number of KCTC 12755BP at the Korea Research Institute of Bioscience and Biotechnology (BRIBB) on Feb. 4, 2015. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
             
            
               
                   
                 SEQ ID NO: 6 
                 ATACCCGGGCATGATTTTAAGGGGGTTAGCAG 
               
               
                   
                   
                 ATGCATAAGTTTAATTTTTTTGTTAAAAAATA 
               
               
                   
                   
                 TTAAACTTTGTGTTTTTTTTAACAAAATATAT 
               
               
                   
                   
                 TGATAAAAATAATAATAGTGGGTATAATTAAG 
               
               
                   
                   
                 TTGTTAGAGAAAACGTATAAATTAGGGATAAA 
               
               
                   
                   
                 CTATGGAACTTATGAAATAGATTGAAATGGTT 
               
               
                   
                   
                 TATCTGTTACCCCGTATCAAAATTTAGGAGGT 
               
               
                   
                   
                 TAGTTTAAACCTGCAGAGATCTCTCGAGGCGG 
               
               
                   
                   
                 CCGCGTCGACTCTAGACCCGGGAATTCACTGG 
               
               
                   
                   
                 CCGTCGTTTTACAACGTCGTGACTGGGAAAAC 
               
               
                   
                   
                 CCTGGCGTTACCCAACTTAATCGCCTTGCAGC 
               
               
                   
                   
                 ACATCCCCCTTTCGCCAGCTGGCGTAATAGCG 
               
               
                   
                   
                 AAGAGGCCCGCACCGATCGCCCTTCCCAACAG 
               
               
                   
                   
                 TTGCGCAGCCTGAATGGCGAATGGCGCCTGAT 
               
               
                   
                   
                 GCGGTATTTTCTCCTTACGCATCTGTGCGGTA 
               
               
                   
                   
                 TTTCACACCGAGCTCATA 
               
               
                   
               
               
                   
                 SEQ ID NO: 7 
                 ATGAACAAAAATATAAAATATTCTCAAAACTT 
               
               
                   
                   
                 TTTAACGAGTGAAAAAGTACTCAACCAAATAA 
               
               
                   
                   
                 TAAAACAATTGAATTTAAAAGAAACCGATACC 
               
               
                   
                   
                 GTTTACGAAATTGGAACAGGTAAAGGGCATTT 
               
               
                   
                   
                 AACGACGAAACTGGCTAAAATAAGTAAACAGG 
               
               
                   
                   
                 TAACGTCTATTGAATTAGACAGTCATCTATTC 
               
               
                   
                   
                 AACTTATCGTCAGAAAAATTAAAACTGAATAC 
               
               
                   
                   
                 TCGTGTCACTTTAATTCACCAAGATATTCTAC 
               
               
                   
                   
                 AGTTTCAATTCCCTAACAAACAGAGGTATAAA 
               
               
                   
                   
                 ATTGTTGGGAGTATTCCTTACCATTTAAGCAC 
               
               
                   
                   
                 ACAAATTATTAAAAAAGTGGTTTTTGAAAGCC 
               
               
                   
                   
                 ATGCGTCTGACATCTATGTGATTGTTGAAGAA 
               
               
                   
                   
                 GGATTCTACAAGCGTACCTTGGATATTCACCG 
               
               
                   
                   
                 AACACTAGGGTTGCTCTTGCACACTCAAGTCT 
               
               
                   
                   
                 CGATTCAGCAATTGCTTAAGCTGCCAGCGGAA 
               
               
                   
                   
                 TGCTTTCATCCTAAACCAAAAGTAAACAGTGT 
               
               
                   
                   
                 CTTAATAAAACTTACCCGCCATACCACAGATG 
               
               
                   
                   
                 TTCCAGATAAATATTGGAACCTATATACGTAC 
               
               
                   
                   
                 TTTGTTTCAAAATGGGTCAATCGAGAATATCG 
               
               
                   
                   
                 TCAACTGTTTACTAAAAATCAGTTTCATCAAG 
               
               
                   
                   
                 CAATGAAACACGCCAAAGTAAACAATTTAAGT 
               
               
                   
                   
                 ACCGTTACTTATGAGCAAGTATTGTCTATTTT 
               
               
                   
                   
                 TAATAGTTATCTATTATTTAACGGGAGGAAAT 
               
               
                   
                   
                 AA 
               
               
                   
               
               
                   
                 SEQ ID NO: 8 
                 ATGAGTATTCAACATTTCCGTGTCGCCCTTAT 
               
               
                   
                   
                 TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT 
               
               
                   
                   
                 TTGCTCACCCAGAAACGCTGGTGAAAGTAAAA 
               
               
                   
                   
                 GATGCTGAAGATCAGTTGGGTGCACGAGTGGG 
               
               
                   
                   
                 TTACATCGAACTGGATCTCAACAGCGGTAAGA 
               
               
                   
                   
                 TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT 
               
               
                   
                   
                 CCAATGATGAGCACTTTTAAAGTTCTGCTATG 
               
               
                   
                   
                 TGGCGCGGTATTATCCCGTATTGACGCCGGGC 
               
               
                   
                   
                 AAGAGCAACTCGGTCGCCGCATACACTATTCT 
               
               
                   
                   
                 CAGAATGACTTGGTTGAGTACTCACCAGTCAC 
               
               
                   
                   
                 AGAAAAGCATCTTACGGATGGCATGACAGTAA 
               
               
                   
                   
                 GAGAATTATGCAGTGCTGCCATAACCATGAGT 
               
               
                   
                   
                 GATAACACTGCGGCCAACTTACTTCTGACAAC 
               
               
                   
                   
                 GATCGGAGGACCGAAGGAGCTAACCGCTTTTT 
               
               
                   
                   
                 TGCACAACATGGGGGATCATGTAACTCGCCTT 
               
               
                   
                   
                 GATCGTTGGGAACCGGAGCTGAATGAAGCCAT 
               
               
                   
                   
                 ACCAAACGACGAGCGTGACACCACGATGCCTG 
               
               
                   
                   
                 TAGCAATGGCAACAACGTTGCGCAAACTATTA 
               
               
                   
                   
                 ACTGGCGAACTACTTACTCTAGCTTCCCGGCA 
               
               
                   
                   
                 ACAATTAATAGACTGGATGGAGGCGGATAAAG 
               
               
                   
                   
                 TTGCAGGACCACTTCTGCGCTCGGCCCTTCCG 
               
               
                   
                   
                 GCTGGCTGGTTTATTGCTGATAAATCTGGAGC 
               
               
                   
                   
                 CGGTGAGCGTGGGTCTCGCGGTATCATTGCAG 
               
               
                   
                   
                 CACTGGGGCCAGATGGTAAGCCCTCCCGTATC 
               
               
                   
                   
                 GTAGTTATCTACACGACGGGGAGTCAGGCAAC 
               
               
                   
                   
                 TATGGATGAACGAAATAGACAGATCGCTGAGA 
               
               
                   
                   
                 TAGGTGCCTCACTGATTAAGCATTGGTAA 
               
               
                   
               
               
                   
                 SEQ ID NO: 9 
                 TCAAAGGATCTTCTTGAGATCCTTTTTTTCTG 
               
               
                   
                   
                 CGGGTAATCTGCTGCTTGCAAACAAAAAAACC 
               
               
                   
                   
                 ACCGCTACCAGCGGTGGTTTGTTTGCCGGATC 
               
               
                   
                   
                 AAGAGCTACCAACTCTTTTTCCGAAGGTAACT 
               
               
                   
                   
                 GGCTTCAGCAGAGCGCAGATACCAAATACTGT 
               
               
                   
                   
                 TCTTCTAGTGTAGCCGTAGTTAGGCCACCACT 
               
               
                   
                   
                 TCAAGAACTCTGTAGCACCGCCTACATACCTC 
               
               
                   
                   
                 GCTCTGCTAATCCTGTTACCAGTGGCTGCTGC 
               
               
                   
                   
                 CAGTGGCGATAAGTCGTGTCTTACCGGGTTGG 
               
               
                   
                   
                 ACTCAAGACGATAGTTACCGGATAAGGCGCAG 
               
               
                   
                   
                 CGGTCGGGCTGAACGGGGGGTTCGTGCACACA 
               
               
                   
                   
                 GCCCAGCTTGGAGCGAACGACCTACACCGAAC 
               
               
                   
                   
                 TGAGATACCTACAGCGTGAGCTATGAGAAAGC 
               
               
                   
                   
                 GCCACGCTTCCCGAAGGGAGAAAGGCGGACAG 
               
               
                   
                   
                 GTATCCGGTAAGCGGCAGGGTCGGAACAGGAG 
               
               
                   
                   
                 AGCGCACGAGGGAGCTTCCAGGGGGAAACGCC 
               
               
                   
                   
                 TGGTATCTTTATAGTCCTGTCGGGTTTCGCCA 
               
               
                   
                   
                 CCTCTGACTTGAGCGTCGATTTTTGTGATGCT 
               
               
                   
                   
                 CGTCAGGGGGGCGGAGCCTATGGAAAAACGCC 
               
               
                   
                   
                 AGCAACG 
               
               
                   
               
               
                   
                 SEQ ID NO: 10 
                 CATGATTTTAAGGGGGTTAGCAGATGCATAAG 
               
               
                   
                   
                 TTTAATTTTTTTGTTAAAAAATATTAAACTTT 
               
               
                   
                   
                 GTGTTTTTTTTAACAAAATATATTGATAAAAA 
               
               
                   
                   
                 TAATAATAGTGGGTATAATTAAGTTGTTAGAG 
               
               
                   
                   
                 AAAACGTATAAATTAGGGATAAACTATGGAAC 
               
               
                   
                   
                 TTATGAAATAGATTGAAATGGTTTATCTGTTA 
               
               
                   
                   
                 CCCCGTATCAAAATTTAGGAGGTTAGTTTAAA 
               
               
                   
                   
                 CCTGCAGAGATCTCTCGAGGCGGCCGCGTCGA 
               
               
                   
                   
                 CTCTAGACCCGGGAATTCACTGGCCGTCGTTT 
               
               
                   
                   
                 TACAACGTCGTGACTGGGAAAACCCTGGCGTT 
               
               
                   
                   
                 ACCCAACTTAATCGCCTTGCAGCACATCCCCC 
               
               
                   
                   
                 TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCC 
               
               
                   
                   
                 GCACCGATCGCCCTTCCCAACAGTTGCGCAGC 
               
               
                   
                   
                 CTGAATGGCGAATGGCGCCTGATGCGGTATTT 
               
               
                   
                   
                 TCTCCTTACGCATCTGTGCGGTATTTCACACC 
               
               
                   
                   
                 GAGCTCACGAAGTCGAGATCAGGGAATGAGTT 
               
               
                   
                   
                 TATAAAATAAAAAAAGCACCTGAAAAGGTGTC 
               
               
                   
                   
                 TTTTTTTGATGGTTTTGAACTTGTTCTTTTTT 
               
               
                   
                   
                 ATCTTGATACATATAGAAATAACGTCATTTTT 
               
               
                   
                   
                 ATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTT 
               
               
                   
                   
                 GGTATGTATGTGTTTTAAAGTATTGAAAACCC 
               
               
                   
                   
                 TTAAAATTGTTTGGACAGAAAAACGCCATGTG 
               
               
                   
                   
                 TTAAAGTTATAAGTGACTAAACAAATAACTAA 
               
               
                   
                   
                 ATAGATGGGGGTTTCTTTTAATATTATGTGTG 
               
               
                   
                   
                 CTAATAGTAGCATTTATTCAGATGAAAAATCA 
               
               
                   
                   
                 AGGGTTTTAGTGGACAAGACAAAAAGTGGAAA 
               
               
                   
                   
                 AGTGAGACCATGGAGAGAAAAGAAAATCGCTA 
               
               
                   
                   
                 ATGTTGATTACTTTGAACTTCTGCATATTCTT 
               
               
                   
                   
                 GAATTTAAAAAGGCTGAAAGAGTAAAAGATTG 
               
               
                   
                   
                 TGCTGAAATATTAGAGTATAAACAAAATCGTG 
               
               
                   
                   
                 AAACAGGCGAAAGAAAGTTGTATCGAGTGTGG 
               
               
                   
                   
                 TTTTGTAAATCCAGGCTTTGTCCAATGTGCAA 
               
               
                   
                   
                 CTGGAGGAGAGCAATGAAACATGGCATTCAGT 
               
               
                   
                   
                 CACAAAAGGTTGTTGCTGAAGTTATTAAACAA 
               
               
                   
                   
                 AAGCCAACAGTTCGTTGGTTGTTTCTCACATT 
               
               
                   
                   
                 AACAGTTAAAAATGTTTATGATGGCGAAGAAT 
               
               
                   
                   
                 TAAATAAGAGTTTGTCAGATATGGCTCAAGGA 
               
               
                   
                   
                 TTTCGCCGAATGATGCAATATAAAAAAATTAA 
               
               
                   
                   
                 TAAAAATCTTGTTGGTTTTATGCGTGCAACGG 
               
               
                   
                   
                 AAGTGACAATAAATAATAAAGATAATTCTTAT 
               
               
                   
                   
                 AATCAGCACATGCATGTATTGGTATGTGTGGA 
               
               
                   
                   
                 ACCAACTTATTTTAAGAATACAGAAAACTACG 
               
               
                   
                   
                 TGAATCAAAAACAATGGATTCAATTTTGGAAA 
               
               
                   
                   
                 AAGGCAATGAAATTAGACTATGATCCAAATGT 
               
               
                   
                   
                 AAAAGTTCAAATGATTCGACCGAAAAATAAAT 
               
               
                   
                   
                 ATAAATCGGATATACAATCGGCAATTGACGAA 
               
               
                   
                   
                 ACTGCAAAATATCCTGTAAAGGATACGGATTT 
               
               
                   
                   
                 TATGACCGATGATGAAGAAAAGAATTTGAAAC 
               
               
                   
                   
                 GTTTGTCTGATTTGGAGGAAGGTTTACACCGT 
               
               
                   
                   
                 AAAAGGTTAATCTCCTATGGTGGTTTGTTAAA 
               
               
                   
                   
                 AGAAATACATAAAAAATTAAACCTTGATGACA 
               
               
                   
                   
                 CAGAAGAAGGCGATTTGATTCATACAGATGAT 
               
               
                   
                   
                 GAGGAAAAAGCCGATGAAGATGGATTTTCTAT 
               
               
                   
                   
                 TATTGCAATGTGGAATTGGGAACGGAAAAATT 
               
               
                   
                   
                 ATTTTATTAAAGAGTAGTTCAACAAACGGGCC 
               
               
                   
                   
                 AGTTTGTTGAAGATTAGATGCTATAATTGTTA 
               
               
                   
                   
                 TTAAAAGGATTGAAGGATGCTTAGGAAGACGA 
               
               
                   
                   
                 GAGATCCTAGCAGCACGCCATAGTGACTGGCG 
               
               
                   
                   
                 ATGCTGTCGGAATGGACGATCAAATTCCCCGT 
               
               
                   
                   
                 AGGCGCTAGGGACCTCTTTAGCTCCTTGGAAG 
               
               
                   
                   
                 CTGTCAGTAGTATACCTAATAATTTATCTACA 
               
               
                   
                   
                 TTCCCTTTAGTAACGTGTAACTTTCCAAATTT 
               
               
                   
                   
                 ACAAAAGCGACTCATAGAATTATTTCCTCCCG 
               
               
                   
                   
                 TTAAATAATAGATAACTATTAAAAATAGACAA 
               
               
                   
                   
                 TACTTGCTCATAAGTAACGGTACTTAAATTGT 
               
               
                   
                   
                 TTACTTTGGCGTGTTTCATTGCTTGATGAAAC 
               
               
                   
                   
                 TGATTTTTAGTAAACAGTTGACGATATTCTCG 
               
               
                   
                   
                 ATTGACCCATTTTGAAACAAAGTACGTATATA 
               
               
                   
                   
                 GCTTCCAATATTTATCTGGAACATCTGTGGTA 
               
               
                   
                   
                 TGGCGGGTAAGTTTTATTAAGACACTGTTTAC 
               
               
                   
                   
                 TTTTGGTTTAGGATGAAAGCATTCCGCTGGCA 
               
               
                   
                   
                 GCTTAAGCAATTGCTGAATCGAGACTTGAGTG 
               
               
                   
                   
                 TGCAAGAGCAACCCTAGTGTTCGGTGAATATC 
               
               
                   
                   
                 CAAGGTACGCTTGTAGAATCCTTCTTCAACAA 
               
               
                   
                   
                 TCAGATAGATGTCAGACGCATGGCTTTCAAAA 
               
               
                   
                   
                 ACCACTTTTTTAATAATTTGTGTGCTTAAATG 
               
               
                   
                   
                 GTAAGGAATACTCCCAACAATTTTATACCTCT 
               
               
                   
                   
                 GTTTGTTAGGGAATTGAAACTGTAGAATATCT 
               
               
                   
                   
                 TGGTGAATTAAAGTGACACGAGTATTCAGTTT 
               
               
                   
                   
                 TAATTTTTCTGACGATAAGTTGAATAGATGAC 
               
               
                   
                   
                 TGTCTAATTCAATAGACGTTACCTGTTTACTT 
               
               
                   
                   
                 ATTTTAGCCAGTTTCGTCGTTAAATGCCCTTT 
               
               
                   
                   
                 ACCTGTTCCAATTTCGTAAACGGTATCGGTTT 
               
               
                   
                   
                 CTTTTAAATTCAATTGTTTTATTATTTGGTTG 
               
               
                   
                   
                 AGTACTTTTTCACTCGTTAAAAAGTTTTGAGA 
               
               
                   
                   
                 ATATTTTATATTTTTGTTCATGTAATCACTCC 
               
               
                   
                   
                 TTCTTAATTACAAATTTTTAGCATCTAATTTA 
               
               
                   
                   
                 ACTTCAATTCCTATTATACAAAATTTTAAGAT 
               
               
                   
                   
                 ACTGCACTATCAACACACTCTTAAGTTTGCTT 
               
               
                   
                   
                 CTAAGTCTTATTTCCATAACTTCTTTTACGTT 
               
               
                   
                   
                 TCCGCCATTCTTTGCTTTTTCGATTTTTATGA 
               
               
                   
                   
                 TATGGTGCAAGTCAGCACGAACACGAACCGTC 
               
               
                   
                   
                 TTATCTCCCATTATATCTTTTTTTGCACTGAT 
               
               
                   
                   
                 TGGTGTATCATTTCGTTTTTCTTTTTATCCCG 
               
               
                   
                   
                 CAAGAGGCCCGGCAGTCAGGTGGCACTTTTCG 
               
               
                   
                   
                 GGGAAATGTGCGCGGAACCCCTATTTGTTTAT 
               
               
                   
                   
                 TTTTCTAAATACATTCAAATATGTATCCGCTC 
               
               
                   
                   
                 ATGAGACAATAACCCTGATAAATGCTTCAATA 
               
               
                   
                   
                 ATATTGAAAAAGGAAGAGTATGAGTATTCAAC 
               
               
                   
                   
                 ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG 
               
               
                   
                   
                 GCATTTTGCCTTCCTGTTTTTGCTCACCCAGA 
               
               
                   
                   
                 AACGCTGGTGAAAGTAAAAGATGCTGAAGATC 
               
               
                   
                   
                 AGTTGGGTGCACGAGTGGGTTACATCGAACTG 
               
               
                   
                   
                 GATCTCAACAGCGGTAAGATCCTTGAGAGTTT 
               
               
                   
                   
                 TCGCCCCGAAGAACGTTTTCCAATGATGAGCA 
               
               
                   
                   
                 CTTTTAAAGTTCTGCTATGTGGCGCGGTATTA 
               
               
                   
                   
                 TCCCGTATTGACGCCGGGCAAGAGCAACTCGG 
               
               
                   
                   
                 TCGCCGCATACACTATTCTCAGAATGACTTGG 
               
               
                   
                   
                 TTGAGTACTCACCAGTCACAGAAAAGCATCTT 
               
               
                   
                   
                 ACGGATGGCATGACAGTAAGAGAATTATGCAG 
               
               
                   
                   
                 TGCTGCCATAACCATGAGTGATAACACTGCGG 
               
               
                   
                   
                 CCAACTTACTTCTGACAACGATCGGAGGACCG 
               
               
                   
                   
                 AAGGAGCTAACCGCTTTTTTGCACAACATGGG 
               
               
                   
                   
                 GGATCATGTAACTCGCCTTGATCGTTGGGAAC 
               
               
                   
                   
                 CGGAGCTGAATGAAGCCATACCAAACGACGAG 
               
               
                   
                   
                 CGTGACACCACGATGCCTGTAGCAATGGCAAC 
               
               
                   
                   
                 AACGTTGCGCAAACTATTAACTGGCGAACTAC 
               
               
                   
                   
                 TTACTCTAGCTTCCCGGCAACAATTAATAGAC 
               
               
                   
                   
                 TGGATGGAGGCGGATAAAGTTGCAGGACCACT 
               
               
                   
                   
                 TCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTA 
               
               
                   
                   
                 TTGCTGATAAATCTCGAGCCGGTGAGCGTGGG 
               
               
                   
                   
                 TCTCGCGGTATCATTGCAGCACTGGGGCCAGA 
               
               
                   
                   
                 TGGTAAGCCCTCCCGTATCGTAGTTATCTACA 
               
               
                   
                   
                 CGACGGGGAGTCAGGCAACTATGGATGAACGA 
               
               
                   
                   
                 AATAGACAGATCGCTGAGATAGGTGCCTCACT 
               
               
                   
                   
                 GATTAAGCATTGGTAACTGTCAGACCAAGTTT 
               
               
                   
                   
                 ACTCATATATACTTTAGATTGATTTAAAACTT 
               
               
                   
                   
                 CATTTTTAATTTAAAAGGATCTAGGTGAAGAT 
               
               
                   
                   
                 CCTTTTTGATAATCTCATGACCAAAATCCCTT 
               
               
                   
                   
                 AACGTGAGTTTTCGTTCCACTGAGCGTCAGAC 
               
               
                   
                   
                 CCCGTAGAAAAGATCAAAGGATCTTCTTGAGA 
               
               
                   
                   
                 TCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC 
               
               
                   
                   
                 AAACAAAAAAACCACCGCTACCAGCGGTGGTT 
               
               
                   
                   
                 TGTTTGCCGGATCAAGAGCTACCAACTCTTTT 
               
               
                   
                   
                 TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGA 
               
               
                   
                   
                 TACCAAATACTGTTCTTCTAGTGTAGCCGTAG 
               
               
                   
                   
                 TTAGGCCACCACTTCAAGAACTCTGTAGCACC 
               
               
                   
                   
                 GCCTACATACCTCGCTCTGCTAATCCTGTTAC 
               
               
                   
                   
                 CAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT 
               
               
                   
                   
                 CTTACCGGGTTGGACTCAAGACGATAGTTACC 
               
               
                   
                   
                 GGATAAGGCGCAGCGGTCGGGCTGAACGGGGG 
               
               
                   
                   
                 GTTCGTGCACACAGCCCAGCTTGGAGCGAACG 
               
               
                   
                   
                 ACCTACACCGAACTGAGATACCTACAGCGTGA 
               
               
                   
                   
                 GCTATGAGAAAGCGCCACGCTTCCCGAAGGGA 
               
               
                   
                   
                 GAAAGGCGGACAGGTATCCGGTAAGCGGCAGG 
               
               
                   
                   
                 GTCGGAACAGGAGAGCGCACGAGGGAGCTTCC 
               
               
                   
                   
                 AGGGGGAAACGCCTGGTATCTTTATAGTCCTG 
               
               
                   
                   
                 TCGGGTTTCGCCACCTCTGACTTGAGCGTCGA 
               
               
                   
                   
                 TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT 
               
               
                   
                   
                 ATGGAAAAACGCCAGCAACGCGGCCTTTTTAC 
               
               
                   
                   
                 GGTTCCTGGCCTTTTGCTGGCCTTTTGCTCAC 
               
               
                   
                   
                 ATGTTCTTTCCTGCGTTATCCCCTGATTCTGT 
               
               
                   
                   
                 GGATAACCGTATTACCGCCTTTGAGTGAGCTG 
               
               
                   
                   
                 ATACCGCTCGCCGCAGCCGAACGACCGAGCGC 
               
               
                   
                   
                 AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCG 
               
               
                   
                   
                 CCCAATACGCAAACCGCCTCTCCCCGCGCGTT 
               
               
                   
                   
                 GGCCGATTCATTAATGCAGGGG 
               
               
                   
               
            
           
         
       
     
     EXPERIMENTAL EXAMPLE 2 
     Evaluation of Segregational Stability of Shuttle Plasmid (pLK1-MCS) 
     &lt;2-1&gt; Construction of Transformants 
     The shuttle plasmid prepared in Experimental Example 1 was introduced into  Clostridium acetobutylicum  to prepare a transformed recombinant microorganism. 
     Detailed methods are as follows.  Clostridium acetobutylicum  was cultured in 100 ml of liquid CGM ( Clostridium  Growth Medium) (0.75 g/L K 2 HPO 4 , 0.75 g/L KH 2 PO 4 , 0.7 g/L, MgSO 4 .7H 2 O, 0.017 g/L MnSO 4 .5H 2 O, 0.01 g/L, FeSO 4 .7H 2 O, 2 g/L (NH 4 ) 2 SO 4 , 1 g/L NaCl, 2 g/L asparagine, 0.004 g/L p-aminobenzoic acid, 5 g/L, yeast extract, and 10 g/L glucose) under anaerobic conditions until OD600 reached 1.0. The culture solution was left on ice for 10 minutes, followed by subjecting to centrifugation at 7000 g for 10 minutes at 4° C., thereby obtaining cell pellets. The obtained cell pellets were washed with a buffer solution three times and suspended in 20 ml of the same buffer solution to prepare cells for transformation. To 500 μl of the prepared cells for transformation, 5.0 μg of shuttle plasmids were added, followed by performing electroporation (4 mm cuvette, 2.5 kV, ∞Ω, 25 uF) using a Gene Pulser II prepared by Bio-Rad Corp. Transformed strains were identified in a medium to which erythromycin was added (Table 3). 
     The plasmids used in the transformation were all methylated in  Escherichia coli  TOP10 strain transformed with pAN1 plasmid (having genes for methylating inner cytosine in case that GCNGC sequence is present) before electroporation so that the plasmids were not affected by the restriction system of  Clostridium acetobutylicum  strain. 
     &lt;2-2&gt; Evaluation of Segregational Stability of pLK1-MCS in  Clostridium    
     The segregational stability of the shuttle plasmid pLK1-MCS was evaluated in  Clostridium acetobutylicum  strain containing the pLK1-MCS shuttle plasmid constructed in Experimental Example 1. The evaluation was performed by adapting the existing evaluation method (Shin M H, Jung M W, Lee J-H, Kim M D, Kim K H. 2008. Strategies for producing recombinant sucrose phosphorylase originating from  Bifidobacterium longum  in  Escherichia coli  JM109. Process Biochemistry 43:822-828). 
     The shuttle plasmid was introduced into  Clostridium acetobutylicum  ATCC 824 strain by electroporation, and then cultured in a solid medium containing erythromycin under anaerobic culture conditions at 37° C. for two days. One colony taken from the culture solution was cultured in a culture tube with 40 ml liquid CGM with no antibiotic present at 37° C. until cell concentration (OD600 nm) reached 1.0. The cell concentration was measured using a spectrophotometer (Hach, USA). 
     The cultured cells were diluted, streaked on solid CGM with no antibiotic present, and cultured at 37° C. for 36 hours. The number of colonies formed was identified. Thereafter, 50 colonies formed were replica plated onto solid CGM containing erythromycin, and the number of cells in which the shuttle plasmid was lost was identified. In case that the shuttle plasmid was lost, colonies could not be formed upon replica plating since there was no erythromycin antibiotic resistance. 
     Further, 40 ml of liquid CGM with no antibiotic present (diluted to 1/1000 of concentration of the initial culture solution) was inoculated with 40 μL of initial liquid culture solution, and then the aforementioned procedures were repeated 10 times to identify the number of cells in which the shuttle plasmid was lost. The stability from shuttle plasmid loss was evaluated over 100 generations. Since cells were inoculated in a concentration of 1:1000 dilution every time, each generation was assumed to have been divided 10 times (2 10 ≈1024). 
     Results are shown in  FIG. 3 . It can be seen that the novel shuttle plasmid pLK1-MCS showed remarkably improved stability from plasmid loss as compared with the existing pMTL500E (including a replication origin of pAM β1 and a base sequence encoding a replication protein) used as  Escherichia coli - Clostridium  shuttle plasmid. Further, the novel shuttle plasmid pLK1-MCS was also found to have better segregational stability as compared with pGS1-MCS shuttle plasmid including a replication origin of pIM13 plasmid and a base sequence encoding a replication protein. As a result, the novel shuttle plasmid pLK1-MCS was found to have better segregational stability as compared with the existing shuttle plasmid (Table 3). 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Plasmid 
                 Number of colony 
                 Temperature (° C.) 
               
               
                   
               
             
            
               
                 pLK1-MCS 
                 4.3 × 10 2   
                 37 
               
               
                 pMTL500E 
                 2.6 × 10 2   
                 37 
               
               
                 pGS1-MCS 
                 4.6 × 10 2   
                 37 
               
               
                   
               
            
           
         
       
     
     &lt;2-3&gt; Evaluation of Segregational Stability of pLK1-MCS in  Escherichia coli    
     The fact that shuttle plasmid pLK1-MCS constructed in Experimental Example 1 is stably replicated in  Escherichia coli  was confirmed by transforming the shuttle plasmid pLK1-MCS with  Escherichia coli  TOP10 containing pAN1 plasmid to obtain methylated pLK1-MCS in Experimental Example &lt;2-1&gt;. 
     EXPERIMENTAL EXAMPLE 3 
     Expression of Foreign Genes Using Shuttle Plasmids 
     &lt;3-1&gt; Construction of pLK1-IPA2 Plasmid 
     According to Korean Patent Publication No. 10-2011-0032375, it is possible for the secondary alcohol dehydrogenase of  Clostridium beijerinckii,  NRRL B593 to convert acetone into isopropanol. Accordingly, it was evaluated whether or not the novel shuttle plasmid obtained by recombining the secondary alcohol dehydrogenase gene (IPA-HydG) as a foreign gene to the novel shuttle plasmid of the present invention expresses the secondary alcohol dehydrogenase gene and thus converts acetone into alcohols. A region encoding secondary alcohol dehydrogenase was obtained by PCR reaction using as a template pTHL1-Cm-IPA2 plasmid used in Korean Patent Publication No. 10-2011-0032375 and using as primer base sequences of SEQ ID NO: 11 and SEQ ID NO: 12 (Table 4). The obtained region encoding secondary alcohol dehydrogenase was cloned into the novel plasmid of the present invention, pLK1-MCS, to construct pLK1-IPA2 plasmid. The constructed pLK1-IPA2 plasmid was introduced into  Clostridium acetobutylicum  PJC4BK strain to obtain a transformed  Clostridium acetobutylicum  PJC4BK (pLK1-IPA) ( FIG. 4 ). 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
             
            
               
                   
                 SEQ ID NO: 11 
                 CACAGGCCTATGAAAGGTTTTGCAA 
               
               
                   
                   
                 TGCTAGGTATTAAT 
               
               
                   
               
               
                   
                 SEQ ID NO: 12 
                 ATATCTAGATTATTTATCACCTCTG 
               
               
                   
                   
                 CAACCACAGCCACC 
               
               
                   
               
            
           
         
       
     
     In this Experimental Example, the plasmids used in the transformation are all methylated in  Escherichia coli  TOP10 strain transformed with pAN1 plasmid (having genes for methylating inner cytosine in case that GCNGC sequence is present) before electroporation so that the plasmids were not affected by the restriction system of  Clostridium acetobutylicum  strain. 
     &lt;3-2&gt; Identification of Production of Isopropanol Using  Clostridium acetobutylicum  PJC4BK (pLK1-IPA2) 
     It was evaluated whether or not the recombined secondary alcohol dehydrogenase was normally expressed to convert acetone into isopropanol in case the recombinant strain prepared in &lt;3-1&gt; is batch cultured. 
     First, the recombinant  Clostridium  PJC4BK (pLK1-IPA2) strain prepared in &lt;3-1&gt; of &lt;Experimental Example 3&gt; was streaked on solid CGM, followed by culturing anaerobically at 37° C. overnight. A single colony was inoculated into a 50 ml disposable tube (Falcon, USA) containing 40 ml of CGM, and then cultured anaerobically until OD600 reached 1 at 37° C. . The seed culture was inoculated into 400 ml of CGM containing 1% glucose, followed by standing, and then culturing anaerobically until OD600 reached 1 at 37° C. The 400 ml culture solution was then inoculated into a a fermenter containing 1.6 L of liquid CGM containing 8% glucose. As a control group,  Clostridium acetobutylicum  PCJ4BK (pTHL-Cm-IPA2) and  Clostridium acetobutylicum  PCJ4BK were used. 
     pH was maintained at 5.0 during anaerobic culture using ammonium hydroxide (NH 4 OH) and anaerobic conditions were maintained by injecting nitrogen at a speed of 20 ml/min The concentration of the produced butanol and mixed solvent was analyzed every three hours after the initiation of glucose culture. The analysis of butanol and mixed solvent was performed using a gas chromatograph (Agilent, USA). The analysis conditions are as summarized in Table 5. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
             
            
               
                   
                 Injector temperature 
                 320° C. 
               
               
                   
                 Detector temperature 
                 320° C. 
               
               
                   
                 Injector split ratio 
                 20/1 
               
            
           
           
               
               
               
               
            
               
                   
                 Injection volume 
                 0.1 
                 ul 
               
            
           
           
               
               
               
            
               
                   
                 Oven condition 
                 80° C./15 min 
               
            
           
           
               
               
               
               
            
               
                   
                 Air flow 
                 300  
                 mL/min 
               
               
                   
                 H 2  flow 
                 30  
                 mL/min 
               
            
           
           
               
               
               
            
               
                   
                 Column 
                 Supelco CarboWAX 
               
               
                   
                   
               
            
           
         
       
     
     As a result, it was confirmed that  Clostridium acetobutylicum  PJC4BK (pLK1-IPA2) had isopropanol producing capability comparable or higher to that of  acetobutylicum  PJC4BK (pTHL-Cm-IPA2) used as a control group (Table 6). 
     Consequently, it was confirmed that the shuttle plasmid pLK1-MCS had segregational stability and foreign gene expression capability comparable or higher to those of prior shuttle plasmids. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                 Acetone 
                 IPA 
                 Ethanol 
                 Butanol 
                 Total 
               
               
                 Strain 
                 Plasmid 
                 (g/L) 
                 (g/L) 
                 (g/L) 
                 (g/L) 
                 (g/L) 
               
               
                   
               
             
            
               
                 
                   Clostridium 
                 
                 — 
                 2.606 
                   
                 2.641 
                 15.296 
                 20.543 
               
               
                 
                   acetobutylicum 
                 
               
               
                 PJC4BK 
               
               
                 
                   Clostridium 
                 
                 pTHL- 
                 0.294 
                 4.397 
                 3.792 
                 15.972 
                 24.455 
               
               
                 
                   acetobutylicum 
                 
                 Cm-IPA2 
               
               
                 PJC4BK 
               
               
                 
                   Clostridium 
                 
                 pLK1- 
                 0.371 
                 4.328 
                 3.953 
                 16.051 
                 24.703 
               
               
                 
                   acetobutylicum 
                 
                 IPA2 
               
               
                 PJC4BK 
               
               
                   
               
            
           
         
       
     
     Although some embodiments have been described herein, it should be understood by those skilled in the art that these embodiments are given by way of illustration only, and that various modifications, variations, and alterations can be made without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be limited only by the accompanying claims and equivalents thereof.