KR101878801B1

KR101878801B1 - Method of genome engineering in clostridia

Info

Publication number: KR101878801B1
Application number: KR1020150190441A
Authority: KR
Inventors: 이상준; 김현주; 김명희; 이상현
Original assignee: 한국생명공학연구원; 지에스칼텍스 주식회사
Priority date: 2015-12-30
Filing date: 2015-12-30
Publication date: 2018-07-16
Also published as: KR20170079649A

Abstract

The present invention relates to a genome engineering plasmid of Clostridium comprising two FRT (flippase recognition target) base sequences and a part of the nucleotide sequence of a target gene, and a genome manipulation method of Clostridium genus microorganism using the plasmid .

Description

[0001] METHOD OF GENOME ENGINEERING IN CLOSTRIDIA [0002]

The present invention relates to a plasmid of genus of Clostridium and a genome manipulation method of Clostridium using the plasmid.

Clostridium microorganisms are industrially useful microorganisms capable of producing biochemicals and biofuels by fermenting biomass. Recently, it is attracting attention as an important microorganism in microbiome research. However, industrial use of Clostridium microorganisms is very limited because there is no gene tool that can insert a gene of interest into a chromosome or effectively inactivate a desired gene in a chromosome. It is also due to the genetic restriction system of Clostridium microorganisms. With the recent discovery of the nucleotide sequence of some Clostridiums, it is expected that the development of tools for genome manipulation of Clostridium for commercial production of biofuels will become more active.

During the last decade, much effort has been made to develop genome manipulation techniques for Clostridia. For example, there is a method of inactivating a target gene of Clostridium microorganism by a single crossover method based on Campbell type of a plasmid containing a nucleotide sequence homologous to the target gene. This method has the limitation that the selective marker (antibiotic resistance marker) in the chromosome of the Clostridium microorganism remains after the inactivation of the target gene, and in order to inactivate another gene, a different kind of antibiotic marker A marker is required. In addition, this method is known to be genetically unstable because the inserted plasmid is naturally cleared to return to wild-type for inactivation of the desired gene.

Second, there is a gene inactivation method by a chromosome double cross over method. This method utilizes a suitable semi-selective marker and a Lamda Red or FlT (Flippase Recognition Target) system, which is designed to replicate the plasmid in a host cell with low double crossing efficiency . However, in order to inactivate the target gene, plasmids for the removal of the gene must be removed from the host cells, which is very difficult. In addition, the available counterselection markers are extremely limited.

The third method utilizes a mobile group II intron derived from Lactococcus lactis microorganism. Although this method is known to be very useful in microorganisms of the genus Clostridium, since this method uses a replicable plasmid as in the second method, it has a disadvantage that it must be removed in the host cell for the second gene inactivation.

Therefore, the inventors of the present invention discovered that while studying a novel gene inactivation method, two FRT (flippase recognition target) nucleotide sequences are inserted in the same direction, and a DNA fragment of a target gene is cloned between the FRT nucleotide sequences , It is introduced into a microorganism of the genus Clostridium. When a specific plasmid containing the gene of the FRT site-specific recombinase is further introduced, the genome of Clostridium is stably manipulated using a single chromosome crossing of the FLP enzyme And completed the present invention.

It is an object of the present invention to provide a method for genome manipulation of Clostridium.

In order to achieve the above object, the present invention provides a dielectric manipulation plasmid of Clostridium and a method for manipulating Clostridium using the same.

The present invention can stably inactivate the gene of interest of the Clostridial genus microorganism. In addition, the recombinant Clostridial microorganism of the present invention is characterized in that the target gene is stably inactivated and does not contain an antibiotic marker.

Fig. 1 shows a process for producing a pHKO1 plasmid.
Fig. 2 shows a process for producing a pHKO1-CaMtlR plasmid.
Fig. 3 shows a process for producing a pHKO1-CaPta plasmid.
FIG. 4A shows a method of inactivating the mtlR gene by sequentially introducing the pHKO1 -CaMtlR plasmid and the pSHL-FLP plasmid into Clostridium acetobutylicum.
Fig. 4B shows the selection step of the strain in which the mtlR gene produced in Fig. 4A is inactivated and a substantial portion of the pHKO1-CaMtlR plasmid is removed. pSHL -FLP plasmid was inserted into the strain in which the mtlR gene was inactivated, and then cultured in Erythromycin medium (left picture of FIG. 4B). Then, the cultured strains were replica plated on a medium containing Thiamphenicol (Th) (FIG. 4B, right), and cultured in medium Th containing Thiamphenicol (Th) Colonies that could not grow were identified in a medium containing Erythromycin (Em) to identify strains in which a substantial portion of the pHKO1-CaMtlR plasmid in the genome was removed.
FIG. 4C shows the gene deletion of the strain by the PCR method using the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20. M is the DNA size marker, 1 is the PCR product of the intact target gene, 2 is the PCR product when the plasmid is inserted into the target gene, and 3 is the PCR product when only the FRT sequence is left after removing the inserted plasmid will be.
(lane 1: As of the Clostridium acetonitrile unit Tilikum △ mtlR :: pHKO1 genomic insert within the pHKO1-CaMtlR, lane 3:: wild-type Clostridium acetonitrile unit Tilikum ATCC 824, lane 2 Clostridium acetonitrile unit Tilly Check gene structure of Qom △ mtlR :: FRT)
4D is to remove much of the pSHL-FLP plasmid from Clostridium acetonitrile unit Tilikum △ mtlR :: FRT strain, and shows the results of this check. A colony growing on a non-antibiotic medium (left picture of FIG. 4D) without growing on a medium containing erythromycin (Em) (right picture of FIG. 4D) is a strain in which the pSHL-FLP plasmid is removed.
Figure 5A shows the results obtained by sequentially introducing a pHKO1-CaPta plasmid and a pSHL-FLP plasmid into Clostridium acetobutylicum pta This shows how to deactivate genes.
5B is a cross-sectional view and pta gene is inactivated, shows a sorting step of pHKO1-CaPta the strain is removed, much of the plasmid (△ pta :: FRT). The pSHL-FLP plasmid was inserted into the strain in which the pta gene was inactivated and then cultured in Erythromycin medium (left picture of FIG. 5B). Subsequently, the cultured strains were replica plated on a medium containing Thiamphenicol (Th) (right picture in FIG. 5B), and cultured in medium Th containing Thiamphenicol (Th) Colonies that did not grow were identified in a medium containing Erythromycin (Em) to identify strains in which a substantial portion of the pHKO1-CaPta plasmid in the genome was removed.
FIG. 5C shows the gene deletion of the strain by the PCR method using the primer pairs of SEQ ID NO: 21 and SEQ ID NO: 22. M is the DNA size marker, 1 is the PCR product of the intact target gene, 2 is the PCR product when the plasmid is inserted into the target gene, and 3 is the PCR product when only the FRT sequence is left after removing the inserted plasmid will be.
(lane 1: wild type Clostridium acetobutylicum ATCC 824, lane 2: Identification of inserted pHKO1-CaPta in the genome of Clostridium acetoobutylicum pta :: pHKO1, lane 3: Clostridium acetobutylicum △ pta :: Identification of the gene structure of FRT)
FIG. 5D shows the results of confirming the removal of a substantial portion of the pSHL-FLP plasmid from Clostridium acetoButylicum Δ pta :: FRT strain. A colony growing on a non-antibiotic medium (left photograph of FIG. 5D) without growing on a medium containing erythromycin (Em) (right picture of FIG. 5D) is a strain in which the pSHL-FLP plasmid is removed.

According to the present invention,

2 > FRT (flippase recognition target) base sequence and a part of the nucleotide sequence of the target gene.

Further, according to the present invention,

Introducing into the recombinant Clostridium into which the genome operative plasmid of Clostridium is introduced to remove at least a part of the genome operative plasmid of Clostridium from the recombinant Clostridium,

Which comprises a base sequence of FRT site-specific recombinase.

Further, according to the present invention,

And introducing the genetic engineering plasmid of Clostridium into Clostridium genus microorganism.

The present invention relates to a method for genome manipulation of microorganisms of the genus Clostridium.

Further, according to the present invention,

Introducing the genome-operative plasmid of Clostridium into Clostridium genus microorganism; and

Introducing a plasmid for the gene deletion of Clostridium into a recombinant clostridial genus microorganism into which a genome operative plasmid of Clostridium has been introduced.

Further, according to the present invention,

The present invention relates to a recombinant Clostridial microorganism which has been manipulated by the above-mentioned genetic manipulation method.

Further, according to the present invention,

Culturing the recombinant Clostridial genus microorganism of the present invention; and

To obtain a fermented product in the culture.

Hereinafter, the present invention will be described in detail.

Two FRT ( flippase recognition target ) Nucleotide sequence

The present invention relates to a plasmid of genus of Clostridium comprising two FRT (flippase recognition target) base sequences and a part of the base sequence of the target gene. It is preferable that the two FRT nucleotide sequences are different base sequences having different sizes, that is, lengths of base sequences.

At this time, the large FRT nucleotide sequence is referred to as a FRT-L (Flippase recognition target-large fragment) nucleotide sequence and the small FRT nucleotide sequence is referred to as a FRT-S (Flippase recognition target-small fragment). At this time, the FRT-S base sequence is preferably the nucleotide sequence of SEQ ID NO: 1. It comprises a nucleotide sequence having a homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more homology with the nucleotide sequence of SEQ ID NO: 1. On the other hand, the FRT-L base sequence is preferably the nucleotide sequence of SEQ ID NO: 2. It comprises a nucleotide sequence having a homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more homology with the nucleotide sequence of SEQ ID NO: 2.

Target gene

The present invention is directed to a genomic plasmid of Clostridium comprising two FRT nucleotide sequences and sequences of some of the nucleotide sequences of the gene of interest.

The target gene refers to a gene that is a target of genome manipulation and more specifically refers to a gene of Clostridium to which a genetic engineering plasmid of the present invention is to be introduced and manipulated. The gene of interest can be determined by a person skilled in the art within the genome of Clostridium and is not particularly limited. In the present invention, pta and < RTI ID = 0.0 > mtlR Genes, but it should be apparent that they are not limited thereto.

The sequence of some of the nucleotide sequences of the gene of interest

The present invention is directed to a genomic plasmid of Clostridium comprising two FRT nucleotide sequences and sequences of some of the nucleotide sequences of the gene of interest. That is, the plasmid of genus of Clostridia of the present invention contains a part of the nucleotide sequence of the target gene. Preferably, a part of the nucleotide sequence of the target gene is located between the two FRT nucleotide sequences. At this time, a restriction enzyme site can be introduced between the two FRT nucleotide sequences in order to introduce a part of the nucleotide sequence of the desired gene.

The sequence of a part of the nucleotide sequence of the target gene can be used to inactivate the target gene by homologous recombination with the target gene when the genetic modification plasmid of the present invention is introduced into Clostridium. In this case, the sequence of a part of the nucleotide sequence of the introduced target gene is preferably inserted between target genes in Clostridium by a single crossing method.

The sequence of a part of the nucleotide sequence of the target gene is a nucleotide sequence of 30% or more, preferably 35% or more, more preferably 40% or more, still more preferably 45% or more in the full length nucleotide sequence of the target gene, or 50% or more. However, the length of a part of the sequence required for homologous recombination may vary depending on the length of the base sequence of the target gene, the desired inactivation efficiency, and the like, and a person skilled in the art will be able to appropriately select it.

In this case, the sequence of a part of the nucleotide sequence of the target gene may be a nucleotide sequence having homology with a part of the nucleotide sequence of the target gene. That is, the sequence of a part of the nucleotide sequence of the target gene may be a sequence having homology with 30% or more of the nucleotide sequence of the desired gene, preferably 35% or more, more preferably 40% or more , Still more preferably 45% or more, or 50% or more. At this time, the nucleotide sequence having the above homology is preferably a nucleotide sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 80% May be a base sequence having 95% or more homology. That is, the plasmid of genus of Clostridia of the present invention may contain a base sequence having homology with some base sequences of the target gene.

Clostridium

The present invention relates to a plasmid for genetic manipulation of Clostridia, a plasmid for removing Clostridia gene, a method for genome manipulation of Clostridium using the plasmids, and the like.

Clostridium of the present invention refers to a microorganism belonging to the genus Clostridium. For example, Clostridium of the present invention is Clostridium acetonitrile unit Tilikum (Clostridium acetobutylicum), Clostridium example linkage by children (Clostridium beijerinckii), part Tilikum (Clostridium saccharide with Clostridium saccharobutylicum), Clostridium saccharide butyl hydroperoxide acetoxy T. glutamicum (Clostridium saccharoperbutylacetonicum), Clostridium puff Lin Regensburg (Clostridium perfringens), Clostridium tetani (Clostridium tetani), Clostridium difficile silica (Clostridium difficile), Clostridium portion Tilikum (Clostridium butyricum), Clostridium Cluj Berry (Clostridium kluyveri), part Tilikum (Clostridium by Clostridium tie tyrobutylicum), cloth may be selected from such a tree Stadium tie butynyl rikum (Clostridium tyrobutyricum), may preferably be particularly Clostridium acetonitrile unit Tilly kumil.

Clostridium Dielectric manipulation plasmid

The plasmid of Clostridium of the present invention is introduced into Clostridium and used to manipulate the desired gene. The manipulation of the target gene means that the activity of the target gene is changed, and preferably the target gene is inactivated.

The genetic engineering plasmid of Clostridium of the present invention may comprise an antibiotic resistance first gene base sequence. The antibiotic resistance first gene is preferably a gene expressed in Escherichia coli or Clostridium. The antibiotic resistance first gene sequence can be selectively labeled and used as an antibiotic marker marker. The antibiotic resistance first gene may be a thiamphenicol antibiotic resistance gene (CmR). However, the present invention is not limited thereto, and it is obvious to those skilled in the art that the first antibiotic resistance gene can be selectively labeled in Clostridia and Escherichia coli, and any antibiotic resistance gene can be used without particular limitation.

The genome operative plasmid of Clostridium of the present invention may contain a replication origin. The replication origin may be a pMB1 replication origin, and the pMB1 replication origin may be a replication origin derived from a pJET2.1 plasmid.

For example, the genetic engineering plasmid of Clostridium of the present invention may include a base sequence of pMB1 replication origin, Thiamphenicol antibiotic marker (CmR), two FRT base sequences and a base sequence of a target gene .

The genome operative plasmid of Clostridium of the present invention may further comprise a sequence for introduction into Clostridium. This may be a sequence other than a part of the nucleotide sequence of the target gene introduced for the purpose of the activity change of the target iodine.

Clostridium Plasmids for gene removal

The present invention provides a recombinant clostridia that is introduced into a recombinant Clostridium into which a genome operative plasmid of Clostridium is introduced to remove at least a part of the genome operative plasmid of Clostridium from the recombinant Clostridium,

Which comprises a base sequence of FRT site-specific recombinase.

The plasmid for removing the gene for Clostridia is introduced into the recombinant Clostridium in which the target gene is inactivated to remove at least a part of the plasmid of the genome of Clostridium. At this time, preferably, the plasmid for removing Clostridium gene removes all of the remaining portions of the genome operative plasmid except for a partial sequence of the introduced target gene and one FRT base sequence in the recombinant Clostridium. This removal is accomplished by introducing the plasmid for removing the gene for Clostridia, thereby expressing the FRT site-specific recombinase in the recombinant Clostridium.

Recombination with a dielectric manipulation plasmid Clostridium

Recombinant Clostridium, into which a genome-operative plasmid of the present invention has been introduced and into which a plasmid for the removal of a gene has not yet been introduced, will be described. The recombinant Clostridium target gene is a recombinant clostridium in which the target gene is in a manipulated state, preferably the target gene is inactivated, and the antibiotic resistance first gene base sequence is included.

FRT The nucleotide sequence of the site-specific recombinase

The plasmid for the gene deletion of Clostridium of the present invention includes the base sequence of the FRT site-specific recombinase.

The plasmid for removing the gene for Clostridia is introduced into Clostridium to induce gene recombination by expressing the FRT site-specific recombinase. In addition, the FRT site-specific recombinase is expressed, and the remaining portions of the genome-modified plasmid, except for a part of the target gene and one FRT nucleotide sequence introduced into the recombinant Clostridium, are removed. Preferably, the clostridial gene deletion plasmid of the present invention comprises an antibiotic resistance first gene sequence (for example, a thiamine penicill antibiotic marker) and a replication origin (for example, pMB1 origin of replication) of the genetic engineering plasmid of Clostridium, .

The FRT site-specific recombinant enzyme is preferably a flippase. The nucleotide sequence of the above Phryphase is preferably a nucleotide sequence of SEQ ID NO: 17 or a sequence having a homology of at least 80% with the nucleotide sequence of SEQ ID NO: 17, preferably 85% or more, More preferably 90% or more, still more preferably 95% or more.

Genome manipulation method of Clostridium genus microorganism

The present invention relates to a method for producing a Clostridial genomic microorganism,

Further, according to the present invention,

Through the above dielectric manipulation method, the present invention can stably inactivate a target gene in Clostridium. Stable inactivation at this time means that some sequence of the target gene sequence introduced for inactivation of the target gene is naturally lost and the target gene is not activated again.

Recombination with plasmid for gene removal Clostridium

The present invention relates to a recombinant Clostridium prepared by sequentially introducing a plasmid for genetic manipulation of Clostridium of the present invention and a plasmid for Clostridia gene deletion.

In the recombinant Clostridium of the present invention, the antibiotic marker marker used for inactivation of the target gene, that is, the antibiotic resistance first gene, is removed due to the introduction of the plasmid for gene deletion. Therefore, in the genome of the recombinant Clostridium of the present invention, there is no antibiotic resistance gene, so that additional genetic manipulation (for example, inactivation of the second target gene) is easy and immediate use is possible for the fermentation or biotransformation process.

Production method of fermented product

The present invention relates to a process for the production of a fermentation product comprising culturing a recombinant Clostridium sp. Microorganism of the present invention and obtaining a fermentation product in the culture.

Recombinant Clostridium prepared by sequentially introducing a plasmid for genetic manipulation of Clostridium and a plasmid for removing Clostridium gene of the present invention can be cultured in an environment free of an antibiotic marker, The process can be carried out on an industrial scale.

The culture may be carried out using a general culture method of Clostridium genus microorganism, and is not particularly limited. The fermentation product may be a fermentation product produced by microorganisms of the genus Clostridium, for example, an alcohol, an organic acid, a ketone, or the like. The alcohol may be an alcohol having 7 or less carbon atoms, a polyhydric alcohol, or the like. For example, the alcohol is not limited to butanol, isopropanol, ethanol, 1,3-propanol, 2,3-butanol, propionic acid, and acetone. Alternatively, the fermentation product may be an expression product of a foreign gene contained in the genome operative plasmid. It is obvious that the recombinant clostridial genus microorganism of the present invention may vary in productivity, concentration, composition and the like depending on the kind of target gene and the like.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

&Lt; Materials and methods >

Clostridium acetobutylicum ATCC 824 and Clostridium baselinkii NCIMB 8052 were distributed from the American Type Culture Collection (ATCC), a strain distributor in the United States.

Example 1 Preparation of pHKO1 plasmid

A fragment of DNA comprising a restriction enzyme site (SEQ ID NO: 1), a fragment of FRT-L (Flippase recognition target-large fragment), and a restriction enzyme site ApaI and StuI Fragment size: 187 nucleotides) was synthesized through IDT (Integrated DNA Technologies, Inc., Coralville, IA, USA) (SEQ ID NO: 3). The synthesized DNA fragment was cloned into a plasmid pJET1.2 (Thermo Fisher Scientific, Waltham, MA) to prepare a pJET-FRT plasmid.

On the other hand, using the primer pairs of SEQ ID NOS: 4 and 5 and using the pGS1-MCS2 plasmid (Lee SH et al., J. Microbiol. Biotechnol. (2015), 25 (10), 1702-1708) The penicill antibiotic resistance gene (CmR) was amplified and digested with MfeI and EcoRI restriction enzymes. Then, it was cloned into the pJET-FRT plasmid digested with EcoRI restriction enzyme to prepare pJET-FRT-CmR.

Then, a part of pJET-FRT-CmR, that is, the ori, FRT and CmR regions were amplified and re-ligation by using the primer pairs of SEQ ID NO: 6 and SEQ ID NO: 7 to amplify the pJET-FRT-CmR plasmid The silenced resistance gene was removed. The transformed pJET-FRT-CmR plasmid in which the ampicillin resistance gene was removed was digested with SmaI and StuI restriction enzymes and ligated to construct a pHKO1 plasmid and registered in GenBank (SEQ ID NO: 8, GenBank accession: KR997562, : April 30, 2015, will be closed until April 30, 2016) (Table 1, Figure 1).

Amplification of the genes was performed using PCR. At this time, 100 μl of the PCR reaction mixture was prepared by adding 250 μM dNTP, 20 pmol each of the primers, 10 μM MgCl 2, 10 × buffer, 10 μl of DNA template, 100 ng of DNA template and 1 unit of pfu polymerase. The amplification was carried out by first denaturing at 95 ° C for 5 minutes, followed by denaturation at 95 ° C for 1 minute, annealing at 58 ° C for 1 minute, and polymerization at 72 ° C for 2 minutes.

The PCR reaction in the following examples proceeded in the same manner as the above method. The amplified DNA fragment was purified on 1% agarose gel, and the DNA fragment was digested with restriction enzyme.

Example 2 Preparation of pHKO1-CaMtlR plasmid

A portion of the mtlR gene was amplified using the chromosome of Clostridium acetoobutylicum ATCC 824 as a template using the primer pair having SEQ ID NO: 9 and SEQ ID NO: 10, and the amplified sequence was digested with ApaI restriction enzyme ).

A portion of the mtIR gene cleaved with the ApaI restriction enzyme was cloned into the pHKO1 plasmid prepared in Example 1 to prepare a pHKO1-CaMtlR plasmid (FIG. 2). For reference, the pHKO1 plasmid was prepared by digesting with ApaI restriction enzyme and treating with CIAP (Calf Intestinal Alkaline Phosphatase) enzyme like the mtlR gene.

At this time, the PCR reaction proceeded under the same conditions as in Example 1 above.

Example 3 Preparation of pHKO1-CaPta plasmid

A part of the pta gene was amplified using a chromosome of Clostridium acetoobutylicum ATCC 824 as a template using a pair of primers having the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12, and the amplified sequence was cut with ApaI restriction enzyme (Table 3).

A portion of the Pta gene cleaved with the ApaI restriction enzyme was cloned into the pHKO1 plasmid prepared in Example 1 to prepare a pHKO1-CaPta plasmid (FIG. 3). For reference, the pHKO1 plasmid was prepared by Like the pta gene, it was digested with ApaI restriction enzyme and treated with CIAP (Calf Intestinal Alkaline Phosphatase) enzyme.

Example 4 Production of pSHL-FLP plasmid

The thiolase promoter and ribosomal binding site sequences were amplified from the chromosome of Clostridium baselinkii NCIMB 8052 using the primer pairs of SEQ ID NO: 13 and SEQ ID NO: 14. The amplified DNA fragment was digested with SmaI and StuI restriction enzymes.

Also, the pSHL-MCS plasmid was used as a template and a part of the pSHL-MCS plasmid, i.e., the ori and EmR regions, was amplified using the primer pairs of SEQ ID NO: 15 and SEQ ID NO: The amplified DNA fragment was then digested with PstI and PmeI restriction enzymes (Table 4).

The amplified DNA fragment digested with SmaI and StuI restriction enzymes and the amplified DNA fragment digested with PstI and PmeI restriction enzymes were ligated to prepare pSHL-StuI plasmid.

On the other hand, a flippase gene which specifically recognizes the FRT nucleotide sequence and recombine the DNA was synthesized by biona (Daejeon, Korea) (SEQ ID NO: 17, Table 5). The synthesized Phryphase gene was digested with StuI and PstI restriction enzymes and cloned into the pSHL-StuI plasmid digested with the same restriction enzymes to prepare pSHL-FLP plasmid. The pSHL-FLP plasmid was registered in Genbank (SEQ ID NO: 18, Genbank accession: KR997561, registered on April 30, 2015, scheduled to be closed until April 30, 2016).

&Lt; Example 5 > mtlR Genetically inactivated recombinant strains

<5-1> mtlR Gene inactivation

The pHKO1-CaMtlR plasmid prepared in Example 2 was introduced into Clostridium acetobutylicum ATCC 824 strain to prepare a transformed recombinant microorganism (Fig. 4A).

The concrete method is as follows. Clostridium acetobutylicum was suspended in a CGM (Clostridium Growth Media) liquid medium (0.75 g / LK 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 The cultures were incubated in anaerobic conditions until the OD600 = 1.0 in 100 ml of the medium, 5 g / L of yeast extract, and 10 g / L of glucose. The cultures were left on ice for 10 minutes and cultured at 7000 g for 10 minutes at 4 ° C Cells were centrifuged. Cell pellets were washed three times with buffer solution and then suspended in 2 ml of the same buffer to prepare transforming cells. 5.0 μg of shuttle plasmids were added to 500 μl of the transformants prepared above, and the cells were subjected to electroporation (4 mm cuvette, 2.5 kV, ∞ Ω, 25 μF) using Bio-Rad Gene pulser II and 5 μg / Thiamphenicol (Th) antibiotic-added 2X YTG solid medium for 72 hours. When the colonies grew on the solid medium, they were cultured in CGM liquid medium for 16 hours. The cultured cells were centrifuged at 7000 g, and the chromosomes were separated and PCR was carried out using the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 20 6), the pHKO1-CaMtlR plasmid was clostridime acetobutylicum ATCC 824 mtlR (Fig. 4C).

<5-2> Removal of pHKO1 plasmid from mtlR gene inactivating strain

In the above Experimental Example 5-1, the pHKO1-CaMtlR plasmid was inserted into the chromosome of Clostridium acetoButylicum ATCC 824 to confirm that the mtlR gene was inactivated. Therefore, a plasmid backbone containing the thiamine penicill resistance gene (CmR) was removed from the chromosome.

The specific method of removal is as follows. First, the pSHL-FLP plasmid prepared in Example 4 was introduced into the clostridium acetyobutiliquum in which the mtlR gene prepared in Example <5-1> was inactivated, and cultured in liquid CGM for 6 hours And then plated on solid CGM medium containing 40 ug / mL of erythromycin. Flippase enzyme was introduced in pSHL-FLP to induce the recombination of FRT nucleotide sequence-specific genes by the flippase enzyme and the plasmid backbone containing the thymine penicill resistance gene (CmR) was removed.

Forty-eight hours later in the smoldered solid medium, 50 colonies were replica plated with CGM solid medium containing Thiamphenicol (Th). At this time, the colonies grown in the erythromycin medium but not growing in the thiamin penicol medium were identified and PCR was performed using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 to confirm the size of the PCR product by electrophoresis. It was confirmed that the plasmid was removed (FIGS. 4B and 4C).

At this time, all of the plasmids used for transformation were methylated in the E. coli TOP10 strain transformed with the pAN1 plasmid (in the presence of the GCNGC sequence, the gene for methylating the internal cytosine) before electroporation and the restriction system of Clostridium strain It is made so that it is not affected by.

<5-3> Removal of pSHL-FLP plasmid

The pSHL-FLP was removed from the strain in which the pHKO1 plasmid backbone was removed in the routine example <5-2>. The pSHL-FLP plasmid was naturally lost due to unstable replication in Clostridium acetobutylicum, but was artificially removed in the present invention for rapid loss.

Process Biochemistry 43: 822- (1986), pp. 179-178, 1982. The present inventors have found that, in the case of the bifidobacterium longum, Escherichia coli JM109, 828). The pSHL-FLP plasmid of Example < 5-2 > One mtlR gene-inactivated colony was cultured in a culture tube containing 40 ml CGM liquid medium at 37 DEG C without antibiotics until the cell concentration reached 1.0 (OD600 nm). At this time, the cell concentration was measured using a spectrophotometer (Hach, USA).

40 uL of the initial liquid culture was again inoculated into 40 ml CGM liquid without antibiotics (diluted to 1/1000 of the initial culture medium concentration), and then repeated 3 times in the same manner as described above to lyse the antibiotic-free 2X YTG solid medium, Of these, 50 colonies were replica plated onto 2X YTG solid medium containing erythromycin. It was confirmed that all colonies did not grow on the erythromycin medium and grew only in the anti-biocompatible medium, confirming that pSHL-FLP was removed (FIGS. 4C and 4D).

&Lt; Example 6 > Production of a recombinant strain in which the pta gene is inactivated

<6-1> pta gene inactivation

The transformed recombinant microorganism was prepared by introducing the pHKO1-CaPta plasmid prepared in Example 3 into Clostridium acetobutylicum ATCC 824 strain.

The concrete method is as follows. Clostridium acetobutylicum ATCC 824 was cultivated in a CGM (Clostridium Growth Media) liquid medium (0.75 g / LK 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揃 5H ₂ O, 0.01 g / L, FeSO ₄揃 7H ₂ O, 2 g / L (NH ₄ ) ₂ SO ₄ , 1 g / L NaCl, 2 g / L asparagine, 0.004 g / L p After incubation for 10 min in ice, the culture was incubated at 7000 g for 10 min at 4 ° C for 10 min. Lt; / RTI > cells were centrifuged. Cell pellets were washed three times with buffer solution and then suspended in 2 ml of the same buffer to prepare transforming cells. 5.0 ug of shuttle plasmid (pHKO1-CaPTA) was added to 500 형 of transformants prepared above, and the cells were subjected to electroporation (4 mm cuvette, 2.5 kV, 25 uF) using Gene pulser II Thiamphenicol (Th) antibiotic-added 2X YTG solid medium for 72 hours. When the colonies grew on the solid medium, they were cultured in CGM liquid medium for 16 hours. The cultured cells were centrifuged at 7000 g, and the chromosomes were separated and subjected to PCR using the primers of SEQ ID NOS: 21 and 22, and pHKO1-CaPTA When the plasmid is selected from the group consisting of Clostridium acetobutylicum pta gene (Fig. 5C).

<6-2> Removal of pHKO1 plasmid from pta gene inactivated strain

In the above Example <6-1>, the pHKO1-CaPta plasmid was inserted into the chromosome of Clostridium acetoobutylicum ATCC 824, pta It was confirmed that the gene was inactivated. Therefore, a plasmid backbone containing the thiamine penicill resistance gene (CmR) was attempted to be removed from the chromosome.

The specific method of removal is as follows. First, the pSHL-FLP plasmid prepared in Example 4 was introduced into the recombinant Clostridium aceticlobutylicum in which the pta gene was inactivated, which was prepared in Example <6-1> And then plated on CGM solid medium containing 40 ug / mL of erythromycin. In the introduced pSHL-FLP, the plasmid backbone containing the thiamepenicol resistance gene (CmR) is removed (FIG. 5A) by inducing the FRT nucleotide sequence-specific gene recombination by expressing the flippase enzyme.

After 48 hours in the solid medium, replicate 50 colonies with CGM solid medium containing thiamine penicol. At this time, the colonies grown in the erythromycin medium but not growing in the thiamin penicol medium were identified and PCR was carried out using the primers of SEQ ID NO: 21 and SEQ ID NO: 22 to confirm the size of the PCR products by electrophoresis. Then, the pHKO1 plasmid (FIG. 5B and FIG. 5C).

<6-3> Removal of pSHL-FLP plasmid

The pSHL-FLP was removed from the strain in which the pHKO1 plasmid backbone was removed in the experiment example <6-2>. pSHL-FLP replicates unstable in Clostridium acetobutylicum and disappears naturally.

Process Biochemistry 43: 822- (1986), pp. 179-178, 1982. The present inventors have found that, in the case of the bifidobacterium longum, Escherichia coli JM109, 828). The pSHL-FLP plasmid of Example < 6-2 > One colony in which the pta gene was inactivated was cultured in a culture tube containing 40 ml CGM liquid medium at 37 DEG C without antibiotics until the cell concentration reached 1.0 (OD600 nm). At this time, the cell concentration was measured using a spectrophotometer (Hach, USA).

40 uL of the initial liquid culture was again inoculated into 40 ml CGM liquid without antibiotics (diluted to 1/1000 of the initial culture medium concentration), and then repeated 3 times in the same manner as described above to lyse the antibiotic-free 2X YTG solid medium, Of these, 50 colonies were replica plated onto 2X YTG solid medium containing erythromycin. At this time, 48 colonies that could not grow on the erythromycin medium but grow only in the non-bioactive medium were identified, and it was confirmed that pSHL-FLP was removed (FIGS. 5C and 5D).

<110> GS CALTEX KRIBB <120> METHOD OF GENOME ENGINEERING IN CLOSTRIDIA <130> DNP150423 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Flippase recognition target-Small fragment <400> 1 gaagttccta tactttctag agaataggaa cttcg 35 <210> 2 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Flippase recognition target-Large fragment <400> 2 gaagttccta tactttctag agaataggaa cttcggaata ggaacttc 48 <210> 3 <211> 187 <212> DNA <213> Artificial Sequence <220> <223> DNA fragments including FRT-S, FRT-L, ApaI and StuI <400> 3 gggatccgta taccgtgtag gctggagctg cttcgaagtt cctatacttt ctagagaata 60 ggaacttcgg aataggaact tcaagatccc cttagatagg cctgggcccg cgctctgaag 120 ttcctatact ttctagagaa taggaacttc gaactgcacg tcgaccgatc cccggtatac 180 gaattcc 187 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggcaattgct ggcacgacag gtttcccgac 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gggaattctt aactatttat caattcctgc 30 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atacccgggc tataaaaata ggcgtatcac gaggcc 36 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctcaggcctg tgagttttcg ttccactgag cgtcagacc 39 <210> 8 <211> 3006 <212> DNA <213> Artificial Sequence <220> <223> pHKO1 plasmid <400> 8 aggcctatct aaggggatct tgaagttcct attccgaagt tcctattctc tagaaagtat 60 aggaacttcg aagcagctcc agcctacacg gtatacggat cccatcttgc tgaaaaactc 120 gagccatccg gaagatctgg cggccgctct ccctatagtg agtcgtatta cgccggatgg 180 atatggtgtt caggcacaag tgttaaagca gttgatttta ttcactatga tgaaaaaaac 240 aatgaatgga acctgctcca agttaaaaat agagataata ccgaaaactc atcgagtagt 300 aagattagag ataatacaac aataaaaaaa tggtttagaa cttactcaca gcgtgatgct 360 actaattggg acaattttcc agatgaagta tcatctaaga atttaaatga agaagacttc 420 agagcttttg ttaaaaatta tttggcaaaa ataatataat tcggctgcag gggcggcctc 480 gtgatacgcc tatttttata gccccctgtg agttttcgtt ccactgagcg tcagaccccg 540 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 600 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 660 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 720 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 780 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 840 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 900 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 960 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1020 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1080 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1140 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1200 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1260 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1320 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1380 aatgcagctg gcacgacagg tttcccgact ggaaagcaat tggcagtgag cgcaacgcaa 1440 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 1500 gtataatgtg tggaattatg agcggataat aatttcacac aggaggttta aactttaaac 1560 atgtcaaaag agacgtcttt tgttaagaat gctgaggaac ttgcaaagca aaaaatggat 1620 gctattaacc ctgaactttc ttcaaaattt aaatttttaa taaaattcct gtctcagttt 1680 cctgaagctt gctctaaacc tcgttcaaaa aaaatgcaga ataaagttgg tcaagaggaa 1740 catattgaat atttagctcg tagttttcat gagagtcgat tgccaagaaa acccacgcca 1800 cctacaacgg ttcctgatga ggtggttagc atagttctta atataagttt taatatacag 1860 cctgaaaatc ttgagagaat aaaagaagaa catcgatttt ccatggcagc tgagaatatt 1920 gtaggatat ttctagaaat gaggaattct taactattta tcaattcctg caattcgttt 1980 acaaaacggc aaatgtgaaa tccgtcacat actgcgtgat gaacttgaat tgccaaaggg 2040 agtataattc tgttatcttc tttataatat ttccccatag taaaaatagg aatcaaataa 2100 tcatatcctt tctgcaaatt cagattaaag ccatcgaagg ttgaccacgg tatcatagat 2160 acattaaaaa tgttttccgg agcatttggc tttccttcca ttctatgatt gtttccatac 2220 cgttgcgtat cactttcata atctgctaaa aatgatttaa agtcagactt acactcagtc 2280 caaaggctgg aaaatgtttc agtatcattg tgaaatattg tatagcttgg tatcatctca 2340 tcatatatcc ccaattcacc atcttgattg attgccgtcc taaactctga atggcggttt 2400 acaatcattg caatataata gagcattgca ggatatagtt tcattccctt ttcctttatt 2460 tgtgtgatat ccactttaac ggtcatgctg tatgtacaag gtacacttgc aaagtagtgg 2520 tcaaaatact cttttctgtt ccaactattt ttatcaattt tttcaaatac catctaagtt 2580 ccctcctttt aaattcaagt ggatcctacg gggtaacaga taaaccattt caatctattt 2640 cataagttcc atagtttatc cctaatttat acgttttctc taacaactta attataccca 2700 ctattattat ttttatcaat atattttgtt aaaaagtcga agcttggcgt aatcatggtc 2760 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 2820 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 2880 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcaattcg tataccgggg 2940 atcggtcgac gtgcagttcg aagttcctat tctctagaaa gtataggaac ttcagagcgc 3000 gggccc 3006 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atagggccct ttaactccta gacagcagta tatactaagt gagtta 46 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atagggcccc cactgtttca tctgtcttat gtccataaag gaa 43 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atagggcccg gttatgttat aaagtatgta tagtaaaatg 40 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cacgggcccg tcaggtatta tcataacaaa gaatcctg 38 <210> 13 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atacccgggt attgtaaaat gagtaaatct ataaatcc 38 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ctcaagcttg tttgacctcc taaaatttta taga 34 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcgctgcaga gatctctcga ggcggccgc 29 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atagtttaaa ccattaatga atcggccaac gcgcggg 37 <210> 17 <211> 1284 <212> DNA <213> Artificial Sequence <220> <223> flippase gene <400> 17 aggcctatgc cacaatttga tatattatgt aaaacaccac ctaaggtgct tgttcgtcag 60 tttgtggaaa ggtttgaaag accttcaggt gagaaaatag cattatgtgc tgctgaacta 120 acctatttat gttggatgat tacacataac ggaacagcaa tcaagagagc cacattcatg 180 agctataata ctatcataag caattcgctg agtttcgata ttgtcaataa atcactccag 240 tttaaataca agacgcaaaa agcaacaatt ctggaagcct cattaaagaa attgattcct 300 gcttgggaat ttacaattat tccttactat ggacaaaaac atcaatctga tatcactgat 360 attgtaagta gtttgcaatt acagttcgaa tcatcggaag aagcagataa gggaaatagc 420 cacagtaaaa aaatgcttaa agcacttcta agtgagggtg aaagcatctg ggagatcact 480 gagaaaatac taaattcgtt tgagtatact tcgagattta caaaaacaaa aactttatac 540 caattcctct tcctagctac tttcatcaat tgtggaagat tcagcgatat taagaacgtt 600 gatccgaaat catttaaatt agtccaaaat aagtatctgg gagtaataat ccagtgttta 660 gtgacagaga caaagacaag cgttagtagg cacatatact tcttttagcgc aaggggtagg 720 atcgatccac ttgtatattt ggatgaattt ttgaggaatt ctgaaccagt cctaaaacga 780 gtaaatagga ccggcaattc ttcaagcaat aaacaggaat accaattatt aaaagataac 840 ttagtcagat cgtacaataa agctttgaag aaaaatgcgc cttattcaat ctttgctata 900 aaaaatggcc caaaatctca cattggaaga catttgatga cctcatttct ttcaatgaag 960 ggcctaacgg agttgactaa tgttgtggga aattggagcg ataagcgtgc ttctgccgtg 1020 gccaggacaa cgtatactca tcagataaca gcaatacctg atcactactt cgcactagtt 1080 tctcggtact atgcatatga tccaatatca aaggaaatga tagcattgaa ggatgagact 1140 aatccaattg aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata 1200 cgataccccg catggaatgg gataatatca caggaggtac tagactacct ttcatcctac 1260 ataaatagac gcatataact gcag 1284 <210> 18 <211> 7792 <212> DNA <213> Artificial Sequence <220> <223> pSHL-FLP plasmid <400> 18 gggtattgta aaatgagtaa atctataaat ccttatggag atggaattgc atctagaaga 60 atagcagatg ctatattaaa atattttggt ttgacaacaa gagaagtaga agaatttaaa 120 agataaagtt tgtttaaatt ttaacaattt atgcttgata aaagaaataa taaaaggtat 180 aatttagtta tgttaataat ttaacaaaag ttaataaaat aatctataaa attttaggag 240 gtcaaacagg cctatgccac aatttgatat attatgtaaa acaccaccta aggtgcttgt 300 tcgtcagttt gtggaaaggt ttgaaagacc ttcaggtgag aaaatagcat tatgtgctgc 360 tgaactaacc tatttatgtt ggatgattac acataacgga acagcaatca agagagccac 420 attcatgagc tataatacta tcataagcaa ttcgctgagt ttcgatattg tcaataaatc 480 actccagttt aaatacaaga cgcaaaaagc aacaattctg gaagcctcat taaagaaatt 540 gattcctgct tgggaattta caattattcc ttactatgga caaaaacatc aatctgatat 600 cactgatatt gtaagtagtt tgcaattaca gttcgaatca tcggaagaag cagataaggg 660 aaatagccac agtaaaaaaa tgcttaaagc acttctaagt gagggtgaaa gcatctggga 720 gatcactgag aaaatactaa attcgtttga gtatacttcg agatttacaa aaacaaaaac 780 tttataccaa ttcctcttcc tagctacttt catcaattgt ggaagattca gcgatattaa 840 gaacgttgat ccgaaatcat ttaaattagt ccaaaataag tatctgggag taataatcca 900 gtgtttagtg acagagacaa agacaagcgt tagtaggcac atatacttct ttagcgcaag 960 gggtaggatc gatccacttg tatatttgga tgaatttttg aggaattctg aaccagtcct 1020 aaaacgagta aataggaccg gcaattcttc aagcaataaa caggaatacc aattattaaa 1080 agataactta gtcagatcgt acaataaagc tttgaagaaa aatgcgcctt attcaatctt 1140 tgctataaaa aatggcccaa aatctcacat tggaagacat ttgatgacct catttctttc 1200 aatgaagggc ctaacggagt tgactaatgt tgtgggaaat tggagcgata agcgtgcttc 1260 tgccgtggcc aggacaacgt atactcatca gataacagca atacctgatc actacttcgc 1320 actagtttct cggtactatg catatgatcc aatatcaaag gaaatgatag cattgaagga 1380 tgagactaat ccaattgagg agtggcagca tatagaacag ctaaagggta gtgctgaagg 1440 aagcatacga taccccgcat ggaatgggat aatatcacag gaggtactag actacctttc 1500 atcctacata aatagacgca tataactgca gagatctctc gaggcggccg cgtcgactct 1560 agacccggga attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 1620 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 1680 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg 1740 cggtattttc tccttacgca tctgtgcggt atttcacacc gggatcttat ttaatcactt 1800 tgactagcaa atactaacaa caagacacac acaccaaaaa tcaaaaattc actactttta 1860 gttaaaaacc acgtaaccac aagaactaat ccaatccatg taatcgggtt cttcaaatat 1920 ttctccaaga ttttcctcct ctaatatgct caacttaaat gacctattca ataaatctat 1980 tatgctgcta aatagtttat aggacaaata agtatactct aatgacctat aaaagataga 2040 aaattaaaaa atcaagtgtt cgcttcgctc tcactgcccc tcgacgtttt agtagccttt 2100 ccctcacttc gttcagtcca agccaactaa aagttttcgg gctactctct ccttctcccc 2160 ctaataatta attaaaatct tactctgtat atttctgcta atcattcgct aaacagcaaa 2220 gaaaaaacaa acacgtatca tagatataaa tgtaatggca tagtgcgggt tttattttca 2280 gcctgtatca tagctaaaca aatcgagttg tgtgtccgtt ttagggcgtt ctgctagctt 2340 gtttaaagtc tcttgaatga atgtatgctc taagtcaaaa gaatttgtca gcgcctttat 2400 atagctttct ttttcttctt tttttacttt aatgatcgat agcaacaatg atttaacact 2460 agcaagttga atgccaccat ttcttcctgg tttaatctta aagaaaattt cctgattcgc 2520 cttcagtacc ttcagcaatt tatctaatgt ccgttcagga atgcctagca cttctctaat 2580 ctcttttttg gtcgtcacta aataaggctt gtatacatcg cttttttcgc taatataagc 2640 cattaaatct tctttccatt ctgacaaatg aacacgttga cgttcgcttc tttttttctt 2700 gaatttaaac cacccttgac ggacaaataa atctttactg gttaaatcac ttgataccca 2760 agctttgcaa agaatggtaa tgtattccct attagcccct tgatagtttt ctgaataggc 2820 acttctaaca attttgatta cttctttttc ttctaagggt tgatctaatc gattattaaa 2880 ctcaaacata ttatattcgc acgtttcgat tgaatagcct gaactaaagt aggctaaaga 2940 gagggtaaac atgacgttat tacgccctat taaacccttt tctcctgaaa atttcgtttc 3000 gtgcaataag agattaaacc agggttcatc tacttgtttt ttgccttctg taccgcttaa 3060 aaccgttaga cttgaacgag taaagccctt attatctgtt tgtttgaaag accaatcttg 3120 ccattctttg aaagaataac ggtaattagg atcaaaaaaat tctacattgt ccgttcttgg 3180 tatgcgagca ataccaaaat gattacacgt tagatcaact ggcaaagact ttccaaaata 3240 ttctcggata ttttgcgaaa ttattttggc tgctttgaca gatttaaatt ctgattttga 3300 agtcacatag actggcgttt ctaaaacaaa atatgcttga taacctttat cagatttgat 3360 aatcatagta ggcataaaac ctaaatcaat agcggttgtt aaaatatcgc ttgctgaaat 3420 agtttctttt gccgtgtgaa tatcaaaatc aataaagaag gtattgattt gtcttaaatt 3480 gttttcagaa tgtcctttcg tgtatgaacg gttttcgtct gcatacgttc cataacgata 3540 aacgttgggt gtccaatgtg taaatgtatc ttgattttct tgaatcgctt cctcggaagt 3600 cagaacaca ccacgaccgc caatcatgct tgattttgag cgatacgcaa aaatagcccc 3660 tttgctttta cctggcttgg tagtgattga gcgaatttta ctatttttaa atttgtactt 3720 taacaagccg tcatgaagca cagtttctac aacaaaaggg atattcattc agctgttctc 3780 ctttcctata aaatcctata aaataggttg tttaattaac ttggtttgct ttttcattca 3840 actgtttcaa tattgcatgt tttgaaaaag atttttttcc tttataagtc aatttttttc 3900 cactaatcga ataaattatt ttgttatttt ctattaactt atatatataa tcttccccct 3960 ccgaagaaaa atacttatct gattttgttt ctaagtagat atttctcttt tctaactctt 4020 tcttaaacgt ttctagtgta tagatatttg ctaattttct tatctccaat aaactatttt 4080 ttatataagt tttacattca tcatgattca tacaaactcc accttctata aatgaataca 4140 aaaaaagcaa tcaaacgatt tccgattgat tgcttaacaa ttcttaaatt cagtagctta 4200 gatacttgaa aactctctga tttccctata taatgatagt acggttatat accgtcttca 4260 aacaaagtta attaaataac ttcttacgag ggaagagttc atctgactaa ctgataagcg 4320 ttggtttggc aatcttatcg ggctatgcat ttataaaatg tcgtcaaaca ttttataaat 4380 gtgtcatggc tcttttttcg tttctattca gttcgttgtt tcgttatatc tagtataccg 4440 cttttaaaaa aaataagcaa cgatttcgtg cattattcac acgaagtcat tgcttttttc 4500 ttcttccatt tctaaatcca atgttacttg ttctgattct gtttctggtt ctggttctgt 4560 tggctcattt gggattaaat ccactactag cgttgagtta gttggggatc ctagcagcac 4620 gccatagtga ctggcgatgc tgtcggaatg gacgatcaaa ttccccgtag gcgctaggga 4680 cctctttagc tccttggaag ctgtcagtag tatacctaat aatttatcta cattcccttt 4740 agtaacgtgt aactttccaa atttacaaaa gcgactcata gaattatttc ctcccgttaa 4800 ataatagata actattaaaa atagacaata cttgctcata agtaacggta cttaaattgt 4860 ttactttggc gtgtttcatt gcttgatgaa actgattttt agtaaacagt tgacgatatt 4920 ctcgattgac ccattttgaa acaaagtacg tatatagctt ccaatattta tctggaacat 4980 ctgtggtatg gcgggtaagt tttattaaga cactgtttac ttttggttta ggatgaaagc 5040 attccgctgg cagcttaagc aattgctgaa tcgagacttg agtgtgcaag agcaacccta 5100 gtgttcggtg aatatccaag gtacgcttgt agaatccttc ttcaacaatc agatagatgt 5160 cagacgcatg gctttcaaaa accacttttt taataatttg tgtgcttaaa tggtaaggaa 5220 tactcccaac aattttatac ctctgtttgt tagggaattg aaactgtaga atatcttggt 5280 gaattaaagt gacacgagta ttcagtttta atttttctga cgataagttg aatagatgac 5340 tgtctaattc aatagacgtt acctgtttac ttattttagc cagtttcgtc gttaaatgcc 5400 ctttacctgt tccaatttcg taaacggtat cggtttcttt taaattcaat tgttttatta 5460 tttggttgag tactttttca ctcgttaaaa agttttgaga atattttata tttttgttca 5520 tgtaatcact ccttcttaat tacaaatttt tagcatctaa tttaacttca attcctatta 5580 tacaaaattt taagatactg cactatcaac acactcttaa gtttgcttct aagtcttatt 5640 tccataactt cttttacgtt tccgccattc tttgctgttt cgatttttat gatatggtgc 5700 aagtcagcac gaacacgaac cgtcttatct cccattatat ctttttttgc actgattggt 5760 gtatcatttc gtttttcttt ttatcccgca agaggcccgg cagtcaggtg gcacttttcg 5820 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 5880 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 5940 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 6000 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 6060 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 6120 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 6180 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 6240 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 6300 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 6360 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 6420 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 6480 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 6540 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 6600 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 6660 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 6720 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 6780 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 6840 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 6900 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 6960 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 7020 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 7080 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 7140 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 7200 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 7260 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 7320 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 7380 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 7440 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 7500 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 7560 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 7620 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 7680 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 7740 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatggt tt 7792 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ttcttagatc gttcaactta tgaagg 26 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ccagctcttt ctattgcagc atcc 24 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ttatatacct ctttgagcct gaacaacagt tata 34 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cctctttgaa caaagtcccc actc 24

Claims

Two FRT (flippase recognition target) nucleotide sequences, an antibiotic resistance first gene sequence and a part of the nucleotide sequence of the target gene,
Wherein a part of the nucleotide sequence of the target gene is located between the two FRT nucleotide sequences.

The method according to claim 1,
Wherein said target gene is a gene of Clostridium.

The method according to claim 1,
Wherein the two FRT nucleotide sequences are different in size from each other.

The method according to claim 1,
Wherein the two FRT nucleotide sequences are the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively.

The method according to claim 1,
Wherein the sequence of a part of the nucleotide sequence of the target gene is inserted between target genes in the clostridia upon introduction of the plasmid.

The method according to claim 1,
Wherein the sequence of a part of the nucleotide sequence of the target gene is at least 30% of the full-length nucleotide sequence of the gene of interest.

delete

12. A method for producing a Clostridial genomic microorganism, comprising the steps of: introducing a plasmid of Clostridium of claim 1 into Clostridial microorganism;
Methods for genomic manipulation of Clostridium genus microorganisms.

13. The method of claim 12,
Further comprising the step of introducing a plasmid for removing the gene of Clostridium into a recombinant Clostridium genus microorganism into which the above DNA-operative plasmid of Clostridium has been introduced,
Wherein the plasmid for removing the gene for Clostridium is a plasmid selected from the group consisting of a recombinant Clostridial microorganism and a recombinant Clostridial microorganism having a nucleotide sequence of FRT site specific recombinase, ,
Methods for genomic manipulation of Clostridium genus microorganisms.

14. A method for manipulating a genome of claim 13,
Recombinant Clostridium microorganism.

15. The method of claim 14,
Wherein the recombinant Clostridial genus microorganism does not contain an antibiotic resistance gene.

Culturing the recombinant Clostridial genus microorganism of claim 14 to produce a culture; and
Thereby obtaining a fermentation product in the culture.