WO2018182361A1 - Method for preparing corynebacterium mutant strain, using crispr/cas system, recombinase, and single-stranded oligodeoxyribonucleic acid - Google Patents

Abstract

The present invention relates to a method for preparing a bacteria mutant strain, comprising the steps of: (a) primarily transforming a normal bacteria into an enzyme-expressing vector which has temperature sensitivity or antibiotic sensitivity and expresses (i) a recombinase; (b) making the primarily transformed bacteria obtained in step (a) to be a competent cell; (c) secondarily transforming the competent cell obtained in step (b) by introducing (i) a single-stranded oligodeoxyribonucleic acid (ssODN) which binds complementarily to the target gene and (ii) a first vector which expresses a guide (RNA) and has antibiotic sensitivity or temperature sensitivity; and (e) removing the inserted enzyme-expressing vector and the first vector from the secondarily transformed bacteria, wherein at least one of the enzyme-expressing vector and the first vector expresses a Cas protein. A method according to the present invention can delete a target gene effectively, select a recombinant microorganism rapidly and at high efficiency, and remove all foreign vectors from the finally selected recombinant microorganism, finding very useful applications in the industrial use of recombinant Corynebacterium.

Description

Method for preparing corynebacterium mutant strain using CRISPR / Cas system, recombinant enzyme and single-stranded oligodioxyribonucleic acid

The present invention uses a CRISPR / Cas system, a recombinant enzyme (Recombinase) and single-stranded oligodioxyribonucleic acid (ssODN) to delete bacterial genes, and the method of producing a mutant strain, characterized in that to quickly select the deleted mutant strains. It is about.

The microbial utilization technology developed in fermented foods is widely used in the field of producing chemicals due to the safety and environment-friendly features of the products, and recently, it is possible to improve the productivity of microorganisms by metabolic engineering. Reached the level.

Microbial metabolic engineering is the introduction of new metabolic circuits or existing metabolism for genetic information obtained by whole genome sequencing of microorganisms to improve target microorganisms by applying genetic recombination and biotechnological techniques. It is a set of technologies that removes, amplifies, and alters circuits to make them industrially useful.

As a useful product produced by microorganisms, amino acids used in food and livestock feed are mainly produced using Corynebacterium glutamicum among various microorganisms. Corynebacterium genus strains are Gram-positive strains, and are widely used for producing amino acids such as glutamate, lysine and threonine and purine-based nucleic acids such as inosic acid. C. glutamicum has easy growth conditions, can be cultured 4 times higher than E. coli, and its genome structure is stable, resulting in low probability of mutation. In addition, it is a non-pathogenic strain and does not make spores, so it does not have a harmful effect on the environment, and has advantages as an industrial strain.

In order to increase the yield of the microorganisms, interest in the development of Corynebacterium to which microbial metabolic engineering (metabolic engineering) is applied is steadily increasing, and the genetic manipulation technology applied thereto is a high performance by producing a mutant strain library or inducing mutations. Labor-intensive and time-consuming traditional methods such as screening strains or manipulating genomes by inducing double crossover have been utilized.

Recently, in order to more efficiently engineer genomes of the genus Corynebacterium , a technique using recombineering based on recombinase on C. glutamicum has been developed (Binder et al., Nucleic Acids Research , 41 (12): 6360-6369, 2013). The technique is characterized in that a specific gene is deleted by using single-stranded oligodeoxyribonucleic acid (ssODN) and RecT of the recombinant enzyme E. coli prophage without preparing a mutant strain library. However, since the mutation efficiency is quite low, the strains are further manipulated to allow biosensoring of the strains again, and then the strains are selected by applying fluorescence-activated cell sorting (FACS). It is cumbersome but has the advantage of being able to quickly select an improved microorganism lacking the desired gene.

In particular, there is a technical limitation that only a few base pairs can be substituted at a time by using RecT and ssODN. Depending on the type of microorganism, the strength of the repair system and the activity of RecT are not the same. There is a problem that is different.

The CRISPR / Cas system, which is an immune system for microorganisms to cope with viruses, can selectively cut target sequences by introducing the CRISPR / Cas system to heterologous cells in 2012. It is known that the genome for various cells can be edited, and as a gene editing technique, it is expected to be utilized more efficiently and conveniently in biological improvement (Jinek et al., Science, 337 (6096): 816-821, 2012).

In gene editing technology, the CRISPR / Cas9 system of the CRISPR / Cas system generates a double-strand break (DSB) on the target DNA by the Cas9 and sgRNA constituting it, and the cell recognizes it as an injury site. This leads to a conventional DNA repair process by non-homologous end joining (NHEJ) or homology-directed repair (HDR), in which normalization by mutation or gene replacement Where possible, this can be used to induce genome editing: the non-homologous end joining (NHEJ) mechanism is used to refine DSBs generated by the action of the CRISPR / Cas system and then to simple conjugation. Frameshift mutations can be introduced in E. coli to easily induce gene deletions, while homologous groups exist in the presence of homologous gene fragments with truncated sites. A mechanism of homology-directed repair (HDR) can occur, which can lead to normalization or deletion by gene replacement.

Because some organisms differ in their inherent recombination capacity, there are microorganisms that cannot repair the DSB with the NHEJ pathway or have difficulty repairing the DSB using HDR. Therefore, while the CRISPR / Cas system is a very convenient means for gene editing, microorganisms that cannot repair DSB by the NHEJ pathway are often observed to die when CRISPR / Cas is introduced to delete target genes. Because of this, it is difficult to apply freely to all microorganisms. However, in most microorganisms, DSB repair via the homologous-based repair (HDR) pathway works even if the DSB repair via the NHEJ pathway is weak, so that additional heterologous recombinant enzymes may be added to delete the target gene. There is a need for an alternative method to enhance the recombining ability of the strain or to inhibit the expression of the target gene using dCas9 (inactive Cas9) from which the nucleic acid cleavage activity of Cas9 has been removed instead of deleting the target gene. As a representative example, a system for controlling the target gene expression level of C. glutamicum using dCas9 (catalytically-dead Cas9) from which nuclease function is removed from Cas9 has been reported (Cleto et al., ACS Synthetic Biology 5). (5): 375-385, 2016).

In the gene editing technology of C. glutamicum , a method of deleting the gene Ncgl1221 by introducing Cas9 and sgRNA (CN105238806A) has recently been attempted. However, the deletion efficiency is considerably low to 5% and the editing efficiency needs to be increased.

The present inventors have found that if we use the results sought, combining the Cas9 and sgRNA and E. coli Rac prophage-derived recombinant enzyme RecT and ssODN want to build a system that can effectively engineered the genome of the bacteria utilize the CRISPR / Cas system, C Efficient deletion of Ncgl1221 (glutamate exporter), gabT (GABA aminotransferase), and gabP (GABA permease), which are involved in the terminal pathway of glutamicum 's γ-aminobutyric acid metabolism, and can rapidly and efficiently select recombinant bacteria. It was also confirmed that using the recombinant glutamate (Glutamate) over-producing strain prepared recombinant C. glutamicum to improve the ability to produce GABA, and completed the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information forming prior art that is known to those skilled in the art. You may not.

Summary of the Invention

It is an object of the present invention to provide a method for producing bacterial strains, wherein the mutant strain is selected using a CRISPR / Cas system in deleting a target gene with a recombinant enzyme (recombinase) and single-stranded oligodioxyribonucleic acid (ssODN). It is.

Another object of the present invention is also to provide a bacterial variant involved in gamma-aminobutyric acid (GABA) metabolism.

Still another object of the present invention is to provide a method for preparing gamma-aminobutyric acid (GABA) using the bacterial mutant strain.

In order to achieve the above object, the present invention comprises the steps of (a) transforming a normal bacteria with an enzyme expression vector expressing a recombinant enzyme (recombinase) having temperature sensitivity or antibiotic sensitivity; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Introducing a first vector expressing RNA (guide RNA) and having antibiotic sensitivity or temperature sensitivity to perform secondary transformation; And (e) removing the inserted enzyme expression vector and the first vector from the secondary transformed bacteria, wherein at least one of the enzyme expression vector and the first vector expresses a Cas protein. Provided are methods for preparing bacterial variant strains.

The present invention also relates to (a) normal bacteria as an enzyme expression vector having a first antibiotic selective marker and at the same time temperature sensitive or antibiotic resistance by the marker (i) expressing a recombinant enzyme (recombinase) Transforming; (b) preparing a competent cell of the transformed bacterium; (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), having a second antibiotic selective marker, and simultaneously transforming by introducing a first vector having antibiotic sensitivity or temperature sensitivity to the second antibiotic; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in a medium containing the first antibiotic and not containing the second antibiotic in a temperature sensitive condition. Selecting strains; (e) a (n) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) a guide RNA that complementarily binds to n (n is an integer of 2 or more) target genes Using the n-th vector, but the remaining components are substantially the same, repeating steps (b) to (d) n-1 times to prepare a multimutant strain having n target genes deleted; And (f) removing the inserted enzyme expression vector and the n-th vector from the multivariate strain prepared in step (e), wherein at least one of the enzyme expression vector, the first vector, and the n-th vector is It provides a method for producing bacterial strains, characterized in that the expression of the Cas protein.

The present invention also provides a bacterial mutant strain having glutamate overproducing ability prepared by the above method, and a method of preparing gamma-aminobutyric acid (GABA) using the same.

1 is a schematic of the pEKEx1 vector.

2 is a schematic of the pEKEx1-Cas9opt vector constructed to express Cas9 protein.

Figure 3 is a SDS-PAGE results confirming the expression of Cas9 protein in recombinant C. glutamicum introduced pEKEx1-Cas9opt.

4 is a schematic of the pUC19-sgRNA vector used as a template to amplify the sgRNA to be introduced into the pCG9-series vector and the pCG9ts-series vector.

5 is a schematic diagram illustrating a process of preparing a pCG9-series vector by combining sgRNAs that cut target genes with pEKEx1-Cas9opt vector.

6 is a schematic diagram of a pCG9-series vector for introducing cas9 gene and sgRNA gene into C. glutamicum .

7 is a schematic of the pEKEx1-sgRNA argR1 vector expressing sgRNA targeting the argR gene in C. glutamicum .

8 is a schematic of the pCG-argR1 vector.

9 is a schematic of the pEKEx1-dCas9 vector for expressing dCas9 protein in C. glutamicum .

10 is a schematic of the pdCG9-srgR1 vector expressing sgRNA and dCas9 protein targeting the argR gene.

Figure 11 is a graph showing the number of colonies formed when the pEKEx1-Cas9opt vector, pEKEx1-sgRNA argR1 vector, pCG9-argR1 vector, pdCG9-argR1 vector were introduced into C. glutamicum using electroporation.

12 is a schematic of the pTacCC1 vector.

13 is a schematic of the pTacCC1-recT vector expressing the recombinant enzyme RecT in C. glutamicum .

14 shows SDS-PAGE results confirming the expression of the recombinant enzyme RecT protein in recombinant C. glutamicum .

15 is a schematic diagram of the pTacCC1-HrT vector expressing the recombinant enzyme RecT with 6xHis bound to the N-terminus in C. glutamicum .

16 is a schematic diagram illustrating a mechanism for deleting target genes using Cas9 and sgRNA and ssODN and RecT.

17 is a schematic diagram illustrating an ssODN binding site targeting the ragR gene.

Figure 18 shows the (a) medium, (b) the thickness of the perforation cuvette, (c) the resistance value during the electroporation method, and (d) to increase the efficiency of the electroporation method for introducing nucleic acids into C. glutamicum . Cell recovery time after electroporation, and (e) a graph measuring the number of colonies formed by changing the type of strain.

19 is a schematic of the pEKTs1 vector showing temperature sensitivity when introduced into C. glutamicum .

20 is a schematic of the temperature sensitive vector pEKTs1-Cas9opt vector expressing Cas9 protein in C. glutamicum .

21 is a schematic of the temperature sensitive vector pCG9ts-series vector expressing Cas9 and sgRNA in C. glutamicum .

Figure 22 is a schematic diagram showing the metabolic pathways and genes involved in producing GABA from l-Glutamate and resolving GABA in recombinant C. glutamicum expressing glutamate decarboxylase GadB.

FIG. 23 is a schematic diagram illustrating a process of sequentially deleting a plurality of genes by repeating the introduction and removal of a temperature sensitive pCG9ts-series vector and ssODN in a C. glutamicum strain expressing a RecT recombinant enzyme.

24 is a schematic diagram of a pGA7 vector for introducing a glutamate decarboxylase gene gadB2 gene derived from Lactobacillus brevis ATCC 367 into C. glutamicum .

FIG. 25 is a graph of GABA concentrations produced after incubating a recombinant C. glutamicum strain with a pGA7 vector in which the Ncgl1221, gabT, and gabP genes were deleted in all combinations in a flask for 96 hours.

Figure 26 is a graph showing the OD, l-glutamage concentration, GABA concentration measured in culture in a flask for 96 hours using recombinant C. glutamicum .

Detailed Description of the Invention and Preferred Embodiments

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

In general, the CRISPR / Cas system works by forming a complex with Cas protein, which is a nucleic acid cleavage enzyme, and guide RNA. In the CRISPR / Cas9 system, single-chain guide RNAs (sgRNAs), which link crRNA and tracrRNA corresponding to guide RNAs, can also be complexed with Cas9 protein.

Since the guide sequence of the guide RNA has a sequence complementary to the target gene, it binds to the target gene and cleaves an adjacent site of the PAM motif (Catosome adjacent motif) by Cas9, a nucleic acid cleavage enzyme. To generate a double strand break (DSB), the target gene is deleted through the DNA repair process inherent in the host cell to repair the cleavage site.

The inventors have found that Ncgl1221 (glutamate exporter), gabT (GABA aminotransferase), and gabP ( Gta ) are involved in the end of the gamma-aminobutyric acid (GABA) metabolic pathway in C. glutamicum , a Corynebacterium genus . In order to delete the GABA permease gene, we tried to use the CRISPR / Cas system.

In a preliminary experiment for, C. The results of applying the CRISPR / Cas system for argR1 the glutamicum, in the case where, introducing pCG9-argR1 As shown in Figure 11 confirmed that the characteristics of the recombinant C. glutamicum colonies is not obtained . Typically, in most microorganisms, DSB repair through the NHEJ pathway is weak, but DSB repair through the homology-directed repair (HDR) pathway works and homologous recombination, particularly if the gene is deleted in C. glutamicum . (homologous recombination) is known (Nesvera et al., Applied Microbiology and Biotechnology , 90 (5): 1641-1645, 2011). Therefore, the preliminary results are C. glutamicum is CRISPR / Cas system by the DSB generated in the genome because it can not be restored through the NHEJ, C. glutamicum this was interpreted as both apoptosis. Therefore, it was confirmed that the simple deletion using the CRISPR / Cas system could not be utilized, and the development strategy was modified by deleting the target gene using the homology-based repair principle.

Homology-based repair principle Lee keombi engineering (Recombineering) to the target gene and having the sequence homologous to the combined system with the template DNA introduced into E. coli phage λ of the λ Red recombination system, E. coli system or Rac prophage RecT / ssODN, which is a recombinant enzyme, may be utilized.

RecT / ssODN binds to a lagging strand that has not yet replicated at the replication junction formed during chromosomal replication of the microorganism, where it acts as a primer for a new Okazaki fragment, acting to modify the target gene. (Murphy, EcoSal Plus , 2016. doi: 10.1128 / ecosalplus.ESP-0011-2015).

In connection with the present invention, the combination of the CRISPR / Cas system and the template DNA, and the λ Red system, which is a recombinant system of E. coli phage λ, was used to combine recombination, but C. glutamicum lacking the gene was not obtained. (Data not shown). On the other hand, Rac prophage-derived RecT and ssODN of E. coli were used together with the CRISPR / Cas system.

Therefore, the present inventors designed a system in which the CRISPR / Cas system and RecT / ssODN were combined to allow the gene deletion to proceed by RecT / ssODN, and the non-deleted strains were killed by the CRISPR / Cas system. Glutamate overproducing C. glutamicum mutant strains lacking Ncgl1221 (glutamate exporter), gabT (GABA aminotransferase), and gabP (GABA permease) resulted in rapid, convenient and high yield of multiple gene mutations. It was confirmed that the modified strain of the genus Corynebacterium ( Coynebacterium ) can be prepared, and completed the present invention

In the present invention, using the Cas protein, single chain guide RNA (sgRNA) constituting the CRISPR / Cas system, a recombinant enzyme (recombinase) represented by RecT and ssODN transformed into Corynebacterium (Coynebacterium) to prepare a mutant strain There is a characteristic. To this end, the present invention delivers a recombinant protein (recombinase) represented by Cas protein, guide RNA, RecT using the 'enzyme expression vector' and 'first vector'.

"Enzyme expression vector" in the present invention is a vector expressing a recombinant enzyme (recombinase) and / or Cas protein, the recombinant enzyme (recombinase) and Cas protein is characterized by being composed of the same vector or separate vectors.

The first vector in the present invention is a vector expressing Cas protein and / or guide RNA acting on the CRISPR / Cas system. When the Cas protein is not composed of the enzyme expression vector, the first vector is used. Can be inserted into a vector. In this case, the Cas protein is characterized by being composed of the same vector or a separate vector and guide RNA (guide RNA).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector and a first vector for deleting the first target gene, and maintaining the enzyme expression vector introduced into the cell without removing them. In this state, only the first vector may be removed, and then the second vector for deleting the second target gene may be introduced.

In a first aspect of the invention, there is provided a method comprising the steps of: (a) first transforming a normal bacterium into an enzyme expression vector having temperature sensitivity or antibiotic sensitivity and (i) expressing a recombinant enzyme (recombinase); (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Introducing a first vector expressing RNA (guide RNA) and having antibiotic sensitivity or temperature sensitivity to perform secondary transformation; And (e) removing the inserted enzyme expression vector and the first vector from the secondary transformed bacteria, wherein at least one of the enzyme expression vector and the first vector expresses a Cas protein. The present invention relates to a method for producing bacterial mutant strains.

In the present invention, step (e) may be characterized by culturing the secondary transformed bacteria of step (d) at 10 ℃ to 42 ℃, but is not limited thereto.

The first aspect of the present invention can be implemented in the following aspects.

In accordance with a first aspect, the present invention provides a method for producing a bacterium comprising the steps of: (a) transforming a normal bacterium into a temperature-sensitive, (i) transforming enzyme with an enzyme expression vector expressing a recombinant protein (recombinase) and (ii) Cas protein; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) a single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Expressing RNA (guide RNA), introducing a first vector having an antibiotic selective marker and having antibiotic sensitivity, and performing secondary transformation; (d) culturing the secondary transformed bacteria in a medium to which an antibiotic corresponding to the antibiotic selective marker is added to primary selection of the mutant strains; And (e) removing the enzyme expression vector and the first vector inserted from the first selected strain strain.

In a second aspect, the present invention provides a method for preparing a bacterium, comprising: (a) transforming a bacterium into an enzyme-sensing vector expressing antibiotic sensitivity and (i) recombinase and (ii) a Cas protein; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) a single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Introducing a first vector expressing RNA (guide RNA) and having temperature sensitivity to perform secondary transformation; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In a third aspect, the present invention provides an antimicrobial susceptibility to the antibiotics, while (a) the bacteria has a first antibiotic selective marker, and (i) the recombinant enzyme and (ii) the Cas protein. First transforming with an expressing enzyme expression vector; (b) preparing a competent cell of the first transformed Corynebacterium sp. strain obtained in step (a); (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to the target gene in the recipient cell obtained in step (b), and (ii) Introducing a Cas protein and a guide RNA, introducing a first vector having a second antibiotic selective marker and having an antibiotic sensitivity to the antibiotic; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In a fourth aspect, the present invention provides a method for producing a bacterium comprising: (a) transforming a bacterium into a temperature-sensitive, (i) transforming enzyme with an enzyme expression vector expressing a recombinant protein (i) recombinase and (ii) Cas protein; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) a single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Introducing a first vector expressing RNA (guide RNA) and having temperature sensitivity to perform secondary transformation; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In accordance with a fifth aspect, the present invention provides a method for producing a bacterium comprising: (a) transforming a bacteria into an enzyme expression vector having temperature sensitivity and expressing a recombinant enzyme; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to the target gene in the recipient cell obtained in step (b), and (ii) Introducing a first vector expressing Cas protein and guide RNA and having antibiotic sensitivity to perform secondary transformation; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In a sixth aspect, the present invention provides a method for preparing a bacterium comprising: (a) first transforming a bacterium into an enzyme expression vector having antibiotic sensitivity and expressing a recombinant enzyme; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to the target gene in the recipient cell obtained in step (b), and (ii) Introducing a Cas vector and a guide RNA, and performing a second transformation by introducing a first vector having a temperature sensitivity; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In accordance with a seventh aspect of the present invention, (a) the bacterium has a first antibiotic selective marker and at the same time has an antibiotic sensitivity to the antibiotic and a primary transformation with an enzyme expression vector expressing a recombinant enzyme (recombinase). Converting; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to the target gene in the recipient cell obtained in step (b), and (ii) Introducing a first protein having a second antibiotic selective marker expressing a Cas protein and guide RNA and having an antibiotic sensitivity to the antibiotic; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In an eighth aspect, the present invention provides a method for preparing a bacterium comprising the steps of: (a) transforming a bacteria into an enzyme expression vector expressing a recombinant enzyme (recombinase) having a first antibiotic selective marker and temperature sensitivity; (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to the target gene in the recipient cell obtained in step (b), and (ii) Introducing a first vector having a temperature sensitivity and expressing a Cas protein and a guide RNA and having a second antibiotic selective marker; And (d) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacterium.

In the present invention, the step of removing the inserted enzyme expression vector and the first vector may be characterized by culturing the secondary transformed bacteria at 10 ℃ to 42 ℃, but is not limited thereto.

In the present invention, the step of removing the inserted enzyme expression vector and the first vector may be characterized by culturing the secondary transformed bacteria at 10 ℃ to 42 ℃, but is not limited thereto.

In the production method of the present invention, the deletion of the target gene is carried out by recombinant enzyme (recombinase) and ssODN, selection of bacterial strains to the production of double stranded break (DSB) by the CRISPR / Cas system and the addition of antibiotics By cell death induction.

In the present invention, the recombinase is selected from the group consisting of RecT, RecET system, Bet, and λ Red system, but is not limited thereto, and the recombinant enzyme in the present invention is a single-stranded oligodioxyribonucleic acid (single). It can bind or act on stranded oligodeoxyribonucleic acid (ssODN) to delete or insert genes.

The term 'enzyme expression vector' in the present invention is a vector expressing Cas protein, which is a recombinant enzyme (recombinase) and / or a nucleic acid cleavage enzyme, not a guide RNA, and is characterized in that it is used when transforming a strain for the first time. have. The enzyme expression vector may be configured to simultaneously or separately express a recombinant enzyme (recombinase), a recombinant enzyme (recombinase) and Cas protein in one vector.

Single-stranded oligodeoxyribonucleic acid (ssODN) of the present invention is characterized in that it is inserted into the strain directly in the deoxyribonucleic acid state, rather than expression by a vector, it may have a length of 80 to 100 nucleotides, The present invention is not limited thereto and can be easily manufactured by those skilled in the art by requesting a manufacturing company based on the target gene information.

Single-stranded olideoxyribonucleic acid (ssODN) of the present invention is sequenced to bind to the lagging strand or leading strand when the chromosome of Corynebacterium is replicated. You can choose. The single-stranded oligodeoxyribonucleic acid (ssODN) is composed of a 5 'homology arm and a 3' homology arm, and may bind complementarily to a target gene. In addition, regions where the homologous sites of the single-stranded oligodeoxyribonucleic acid (ssODN) in the target gene bind to each other may be spaced apart from each other, in which case, each homologous region of the ssODN in the target gene sequence binds. The non-inner region may form a loop structure.

For example, in the present invention, the 5 'homology arm and the 3' homology arm of the single-stranded oligodeoxyribonucleic acid (ssODN) having a length of 80 nucleotides are 40 nucleotides, respectively. It was prepared to have a length, the inner sequence that is not bound to the homologous region in the target gene sequence, that is, the loop region may have a length of 100 to 400 nucleotides, but is not limited thereto.

When the single-stranded oligodeoxyribonucleic acid (ssODN) consists of only 5 'homology arm and 3' homology arm, the binding to the target gene removes the loop region, When inserted into a strain, the target gene is deleted, and the foreign gene or foreign gene regulator is placed in a region between each homologous region, that is, a loop region, and when inserted into the strain, the foreign gene or regulator Can be introduced, in which case overexpression of the target gene is possible.

The single-stranded oligodeoxyribonucleic acid (ssODN) of the present invention is characterized by introducing into a strain directly in the ssODN state without using a vector when inserted into the strain.

In the present invention, the guide RNA (guide RNA, gRNA) includes a guide sequence having a sequence complementary to the sequence of the target gene.

In the present invention, the guide RNA (i) is a crRNA comprising a guide sequence (i) complementary to the sequence of the target gene, (ii) a guide complementary to the sequence of the target gene It may be a dual RNA comprising a crRNA and a tracrRNA comprising a guide sequence, or (iii) a single-strand guide RNA (sgRNA) consisting of a single strand of the crRNA and tracrRNA. .

The guide sequence of the guide RNA of the present invention is not limited to 20 nucleotides in length, and has a sequence that complementarily binds to a region to be deleted in the sequence of a target gene. . The PAM sequence (protospacer adjacent motif) is present in the complementary sequence immediately adjacent to the 3 'end of the sequence complementary to the guide sequence on the target gene.

For guide RNA, the guide sequence is sgRNA Designer (Doench et al., Nature Biotechnology 34 (2): 184-191, 2016); E-CRISP (http://www.e-crisp.org/E-CRISP/Heigwar et al., Nature Methods 11 (2): 122-123, 2014); Benchling (https://benchling.com); sgRNA scorer 2.0 (https://crispr.med.harvard.edu/sgRNAScorerV2; Chari et al., ACS Synthetic Biology, 2017.doi: 10.1021 / acssynbio.6b00343), CRISPy-web (Blin et al., Synthetic and Systems Biotechnology , 1 (2): 118-121, 2016) can be easily selected by those skilled in the art.

In the present invention, the guide RNA is composed of crRNA, or crRNA and tracrRNA. The crRNA and / or tracrRNA, except for the guide sequence region, is characterized by the formation of an RNA scaffold to which the Cas protein binds. The Cas protein is an essential nucleic acid cleavage enzyme in the CRISPR / Cas system and generates a double strand break (DSB). CRISPR / Cas systems can be classified into CRISR / Cas type I, CRISPR / Cas type II, CRISPR / Cas type III, CRISPR / Cas type IV, CRISPR / Cas type V, and CRISPR / Cas type VI, Cas protein may be Cas3, Cas9, Cpf1, Cas6, C2c2 and the like.

Accordingly, the Cas protein of the present invention may be a nucleic acid cleavage enzyme selected from Cas3, Cas9, Cpf1, Cas6, or C2c2, and more preferably, it is Cas9 of CRISPR / Cas type II.

Gene and protein information of Cas protein can be obtained from GenBank of the National Center for Biotechnology Information (NCBI).

In the present invention, the Cas protein, for example, Cas9 (CRISPR associated protein 9), binds to and forms a complex with the RNA scaffold portion of the guide RNA, recognizes the PAM sequence present in the sequence of the target gene, and targets it. The gene guides the CRISPR / Cas system, identifies the target gene through complementary binding between the guide sequence of the guide RNA and the target gene, and finally exhibits nucleic acid cleavage activity by the HNH and RuvC domains, which are active sites.

It is known that if one mutation is introduced into a specific amino acid in two domains involved in nucleotide cleavage among several domains of Cas9, nucleic acid cleavage ability is lost. For example, in case of Cas9 of Streptococcus pyogenes , amino acid 10 and 840 are transformed into alanine (D10A and H840A), respectively, to lose DNA cleavage ability, commonly called dCas9. It is also known that Cas9 enzyme, in which the amino acid of any of No. 10 and No. 840 is changed to alanine (D10A or H840A), has a nickase activity that cleaves only one of the double-stranded bases.

In the present invention, when the Cas protein and the guide RNA (guide RNA) is introduced into the strain at the same time can be configured to be expressed by a single vector or different vectors, the expressed Cas protein and guide RNA (guide RNA) in the strain Once expressed, it can spontaneously form a complex. The complex may be used interchangeably with terms such as' CRISPR / Cas system ',' CRISPR complex ', Cas9-gRNA complex', 'CRISPR / Cas complex', and 'Cas protein complex'.

Cas protein in the present invention, preferably Cas9 is Corynebacter ( Syneella ), Sutterella , Legionella ( Legionella ), Treponema ( Treponema ), Pilifactor ( Filifactor ), Eubacterium ( Eubacterium ), Streptococcus (Streptococcus), Lactobacillus bacteria (Lactobacillus), Miko plasma (Mycoplasma), bakteo Lloyd (Bacteroides), flaviviruses Plastic non carambola (Flaviivola), Flavobacterium (Flavobacterium), azo RY rilrum (Azospirillum), glucoside or Gluconacetobacter , Neisseria , Roseburia , Parvibaculum , Staphylococcus , Nitratifractor , Corynebacterium , and Campylobacter (Campylobacter) can be derived from a microorganism containing the erroneous log in (ortholog) of the Cas protein is selected from the group consisting of, preferably Streptococcus It may be isolated from Streptococcus pyogenes , or may be a recombinant protein, but is not limited thereto.

Cas protein of the present invention is characterized in that the codon optimization, so that it can be smoothly expressed in the transformed strain.

In the present invention, the recombinant enzyme (Recombinase) and Cas protein can be inserted into the vector by improving the enzyme gene so that hexahistidine is artificially included in the amino acid sequence to improve the expression ability, which is a technique well known to those skilled in the art .

In the present invention, the guide RNA and the Cas protein form a complex to represent a gene editing function, and the Cas protein is identical if the sequence of the RNA scaffold in the guide RNA is the same regardless of the guide sequence. It can be combined with guide RNAs that recognize different targets. Therefore, to delete one or more genes, the strain is transformed to simultaneously express one or more guide RNAs having a guide sequence complementary to a different target gene and a gene expressing one Cas protein. Multivariate editing effects can be induced.

In the present invention, each homologous arm of ssODN binds to a target gene having a corresponding sequence, and homologous to single-stranded oligodeoxyribonucleic acid (ssODN) in the target gene. the inner sequence at the 3 'end and 5' end of the target gene that do not bind to arm) forms a loop (FIG. 17), and the guide RNA binds to the sequence on the loop region. There is this. In this case, the 'sequence on the loop region' may be a sequence of the strand to which the ssODN is bound, or a sequence complementary thereto.

As used herein, the term “homology” refers to a degree similar to the amino acid sequence of a protein or a nucleotide sequence encoding the same.

In the preparation method of the present invention, one guide RNA and two or more ssODNs can be used to delete two or more target genes simultaneously. In this case, each ssODN is terminated at each end of each region to be deleted. Simultaneously bind to the outside, the guide RNA selects a sequence to complementarily bind to the sequence of the loop region formed when ssODN binds to one of the target genes to which ssODN binds. There is a characteristic.

In the production method of the present invention, the single-stranded oligodeoxyribonucleic acid (single-stranded oligodexoyribonucleic acid, ssODN) is characterized in that it is inserted into the strain as the number of target genes. On the other hand, since guide RNAs cause an effect that the strain is killed by cleavage regardless of the deletion of the target gene, the number of target genes to be deleted There is no need to introduce the strain in the same number. For example, in the present invention, when the gapT and gapP are deleted at the same time, simultaneous ssODN and guide RNA having a binding capacity to one of the two genes can be easily co-deleted even when introduced into the strain. It was confirmed in Example 4-2. However, in order to have such co-deletion effect, there is a limit that target genes of co-deletion should be adjacent to each other. In this case, the 'adjacent' range is 100 kb or less, preferably 10 kb or less, and more preferably 5 kb or less.

In the production method of the present invention, the recipient cell (competent cell) is a medium containing the transgenic strain of step (a) Tween-20, DL-threonine (Theronine), isoniazid and glycine (Glycine) It is characterized in that the OD ₆₀₀ is produced by culturing to the range of 0.3 to 0.5. The culture is a pretreatment for further improving the transformation efficiency of the vector expressing ssODN, Cas protein and guide RNA, or ssODN and guide RNA to bacteria in a later step, more specifically Ruan et al., 2015 (Biotechnology Lett , 37: 2445, 2015).

The bacterium of the present invention may be a strain of the genus Corynebacterium , preferably C. glutamicum ATCC 13032, but is not limited thereto.

The term 'transformation' in the present invention may be used in the same sense as 'insertion' or 'introduction' of a gene, and the DNA is introduced into a host so that the DNA can be reproduced as an extrachromosomal factor or by chromosomal integration. It means to be. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. These methods include electroporation, precipitation using calcium phosphate (CaPO ₄ ) or calcium chloride (CaCl ₂ ), microinjection, polyethylene glycol (PEG), cationic liposomes, DEAE (diethylaminoethyl) dextran Method, and lithium acetate-DMSO method, and the like, but is not limited thereto. Preferably, the method is characterized in that it is prepared by electroporation.

In the present invention, the term 'electroporation' refers to inserting a gene into a microorganism by using a principle that DNA molecules are introduced into a cell while a cell membrane through which DNA molecules pass through is opened by an electric pulse in an organism having a cell membrane. That's how.

Electroporation (electroporation) in the present invention is characterized by applying an electric field of 10 to 20 kv / cm for an impact time of 3 to 5 ms, using a 1mm cuvette, but is not limited thereto.

The term 'selective marker' in the present invention may be used interchangeably with terms such as 'selective marker' and 'selective marker', and expresses genes that impart characteristics that can be selected by chemical methods. As the nucleotide sequence, all genes capable of distinguishing the transformed cells from the non-transformed cells are applicable thereto, and may be antibiotic resistance genes, but are not limited thereto.

In the present invention, the 'selection marker' is an antibiotic resistance gene, and the antibiotic may be kanamycin, spectamyomycin, streptomycin, chloramphenicol, or apramycin. It is not limited thereto.

The term 'deletion' in the present invention refers to inhibiting the activity of a gene, and by this process, the gene is said to be in a 'deleted' state. Inhibition of the activity may comprise i) inactivating the expression product of the relevant gene by appropriate methods such that it is not expressed or the expression product loses its function, ii) inhibiting expression of the related gene, iii) at least the related gene It may be to remove a part. Preferably, deletion of the gene essentially inhibits the gene, which may be replaced with a selection marker gene that facilitates identification, isolation and purification of the improved strains according to the invention.

The term 'vector' in the present invention can be used in combination with 'plasmid' and is a cyclic DNA that can be replicated while being present independently of the main chromosome in a microorganism. In general, a vector is the origin of replication (ori) to maintain a vector in a microorganism, a selectable marker gene for screening microorganisms such as antibiotic resistance genes, and multicloning for cloning foreign genes. Multi-cloning site (MCS).

As used herein, the term 'shuttle vector' generally includes a vector that can be maintained in a plurality of strains. C. glutamicum-The E. coli shuttle vector contains both the origin of replication of C. glutamicum and the origin of replication of E. coli . By using the shuttle vector, a desired trait can be easily introduced into the target strain.

The target gene is deleted from the mutant strains of the genus Corynebacterium , and then the inserted foreign vector must be removed to be used as an industrial strain. In addition, in order to develop an optimal strain, in most cases, multiple genes must be deleted. In order to delete one target gene, the first introduced vector must be removed. As a result, it is essential to easily remove the previously inserted vector from the genus Corynebacterium .

Therefore, in the present invention, the inserted foreign vector was artificially manufactured to have characteristics of 'temperature sensitivity' or 'antibiotic sensitivity' so that the inserted foreign vector can be removed in a strain under specific culture conditions.

In addition, the vector produced in the present invention has one antibiotic selective marker.

The 'temperature sensitive' vector in the present invention is a vector artificially engineered to have a single base mutation on the Rep protein, which has a pBL1 origin of replication and acts on it. The Rep protein is involved in the replication process of the pBL1 origin of replication (ori). When a single nucleotide mutation (C → T) is introduced into the Rep protein gene, amino acid substitution of P47S occurs, and the strain containing the vector is absent. When the culture temperature rises above 34 ° C. in antibiotic medium, the pBL1 replication origin (ori) is known to lose its function (Nakamura et al., Plasmid 56 (3): 179-186, 2006). Therefore, bacterial strains prepared using the vector having the origin of replication are stable when cultured at 10 ° C. to 30 ° C. using a medium to which antibiotics corresponding to antibiotic selective markers present in the vector are added. However, when incubated at 34 ° C. or higher, typically 37 ° C. to 42 ° C. or lower, in an antibiotic-free medium, the vector is not maintained. Therefore, by controlling the presence of antibiotics and the incubation temperature, the vector can be maintained or removed. have.

The antibiotic susceptibility vector of the present invention is a vector of the pTacCC1 family in the process of developing the present technology, when the concentration of antibiotics corresponding to the resistance gene inserted into the vector, that is, the antibiotic selective marker, is lost. It was found that these features were used, and this feature was used for vector removal.

In addition, the 'antibiotic susceptibility' vector and the 'antibiotic susceptibility' vector are vectors inserted with different resistance genes. When the antibiotic concentration is lowered and cultured at 10 ° C. to 42 ° C., only the vector is lost. There is this.

Thus, in the present invention, the 'temperature sensitive' vector is artificially engineered to have a single base mutation on the Rep protein which has the origin of pBL1 replication and acts on it, and is a vector having an antibiotic selective marker.

In addition, the 'antibiotic sensitive' vector in the present invention is an antibiotic selective marker, that is, a vector having an antibiotic resistance gene and having a pCC1 origin of replication, in particular acting on 'first antibiotic susceptibility' and 'second antibiotic susceptibility'. Antibiotics have different characteristics.

The antibiotic in the vector may be kanamycin (kanamycin), spectinomycin (spectinomycin), streptomycin (streptomycin), chloramphenicol (chloramphenicol), or apramycin (apramycin), but is not limited thereto.

In the method for producing a variant strain of the present invention, an enzyme expression vector expressing a recombinant enzyme (recombinase) and / or Cas protein can be prepared as a vector having the characteristics of temperature sensitivity or antibiotic sensitivity.

In the method for producing a variant strain of the present invention, the first vector expressing the Cas protein and / or sgRNA can be prepared as a vector having characteristics of temperature sensitivity or antibiotic sensitivity.

By way of example, with respect to the first aspect of the invention, antibiotics in a primary transformed strain using a temperature sensitive vector (pEKTs-series, such as a pBL1ts replication origin based vector) expressing a recombinase and Cas protein, After secondary transformation using a guide RNA expression vector of sensitivity (based on pCC1 replication origin, such as pTacCC1-series), (a) the temperature sensitive vector and the antibiotic sensitivity at 10 ° C to 34 ° C. When cultured using a medium containing two antibiotics corresponding to the antibiotic selective marker of the vector ', both vectors remain in the strain, while (b) antibiotic selection of the antibiotic-sensitizing vector is selected. When antibiotics are not added to the marker (selective marker) but incubated using a medium to which antibiotics are added to the antibiotic selective marker of the 'temperature sensitive vector'. Sensitive vector RNA guide (guide RNA) expression vector is characterized in that is removed in the strain is maintained only recombinase (recombinse), and Cas protein expression vector.

By way of example, in relation to a second aspect of the invention, a temperature is transformed into a strain transformed primarily using an antibiotic sensitive vector (pCC1 replication origin based vector such as pTacCC1-series) that expresses a recombinant enzyme (Recombinase) and Cas protein. Secondary transformation using a guide RNA expression vector of a sensitivity vector (pBL1ts replication origin-based vector, such as pCG9ts-series), followed by (a) the 'temperature sensitive vector' and the ' When culturing with the addition of two antibiotics corresponding to the antibiotic selective marker of an antibiotic sensitive vector, both vectors remain in the strain, while (b) the temperature sensitive vector at 34 ° C to 42 ° C. Using a medium in which no antibiotic was added to the antibiotic selective marker of 'but only the antibiotic was added to the antibiotic selective marker of the antibiotic sensitivity vector' When cultured, the temperature sensitive vector, that is, a guide RNA expression vector, is removed from the strain and is characterized by maintaining only an enzyme expression vector expressing a recombinant enzyme (Recombinse) and Cas protein.

Illustratively, in connection with a third aspect of the present invention, a strain transformed primary using a first antibiotic-sensitized vector (pCC1 replication origin based vector such as pTacCC1-series) expressing a recombinant enzyme (Recombinase) and Cas protein Secondary transformation using a guide RNA expression vector of a second antibiotic-sensitized vector (pCC1 replication origin-based vector, such as pTacCC1-series), followed by (a) 'first antibiotic-sensitive vector' and 'agent' In the case of incubation with two antibiotics (hereinafter referred to as 'first antibiotic' and 'second antibiotic') corresponding to the antibiotic selective marker of the antibiotic-sensitizing vector, both vectors are strains. (B) when cultured in a medium containing only 'first antibiotic' but not 'second antibiotic', the guide RNA expression vector is removed in the strain and the recombinant enzyme (recombinse) and Cas protein It is characterized in that keeping only small expression vector.

By way of example, in connection with a fourth aspect of the present invention, a temperature sensitive vector (pEKTs-series, such as pBLTs-series, origin of replication) that expresses a recombinant protein and a Cas protein and has an antibiotic selective marker for the first antibiotic Guide RNA expression vector of a temperature-sensitive vector (pEKTs-series, such as pBL1ts replication origin-based vector) having an antibiotic selective marker for a second antibiotic, in a strain transformed firstly using the vector). After the second transformation using (a) when cultured in a medium in which the first antibiotic and the second antibiotic were added together at 10 ° C. to 34 ° C., both vectors were maintained in the strain, (b) Both vectors may be removed when cultured in an antibiotic free medium at 34 ° C. to 42 ° C., but (c) medium at 34 ° C. to 42 ° C. where the first antibiotic is added and does not contain the second antibiotic. When cultured in the strain can be selected with only the enzyme expression vector.

By way of example, in connection with a fifth aspect of the present invention, antibiotic sensitivity (pTacCC1-to-pTacCC1- After the second transformation using a Cas protein / guide RNA expression vector of pCC1 origin of replication) such as series, and (a) the temperature sensitive vector and the antibiotic sensitive vector at 10 ° C. to 34 ° C. When cultured using a medium to which antibiotics were added, both vectors were maintained in the strain, while (b) no antibiotics corresponding to the 'antibiotic sensitivity vector' were added to the 'temperature sensitive vector'. When cultured using a medium with the corresponding antibiotics, the antibiotic sensitive vector, ie, the guide RNA expression vector, is removed in the strain and the recombinant enzyme and Cas protein Vector is characterized in that only the maintenance.

In addition, as shown in the example of FIG. 23, illustratively, in connection with the sixth aspect of the present invention, an antibiotic-sensitive vector (pCC1 replication origin-based vector such as pTacCC1-series) expressing a recombinant enzyme (recombinase) is used. The first transformed strain was subjected to secondary transformation using a Cas9 / guide RNA expression vector of a temperature sensitive vector (pBL1ts replication origin-based vector such as pCG9ts-series), followed by (a) 30 ° C to 34 ° C. In the case of culturing by adding antibiotics corresponding to the 'temperature sensitive vector' and the 'antibiotic sensitive vector', both vectors are maintained in the strain, while (b) the 'temperature sensitive vector' at 34 ° C to 42 ° C. When no antibiotics were added but cultured using a medium with antibiotics corresponding to the 'antibiotic sensitive vector', the temperature sensitive vector, ie, the guide RNA expression vector, was expressed in the strain. Document removal, and is characterized in that the enzyme maintained the expression vector only expressing the recombinase (recombinse), and Cas protein.

By way of example, in connection with a seventh aspect of the present invention, a second transformed strain may be subjected to a first transformed strain using a first antibiotic-sensitized vector (pCC1 replication origin-based vector such as pTacCC1-series, etc.) expressing a recombinase. After secondary transformation using a Cas protein / guide RNA expression vector of an antibiotic sensitive vector (pCC1 replication origin-based vector, such as pTacCC1-series), (a) 'first antibiotic sensitivity' and 'second When cultured with antibiotics corresponding to antibiotic susceptibility (hereinafter referred to as 'first antibiotic' and 'second antibiotic', respectively, both vectors remain in the strain, while (b) 'first antibiotic' When cultured using a medium containing only 'second antibiotic', the guide RNA expression vector is removed in the strain and the enzyme expression vector expresses the recombinant enzyme and Cas protein. Only features are maintained.

By way of example, in connection with an eighth aspect of the present invention, a temperature sensitive vector (pEKTs) is used for a strain transformed first using a temperature sensitive vector (pEKTs-series, such as a pBL1ts replication origin-based vector) expressing a recombinase. After a second transformation using a Cas protein / guide RNA expression vector of pBL1ts replication origin-based vector), such as -series, and the like, (a) the recombinant enzyme and Cas protein expression vector at 10 ° C to 34 ° C. And both vectors are maintained in the strain when cultured with an antibiotic corresponding to the 'guide RNA expression vector', (b) the recombinant enzyme and Cas protein expression vector and the protein at 34 ° C to 42 ° C. When cultured in a medium that does not contain an antibiotic corresponding to the 'guide RNA expression vector' both vectors can be removed, but corresponding to the 'recombinant enzyme and Cas protein expression vector' at 34 ℃ to 42 ℃ Antibiotics may be cultured in the case of antibiotics are not included medium corresponding to "guide RNA expression vectors, the strains having only the enzyme expression vectors may be selected but included.

In this case, the 'first antibiotic' and the 'second antibiotic' are characterized by being different antibiotics.

In the production method of the present invention, when one target gene is deleted, a mutant strain may be obtained by simultaneously introducing one ssODN and one guide RNA having a binding capacity complementary to the target gene into a strain. Can be.

In the production method of the present invention, the target gene is characterized in that two or more, the two or more target genes can be deleted simultaneously or sequentially. In the present invention, in order to delete two or more target genes simultaneously, two or more ssODNs having complementary binding capacities to two or more target genes and guide RNAs having complementary binding capacities to one of the genes to which the ssODNs bind are used. Multiple genes can be deleted. In this case, two or more ssODNs and one guide RNA are characterized in that they are introduced into the strain at the same time, and in the simultaneous deletion, the distance between different adjacent genes to which two or more ssODNs simultaneously bind is 100 kb or less, preferably May be 10 kb or less, more preferably 3 kb or less, but is not limited thereto.

In the preparation method of the present invention, when deleting two or more target genes sequentially, the culture conditions are adjusted to maintain only the enzyme expression vector, which is a recombinant enzyme (recombinase) or an expression vector of the recombinant enzyme and Cas protein, and the first target. The first vector expressing ssODN and guide RNA, or ssODN and Cas protein / guide RNA, for the gene was removed by the temperature sensitive or antibiotic sensitive culture conditions, followed by By repeating the process of additionally introducing a vector expressing ssODN and guide RNA or ssODN and Cas protein / guide RNA, multivariate strains can be obtained.

In the present invention, using a Cas protein constituting the CRISPR / Cas system, guide RNA (guide RNA), recombinant enzyme (recombinase) represented by RecT and ssODN transformed into Corynebacterium (Coynebacterium) to prepare a multivariate strain There is a characteristic. To this end, the present invention delivers a recombinant protein (recombinase) represented by Cas protein, guide RNA, RecT using the 'enzyme expression vector' and 'first vector'.

"Enzyme expression vector" in the present invention is a vector expressing a recombinant enzyme (recombinase) and / or Cas protein, the recombinant enzyme (recombinase) and Cas protein is characterized by being composed of the same vector or separate vectors.

The first vector in the present invention is a vector expressing Cas protein and / or guide RNA acting on the CRISPR / Cas system. When the Cas protein is not composed of the enzyme expression vector, the first vector is used. Can be inserted into a vector. In this case, the Cas protein is characterized by being composed of the same vector or a separate vector and guide RNA (guide RNA).

The first antibiotic sensitivity vector in the present invention has a first antibiotic selective marker and at the same time has an antibiotic sensitivity to the first antibiotic.

In the present invention, the 'second antibiotic sensitivity vector' has a characteristic of having antibiotic sensitivity to the second antibiotic while having a second antibiotic selective marker.

Considering only the vector to be introduced into the transformation in the present invention, the method for deleting one or more target genes to prepare a mutant strain using an enzyme expression vector and a first vector for deleting the first target gene, and introduced into the cell In the state where the enzyme expression vector is maintained without being removed, only the first vector is removed, and then, the second vector for deleting the second target gene is introduced again, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, in the present invention, when deleting one target gene, only the first vector may be used, and when deleting two target genes, the first vector and the second vector may be used, and include one vector removal step. When n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included.

In the present invention, in order to delete the target gene, ssODN and the vector must be introduced into the strain together. In particular, since ssODN is introduced as a nucleic acid molecule rather than a vector, it is diluted during cell division and removed naturally in the cell without special treatment. There is this.

In a second aspect, the present invention provides an enzyme that (a) a normal bacterium having a first antibiotic selective marker, and at the same time having temperature sensitivity or antibiotic resistance by the marker, and (i) expressing a recombinase. Transforming with an expression vector; (b) preparing a competent cell of the transformed bacterium; (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), having a second antibiotic selective marker, and simultaneously transforming by introducing a first vector having antibiotic sensitivity or temperature sensitivity to the second antibiotic; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in a medium containing the first antibiotic and not containing the second antibiotic in a temperature sensitive condition. Selecting strains; (e) a (n) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) a guide RNA that complementarily binds to n (n is an integer of 2 or more) target genes Using the n-th vector, but the remaining components are substantially the same, repeating steps (b) to (d) n-1 times to prepare a multimutant strain having n target genes deleted; And (f) removing the inserted enzyme expression vector and the n-th vector from the multivariate strain prepared in step (e), wherein at least one of the enzyme expression vector, the first vector, and the n-th vector is It relates to a method for producing bacterial mutant strains, which expresses Cas protein.

The second aspect may be implemented in the following aspects.

In accordance with a ninth aspect of the present invention, an enzyme expression vector expressing (a) a bacterium having a first antibiotic selective marker and temperature sensitivity and (i) a recombinase and (ii) a Cas protein is expressed. Transforming with; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), having a second antibiotic selective marker, and simultaneously transforming the first vector by introducing a first vector having antibiotic sensitivity to the second antibiotic; (d) A mutant strain in which the first target gene is deleted by culturing the first transformed strain in a medium containing the first antibiotic and not containing the second antibiotic and removing the inserted first vector. Screening; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, And, the remaining components are substantially the same to repeat the steps (b) to (d) n-1 times to prepare a multivariant strain having the deletion of n target genes; And (f) removing the enzyme expression vector and the n-th vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector, ssODN and a first vector for deleting the first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, in consideration of only the vector introduced into the transformation, only the first vector can be used when deleting one target gene in the present invention, and the first vector and the second when the two target genes are deleted. The vector is used, and includes one vector removal step. When n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. It features.

In accordance with a tenth aspect, the present invention provides an antimicrobial susceptibility to (a) a bacterium having a first antibiotic selective marker and at the same time having an antibiotic sensitivity to the first antibiotic (i) a recombinase and (ii) a Cas protein. Transforming with an enzyme expression vector expressing a; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), introducing a first vector having a second antibiotic selective marker and having a temperature sensitivity, and performing primary transformation; (d) A mutant strain in which the first target gene is deleted by culturing the first transformed strain in a medium containing the first antibiotic and not containing the second antibiotic and removing the inserted first vector. Screening; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, Repeating steps (b) to (d) n-1 times to prepare a multivariant strain in which n genes are deleted; And (f) removing the enzyme expression vector and the n-th vector inserted into the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector, ssODN and a first vector for deleting the first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, in consideration of only the vector introduced into the transformation, only the first vector can be used when deleting one target gene in the present invention, and the first vector and the second when the two target genes are deleted. The vector is used, and includes one vector removal step. When n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. It features.

In accordance with an eleventh aspect, the present invention provides (a) a bacterium having a first antibiotic selective marker and at the same time an antibiotic sensitivity to the first antibiotic, (i) a recombinase and (ii) a Cas protein. Transforming with an enzyme expression vector expressing a; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), introducing a first vector having a second antibiotic selective marker and having an antibiotic sensitivity to the second antibiotic, and performing primary transformation; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in the medium containing the first antibiotic and not containing the second antibiotic and in a temperature sensitive condition. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, And, the remaining components are substantially the same to repeat the steps (b) to (d) n-1 times to prepare a multivariant strain having the deletion of n target genes; And (f) removing the enzyme expression vector and the n-th vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector and a first vector for deleting the first target gene, and maintaining the enzyme expression vector introduced into the cell without removing them. In this state, only the first vector is removed, and then, a multivariate strain is produced by introducing a second vector for deleting the second target gene. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, when considering only the vector to be transformed, only the first vector can be used when deleting one target gene in the present invention, the first vector and the second vector is deleted when two target genes are deleted. And a vector removal step, wherein n (n is an integer of 2 or more), and when the target gene is deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. do.

In accordance with a twelfth aspect of the present invention, an enzyme expression vector expressing (a) a bacterium having a first antibiotic selective marker and temperature sensitivity and (i) a recombinase and (ii) a Cas protein is expressed. Transforming with; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), introducing a first vector having a second antibiotic selective marker and having a temperature sensitivity, and performing primary transformation; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in the medium containing the first antibiotic and not containing the second antibiotic and in a temperature sensitive condition. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, (B) to (d), repeating the steps n-1 times to prepare the multivariant strain of Corynebacterium, which has deleted n target genes; And (f) removing the enzyme expression vector and the n-th vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector, ssODN and a first vector for deleting the first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, when considering only the vector to be transformed, only the first vector can be used when deleting one target gene in the present invention, the first vector and the second vector is deleted when two target genes are deleted. And a vector removal step, wherein n (n is an integer of 2 or more), and when the target gene is deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. do.

In a thirteenth aspect, the present invention provides a method for preparing a bacterium comprising: (a) transforming a bacterium into an enzyme expression vector having a first antibiotic selective marker and having temperature sensitivity and expressing a recombinant enzyme; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) Cas, complementarily binding to a first target gene to a competent cell obtained in step (b). Expressing a protein and a guide RNA, introducing a first vector having a second antibiotic selective marker and having an antibiotic sensitivity to the second antibiotic; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in the medium containing the first antibiotic and not containing the second antibiotic and in a temperature sensitive condition. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, (B) to (d), repeating steps n-1, to prepare a multivariant strain of Corynebacterium , in which n target genes are deleted; And (f) removing the enzyme expression vector and the first vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector, ssODN and a first vector for deleting the first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, in consideration of only the vector introduced into the transformation, only the first vector can be used when deleting one target gene in the present invention, and the first vector and the second when the two target genes are deleted. The vector is used, and includes one vector removal step. When n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. It features.

In accordance with a fourteenth aspect of the present invention, (a) bacteria are transformed with an enzyme expression vector having a first antibiotic selective marker and at the same time having an antibiotic sensitivity to the first antibiotic and expressing a recombinant enzyme (recombinase) Making a step; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to one target gene in a competent cell obtained in step (b), and (ii) Introducing a Cas protein and a guide RNA, first transforming the first vector having a temperature-sensitive first vector with a second antibiotic selective marker; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in a medium and temperature sensitive condition containing the first antibiotic but not the second antibiotic. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, And, the remaining components are substantially the same to repeat the steps (b) to (d) n-1 times to prepare a multivariant strain having the deletion of n target genes; And (f) removing the enzyme expression vector and the first vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a variant strain using an ssODN and a first vector for deleting an enzyme expression vector and a first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, in consideration of only the vector introduced into the transformation, only the first vector can be used when deleting one target gene in the present invention, and the first vector and the second when the two target genes are deleted. The vector is used, and includes one vector removal step. When n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. It features.

In accordance with a fifteenth aspect of the present invention, (a) bacteria are transformed with an enzyme expression vector that has a first antibiotic selective marker and at the same time has an antibiotic sensitivity to the first antibiotic and expresses a recombinant enzyme (recombinase) Making a step; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to one target gene in a competent cell obtained in step (b), and (ii) Expressing Cas protein and guide RNA, first transforming with a first vector having a second antibiotic selective marker and having an antibiotic sensitivity to the second antibiotic; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in the medium containing the first antibiotic and not containing the second antibiotic and in a temperature sensitive condition. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, And, the remaining components are substantially the same to repeat the steps (b) to (d) n-1 times to prepare a multivariant strain having the deletion of n target genes; And (f) removing the enzyme expression vector and the n-th vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector, ssODN and a first vector for deleting the first target gene, without removing the enzyme expression vector introduced into the cell. In the maintained state, only the first vector is removed, and then the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, when considering only the vector to be transformed, only the first vector can be used when deleting one target gene in the present invention, the first vector and the second vector is deleted when two target genes are deleted. And a vector removal step, wherein n (n is an integer of 2 or more), and when the target gene is deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. do.

In accordance with a sixteenth aspect, the present invention provides a method for preparing a bacterium comprising: (a) transforming a bacterium into an enzyme expression vector having a first antibiotic selective marker and having a temperature sensitivity and expressing a recombinase; (b) preparing a competent cell of the transformed bacterium; (c) (i) single-stranded oligodeoxyribonucleic acid, ssODN, which complementarily binds to one target gene in a competent cell obtained in step (b), and (ii) Introducing a Cas protein and a guide RNA, first transforming the first vector having a temperature-sensitive first vector with a second antibiotic selective marker; (d) Mutation in which the first target gene is deleted by removing the inserted first vector by culturing the first transformed strain in the medium containing the first antibiotic and not containing the second antibiotic and in a temperature sensitive condition. Selecting strains; (e) using (i) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guide RNA that complementarily binds to n (n is an integer greater than or equal to) target genes, And, the remaining components are substantially the same to repeat the steps (b) to (d) n-1 times to prepare a multivariant strain having the deletion of n target genes; And (f) removing the enzyme expression vector and the n-th vector inserted from the multivariate strain prepared in step (e).

In the present invention, a method for deleting one or more target genes may be performed by preparing a mutant strain using an enzyme expression vector and a first vector for deleting the first target gene, and maintaining the enzyme expression vector introduced into the cell without removing them. In this state, only the first vector is removed, and then, a multivariate strain is produced by introducing a second vector for deleting the second target gene. In this case, after the introduction and removal of the first vector to the first target gene to delete the two target genes, the process of introducing the second vector is performed, and to delete the three target genes, the first target gene is deleted. Introduction and removal of one vector, introduction and removal of a second vector to a second target gene, and introduction of a third vector should be performed a manufacturing method including two vector removal steps.

Therefore, when considering only the vector introduced into the transformation, in the present invention, when deleting one target gene, only the first vector can be used, and when deleting two target genes, the first vector and the second vector Is used, and includes one vector removal step, wherein when n (n is an integer of 2 or more) target genes are deleted, the first vector to the nth vector are used, and n-1 vector removal steps are included. It is done.

In the present invention, in order to delete the target gene, the ssODN and the vector must be introduced into the strain together. In particular, since the ssODN is introduced into the nucleic acid molecule rather than the vector, it is diluted during cell division and removed naturally in the cell without special treatment. There is this.

In the present invention, in step (c), single-stranded oligodeoxyribonucleic acid (ssODN) may be introduced at least one, and guide RNA (guide) expressed in the first vector of step (c) RNA) is characterized in that at least one.

In order to delete one or more target genes in the present invention, it is possible by introducing one or more ssODNs and guide RNAs into the strain. In this case, ssODN / guide RNA for one target gene is inserted, followed by ssODN and guide RNA for another target gene, as well as a plurality of ssODN and guide RNAs. By introducing a plurality of expressing vectors into the strain, and then repeating the above method, it is possible to additionally delete another target gene sequentially while simultaneously deleting one or more target genes.

In addition, in the present invention, when simultaneously deleting two or more target genes, two or more ssODN having a complementary binding capacity to each of the two or more target genes and a guide RNA having a binding capacity complementary to one of the genes to which the ssODN binds Can be used to delete multiple genes. In this case, two or more ssODN and one guide RNA (guide RNA) is characterized by introducing into the strain at the same time, the target gene to which each ssODN binds is characterized by being an adjacent gene. In the co-deletion, the distance between different adjacent genes to which two or more ssODNs simultaneously bind may be 100 kb or less, preferably 10 kb or less, more preferably 3 kb or less, but is not limited thereto.

The bacterium of the present invention may be a strain of the genus Corynebacterium , preferably C. glutamicum ATCC 13032, but is not limited thereto.

When the mutant strain finally selected by the production method of the present invention is cultured using a medium containing no antibiotics corresponding to antibiotic sensitivity at 34 ° C to 42 ° C, all introduced vectors may be removed and used industrially. .

The term 'mutant strain' of the present invention refers to a microorganism having a genotype different from the wild type by artificially modifying or mutating a gene of a wild type microorganism, and when the mutant strain, a transformant, or one or more genes are mutated or deleted It can be commonly used as 'multivariate strain'.

The 'temperature sensitive' vector in the present invention is a vector artificially engineered to have a single base mutation on the Rep protein, which has a pBL1 origin of replication and acts on it. The rep protein is involved in the replication process of the pBL1 origin of replication (ori), and when a single base mutation (C → T) is introduced into the Rep protein gene, amino acid substitution of P47S occurs, and culture of the strain containing the vector. When the temperature rises above 34 ° C. without the addition of antibiotics, the pBL1 replication origin (ori) is known to lose its function (Nakamura et al., Plasmid 56 (3): 179-186, 2006). Thus, bacterial mutants prepared using the vector having the origin of replication are stable when cultured at 10 ° C. to 30 ° C. in a medium containing antibiotics, but at 34 ° C. or higher, typically 37 ° C. in an antibiotic-free medium. When cultured at less than 42 ℃, the vector is not maintained, therefore, by adjusting the culture temperature in the antibiotic-free culture conditions, it is possible to maintain or remove the vector.

The antibiotic susceptibility vector of the present invention is a vector of the pTacCC1 family in the course of developing the present technology, when the concentration of antibiotics corresponding to the resistance gene inserted into the vector, that is, the antibiotic selective marker, becomes low. It was found to be missing, and this feature was used for vector removal.

In addition, the first antibiotic susceptibility vector and the second antibiotic susceptibility vector are vectors having a first antibiotic selective marker or a first antibiotic selective marker, and having different resistance genes inserted therein. This, when the concentration of the antibiotic is lowered, there is a feature that only the corresponding vector is lost.

Thus, in the present invention, the 'temperature sensitive' vector is a vector which has been artificially engineered to have a single base mutation on the Rep protein which has the origin of pBL1 replication and acts on it.

In addition, the "antibiotic sensitive" vector in the present invention is a vector having an antibiotic resistance gene and having a pCC1 origin of replication, in particular, the antibiotics acting on 'first antibiotic sensitivity' and 'second antibiotic sensitivity' have different characteristics.

The antibiotic in the vector may be kanamycin (kanamycin), spectinomycin (spectinomycin), streptomycin (streptomycin), chloramphenicol (chloramphenicol), or apramycin (apramycin), but is not limited thereto.

In the method of the present invention, when the vector has an antibiotic selective marker and at the same time is characterized by antibiotic sensitivity, the antibiotic corresponding to the 'selective marker' and the antibiotic acting on the 'antibiotic sensitivity' are the same. You can also use different antibiotics. In the latter case, two antibiotic selective markers can be implemented by present in the vector.

In the present invention, when the 'temperature sensitive' vector is removed, at a temperature of 34 ° C. or higher, typically 37 ° C. to 42 ° C. or lower, it does not include an antibiotic corresponding to the antibiotic selective marker included in the vector. Vectors can only be completely removed if cultured in medium

As used herein, the term 'recombinant' refers to, for example, cells, nucleic acids, proteins or vectors, etc., when used to introduce heterologous nucleic acids or proteins or to alterations of native nucleic acids or proteins, or from modified cells. Cell, nucleic acid, protein, or vector modified by the cell from which it is derived.

The present invention can also delete genes involved in the metabolism of gamma-aminobutyric acid (GABA) synthetic target genes.

The genes involved in the metabolism of gamma-aminobutyric acid (γ-aminobutyric acid, GABA) are Ncgl1221, gabT, and gabP .

The present invention, in a seventeenth aspect, relates to a bacterial mutant having glutamate overproduction capacity in which Ncgl1221, gabT, and gabP are deleted.

In the present invention, the mutant strain may be prepared by simultaneously or sequentially deleting three genes through the preparation method of the present invention: For example, a bacterial mutant having glutamate overproduction ability of the present invention may be prepared. By sequential deletion, bacteria can be transformed by inserting an enzyme expression vector, followed by (a) primary transformation with a first vector expressing ssODN and guide RNA targeting Ncgl1221 . By removing the first vector by adjusting the culture conditions according to the characteristics of the temperature sensitivity or antibiotic sensitivity of the first vector, only a recombinant protein and / or a Cas protein are expressed and the first transformed Ncgl1221 is deleted. Strains can be obtained. Then, (b) ssODN and guide the gabT a target RNA (guide RNA) expression switched to insert the second vector, the second repeat the vector removal process Ncgl1221 and secondary traits gabT a deletion of Strains can be prepared. Then again, (c) is the Ncgl1221 and gabT is to re-insert to the gabP the converted secondary transformants deletion strain expressing ssODN and a guide RNA (guide RNA) to target the third vector Ncgl1221, gabT and gabP By preparing a deleted tertiary transformation strain, all target genes can be deleted. Finally, the mutant strain was completed by sequential deletion by removing all the foreign vectors introduced into the mutant strain using culture conditions for removing the vector expressing the recombinant enzyme (recombinase) and / or Cas protein.

In addition, bacterial mutants having glutamate overproduction ability of the present invention can be prepared by co-deletion: transformation by inserting an enzyme expression vector expressing a recombinant recombinase and / or Cas protein, (a) gabT and gabP and the switching primary transformed with a first vector that expresses a guide RNA (guide RNA) as ssODN, and gabT or gabP target to target, and finally, the recombinant enzyme (recombinase) and / or a Cas protein By removing all the foreign vectors introduced into the mutant strain using culture conditions for removing the vector expressing the mutant strain, the mutant strain may be prepared by simultaneous deletion.

In the production of mutant strains by simultaneous deletion of two or more target genes, adjacent genes may be deleted by two or more ssODNs that complementarily bind to each target and guide RNAs that complementarily bind to one target gene. Therefore, when applied to gabT and gabP separated about 3kb, there is a characteristic that the deletion effect can be obtained only by two kinds of ssODN and one type of guide RNA.

The 'culture condition for removing the vector' is a temperature sensitive vector (pBL1ts replication origin-based vector, such as pEKTs-series), while the vector is maintained when incubated at 10 ° C. to 34 ° C. in the presence of antibiotics, whereas no antibiotic medium is used. When incubated at 34 ℃ to 37 ℃ is characterized in that the vector is removed. In addition, antibiotic sensitive vectors (based on pCC1 origin of replication, such as pTacCC1-series) are maintained when the antibiotics corresponding to antibiotic sensitivity are cultured at 10 ° C. to 42 ° C. using a medium. Vector may be removed when incubated at 10 ℃ to 42 ℃ using.

In accordance with an aspect of the present invention, there is provided a method for producing a gamma-aminobutyric acid (GABA) by culturing the strain; And it relates to a method for producing gamma-aminobutyric acid (γ-aminobutyric acid, GABA) comprising recovering the generated gamma-aminobutyric acid (γ-aminobutyric acid, GABA).

In the present invention, the 'cultivation' of the strain, the mutant strain or the transformant may be made according to a suitable medium and culture conditions known in the art. This culture process can be easily adjusted and used according to the microorganism selected.

Specifically, the medium used for culturing may include, but is not limited to, one selected from the group consisting of various carbon sources, nitrogen sources, trace element components, and combinations thereof to suitably satisfy the requirements of specific microorganisms. Various microbial culture media are described, for example, in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology, Washington D.C., USA, 1981.

The carbon source may include a carbon source selected from the group consisting of carbohydrates, fats, fatty acids, alcohols, organic acids, and combinations thereof. The carbohydrate may include one selected from the group consisting of glucose, sucrose, lactose, fructose, maltose, starch, cellulose, and combinations thereof. The fat may include selected from the group consisting of soybean oil, sunflower oil, pajama oil, coconut oil, and combinations thereof. The fatty acid may include one selected from the group consisting of palmitic acid, stearic acid, linoleic acid, or a combination thereof. The alcohol may include one selected from the group consisting of glycerol, ethanol or a combination thereof. The organic acid may comprise acetic acid.

The nitrogen source may include an organic nitrogen source, an inorganic nitrogen source, or a combination thereof. The organic nitrogen source may include one selected from the group consisting of peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), soybean wheat, and combinations thereof. The inorganic nitrogen source may include one selected from the group consisting of urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, and combinations thereof.

The medium may include one selected from the group consisting of phosphorus, metal salts, amino acids, vitamins, precursors and combinations thereof. The source of phosphorus may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt corresponding thereto. The metal salt may include magnesium sulfate or iron sulfate.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Restriction enzymes used in this example and the following examples were purchased from New England Biolabs (USA) and Enzynomics (Korea), PCR polymerase from BIOFACT (Korea), and DNA ligase (DNA ligase) from Elpis Biotech (Korea). . Other things were marked separately.

Example 1 Expression and Performance of Cas9 and Guide RNA in C. glutamicum

1-1: Preparation of a Cas9 Expressing Vector (pEKEx1-Cas9opt)

A vector pEKEx1-Cas9opt, which can effectively express Cas9 protein in C. glutamicum, was prepared by the following procedure.

To extract the Cas9 gene from the pCRISPR-Cas9 vector (Tong et al., ACS Synthetic Biology , 4 (9): 1020-1029, 2015), the vector was used as a template and primers of SEQ ID NO: 1 and SEQ ID NO: PCR was performed to amplify cas9 gene, which was codon optimized according to the codon usage of Actinomycetes .

At this time, the hexahistidine tag sequence was added to the 5 'end of the amplified cas9 gene by the sequence of the hexahistidine tag included in the primer of SEQ ID NO: 1 used in the amplification process.

The amplified sequence is a one-step sequence and ligation-independent cloning (SLIC) protocol (Jeong et al. ) In pEKEx1 digested with EcoRI and BamHI (FIG. 1; Eikmanns et al., Gene 102 (1): 93-98, 1991). , Applied and Environmental Microbiology , 78 (15): 174, 2012), and finally pEKEx1-Cas9opt was prepared (FIG. 2).

T4 DNA polymerase used for SLIC was purchased from New England Biolabs (USA).

1-2: Cas9 expression confirmed in pEKEx1-Cas9opt

To confirm whether Cas9 is expressed in C. glutamicum from pEKEx1-Cas9opt with cas9 gene inserted, the vector was introduced into C. glutamicum ATCC 13032 by electroporation.

The transformation through the electroporation, first, for C. glutamicum cultured in the early exponential phase, washed twice with 10% glycerol cooled to 0 ~ 4 ℃ to transform into a soluble cell (competent cell), to introduce After mixing with the vector, it was transferred to an electroporation cuvette cooled to 0-4 DEG C and having a distance of 1 mm, and subjected to electric shock with a capacitance of 25 µF, a voltage of 1.8 kV, and a 200 Ω resistor. .

The cells undergoing electroporation participated in RG medium (10 g / L glucose, 40 g / L BHI, 10 g / L Beef extract, 30 g / L sorbitol), and recovered at 30 ° C. at 200 rpm. Smear a portion of RG solid medium (10 g / L glucose, 40 g / L BHI, 10 g / L beef extract, 30 g / L sorbitol, 1.5 g / L agarose) to which 25 μg / mL kanamycin was added. Cultured at 30 ° C. yielded transformed colonies.

In order to confirm whether Cas9 is expressed from pEKEx1-Cas9opt into which the cas9 gene has been inserted for the transformed C. glutamicum colony, the transformed C. glutamicum strain is again RG medium (10 g / L glucose, 40 g / L BHI, 10). Inoculated with g / L beef extract, 30 g / L sorbitol and BHI (brain heart infusion, Becton, Dickinson and Company (USA)) and incubated at 30 rpm for 24 hours at 200 rpm. 0, 0.5, 1, and 2 mM IPTG was added during the incubation, and when the incubation was completed, the cells were recovered by centrifugation, and then the buffer solution (20 mM Tris) was made to have a final optical density (OD ₆₀₀ ) of 7 at 600 nm. Cells were suspended using -HCl (pH8.0), 300 mM NaCl, 5 mM Imidazole, and then lysed by sonication.

Cell-derived proteins obtained by fusion by ultrasonic grinding were developed using 10% Tricine-SDS-PAGE (Schagger et al., Nature Protocols , 1 (1): 16-22, 2006) and Coomassie Briliant Blue R-250 The expression of Cas9 was confirmed by staining in a conventional manner using (Lawrence et al., Journal of Visualized Experiments , (30): e1350, 2009. doi: 10.3791 / 1350).

As a result, when the C. glutamicum strain transformed with pEKEx1-Cas9opt containing the cas9 gene was cultured in RG medium, Cas9 protein was not expressed without adding IPTG, and Cas9 protein when IPTG was added at a concentration of 0.5 mM or more. It was confirmed that this was strongly expressed. On the other hand, when the strain was cultured in BHI it was confirmed that Cas9 protein is expressed even without addition of IPTG (Fig. 3).

1-3: Preparation of a vector (pCG9-series) expressing Cas9 and guide RNA simultaneously

The vector pCG9-series was prepared as follows to further clone the gene encoding sgRNA as a guide RNA into pEKEx1-Cas9opt to simultaneously express Cas9 and sgRNA in C. glutamicum .

pWAS vector (Na et al., Nature Biotechnology , 31 (2): 170-174, 2013) as a template and amplifying the T1 / TE terminator using primers of SEQ ID NO: 3 and SEQ ID NO: 4 A fragment of the sgRNA-T1 / TE DNA to which the sequence of the sgRNA was bound was amplified except for a guide sequence that complementarily binds to the target gene. Thereafter, the amplified sgRNA-T1 / TE DNA was inserted into pUC19 digested with Eco RI and Hind III to prepare pUC19-sgRNA (FIG. 4).

In order to extract the DNA fragment encoding the sgRNA to be inserted into pEKEx1-Cas9opt and the sgRNA of the pUC19-sgRNA as a template and the nucleotide sequence of the T1 / TE termination site, the primers of SEQ ID NO: 5 and SEQ ID NO: 6 are used. The sgRNA-T1 / TE DNA fragment containing this was amplified. However, 20 N in SEQ ID NO: 5 refers to 20 base sequences capable of complementarily binding to the target gene.

Subsequently, the amplified DNA fragment was used as a template to amplify the final sgRNA-T1 / TE DNA to be introduced into pEKEx1-Cas9opt using primers SEQ ID NO: 7 and SEQ ID NO: 8.

The amplified fragment was cloned using a Gibson assembly (Gibson et al., Nature Methods, 6 (5): 343-345, 2009) at the StuI site of pEKEx1-Cas9opt (FIG. 5). As a result, pCG9-series was prepared that can simultaneously express Cas9 protein and sgRNA in C. glutamicum (FIG. 6).

1-4: Confirmation of co-expression of Cas9 and sgRNA in pCG9-series

To determine whether the Cas9-sgRNA complex is expressed in C. glutamicum transformed with pCG9-series and has the ability to cleave the target gene, we targeted the arginine repressor argR gene of C. glutamicum . PCG9-argR1 expressing Cas9 and sgRNA on C. glutamicum was introduced into the C. glutamicum by electroporation.

In this experiment, pEKEx1-Cas9opt, pEKEx1-sgRNA argR1 and pdCG9-argR1 were used as controls, and the necessary vectors, pEKEx1-sgRNA argR1 and pdCG9-argR1 were further prepared as follows.

(a) Preparation of pEKEx1-sgRNA argR1

5′-TGG targeting the arginine repressor argR gene of C. glutamicum to the primer of SEQ ID NO: 5 mentioned in Examples 1-3 to amplify sgRNA-T1 / TE DNA using pUC19-sgRNA as a template SgRNA-T1 / TE DNA fragments were first amplified using primers of SEQ ID NO: 9 and SEQ ID NO: 6 containing a guide sequence (5'-AGCTCTCATT TTGCAGATTT-3 ') with -3' as the PAM sequence.

The guide sequence was selected using CRISPy-web to determine the sequence near the start codon.

Next, the amplification products were amplified secondly using primers of SEQ ID NO: 7 and SEQ ID NO: 8. DNA fragments encoding the sgRNA-T1 / TE sequence targeting the amplified argR gene were coupled to pEKEx1 via a Gibson assembly to be inserted into the Stu I cleavage site of pEKEx1. As a result, pEKEx1-sgRNA argR1 expressing sgRNA targeting C. glutamicum argR was produced (FIG. 7).

(b) Preparation of pCG9-argR1

PCG9-argR1 simultaneously expressing sgRNA targeting the C. glutamicum argR gene with Cas9 protein was prepared as follows.

Using pEKEx1-sgRNA argR1 as a template, primers of SEQ ID NO: 7 and SEQ ID NO: 8 were used to amplify DNA fragments encoding sgRNA-T1 / TE sequences targeting the C. glutamicum argR gene. PEKEx1-Cas9opt was inserted into the cleavage site of StuI through the Gibson assembly, thereby preparing pCG9-argR1 expressing sgRNA targeting the C. glutamicum argR gene together with Cas9 protein (FIG. 8). .

(c) preparation of pEKEx1-dCas9 and pdCG9-argR1 comprising dCas9

PEKEx1-dCas9 was first constructed to produce pdCG9-argR1 expressing dCas9 protein that binds to sgRNA targeting C. glutmaicum argR and loses DNA cleavage activity.

To extract the dCas9 gene from the pCRISPR-dCas9 vector (Tong et al., ACS Synthetic Biology , 4 (9): 1020-1029, 2015), the vector was used as a template, and SEQ ID NO: 1 and SEQ ID NO: PCR was carried out using the primers to amplify the codon optimized cas9 according to the codon usage of Actinomycetes .

At this time, it was prepared such that the 5 'end of the cas9 amplified by the sequence of the hexahistidine tag with the primer of SEQ ID NO: 1 used in the step of amplifying comprises the sequence of the hexahistidine tag. The amplified sequence was inserted into the cleavage sites of Eco RI and Bam HI of pEKEx1 using the SLIC protocol to prepare pEKEx1-dCas9opt (FIG. 9).

Next, the DNA fragment encoding the sgRNA-T1 / TE sequence targeting the C. glutamicum argR gene was amplified using pEKEx1-sgRNA argR1 as a template and primers of SEQ ID NO: 7 and SEQ ID NO: 8. The amplified DNA fragment was inserted into the Stu I cleavage site of pEKEx1-dCas9opt through a Gibson assembly to finally produce pdCG9-argR1 co-expressing dCas9 and argR1 sgRNA (FIG. 10).

(d) Confirmation of Cas9-sgRNA complex formation in C. glutamicum

pEKEx1-sgRNA argR1, pEKEx1-Cas9opt, and pCG9-argR1, and pdCG9-argR1 were introduced into C. glutamicum through the same electroporation method as in Example 1-2 to confirm the complex-forming ability.

As a result, when sgRNA or Cas9 alone was expressed or when dCas9 and sgRNA were expressed together, colonies of transformed C. glutamicum were confirmed, but when sgRNA and Cas9 were simultaneously expressed by pCG9-argR1 introduction, C. glutamicum Colonies could not be confirmed and all were confirmed to have died (FIG. 11).

Cas9-sgRNA is expressed in microorganisms, cutting genes in the genome of microorganisms to create double-strand breaks (DSBs), which facilitates DSB repair through non-homologous end joining (NHEJ). Most microorganisms that have not been reported have been killed. That is, the same result was obtained in FIG. 11, and C. glutamicum is a microorganism having a weak non-homologous end joining (NHEJ) mechanism, and Cas9 expressed in pCG9-argR1 according to the present invention. And it was confirmed that sgRNA can form Cas9-sgRNA complex in the cells.

According to the experimental results so far, when the CRISPR / Cas system is introduced into C. glutamicum , all microorganisms are killed, so simply inserting sgRNA and Cas9 targeting a specific gene results in the deletion of the target gene. It was confirmed that this is not possible, and the following experiment was carried out to transform microorganisms using the HDR mechanism, which is a homologous recombination principle instead of NHEJ.

Example 2 Preparation of Recombinant Microorganisms Using Cas9-sgRNA and Homologous Recombination Principle

2-1: Gene editing effect using RecT, ssODN and pCG9-argR1 derived from E. coli Rac prophage

Recently, in C. glutamicum , the use of RecT protein and ssODN derived from Rac prophage of E. coli is known to be effective in chromosomal engineering of C. glutamicum (Blinder et al., Nucleic Acids Research , 41 (12): 6360-6369, 2013). Therefore, in the present invention, a system in which RecT is combined with Cas9, sgRNA, and ssODN is attempted to delete a target gene in C. glutamicum .

(a) C. glutamicum-E. Preparation of the coli shuttle vector pTacCC1

Before the production of E. coli-derived RecT prophage recombinase expression vector in C. glutamicum, because Cas9-sgRNA and each to be expressed by using a different vector RecT, the vector preferentially is expressed in C. glutamicum Cas9-sgRNA There is a need for shuttle vector production with different origins of replication (ori) that can coexist with the backbone pEKEx1. In C. glutamicum , pCC1 replication origin (ori) is known as a replication origin that can coexist without mutual interference with pBL1 replication origin (ori) -based vectors such as pEKEx1. We tried to construct a C. glutamicum-E. coli shuttle vector based on the pCC1 origin of replication (ori).

First, using the primers of SEQ ID NO: 10 and SEQ ID NO: 11 using pACYC184 (Chang et al., Journal of Bacteriology, 134 (3): 1141-1156, 1978) as a template for E. coli , ori) was amplified. In addition, pKCA212-MCS (Shin et al., Microbial Cell Factories , 15 (1): 174, 2016) was used as a template to amplify the pCC1 origin of replication (ori) using the primers of SEQ ID NO: 12 and SEQ ID NO: 13. In addition, Ptac-rrnB T1T2 was amplified using primers of SEQ ID NO: 14 and SEQ ID NO: 15 using the E. coli expression vector pTac15K (Lee et al. , US20110269183A) as a template. Using the E. coli expression vector pCDFDuet-1 (Novagen, Germany) as a template, the primers of SEQ ID NO: 16 and SEQ ID NO: 17 were used to amplify the resistance gene aadA expressing spectinomycin.

By inserting the four amplified DNA fragments into one vector through a Gibson assembly, pTacCC1, a C. glutamicum-E. coli shuttle vector, was prepared (FIG. 12).

(b) Preparation of RecT Expression Vector (pTacCC1-recT)

The following experiment was performed to insert the recT gene into pTacCC1 to express RecT in C. glutamicum .

The recT gene was amplified using the primers of SEQ ID NO: 18 and SEQ ID NO: 19 using the genomic DNA of E. coli K-12 MG1655 as a template. The DNA fragment encoding the amplified recT gene was inserted into pTacCC1 digested with Eco RI and Sal I through SLIC, finally preparing pTacCC1-recT (FIG. 13).

The pTacCC1-recT was introduced into C. glutamicum ATCC 13032 by electroporation, followed by the RecT recombinase via Tricine-SDS-PAGE (Schagger et al., Nature Protocols, 1 (1): 16-22, 2006). It was confirmed that is expressed (Fig. 14).

(c) Preparation of RexT Expression Vector (pTacCC1-HrT) with 6xHis Attached

Conventional reports show that the addition of six histidine (hexahistidine (6xHis)) sequences to the N-terminus of protein in C. glutamicum increases protein expression (Shin et al., Microbial Cell Factories , 15 (1): 174, 2016 ), The base sequence encoding 6xHis in the recT gene upstream of pTacCC1-recT was added through the following process.

To add the base sequence encoding 6xHis to pTacCC1-recT, using the primers of pTacCC1-recT as the template and the primers of SEQ ID NO: 20 and SEQ ID NO: 21, a DNA fragment having the 6xHis coding sequence added to the top of the recT gene was added. Amplified.

The amplified DNA fragments were phosphorylated at 5 ′ ends using T4 polynucleotide kinase (T4 PNK; Enzynomics, Korea) and simultaneously conjugated at both ends using T4 DNA conjugated enzyme to prepare pTacCC1-HrT ( 15).

The prepared pTacCC1-HrT was introduced into C. glutamicum ATCC 13032 by electroporation to obtain a recombinant C. glutamicum strain transformed with pTacCC1-HrT.

The expression level of RecT recombinant enzyme added with 6xHis in the recombinant C. glutamicum in which pTacCC1-HrT was introduced was confirmed by the same method as in the case of confirming the expression level of RecT recombinant enzyme in recombinant C. glutamicum in which pTacCC1-recT was introduced. When the nucleotide sequence encoding 6xHis was added to the top of the recT gene, it was confirmed that the expression level of the RecT recombinant enzyme was slightly increased (FIG. 14).

(d) Confirmation of transformation efficiency using recT, ssODN and Cas9 / sgRNA

In the case of recombination using bacteriophage or prophage-derived recombination enzymes including RecT and ssODN, ssODN is complementarily bound to the lagging strand before replication at the replication junction formed during chromosomal replication of the host microorganism. It is known to modify the target gene by acting as a primer of a new Okazaki fragment (Murphy, EcoSal Plus , 2016. doi: 10.1128 / ecosalplus.ESP-0011-2015).

Therefore, in the present invention, the ssODN, which forms a complex with RecT, binds to the target gene by complementary use thereof, thereby deleting the target gene, and the Cas9-sgRNA complex that cuts the sequence to be deleted of the target gene, deletes the gene. It is a system for introducing a DSB into a chromosome that is not ssODN and RecT to kill a host whose target gene is not deleted (FIG. 16).

In addition, the ssODN targeting the argR gene is composed of a 5'-homology arm and a 3'-homology arm, which bind to both outer sides of the site to which the guide sequence of the sgRNA binds. When combined with the target, there is a feature to form a loop structure (Fig. 17).

In addition, the resistance gene on the Cas9-sgRNA vector cannot be survived so that the cells without the Cas9-sgRNA vector introduced cannot be survived in order to remove the surviving cells without introducing the Cas9-sgRNA vector (pCG9-series vector) even though the target gene is not deleted. The system was designed to further screen with antibiotics kanamycin present.

For recombinant strain C. glutamicum ATCC 13032 containing pTacCC1-HrT, pCG9-argR1 and ssODN of SEQ ID NO: 22 were introduced together through electroporation to determine the number of colonies. Finally, cells were screened in RG medium containing 25 μg / mL of kanamycin and 200 μg / mL of spectinomycin. When transformed with ssODN alone, the number of colonies was about 6200. When transformed, about one colony was obtained. In addition, as a result of selecting colonies grown by colony PCR using the primers of SEQ ID NO: 23 and SEQ ID NO: 24, it was possible to confirm the colonies from which the target argR gene was deleted.

2-2: Gene deletion efficiency using Cas9-sgRNA, RecT and ssODN

In Example 2-1, Cas9-sgRNA argR1, RecT and ssODN were used to delete the target gene argR in C. glutamicum , but at the same time there was a problem that the argR gene deletion was not consistently observed. This problem was analyzed due to the poor efficiency of electroporation used for transformation of microorganisms in this experiment. Therefore, the following experiment was conducted to solve the problem (Choi et al., Microbial Cell Factories , 14: 21, 2015).

In preparing the water-soluble cells (competent cells) of C. glutamicum for electroporation, C. glutamicum by interfering with cell wall formation of C. glutamicum during the culturing process, a method for increasing the efficiency of electroporation is known and (Ruan et al., Biotechnology Letters, 37 (12): 2445-2452, 2015), applying this system to antibiotic resistant recombinant C. formed against the conventional electroporation method used for argR gene deletion in C. gltuamicum . The colony number of glutamicum was increased by about 13,000 times (FIG. 18).

10 μg and 1 μg of ssODN and pCG9-argR1 of SEQ ID NO: 30 were respectively introduced into C. glutamicum ATCC13032 expressing RecT using the electroporation method, and 200 μg / mL spectinomycin and 25 μg / mL kanamycin were added. After culturing in the solid medium, colony PCR was performed using the primers of SEQ ID NO: 23 and SEQ ID NO: 24 for the colonies obtained therefrom, and the colonies in which the intended 400 bp sequence was deleted in argR were repeatedly identified. (Table 9). In the gene deletion experiment, the optimized electroporation method was applied to introduce the vector and ssODN into the recombinant C. glutamicum strain.

Example 3: Vector Removal from Gene Deleted Recombinant C. glutamicum

Recombinant C. glutamicum expressing RecT deletes the target gene using ssODN and pCG9-series and needs to remove the vector to be used as an industrial strain. In addition, in order to develop an optimal strain, in most cases, several genes must be deleted. Therefore, to delete a target gene after deleting one target gene, the first introduced vector must be removed to introduce another vector to be used for the next gene deletion. Therefore, it is essential to easily remove vectors from C. glutamicum after gene deletion.

3-1: Construction and Removal of Temperature Sensitive Cas9-sgRNA Vectors

In order to prepare pCG9-series expressing Cas9 and sgRNA simultaneously disclosed in Example 1-3 as a temperature sensitive vector, the following experiment was carried out.

In C. glutamicum , the replication origin that maintains the pCG9-series is stable. A single base mutation (C → T) is introduced into the Rep protein gene involved in the replication of the pBL1 replication origin (ori) to replace the P47S amino acid in the Rep protein. If this occurs, it can be converted to a temperature sensitive replication origin (ori), where the function as the replication origin (ori) is neutralized simply by raising the culture temperature above 34 ° C (Nakamura et al., Plasmid 56 (3): 179-186, 2006). In order to convert the pCG9-series into a temperature sensitive vector, it was decided to introduce the temperature sensitive replication origin in the pCG9-series.

First, in order to convert the pBL1 replication origin of pEKEx1-Cas9opt, a backbone vector of pCG9-series, into a temperature sensitive replication origin, pEKEx1-Cas9opt was used as a template and the entire vector was amplified using primers of SEQ ID NO: 25 and SEQ ID NO: 26. The amplified linear DNA fragments were phosphorylated at 5 ′ ends using T4 PNK and T4 DNA conjugation enzymes, respectively, followed by conjugation of both ends of the DNA fragments to prepare a temperature sensitive vector pEKTs1 (FIG. 19).

Then, using the primers of SEQ ID NO: 27 and SEQ ID NO: 28 using the pEKTs1 as a template, DNA fragments containing the temperature-sensitive pBL1 replication origin were amplified, and using pEKEx1-Cas9opt of FIG. The DNA fragment containing the cas9 gene was amplified using the primer of SEQ ID NO: 28. The two amplified DNA fragments were combined into one vector by Gibson assembly, and as a result, pEKTs1-Cas9opt was prepared (FIG. 20).

Thereafter, pCG9ts-series, a temperature sensitive vector expressing Cas9 and sgRNA, was prepared in the same manner as in Example 1-3 from pEKTs1-Cas9opt (FIG. 21).

The recombinant C. glutamicum strain containing the pCG9ts-series was streaked with one letter in a solid medium without antibiotics (kanamycin) and cultured at 37 ° C. 11).

3-2: Verification of pTacCC1-HrT Removal Efficiency Using Antibiotic Dependent Properties

The origin of pCC1 replication, the origin of replication for C. glutamicum in the pTacCC1-HrT vector, is not characterized by temperature sensitivity. However, as the backbone of pTacCC1 itself was unstable and the concentration of antibiotics was lowered, it was found that the vector of the pTacCC1 family gradually disappeared from recombinant C. glutamicum . To apply this vector removal, the removal efficiency by changing the antibiotic concentration was as follows. It was verified as well.

For recombinant C. glutamicum containing pTacCC1-HrT, since the vector contains a spectinomycin expression gene as a selective marker gene, an antibiotic was used spectinomycin.

Thus, streaking recombinant C. glutamicum containing the pTacCC1-HrT vector into a single medium in a culture medium without the addition of spectinomycin, followed by culturing, and confirming that C. glutamicum without the spectinomycin resistance was obtained by removing the pTacCC1-HrT vector. (Table 12).

3-3: Simultaneous Removal of pCG9ts-series and pTacCC1-HrT

In addition, to determine whether it is possible to simultaneously remove two vectors, pCG9ts-series and pTacCC1-HrT, C. glutamicum transformed with the two vectors was used as both spectinomycin and kanamycin which showed the resistance of the selective marker gene of each vector. After streaking in a solid medium not containing one character and incubated at 37 ° C., the same culture method was repeated once for reliable screening. As a result, a recombinant C. glutamicum strain from which both vectors were removed was obtained. 13).

Example 4 Preparation of Recombinant C. glutamicum with GABA Production Capacity

4-1: Preparation of Vector and ssODN for Ncgl1221, gabT, gabP Gene Deletion

I) to be inserted into C. glutamicum transformed with pTacCC1-HrT to prepare recombinant C. glutamicum , in which three genes Ncgl1221, gabT, gabP, which are involved in the GABA (γ-aminobutyric acid) biosynthetic terminal pathway of FIG. PCG9ts-series, each containing the sgRNA sequences of the three genes, ii) ssODNs respectively binding to the three genes were prepared as follows. As described in FIG. 23, gene deletion for C. glutamicum was attempted.

(a) Preparation of pCG9ts-series

First, using the online program CRISPy-web (Blin et al., Synthetic and Systems Biotechnology, 1 (2): 118-121, 2016), which analyzes the nonspecific target of the sgRNA guide sequence and recommends the optimal sgRNA guide sequence, The optimal guide sequence with low off-target effect was selected as follows (Table 14).

PCG9ts-Ncgl1221, pCG9ts- using pEKEx1-Cas9opt to include the sgRNA of each gene described in Table 15, in the same manner as described in Examples 1-3, to prepare a vector comprising the guide sequence. gabT, and pCG9ts-gabP were produced.

(b) Preparation of ssODNs for each gene

As described in Example 2-1, the ssODN for deleting the target gene was mechanically selected such that the position where the guide sequence of the sgRNA binds was located in the section between the two binding sequences of the ssODN, and was designed to be 80 nt in total. The ssODN consists of a 5'-homology arm and a 3'-homology arm, each homology arm is a 40 base pair, designed to bind to both ends of the target gene region containing the complementary sequence of the sgRNA guide sequence. It was. When ssODN binds to the target, a loop structure is formed and the region is deleted. The length of the deletion region is designed to be 100-400 bp so that the target gene can be easily identified through PCR.

4-2: Preparation of Recombinant C. glutamicum

Genotypes of the transformed strains obtained through the sequential deletion or simultaneous deletion described below are shown in Table 17. For example, among the strains disclosed in Table 17, WH8 prepared using the simultaneous deletion technology of gabT and gabP genes for strain WH2 lacking Ncgl1221 and W2 to which pTacCC1-HrT was removed using antibiotic dependence A strain of W8 was obtained.

(a) Preparation of recombinant microorganisms by sequential gene deletion

Optimized electroporation of 2-2 in sequence to C. glutamicum expressing RecT using a vector comprising pCG9ts-Ncgl1221, pCG9ts-gabT, and pCG9ts-gabP prepared in Example 4-1 and ssODN for each gene The target gene deletion was attempted by transformation using the method, and the deletion of each gene was confirmed using the primers of Table 18.

Random selection or analysis of all eight colonies cultured on the plate revealed that the deletion efficiency was excellent, as shown in Table 19.

(b) Preparation of recombinant microorganisms by simultaneous deletion of genes

When 1 μg of a mixture of ssODN, ssODNgabT_100 ssODNgabP_150 and 10 μg each pCG9ts-gabT and Table 19, have been introduced at the same time, the C. glutamicum strain expressing RecT via electroporation, gabT gene and both a gene gabP deletion colonies Could be screened. Similarly, 1 μg of pCG9ts-gabP and ssODN ssODNgabT_100 ssODNgabP_150 and each 10 μg were mixed to be a screening all gabT gene and gene deletion gabP colonies when introduced at the same time the C. glutamicum strain expressing RecT via electroporation (Table 20).

In contrast, no simultaneous deletion of Ncgl1221 and gabT or Ncgl1221 and gabP was observed. Considering that the distance between the regions deleted in gabT and gabP is about 3 kb, the distance between Ncgl1221 and gabT or gabP is about 830 kb. It seems to be.

4-3: GABA Production of Gene Deleted C. glutamicum

The microorganism of Example 4-2 is a glutamate overexpressing microorganism, in which a glutamate decarboxylase ( gadB2 ) gene derived from Lactobacillus brevis ATCC 367 should be additionally introduced in order to convert to a GABA-producing ability. Thus, the expression vector pGA7 Was prepared as follows.

(a) Preparation of pGA7 and the transformed strain comprising the same

First, the gadB2 gene was amplified using the primers of SEQ ID NO: 44 and SEQ ID NO: 45 using the genomic DNA of L. brevis ATCC 13032 as a template. DNA fragments encoding the amplified gadB2 gene and pEKEx1 were digested with Bam HI and Sal I, and then conjugated to each other to prepare pGA7 (FIG. 24).

The prepared pGA7 was introduced into the strains of WT, W2, W3, W4, W5, W6, W7 and W8 disclosed in Table 23 by electroporation, and GAB1, GAB2, GAB3, GAB4, GAB5, GAB6, GAB7, and GAB8, respectively. Strains were prepared, and GAB0 strains into which pEKEx1 was introduced were prepared as negative controls (Table 22).

(b) determine GABA-producing ability of the recombinant C. glutamicum

The strains described in Table 25 were freshly streaked in solid RG medium to which 25 μg / mL kanamycin was added, respectively, inoculated in 10 mL GAP-seed medium, and then incubated in a baffled flask (erlenmeyer flask) for 30 hours at 200 ° C and 200 rpm. In the condition of OD ₆₀₀ = 20 ~ 60 to increase the cells, and used as a seed for the culture for GABA production. The composition of the GAP-Seed medium is as shown in Table 23.

In order to produce GABA using the strain, the cultured cells were inoculated into a baffled flask (erlenmeyer flask) containing 10 mL of GAP-main medium so that the final OD ₆₀₀ to 0.2 and 200 rpm at 30 ° C. for 96 hours. The main culture was carried out under the conditions of. The composition of the GAP-main medium is as shown in Table 24.

Samples were taken once every 24 hours and 96 hours for analysis in the main culture process, centrifuged at 16,100 × g for 10 minutes, then only the supernatant was diluted with distilled water, and the impurities were removed using a 0.2-μm filter. The concentration was determined by the method previously reported via HPLC (Agilent Technologies) (Shin et al., Microbial Cell Factories , 15 (1): 174, 2016).

As a result, the total of seven combinations of gene deletions gabT (GAB3), gabT and gabP (GAB7), and Ncgl1221 with gabT, gabP 27.5 ± 2.5 (GAB8 ) , each gene is deletion of the GABA production of recombinant C. glutamicum g / L , 28.7 ± 0.1 g / L, 27.5 ± 0.3 g / L was shown to be the best (Figs. 25 and 26).

Example 5: further verification of the performance of the present gene deletion system

In order to show that the utilization of the gene deletion system of the present invention is not limited to the above examples, the following experiment was further conducted.

5-1: Various gene deletions in various strains

To show the range of genetic strains and the target gene that can be applied to the fruit machine of the present invention is not limited to strains and Ncgl1221, and gabP gabT gene of C. glutamicum ATCC 13032 derived using in Example C. glutamicum ATCC 21831 from C. glutamicum ATCC 13032 (hereinafter WT) based on the origin of the C. glutamicum AR4ΔargF, industrial strain C. glutamicum strain S112 and derived S112ΔargR and example was performed to snaA, argF, and hmuO crtEb gene deletion.

PCG9ts-series vector pCG9ts-snaA, pCG9ts- containing the sequence of Table 25 as a guide sequence according to the method described in Example 4-1 (a) to construct a pCG9ts-series vector required for gene deletion argF, pCG9ts-crtEb and pCG9ts-hmuO were produced.

In addition, ssODN required to delete the target gene was designed as described in Example 4-1 (b), but was designed as shown in Table 26 so that the length of the sequence to be deleted is 100 bp.

PTacCC1-to the strains C. glutamicum AR4ΔargF, C. glutamicum S112, C. glutamicum S112ΔargR and C. glutamicum WT using the method described in Example 4-2 to proceed with gene deletion using the pCG9ts-series vector and ssODN. The strain of Table 27 was obtained by introducing the HrT vector.

Using the optimized electroporation method of Example 2-2, the strains disclosed in Table 27 were transformed using the vector of Table 25 and ssODN of Table 26, and a target gene deletion was attempted. It was confirmed using the primer of 28.

Analysis of all colonies cultured on the plate confirmed that the deletion efficiency was good for both various strains and various genes as shown in Table 29.

5-2: Gene Deletion of Various Lengths

The length of genes that can be deleted using the gene deletion system of the present invention is not limited to the 100-400 bp disclosed in the above examples, and the ssODN used for gene deletion is not limited to complementary to the delayed strand of the target gene and is the lead strand. The gene deletion of the present embodiment was carried out to show that the gene deletion can be successfully caused even if complementary to.

SsODN was designed as shown in Table 30 to delete a sequence of 100-5000 bp long around the gabT gene as a target gene.

The gene deletion was performed by introducing the pCG9ts-gabT vector prepared in Example 4-1 and the ssODN described in Table 30 to the WT-HrT strain described in Table 17 using the optimized electroporation method of Example 2-2. Gene deletion for each ssODN was confirmed using the primers in Table 31.

As a result of analyzing all colonies cultured on the plate, it was confirmed that the 5 kb long sequence could be deleted using the gene deletion system of the present invention as shown in Table 32. It is also assumed that RecT / ssODN binds to a lagging strand that has not yet replicated at the replication junction formed during chromosomal replication of the microorganism, where it acts as a primer for a new Okazaki fragment to modify the target gene. report (Murphy, EcoSal Plus, 2016. doi : 10.1128 / ecosalplus.ESP-0011-2015) , unlike, RecT / ssODN binding complementarily to the leading strand of the target gene or (Table 32, trial 1-3) delay Complementary binding to the strands (Table 32, trial 4-6) was confirmed that no difference in gene deletion efficiency.

5-3: Another case of gene deletion

Gene deletion of the present example was performed to show that the simultaneous deletion of two genes using the gene deletion system of the present invention is not limited to the gabT and gabP genes of Example 4-2.

PCG9ts-series vector pCG9ts-Ncgl0595 comprising the sequence shown in Table 33 as a guide sequence was constructed in accordance with the method described in Example 4-1 (a) to construct the pCG9ts-series vector required for gene deletion. .

In addition, the ssODN required to delete the target gene was designed as described in Example 4-1 (b), but designed as shown in Table 34 so that the length of the sequence to be deleted is 100 bp.

In order to proceed with gene deletion using the pCG9ts-Ncgl0595 vector and ssODN, the gene was prepared by deleting the crtEb gene from the WT-HrT strain in Example 5-1 using the method described in Example 4-2 (Table 29, trial 6). ) The pCG9ts-Ncgl0595 vector and ssODN described in Table 34 were introduced into the WTΔcrtEb-HrT strain to co-delet the Ncgl0595 and Ncgl0596 genes. Gene deletion was first confirmed using the primers in Table 35 and finally confirmed by sequencing.

As a result of analyzing all colonies cultured on the plate, as shown in Table 36, in the gene deletion system of the present invention, the NCgl0595 gene and the Ncgl0596 gene were simultaneously deleted by targeting only one Ncgl0595 gene using the pCG9ts-Ncgl0595 vector. It was confirmed that this is not applicable to one specific case but applicable to various cases.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The method according to the present invention can effectively delete target genes and select recombinant microorganisms quickly and with high efficiency, in particular, vectors expressing Cas proteins, sgRNAs and RecTs are characterized by temperature sensitivity or antibiotic sensitivity. Since it is improved, it can be easily removed in cells by adjusting the culture temperature and changing the culture conditions with or without antibiotics.

This feature can be used for (a) introducing a new vector to delete several genes sequentially or simultaneously, since the introduced vector can be easily removed within the strain; (b) can readily select for transformed bacteria; (C) It is also very useful for industrial applications related to the production of useful products, since there is no foreign vector in the finally selected mutants.

Electronic file attached.

Claims (20)

A method for preparing a bacterial strain comprising a target gene deleted comprising the following steps: (a) first transforming normal bacteria with an enzyme expression vector that is temperature sensitive or antibiotic sensitive and expresses a recombinant enzyme (recombinase); (b) preparing a recipient cell of the primary transformed bacteria obtained in step (a); (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides that complementarily bind to the target gene to the recipient cell obtained in step (b). Introducing a first vector expressing RNA (guide RNA) and having antibiotic sensitivity or temperature sensitivity to perform secondary transformation; And (e) culturing the secondary transformed bacteria in an antibiotic free medium to remove the inserted enzyme expression vector and the first vector. Wherein at least one of the enzyme expression vector and the first vector expresses a Cas protein. Method for preparing bacterial strains comprising the following steps: (a) transforming normal bacteria with an enzyme expression vector having a first antibiotic selective marker and at the same time having temperature sensitivity or antibiotic resistance by the marker (i) expressing a recombinant enzyme; (b) preparing a competent cell of the transformed bacterium; (c) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) guides, which complementarily bind to a first target gene in a competent cell obtained in step (b). Expressing RNA (guide RNA), having a second antibiotic selective marker, and simultaneously transforming by introducing a first vector having antibiotic sensitivity or temperature sensitivity to the second antibiotic; (d) The first transformed strain is cultured in a medium containing the first antibiotic but not the second antibiotic, but cultured under a temperature sensitive condition to remove the inserted first vector, thereby deleting the first target gene. Selecting the modified strains; (e) a (n) single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) a guide RNA that complementarily binds to n (n is an integer of 2 or more) target genes Using the n-th vector, but the remaining components are substantially the same, repeating steps (b) to (d) n-1 times to prepare a multimutant strain having n target genes deleted; And (f) culturing the multivariant strain prepared in step (e) in an antibiotic free medium to remove the inserted enzyme expression vector and the nth vector, Here, at least one of the enzyme expression vector, the first vector and the n-th vector expresses the Cas protein. According to claim 1, wherein the step (e) is a method for producing bacterial strains, characterized in that the secondary transformed bacteria of step (d) incubated at 10 ℃ to 42 ℃. According to claim 2, wherein (d) the temperature-sensitive condition of the daze is 10 ℃ to 42 ℃, or (f) step is characterized in that the culture of the transformed bacteria of step (e) at 10 ℃ to 42 ℃ Method of producing bacterial mutants. According to claim 1 or 2, wherein the step (b) is a bacterial strain, characterized in that the culture is prepared by culturing in a medium containing Tween-20, DL-Theronine isoniazid and glycine (Glycine) Manufacturing method. The method of claim 1 or 2, wherein the recombinant enzyme (recombinase) is selected from the group consisting of RecT, RecET system, Bet, and λ Red system. The method of claim 1 or 2, wherein the recombinant enzyme (Recombinase) and Cas protein of step (a) is characterized in that the expression of the same or different vectors. The method of claim 1, wherein the Cas protein and guide RNA of step (c) are expressed by the same or different vectors. The method of claim 1 or 2, wherein the single-stranded oligodeoxyribonucleic acid (ssODN) is introduced into the cell in a deoxyribonucleic acid state. The method of claim 1, wherein the target gene is two or more. 11. The method of claim 10, wherein the two or more target genes (i) two or more single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) the sequence of the gene, each complementary to the sequence of the gene Method of producing a bacterial strain, characterized in that the deletion at the same time using a guide RNA (binding complementary to) complementary. 3. The method of claim 1, wherein the enzyme expression vector or the first vector comprises different antibiotic selective markers. 4. The method of claim 1, wherein the target gene is a gene involved in the metabolic pathway of gamma-aminobutyric acid (GABA) synthesis. The method of claim 1, wherein the target gene is selected from the group consisting of Ncgl1221, gabT, gabPsnaA, argF, crtEb, and hmuO . The method of claim 1 or 2, wherein the single-stranded oligodeoxyribonucleic acid (ssODN) is introduced into the cell in the state of ribonucleic acid. The method of claim 2, wherein the first antibiotic and the second antibiotic are different antibiotics. The method of claim 1 or 2, wherein in step (c), single-stranded oligodeoxyribonucleic acid (ssODN) is introduced. The method according to claim 1 or 2, wherein the guide RNA (guide RNA) expressed in the first vector of step (c) is one or more of the production method of bacterial strains. A bacterial variant produced by the method of claim 2 and having glutamate overproduction capacity in which Ncgl1221, gabT, and gabP are deleted. Culturing the bacterial variant strain of claim 19 to produce gamma-aminobutyric acid (GABA); And recovering the generated gamma-aminobutyric acid (γ-aminobutyric acid, GABA).

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