WO2018182361A1 - Procédé de préparation d'une souche mutante de corynebacterium, à l'aide d'un système crispr/cas, d'une recombinase et d'un acide oligodésoxyribonucléique simple brin - Google Patents

Procédé de préparation d'une souche mutante de corynebacterium, à l'aide d'un système crispr/cas, d'une recombinase et d'un acide oligodésoxyribonucléique simple brin Download PDF

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WO2018182361A1
WO2018182361A1 PCT/KR2018/003784 KR2018003784W WO2018182361A1 WO 2018182361 A1 WO2018182361 A1 WO 2018182361A1 KR 2018003784 W KR2018003784 W KR 2018003784W WO 2018182361 A1 WO2018182361 A1 WO 2018182361A1
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antibiotic
gene
ssodn
strain
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이상엽
조재성
최경록
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한국과학기술원
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Definitions

  • 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.
  • Recombinase a recombinant enzyme
  • ssODN single-stranded oligodioxyribonucleic acid
  • 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.
  • Corynebacterium glutamicum 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.
  • 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.
  • FACS fluorescence-activated cell sorting
  • 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).
  • 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.
  • DSB double-strand break
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • 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.
  • recombinase recombinant enzyme
  • ssODN single-stranded oligodioxyribonucleic acid
  • Another object of the present invention is also to provide a bacterial variant involved in gamma-aminobutyric acid (GABA) metabolism.
  • GABA gamma-aminobutyric acid
  • Still another object of the present invention is to provide a method for preparing gamma-aminobutyric acid (GABA) using the bacterial mutant strain.
  • GABA gamma-aminobutyric acid
  • 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).
  • recombinase a recombinant enzyme having temperature sensitivity or antibiotic sensitivity
  • 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).
  • a recombinant enzyme recombinase
  • RNA 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.
  • 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.
  • GABA gamma-aminobutyric acid
  • 1 is a schematic of the pEKEx1 vector.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 6 is a schematic diagram of a pCG9-series vector for introducing cas9 gene and sgRNA gene into C. glutamicum .
  • FIG. 7 is a schematic of the pEKEx1-sgRNA argR1 vector expressing sgRNA targeting the argR gene in C. glutamicum .
  • FIG. 9 is a schematic of the pEKEx1-dCas9 vector for expressing dCas9 protein in C. glutamicum .
  • FIG. 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.
  • FIG. 12 is a schematic of the pTacCC1 vector.
  • FIG. 13 is a schematic of the pTacCC1-recT vector expressing the recombinant enzyme RecT in 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.
  • FIG. 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 .
  • the CRISPR / Cas system works by forming a complex with Cas protein, which is a nucleic acid cleavage enzyme, and guide RNA.
  • Cas protein which is a nucleic acid cleavage enzyme
  • guide RNA guide RNA
  • sgRNAs single-chain guide RNAs
  • the guide sequence of the guide RNA 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.
  • PAM motif Catosome adjacent motif
  • Ncgl1221 glutamicum
  • gabT GABA aminotransferase
  • gabP Gta
  • C. glutamicum C. glutamicum , a Corynebacterium genus
  • CRISPR / Cas system In order to delete the GABA permease gene, we tried to use the CRISPR / Cas system.
  • 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.
  • 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.
  • 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
  • the present invention uses 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.
  • 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.
  • the first vector is used. Can be inserted into a vector.
  • the Cas protein is characterized by being composed of the same vector or a separate vector and guide RNA (guide RNA).
  • 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.
  • 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).
  • recombinase a recombinant enzyme
  • the present invention relates to a method for producing bacterial mutant strains.
  • step (e) may be characterized by culturing the secondary transformed bacteria of step (d) at 10 °C to 42 °C, but is not limited thereto.
  • the first aspect of the present invention can be implemented in the following aspects.
  • 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).
  • recombinase transforming enzyme with an enzyme expression vector expressing a recombinant protein
  • Cas protein Cas protein
  • RNA 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.
  • 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.
  • 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.
  • 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.
  • ssODN single-stranded oligodeoxyribonucleic acid
  • 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.
  • 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.
  • 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.
  • 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).
  • step (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.
  • ssODN single-stranded oligodeoxyribonucleic acid
  • 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.
  • recombinase a recombinant enzyme having a first antibiotic selective marker and temperature sensitivity
  • the step of removing the inserted enzyme expression vector and the first vector may be characterized by culturing the secondary transformed bacteria at 10 °C to 42 °C, but is not limited thereto.
  • the step of removing the inserted enzyme expression vector and the first vector may be characterized by culturing the secondary transformed bacteria at 10 °C to 42 °C, but is not limited thereto.
  • 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.
  • recombinase recombinant enzyme
  • ssODN selection of bacterial strains to the production of double stranded break
  • the recombinase is selected from the group consisting of RecT, RecET system, Bet, and ⁇ 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.
  • single-stranded oligodioxyribonucleic acid 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.
  • 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.
  • 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.
  • 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.
  • the binding to the target gene removes the loop region,
  • 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.
  • the guide RNA (guide RNA, gRNA) includes a guide sequence having a sequence complementary to the sequence of the target gene.
  • the guide RNA 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. .
  • sgRNA single-strand guide RNA
  • 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
  • the PAM sequence is present in the complementary sequence immediately adjacent to the 3 'end of the sequence complementary to the guide sequence on the target gene.
  • 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.
  • 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.
  • 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).
  • the Cas protein for example, Cas9 (CRISPR associated protein 9)
  • Cas9 CRISPR associated protein 9
  • 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.
  • the Cas protein and the guide RNA (guide RNA) when 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 (orth
  • Cas protein of the present invention is characterized in that the codon optimization, so that it can be smoothly expressed in the transformed strain.
  • 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 .
  • the guide RNA and the Cas protein form a complex to represent a gene editing function
  • 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.
  • 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.
  • the 'sequence on the loop region' may be a sequence of the strand to which the ssODN is bound, or a sequence complementary thereto.
  • the term “homology” refers to a degree similar to the amino acid sequence of a protein or a nucleotide sequence encoding the same.
  • one guide RNA and two or more ssODNs can be used to delete two or more target genes simultaneously.
  • each ssODN is terminated at each end of each region to be deleted.
  • 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.
  • 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.
  • ssODN single-stranded oligodexoyribonucleic acid
  • 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.
  • 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.
  • the 'adjacent' range is 100 kb or less, preferably 10 kb or less, and more preferably 5 kb or less.
  • 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.
  • 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.
  • the method is characterized in that it is prepared by electroporation.
  • 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.
  • '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.
  • 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.
  • the 'selection marker' is an antibiotic resistance gene, and the antibiotic may be kanamycin, spectamyomycin, streptomycin, chloramphenicol, or apramycin. It is not limited thereto.
  • '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.
  • 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.
  • '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.
  • 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.
  • MCS Multi-cloning site
  • 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.
  • multiple genes must be deleted.
  • the first introduced vector 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 .
  • 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.
  • 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).
  • a single nucleotide mutation C ⁇ T
  • amino acid substitution of P47S occurs, and the strain containing the vector is absent.
  • the pBL1 replication origin (ori) is known to lose its function (Nakamura et al., Plasmid 56 (3): 179-186, 2006).
  • 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.
  • the vector 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.
  • the 'antibiotic susceptibility' vector and the 'antibiotic susceptibility' vector are vectors inserted with different resistance genes.
  • the antibiotic concentration is lowered and cultured at 10 ° C. to 42 ° C., only the vector is lost. There is this.
  • 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.
  • 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.
  • 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.
  • the first vector expressing the Cas protein and / or sgRNA can be prepared as a vector having characteristics of temperature sensitivity or antibiotic sensitivity.
  • antibiotics in a primary transformed strain using a temperature sensitive vector pEKTs-series, such as a pBL1ts replication origin based vector
  • a temperature sensitive vector based on pCC1 replication origin, such as pTacCC1-series
  • pCC1 replication origin such as pTacCC1-series
  • 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.
  • 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.
  • an antibiotic sensitive vector pCC1 replication origin based vector such as pTacCC1-series
  • Recombinase a recombinant enzyme
  • Cas protein Cas protein
  • the temperature sensitive vector that is, a guide RNA expression vector
  • Recombinse a recombinant enzyme
  • a strain transformed primary using a first antibiotic-sensitized vector pCC1 replication origin based vector such as pTacCC1-series
  • a recombinant enzyme Recombinase
  • 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)
  • pCC1 replication origin-based vector such as pTacCC1-series
  • pCC1 replication origin-based vector such as pTacCC1-series
  • 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).
  • pEKTs-series such as pBLTs-series, origin of replication
  • 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.
  • cultured in the strain can be selected with only the enzyme expression vector.
  • antibiotic sensitivity pTacCC1-to-pTacCC1- After the second transformation using a Cas protein / guide RNA expression vector of pCC1 origin of replication
  • both vectors were maintained in the strain, while (b) no antibiotics corresponding to the 'antibiotic sensitivity vector' were added to the 'temperature sensitive vector'.
  • the antibiotic sensitive vector ie, the guide RNA expression vector
  • the recombinant enzyme and Cas protein Vector is characterized in that only the maintenance.
  • an antibiotic-sensitive vector pCC1 replication origin-based vector such as pTacCC1-series
  • a recombinant enzyme recombinase
  • 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.
  • both vectors are maintained in the strain, while (b) the 'temperature sensitive vector' at 34 ° C to 42 ° C.
  • the temperature sensitive vector ie, the guide RNA expression vector
  • the enzyme maintained the expression vector only expressing the recombinase (recombinse), and Cas protein.
  • 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.
  • a first antibiotic-sensitized vector pCC1 replication origin-based vector such as pTacCC1-series, etc.
  • 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.
  • an antibiotic sensitive vector pCC1 replication origin-based vector, such as pTacCC1-series
  • a temperature sensitive vector 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.
  • pEKTs-series such as a pBL1ts replication origin-based vector
  • Cas protein / guide RNA expression vector of pBL1ts replication origin-based vector such as -series, and the like.
  • 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.
  • both vectors can be removed, but corresponding to the 'recombinant enzyme and Cas protein expression vector' at 34 °C to 42 °C
  • 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.
  • the 'first antibiotic' and the 'second antibiotic' are characterized by being different antibiotics.
  • a mutant strain 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.
  • the target gene is characterized in that two or more, the two or more target genes can be deleted simultaneously or sequentially.
  • 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.
  • 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.
  • 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.
  • the present invention uses 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.
  • 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.
  • the first vector is used. Can be inserted into a vector.
  • 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.
  • the 'second antibiotic sensitivity vector' has a characteristic of having antibiotic sensitivity to the second antibiotic while having a second antibiotic selective marker.
  • 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.
  • 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.
  • the first vector 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.
  • n n is an integer of 2 or more
  • the first vector to the nth vector are used, and n-1 vector removal steps are included.
  • ssODN and the vector in order to delete the target gene, ssODN and the vector must be introduced into the strain together.
  • 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.
  • 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).
  • ssODN single-stranded oligodeoxyribonucleic acid
  • RNA 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.
  • the second aspect may be implemented in the following aspects.
  • 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.
  • ssODN single-stranded oligodeoxyribonucleic acid
  • RNA 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • ssODN single-stranded oligodeoxyribonucleic acid
  • RNA 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.
  • 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.
  • the first vector and the second vector is deleted when two target genes are deleted.
  • 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.
  • 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.
  • RNA 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.
  • 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.
  • 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.
  • 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.
  • the first vector and the second vector is deleted when two target genes are deleted.
  • 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.
  • 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).
  • ssODN single-stranded oligodeoxyribonucleic acid
  • Cas Cas
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • recombinant enzyme recombinase
  • 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.
  • the ssODN and the second vector are introduced again to delete the second target gene, thereby producing a multivariate strain.
  • 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.
  • 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.
  • 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.
  • 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.
  • recombinant enzyme recombinase
  • 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.
  • 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.
  • 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.
  • the first vector and the second vector is deleted when two target genes are deleted.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ssODN and the vector in order to delete the target gene, the ssODN and the vector must be introduced into the strain together.
  • the ssODN 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.
  • 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.
  • ssODN single-stranded oligodeoxyribonucleic acid
  • ssODNs and guide RNAs are introduced into the strain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • '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.
  • the pBL1 replication origin (ori) is known to lose its function (Nakamura et al., Plasmid 56 (3): 179-186, 2006).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the 'temperature sensitive' vector 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
  • 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.
  • the present invention can also delete genes involved in the metabolism of gamma-aminobutyric acid (GABA) synthetic target genes.
  • GABA gamma-aminobutyric acid
  • ⁇ -aminobutyric acid gamma-aminobutyric acid
  • GABA gamma-aminobutyric acid
  • the present invention in a seventeenth aspect, relates to a bacterial mutant having glutamate overproduction capacity in which Ncgl1221, gabT, and gabP are deleted.
  • the mutant strain may be prepared by simultaneously or sequentially deleting three genes through the preparation method of the present invention:
  • a bacterial mutant having glutamate overproduction ability of the present invention may be prepared.
  • 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 .
  • 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.
  • 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
  • guide RNA guide RNA
  • the mutant strain may be prepared by simultaneous deletion.
  • 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.
  • pBL1ts replication origin-based vector such as pEKTs-series
  • antibiotic sensitive vectors 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 °C to 42 °C using.
  • a method for producing a gamma-aminobutyric acid (GABA) by culturing the strain and it relates to a method for producing gamma-aminobutyric acid ( ⁇ -aminobutyric acid, GABA) comprising recovering the generated gamma-aminobutyric acid ( ⁇ -aminobutyric acid, GABA).
  • GABA gamma-aminobutyric acid
  • 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.
  • 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.
  • 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.
  • a vector pEKEx1-Cas9opt which can effectively express Cas9 protein in C. glutamicum, was prepared by the following procedure.
  • 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 .
  • 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).
  • SLIC ligation-independent cloning
  • T4 DNA polymerase used for SLIC was purchased from New England Biolabs (USA).
  • 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 ⁇ g / mL kanamycin was added. Cultured at 30 ° C. yielded transformed colonies.
  • RG medium 10 g / L glucose, 40 g / L BHI, 10 g / L Beef extract, 30 g / L sorbitol
  • 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
  • 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.
  • 0.5, 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.
  • 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).
  • the primers of SEQ ID NO: 5 and SEQ ID NO: 6 are used.
  • the sgRNA-T1 / TE DNA fragment containing this was amplified.
  • 20 N in SEQ ID NO: 5 refers to 20 base sequences capable of complementarily binding to the target gene.
  • 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).
  • pCG9-series was prepared that can simultaneously express Cas9 protein and sgRNA in C. glutamicum (FIG. 6).
  • 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.
  • 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.
  • 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.
  • pEKEx1-sgRNA argR1 expressing sgRNA targeting C. glutamicum argR was produced (FIG. 7).
  • PCG9-argR1 simultaneously expressing sgRNA targeting the C. glutamicum argR gene with Cas9 protein was prepared as follows.
  • 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). .
  • 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.
  • 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 .
  • 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).
  • 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).
  • 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.
  • 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.
  • NHEJ non-homologous end joining
  • RecT protein and ssODN derived from Rac prophage of E. coli are 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 .
  • coli expression vector pTac15K (Lee et al. , US20110269183A) as a template.
  • 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.
  • pTacCC1 a C. glutamicum-E. coli shuttle vector
  • 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).
  • 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).
  • T4 PNK T4 polynucleotide kinase
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • a loop structure Fig. 17
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • the origin of pCC1 replication 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.
  • 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).
  • C. glutamicum transformed with pTacCC1-HrT to prepare recombinant C. glutamicum , in which three genes Ncgl1221, gabT, gabP, which are involved in the GABA ( ⁇ -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.
  • 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.
  • 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.
  • Genotypes of the transformed strains obtained through the sequential deletion or simultaneous deletion described below are shown in Table 17.
  • Table 17 Genotypes of the transformed strains obtained through the sequential deletion or simultaneous deletion described below are shown 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.
  • 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.
  • a glutamate decarboxylase ( gadB2 ) gene derived from Lactobacillus brevis ATCC 367 should be additionally introduced in order to convert to a GABA-producing ability.
  • gadB2 glutamate decarboxylase
  • 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).
  • the composition of the GAP-Seed medium is as shown in Table 23.
  • 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.
  • 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
  • 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 AR4 ⁇ argF, industrial strain C. glutamicum strain S112 and derived S112 ⁇ argR and example was performed to snaA, argF, and hmuO crtEb gene deletion.
  • 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.
  • Example 2-2 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.
  • 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.
  • 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. .
  • 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.
  • 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 WT ⁇ crtEb-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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé de préparation d'une souche mutante de bactérie, comprenant les étapes consistant : (a) à transformer en premier lieu une bactérie normale en un vecteur exprimant une enzyme qui présente une sensibilité à la température ou une sensibilité à un antibiotique et exprime (i) une recombinase ; (b) à amener la bactérie transformée en premier lieu obtenue à l'étape (a) à être une cellule compétente ; (c) à transformer en second lieu la cellule compétente obtenue à l'étape (b) en introduisant (i) un acide oligodésoxyribonucléique simple brin (ADNsb) qui se lie de manière complémentaire au gène cible et (ii) un premier vecteur qui exprime un guide (ARN) et qui présente une sensibilité à un antibiotique ou une sensibilité à la température ; et (e) à éliminer le vecteur d'expression d'enzyme inséré et le premier vecteur à partir de la bactérie transformée en second lieu, le vecteur d'expression d'enzyme et/ou le premier vecteur exprimant une protéine Cas. Un procédé selon la présente invention permet de supprimer efficacement un gène cible, de sélectionner un micro-organisme recombinant rapidement et à un haut rendement, et d'éliminer tous les vecteurs étrangers du micro-organisme recombinant finalement sélectionné, trouvant ainsi des applications très utiles dans l'utilisation industrielle du Corynebacterium recombinant.
PCT/KR2018/003784 2017-03-31 2018-03-30 Procédé de préparation d'une souche mutante de corynebacterium, à l'aide d'un système crispr/cas, d'une recombinase et d'un acide oligodésoxyribonucléique simple brin WO2018182361A1 (fr)

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KR102201720B1 (ko) 2018-05-24 2021-01-12 한화솔루션 주식회사 1,3-pdo 생성능을 가지고 3-hp 생성능이 저해된 재조합 코리네박테리움 및 이를 이용한 1,3-pdo의 제조방법
KR102223521B1 (ko) 2019-02-26 2021-03-05 한국과학기술원 안트라닐산 메틸 생성능을 가지는 재조합 미생물 및 이를 이용한 안트라닐산 메틸의 제조방법

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CN117264990B (zh) * 2023-11-20 2024-02-06 中国农业科学院北京畜牧兽医研究所 一种时序性调控RecET和Cas12a表达的谷氨酸棒杆菌基因编辑质粒及编辑方法

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