WO2020246845A1

WO2020246845A1 - Mutant strain for improving genome of microorganism, method for preparing same, and method for improving genome of microorganism by using same

Info

Publication number: WO2020246845A1
Application number: PCT/KR2020/007332
Authority: WO
Inventors: 최용준; 정선욱
Original assignee: 서울시립대학교 산학협력단
Priority date: 2019-06-05
Filing date: 2020-06-05
Publication date: 2020-12-10
Also published as: KR20200140442A; KR102194368B1

Abstract

The present invention relates to a mutant strain for improving the genome of a microorganism, a method for preparing same, and a method for improving the genome of a microorganism by using same, wherein the mutant strain for improving the genome of a microorganism prepared by the method of the present invention does not require re-introduction of a plasmid capable of expressing a site-specific recombinant enzyme such as a Cre recombinant enzyme in a strain to remove the sequence encoding an antibiotic resistance-related protein, which is a selective marker artificially inserted into the genome, and therefore, by utilizing the mutant strain, it is possible to efficiently replace or delete a target gene sequence in the strain or insert a target sequence into the strain, such that a target gene of the strain can be efficiently modified, and furthermore the mutant strain can be utilized to prepare a mutant strain of the genome of a microorganism for basic research.

Description

Mutant strain for improving the genome of microorganisms, manufacturing method thereof, and method of improving genome of microorganisms using the same

The present invention relates to a mutant strain for improving the genome of a microorganism, a method for preparing the same, and a method for improving the genome of a microorganism using the same.

Microbial metabolic engineering is a technology that improves the genome of microorganisms and uses them for a desired purpose. Useful metabolites can be obtained from the genetic information and metabolic circuits of microorganisms, and more useful metabolites or technologies that can improve their properties are being developed through manipulation of microbial genes. In particular, in the field of metabolic engineering, site-specific recombination technologies such as CRISPR/Cas9, Flp/FRT, and cre/lox are applied to various models of living organisms such as plants, yeast, and microorganisms. It is possible to produce mutant strains (Datsenko, K, A et al, PNAS 97:6640-6645, 2000; Gilbertson, L Trends in biotechnology 21:550-555, 2003; Baba, T et al. Molecular systems biology 2, 2006; Yan, X et al, Applied and environmental microbiology 74:5556-5562, 2008).

Meanwhile, in order to improve the microbial genome, a plasmid capable of self-replicating in the microorganism to be improved is essential for the introduction of foreign genes and overexpression of a specific protein. The system is being introduced. Examples thereof include a promoter for expressing the Cre recombinant enzyme, an Origin for self-replication in cells, and a plasmid containing an antibiotic resistance gene. However, it may be host specific because it must be a plasmid capable of self-replicating in cells. To overcome this, a broad-host-range plasmid has been developed to overexpress a specific gene in a variety of microbial organisms, but the stability in cells is different for each microbial strain. It is difficult to apply to microorganisms. In addition, when the gene introduced by the plasmid is overexpressed, the cell mass and productivity may finally decrease depending on the plasmid copy number and the expression strength of the promoter in the cell. Accordingly, methods have been devised to increase the expression of specific genes according to a specific time and growth phase of the cells through treatment of an inducer such as arabinose, tryptophan, and IPTG, but the above method Depending on the type and concentration of the inducer, the expression level of the target gene is large, it is difficult to continuously and stably express the gene, and there are difficulties in industrial application due to the cost of the inducer to be processed. In addition, since these inducers may be toxic to the cells, there is a disadvantage in that they may affect the amount of cells.

On the other hand, Deinococcus radiodurans is known to have resistance to ionizing radiation that can directly damage the DNA or protein of an organism (Makarova, KS et al Microbiology and molecular biology reviews 65:44-79, 2001). . The strain can recover DNA damage caused by ionizing radiation in a short time (Grimsley, JK et al, International journal of radiation biology 60:613-626, 1991), and is produced in cells by enzymatic or non-enzymatic systems. It has the ability to effectively remove the activated oxygen species (ROS) (Shashidhar, R et al, Canadian journal of microbiology 56:195-201, 2010). Due to these characteristics, Deinococcus radiodurans are used for the purification of radioactive waste. In addition, cases of technology applying Deinococcus radiodurance are expected to increase in various fields such as medicine, health care, and biotechnology.

As reported so far, for metabolic engineering control of Deinococcus radiodurans, a technique for producing a mutant strain by introducing a gene of another microorganism into cells or using a homologous recombination method has been devised. However, this mutant production technique not only takes a lot of time due to the repetitive gene cloning process, but also has difficulty in producing multiple mutant strains due to the limitation of antibiotic markers for selecting mutant strains. In other words, in order to remove the antibiotic resistance gene, which is a selective marker artificially inserted into the genome, a plasmid capable of expressing a site-specific recombinant enzyme such as Cre recombinase is re-introduced in the strain and removed from the strain again. It requires repetitive work.

Therefore, the inventors of the present invention cre-lox By applying a position-specific recombination technology such as the system , an antibiotic-resistance-related protein containing a trc promoter , a sequence encoding a cre recombinant enzyme, and a lox nucleotide sequence at both ends Preparation of a recombinant plasmid containing the encoding sequence and the sequence encoding the lacΙq protein, and introducing the recombinant plasmid into the strain to include a trc promoter , a sequence encoding a cre recombinant enzyme, and a lox nucleotide sequence at both ends related to antibiotic resistance Protein The nucleic acid fragment containing the encoding sequence and the sequence encoding the lacΙq protein was successfully inserted into the DH-1 strain of the genus Deinococcus radiodurans and Methylomonas. Thereafter, Cre recombinase is conditionally expressed by an IPTG inducer, and the antibiotic resistance-related protein inserted in the strain is It was confirmed that the encoding sequence was completely removed, and a mutant strain in which the above process was stably maintained was obtained. Then, it was confirmed that the target gene was efficiently deleted using the mutant strain prepared above. Accordingly, it is possible to provide a method of efficiently replacing, deleting, or inserting a target sequence of a target gene sequence for various strains using the method of preparing the mutant strain to modify a target gene in the strain.

It is an object of the present invention to provide a method for preparing a mutant strain for improving the genome of a microorganism and a mutant strain prepared by the method.

To achieve the above object,

The present invention relates to a sequence encoding an upper fragment sequence of a target intergenic region, a trc promoter, and a cre protein regulated by it, a sequence encoding an antibiotic resistance-related protein including a lox nucleotide sequence at both ends, a lacIq protein A polynucleotide is provided in which a sequence encoding a sequence and a sequence of a lower fragment of a target intergenic region are sequentially linked.

In addition, the present invention comprises the steps of 1) introducing the polynucleotide of claim 1 into a strain; And

2) The sequence encoding the trc promoter and the cre protein regulated by the trc promoter in the target intergenic region of the strain, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends and the lacIq protein It provides a method for producing a mutant strain comprising; the step of inserting the sequence.

In addition, the present invention comprises the steps of 1) introducing the polynucleotide of claim 1 into a strain;

2) The sequence encoding the trc promoter and the cre protein regulated by the trc promoter in the target intergenic region of the strain, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends and the lacIq protein The step of inserting the sequence; And

3) It provides a method of producing a mutant strain further comprising the step of removing a sequence encoding an antibiotic resistance-related protein by culturing in a medium to which isopropyl β-D-1-thiogalactopyranoside (IPTG) is added.

In addition, the present invention provides a mutant strain in which the trc promoter prepared by the above method and the sequence encoding the cre protein and the sequence encoding the lacIq protein are inserted.

In addition, the present invention is the purpose of the mutant strain by introducing a polynucleotide in which the upper and lower fragment sequences of the target sequence are combined at both ends of the lox nucleic acid fragment containing the sequence encoding the antibiotic resistance-related protein to the mutant strain. It provides a way to modify genes.

The mutant strain for the improvement of the genome of the microorganism produced by the method of the present invention has a specific location such as cre recombinant enzyme in the strain to remove the sequence encoding the antibiotic resistance-related protein, which is a selective marker artificially inserted into the genome. Since there is no need to re-introduce a plasmid capable of expressing an enemy recombinant enzyme, the mutant strain can be used to efficiently replace, delete, or insert the target sequence into the strain, thereby efficiently modifying the target gene of the strain. In addition, it can be usefully used to produce a genome mutant strain of microorganisms for basic research.

1 is a diagram showing a process of preparing a mutant strain into which a trc promoter and a sequence encoding a cre protein regulated by the trc promoter and a sequence encoding a lacIq protein are inserted.

2A is a diagram showing a pAM1 plasmid comprising a sequence encoding a lox nucleotide sequence, a kat promoter, and a kanamycin resistance-related protein.

2B is a diagram showing a pAM3 plasmid comprising a trc promoter, a sequence encoding a cre protein, and a sequence encoding a lacIq protein.

3 is a process for preparing a pAM4 plasmid comprising a trc promoter, a sequence encoding a cre protein, a kat promoter including a lox nucleotide sequence at both ends, a sequence encoding a kanamycin resistance-related protein, and a sequence encoding a lacIq protein. It is a diagram shown.

Figure 4 is a kanamycin resistance including a partial nucleotide sequence of the Dinococcus radiodurans dr_C0022 gene and an upper fragment sequence of the target intergenic region, a trc promoter and a sequence encoding a cre protein regulated by it, and a lox nucleotide sequence at both ends. It is a diagram showing a pAM5 plasmid including a sequence encoding a related protein, a sequence encoding a lacIq protein, a lower fragment sequence of a target intergenic region, and a partial nucleotide sequence of a dr_C0023 gene.

Figure 5 is a mutant strain in which the sequence encoding the trc promoter and the cre protein regulated by it, the sequence encoding the kanamycin resistance protein including the lox nucleotide sequence at both ends, and the sequence encoding the lacIq protein are inserted, including IPTG. It is a diagram showing a process of producing a Deinococcus radiodurans mutant strain (DrCre) from which the sequence encoding a kanamycin resistance-related protein has been removed by culturing in a medium.

Figure 6a is a wild-type Deinococcus radiodurans strain (WT), a sequence encoding a trc promoter and a cre protein regulated by the introduction of the pAM5 plasmid, a sequence encoding a kanamycin resistance protein including a lox nucleotide sequence at both ends And Deinococcus radiodurans strain (DrCreKm) in which the sequence encoding the lacIq protein was inserted and the cre recombinant enzyme was expressed by IPTG and the sequence encoding the kanamycin-resistant protein was removed (DrCre). It is a diagram showing the result of PCR amplification of the mutation site of.

Figure 6b is a diagram showing the results of culturing the strain of Figure 6a in TGY medium containing kanamycin and TGY medium not containing kanamycin.

7A is a Deinococcus radiodurans strain (DrCre) from which the cre recombinant enzyme was expressed by IPTG and the sequence encoding the kanamycin resistance-related protein was removed, the crtB and crtI genes were deleted, and the kanamycin resistance-related protein was encoded. TGY medium containing kanamycin and kanamycin are not included in the strain in which the sequence was not removed (ΔcrtBI_Km) and the strain in which the crtB and crtI genes were deleted (deletion) and the sequence encoding the kanamycin resistance-related protein was removed (ΔcrtBI). It is a diagram showing the result of culturing in a non-TGY medium.

Figure 7b is the color of the Deinococcus radiodurans strain (DrCre) from which the cre recombinant enzyme was expressed by IPTG and the sequence encoding the kanamycin resistance-related protein was removed, and the strain in which the crtB and crtI genes were deleted. This is a comparison diagram.

Figure 8a is a wild-type methylomonas sp. DH-1 strain (WT), a sequence encoding a trc promoter and a cre protein regulated by the introduction of the pAM6 plasmid, encoding a kanamycin resistance-related protein including lox nucleotide sequences at both ends DH-1 strain (DH-1:Km) of the genus Methylomonas into which the sequence and the sequence encoding the lacIq protein was inserted, and the cre recombinant enzyme was expressed by IPTG, and the sequence encoding the kanamycin resistance-related protein was removed. It is a diagram showing the results of electrophoresis of the DH-1 strain (DH-1:cre) of the genus Monas.

1: Wild type methylomonas sp. DH-1 strain (WT)

2: DH-1:Km

3: DH-1:cre

8B is a diagram showing the results of culturing the strain of FIG. 8A in an NMS medium containing kanamycin and an NMS medium not containing kanamycin.

1: Wild type methylomonas sp. DH-1 strain (WT)

2: DH-1:Km

3: DH-1:cre

9 is a partial nucleotide sequence of the DH-1 AYM39_00230 gene of the genus Methylomonas and an upper fragment sequence of the target intergenic region, a trc promoter and a sequence encoding a cre protein regulated thereby, kanamycin including a lox nucleotide sequence at both ends. It is a diagram showing a pAM6 plasmid including a sequence encoding a resistance-related protein, a sequence encoding a lacIq protein, a lower fragment sequence of a target intergenic region, and a partial nucleotide sequence of the AYM39_00235 gene.

Hereinafter, the present invention will be described in detail.

The present invention relates to a sequence encoding an upper fragment sequence of a target intergenic region, a trc promoter, and a Cre protein regulated by it, a sequence encoding an antibiotic resistance-related protein comprising a lox nucleotide sequence at both ends, a lacIq protein A polynucleotide is provided in which a sequence encoding a sequence and a sequence of a lower fragment of a target intergenic region are sequentially linked.

The term "intergenic region" used in the present invention refers to a DNA sequence located between portions where genes are clustered, and refers to a DNA sequence that does not encode a protein. This is different from introns (non-expression sites, introns, intragenic regions), which are short, protein-free regions found in genes in eukaryotes. Some intergenic regions control the expression of adjacent genes, but most of them do not function. Not known The intergenic region is one of the DNA sequences collectively called junk DNA, but recently it is known to contain the DNA sequence of a promoter or enhancer, and it has been revealed that not all intergenic regions are junk DNA.

The intergenic region may be a DNA sequence located between the dr_C0021 gene and the dr_C0022 gene of the Deinococcus radiodurans strain, but is not limited thereto.

The intergenic region may be a DNA sequence located between the AYM39_00230 gene and the AYM39_00235 gene of the DH-1 strain of the genus Methylomonas, but is not limited thereto.

The upper fragment sequence of the target intergenic region may be a 770 bp nucleotide sequence including a part of the nucleotide sequence of the dr_C0022 gene and a 5′ upstream region of the target intergenic region, but is not limited thereto.

The upper fragment sequence of the target intergenic region may be a 820 bp nucleotide sequence including a part of the nucleotide sequence of the AYM39_00230 gene and a 5′ upstream region of the target intergenic region, but is not limited thereto.

The lower fragment sequence of the target intergenic region may be a 710 bp nucleotide sequence including a downstream region of the target intergenic region and a part of the nucleotide sequence of the dr_C0023 gene, but is not limited thereto.

The lower fragment sequence of the target intergenic region may be a 810 bp nucleotide sequence including a part of the nucleotide sequence of the AYM39_00235 gene and a downstream region of the target intergenic region, but is not limited thereto.

The term "promoter" as used in the present invention is an untranslated nucleic acid sequence upstream of the coding region that includes a binding site for RNA polymerase and has an activity to initiate transcription of a lower gene of the promoter into mRNA. Means. Specifically, "promoter" refers to a TATA box or TATA box-like, which is located upstream from the transcription initiation point (+1) to the 20th to 30th base, and is responsible for initiating transcription to RNA polymerase from the correct position. Regions are included, but are not necessarily limited before and after these regions, and regions other than these regions may include regions necessary for association of proteins other than RNA polymerase for expression regulation. The promoter is operably linked to induce the expression of a target gene, which is a foreign gene, wherein "operably linked" refers to a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. It refers to a functional connection. The operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.

The promoter used in the present invention may be a Kat promoter or a trc promoter, but is not limited thereto.

The term "Kat promoter" used in the present invention is preferably a base sequence (SEQ ID NO: 1) containing 101bp of an upstream region of the KatE1 (NCBI acc. number: 1800077) gene.

The term "trc promoter" used in the present invention is preferably a 74bp base sequence (SEQ ID NO: 2) contained in the ptrc99a plasmid (Pharmarcia Biotech, Uppsala, Sweden).

The term "sequence encoding Cre protein" as used herein refers to a DNA sequence expressing a Cre recombinant enzyme, and pAM2 plasmid (Jeong et al., Korean J. Chem. Eng. 34:1728-1733, 2017) It is preferably a base sequence of 1032 bp (SEQ ID NO: 25) contained in.

The term "lox nucleotide sequence" used in the present invention may be lox 66 (SEQ ID NO: 26) or lox71 (SEQ ID NO: 27), but is not limited thereto.

In the present invention, the term "selective marker" means a product of a specific gene. Microorganisms containing the product have special traits that do not appear in microorganisms that do not contain them, thereby enabling the microbial to be distinguished.

The selection marker may be a sequence encoding an antibiotic resistance-related protein, but is not limited thereto.

The term "antibiotic resistance-related protein" used in the present invention is a protein that allows bacteria to proliferate without being affected by a specific antibiotic, and may be an antibiotic degrading enzyme, but is not limited thereto. An example of an antibiotic degrading enzyme is the beta-lactamase (β-lactamase) possessed by Streptomyces clavuligerus. Beta-lactamase (β-lactamase) is a hydrolytic enzyme that cleaves the beta-lactam ring of beta-lactams antibiotics.

The term "sequence encoding an antibiotic resistance-related protein" as used herein means a DNA sequence expressing the antibiotic resistance-related protein.

The antibiotic may be one selected from the group consisting of kanamycin, chloramphenicol, spectinomycin, and streptomycin, but is not limited thereto.

The term "sequence encoding lacΙq protein" as used in the present invention is a DNA sequence expressing a repressor protein. It binds to the trc promoter under normal culture conditions to prevent binding to RNA polymerase, and is linked to the trc promoter. It is a regulatory protein that plays a role in inhibiting the transcription of genes, and is preferably a nucleotide sequence of 1,092 bp (SEQ ID NO: 3) contained in the ptrc99a plasmid.

As used herein, the term "Cre-lox system" refers to a site-specific recombinant enzyme technology used to perform deletion, insertion, translocation, and inversion at a specific site of DNA. The Cre-lox system can be used to mutate genes in eukaryotic and prokaryotic organisms. It consists of a single enzyme, Cre recombinase, and recombines the short target sequence, lox sequence. By appropriately arranging the lox sequence at a position to cause mutation, the target gene may be activated or suppressed, or may be substituted with another gene. In general, lox P is a bacteriophage P1 site composed of 34 bases, contains an asymmetric 8 bp nucleotide sequence, and has 2 pairs of palimdromics with a size of 13 bp except for 2 bases in the center ( ATAACTTCGTATANNNTANNN-TATACGAAGTTAT).

The term "transformation" as used in the present invention means introducing a specific foreign DNA strand from outside the cell into the cell. Host microorganisms including the introduced DNA strands are called "transformed microorganisms." 'Transformation', which means that DNA can be replicated as an extrachromosomal factor or by the completion of chromosomal integration by introducing DNA into a host cell, or introducing a vector containing a polynucleotide encoding a target protein into a host cell. It means to integrate and complete the encoding polynucleotide into the chromosome of the host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides include all of them, whether inserted into the host cell's chromosome or outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette usually includes a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.

Further, the present invention provides a vector comprising the polynucleotide.

The term "vector" as used herein refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles, or simply potential genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are currently the most commonly used form of vectors, plasmids and vectors are sometimes used interchangeably in the specification of the present invention. However, the present invention encompasses other forms of vectors that have functions equivalent to those known or become known in the art.

Vectors containing the polynucleotide can be prepared by gene recombination techniques well known in the art, and cleavage and linkage of site-specific DNA sequences are generally known in the art and DNA ligase can be used.

The vector is a plasmid vector, cosmid vector, HIV (Human immunodeficiency virus) vector, MLV (Murineleukemia virus) vector, ASLV (Avian sarcoma/leukosis) vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, It may be any one selected from the group consisting of a mouse mammary tumor virus (MMTV) vector, an adenovirus vector and a herpes simplex virus vector, a lentivirus vector, and an episomal vector, and , Preferably, it may be a plasmid vector, but is not limited thereto.

The restriction enzyme collectively refers to an enzyme that cleaves a DNA sequence by binding to a specific DNA sequence.

2) In the target intergenic region of the strain, the trc promoter and the sequence encoding the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, and the lacIq protein It provides a method for producing a mutant strain comprising; the step of inserting the sequence.

The strains include Methylomonas sp., Escherichia coli, Bacillus subtilis, D. radiodurans, D. indicus, D. indicus, and Deinococcus. D. caeni, D. aquaticus, D. depolymerans, D. grandis, Deinococcus dezonesis (D. daejeonensis), D. radiotolerans, D. geothermalis, D. ruber, D. antarcticus, D. antarcticus ), D. proteolyticus, D. radiopugnans, D. radiophilus, D. proteolyticus, D. cellulosilyticus) and Deinococcus swuensis (D. swuensis) may be any one or more selected from the group consisting of, but is not limited thereto, and may be any microorganism whose entire nucleotide sequence is known.

2) In the target intergenic region of the strain, the trc promoter and the sequence encoding the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, and the lacIq protein The step of inserting the sequence; And

The removal of the sequence encoding the antibiotic resistance-related protein may be performed through expression of the Cre recombinant enzyme.

The concentration of IPTG may be 0.5mM to 1.0mM, preferably 1.0mM, but is not limited thereto.

The term "target sequence" as used in the present invention refers to a portion of a target gene, for example, one or more exon sequences of a target gene, an intron sequence or a regulatory sequence of a target gene, or an exon of a target gene, unless specifically specified otherwise. And intron sequences, introns and regulatory sequences, exons and regulatory sequences, or combinations of exons, introns and regulatory sequences.

The target gene may be crtB and crtI , but is not limited thereto.

The upper fragment sequence of the target sequence may be 0.5 to 1 kb, preferably 1 kb, but is not limited thereto.

The lower fragment sequence of the target sequence may be 0.5 to 1 kb, preferably 1 kb, but is not limited thereto.

The lox nucleic acid fragment containing the sequence encoding the antibiotic resistance-related protein may sequentially include a sequence encoding a lox66, a kat promoter, a kanamycin resistance-related protein, and lox71, but is not limited thereto.

In addition, the present invention provides a method for producing a mutant strain in which the sequence encoding an antibiotic resistance-related protein is removed by culturing the mutant strain in which the target gene is modified in a medium to which Isopropyl β-D-1-thiogalactopyranoside (IPTG) is added. do.

The modification may be a substitution, deletion of the target gene sequence, or insertion of the target sequence, but is not limited thereto.

In a specific embodiment of the present invention, a sequence encoding a trc promoter and a Cre protein regulated by the trc promoter in the deinococcus radiodurans dr_C0022 gene and the dr_C0023 intergenic region, and an antibiotic comprising a lox nucleotide sequence at both ends To prepare a mutant strain in which a sequence encoding a resistance-related protein and a sequence encoding a lacIq protein were inserted (FIG. 5), the strain was cultured in a medium to which Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, thereby kanamycin resistance. A mutant strain from which a sequence encoding a related protein was removed was obtained and named as a basic mutant strain (DrCre) for genome improvement. Using the mutant strain, a polynucleotide in which a partial sequence of crtB and a partial sequence of crtI are conjugated to both ends of a lox nucleic acid fragment containing a sequence encoding a kanamycin resistance-related protein, respectively, and the crtB and crtI genes of the mutant strain Was deleted, and cultured in a medium to which IPTG was added to remove the sequence encoding the kanamycin resistance-related protein, thereby obtaining a Deinococcus radiodurans strain from which all the sequences encoding the crtB and crtI genes and the kanamycin resistance-related protein were removed. First, it was confirmed that the genome of the strain can be improved using the basic mutant strain (FIGS. 7A and 7B). In addition, it was confirmed that the DH-1 mutant strain of the genus Methylomonas stably expressing the Cre recombinant enzyme was obtained, and the genome improvement method of the present invention can be applied regardless of the type of the strain (FIGS. 8A and 8B ).

Therefore, when the basic mutant strains (DrCre and DH-1:Cre) of the present invention are used in a method of modifying a target gene, in order to remove a selective marker artificially introduced to select the transformed strain Since there is no need to introduce a separate plasmid and go through the process to remove it, the target gene can be easily and efficiently modified, and furthermore, it can be usefully used to create a genome mutant strain of microorganisms for basic research. .

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.

<실시예 1> trc 프로모터, Cre 단백질을 암호화하는 서열 및 lacΙq 단백질을 암호화하는 서열을 포함하는 플라스미드(pAM3)의 제작<Example 1> Preparation of a plasmid (pAM3) containing a trc promoter, a sequence encoding a Cre protein, and a sequence encoding a lacΙq protein

A plasmid (pAM3) containing a trc promoter, a sequence encoding a Cre protein, and a sequence encoding a lacΙq protein (pAM3) was prepared by the following method.

First, CreF having the nucleotide sequence shown in Table 1 below using the pAM2 plasmid (Jeong et al., Korean J. Chem. Eng. 34:1728-1733, 2017; patent registration number, 10-1835852) as a template (SEQ ID NO: 5) and CreR (SEQ ID NO: 6) primers were used to amplify the sequence encoding the Cre protein. Specifically, a mixture of 1 µl of pAM2, 1 µl of each of CreF and CreR primers (10 pmole/µl) and 17 µl of sterile distilled water was added to the pfu polymerase mix (Bioneer), followed by T-100 Thermal cycler. PCR was performed using a DNA amplifier (Bio-rad). PCR was performed by reacting at 95° C. for 5 minutes, then repeating the reaction process of 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes once and repeating this 25 times.

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호 5SEQ ID NO: 5	CreFCreF	gatcgaattcatgtccaatttactgcccgtagatcgaattcatgtccaatttactgcccgta
서열번호 6SEQ ID NO: 6	CreRCreR	gatcgtcgacctaatcgccatcttccagcagatcgtcgacctaatcgccatcttccagca

The obtained PCR product was confirmed by applying a voltage of 120V on a 1% agarose gel and electrophoresis for 20 minutes, put it in a 1.5ml tube, and purified using a DNA fragment purification kit (Intron lifetechnology). The obtained PCR product and pTrc99a plasmid (Invitrogen) were added to 5 µl of buffer solution, 1 µl of EcoR I, 1 µl of Sal I, 20 µl of PCR product or pTrc99a DNA solution, and 23 µl of sterile distilled water to a 1.5 ml tube. It was reacted at for 6 hours and digested with EcoRI and SalI restriction enzymes, respectively. Then, 7 µl of restriction enzyme-treated PCR product, 1 µl of pTrc99a, 1 µl of T4 DNA ligase (Enzynomics), and 1 µl of a buffer solution were added to a 1.5 ml tube, followed by reaction at 16° C. for 8 hours for ligation. The DNA ligation mixed solution was transformed into E. coli ( E. coli DH5α), and plasmid DNA was extracted from the transformed E. coli using a plasmid preparation kit (intron lifetechnology) according to the method recommended by the manufacturer, and the corresponding nucleotide sequence was analyzed. Finally, the cloned plasmid was confirmed, and it was named pAM3 (SEQ ID NO: 7) plasmid (Fig. 2b).

<실시예 2> trc 프로모터, Cre 단백질을 암호화하는 서열, 양 말단에 loxP 염기 서열을 포함하는 카나마이신 (kanamycin) 저항성 관련 단백질을 암호화하는 서열 및 lacΙq 단백질을 암호화하는 서열을 포함하는 플라스미드(pAM4)의 제작<Example 2> of a plasmid (pAM4) comprising a trc promoter, a sequence encoding a Cre protein, a sequence encoding a kanamycin resistance-related protein including a loxP nucleotide sequence at both ends, and a sequence encoding a lacΙq protein making

A plasmid (pAM4) including a sequence encoding a trc promoter, a sequence encoding a Cre protein, a kanamycin resistance-related protein including a loxP nucleotide sequence at both ends and a sequence encoding a lacΙq protein (pAM4) was prepared in the following manner. Was prepared.

<2-1> Obtaining a sequence encoding a Cre protein containing a trc promoter

In order to obtain a sequence encoding a Cre protein containing a trc promoter, the pAM3 plasmid prepared by the method of Example 1 was used as a template, and trcCreF (SEQ ID NO: 8) and trcCreR having the nucleotide sequences shown in Table 2 below. (SEQ ID NO: 9) A sequence encoding a Cre protein containing a trc promoter was amplified using a primer. Specifically, PCR was performed using 2 µl of pAM3, 1 µl of trcCreF and 2 µl of trcCreR primers (10 pmole/µl) and 16 µl of sterile distilled water using AccuPower ^™ pfu PCR premix mix (Bioneer). As a result, a PCR product of the sequence encoding the Cre protein containing the trc promoter was obtained.

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호 8SEQ ID NO: 8	trcCreFtrcCreF	ctagggtaccgccgacatcataacggttctctagggtaccgccgacatcataacggttct
서열번호 9SEQ ID NO: 9	trcCreRtrcCreR	gatcctcgagtgtcctactcaggagagcgtgatcctcgagtgtcctactcaggagagcgt

<2-2> Obtaining a sequence encoding a kanamycin resistance-related protein including lox nucleotide sequences at both ends

In order to obtain a sequence encoding a kanamycin resistance-related protein comprising lox nucleotide sequences at both ends, pAM1 in Fig. 2A (Jeong et al., 2017 Korean J. Chem. Eng ., 4June 2017, Volume 34, Issue 6, pp 1728-1733, and Patent Registration No. 10-1835852) as a template, except that the loxKmF (SEQ ID NO: 10) and loxKmR (SEQ ID NO: 11) primers having the nucleotide sequence shown in Table 3 were used. PCR was performed under the same conditions and methods as in 1. As a result, a PCR product of a sequence encoding a kanamycin resistance-related protein including a lox nucleotide sequence was obtained.

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호 10SEQ ID NO: 10	loxKmFloxKmF	ggtaccgggccccccctcgaggtcggtaccgggccccccctcgaggtc
서열번호 11SEQ ID NO: 11	loxKmRloxKmR	ctctagaggatcctaccgttcgtactctagaggatcctaccgttcgta

<2-3> Obtaining a sequence encoding lacΙq protein

In order to obtain a sequence encoding a lacΙq protein, the pAM3 plasmid prepared in Example 1 was used as a template, except that the primers of lacΙqF (SEQ ID NO: 12) and lacΙqR (SEQ ID NO: 13) described in Table 4 were used. Then, PCR was performed under the same conditions and methods as in Example 1. As a result, a PCR product of the sequence encoding the lacΙq protein was obtained.

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호 12SEQ ID NO: 12	lacΙqFlacΙqF	ctagggatcctcctgcgttatcccctgattctagggatcctcctgcgttatcccctgatt
서열번호 13SEQ ID NO: 13	lacΙqRlacΙqR	taacgtcgactctagatcactgcccgctttccagtctaacgtcgactctagatcactgcccgctttccagtc

<2-4> of a plasmid (pAM4) comprising a trc promoter, a sequence encoding a Cre protein, a sequence encoding a kanamycin resistance-related protein including a lox nucleotide sequence at both ends, and a sequence encoding a lacΙq protein making

The PCR product of the sequence encoding the Cre protein containing the trc promoter obtained in Example 2-1 was digested with restriction enzymes Kpn I and Xho I, and lox nucleotide sequences at both ends obtained in Example 2-2. The PCR product of the sequence encoding the kanamycin resistance-related protein including, was digested with restriction enzymes Kpn I and Xba I, and the PCR product of the sequence encoding the lacΙq protein obtained in Example 2-3 was restriction enzyme B amH I A plasmid prepared by digesting with Sal I and ligated to pUC19 plasmid (SEQ ID NO: 36) was named pAM4 (SEQ ID NO: 4) (FIG. 3).

<실시예 3> trc 프로모터, Cre 단백질을 암호화하는 서열, 양 말단에 lox 염기 서열을 포함하는 카나마이신 저항성 관련 단백질을 암호화하는 서열 및 lacΙq 단백질을 암호화하는 서열이 삽입된 데이노코쿠스 라디오두란스 변이 균주(DrCreKm)의 제조<Example 3> Deinococcus radiodurans mutant strain in which a sequence encoding a kanamycin resistance-related protein including a trc promoter, a Cre protein encoding sequence, a lox nucleotide sequence at both ends, and a sequence encoding a lacΙq protein were inserted Preparation of (DrCreKm)

<3-1> the sequence encoding the upper fragment sequence of the target intergenic region, the trc promoter and the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, Construction of a plasmid (pAM5) containing the sequence encoding the lacIq protein and the lower fragment sequence of the target intergenic region

Using the DNA of Deinococcus radiodurans as a template, the nucleotide sequence of 770 bp including a partial sequence of the drC0022 gene and the intergenic region is the primers of drc0022upF (SEQ ID NO: 14) and drc0022upR (SEQ ID NO: 15) in Table 5 below, In the same conditions as in Example 1, except for using the primers of drc0023dnF (SEQ ID NO: 16) and drc0023dnR (SEQ ID NO: 17) shown in Table 5 below, and the nucleotide sequence of 710bp including a partial sequence of the intergenic region and the drC0023 gene. PCR was performed by the method. As a result, a PCR product of a nucleotide sequence of 770 bp including a partial sequence of the drC0022 gene and an intergenic region and a nucleotide sequence of 710 bp including a partial sequence of the intergenic region and the drC0023 gene was obtained.

The 770 bp nucleotide sequence PCR product containing the drC0022 gene and some intergenic regions was digested with restriction enzymes Nde I and Kpn I, and the 710 bp nucleotide sequence containing the drC0023 gene was digested with restriction enzymes Nde I and Kpn I. Silver restriction enzymes Sal I and Sph I were digested and ligated to the pAM4 plasmid prepared by the method of Example 2-4 to prepare a pAM5 plasmid (SEQ ID NO: 18) (FIG. 4).

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호14SEQ ID NO: 14	drc0022upFdrc0022upF	atcgcatatgggtgcgcatctcaagtcttgatcgcatatgggtgcgcatctcaagtcttg
서열번호15SEQ ID NO: 15	drc0022upRdrc0022upR	ctagggtaccggcctgtcactgtgattaagctagggtaccggcctgtcactgtgattaag
서열번호16SEQ ID NO: 16	drc0023dnFdrc0023dnF	ctgggtcgaccggagcatgacctaagaggtctgggtcgaccggagcatgacctaagaggt
서열번호17SEQ ID NO: 17	drc0023dnRdrc0023dnR	tacggcatgcggtgatcgcgagttggcgtttacggcatgcggtgatcgcgagttggcgtt

<3-2> Preparation of a mutant strain in which the sequence encoding the trc promoter, the Cre protein, the kanamycin resistance-related protein including the lox nucleotide sequence at both ends, and the sequence encoding the lacΙq protein are inserted

First, deinococcus radiodurans (ATCC 13939, Agricultural Genetic Resource Information Center, Korea) strain was used in TGY medium (0.5% (w/v) tryptone, 0.1% (w/v) glucose and 0.3% (w). /v) yeast extract) was incubated at a temperature of 30°C until a value of 600 nm of OD (optical density) reached 0.3. The cultured cells were centrifuged for 15 minutes at 4000 rpm and 4° C., and suspended in 2x TGY medium containing 30 mM calcium chloride (CaCl ₂ ), and the suspension was allowed to stand on ice for 1 hour. This was again centrifuged at 4000rpm and 4℃ for 15 minutes to remove the supernatant, and resuspended by adding TGY medium containing 30 mM calcium chloride (CaCl ₂ ) and 10% (v/v) glycerol, and then sterilized. It was prepared by dispensing 50 µl into a 1.5 ml tube.

To 50 µl of the prepared Deinococcus riodurans strain, 10 µl of the pAM5 plasmid DNA prepared in Example 3-1 and 2 X TGY solution containing 30 mM calcium chloride (CaCl ₂ ) were added, and left to stand on ice for 30 minutes. After that, it was reacted at 32° C. for 1.5 hours. Then, 900 µl of 2 X TGY solution was added and incubated for 16 hours at 30° C., and then 2 X TGY agar medium (1% (w/v) tryptone, 0.2%) containing 25 µg/ml kanamycin antibiotic (w/v) Glucose, 0.6% (w/v) yeast extract and 1.5% (w/v) Bactoagar) were plated 200 µl and cultured in a 30° C. incubator for 3 to 4 days. Finally, in 2 X TGY agar medium containing 25 μg/ml kanamycin antibiotic, the trc promoter that formed a colony, the sequence encoding the Cre protein, and the kanamycin resistance-related protein including the lox nucleotide sequence at both ends were encoded. The mutant strain into which the sequence and the sequence encoding the lacΙq protein were inserted was confirmed by diagnostic PCR and sequencing, and the final selected strain was named DrCreKm (FIG. 5).

<실시예 4> trc 프로모터, Cre 단백질을 암호화하는 서열, 양 말단에 lox 염기 서열을 포함하는 카나마이신 저항성 관련 단백질을 암호화하는 서열 및 lacΙq 단백질을 암호화하는 서열이 삽입된 변이 균주로부터 카나마이신 저항성 관련 단백질을 암호화하는 서열의 제거<Example 4> A kanamycin resistance-related protein was prepared from a mutant strain in which a trc promoter, a sequence encoding a Cre protein, a sequence encoding a kanamycin resistance-related protein including a lox nucleotide sequence at both ends, and a sequence encoding a lacΙq protein were inserted. Removal of the coding sequence

The following method was performed in order to remove the sequence encoding the kanamycin resistance related protein from the DrCreKm strain prepared in Example 3-2.

Specifically, the DrCeKm strain prepared in Example 3-2 was cultured with shaking at 30° C. for 16 hours in 3 mL of TGY medium to which kanamycin was not added, and then until the value of OD (optical density) 600 nm became 0.25 1 Mm isoprophyl-β-D-1-thiogalactopyranoside (IPTG) was added and incubated for 16 hours under the same culture conditions. Thereafter, the culture solution was diluted to 1/1000, spread on a TGY agar medium containing 10 mM IPTG, and cultured at 30° C. until colonies were formed. Each single colony formed on the TGY agar medium was simultaneously inoculated into TGY agar medium containing kanamycin and TGY agar medium not containing kanamycin using a sterilized tip. Thereafter, strains that could not be grown in a medium containing kanamycin and grown only in a TGY agar medium not containing kanamycin were finally selected (FIGS. 5, 6A and 6B ).

The final selected strain was confirmed that the sequence encoding the kanamycin resistance-related protein was removed from the Deinococcus radiodurans mutant strain through diagnostic PCR, sequencing analysis, and plate assay, and the strain was Deinococcus radiodurans genome It was named as a basic mutant strain (DrCre) for improvement (FIGS. 5, 6A and 6B).

<실시예 5> <Example 5> crtBcrtB 및 And crtIcrtI 유전자가 결실된 데이노코쿠스 라디오두란스 균주의 제조 Preparation of Deinococcus radiodurans strain in which the gene was deleted

Example using a mutant strain obtained in fourth deleting the crtI gene to verify that can improve the genome of the strain, encoding a crtB gene and payito yen unsaturated enzyme encoding a synthase involved payito yen the carotenoid biosynthesis pathway ( deletion) was performed.

<5-1> obtaining crtB and crtI base sequences

Using Deinococcus radiodurans DNA as a template, the base sequence 1010bp (SEQ ID NO: 28) containing the crtB gene is the primers of dr6162-1 (SEQ ID NO: 19) and dr6162-2 (SEQ ID NO: 20) in Table 6 below, The nucleotide sequence 820bp (SEQ ID NO: 29) containing the crtI gene was the same conditions as in Example 1, except that the primers of dr6162-5 (SEQ ID NO: 23) and dr6162-6 (SEQ ID NO: 24) in Table 6 were used. And PCR was performed by the method. As a result, PCR products of the crtB and crtI genes were obtained, respectively.

서열번호Sequence number	이름name	서열(5'→3')Sequence (5'→3')
서열번호19SEQ ID NO: 19	dr6162-1dr6162-1	aagtagtcgggcgtgatgaaaagtagtcgggcgtgatgaa
서열번호20SEQ ID NO: 20	dr6162-2dr6162-2	agcttatcgataccgtcgacggcacgtatttcgactacgaagcttatcgataccgtcgacggcacgtatttcgactacga
서열번호21SEQ ID NO: 21	dr6162-3dr6162-3	tcgtagtcgaaatacgtgccgtcgacggtatcgataagcttcgtagtcgaaatacgtgccgtcgacggtatcgataagct
서열번호22SEQ ID NO:22	dr6162-4dr6162-4	attctggacgacctcgaacgcatgcctgcaggtcgactctattctggacgacctcgaacgcatgcctgcaggtcgactct
서열번호23SEQ ID NO: 23	dr6162-5dr6162-5	agagtcgacctgcaggcatgcgttcgaggtcgtccagaatagagtcgacctgcaggcatgcgttcgaggtcgtccagaat
서열번호24SEQ ID NO: 24	dr6162-6dr6162-6	tcaccgtgacggactattcatcaccgtgacggactattca

<5-2> Amplification of a sequence encoding a kanamycin resistance-related protein containing lox nucleotide sequences at both ends

Using the pAM1 plasmid as a template, a sequence encoding a kanamycin resistance-related protein containing lox71 and lox66 genes at both ends was obtained using dr6162-3 (SEQ ID NO: 21) and dr6162-4 (SEQ ID NO: 22) described in Table 6 above. Except that, PCR was performed under the same conditions and methods of Example 1. As a result, a PCR product of a sequence encoding a kanamycin resistance-related protein containing lox71 and lox66 genes at both ends was obtained.

<5-3> Linkage of a sequence encoding a kanamycin resistance-related protein containing crtB and crtI nucleotide sequences and lox nucleotide sequences at both ends

First, the PCR products obtained in Examples 5-1 and 5-2 were confirmed by electrophoresis on a 1% agarose gel, and these were respectively purified using a DNA fragment purification kit (Intron lifetechnology). The purified PCR product was mixed and used as a template, except that the primers dr6162-1 (SEQ ID NO: 19) and dr6162-6 (SEQ ID NO: 24) described in Table 6 were used and reacted for 4 minutes at 72°C in the PCR process. Then, PCR was performed under the same conditions and methods as in Example 1. As a result, a PCR product of 2450 bp was obtained in which a sequence encoding a kanamycin resistance-related protein containing crtB and crtI nucleotide sequences and lox nucleotide sequences at both ends was linked.

<5-4> Preparation of a mutant strain in which the crtB and crtI genes are deleted

First, the basic mutant strain (DrCre) prepared in Example 4 was TGY medium (0.5% (w/v) tryptone, 0.1% (w/v) glucose, and 0.3% (w/v) yeast extract. ) Was incubated at a temperature of 30° C. until a value of 600 nm of OD (optical density) reached 0.3. The cultured cells were centrifuged for 15 minutes at 4000 rpm and 4° C., and suspended in 2x TGY medium containing 30 mM calcium chloride (CaCl2), and the suspension was allowed to stand on ice for 1 hour. This was again centrifuged at 4000 rpm for 15 minutes at 4°C to remove the supernatant and resuspended by adding 50 µl of TGY medium containing 30 mM CaCl2 and 10% (v/v) glycerol, and then resuspended in a sterile 15 ml tube. Prepared by dispensing 50 µl into each.

Meanwhile, the PCR product obtained in Example 5-3 was electrophoresed on a 1% agarose gel, and this was purified using a DNA fragment purification kit (Intron lifetechnology).

To 50 µl of the prepared basic mutant strain (DrCre) prepared in Example 4, 10 µl of the DNA prepared in Example 5-3 and 2 X TGY solution containing 30 mM calcium chloride (CaCl ₂ ) were added, and then in ice. After allowing it to stand for 30 minutes, it was reacted at 32°C for 1.5 hours. Then, 900 µl of 2 X TGY solution was added and incubated for 16 hours at 30° C., and then 2 X TGY agar medium (1% (w/v) tryptone, 0.2%) containing 25 µg/ml kanamycin antibiotic (w/v) Glucose, 0.6% (w/v) yeast extract and 1.5% (w/v) Bactoagar) were plated 200 µl and cultured in a 30° C. incubator for 3 to 4 days. Finally, in 2 X TGY agar medium containing 25 μg/ml of kanamycin antibiotic, the crtB and crtI genes that formed colonies were deleted, and the sequence encoding the kanamycin resistance-related protein containing the lox nucleotide sequence at both ends was The included strain was obtained (Fig. 7a).

As a result, as shown in Fig. 7b, a mutant strain in which the crtB and crtI genes were deleted and carotenoids were not synthesized and thus did not appear orange was obtained, and this was designated as ΔcrtBI_Km (Fig. 7b).

<5-5> Preparation of a Deinococcus radiodurans mutant strain from which all sequences encoding crtB , crtI and kanamycin resistance-related proteins have been removed

Removal of the sequence encoding the kanamycin resistance-related protein contained in the ΔcrtBI_Km strain obtained in Example 5-4 was performed by expressing the sequence encoding the Cre protein introduced in the DrCre mutant strain prepared in Example 4, thereby recombining Cre As the enzyme is produced, it is removed at the same time.

Specifically, the ΔcrtBI_Km strain prepared in Example 5-2 was incubated for 16 hours or longer in a TY (trypton 0.5%, yeast extract 0.3%) liquid medium, and a new 3 mL TY liquid medium to which 1 mM IPTG was added. 30 µl of the culture solution of the ΔcrtBI_Km strain was added to the mixture, followed by further culturing at 37°C for 16 hours or longer. After diluting the culture solution in sterilized water at a ratio of 1:10000, 50 µl of the diluted solution was uniformly spread on TY solid medium containing 1 mM IPTG, followed by stationary culture in a 37°C incubator for 3 days. Each single colony formed on the TY solid medium was simultaneously inoculated into the TY solid medium containing 25 μg/ml of kanamycin and the TY solid medium not containing kanamycin using a sterile tip, and then at 37°C. Incubated for 1 day. Thereafter, as shown in Figure 7a, a single colony growing only in a medium containing no kanamycin was finally selected, and the sequence encoding crtB , crtI, and kanamycin resistance-related proteins were all removed, and the Deinococcus radiodurans mutant strain △ Named as crtBI (Fig. 7b). Through the above results, it was confirmed that using the basic mutant strain (DrCre) for genome improvement prepared in Example 4, a mutant strain in which both the sequence encoding the antibiotic resistance-related protein and the target gene were removed can be obtained.

<실시예 6> Cre 재조합 효소를 발현하는 메틸로모나스 속<Example 6> Genus Methylomonas expressing Cre recombinant enzyme (Methylomonas sp.)(Methylomonas sp.) DH-1 변이 균주의 제조 Preparation of DH-1 mutant strain

A mutant strain expressing Cre recombinase (DH-1) using a gram-positive strain of Deinococcus radiodurans as well as a gram-negative strain of methylomonas genus (DH-1: Cre) was prepared by the following method.

<6-1> Construction of a plasmid (pAM6) containing the gene of the DH-1 strain of the genus Methylomonas

<6-1-1> Construction of a plasmid containing AYM39_00230 gene and AYM39_00235 gene

Using the DNA of DH-1 of the genus Methylomonas as a template, the 820bp nucleotide sequence (SEQ ID NO: 38) including the AYM39_00230 gene and some sequences of the intergenic region is shown in Table 7 below: DH1 intRG-1 (SEQ ID NO: 30) and The primer of DH1 intRG-2 (SEQ ID NO: 31), a 810bp nucleotide sequence (SEQ ID NO: 39) including a partial sequence of the intergenic region and the AYM39_00235 gene is shown in Table 7 below: DH1 intRG-3 (SEQ ID NO: 32) and DH1 intRG PCR was performed in the same conditions and methods as in Example 1, except that a primer of -4 (SEQ ID NO: 33) was used.

As a result, a PCR product of a 820 bp nucleotide sequence including the AYM39_00230 gene and a partial sequence of the intergenic region and a 810 bp nucleotide sequence including the AYM39_00235 gene was obtained.

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호30SEQ ID NO: 30	DH1 intRG-1DH1 intRG-1	ctagggtacctgagttcgtattctgagccgctagggtacctgagttcgtattctgagccg
서열번호31SEQ ID NO: 31	DH1 intRG-2DH1 intRG-2	cgttatgatgcagcgcttcaaggcctagtgacgcaagttcggctatcgttatgatgcagcgcttcaaggcctagtgacgcaagttcggctat
서열번호32SEQ ID NO: 32	DH1 intRG-3DH1 intRG-3	atagccgaacttgcgtcactaggccttgaagcgctgcatcataacgatagccgaacttgcgtcactaggccttgaagcgctgcatcataacg
서열번호33SEQ ID NO: 33	DH1 intRG-4DH1 intRG-4	ctagggtaccacgaacacgcctgtgatactctagggtaccacgaacacgcctgtgatact

Using the obtained two PCR products as a template, the same conditions and methods as in Example 1, except that the DH1 intRG-1 primer (SEQ ID NO: 30) and DH1 intRG-4 primer (SEQ ID NO: 33) of Table 7 were used. PCR was performed to link the two PCR products to finally obtain a PCR product having a nucleotide sequence of 1,656 bp. The PCR product having a nucleotide sequence of 1,656 bp was digested with restriction enzyme Kpn I and ligated to pUC19 plasmid digested with the same restriction enzyme (SEQ ID NO: 34), and a plasmid containing a partial sequence of the AYM39_00230 gene and a partial sequence of the AYM39_00235 gene (sequence No. 40) was produced.

<6-1-2> The sequence encoding the upper fragment sequence of the target intergenic region, the trc promoter, and the Cre protein regulated by it, the antibiotic resistance-related protein including the lox nucleotide sequence at both ends Construction of a plasmid (pAM6) containing the sequence, the sequence encoding the lacIq protein, and the lower fragment sequence of the target intergenic region

In order to insert the sequence encoding the Cre protein, the sequence encoding the kanamycin resistance-related protein, and the sequence encoding the lacIq protein between the partial sequence of the AYM39_00230 gene and the partial sequence of the AYM39_00235 gene, trcCreF2 (sequence) in Table 8 below as a template No. 35) and lacIqR2 (SEQ ID No. 36) primers were used to perform PCR in the same manner as in Example 1 to obtain a PCR product of 4,118 bp. The resulting PCR product of 4118 bp and a plasmid containing a partial sequence of the AYM39_00230 gene and a partial sequence of the AYM39_00235 gene prepared in Example 6-1-1 were treated with Stu I restriction enzyme and ligated to pAM6 plasmid (SEQ ID NO: 37 ) Was produced (FIG. 9).

서열번호Sequence number	이름name	서열(5'-3')Sequence (5'-3')
서열번호35SEQ ID NO:35	trcCreF2trcCreF2	ctagaggcctgccgacatcataacggttctctagaggcctgccgacatcataacggttct
서열번호36SEQ ID NO: 36	lacIqR2lacIqR2	taacaggccttcactgcccgctttccagtctaacaggccttcactgcccgctttccagtc

<6-2> DH-1 mutation in the genus Methylomonas into which the trc promoter, the sequence encoding the Cre protein, the sequence encoding the kanamycin resistance-related protein including the lox nucleotide sequence at both ends, and the sequence encoding the lacΙq protein are inserted Preparation of strain (DH-1:Km)

First, methylomonas DH-1 (Hur et al., J Chem Technol Biotechnol 2017; 92: 311-318, registered patent 1017149670000) containing the NMS medium (Nitrate mineral salts) composed of the composition of Table 9 below. After inoculation into the flask, the flask was sealed with a screw cap. The flask was filled with methane gas to a concentration of 30% using a vacuum pump, and incubated at 30° C. until a value of 600 nm OD (optical density) reached 0.5. The cultured cells were centrifuged at 4,000 rpm and 4° C. for 15 minutes and then suspended in sterilized tertiary distilled water. The suspension was centrifuged again under the same conditions to remove the supernatant, resuspended in sterilized tertiary distilled water, and dispensed 200 µl into a sterilized 1.5 ml tube.

After adding 1.0 μg of pAM6 plasmid DNA prepared in Example 6-1-2 to 200 μl of the prepared DH-1 strain of the genus Methylomonas, Electroporation cuvette (Gene Pulser®™, MicroPulser™, Bio -Rad, USA). DNA was introduced by electroporation at 2.5 kV using a Micropulser electroporator (Bio-Rad, USA), and then transferred to a sterilized serum bottle containing 10 ml NMS medium and sealed. The serum bottle was filled with methane gas to a concentration of 30% and incubated with shaking at 30° C. for 24 hours. The cultured cells were centrifuged at 4,000 rpm and 4°C for 15 minutes to remove the supernatant, resuspended in 200 µl NMS medium, and plated on NMS agar medium containing 2.5 mg/L of kanamycin antibiotic. The NMS agar medium was put in an anaerobic jar (Oxoid, UK) used for culturing anaerobic bacteria and sealed, and 30% of the gas inside the anaerobic jar was removed using a vacuum pump, and then the same amount of sterilized methane gas was filled. The trc promoter that formed a colony in NMS agar medium containing 2.5 mg/L of kanamycin after stationary culture in an incubator at 30° C. for 6 to 7 days, a sequence encoding a Cre protein, and a lox nucleotide sequence at both ends The mutant strain into which the sequence encoding the kanamycin resistance-related protein and the sequence encoding the lacΙq protein was inserted was confirmed by diagnostic PCR and nucleotide sequence analysis. The final selected strain was named DH-1:Km (FIGS. 8A and 8B ).

성분 (입수처)Ingredients (from)	최종 농도Final concentration
MgSO ₄7H ₂O (DAEJUNG, 대한민국)MgSO ₄ 7H ₂ O (DAEJUNG, Korea)	1 g/ℓ1 g/ℓ
KNO ₃(JUNSEI, 일본)KNO ₃ (JUNSEI, Japan)	1 g/ℓ1 g/ℓ
CaCl ₂H ₂O (JUNSEI, 일본)CaCl ₂ H ₂ O (JUNSEI, Japan)	0.2 g/ℓ0.2 g/ℓ
3.8% (w/v) solution Fe-EDTA (DUKSAN, 대한민국)3.8% (w/v) solution Fe-EDTA (DUKSAN, Korea)	0.1 ㎖/ℓ0.1 ml/ℓ
0.1% (w/v) NaMo4H ₂O (DUKSAN, 대한민국)0.1% (w/v) NaMo4H ₂ O (DUKSAN, Korea)	0.5 ㎖/ℓ0.5 ml/ℓ
Trace element solution (표 10)Trace element solution (Table 10)	1.0 ㎖/ℓ1.0 ml/ℓ
Phosphate stock solution (표 11)Phosphate stock solution (Table 11)	10 ㎖/ℓ10 ml/ℓ
Vitamin stock solution (표 12)Vitamin stock solution (Table 12)	10 ㎖/ℓ10 ml/ℓ
CuCl ₂2H ₂O (DAEJUNG, 대한민국)CuCl ₂ 2H ₂ O (DAEJUNG, Korea)	1.4 mg/ℓ1.4 mg/ℓ
Bacto agar (필요시) (BD, 미국)Bacto agar (if needed) (BD, USA)	15 g/ℓ15 g/ℓ

성분 (입수처)Ingredients (from)	최종 농도Final concentration
FeSO ₄7H ₂O (JUNSEI, 일본)FeSO ₄ 7H ₂ O (JUNSEI, Japan)	500 mg/ℓ500 mg/ℓ
ZnSO ₄7H ₂O (JUNSEI, 일본)ZnSO ₄ 7H ₂ O (JUNSEI, Japan)	400 mg/ℓ400 mg/ℓ
MnCl ₂7H ₂O (SIGMA, 미국)MnCl ₂ 7H ₂ O (SIGMA, USA)	20 mg/ℓ20 mg/ℓ
CoCl ₂6H ₂O (DAEJUNG, 대한민국)CoCl ₂ 6H ₂ O (DAEJUNG, Korea)	50 mg/ℓ50 mg/ℓ
NiCl ₂6H ₂O (SAMCHUN, 대한민국)NiCl ₂ 6H ₂ O (SAMCHUN, Korea)	10 mg/ℓ10 mg/ℓ
H ₃BO ₃ (boric acid) (KANTO, 일본)H ₃ BO ₃ (boric acid) (KANTO, Japan)	15 mg/ℓ15 mg/ℓ
EDTA (SAMCHUN, 대한민국)EDTA (SAMCHUN, Korea)	250 mg/ℓ250 mg/ℓ

성분 (입수처)Ingredients (from)	최종 농도Final concentration
KH ₂PO ₄(JUNSEI, 일본)KH ₂ PO ₄ (JUNSEI, Japan)	26 g/ℓ26 g/ℓ
Na ₂HPO ₄7(H ₂O) (JUNSEI, 일본)Na ₂ HPO ₄ 7(H ₂ O) (JUNSEI, Japan)	62 g/ℓ62 g/ℓ

성분 (입수처)Ingredients (from)	최종 농도Final concentration
Biotin (ACROS, 미국)Biotin (ACROS, USA)	2.0 mg/ℓ2.0 mg/ℓ
Folic acid (SIGMA, 미국)Folic acid (SIGMA, USA)	2.0 mg/ℓ2.0 mg/ℓ
Thiamine HCl (DAEJUNG, 대한민국)Thiamine HCl (DAEJUNG, Korea)	5.0 mg/ℓ5.0 mg/ℓ
Ca pantothenate (KANTO, 일본)Ca pantothenate (KANTO, Japan)	5.0 mg/ℓ5.0 mg/ℓ
Vitamin B12 (SAMCHUN, 대한민국)Vitamin B12 (SAMCHUN, Korea)	0.1 mg/ℓ0.1 mg/ℓ
Riboflavin (JUNSEI, 일본)Riboflavin (JUNSEI, Japan)	5.0 mg/ℓ5.0 mg/ℓ
Nicotiamide (SIGMA, 미국)Nicotiamide (SIGMA, USA)	5.0 mg/ℓ5.0 mg/ℓ

<6-3> DH-1 mutation of the genus Methylomonas into which the trc promoter, the sequence encoding the Cre protein, the sequence encoding the kanamycin resistance-related protein including the lox nucleotide sequence at both ends, and the sequence encoding the lacΙq protein are inserted Removal of the sequence encoding the kanamycin resistance-related protein from the strain

The following method was performed in order to remove the sequence encoding the kanamycin resistance related protein from the DH-1:Km strain prepared in Example 6-2.

Specifically, 50 mL of NMS medium without kanamycin was added to a flask sealed with a screw lid, 1 mM isoprophyl-β-D-1-thiogalactopyranoside (IPTG) was added, and then the DH prepared in Example 6-2. 500 µl of -1:Km strain was inoculated and cultured with shaking at 30° C. for 24 hours. At this time, 30% of the gas inside the flask was replaced with sterilized methane gas. The cultured cells were diluted to 1/10000, plated on NMS agar medium containing 1 mM IPTG, and cultured at 30° C. using anaerobic jar containing 30% methane gas until colonies were formed. Each single colony formed on the NMS agar medium was simultaneously inoculated into the NMS agar medium containing kanamycin and the NMS agar medium not containing kanamycin using a sterilized tip. Thereafter, strains that could not be grown in a medium containing kanamycin and only grown in an NMS agar medium not containing kanamycin were finally selected (FIG. 8B).

The final selected strain was confirmed that the sequence encoding the kanamycin resistance-related protein was removed from the DH-1 mutant strain of the genus Methylomonas through diagnostic PCR, sequencing analysis, and plate assay, and the strain was DH- 1 It was named as a basic mutant strain (DH-1:Cre) for genome improvement (FIGS. 8A and 8B ).

Therefore, it was confirmed that the method of preparing a basic mutant strain for genome improvement can be applied to the DH-1 strain of the genus Methylomonas.

Claims

The sequence encoding the upper fragment sequence of the target intergenic region, the trc promoter and the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, the sequence encoding the lacIq protein A polynucleotide in which the sequence and the sequence of the lower fragment of the target intergenic region are sequentially linked.
A vector comprising the polynucleotide of claim 1.
The polynucleotide of claim 1, wherein the trc promoter is composed of the nucleotide sequence of SEQ ID NO: 2.
The polynucleotide of claim 1, wherein the sequence encoding the Cre protein is composed of the nucleotide sequence of SEQ ID NO: 25.
The polynucleotide according to claim 1, wherein the lox base sequence is lox66 consisting of the nucleotide sequence of SEQ ID NO: 26 or lox71 consisting of the nucleotide sequence of SEQ ID NO: 27.
The polynucleotide of claim 1, wherein the sequence encoding the lacIq protein is composed of the nucleotide sequence of SEQ ID NO: 3.
1) introducing the polynucleotide of claim 1 into a strain; And

2) In the target intergenic region of the strain, the trc promoter and the sequence encoding the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, and the lacIq protein The method for producing a mutant strain comprising; the step of inserting the sequence.
1) introducing the polynucleotide of claim 1 into a strain;

2) In the target intergenic region of the strain, the trc promoter and the sequence encoding the Cre protein regulated thereby, the sequence encoding the antibiotic resistance-related protein including the lox nucleotide sequence at both ends, and the lacIq protein The step of inserting the sequence; And

3) A method of producing a mutant strain further comprising the step of removing a sequence encoding an antibiotic resistance-related protein by culturing it in a medium to which isopropyl β-D-1-thiogalactopyranoside (IPTG) is added.
A mutant strain prepared by the manufacturing method of claim 8.
The target gene of the mutant strain is modified by introducing polynucleotides in which the upper and lower fragment sequences of the target sequence are combined at both ends of the lox nucleic acid fragment containing the sequence encoding the antibiotic resistance-related protein to the mutant strain of claim 9 How to.
The target gene of the mutant strain according to claim 10, wherein a sequence encoding an antibiotic resistance-related protein is removed by culturing the mutant strain in which the target gene is modified in a medium to which Isopropyl β-D-1-thiogalactopyranoside (IPTG) is added. How to transform it.
The method of claim 10, wherein the modification is a substitution, deletion or insertion of the target gene sequence.