Microorganism with increased colonization in host and method for producing same

Abstract

The invention relates to improving colonization of a bacterial preparation in a host. More specifically, the present application relates to a microorganism in which at least one gene is modified, the modification improving the colonization of the microorganism in a host compared to a microorganism having an unmodified gene.

Description

Technical Field

[0001] The present application relates to a microorganism whose colonization in a host is increased by modifying a gene, as compared with a microorganism that has not been modified for the gene, and a method for producing the microorganism. Background Art

[0002] In recent years, in order to treat and prevent a wide range of diseases such as various infectious diseases, ulcerative colitis, and cancer as targets, attempts have been made to develop preparations composed of single or multiple bacteria as drugs, and a large number of bacterial species have been tested. On the other hand, since it is known that the gut microbiota is optimized according to individuals and such bacterial preparations are excreted even when ingested, it is necessary to improve the colonization of microorganisms. Summary of the Invention Means for Solving the Problem

[0003] Therefore, the present application provides the following. (Item 1) A microorganism in which at least one gene is modified, and the modification increases the colonization of the microorganism in a host, as compared with a microorganism having the unmodified gene. (Item 2) The microorganism according to the above item, wherein the increase in colonization includes a decrease in motility. (Item 3) The microorganism according to any one of the above items, wherein the gene is selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes. (Item 4) The microorganism according to any one of the above items, wherein the gene is selected from the group consisting of flhD, flhD / C, fur, srlR, lpxM, ntrC, cytR, malI, nagC, flhC, gntT, frlR, kdgR, oxyR, envZ, fliC, gatZ, ompR, malT, and lrp. (Item 5) The microorganism according to any one of the above items, wherein the modification includes a point mutation in the gene. (Item 6) The microorganism according to any one of the above items, wherein the modification includes a mutation that generates a stop codon. (Item 7) The microorganism according to any one of the above items, wherein the modification includes at least two mutations in the at least 1 gene. (Item 8) The microorganism according to any one of the above items, wherein the modification includes at least one mutation respectively in at least two genes. (Item 9) The microorganism according to any one of the above items, wherein the modification includes at least two mutations respectively in at least two genes. (Item 10) The microorganism according to any one of the above items, wherein the at least two genes are each independently selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes. (Item A1) A method, which is a method for producing a microorganism in which at least 1 gene is modified, comprising: (A) A modification step, which is a step for modifying the at least 1 gene. In this step, in the microorganism, one or more nucleotides of the target nucleic acid sequence of the gene are replaced with other nucleotides, or deleted, or one or more nucleotides are inserted into the target nucleic acid sequence of the gene; (B) A step of testing the colonization ability of the microorganism in the host for the microorganism having the modified gene, and selecting a microorganism whose colonization ability is increased compared to the microorganism having the unmodified gene; (C) When a microorganism with increased colonization ability cannot be selected, repeating steps (A) and (B) as needed. (Item A2) According to the method described in the above item, the improvement of the colonization ability includes a decrease in motility. (Item A3) According to any one of the above items of the method, the gene is selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes. (Item A4) The method according to any one of the above items, wherein the gene is selected from the group consisting of flhD, flhD / C, fur, srlR, lpxM, ntrC, cytR, malI, nagC, flhC, gntT, frlR, kdgR, oxyR, envZ, fliC, gatZ, ompR, malT, and lrp. (Item A5) The method according to any one of the above items, wherein the modification includes a point mutation in the gene. (Item A6) The method according to any one of the above items, wherein the modification includes a mutation that generates a stop codon. (Item A7) The method according to any one of the above items, wherein the modification includes at least two mutations on the at least one gene. (Item A8) The method according to any one of the above items, wherein the modification includes at least one mutation each on at least two genes. (Item A9) The method according to any one of the above items, wherein the modification includes at least two mutations each on at least two genes. (Item A10) The method according to any one of the above items, wherein the at least two genes are each independently selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes.

[0004] In the present application, it is intended to provide, in addition to the explicitly described combinations, one or more of the above features in further combinations. Additionally, more embodiments and advantages of the present application can be recognized by those skilled in the art if they read the following detailed description and understand it as needed.

[0005] Furthermore, for those skilled in the art, the features, significant functions, and effects of the present application other than the above can be clarified by referring to the items of the embodiments of the invention and the drawings below. Effects of the Invention

[0006] By using genome editing technology (base editing technology), it is possible to produce strains that can continuously colonize in the intestine for microorganisms. In addition, since the microorganisms can be modified to colonize for a long time by this method, it is related to the development of effective treatment methods mediated by microorganisms. Brief Description of the Drawings

[0007] Figure 1 Figure 1 In one embodiment of the present application, the results of the motility test and the growth curve test of the genome-edited strains of the ATCC25922 system are summarized. Figure 2 Figure 2 In one embodiment of the present application, the results of the motility test and the growth curve test of the genome-edited strains of the ATCC700926 system are summarized. Figure 3 Figure 3 In one embodiment of the present application, a graph of the motility rate of the genome-edited strains of the ATCC25922 system is summarized. Figure 4 Figure 4 In one embodiment of the present application, a graph of the motility rate of the genome-edited strains of the ATCC700926 system is summarized. Figure 5 Figure 5 In one embodiment of the present application, a graph of the motility inhibition rate of the genome-edited strains of the ATCC25922 system is summarized. Figure 6 Figure 6 In one embodiment of the present application, a graph of the motility inhibition rate of the genome-edited strains of the ATCC700926 system is summarized. Figure 7 Figure 7 In one embodiment of the present application, a graph showing the results of the cell attachment test of the genome-edited strains to human T84 cells is presented. Figure 8 Figure 8 In one embodiment of the present application, a graph summarizing the attachment rate of the genome-edited strains of the ATCC700926 system to human cells is presented. Figure 9 Figure 9 In one embodiment of the present application, a graph summarizing the relative value of the attachment rate of the genome-edited strains of the ATCC700926 system to human cells is presented. Figure 10 Figure 10 In one embodiment of the present application, a graph showing the correlation between the relative value of the attachment rate and the motility rate of the genome-edited strains is presented. Detailed implementation manners

[0008] ​​​​​​​​​​​​​​​​​​​​The following description will be given while presenting the best mode of the present application. Throughout this specification, unless otherwise specifically mentioned, expressions in the singular form are also understood to include the concept of their plural forms. Therefore, unless otherwise specifically mentioned, articles in the singular form (such as "a", "an", "the", etc. in the case of English) should be understood to also include the concept of their plural forms. In addition, unless otherwise specifically mentioned, the terms used in this specification should be understood to be used in the meanings commonly used in the art. Therefore, unless otherwise defined, all technical terms and scientific and technological terms used in this specification have the same meanings as those commonly understood by those skilled in the art to which the present application belongs. In case of contradiction, this specification (including definitions) shall prevail.

[0009] The definitions and / or basic technical contents of the terms specifically used in this specification will be appropriately described below.

[0010] In this specification, "about" means ±10% of the numerical value that follows.

[0011] In this specification, "microorganism" refers to minute organisms, such as prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and molds, lower algae, fungi, viruses, etc. In addition, it also includes cells that exist individually and separately even in multicellular organisms such as animals and plants. In addition, among microorganisms, in addition to natural microorganisms, it also includes microorganisms that are artificially propagated after culturing these, mutated microorganisms, or microorganisms artificially modified by transformation and other methods.

[0012] In this specification, the "colonization property" of a microorganism means that the microorganism exists in the environment or organism as the object, or continuously exists in the tissue or organ as the target or purpose after being administered into the body, and there is substantially no outflow or movement to the outside of the environment or organism. It is also called adhesiveness or attachment property.

[0013] In this specification, the "migratory property" of a microorganism means that the microorganism has the property of being able to move from a certain position in the environment or organism as the object to other positions, and also includes the property of being able to flow out to the outside. The migratory property of a microorganism mainly originates from the motility derived from flagella. Flagella are cell organs involved in the movement of cells, and they are composed of flagellin that extends outside the cell, hook protein, and lipopolysaccharide membrane, peptidoglycan layer, periplasm layer, and various proteins that form rings and rods existing in the lipid membrane in the cell surface membrane. Among microorganisms, genera having several flagella such as Escherichia coli, Salmonella, Bacillus subtilis, Enterococcus, and Cupriavidus bacteria, and bacteria having a single flagellum such as Vibrio bacteria are known.

[0014] In this specification, "modification" of a gene refers to the substitution of a certain nucleotide (such as dC) on a DNA strand with another nucleotide (such as dT, dA, or dG), or deletion, or insertion or addition of nucleotides or nucleotide sequences between certain nucleotides on the DNA strand. The "modification" in this specification includes substitution of one or more nucleotides at the targeting site of double-stranded DNA, deletion, or insertion or addition of one or more nucleotides to the targeting site of double-stranded DNA. Among them, the double-stranded DNA to be modified is not particularly limited, but genomic DNA is preferred. In addition, the "targeting site" of double-stranded DNA refers to all or part of the "target nucleotide sequence" specifically recognized and bound by the nucleic acid sequence recognition module, or its vicinity (either one or both sides of the 5' upstream and 3' downstream) of the target nucleotide sequence, and its range can be appropriately adjusted between 1 base and several hundred base lengths according to its purpose.

[0015] In this specification, "gene" is interpreted in the broadest sense, which is the literal sequence of nucleic acids or the sequence of substances carrying it (such as nucleotides such as DNA, RNA, etc.), preferably a sequence or a substance containing a sequence that performs a certain function. For example, in addition to encoding proteins, it also includes adjacent transcriptional regulatory regions such as promoters and enhancers that regulate the transcription period and production amount of transcriptional products as transcription factor binding sites, and adjacent transcriptional regulatory regions such as promoters and enhancers that regulate the transcription period and production amount of transcriptional products as transcription factor binding sites.

[0016] In this specification, "flagella-related gene" refers to genes related to flagella formation, flagella motility, etc., and also includes genes encoding proteins that make up flagella. As exemplary flagella-related genes, flhD, flhC, fliC, etc. can be cited.

[0017] In this specification, "iron metabolism-related gene" refers to genes related to iron absorption, utilization process in the bacterial cell, iron excretion, etc., and also includes genes whose expression is regulated depending on the iron concentration inside and outside the bacterial cell. As exemplary iron metabolism-related genes, fur, oxyR, tonB, etc. can be cited.

[0018] In this specification, "carbohydrate metabolism-related gene" refers to genes related to energy production from carbohydrates, etc., and also includes genes related to carbohydrate absorption / decomposition and regulatory factors (regulators) or transcription factors that regulate the expression of these genes. As exemplary carbohydrate metabolism-related genes, srlR, mall, nagC, gntT, malT, frlR, kdgR, gatZ, etc. can be cited.

[0019] In this specification, "phospholipid-related genes" refer to genes related to the biosynthesis of components that constitute the outer membrane of the cell wall specific to Gram-negative bacteria, and also include genes encoding enzymes for synthesizing lipopolysaccharides. As exemplary phospholipid-related genes, there can be mentioned: lpxM, waaC, etc.

[0020] In this specification, "nitrogen signal transduction-related genes" refer to genes related to factors that sense the nitrogen concentration outside the bacterium and constitute a pathway for transducing this signal to downstream genes, and also include downstream genes whose expression is regulated by the nitrogen signal. As exemplary nitrogen signal transduction-related genes, there can be mentioned: ntrC, ntrB, etc.

[0021] In this specification, "two-component regulatory system-related genes" refer to gene clusters composed of two types of proteins, a sensor protein that senses environmental changes inside and outside the bacterium and a regulator protein that receives its signal and regulates downstream genes, and also include downstream genes that are regulated upon receiving the signal. As exemplary two-component regulatory system-related genes, there can be mentioned: ntrC, ntrB, envZ, ompR, etc.

[0022] In this specification, "nucleic acid-related genes" refer to genes encoding a series of proteins responsible for nucleic acid metabolism in bacteria, and also include genes encoding transcription factors that control these series of proteins. As exemplary nucleic acid-related genes, there can be mentioned cytR, etc.

[0023] In this specification, "amino acid metabolism-related genes" refer to genes related to pathways for synthesizing various amino acids from substrates, and also include genes encoding coenzymes involved in amino acid biosynthesis. As exemplary amino acid metabolism-related genes, there can be mentioned: nagC, Lrp, etc.

[0024] In this specification, "oxidative stress-related genes" refer to genes related to the detoxification of reactive oxygen species generated in the bacterium through biological activities, and also include genes encoding proteins responsible for repairing the bacterium damaged by reactive oxygen species. As exemplary oxidative stress-related genes, there can be mentioned: oxyR, sodA / B / C, katE / G, AhpCF, etc.

[0025] In this specification, "signal transduction-related genes" refer to genes related to sensing environmental changes inside and outside the bacterium (such as temperature, osmotic pressure, nutrient source depletion, etc.) and signal transduction to downstream genes, and also include two-component regulatory system-related genes. As exemplary signal transduction-related genes, there can be mentioned: ompR, ntrC, ntrB, envZ, etc.

[0026] In this specification, the "osmotic stress-related gene" refers to a gene that functions to protect the cell structure of the bacterium from the osmotic pressure outside the bacterium. As an exemplary osmotic stress-related gene, envZ etc. can be cited.

[0027] (Preferred embodiment) The preferred embodiments of the present application are described below. The embodiments provided below are provided for better understanding of the present application, and the scope of the present application should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate changes within the scope of the present application with reference to the descriptions in this specification. In addition, the following embodiments of the present application can be used alone or in combination.

[0028] In one aspect of the present application, a microorganism is provided in which at least one gene is modified, and the modification enhances the colonization ability of the microorganism in the host compared to a microorganism having the unmodified gene. The microorganism of the present application is useful as a technique for preventing the spread of bacterial therapeutic agents in the environment.

[0029] As a microbiome therapy targeting the gut microbiota, for example, fecal microbial transplantation (FMT) mainly targeting infectious diseases is in clinical development. FMT aims only at the short-term normalization of the gut microbiota composition and cannot achieve the long-term colonization of transplanted bacteria accompanied by the improvement of human physiological changes and pathological phenotypes. In contrast, live biotherapeutic products (LBPs) are expected to continuously provide functions beneficial to the host as a result of modifying the functions of microorganisms and can treat various diseases. Therefore, it has been reported that the therapeutic targets of LBPs are not only inflammatory bowel disease, cancer, and neurodegenerative diseases, but also the possibility of adapting to chronic diseases with generally persistent symptoms such as obesity, non-alcoholic fatty liver disease, and type II diabetes. In the treatment of such diseases, it is necessary to long-term colonize the strain in the intestine, and it is necessary to have the characteristic of long-term colonization in the lumen environment hostile to the administered bacteria after transplantation into the intestine.

[0030] Microorganisms, especially many microorganisms present in the intestine, form colonies and colonize after adhering to the mucus layer in the highly fluid intestinal environment. The mucus layer covering the intestinal epithelium consists of two layers: a high-density inner layer in contact with epithelial cells and a low-density and highly viscous outer layer. It has been reported that the mucus layer of the human small intestine consists of a thin inner layer of about 30 μm or less and an outer layer of about 100 μm to about 400 μm, forming an uneven layer, while the inner mucus layer of the large intestine reaches a thickness of about 100 μm and the outer layer reaches a thickness of about 700 μm. In these two layers, the inner layer is known to have a high mucus density and plays a barrier function to defend against the invasion of pathogenic bacteria into cells. In contrast, the outer layer comes into daily contact with resident bacteria and provides a colonization site.

[0031] As an example, although not intended to be limiting, in the present application, when the function of a protein related to the movement and migration of microorganisms is lost, microorganisms with increased colonization ability are produced. Since the colonization ability of the microorganisms used as bacterial preparations increases and the outflow to the external environment decreases, it can be applied to the control of environmental pollution and the like.

[0032] As an example, although not intended to be limiting, in the case of the intestinal environment, mucin, which is the main constituent of mucus, is a huge glycoprotein with a molecular weight of millions that is highly O-glycosylated, and many bacteria that obtain a colonization site in the mucus layer are considered to have adhesiveness to mucin.

[0033] In addition, as an example, although not intended to be limiting, the adhesion factors on the bacterial cell side that exhibit binding to the receptor are called adhesins. For the adhesins of mucin in lactic acid bacteria and bifidobacteria, they are roughly classified into three types in the protein components of the bacterial cell: S-layer proteins with a secretion signal, cell wall-anchored proteins, and housekeeping proteins. Among the cell wall-anchored proteins, MubB initially discovered in Lactobacillus reuteri, MucBP with the same sequence found in more than 10 types of bacteria, and Msa showing mannose-binding properties found in Lactobacillus plantarum have been reported. In addition, pili, whose existence has been unknown in lactic acid bacteria and bifidobacteria, have been found in the Lactobacillus casei group, Bifidobacterium longum group, Bifidobacterium bifidum, Bifidobacterium animalis, and Bifidobacterium adolescentis. SpaC, a pilus component of Lactobacillus rhamnosus GG, and FimA of Bifidobacterium longum are adhesins that exhibit binding to mucin. Among the housekeeping proteins of lactic acid bacteria, GroEL as a chaperonin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constituent factor of the ATP-binding cassette (ABC) transporter, and elongation factor Tu (EF-Tu) as a translation elongation factor have been reported as adhesins to mucin. On the other hand, the blood group antigen seen in the mucin sugar chain is the binding epitope of GAPDH of Lactobacillus plantarum LA318, while the sulfated sugar chain structure of mucin has been clarified as the binding epitope of EF-Tu of Lactobacillus reuteri. As other adhesion factors, polysaccharides related to lipoprotein, lipoteichoic acid, and biofilm formation have been reported.

[0034] In one embodiment, although not intended to be limiting, by enhancing the functions and activities of the adhesins as described above, the colonization ability can also be improved.

[0035] In addition, it is also known that when lpxF is knocked out, the intestinal colonization ability of Bacteroides thetaiotaomicron decreases over time. Since lpxF is one of the enzymes responsible for the lipopolysaccharide (LPS) synthesis pathway, it is considered that the intestinal colonization ability of microorganisms can be improved by modifying the LPS structure of the microorganisms to the same structure as that of Bacteroides thetaiotaomicron.

[0036] In one embodiment, although not intended to be limiting, it is considered that since bacteria belonging to the genus Bacteroides, which are representative bacteria of intestinal resident bacteria, do not have a partial structure of LPS, in order to mimic the colonization ability of intestinal resident bacteria, it is necessary to inactivate the functions of LpxK or LpxM. Since LpxK is an essential gene, it is considered that the colonization ability can be improved by targeting the modification of LpxM. In addition, a change in the LPS structure is also expected to reduce the ability to induce inflammation.

[0037] In one embodiment, although not intended to be limiting, since bacteria absorb iron when iron is deficient and sequester / export iron when iron is in excess, for example, for a lesion site accompanied by bleeding, such as inflammatory bowel disease, it is expected that bacteria with enhanced ability to obtain iron contained in red blood cells will easily preferentially aggregate at the desired site. Since the iron homeostasis of bacteria is regulated by the transcriptional repressor Fur and the translational repressor ryhB, it is considered that the uptake of iron can be enhanced by mutation of Fur.

[0038] In one embodiment, although not intended to be limiting, it is considered that the ability to uptake sorbitol can be enhanced by knocking out (genome editing) SrlR, a transcriptional factor that acts as a repressor.

[0039] In one embodiment, although not intended to be limiting, by enhancing the function of oxyR (a hydrogen peroxide-responsive transcriptional activator) in Escherichia coli, it is possible to expect improvement in the proliferation and survival of bacteria caused by neutralization of the hyperactivation of oxidative stress accompanying enhanced iron uptake, and to confer an advantage in competitive binding with other bacteria due to enhanced stress tolerance.

[0040] In one embodiment of the present application, in a microorganism in which at least one gene is modified, as an improvement in colonization ability, a decrease in motility can be cited.

[0041] In one embodiment, as at least one gene modified in the microorganism of the present application, any gene that can enhance the colonization of the microorganism in the host through the modification compared to a microorganism having an unmodified gene may be used. For example, genes selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes may be mentioned, but are not limited to these.

[0042] In addition, in one embodiment, examples of such genes include: DNA-binding transcriptional activator (Gene ID: 945442) flhD, DNA-binding ferric uptake regulator (Gene ID: 945295) fur, DNA-binding glucitol operon transcriptional repressor (Gene ID: 948942) srlR, myristoyl-acyl carrier protein (acp)-dependent acyltransferase (Gene ID: 945143) lpxM, DNA-binding transcriptional activator (Gene ID: 948361) ntrC, DNA-binding transcriptional repressor (Gene ID: 948427) cytR, DNA-binding mal regulon transcriptional repressor (Gene ID: 947104) malI, DNA-binding transcriptional dual regulator (Gene ID: 945285) nagC, DNA-binding transcriptional activator (Gene ID: 947280) flhC, high-affinity gluconate transporter (Gene ID: 947924) gntT, DNA-binding fructoselysine utilization operon transcriptional repressor (Gene ID: 947885) frlR, DNA-binding transcriptional repressor (DNA-binding transcriptional repressor;GeneID: 946129) kdgR, DNA-binding transcriptional regulator (Gene ID: 948462) oxyR, osmolarity sensor protein (Gene ID: 947272) envZ, flagellar filament structural protein (Gene ID: 949101) fliC, Tagatose 6-phosphate kinase (Gene ID: 946641) gatZ, DNA-binding transcriptional regulator (Gene ID: 947913) ompR, and Leucine-responsive regulatory protein (Gene ID: 949051) lrp, but not limited to these.;

[0043] In one embodiment, the modification of at least one gene in the microorganism of the present application can be carried out using standard molecular biology methods. In one embodiment, the modification may include point mutations in the gene. As a method for site-specifically and correctly modifying a target double-stranded polynucleotide, for example, a method of contacting the target double-stranded polynucleotide with a Cas protein and a guide RNA, or a method of contacting a complex of the target double-stranded polynucleotide, a Cas protein, and a nucleic acid base conversion enzyme with a guide RNA can be used. In such a method, the Cas9 protein forms a complex with the guide RNA and binds to the target double-stranded polynucleotide. Among them, the Cas9 protein does not cleave the above-mentioned target double-stranded polynucleotide or only cleaves one strand, that is, it does not cause double-strand cleavage, and modifies the base sequence within the target polynucleotide. In one embodiment, the modification is preferably carried out in single-base units.

[0044] In one embodiment, the specific and correct modification (single-base editing) of the above single-base unit is preferably carried out using a nucleic acid base conversion enzyme in the complex. As the nucleic acid base conversion enzyme, deaminases (deaminating enzymes) can be mentioned. As deaminases, for example, cytosine deaminase, cytidine deaminase, adenosine deaminase, etc. can be used. In the complex in one embodiment, in addition to containing the relevant nucleic acid base conversion enzyme, since the formation of Indels is inhibited, an Indel formation inhibitor called uracil DNA glycosylase inhibitor (UGI) can also be included.

[0045] In one embodiment, the specific and correct modification (single-base editing) of the above single-base unit can also be achieved by using a method that employs a complex of a nucleic acid sequence recognition module and a DNA glycosylase. When the complex of the nucleic acid sequence recognition module and the DNA glycosylase is expressed from an expression vector or an RNA molecule introduced into a cell, the nucleic acid sequence recognition module specifically recognizes and binds to a target nucleotide sequence within a target double-stranded DNA (such as genomic DNA). Through the action of the DNA glycosylase linked to the nucleic acid sequence recognition module, a depurination reaction is induced on the sense strand or the antisense strand of the targeted site (which can be appropriately adjusted within a range of several hundred bases including all or part of the target nucleotide sequence or in their vicinity), generating an abasic site (AP site) on one strand of the double-stranded DNA. Then, the base excision repair (BER) system in the cell is activated. First, the AP endonuclease recognizes the AP site and cleaves the phosphodiester bond of the DNA single strand, and the exonuclease removes the depurinated nucleotide. Subsequently, DNA polymerase inserts a new nucleotide using the negative-strand DNA as a template, and finally, DNA ligase repairs the nick. By inducing repair errors at any stage of this BER, various mutations can be introduced. As described above, although the enzyme activity is lost, by co-using a mutant AP endonuclease that retains the binding ability to the AP site, the BER structure in the cell is inhibited, and the frequency of repair errors, thereby enhancing the mutation introduction efficiency.

[0046] Since the CRISPR-Cas system recognizes the target double-stranded DNA sequence through a guide RNA complementary to the target nucleotide sequence, any sequence can be targeted simply by synthesizing an oligo RNA and / or oligo DNA that can specifically hybridize with the target nucleotide sequence. Moreover, at the targeted site, a region where the double-stranded DNA is unwound to form a single-stranded structure and an adjacent region with a relaxed double-stranded DNA structure are generated. Therefore, it is possible to efficiently utilize a nucleic acid base-converting enzyme and a DNA glycosylase specifically at the targeted site without combining factors that change the double-stranded DNA structure. Thus, in a more preferred embodiment of the present application, as the nucleic acid sequence recognition module, a CRISPR-Cas system without at least one DNA cleavage ability of Cas (CRISPR-mutant Cas) or a CRISPR-Cas system without two DNA cleavage abilities of Cas (CRISPR-mutant Cas) can be preferably used.

[0047] The nucleic acid sequence recognition module of the present application using CRISPR-mutant Cas is provided as a complex of an RNA molecule composed of a guide RNA complementary to the target nucleotide sequence and a tracrRNA required for the recruitment of the mutant Cas protein and the mutant Cas protein.

[0048] In one embodiment, the modification can be carried out in any environment, either in vivo or in vitro. In one embodiment, the modification can also be carried out outside the organism, that is, ex vivo or in vitro.

[0049] In one embodiment of the present application, by the method as described above, the modification of at least one gene in the microorganism of the present application may include mutations that generate stop codons and mutations that generate amino acid substitutions. Thereby, the colonization ability of the microorganism in the host can be enhanced.

[0050] In one embodiment of the present application, the modification of at least one gene in the microorganism of the present application can contain at least 2, 3, 4, 5, 6, 7, or 8 mutations in a certain gene. By generating at least 2 mutations in a certain gene, compared with the case of generating 1 mutation, the colonization ability of the microorganism in the host can be enhanced.

[0051] In one embodiment of the present application, the modification of at least one gene in the microorganism of the present application may include at least 1 mutation in each of at least 2 genes. For example, the at least 2 genes can be independently selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes. In other embodiments, the modification of at least one gene in the microorganism of the present application may include at least 2 mutations in each of at least 2 genes. By generating at least 1 or at least 2 mutations in each of at least 2 genes, compared with the case of modifying 1 gene, the colonization ability of the microorganism in the host can be enhanced.

[0052] Regarding the proliferation ability of the microorganism, any method can be used as long as it is a method well-known in the art. For example, the proliferation curve can be evaluated by inoculating a single colony of the strain into an LB liquid medium without the agent and culturing it statically at 37°C for 24 hours, and then plotting its proliferation curve. At this time, for example, 24 hours after the start of the culture, the turbidity (O.D. 600 ) of the culture solution can be measured using a plate reader to plot the proliferation curve.

[0053] In one embodiment, the microorganisms of the present application may include, for example: Escherichia coli, Lactococcusmembers of the phyla Lactobacillales, Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria or Verrucomicrobia and the genera Bacteroides, Alistipes, Faecalibacterium, Parabacteroides, Prevotella, Roseburia, Ruminococcus, Clostridium, Oscillibacter, Gemmiger, Barnesiella, Dialister, Parasutterella, Phascolarctobacterium, Propionibacterium, Sutterella, Blautia, Paraprevotella, Coprococcus, Odoribacter, Spiroplasma, Anaerostipes, Akkermansia, Lactobacillus, Streptococcus, Bifidobacterium, Akkermansia, Megasphaera, Eubacterium, Bariatricus, Erysipelatoclostridium, Pediococcus, Enterococcus, Pseudoflavonifractor, Lachnospiraceae, Erysipelotrichaceae, Oscillospiraceae, bacteria of the genus Faecalicatena, Enterococcus casseliflavus, Enteroccocusgallinarum), Enterococcus faecalis, Enterococcus hirae, Enterococcus mundtii, Lactiplantibacillus plantarum, Lactobacillus gasseri, Akkermansia muciniphila, Faecalibacterium prausnitzii, Bifidobacterium spp., Bifidobacterium animalis, Bifidobacterium breve, Streptococcus cristatus, Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, Staphylococcus aureus, Staphylococcus epidermidis, Porphyromonas gingivalis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium sporogenes, Clostridium cocleatum, Clostridium saccharogumia, Clostridium spiroforme, Clostridium innocuum, Clostridium ramosum, Clostridium hathewayi, Clostridium saccharolyticum, Clostridium scindens, Clostridium sp., Clostridium bolteae, Clostridium indolis, Clostridium lavalense, Clostridium asparagiformeClostridium asparagiforme, Clostridium symbiosum, Clostridiales bacterium, Fusobacterium nucleatum, Blautia hydrogenotrophica, Blautia stercoris, Blautia wexlerae, Blautia producta, Blautia coccoides, Blautia hansenii, Blautia faecis, Blautia glucerasea, Blautia luti, Blautia schinkii, Megasphaera massiliensis, Megasphaera elsdenii, Megasphaera cerevisiae, Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis, Megasphaera micronuciformis, Megasphaera hexanoica, Eubacterium contortum, Eubacterium fissicatena, Eubacterium limosum, Eubacterium rectale, Eubacterium aerofaciens, Eubacterium callanderi, Eubacterium eligens, Eubacterium hallii, Bariatricus massiliensis, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes rhamnosivorans, Anaerostipescaccae), Anaerotruncus colihominis, Faecalicatena fissicatena, Faecalicatena contorta, Erysipelatoclostridium ramosum, Parabacteroides distasonis, Parabacteroides merdae, Parabacteroides goldsteinii, Parabacteroides johnsonii, Pediococcus acidilactici, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, Pediococcus pentosaceus, Pediococcus stilesii, Roseburia hominis, Roseburia intestinalis, Roseburia faecis, Roseburia massiliensis, Roseburia cecicola, Roseburia inulinivorans, Bacteroides coprocola, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides sp., Pseudoflavonifractor capillosus, Lachnospiraceae bacterium, Lachnospiraceae sp., Erysipelotrichaceaebacterium), Ruminococcus sp., Oscillospiraceae bacterium, Oscillibacter valericigenes, and Lactobacillus reuteri, etc.

[0054] In one embodiment, the microorganisms of the present application can inhabit the intestine, oral cavity, and / or skin of an object, etc. For example, they are genera that constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40% of the total culturable microorganisms in the feces of the object. For the microorganisms in the intestine or feces of an object, they can be analyzed by any technique known in the art including determining 16S ribosomal sequences. For example, the genus Bacteroides is the most naturally abundant genus in the human intestine. As an illustrative species of the genus Bacteroides, examples include: B. acidifaciens, B. amylophilus, B. asaccharolyticus, B. barnesiaes, B. bivius, B. buccae, B. buccalis, B. caccae, B. caecicola, B. caecigallinarum, B. capillosus, B. capillus, B. cellulosilyticus, B. cellulosolvens, B. chinchilla, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. corporis, B. denticola, B. disiens, B. distasonis, B. dorei, B. eggerthii, B. endodontalis, B. faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. forsythus, B. fragilis, B. furcosus, B. galacturonicus, B. gallinaceum, B. gallinarum, B. gingivalis, B. goldsteinii, B.gracilis), B. graminisolvens, B. helcogenes, B. heparinolyticus, B. hypermegas, B. intermedius, B. intestinalis, B. johnsonii, B. levvi, B. loescheii, B. luti, B. macacae, B. massiliensis, B. melaninogenicus, B. merdae, B. microfusus, B. multiacidus, B. nodosus, B. nordii, B. ochraceus, B. oleiciplenus, B. oralis, B. oris, B. oulorum, B. ovatus, B. paurosaccharolyticus, B. pectinophilus, B. pentosaceus, B. plebeius, B. pneumosintes, B. polypragmatus, B. praeacutus, B. propionicifaciens, B. putredinis, B. pyogenes, B. reticulotermitis, B. rodentium, B. ruminicola, B. salanitronis, B. salivosus, B. salyersiae, B. sartorii, B. sediment, B. splanchnicus, B. stercorirosoris, B. stercoris, B. succinogenes, B. suis, B. tectus, B. termitidis, B.Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, Bacteroides veroralis, Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides xylanolyticus, or Bacteroides zoogleofonnans. In addition, as oral bacteria, examples include Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, etc.; as skin resident bacteria, examples include Staphylococcus aureus, Staphylococcus epidermidis, etc.

[0055] In one embodiment, the microorganism of the present application can stably form colonies in the human intestine, oral cavity, and / or skin. The microorganism of the present application, for example, in terms of the increased abundance, stability, or ease of initial colony formation in the intestine, can form colonies in the intestine of the subject compared to the unmodified same or similar microorganisms.

[0056] In one embodiment, the microorganism of the present application can further contain relevant genes for treatment. Such relevant genes for treatment can be genes originally possessed by the microorganism or genes that exert the desired effect can be directly introduced or introduced after modifying a part thereof. In one embodiment, examples of relevant genes for treatment include Type 1 fimbrin D-mannose specific adhesin (fimH), etc. In one embodiment, the microorganism of the present application can further contain relevant genes for diagnosis. Such relevant genes for diagnosis can be genes originally possessed by the microorganism or genes that exert the desired effect can be directly introduced or introduced after modifying a part thereof. In one embodiment, examples of relevant genes for diagnosis include Cell shape-determining protein: mreB, etc. In one embodiment, the microorganism of the present application can further contain genes essential for survival. Such genes essential for survival can be genes originally possessed by the microorganism or genes that exert the desired effect can be directly introduced or introduced after modifying a part thereof. In one embodiment, examples of genes essential for survival include the gene encoding lysine biosynthetic enzyme (dapA), etc.

[0057] In one embodiment, as a result of single-base editing, the functional expression of the target protein can be inhibited by the influence of the introduced stop codon. When the microorganism contains genes or nucleic acids encoding multiple proteins, open reading frames intended to encode two or more proteins can, for example, exist in a single operon.

[0058] In one embodiment of the present application, the microorganism of the present application can also be used as a therapeutic agent. For such a therapeutic agent, for example, based on the weight ratio of the microorganism, it may contain at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0% or more of the microorganism of the present application in a therapeutically effective amount.

[0059] In other aspects of the present application, a method is provided, which is a method for producing a microorganism in which at least one gene is modified, including: (A) a modification step, which is a step for modifying the at least one gene. In this step, within the microorganism, one or more nucleotides of the target nucleic acid sequence of the gene are replaced with other nucleotides, or deleted, or one or more nucleotides are inserted into the target nucleic acid sequence of the gene; (B) a step of testing the colonization ability of the microorganism in the host for the microorganism having the modified gene, and selecting a microorganism with increased colonization ability compared to the microorganism having the unmodified gene; (C) a step of repeating steps (A) and (B) as needed when a microorganism with increased colonization ability cannot be selected. In one embodiment of the present application, the method of the present application may have any feature described in other positions of this specification.

[0060] (General technology) The molecular biology techniques, biochemical techniques, and microbiological techniques that can be used in this specification are well-known and commonly used in the art, such as those described in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J.J. et al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press, and "Experimental Methods for Gene Transfer & Expression Analysis" of Bunsha, 1997, etc. Relevant parts (which may be all) of these are incorporated herein by reference.

[0061] Regarding DNA synthesis techniques and nucleic acid chemistry for producing synthetic genes, for example, gene synthesis and fragment synthesis services of GeneArt, GenScript, Integrated DNA Technologies (IDT), etc. can also be used. Others, for example, are described in Gait, M.J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M.J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.T. (1996). Bioconjugate Techniques, Academic Press, etc. Relevant parts of these are incorporated herein by reference.

[0062] In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". When it is clearly stated in this specification that it is within the "range" of "two values", this range also includes the two values themselves. Regarding the references such as scientific literatures, patents, patent applications, etc. cited in this specification, the whole of them is incorporated herein by reference to the same extent as their respective specifically described contents.

[0063] The preferred embodiments are shown above for the purpose of easily understanding the present application to illustrate the present application. The present application will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present application. Therefore, the scope of the present application is not limited to the embodiments and examples specifically described in this specification, but is only limited by the claims. Example

[0064] (Example 1: Study on the Colonization of Genome-Edited Strains) Using genome-edited strains prepared by mutating the target genes and their editing positions as shown in Figure 1 and 2 , the colonization of each strain was studied. The target genes subjected to editing were comprehensively selected from gene clusters classified into the following seven protein functions: (1) factors related to bacterial motility (bacterial swimming), (2) nucleic acid-related factors, (3) amino acid metabolism-related factors, (4) sugar metabolism-related factors, (5) metal metabolism-related factors, (6) signal transduction systems (especially two-component regulatory systems) related factors, and (7) phospholipid synthesis-related factors. For the design of gRNA for each target gene, a stop codon was mainly introduced into the 5'-end side to induce a complete loss of protein function, and it was set to induce amino acid substitutions at the active site and the like predicted according to the protein structure.

[0065] For two strains of Escherichia coli ATCC 25922 and ATCC 700926, after genome editing of the genes encoding the above factors (a total of 20 genes), in vitro experiments (proliferation tests in aerobic / anaerobic environments, motility tests, and colonization tests on cultured cells derived from the human intestine) were carried out.

[0066] <Motility Test> Single colonies of Escherichia coli ATCC 25922 strain, Escherichia coli ATCC 700926 strain, Escherichia coli genome-edited strain, and Staphylococcus aureus JCM 2413 strain were inoculated into LB liquid medium without drugs and cultured with shaking at 37 °C and 1,500 rpm min -1 for 18 to 24 hours. After measuring the concentration of each culture solution using a spectrophotometer, 3 - 5 × 10 6 cells were spotted on 0.25% (w / v) LB agar medium having a hole with a depth of about 2 - 3 mm in the center. After spotting, it was incubated at 30 °C for 10 - 16 hours to perform the motility test of each strain.

[0067] After a specified time, the diameter of the bacterial colonies that spread (migrate) concentrically from the inoculation site was measured. For the migration rate, the measured diameter values were used to calculate the relative values of each genome-edited strain relative to the parental strain. In addition, the migration inhibition rate was determined by subtracting the migration rate from 100%.

[0068] <Proliferation curve test> Single colonies of each genome-edited strain prepared using Escherichia coli ATCC 25922 and ATCC 700926 as parental strains were inoculated into LB liquid medium without drugs and statically cultured at 37 °C for 24 hours. Twenty-four hours after the start of the culture, the turbidity (O.D. 600 ) of the culture broth was measured using a microplate reader. The 24-hour culture broth was diluted 100-fold with fresh LB liquid medium (when performing the proliferation test under a hypoxic environment, it was fresh LB liquid medium supplemented with 2% (v / v) Oxyrase), and then injected into a U-bottom 96-well plate. The turbidity was measured every 10 minutes using a microplate reader until 18 hours (in an aerobic environment) or 24 hours (in a hypoxic environment). The set temperature during the measurement was 37 °C. When performing the measurement under a hypoxic environment, in an anaerobic chamber, sterilized deoxygenated mineral oil was layered on top of the culture broth.

[0069] The transition ability to the logarithmic phase in each strain was defined as 3 hours or 4 hours after the start of turbidity measurement (3 hours after for strains derived from ATCC 25922 and 4 hours after for strains derived from ATCC 700926), and 18 hours after was defined as an index of the total bacterial count in each strain. Using the O.D. 600 values at each time point, the relative values relative to the parental strain were calculated.

[0070] Regarding the migration test and the proliferation curve test, the results of the ATCC25922 system and the results of the ATCC700926 system are as follows Figure 1 and Figure 2 shown.

[0071] In addition, the graph summarizing the migration rates of the genome-edited strains of the ATCC25922 system and the graph summarizing the migration rates of the genome-edited strains of the ATCC700926 system are as follows Figure 3 , Figure 4 shown. In addition, the graph summarizing the migration inhibition rates of the genome-edited strains of the ATCC25922 system and the graph summarizing the migration inhibition rates of the genome-edited strains of the ATCC700926 system are as follows Figure 5 , Figure 6 shown.

[0072] In the in vitro test evaluation of the ATCC 25922 reference strain, a delay in migration during the logarithmic growth phase was confirmed in almost all genome-edited strains under aerobic / anaerobic conditions, but some strains showed faster proliferation ability compared to the parental strain. In addition, in the motility test, there was no difference in specific protein functions, and motility inhibition occurred in almost all strains.

[0073] Even in the in vitro evaluation of the ATCC 700926 reference strain, almost all genome-edited strains showed a delay in proliferation compared to the parental strain under aerobic conditions. On the other hand, strains with disrupted signal transduction-related factors showed very early proliferation. In addition, in the motility test, significant motility inhibition was confirmed in disrupted strains of nucleic acid-related factors and sugar metabolism-related factors, in addition to signal transduction-related factors.

[0074] (Example 2: Cell attachment test 1 on human cells) The attachment rate of genome-edited strains to human T84 cells was investigated. The following strains were used as genome-edited strains. Information on edited strains (ID number, target gene, base editing information) · ID number: BP1021 · Target genes: flhD, flhC (flagellar transcriptional activator) · Base editing information: flhD A21V, Q22*, R23C, flhC W67*, E68K In addition, the experimental conditions are as described below. · Experimental conditions (bacterial culture, co-culture with cells, other conditions including temperature, etc.) · Bacterial culture: Shake culture at 37°C for 4 hours · Co-culture with cells: For T84 cells, after adding to MOI = 10, centrifuge at 600×g for 5 minutes at room temperature, and co-culture in a CO 2 incubator (37°C) for 2 hours · Counting of the number of bacteria attached to cells: To calculate the number of attached bacteria, wash 5 times with 1 mL PBS and then count the number of viable bacteria recovered.

[0075] The results are as Figure 7 shown. Figure 7 The vertical axis represents the attachment rate (%), which is calculated as the number of attached bacteria per well / the total number of bacteria per well × 100. The experiment was carried out using a 24-well plate, and the results under duplicate are shown. As can also be seen from these results, it is known that the genome-edited strain (BP1021) has an increased attachment rate to human T84 cells compared to the parental strain.

[0076] (Example 3: Cell attachment test 2 for human cells) Performed in the same manner as in Example 2, the attachment rate to human T84 cells was also investigated for genome-edited strains other than BP1021. The relative values of the attachment rate for each genome-edited strain are as shown in Figure 1 and 2 . In addition, the graphs of the attachment rates of each ATCC700926 system genome-edited strain and the relative values of the attachment rates of the ATCC700926 system genome-edited strains summarized are as shown in Figure 8 and Figure 9 .

[0077] The graph showing the correlation between the relative values of the attachment rate and the migration rate of each of the above genome-edited strains is Figure 10 . The "1" on the vertical axis represents the cell attachment ability of the Escherichia coli wild strain. As can also be seen from Figure 10 , a negative correlation (r = -0.6) was confirmed between the relative value of the attachment rate and the migration rate of the genome-edited strains. This time, by utilizing the base editing technology to disrupt the genes related to colonization, a decrease in migration was first discovered.

[0078] (Example 4: Administration test to germ-free mice in the intestine) (Administration of Escherichia coli to a germ-free mouse model in the intestine) An experiment was conducted on the intestinal colonization persistence of genome-edited Escherichia coli strains in mice that had been germ-free in the intestine by administration of antibiotics. For C57BL / 6J mice (8 weeks old, male, Jackson Laboratory Japan Co., Ltd.), levofloxacin (Tokyo Chemical Industry; dissolved in phosphate-buffered saline at a concentration of 10 - 30 mg / mL) was forcibly administered orally at a dose of 10 μL per 1 g body weight (100 - 300 μg of levofloxacin per 1 g body weight) to perform intestinal decontamination. One to five days after levofloxacin was excreted from the intestine, a phosphate-buffered saline suspension of genome-edited Escherichia coli or wild-type Escherichia coli as its parental strain at a dose of 10^9 CFU per individual was forcibly administered orally to each individual at a volume of 100 μL.

[0079] (Administration of Escherichia coli to a natural proliferation mouse model of Escherichia coli) Conduct an experiment on the intestinal colonization persistence of a genome-edited Escherichia coli strain in Escherichia coli-naturally proliferated mice. As reported in Bolick et al. (Enteroaggregative Escherichia coli strain in a novel weaned mouse model: Exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide, J. Med. Microbiol. (2013) 62:896-905) or Brown et al. (Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model, Nat. Commun. (2015) 6:7806), etc., by immediately exposing weaned mice to a low-nutrition state, Escherichia coli in the intestine proliferates, and in addition, the colonization of an Escherichia coli strain administered externally is promoted. Immediately after weaning, C57BL / 6J mice (4 weeks old, male, Jackson Laboratory Japan Co., Ltd.) were randomly fed a low-nutrition diet (low-protein, low-fat, reduced-mineral diet; Research Diets, Inc.). After 3 to 6 days, 100 μL of a phosphate-buffered saline suspension of the genome-edited Escherichia coli or the wild-type Escherichia coli as its parental strain was orally administered to each individual at a dose of 10^9 CFU per individual.

[0080] <Evaluation of the intestinal colonization persistence of the administered Escherichia coli strain> Since it is known that the number of Escherichia coli in the intestine is related to the number of Escherichia coli in the excreted feces, the intestinal colonization persistence of the administered Escherichia coli strain can be evaluated by measuring the number of Escherichia coli administered in the feces over time. For the number of Escherichia coli in the feces, it can be measured by quantitative analysis of the number of Escherichia coli genome copies per unit weight of feces, etc. For example, fresh feces are collected, and genomic DNA of bacteria in the feces is extracted using a Nucleospin DNA Stool Kit (Macherey-Nagel), etc. Using a primer set that amplifies the genomic sequence specific to the administered Escherichia coli strain for the extracted DNA, QPCR is performed to quantitatively analyze the number of Escherichia coli administered in each feces sample.

[0081] For example, when administering the ATCC 700926 strain or its genome-edited strain, a primer set such as 5'-GGAGAGATCTCACTAAAAACTGGGG-3' (SEQ ID NO: 75) and 5'-ATCCTGCGCACCAATCAACAA-3' (SEQ ID NO: 76) that amplifies the region around the wbbL gene with a genomic insertion sequence specific to the system strain can be used as a primer set capable of specifically detecting the administered strain; when administering the ATCC 25922 strain or its genome-edited strain, a primer set such as 5'-CCGGAAACTGAGCGTCTTGA-3' (SEQ ID NO: 77) and 5'-GCCCTAGCCAATCATCTCCC-3' (SEQ ID NO: 78) that amplifies the region around the kpsM gene with a genomic sequence specific to the system strain can be used as a primer set capable of specifically detecting the administered strain.

[0082] (Annotation) As described above, it can be understood that although the preferred embodiments of the present application are used to illustrate the present application, the scope of the present application should be interpreted only by the claims. For the patents, patent applications, and other documents cited in this specification, it can be understood that their contents themselves should be cited as references for this specification in the same way as the contents specifically described in this specification. The present application claims priority from Japanese Patent Application No. 2022-177704 filed on November 4, 2022, and the entire contents thereof are incorporated herein by reference. Industrial Applicability

[0083] Since the microorganisms of the present application can protect the quality and safety of bacterial preparations and prevent their transmission and / or spread in the environment or to humans, wide applications can be expected in the medical field. Sequence Listing Free Text

[0084] SEQ ID NO: 1: Nucleic acid sequence of flhD of Escherichia coli ATCC 25922 strain SEQ ID NO: 2: Amino acid sequence of flhD of Escherichia coli ATCC 25922 strain SEQ ID NO: 3: Nucleic acid sequence of fur of Escherichia coli ATCC 25922 strain SEQ ID NO: 4: Amino acid sequence of fur of Escherichia coli ATCC 25922 strain SEQ ID NO: 5: Nucleic acid sequence of srlR of Escherichia coli ATCC 25922 strain Sequence number 6: Amino acid sequence of srlR of Escherichia coli ATCC 25922 strain Sequence number 7: Nucleic acid sequence of lpxM of Escherichia coli ATCC 25922 strain Sequence number 8: Amino acid sequence of lpxM of Escherichia coli ATCC 25922 strain Sequence number 9: Nucleic acid sequence of ntrC of Escherichia coli ATCC 25922 strain Sequence number 10: Amino acid sequence of ntrC of Escherichia coli ATCC 25922 strain Sequence number 11: Nucleic acid sequence of cytR of Escherichia coli ATCC 25922 strain Sequence number 12: Amino acid sequence of cytR of Escherichia coli ATCC 25922 strain Sequence number 13: Nucleic acid sequence of malI of Escherichia coli ATCC 25922 strain Sequence number 14: Amino acid sequence of malI of Escherichia coli ATCC 25922 strain Sequence number 15: Nucleic acid sequence of nagC of Escherichia coli ATCC 25922 strain Sequence number 16: Amino acid sequence of nagC of Escherichia coli ATCC 25922 strain Sequence number 17: Nucleic acid sequence of flhC of Escherichia coli ATCC 25922 strain Sequence number 18: Amino acid sequence of flhC of Escherichia coli ATCC 25922 strain Sequence number 19: Nucleic acid sequence of gntT of Escherichia coli ATCC 25922 strain Sequence number 20: Amino acid sequence of gntT of Escherichia coli ATCC 25922 strain Sequence number 21: Nucleic acid sequence of kdgR of Escherichia coli ATCC 25922 strain Sequence number 22: Amino acid sequence of kdgR of Escherichia coli ATCC 25922 strain Sequence number 23: Nucleic acid sequence of oxyR of Escherichia coli strain ATCC 25922 Sequence number 24: Amino acid sequence of oxyR of Escherichia coli strain ATCC 25922 Sequence number 25: Nucleic acid sequence of envZ of Escherichia coli strain ATCC 25922 Sequence number 26: Amino acid sequence of envZ of Escherichia coli strain ATCC 25922 Sequence number 27: Nucleic acid sequence of fliC of Escherichia coli strain ATCC 25922 Sequence number 28: Amino acid sequence of fliC of Escherichia coli strain ATCC 25922 Sequence number 29: Nucleic acid sequence of gatZ of Escherichia coli strain ATCC 25922 Sequence number 30: Amino acid sequence of gatZ of Escherichia coli strain ATCC 25922 Sequence number 31: Nucleic acid sequence of ompR of Escherichia coli strain ATCC 25922 Sequence number 32: Amino acid sequence of ompR of Escherichia coli strain ATCC 25922 Sequence number 33: Nucleic acid sequence of lrp of Escherichia coli strain ATCC 25922 Sequence number 34: Amino acid sequence of lrp of Escherichia coli strain ATCC 25922 Sequence number 35: Nucleic acid sequence of malT of Escherichia coli strain ATCC 25922 Sequence number 36: Amino acid sequence of malT of Escherichia coli strain ATCC 25922 Sequence number 37: Nucleic acid sequence of flhD of Escherichia coli strain ATCC 700926 Sequence number 38: Amino acid sequence of flhD of Escherichia coli strain ATCC 700926 Sequence number 39: Nucleic acid sequence of fur of Escherichia coli strain ATCC 700926 Sequence number 40: Amino acid sequence of fur of Escherichia coli strain ATCC 700926 Sequence number 41: Nucleic acid sequence of srlR of Escherichia coli strain ATCC 700926 Sequence number 42: Amino acid sequence of srlR of Escherichia coli strain ATCC 700926 Sequence number 43: Nucleic acid sequence of lpxM of Escherichia coli strain ATCC 700926 Sequence number 44: Amino acid sequence of lpxM of Escherichia coli strain ATCC 700926 Sequence number 45: Nucleic acid sequence of ntrC of Escherichia coli strain ATCC 700926 Sequence number 46: Amino acid sequence of ntrC of Escherichia coli strain ATCC 700926 Sequence number 47: Nucleic acid sequence of cytR of Escherichia coli strain ATCC 700926 Sequence number 48: Amino acid sequence of cytR of Escherichia coli strain ATCC 700926 Sequence number 49: Nucleic acid sequence of malI of Escherichia coli strain ATCC 700926 Sequence number 50: Amino acid sequence of malI of Escherichia coli strain ATCC 700926 Sequence number 51: Nucleic acid sequence of nagC of Escherichia coli strain ATCC 700926 Sequence number 52: Amino acid sequence of nagC of Escherichia coli strain ATCC 700926 Sequence number 53: Nucleic acid sequence of flhC of Escherichia coli strain ATCC 700926 Sequence number 54: Amino acid sequence of flhC of Escherichia coli strain ATCC 700926 Sequence number 55: Nucleic acid sequence of gntT of Escherichia coli strain ATCC 700926 Sequence number 56: Amino acid sequence of gntT of Escherichia coli strain ATCC 700926 Sequence number 57: Nucleic acid sequence of frlR of Escherichia coli ATCC 700926 strain Sequence number 58: Amino acid sequence of frlR of Escherichia coli ATCC 700926 strain Sequence number 59: Nucleic acid sequence of kdgR of Escherichia coli ATCC 700926 strain Sequence number 60: Amino acid sequence of kdgR of Escherichia coli ATCC 700926 strain Sequence number 61: Nucleic acid sequence of oxyR of Escherichia coli ATCC 700926 strain Sequence number 62: Amino acid sequence of oxyR of Escherichia coli ATCC 700926 strain Sequence number 63: Nucleic acid sequence of envZ of Escherichia coli ATCC 700926 strain Sequence number 64: Amino acid sequence of envZ of Escherichia coli ATCC 700926 strain Sequence number 65: Nucleic acid sequence of fliC of Escherichia coli ATCC 700926 strain Sequence number 66: Amino acid sequence of fliC of Escherichia coli ATCC 700926 strain Sequence number 67: Nucleic acid sequence of gatZ of Escherichia coli ATCC 700926 strain Sequence number 68: Amino acid sequence of gatZ of Escherichia coli ATCC 700926 strain Sequence number 69: Nucleic acid sequence of ompR of Escherichia coli ATCC 700926 strain Sequence number 70: Amino acid sequence of ompR of Escherichia coli ATCC 700926 strain Sequence number 71: Nucleic acid sequence of lrp of Escherichia coli ATCC 700926 strain Sequence number 72: Amino acid sequence of lrp of Escherichia coli ATCC 700926 strain Sequence number 73: Nucleic acid sequence of malT of Escherichia coli ATCC 700926 strain Sequence number 74: Amino acid sequence of malT of Escherichia coli ATCC 700926 strain Sequence number 75: Primers for amplifying the region around the wbbL gene Sequence number 76: Primers for amplifying the region around the wbbL gene Sequence number 77: Primers for amplifying the region around the kpsM gene Sequence number 78: Primers for amplifying the region around the kpsM gene.

Claims

1. A microorganism in which at least one gene is modified, and the modification is a modification that enhances the colonization ability of the microorganism in a host as compared to a microorganism having an unmodified version of the gene.

2. The microorganism according to claim 1, wherein the enhancement of the colonization ability includes a decrease in motility.

3. The microorganism according to claim 1 or 2, wherein the gene is selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes.

4. The microorganism according to any one of claims 1 to 3, wherein the gene is selected from the group consisting of flhD, flhD / C, fur, srlR, lpxM, ntrC, cytR, malI, nagC, flhC, gntT, frlR, kdgR, oxyR, envZ, fliC, gatZ, ompR, malT, and lrp.

5. The microorganism according to any one of claims 1 to 4, wherein the modification includes a point mutation in the gene.

6. The microorganism according to any one of claims 1 to 5, wherein the modification includes a mutation that generates a stop codon.

7. The microorganism according to any one of claims 1 to 6, wherein the modification includes at least two mutations on the at least one gene.

8. The microorganism according to any one of claims 1 to 7, wherein the modification includes at least one mutation each on at least two genes.

9. The microorganism according to any one of claims 1 to 8, wherein the modification includes at least two mutations each on at least two genes.

10. The microorganism according to claim 8 or 9, wherein the at least two genes are each independently selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes.

11. A method, which is a method for producing a microorganism in which at least one gene is modified, comprising: (A) A modification step, which is a step of modifying the at least one gene, in which in the microorganism, one or more nucleotides of the target nucleic acid sequence of the gene are replaced with other nucleotides, or deleted, or one or more nucleotides are inserted into the target nucleic acid sequence of the gene; (B) A step of testing the colonization ability of the microorganism in a host for a microorganism having the modified gene, and selecting a microorganism in which the colonization ability is increased as compared to a microorganism having an unmodified version of the gene; (C) A step of repeating steps (A) and (B) as needed when a microorganism with increased colonization ability cannot be selected.

12. The method according to claim 11, wherein the enhancement of the colonization ability includes a decrease in motility.

13. The method according to claim 11 or 12, wherein the gene is selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes.

14. The method according to any one of claims 11 to 13, wherein the gene is selected from the group consisting of flhD, flhD / C, fur, srlR, lpxM, ntrC, cytR, malI, nagC, flhC, gntT, frlR, kdgR, oxyR, envZ, fliC, gatZ, ompR, malT, and lrp.

15. The method according to any one of claims 11 to 14, wherein the modification comprises point mutations in the gene.

16. The method according to any one of claims 11 to 15, wherein the modification comprises mutations that generate stop codons.

17. The method according to any one of claims 11 to 16, wherein the modification comprises at least two mutations on the at least 1 gene.

18. The method according to any one of claims 11 to 17, wherein the modification comprises at least one mutation on each of at least two genes.

19. The method according to any one of claims 11 to 18, wherein the modification comprises at least two mutations on each of at least two genes.

20. The method according to claim 18 or 19, wherein the at least two genes are each independently selected from the group consisting of flagella-related genes, iron metabolism-related genes, sugar metabolism-related genes, phospholipid-related genes, nitrogen signal transduction-related genes, two-component regulatory system-related genes, nucleic acid-related genes, amino acid metabolism-related genes, oxidative stress-related genes, signal transduction-related genes, and osmotic stress-related genes.

Images