Microorganisms expressing vasoactive intestinal peptide and uses thereof

Abstract

A recombinant microorganism expressing the VIP gene and a composition containing the microorganism for preventing or treating diseases that cause damage to the gastrointestinal tract are provided.

Description

[Technical field]

[0001] The present invention relates to a microorganism expressing vasoactive intestinal peptide (VIP) and a composition containing the same for preventing or treating a disease causing damage to the gastrointestinal tract. [Background technology]

[0002] VIP is a peptide hormone that is vasoactive in the intestine. It is a 28 amino acid peptide that belongs to the glucagon / secretin superfamily, a ligand of class II G protein-coupled receptors.

[0003] Korean Patent Publication No. 2014-0000620 discloses a Lactobacillus that increases VIP levels in the enteric nervous system and its use in treating irritable bowel syndrome (IBD). It is a strain screened in nature and is not a recombinant microorganism.

[0004] China Patent Publication No. 108753670 discloses a recombinant microorganism Lactococcus lactis NZ9000 that expresses VIP and is rich in selenium nanoparticles (SeNPs) in its cells. It is described that the recombinant microorganism has selenium present in the form of nanoparticles in its cells, and therefore when used as a selenium additive, it can solve the problems of selenium's strong toxicity, low bioavailability, high environmental pollution, and high production costs, and also secretes the antibacterial peptide VIP, solving the antibiotic residue problem. In addition, the pNZ8148 plasmid of the NICE® Lactococcus lactis expression system was used to express and secrete VIP.

[0005] Recombinant Lactobacillus microorganisms expressing exogenous VIP, recombinant lactic acid bacteria expressing exogenous VIP under a constitutive promoter, and the use of such microorganisms to prevent or treat diseases causing gastrointestinal damage, such as IBD, in humans, are not known. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present disclosure is to provide a recombinant Lactobacillus microorganism expressing an exogenous VIP.

[0007] Another object is to provide a recombinant lactic acid bacterium expressing an exogenous VIP under a constitutive promoter.

[0008] Yet another object is to provide a composition for preventing or treating diseases causing damage to the gastrointestinal tract in humans, comprising said microorganism. [Means for solving the problem]

[0009] One aspect of the present disclosure is a method for producing a promoter and a method for producing an activator of said promoter. Possible The present invention provides a recombinant microorganism of the genus Lactobacillus, into which a foreign gene encoding vasoactive intestinal peptide (VIP) linked to a ribozyme has been introduced and into which a protease has been inactivated.

[0010] Another aspect of the present disclosure is a constitutive promoter and a gene that functions in conjunction with said promoter. Possible The present invention provides a recombinant microorganism having an exogenous gene encoding vasoactive intestinal peptide (VIP) linked to a lactic acid bacterium, in which proteases are inactivated, the recombinant microorganism also being a lactic acid bacterium.

[0011] Yet another aspect of the present disclosure provides a composition for preventing or treating a disease causing damage to the gastrointestinal tract in humans, comprising a recombinant microorganism having a promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter, the recombinant microorganism having inactivated proteases, and the recombinant microorganism is also a lactic acid bacterium.

[0012] The details will be explained below.

[0013] As used herein, the term "VIP," "VIP protein" or "VIP polypeptide" refers to a biologically active polypeptide having one or more of the biological activities as described herein.

[0014] In humans, the q25 region of chromosome 6 on the human genome encodes a secretin family member that is 170 amino acids in length. This 170 amino acid member is post-translationally cleaved to form vasoactive intestinal peptide (VIP). The active form of VIP polypeptide performs functions such as lowering blood pressure, increasing vasodilation of blood vessel walls, relaxing smooth muscle in respiratory and gastrointestinal tissues, reducing immune responses through the promotion of Th2 as well as reducing Th1 responses, regulating innate and adaptive immune responses, or promoting electrolyte secretion in visceral organs. It has also been shown that VIP is a neurotransmitter and is active in the central nervous system and in communicating with lymphocytes. The biological activity of VIP is transmitted through three known receptors: VIP1R, VIP2R, and PAC1R. These receptors are known to cause cAMP concentration as well as intracellular calcium production. Like VIP, their affinity for secretin varies depending on the subtype and amino acid sequence of the ligand. Human native VIP has a short half-life in the bloodstream, about 2 minutes.

[0015] The VIPs referred to herein include variants that mimic the function of native VIP, including variants that have an increased half-life relative to native VIP and have equal or greater biological activity than native VIP.

[0016] In the present specification, VIP includes, but is not limited to, human VIP, recombinant human VIP, murine VIP and / or recombinant mouse VIP. The VIP may have 50% or more, 60% or more, 70% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. The VIP may have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 9, respectively. The VIP of SEQ ID NO: 1 is a natural VIP of human origin. The VIPs having the amino acid sequences of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 and 9 are natural VIP variants of human origin VIP1, VIP2, VIP3, VIP4, VIP5, VIP6, VIP7 and VIP8.

[0017] The VIP polypeptides used herein may be isolated from a variety of sources, such as from human tissue, or from other sources, or may be produced by recombinant or synthetic methods.

[0018] The term "VIP polypeptide" also includes variants of VIP polypeptides. In the present application, VIP is also modified in a manner that forms chimeric molecules that include VIP fused to another, heterologous polypeptide or amino acid sequence.

[0019] As used herein, the term "protease" is a general term for enzymes that degrade proteins, and includes, without particular limitation, any type of protease known in the art.

[0020] As used herein, the term "auxotroph" refers to a strain that cannot grow in a synthetic medium consisting of only inorganic salts and a carbon source, and can only grow when supplemented with one or more nutrients, and includes, without limitation, any type of strain known in the art.

[0021] The microorganism according to one aspect of the present disclosure is a recombinant microorganism of the genus Lactobacillus, into which a promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter have been introduced and in which proteases have been inactivated.

[0022] According to another aspect of the present disclosure, the microorganism is a recombinant microorganism into which a constitutive promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter have been introduced, and in which proteases have been inactivated, and which is also a recombinant microorganism that is a lactic acid bacterium.

[0023] The lactic acid bacteria may be of the genera Lactobacillus, Lactococcus, Bifidobacteria, Streptococcus, Leuconostoc, Weissella, Pediococcus or Enterococcus, in particular, of the genera Lactobacillus and Lactococcus, such as Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum or Lactococcus lactis.

[0024] The lactobacillus microorganism may be Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus plantarum, such as Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP) or Lactobacillus brevis LMT1-46 (KCTC 13423BP).

[0025] The promoter may also be a constitutive promoter. The promoter may also have a transcription initiation efficiency in lactic acid bacteria that is equal to or higher than that of a conventional promoter, such as the P11 promoter. The promoter may also be an inducible promoter. The inducible promoter may also be, for example, the nisin inducible promoter. The promoter may also be PR4 from Lactobacillus paracasei, which comprises the polynucleotide of SEQ ID NO: 10. The PR4 may also be constituted by the nucleotide sequence of SEQ ID NO: 11.

[0026] The microorganism according to the present disclosure is characterized in that an exogenous gene encoding a VIP has been introduced therein. The introduction may be by transformation, transduction, transfection or electroporation. The introduced exogenous gene may be integrated into the genome of the host cell or may be present as a non-integrated gene. For example, the exogenous gene may be integrated into the genome of the host cell.

[0027] The microorganism according to the present disclosure is also characterized in that the protease is inactivated. In one embodiment, the gene encoding the protease is deleted. "Deletion" includes at least a part of the gene, e.g., the whole or a part, is deleted.

[0028] According to one embodiment, the gene encoding the protease is replaced with an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to a promoter, thereby realizing both introduction of an exogenous VIP gene and deletion of the protease gene at the same time. "Replacement" includes replacement of at least a portion of the gene, e.g., the whole or a part.

[0029] In the present specification, the term "protease" includes any type known in the art, without any particular limitation. For example, it is one or more selected from the group consisting of HtrA (high temperature requirement A), PepN (Aminopeptidase N), ClpP (Caseinolytic protease P) and Lon (Lon protease), for example, HtrA and / or PepN, for example, HtrA. The htrA gene is also L. brevis htrA gene, particularly SEQ ID NO: 14. The pepN gene is also L. brevis pepN gene, particularly SEQ ID NO: 15. The clpP gene is also L. brevis clpP gene, particularly SEQ ID NO: 16. The lon gene is also L. brevis lon gene, particularly SEQ ID NO: 17. In such a microorganism, the protease is not expressed, and the foreign VIP protein is prevented from being decomposed, thereby increasing the yield or productivity of the VIP protein. According to one embodiment, when HtrA is not expressed, the productivity of the VIP protein can be significantly increased.

[0030] The recombinant microorganism further comprises a signal sequence operably linked between the promoter and the foreign gene. The signal sequence, when linked in frame with a foreign protein gene, has a higher ability to cause extracellular secretion than a conventional signal sequence, for example, the USP45 signal sequence. The signal sequence may code for SP4 (SEQ ID NO: 12) from Lactobacillus paracasei. For example, SP4 may have the nucleotide sequence of SEQ ID NO: 13. The microorganism may secrete VIP extracellularly.

[0031] In one embodiment, the recombinant microorganism is also an auxotroph. The auxotroph may be, for example, one or more genes selected from the group consisting of ribB (Riboflavin biosynthesis protein gene), thyA (Thymidylate synthase gene) and glmS (Glutamine-fructose-6-phosphate aminotransferase gene), such as ribB and / or thyA, for example, ribB, but is not limited thereto. ribB may be L. brevis ribB, particularly SEQ ID NO: 18. thyA may be L. brevis thyA, particularly SEQ ID NO: 19. glmS may be L. brevis glmS, particularly SEQ ID NO: 20. In this case, the growth and survival of the recombinant microorganism may be controlled to enhance environmental safety. "Deletion" includes at least a part of the gene, for example, the whole or a part, is deleted.

[0032] Yet another aspect of the present disclosure provides compositions for use in preventing or treating diseases that cause damage to the gastrointestinal tract involving the aforementioned microorganisms.

[0033] The disease may also cause gastrointestinal inflammation. The disease may also be one or more selected from the group consisting of inflammatory bowel disease (IBD) and colitis. The inflammatory bowel disease may also be ulcerative colitis or Crohn's disease.

[0034] The composition may also include a pharma- ceutically or food-acceptable carrier, excipient or diluent. The pharma- ceutically or food-acceptable carrier refers to any standard pharmaceutical or food carrier, such as phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include auxiliary agents, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavors, and colorants. The desired pharmaceutical or food carrier depends on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) administration. The composition may also be in unit dosage form. The composition may have an oral dosage form. The composition may also be a food or pharmaceutical composition. The composition may also include a dry matter of the microorganism. The composition may also include a culture medium of the microorganism.

[0035] As used herein, "pharmaceutical acceptable or acceptable for food" refers to something that does not substantially cause adverse reactions when administered to an individual, such as toxic, allergic or immune reactions.

[0036] Another aspect provides a kit for use in preventing or treating a disease that causes gastrointestinal damage, comprising the recombinant microorganism as described above and another drug that treats the gastrointestinal damage disease. The recombinant microorganism is as described above. The recombinant microorganism may also be in the form of a composition as described above. The kit also includes written instructions for using the recombinant microorganism and the drug that treats the gastrointestinal damage disease to prevent or treat a disease that causes gastrointestinal damage.

[0037] The aforementioned other drugs for treating gastrointestinal damage diseases include any drug that treats gastrointestinal damage diseases, such as inflammatory bowel disease. Drugs for treating gastrointestinal damage diseases also include the following drugs: (i) Steroidal anti-inflammatory agents dexamethasone, hexestrol, methimazole, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fluocinolone acetonide, predonisolone, methylpredonisolone, cortisone acetate, hydrocortisone, fluorometholone, beclomethasone dipropionate, estriol, paramethasone acetate, fludrocortisone acetate, clobetasol propionate, diflorasone acetate, dexamethasone propionate, difluprednate, betamethasone dipropionate, budesonide, diflucortolone valerate, amcinonide, halcinonide, mometasone furoate, hydrocortisone butyrate propionate, flumetasone pivalate, clobetasone butyrate, dexametasone acetate etc. (ii) 5-aminosalicylic acid sulfasalazine, mesalazine, olsalazine, balsalazide etc. (iii) Immunomodulators or immunosuppressants methotrexate, cyclophosphamide, MX-68, atiprimod dihydrochloride, BMS-188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropoietin, colony stimulating factor, interleukin, interferon, etc. (iv) JAK inhibitor tofacitinib, ruxolitinib, etc. (v) TNF inhibitor Recombinant TNF-alpha-receptor IgG-Fc fusion protein (etanercept), infliximab, adalimumab, certolizumab pegol, golimumab, PASSTNF-α, soluble TNF-α receptor, TNF-α binding protein, anti-TNF-α antibody, CDP571, etc. (vi) integrin inhibitor natalizumab, vedolizumab, AJM300, TRK-170, E-6007, etc. (vii) interleukin-12 / 23 inhibitor Ustekinumab, briakinumab (anti-interleukin-12 / 23 antibody), etc. (viii) Nonsteroidal anti-inflammatory drugs (NSAIDs) (a) Classic NSAIDs alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lornoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, ketophenylbutazone, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafenine, piroxicam, tenoxicam, epirizole, tiaramide hydrochloride, zaltoprofen, gabexate mesylate, camostat mesylate, ulinastatin, Colchicine, probenecid, sulfinpyrazone, bucolome, benzbromarone, allopurinol, sodium aurothiomalate, sodium hyaluronate, sodium salicylate, salicylic acid, atropine, scopolamine, levorphanol, oxymorphone, or salts thereof (b) Cyclooxygenase inhibitors (COX-1 selective inhibitors, COX-2 selective inhibitors, etc.) Salicylic acid derivatives (e.g., celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac sodium, indomethacin, loxoprofen, etc. (c) Nitric oxide-releasing NSAIDs. The matters described above regarding the microorganisms are also applicable to the compositions.

[0038] Another aspect provides a method for preventing or treating a disease causing gastrointestinal tract damage comprising administering to an individual an amount of said microorganism that is prophylactically or therapeutically effective for the disease causing gastrointestinal tract damage.

[0039] The method may further include administering to the individual a TNF-alpha blocking agent in an amount effective to prevent or treat a disease causing damage to the gastrointestinal tract. The administering of the TNF-alpha blocking agent to the individual may be performed simultaneously or before or after administering the microorganism to the individual. The TNF-alpha blocking agent may be administered by a route that is the same as or different from the route by which the microorganism is administered. For example, the TNF-alpha blocking agent may be administered orally or parenterally. The parenteral route may be an injection, such as an intravenous injection, an intraperitoneal injection, or a subcutaneous injection. For example, the TNF-alpha blocking agent may be administered intraperitoneally and the microorganism may be administered orally. The TNF-alpha blocking agent may be administered intraperitoneally after the microorganism is administered orally. The TNF-alpha blocking agent may be administered in an amount of 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 40 mg, 10 mg to 50 mg, or 20 mg to 50 mg per dose.

[0040] In the method, the disease may be one that causes gastrointestinal inflammation. The disease may be one or more selected from the group consisting of inflammatory intestinal disease, autoimmune disease, gastrointestinal damage caused by radiation or graft versus host disease (GVHD), inflammatory bowel disease (IBD), and colitis, including chronic colitis. The inflammatory bowel disease may be ulcerative colitis or Crohn's disease.

[0041] As used herein, the term "administering" or "administration" refers to the act of administering the microorganism, a composition comprising the same, or a therapeutic treatment to a physiological system (individual or in vivo, in vitro, or ex vivo cells, tissues, and organs). Thus, in the method, the microorganism is also in the form of a composition as described above. Acceptable routes of administration to the human body are also via the mouth or mucosa (e.g., intestinal mucosa, oral mucosa, or buccal). The administration may also be in combination with a further therapeutic agent. The combined administration may be simultaneous or sequential, in any order.

[0042] As used herein, the term "treatment" refers to prophylactic or therapeutic treatment. In certain embodiments, "treatment" refers to the administration of a microorganism or composition to an individual for therapeutic or prophylactic purposes.

[0043] A "therapeutic" treatment is a treatment administered to an individual exhibiting pathological signs or symptoms to reduce or eliminate the signs or symptoms, which may be biochemical, cellular, histological, functional, or physical, subjective or objective.

[0044] A "prophylactic" treatment is a treatment administered to an individual who shows no signs of disease or who shows only early signs of disease, in order to reduce the risk of developing a pathology. The microorganisms or compositions described herein are also provided as prophylactic treatments, in order to reduce the likelihood of developing a pathology or, if it does develop, to minimize the severity of said pathology.

[0045] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a stated purpose. The effective amount is also determined experimentally. The effective amount is also determined, for example, by the degree to which the microorganism secretes VIP extracellularly. For example, the effective amount is the number of the microorganisms capable of secreting 0.01 to 300 mg, or 0.5 to 100 mg, of VIP per day per human having a body weight of 60 kg. For example, 1x10 5 Or 1x10 13 cfu, 1x10 6 Or 1x10 13 cfu, 1x10 7 Or 1x10 13 cfu, 1x10 7 Or 1x10 12 cfu, 1x10 7 Or 1x10 11 cfu, 1x10 7 Or 1x10 10 cfu, 1x10 8 Or 1x10 11 cfu, 1x10 8 Or 1x10 10 cfu, 2.5x10 8Or 7.5x10 9 cfu, 5.0x10 8 Or 5.0x10 9 cfu, 7.5x10 8 Or 2.5x10 9 cfu, or approximately 1x10 9 It may also be administered in cfu.

[0046] In the method, the individual is also a mammal. The mammal may be a human, a horse, a pig, a cow, a dog, a cat, a monkey, a chimpanzee, a sheep or a goat.

[0047] The statements made regarding the microorganisms and / or compositions are also applicable to the methods. Effect of the Invention

[0048] The recombinant microorganisms disclosed herein not only produce and secrete VIP with higher productivity, but also exhibit higher environmental safety and can more effectively prevent or treat diseases that cause damage to the gastrointestinal tract. [Brief description of the drawings]

[0049] [Figure 1A] FIG. 1 is a schematic diagram showing gene manipulation sites of a recombinant microorganism according to one embodiment. [Figure 1B] FIG. 1 is a schematic diagram showing gene manipulation sites of a recombinant microorganism according to one embodiment. [Figure 2A] FIG. 1 shows a diagram confirming the production of a recombinant microorganism with improved VIP productivity according to one embodiment. [Figure 2B] FIG. 1 shows a diagram confirming the production of a recombinant microorganism with improved VIP productivity according to one embodiment. [Figure 3A] 1 is a diagram showing improved VIP productivity of a recombinant microorganism according to an embodiment. [Figure 3B] 1 is a diagram showing improved VIP productivity of a recombinant microorganism according to an embodiment. [Figure 4A]FIG. 1 is a diagram confirming the production of a recombinant microorganism with improved environmental safety according to one embodiment. [Figure 4B] FIG. 1 is a diagram confirming the production of a recombinant microorganism with improved environmental safety according to one embodiment. [Figure 5A] 1 is a diagram showing the enhanced environmental safety of a recombinant microorganism according to an embodiment. [Figure 5B] 1 is a diagram showing the enhanced environmental safety of a recombinant microorganism according to an embodiment. [Figure 6] 1 is a diagram showing that a recombinant microorganism according to an embodiment exhibits therapeutic effects on IBD in an animal model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples in any way.

[0051] Example 1: Construction of recombinant microorganisms Using Lactobacillus brevis LMT1-46 (KCTC 13423BP), we generated recombinant microorganisms expressing exogenous VIP by a homologous recombination gene manipulation method commonly used in lactic acid bacteria (Zhang et al., D-Ala-D-Ala ligase as a broad host-range counterselection marker in vancomycin resistant lactic acid bacteria, J. Bacteriol., 2018).

[0052] 1A and 1B show schematic diagrams of the genetic manipulation sites of the microorganism according to this embodiment. FIG. 1A shows the process of replacing the protease gene (htrA or pepN) with the VIP gene to improve VIP productivity. A signal peptide to aid in secretion and a tag for detection were included as a single cassette after the VIP. The protease gene is L. brevis htrA (SEQ ID NO: 14) or L. brevis pepN (SEQ ID NO: 15). FIG. 1B shows the process of forming an auxotrophic strain to improve environmental safety, which is a method of removing a partial region of the target gene. The target gene is L. brevis ribB (SEQ ID NO: 18). In Figures 1A and 1B, the abbreviations have the following meanings: P: promoter, SP: signal peptide, UTR: untranslated region, VIP: vasoactive intestinal peptide, A: L. brevis ribB, B: L. brevis htrA or L. brevis pepN.

[0053] Example 2: Confirmation of the production of recombinant microorganisms PCR was performed to confirm whether the recombinant microorganism was produced as intended. Lactobacillus colonies statically cultured at 37°C for 16 hours on MRS plates were used. A single colony was peaked and suspended in a tube containing 100 μl of distilled water. After bead-beating, the resulting mixture was heated at 98°C for 10 minutes and used as a DNA template. 1 μl of DNA template, 1 μl of forward primer, 1 μl of reverse primer, and 25 μl of DNA polymerase mix were mixed to prepare a PCR mixture. PCR was performed under the following conditions: (a) denaturation step, 98°C for 10 seconds, (b) annealing step, 55°C for 15 seconds, and (c) extension step, 72°C for 10 seconds per cycle, and a final extension step at 72°C for 2 minutes. The primers used were designed in the ORF outside region of the gene site to be manipulated, and the sequences are described in Table 1 below. [Table 1]

[0054] The results are shown in Figures 2A and 2B, respectively. More specifically, Figures 2A and 2B are schematic diagrams (left side) showing the replacement of the protease genes htrA (Figure 2A) and pepN (Figure 2B) with the VIP expression cassette, respectively, and gel images (right side) showing the recombinant microorganisms generated therefrom and the band sizes changed as intended. As shown in Figures 2A and 2B, it was confirmed that recombinant microorganisms in which the protease genes htrA and pepN were replaced with the VIP expression cassette, respectively, were generated. In the case of htrA, the band size of the PCR product was shown to be 1.5 kb for the wild type, and the band size was shown to be 920 bp when the htrA region was replaced with the VIP expression cassette. In the case of pepN, the band size of the PCR product was shown to be 2 kb for the wild type, and the band size was shown to be 1.3 kb when the pepN region was replaced with the VIP expression cassette. In the schematic diagrams of FIG. 2A and FIG. 2B, the abbreviations are as defined in Example 1, and WT in the gel images means wild type.

[0055] Example 3: Evaluation of VIP productivity of recombinant microorganisms In order to confirm whether the VIP productivity of the recombinant microorganism was improved, the strain prepared in Example 1 was evaluated to confirm the VIP expression level. The stack of the prepared strain was streaked on an MRS plate and cultured at 37°C for 3 days. A single colony was selected from the stack and cultured in MRS liquid medium at 37°C for 24 hours with shaking. The resulting colony was transferred to 25 ml of MRS and incubated at OD 600After inoculation to a pH of 0.1, the mixture was shaken at 37°C for 16 hours. 10 ml of the culture solution was extracted, centrifuged at 4,000 rpm for 10 minutes, and only the supernatant was extracted. 1 ml of TCA (tricholoacetic acid) was added and treated at 4°C for 30 minutes or more. The mixture was centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the pellet was suspended in 1 ml of acetone stored at 4°C. The mixture was centrifuged at 13,000 rpm for 10 minutes, the supernatant was removed, and 0.5 ml of acetone was added to suspend the pellet. The mixture was centrifuged again at 13,000 rpm for 10 minutes and dried at 60°C for 5 minutes. Sample buffer (reducing buffer) was added, the pellet was thoroughly mixed, and treated at 98°C for 10 minutes. Western blotting was performed using the mixture. Based on the results of the Western blotting, the intensity of the bands measured by the same size area was quantified using a program called ImageJ.

[0056] The results are shown in Figures 3A and 3B, respectively. More specifically, Figure 3A shows a gel image (left) of VIP expressed from a recombinant strain in which htrA and / or ribB were deleted, and a graph (right) showing the quantification of the expression level of VIP. Figure 3B is a graph showing the expression level of VIP expressed from a recombinant strain in which pepN was deleted. As can be seen from Figures 3A and 3B, when the protease gene of L. brevis was deleted, the expression level of VIP, an active substance, increased. In particular, when htrA was deleted, the expression level of VIP, a target substance, increased by more than three times compared to when other genes were deleted.

[0057] Example 4: Confirmation of the generation of auxotrophic strains PCR was performed to confirm whether the auxotrophic strain was generated as intended. Lactobacillus colonies statically cultured on MRS plates at 37°C for 16 hours were used. A single colony was picked and suspended in a tube containing 100 μl of distilled water. After bead-biting, it was heated at 98°C for 10 minutes and used as a DNA template. 1 μl of DNA template, 1 μl of forward primer, 1 μl of reverse primer, and 25 μl of DNA polymerase mix were mixed to prepare a PCR mixture. PCR was performed under the following conditions: (a) denaturation step, 98°C for 10 seconds, (b) annealing step, 55°C for 15 seconds, and (c) extension step, 72°C for 10 seconds per cycle, and a final extension step at 72°C for 2 minutes. The primers used were designed in the ORF inside and ORF outside regions of the gene site to be manipulated, and the sequences are described in Table 2 below. [Table 2]

[0058] The results are shown in Figures 4A and 4B, respectively. More specifically, Figures 4A and 4B are schematic diagrams (left) of the construction of recombinant microorganisms in which rib and thyA have been deleted, and gel images (right) showing the recombinant microorganisms generated therefrom and the band sizes changed as intended. In Figures 4A and 4B, the abbreviations are as defined in Example 1, and WT in the gel images means wild type. As shown in Figures 4A and 4B, it was confirmed that recombinant microorganisms in which ribB and thyA have been deleted, respectively, were generated. In the case of ribB, the band size of the PCR product of the wild type was shown to be 1.0 kb, and when the ribB region was deleted, the band size was shown to be 500 bp. When PCR was performed using primers that bind to the inside of the ribB region, the wild type was shown to be 250 bp, and no band was observed in the strain in which ribB was deleted. In the case of thyA, the band size of the PCR product was shown to be 700 bp for the wild type, and the band size was shown to be 500 bp when a part of the thyA region was deleted.

[0059] Example 5: Environmental safety assessment of auxotrophs A growth test was carried out to determine the growth pattern of the strain prepared in Example 1. A stack of the prepared strain was streaked on an MRS plate and cultured at 37°C for 3 days. A single colony was selected from the plate and cultured in MRS liquid medium at 37°C for 24 hours with shaking (230 rpm). 1 ml of the culture was extracted and centrifuged at 13,000 rpm for 1 minute. The cell pellet was suspended in 1 ml of 1x PBS solution, and the OD 600 The OD was measured and the OD was measured in 25 ml of SDM (semi-defined media), a minimal medium for lactic acid bacteria, which is an environment where growth can be inhibited. 600 The OD was 0.0025. The mixture was cultured at 37°C with shaking (230 rpm) for 40 hours, and the OD was measured at 15, 17, 20, 22, 24, and 40 hours after inoculation. 600 The measured values ​​are shown in the graph.

[0060] The results are shown in Figures 5A and 5B, respectively. More specifically, Figures 5A and 5B are graphs showing the growth patterns of recombinant lactic acid bacteria lacking ribB (Figure 5A) and thyA (Figure 5B). In Figures 5A and 5B, WT means wild type. In Figures 5A and 5B, it was confirmed that auxotrophic lactic acid bacteria were inhibited in growth under nutrient-deficient medium conditions.

[0061] Example 6: In vivo efficacy: IBD animal model studies To evaluate the therapeutic efficacy of VIP protein derived from the transformed strain, the transformed strain was orally administered to a mouse model of intestinal inflammation induced by DSS (dextransulfate sodium), and the survival rate or DAI (disease activity index) score was observed. After primary culture in 5 ml of MRS medium for one day, the cultured strain was transferred to 50 ml of MRS medium at OD 600 After 16 to 18 hours, the OD of the culture was 0.1. 600 When the number of mice was 4-5, the strains were collected according to the total number of mice administered and the number of feedings. The strains were collected by centrifugation at 4,000 rpm for 10 minutes and removing the culture supernatant. 9 The strains were suspended in 1x PBS and administered at cfu levels once daily for a total of 16 days.

[0062] The first administration of the strain was set as Day 9, and the therapeutic efficacy experiment began. From Day 0 to Day 5, 2% DSS was administered to all groups except the PBS group in drinking water, and the animals were euthanized on Day 8. After DSS treatment, weight loss, hair growth, animal movement, and diarrhea were checked at 2-day intervals, and the DAI score was calculated.

[0063] The results are shown in Figure 6. As shown in Figure 6, it was confirmed that the strain with improved productivity and environmental safety showed superior efficacy compared to the control group in IBD animal model experiments. [Accession number] Depository institution: Korea Institute of Bioscience and Biotechnology Accession number: KCTC 13421BP Date of acceptance: 20171212 Depository institution: Korea Institute of Bioscience and Biotechnology Accession number: KCTC 13422BP Date of acceptance: 20171212 Depository institution: Korea Institute of Bioscience and Biotechnology Accession number: KCTC 13423BP Date of acceptance: 20171212

[0064] Deposit of microorganisms for patent applications Budapest Treaty on International Recognition International Style To. Medytox Inc. Medytox Inc. 114 Central Town-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea 16506 The original receipt issued pursuant to Rule 7.1 by the following international depositary institution: TIFF0007623019000003.tif107170 Form BP / 4 (KCTC form 17)

[0065] Deposit of microorganisms for patent applications Budapest Treaty on International Recognition International Style To. Medytox Inc. Medytox Inc. 114 Central Town-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea 16506 The original receipt issued pursuant to Rule 7.1 by the following international depositary institution: TIFF0007623019000004.tif106170 Form BP / 4 (KCTC form 17)

[0066] Deposit of microorganisms for patent applications Budapest Treaty on International Recognition International Style To. Medytox Inc. Medytox Inc. 114 Central Town-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea 16506 The original receipt issued pursuant to Rule 7.1 by the following international depositary institution: TIFF0007623019000005.tif106170 Form BP / 4 (KCTC form 17)

Claims

1. A recombinant microorganism of the genus Lactobacillus, wherein a promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter have been introduced, proteases have been inactivated, and the microorganism is Lactobacillus paracasei, Lactobacillus brevis, or Lactobacillus plantarum.

2. A recombinant microorganism that is a lactic acid bacterium, into which a constitutive promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the constitutive promoter have been introduced, and in which proteases have been inactivated, the recombinant microorganism being Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum or Lactococcus lactis.

3. The recombinant microorganism of claim 1 or 2, further comprising a signal sequence operably linked between the promoter and the exogenous gene.

4. 3. The recombinant microorganism of claim 1 or 2, wherein the gene encoding the protease has been deleted.

5. 5. The recombinant microorganism of claim 4, wherein the gene encoding the protease is replaced with an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to a promoter.

6. 3. The recombinant microorganism of claim 1, wherein the protease is one or more selected from the group consisting of HtrA, PepN, and ClpP.

7. 3. The recombinant microorganism of claim 1 or 2, which is an auxotroph.

8. 8. The recombinant microorganism of claim 7, wherein one or more genes selected from the group consisting of ribB, thyA and glmS have been deleted.

9. A composition for preventing or treating a disease causing damage to the gastrointestinal tract in humans, comprising a recombinant microorganism which is a lactic acid bacterium into which a promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter have been introduced and in which proteases have been inactivated, the lactic acid bacterium being of the genus Lactobacillus, and the microorganism being Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus plantarum.

10. The composition of claim 9, wherein the disease causes gastrointestinal inflammation.

11. The composition of claim 10, wherein the disease is one or more selected from the group consisting of inflammatory bowel disease (IBD) and colitis.

12. The composition of claim 11, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

13. A composition for preventing or treating a disease causing damage to the gastrointestinal tract in humans, comprising a recombinant microorganism that is a lactic acid bacterium, into which a constitutive promoter and an exogenous gene encoding vasoactive intestinal peptide (VIP) operably linked to the constitutive promoter have been introduced and in which proteases have been inactivated, the lactic acid bacterium being Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum or Lactococcus lactis.

Images