MICROORGANISM EXPRESSING VASOACTIVE INTESTINAL PEPTIDE, AND USE THEREOF

Abstract

Provided are a recombinant microorganism expressing a vasoactive intestinal peptide (VIP) gene, and a composition including the microorganism for preventing or treating a disease causing damage to the gastrointestinal tract.

Claims

1. A recombinant microorganism of the genus Lactobacillus, in which a promoter and an exogenous gene are introduced and a protease is inactivated, wherein the exogenous gene is operably linked to the promoter and encodes vasoactive intestinal peptide (VIP).

2. The microorganism of claim 1, wherein the microorganism is Lactobacillus paracasei, Lactobacillus brevis, or Lactobacillus plantarum.

3. A recombinant microorganism, which is a lactic acid bacterium, in which a constitutive promoter and an exogenous gene are introduced and a protease is inactivated, wherein the exogenous gene is operably linked to the promoter and encodes vasoactive intestinal peptide (VIP).

4. The microorganism of claim 3, wherein the lactic acid bacterium is of the genus Lactobacillus or the genus Lactococcus.

5. The microorganism of claim 4, wherein the lactic acid bacterium is Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, or Lactococcus lactis.

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

7. The microorganism of claim 1, wherein a gene encoding a protease is deleted.

8. The microorganism of claim 7, wherein the gene encoding a protease is replaced with an exogenous gene operably linked to the promoter and encoding vasoactive intestinal peptide (VIP).

9. The microorganism of claim 1, wherein the protease is at least one selected from the group consisting of HtrA, PepN, ClpP, and Lon.

10. The microorganism of claim 1, wherein the microorganism is an auxotroph.

11. The microorganism of claim 10, wherein at least one gene selected from the group consisting of ribB, thy A, and glmS is deleted.

12. 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, in which a promoter and an exogenous gene are introduced and a protease is inactivated, wherein the exogenous gene is operably linked to the promoter and encodes vasoactive intestinal peptide (VIP).

13. The composition of claim 12, wherein the disease is one causing inflammation of the gastrointestinal tract.

14. The composition of claim 13, wherein the disease is at least one selected from the group consisting of inflammatory bowel disease (IBD) and colitis.

15. The composition of claim 14, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0059] FIGS. 1A and 1B are schematics showing genetically engineered sites of recombinant microorganisms according to an example.

[0060] FIGS. 2A and 2B are figures confirming the generation of recombinant microorganisms with improved VIP productivity, according to an example.

[0061] FIGS. 3A and 3B are figures showing that recombinant microorganisms according to an example had improved VIP productivity.

[0062] FIGS. 4A and 4B are figures confirming the generation of recombinant microorganisms with improved environmental safety, according to an example.

[0063] FIGS. 5A and 5B are graphs showing recombinant microorganisms according to an example had improved environmental safety.

[0064] FIG. 6 is a graph showing that a recombinant microorganism according to an example exhibits a therapeutic effect on IBD in an animal model.

BEST MODE

[0065] Hereinafter, the disclosure will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited in any way by these examples.

Example 1: Preparation of Recombinant Microorganism

[0066] A recombinant microorganism expressing exogenous VIP was prepared, according to the homologous recombination genetic engineering method commonly used for 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), using Lactobacillus brevis LMT1-46 (KCTC 13423BP).

[0067] FIGS. 1A and 1B schematically show the genetically engineered site of the microorganism according to the example. FIG. 1A shows the process of replacing the protease gene (htrA or pepN) with the VIP gene to improve VIP productivity. A signal peptide that assists in secretion on 5-side of the VIP gene and a tag for detection on 3-side of the VIP gene were included in one cassette. The protease gene is L. brevis htrA (SEQ ID NO: 14) or L. brevis pepN (SEQ ID NO: 15). FIG. 1B shows a process of generating an auxotroph to promote environmental safety, by removing some regions of a target gene. The target gene is L. brevis ribB (SEQ ID NO: 18). Each abbreviation in FIGS. 1A and 1B has the following meaning: P: Promoter, SP: Signal peptide, UTR: Untranslated region, VIP: Vasoactive intestinal peptide), A: L. brevis ribB, B: L. brevis htrA or L. brevis pepN.

Example 2: Confirmation of Generation of Recombinant Microorganism

[0068] PCR was proceeded to confirm that the recombinant microorganism was produced as intended. Lactic acid bacteria colonies stationary cultured at 37? C. for 16 hours on a De Man, Rogosa and Sharpe agar (MRS) plate were used. A single colony was picked and suspended in a tube containing 100 ?l of distilled water. After bead-beating, the suspension was heated at 98? C. for 10 minutes to use as a DNA template. 1 ?l of DNA template, 1 ?l of forward primer, 1 ?l of reverse primer, and 25 ?l of DNA polymerase mix were mixed to prepare a PCR mixture. PCR was performed under the conditions of 40 cycles of amplification of (a) denaturation, 10 seconds at 98? C.; (b) annealing, 15 seconds at 55? C.; and (c) extension, 10 seconds at 72? C. per cycle, and a final extension of 2 minutes at 72? C. The primers used were designed to target regions outside the open reading frame (ORF) of the gene region to be engineered, and the sequences are described in Table 1 below.

TABLE-US-00001 TABLE1 SEQ ID Name Sequence NO 1-46pepNicF AGCAACCTT 21 TGACCTAGC 1-46pepNicR AATTCCATA 22 TCACCACCC AC pepNorfcF CCCGATGGC 23 CTTACAAC pepNorfcR GGAACGGCT 24 GTCCATTAG 1-46htrA CTAAATGAG 25 insertcF GAGGGTTCG CG 1-46htrA AAGCTGGCG 26 insertcR CTTTCATTC C 1-46htrA ACCTCTAAC 27 ORFcF GTCAACGTC 1-46htrA AGTTCCCAG 28 ORFcR GGTTAATCG

[0069] The results are each shown in FIGS. 2A and 2B. More specifically, FIGS. 2A and 2B are schematics (left) showing that the protease genes htrA (FIG. 2A) and pepN (FIG. 2B) are each replaced with a VIP expression cassette, and gel images (right) showing that the band sizes were changed as intended due to generation of recombinant microorganisms. As shown in FIGS. 2A and 2B, it was confirmed that recombinant microorganisms were generated in which the protease genes htrA and pepN were each replaced with a VIP expression cassette. In case of htrA, the band size of the PCR product was 1.5 kb in the wild-type, and when the htrA region was replaced with a VIP expression cassette, the band size was 920 bp. In case of pepN, the band size of the PCR product was 2 kb in the wild-type, and when the pepN region was replaced with a VIP expression cassette, the band size was 1.3 kb. In the schematics of FIGS. 2A and 2B, each abbreviation is as defined in Example 1, and WT in the gel image refers to the wild-type.

Example 3: Evaluation of VIP Productivity of Recombinant Microorganism

[0070] In order to identify whether the VIP productivity of the recombinant microorganism was improved, an evaluation was performed to identify the VIP expression level of the strains prepared in Example 1. The stock of the prepared strain was streaked on an MRS plate and stationary cultured for 3 days at 37? C. Here, a single colony was picked and cultured with shaking at 37? C. in a MRS liquid medium for 24 hours. The cultured medium was inoculated into 25 ml of MRS so that OD.sub.600 became 0.1, and then cultured with shaking at 37? C. for 16 hours. 10 ml of the strain culture medium was extracted and centrifuged at 4,000 rpm for 10 minutes, and only the supernatant was extracted, 1 ml of tricholoacetic acid (TCA) was added, and treated at 4? C. for 30 minutes or more. This was centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed and the pellet was suspended using 1 ml of acetone stored at 4? C. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was removed, and 0.5 ml of acetone was added to suspend the pellet. The suspension was again centrifuged at 13,000 rpm for 10 minutes, and dried at 60? C. for 5 minutes. A sample buffer (reducing buffer) was added and the pellet was mixed well and treated at 98? C. for 10 minutes. Western blotting was performed using the obtained solution. Based on the results of the western blotting, the intensities of the bands measured according to regions of the same size were quantified by using a program called ImageJ.

[0071] The results are each shown in FIGS. 3A and 3B. More specifically, FIG. 3A is a gel image (left) showing the generated recombinant strain in which htrA and/or ribB is deleted, and VIP expressed therefrom, and a graph (right) quantifying expression level of VIP. FIG. 3B is a graph showing expression level of VIP expressed from a recombinant strain in which pepN is deleted. As may be identified from FIGS. 3A and 3B, when the protease gene was deleted from L. brevis, the expression level of VIP, an effective substance, was increased. In particular, when htrA was deleted, the expression level of the target substance, VIP, was increased more than 3 times compared to those when other genes were deleted.

Example 4: Confirmation of Generation of Auxotrophs

[0072] PCR was performed to confirm that the auxotroph was generated as intended. Lactic acid bacteria colonies, which were stationary cultured at 37? C. for 16 hours on a De Man, Rogosa and Sharpe agar (MRS) plate, were used. A single colony was picked and suspended in a tube containing 100 ?l of distilled water. After bead-beating, the suspension was heated at 98? C. for 10 minutes to use as a DNA template. 1 ?l of DNA template, 1 ?l of forward primer, 1 ?l of reverse primer, and 25 ?l of DNA polymerase mix were mixed to prepare a PCR mixture. PCR was performed under the conditions of 40 cycles of amplification of (a) denaturation, 10 seconds at 98? C.; (b) annealing, 15 seconds at 55? C.; and (c) extension, 10 seconds at 72? C. per a cycle, and a final extension of 2 minutes at 72? C. The primers used were designed to target regions inside and outside the ORF of the gene region to be engineered, and the sequences are described in Table 2 below.

TABLE-US-00002 TABLE2 SEQID Name Sequence NO 1-46ribB TAACCGCAG 29 orfcF TGACTGAC 1-46ribB AGCTGATAC 30 orfcR ATCAAAGGT C 1-46ribBicF AGCATTGTG 31 TTATCAGC 1-46ribBicR GCAGCATTG 32 GTAGCAAC LbthyA-UP-cF3 GTGTGGCAA 33 GGTGGCAAA GCCA LbthyA-DN-cR1 CCGATCTAC 34 AGGCCCAAC TCGATGA

[0073] The results are each shown in FIGS. 4A and 4B. More specifically, FIGS. 4A and 4B are schematics (left) of recombinant microorganisms in which rib and thyA are deleted, respectively, and gel images (right) showing that the band sizes were changed as intended due to the generation of the recombinant microorganisms. In FIGS. 4A and 4B, each abbreviation is as defined in Example 1, and WT in the gel image refers to the wild-type. As shown in FIGS. 4A and 4B, it was confirmed that recombinant microorganisms were generated in which ribB and thyA were deleted, respectively. In case of ribB, the band size of the PCR product was 1.0 kb in the wild-type, and when the ribB region was deleted, the band size was 500 bp. When PCR was performed using a primer that binds to the inside of the ribB region, the wild-type was found to be 250 bp, and no band was observed in the strain in which ribB was deleted. In case of thyA, the band size of the PCR product was 700 bp in the wild-type, and when a part of the thyA region was deleted, the band size was 500 bp.

Example 5: Evaluation of Environmental Safety of Auxotroph

[0074] A growth test was performed to determine the growth pattern of the strain prepared in Example 1. The stock of the prepared strain was streaked on an MRS plate and stationary cultured for 3 days at 37? C. From the plate, a single colony was picked and cultured with shaking (230 rpm) in an MRS liquid medium at 37? C. for 24 hours. 1 ml of the culture medium was extracted and centrifuged at 13,000 rpm for 1 minute. After suspending the cell pellet in 1 ml of 1x PBS solution, the cell was inoculated into 25 ml of a minimum medium for lactic acid bacteria which could limit the growth of lactic acid bacteria called semi-defined media (SDM), so that the measured OD.sub.600 became 0.0025. It was cultured for 40 hours under a shaking culture (230 rpm) condition at 37? C., and the measured OD.sub.600 values at 15, 17, 20, 22, 24, and 40 hours after inoculation are shown as a graph.

[0075] The results are each shown in FIGS. 5A and 5B. More specifically, FIGS. 5A and 5B are graphs showing the growth patterns of ribB-deleted recombinant lactic acid bacteria (FIG. 5A) and thyA-deleted recombinant lactic acid bacteria (FIG. 5B). In FIGS. 5A and 5B, WT means wild-type. It was confirmed from FIGS. 5A and 5B that the growth of the auxotrophic lactic acid bacteria has been inhibited in nutrient-deficient medium conditions.

Example 6: In Vivo Efficacy: IBD Animal Model Experiment

[0076] In order to evaluate therapeutic efficacy of VIP protein derived from the transformed strain, the transformed strain was orally administered to mouse intestinal inflammation models induced by dextran sulfate sodium (DSS), and then the survival rate or disease activity index (DAI) score were identified. After primarily culturing in 5 ml of MRS medium for one day, the cultured strain was inoculated into 50 ml of MRS medium so that OD.sub.600 became 0.1. When the OD.sub.600 of the culture medium reached 4 to 5 after 16 to 18 hours, the strain was recovered according to the total number of mice to be administered and the number of feedings. The culture supernatant was removed by centrifugation at 4,000 rpm for 10 minutes, and the recovered strain was suspended in 1?PBS so that it could be administered at 1?109 cfu per mouse per administration. The administration of the strain was performed once a day, for a total of 16 days.

[0077] The treatment efficacy experiment was started by setting the first day of administrating the strain to Day-9, and from Day 0 to Day 5, 2% DSS was in drinking water in all groups except for the PBS group, and euthanasia was performed on Day 8. After DSS treatment, the DAI score was calculated by checking the weight loss, bristled hair, the degree of movement of the animals and the presence of diarrhea every two days.

[0078] The results are shown in FIG. 6. As shown in FIG. 6, it was confirmed that the strain having improved productivity and improved environmental safety exhibited superior efficacy compared to the control groups in the IBD animal model experiment.

ACCESSION NUMBER

[0079] Depository institute: Korea Research Institute of Bioscience and Biotechnology [0080] Accession number: KCTC13421BP [0081] Deposition date: 2017 Dec. 12 [0082] Depository institute: Korea Research Institute of Bioscience and Biotechnology [0083] Accession number: KCTC13422BP [0084] Deposition date: 2017 Dec. 12 [0085] Depository institute: Korea Research Institute of Bioscience and Biotechnology [0086] Accession number: KCTC13423BP [0087] Deposition date: 2017 Dec. 12