Intestinal probiotic genetically engineered bacterium as well as construction method and application thereof

Abstract

The invention relates to an intestinal probiotic genetically engineered bacterium as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. According to the invention, Escherichia coli is used as an expression host, endogenous recessive plasmid pMUT1 gene and pMUT2 gene are knocked out, an exo-algin lyase gene and an open-loop uronic acid monomer dehydrogenase gene are subjected to heterologous expression, and a 2-keto-3-deoxy-gluconate kinase gene and a 2-keto-3-deoxy-6-phosphate-gluconate aldolase gene are subjected to overexpression. According to the invention, an endogenous recessive plasmid pMUT2 of Escherichia coli Nissle 1917 is utilized, metabolic pathways of algin and degradation products thereof are introduced, and a stable passage engineered strain EcNc-pMUT2 '-4 which can be proliferated by utilizing algin and degradation products thereof and is free of antibiotic markers is obtained, and the engineered strain has good intestinal tract probiotic performance.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a strain of intestinal probiotic genetically engineered bacteria, a construction method thereof, and an application thereof. Background Art

[0002] Escherichia coli Nissle 1917 (EcN) is a high-quality probiotic that has been widely used in the prevention and treatment of intestinal inflammatory diseases. However, EcN's ability to colonize the intestine for a long time is not strong, and the different gastrointestinal environments of clinical populations also make its colonization complicated and difficult, affecting the intestinal probiotic and therapeutic effects of EcN. Targeted modification of EcN through synthetic biology strategies to enhance its intestinal colonization ability and its probiotic effects in the intestine has become a current research hotspot. Previous studies have inserted the epidermal growth factor EGF gene (JCI Insight, 2019, 4, e125166) and the anti-inflammatory factor SJ16 gene (Proc Natl Acad Sci U SA, 2012, 109, 12734) into the EcN genome. The modified engineered EcN has more significant effects in models such as intestinal ulcers and inflammation. In addition, there are also documents that construct metabolic pathways of exogenous effectors in EcN to obtain the synthesis of specific intestinal-targeted substances, such as 3-hydroxybutyric acid (Nat Commun, 2021, 12, 1513), which also show good effects in enhancing therapeutic effects. However, the shortcomings of the existing technology are: generally plasmid expression and integrated expression. Plasmid expression is efficient but requires the presence of antibiotics to maintain separation stability; integrated expression is stable but the expression effect is poor.

[0003] In synthetic biology applications, plasmids are easy to operate as expression vectors, and plasmids with different copy numbers can be selected according to the expression requirements of the target gene. However, compared to genome integration, traditional plasmid systems usually rely on antibiotics to maintain separation stability, which causes limitations in application. EcN itself has two free cryptic plasmids pMUT1 and pMUT2 (NCBI accession numbers are NZ_MW240712 and NZ_CP023342, respectively). Both of these cryptic plasmids have multiple copies and exist naturally and stably in EcN. Therefore, the use of stealth plasmids as functional gene expression vectors can not only utilize their high copy characteristics to achieve efficient expression of target genes, but also avoid the ecological risks brought about by the use of antibiotics, providing a new technical path for the construction of safe and efficient engineered strains. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a genetically engineered intestinal probiotic strain, its construction method, and its application. This invention uses the endogenous cryptic plasmid pMUT2 as a vector in an EcN derivative strain to introduce an exogenous pathway for metabolizing alginate and its degradation products. This results in the construction of an engineered strain, EcNc-pMUT2'-4, which can utilize alginate and its degradation products, is stable for passage, and is antibiotic-free. This strain exhibits excellent intestinal probiotic properties.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first objective of the present invention is to provide a genetically engineered intestinal probiotic bacterium, wherein the genetically engineered bacterium uses Escherichia coli as an expression host, knocks out the endogenous cryptic plasmid pMUT1 gene and pMUT2 gene, heterologously expresses an exo-acting alginate lyase gene and a ring-opening uronic acid monomer dehydrogenase gene, and overexpresses a 2-keto-3-deoxy-gluconokinase gene and a 2-keto-3-deoxy-6-phosphogluconate aldolase gene.

[0007] Furthermore, the genetically engineered Escherichia coli is Escherichia coli Nissle 1917.

[0008] Furthermore, the nucleotide sequence of the exo-acting alginate lyase is as shown in SEQ ID NO. 1, or has at least 90% homology to the sequence shown in SEQ ID NO. 1, and encodes a nucleotide sequence having exo-acting alginate lyase activity;

[0009] And / or, the nucleotide sequence of the ring-opening uronic acid monomer dehydrogenase is as shown in SEQ ID NO. 2, or has at least 90% homology to the sequence shown in SEQ ID NO. 2, and encodes a nucleotide sequence having ring-opening uronic acid monomer dehydrogenase activity.

[0010] Furthermore, the exo-acting alginate lyase gene and the ring-opening uronic acid monomer dehydrogenase gene are derived from marine Vibrio, Flavobacterium or Hydrogenophage.

[0011] Furthermore, the 2-keto-3-deoxy-gluconokinase gene and the 2-keto-3-deoxy-6-phospho-gluconoyl aldolase gene are derived from Escherichia coli.

[0012] Furthermore, the NCBI accession number of the pMUT1 is NZ_MW240712; the NCBI accession number of the pMUT2 is NZ_CP023342.

[0013] Furthermore, plasmid pMUT2 was used as an expression vector.

[0014] Furthermore, the nucleotide sequence of the exo-acting alginate lyase is shown in SEQ ID NO.1, the nucleotide sequence of the ring-opening uronic acid monomer (DEH) dehydrogenase is shown in SEQ ID NO.2, the nucleotide sequence of the 2-keto-3-deoxy-gluconate (KDG) kinase is shown in SEQ ID NO.3, and the nucleotide sequence of the 2-keto-3-deoxy-6-phospho-gluconate (KDPG) aldolase is shown in SEQ ID NO.4.

[0015] The second object of the present invention is to provide a method for constructing the genetically engineered bacteria, comprising the following steps:

[0016] (1) The exo-acting alginate lyase gene, the ring-opening uronic acid monomer dehydrogenase gene, the 2-keto-3-deoxy-gluconokinase gene, and the 2-keto-3-deoxy-6-phospho-gluconoyl aldolase gene were sequentially connected to the plasmid backbone to construct a recombinant plasmid;

[0017] (2) The recombinant plasmid obtained in step (1) is transformed into an Escherichia coli host in which the endogenous cryptic plasmid pMUT1 gene and pMUT2 gene are knocked out to obtain the genetically engineered bacteria.

[0018] The third object of the present invention is to provide a microbial preparation comprising the genetically engineered bacteria.

[0019] Furthermore, the microbial preparation is in solid or liquid form.

[0020] The fourth object of the present invention is to provide the use of the genetically engineered bacteria or the microbial preparation in the preparation of intestinal probiotic products.

[0021] Furthermore, the product also includes alginate and / or alginate degradation products.

[0022] The genetically engineered bacteria of the present invention are fermented and cultured in an M9 liquid culture medium with alginate and its degradation products as the sole carbon source. The liquid culture medium is alginate degradation product (AOS)-M9 culture medium (g / L).

[0023] The genetically engineered bacteria provided by the present invention have the following probiotic characteristics:

[0024] After 3 hours of treatment with artificial simulated gastric fluid, the survival rate exceeded 65%; after 6 hours of treatment with artificial simulated intestinal fluid, the survival rate exceeded 40%.

[0025] The cells could survive for 24 hours in a medium containing 1.2 g / L bile salts, with a survival rate exceeding 60%.

[0026] The hydrophobicity rate in organic solvents such as chloroform, ethyl acetate and n-hexane exceeds 40%.

[0027] In the co-culture system, it has a promoting effect on the probiotic Clostridium butyricum ATCC 19398 and an inhibitory effect on the opportunistic pathogen Enterobacter hallii ATCC 700323.

[0028] The recombinant Escherichia coli of the present invention has good probiotic properties such as resistance to gastrointestinal stress and bile salt tolerance.

[0029] The above technical solution of the present invention has the following advantages over the prior art:

[0030] The present invention provides a genetically engineered intestinal probiotic strain and its construction method and application. The present invention constructs an engineered strain EcNc-pMUT2'-4 that can efficiently metabolize alginate and its degradation products. The cryptic plasmid pMUT2 is used to express exogenous proteins in the EcN derivative strain EcNc with the cryptic plasmid knocked out, that is, heterologously express exo-type alginate lyase and DEH dehydrogenase, and overexpress the glycolysis pathway inherent to EcN, so that the strain can grow under antibiotic-free conditions and the OD value is 0.05 in M9 medium with alginate degradation products as the sole carbon source. 600 The cell cycle efficiency was 0.77, and the cell cycle could be stably propagated.

[0031] The engineered strain EcNc-pMUT2'-4 provided by the present invention has good intestinal probiotic effects, has high resistance to gastrointestinal stress and bile salt tolerance, and can promote the growth of the probiotic Clostridium butyricum ATCC 19398 and inhibit the proliferation of the pathogenic bacteria Enterobacter hallii ATCC 700323. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 This is the growth curve of the engineered strain EcNc-pMUT2'-4 constructed in Example 3 of the present invention in M9-AOS liquid culture medium;

[0034] Figure 2 The stability of the EcNc-pMUT2'-4 plasmid constructed in Example 3 of the present invention was determined and compared.

[0035] Figure 3 is the tolerance of the engineered strain EcNc-pMUT2'-4 constructed in Example 3 of the present invention to simulated artificial gastrointestinal fluid;

[0036] Figure 4 The tolerance of the engineered strain EcNc-pMUT2'-4 constructed in Example 3 of the present invention to different concentrations of bile salts;

[0037] Figure 5 The total OD of the engineered strain EcNc-pMUT2'-4 constructed in Example 7 of the present invention was in a co-culture system with Clostridium butyricum ATCC 19398. 600 and changes in the biomass of each strain; among them, Figure 5 a in the figure is the total growth curve; Figure 5 b in the table is the biomass of Clostridium butyricum; Figure 5 c in the equation is the biomass of E. coli;

[0038] Figure 6 The total OD of the engineered strain EcNc-pMUT2'-4 constructed in Example 8 of the present invention was in a co-culture system with Enterobacter hallii ATCC700323. 600 and changes in the biomass of each strain; among them, Figure 6 a in the figure is the total growth curve; Figure 6 b in the equation is the biomass of Enterobacter hallii; Figure 6 Where c is the biomass of Escherichia coli. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0040] The present invention provides a genetically engineered bacterium with intestinal probiotic properties, its construction method, and application. The genetically engineered bacterium is an engineered strain EcNc-pMUT2'-4 that can utilize alginate and its degradation products in a targeted manner. The endogenous cryptic plasmids pMUT1 and pMUT2 in EcN are knocked out to obtain the engineered host EcNc. The endogenous cryptic plasmid pMUT2 is used as a vector to construct the exo-type alginate lyase and the ring-opening uronic acid monomer (DEH) dehydrogenase missing in the metabolic pathway of alginate and its degradation products into EcNc. At the same time, 2-keto-3-deoxy-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phospho-gluconate (KDPG) aldolase are overexpressed to form a complete metabolic pathway of alginate and its degradation products. The exo-type alginate lyase and DEH dehydrogenase are both mined from the autophagic bacterium UMI-18, and the KDG kinase and KDPG aldolase are derived from EcN itself.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0042] (1) Primers

[0043] The primer sequences used in the present invention are shown in Table 1.

[0044] Table 1. Primers used in the present invention

[0045]

[0046]

[0047] (2) Culture medium

[0048] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, solid medium containing 20.0 g / L agar; when screening transformants, add kanamycin to the medium at a final concentration of 50 μg / mL as needed.

[0049] The engineered strain was cultured in LB liquid or solid medium (with corresponding antibiotics added as needed) at 37° C. for 12 h, with a liquid culture speed of 220 rpm.

[0050] AOS-M9 medium (g / L): Na2HPO4 6.78, KH2PO4 3.0, NaCl 0.5, NH4Cl 1.0, MgSO4 0.241, CaCl2 0.011, AOS 4.0.

[0051] The engineered strain was cultured in AOS-M9 medium at 37°C, 220 rpm for 60 h to evaluate its ability to metabolize alginate degradation products.

[0052] Molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0053] (3) The cell genome extraction kit used in the following examples was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; the one-step cloning kit ClonExpress Ultra One Step Cloning Kit V2 C116 was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; Clostridium butyricum ATCC 19398 and Enterobacter hallii ATCC 700323 were purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0054] Example 1: Construction of recombinant cryptic plasmid

[0055] Using EcN as a template and anti-pMUT1-F / R primers, PCR amplification was performed to generate the pMUT1 linear vector. Using the pCOLADuet-1 plasmid as a template and KanR-1-F / R primers, PCR amplification was performed to generate the kanR fragment. The pMUT1 linear vector and kanR fragment were then homologously ligated to generate the plasmid pMUT1' for transformant screening. Similarly, using anti-pMUT2-F / R primers, PCR amplification was performed to generate the pMUT2 linear vector. This was then used to construct the plasmid pMUT2' containing the kanR fragment.

[0056] The PCR reaction system is shown in Table 2, and the amplification conditions are shown in Table 3.

[0057] Table 2 PCR reaction system

[0058]

[0059] Table 3 PCR amplification conditions

[0060]

[0061] Example 2: Construction of metabolic pathways for alginate and its degradation products

[0062] The coding sequence gene fragments of the exo-acting alginate lyase HyAlyI (nucleotide sequence shown in SEQ ID NO. 1) and DEH dehydrogenase HyDEHI (nucleotide sequence shown in SEQ ID NO. 2) from Hydrogenophage UMI-18 were commissioned to be synthesized by a company. PCR amplification was performed using primers HyalyI-F / R and HyDEHI-F / R. The fragments were then homologously recombined with pMUT1' and pMUT2' using a one-step cloning kit (ClonExpress Ultra One Step Cloning Kit V2 C116), respectively, to obtain the recombinant plasmids pMUT1'-hyalyI-hydehI and pMUT2'-hyalyI-hydehI.

[0063] Example 3: Enhancement of the intrinsic glycolytic pathway of EcN

[0064] EcN genomic DNA was extracted using a cell genome extraction kit. Primers were designed to amplify the kdgk and kdpga genes. Homologous recombination was performed on the recombinant plasmids pMUT1'-hyalyI-hydehI and pMUT2'-hyalyI-hydehI using a one-step cloning kit (ClonExpress Ultra One Step Cloning Kit V2C116). The recombinant plasmids pMUT1'-hyalyI-hydehI-kdgk-kdpga and pMUT2'-hyalyI-hydehI-kdgk-kdpga, respectively, containing four gene segments, were constructed, abbreviated as pMUT1'-4 and pMUT2'-4, respectively. The plasmids were transformed into Escherichia coli BL21(DE3) and transformed into plasmids extracted and sequenced. pMUT1'-4 and pMUT2'-4 plasmids were successfully constructed. The above recombinant plasmids were transformed into the EcN derivative strain EcNc in which the cryptic plasmids pMUT1 and pMUT2 were removed to obtain the engineered strains EcNc-pMUR1'-4 and EcNc-pMUT2'-4.

[0065] Example 4: Ability of engineered bacteria to metabolize alginate degradation products

[0066] The engineered strain constructed in Example 3 was inoculated into 10 mL of LB medium and cultured overnight at 220 rpm at 37°C. The cells were collected by centrifugation at 5000 rpm at 4°C, resuspended in AOS-M9 medium, and inoculated into 50 mL of AOS-M9 medium at a 2% concentration. The fermentation broth was then fermented at 37°C for 60 h. Samples were taken every 6 h to determine the bacterial concentration. Figure 1 As shown, the engineered strain EcNc-pMUT2'-4 has a strong ability to metabolize alginate degradation products, with the highest bacterial concentration reaching 0.77, while the highest bacterial concentration of the engineered strain EcNc-pMUT1'-4 is only 0.59. EcN hardly grows.

[0067] Comparative Example 1:

[0068] The engineered bacteria EcN-MED constructed in the China Science and Technology Paper Online "Construction of Engineering Bacteria for Directed Utilization of Alginate Oligosaccharides and Evaluation of Their Probiotic Performance" was cultured in a culture medium with AOS as the sole carbon source for 60 hours and reached a stable period after 36 hours. The final OD 600 About 0.25.

[0069] The biomass of the engineered bacteria EcNc-pMUT2'-4 mentioned in the present invention is more than three times that of the engineered bacteria EcN-MED in Example 1 under the same culture conditions, indicating that its metabolic capacity for AOS is significantly enhanced and it can more efficiently convert AOS into energy and substances required for bacterial growth.

[0070] Example 5: Stability of engineered bacteria EcNc-pMUT2'-4 through passage

[0071] The engineered bacteria EcNc-pMUT2'-4 were inoculated into LB medium with and without antibiotics, respectively. The bacterial solution was diluted every 12 hours and then spread onto LB plates without antibiotics and LB plates with antibiotics. Plasmid stability was expressed as the ratio of the number of colonies on the plate with antibiotics to the number of colonies on the plate without antibiotics. The engineered bacteria EcNc-pMUT1'-4 constructed with the cryptic plasmid pMUT1' and the engineered bacteria EcNc-pET3b-4 constructed with the commonly used plasmid pET3b were used as controls. The results are shown in Figure 2. Figure 2 After 7 days of continuous passage, more than 90% of the plasmid could still be detected in the engineered bacteria EcNc-pMUT2'-4, but the plasmid stability in EcNc-pMUT1'-4 and EcNc-pET3b-4 decreased to 63% and 31%, respectively.

[0072] Example 6: Tolerance of engineered bacteria to artificial simulated gastrointestinal fluid

[0073] Artificial simulated gastric fluid formulation: Take 1.0 mL of 0.1 mol / L hydrochloric acid and dilute it with distilled water to adjust its pH to 2.0. Then, add 0.01 g / mL of pepsin, dissolve evenly, and sterilize with a microporous filter.

[0074] Formula for artificial simulated intestinal fluid: Take 2.04 g of KH2PO4, add 150 mL of distilled water to dissolve it, adjust the pH value to 6.8 with 4 g / L NaOH solution, and then add trypsin at a concentration of 0.01 g / mL. After dissolving evenly, sterilize it with a microporous filter membrane and set aside.

[0075] The fermentation broth of the engineered bacteria was collected after 12 hours of fermentation, and the cells were collected by centrifugation. The OD value was adjusted with PBS buffer. 600 =1.0, inoculate into artificial simulated gastric fluid at a volume ratio of 1:10, culture at 37°C for 3 hours, then inoculate the mixture into artificial simulated intestinal fluid at the same inoculation ratio and culture for 6 hours, sample and count the number of viable bacteria every hour, and calculate its survival rate. The formula survival rate (%) = (N i / N0)×100% to evaluate the strain's tolerance to artificial simulated gastrointestinal fluid. i is the number of viable bacteria in the samples at different time points; N0 is the initial number of viable bacteria.

[0076] The results are as follows Figure 3As shown, the survival rate of EcNc-pMUT2'-4 was 67.31% after 3 hours of treatment with artificial simulated gastric juice, and the survival rate was 43.32% after 6 hours of treatment with simulated artificial intestinal juice; the survival rate of the wild-type EcN in the control group was 63.31% after 3 hours of treatment with artificial simulated gastric juice, and the survival rate was 40.12% after 6 hours of treatment with simulated artificial intestinal juice, indicating that both wild-type EcN and recombinant Escherichia coli EcNc-pMUT2'-4 have good tolerance in the artificial simulated digestive tract fluid environment.

[0077] Example 7: Tolerance of engineered bacteria to bile salts

[0078] The E. coli EcN and EcNc-pMUT2'-4 before and after transformation were inoculated into LB medium containing 0.03%, 0.06%, 0.09% and 0.12% bile salts, respectively. The LB medium without bile salts was used as the control. The cells were cultured at 37°C for 24 h. Samples were taken every 8 h for viable bacteria count. The survival rate (%) was calculated according to the formula: (N ii / N 0i )×100% to calculate the survival rate. ii is the number of viable bacteria in different bile salt concentration groups at different times, N 0i is the number of viable bacteria in the control group at different times. Figure 4 As shown, when the bile salt concentration was 0.12%, the survival rate of the strain remained above 60% after 24 hours, indicating that the engineered bacteria had a good ability to tolerate bile salts.

[0079] Example 8: Analysis of the probiotic properties of co-cultured engineered bacteria and probiotic Clostridium butyricum

[0080] The engineered bacteria EcNc-pMUT2'-4 and the probiotic Clostridium butyricum ATCC 19398 (Clostridium butyricum, CB) were inoculated into LB liquid medium containing 5% AOS. The inoculum size of the engineered bacteria EcNc-pMUT2'-4 was 1% by volume, and the inoculum size of the co-cultured bacteria was 0.5% by volume. The cultures were anaerobically cultured at 37°C. The co-culture system of EcN and the strain was used as a control. The OD values ​​of the co-culture system were measured at 0, 4, 8, 12, 16, 20, and 24 hours. 600 .

[0081] RT-qPCR was used to determine the copy number of different microorganisms in the co-culture system. Total RNA was extracted from the co-culture fermentation broth and used as a template. The RT-qPCR reaction system and conditions were as follows: 25 μL reaction system, 1 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 12 μL real-time fluorescence premix (SYBR Green Master Mix), and 10 μL ddH2O. The RT-qPCR amplification program was as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 10 s, 60°C annealing and extension temperature for 30 s, 40 cycles, and the instrument default settings for the melting curve stage.

[0082] The bacterial concentration changes of the co-culture system of bacteria and Clostridium butyricum ATCC 19398 are as follows Figure 5 As shown in a, the growth of the engineered bacteria EcNc-pMUT2'-4 co-cultured with Clostridium butyricum was significantly better than that of Clostridium butyricum cultured alone, and the co-culture of the strain before transformation with Clostridium butyricum was significantly better than that of the engineered bacteria EcNc-pMUT2'-4 co-cultured with Clostridium butyricum. 600 Reached 2.79; 12 hours later, the OD of the co-culture system of the engineered bacteria EcNc-pMUT2'-4 and Clostridium butyricum 600 Continuous and stable proliferation.

[0083] The biomass changes of each microorganism in the co-culture system are as follows Figure 5 As shown in Figure 5b and Figure 5c. After 24 hours of co-culture, the biomass of C. butyricum reached 4.3 copies / mL, and the biomass of the engineered EcNc-pMUT2'-4 reached 1.1 copies / mL. Compared to the unmodified EcN-pMUT2'-4 co-culture system, the colony counts of C. butyricum and the engineered EcNc-pMUT2'-4 increased by 31% and 10%, respectively.

[0084] Example 9: Analysis of the probiotic properties of co-culture of engineered bacteria and conditionally pathogenic Enterobacter hormaechei The engineered bacteria EcNc-pMUT2'-4 and the probiotic pathogen Enterobacter hormaechei ATCC 700323 (Enterobacter hormaechei, EH) were inoculated into LB liquid culture medium containing 5% AOS. The inoculation amount of the engineered bacteria EcNc-pMUT2'-4 was 1% by volume, and the inoculation amount of the co-cultured bacteria was 0.5% by volume. The culture was anaerobically statically cultured at 37°C. The co-culture system of EcN and the strain was used as a control. The OD of the co-culture system was measured at 0, 4, 8, 12, 16, 20 and 24 h. 600 .

[0085] The bacterial concentration changes of the co-culture system with Enterobacter hallii ATCC 700323 are as follows Figure 6As shown in the figure, the growth of the engineered bacteria EcNc-pMUT2'-4 co-cultured with E. coli was significantly worse than that of E. coli cultured alone, and the OD 600 From 2.5 to 1.8.

[0086] Further analysis revealed changes in the biomass of each microorganism in the co-culture system. After 24 hours of co-culture with E. hallii ATCC 700323, the biomass of E. hallii reached 2.1 copies / mL, while that of E. hallii reached 1.3 copies / mL. Compared to the co-culture system of EcN and E. hallii, the colony count of E. hallii decreased by 8%, while the colony count of E. hallii increased by 41%. These results demonstrate that the engineered strain EcNc-pMUT2'-4 effectively promotes the proliferation of beneficial bacteria and inhibits opportunistic pathogens.

[0087] The present invention demonstrates the superiority of the constructed engineered bacteria EcNc-pMUT2'-4 in utilizing alginate and its degradation products, and also provides a better engineering strain selection for its application in the fields of probiotic preparations and intestinal microecological regulation.

[0088] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A strain of intestinal probiotic genetically engineered bacteria, characterized by: The genetically engineered bacteria uses Escherichia coli as an expression host, knocks out the endogenous cryptic plasmid pMUT1 gene and pMUT2 gene, heterologously expresses the exo-algin lyase gene and the ring-opening uronic acid monomer dehydrogenase gene, and overexpresses the 2-keto-3-deoxy-gluconokinase gene and the 2-keto-3-deoxy-6-phospho-gluconoyl aldolase gene.

2. The genetically engineered bacterium according to claim 1, characterized in that The genetically engineered Escherichia coli is Escherichia coli Nissle 1917.

3. The genetically engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the exo-acting alginate lyase is shown in SEQ ID NO. 1, or has at least 90% homology to the sequence shown in SEQ ID NO. 1, and encodes a nucleotide sequence having exo-acting alginate lyase activity; And / or, the nucleotide sequence of the ring-opening uronic acid monomer dehydrogenase is as shown in SEQ ID NO. 2, or has at least 90% homology to the sequence shown in SEQ ID NO. 2, and encodes a nucleotide sequence having ring-opening uronic acid monomer dehydrogenase activity.

4. The genetically engineered bacterium according to claim 1, characterized in that The 2-keto-3-deoxy-gluconokinase gene and the 2-keto-3-deoxy-6-phospho-gluconoyl aldolase gene are derived from Escherichia coli.

5. The genetically engineered bacterium according to claim 1, characterized in that The NCBI accession number of the pMUT1 is NZ_MW240712; the NCBI accession number of the pMUT2 is NZ_CP023342.

6. The genetically engineered bacterium according to claim 1, characterized in that Plasmid pMUT2 was used as the expression vector.

7. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) The exo-acting alginate lyase gene, the ring-opening uronic acid monomer dehydrogenase gene, the 2-keto-3-deoxy-gluconokinase gene, and the 2-keto-3-deoxy-6-phospho-gluconoyl aldolase gene were sequentially connected to the plasmid backbone to construct a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) is transformed into an Escherichia coli host in which the endogenous cryptic plasmid pMUT1 gene and pMUT2 gene are knocked out to obtain the genetically engineered bacteria.

8. A microbial preparation, characterized in that Comprising the genetically engineered bacteria according to any one of claims 1 to 6.

9. Use of the genetically engineered bacteria according to any one of claims 1 to 6 or the microbial preparation according to claim 8 in the preparation of intestinal probiotic products.

10. The use according to claim 9, characterized in that The product also includes alginate and / or alginate degradation products.

Images