L-tryptophan-producing engineered strain of clostridium butyricum and use thereof in preparation of drug for treating tumors

Abstract

The present invention relates to an L-tryptophan-producing engineered strain of Clostridium butyricum and use thereof in the preparation of a drug for treating tumors, and pertains to the technical field of biomedical engineering. The tryptophan operon gene trpEDCBA is overexpressed in Clostridium butyricum by means of a thl promoter and a pMTL82151 shuttle vector to construct an engineered strain of Clostridium butyricum with a high L-tryptophan yield. The strain can specifically target and colonize tumors in mice without additional treatment. The strain secretes butanoic acid and tryptophan metabolites to inhibit IDO enzyme expression, regulate CD8+ T cell metabolism, and change the immune microenvironment of tumors, thereby achieving the purpose of inhibiting tumor growth. In addition, the strain will not affect the physiological structures of the major organs in the body, showing a favorable safety profile.

Description

An engineered clostridium butyricum producing l-tryptophan and application thereof in preparation of tumor treatment drugs TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, and particularly discloses an engineered clostridium butyricum producing l-tryptophan and application thereof in preparation of tumor treatment drugs. BACKGROUND

[0002] Cancer is a disease that seriously threatens human health. In recent years, with the exploration of cancer treatment methods, immunotherapy has attracted more and more attention due to its significant clinical treatment effect. Although it can make patients with response have a high probability of long-term survival, the overall response rate of tumor patients is relatively low, which greatly limits the clinical effect of tumor immunotherapy. Therefore, it is necessary to develop new tumor immunotherapy strategies to inhibit tumors and improve treatment effect. In the past decade, microbiota has played an important role in cancer immunotherapy. In preclinical and clinical trials, targeting tumor microenvironment with live bacteria is a very promising immunotherapy strategy. Some bacteria, especially obligate and facultative anaerobic strains, can preferentially colonize the hypoxic and vascular-rich tumor microenvironment to trigger an immune response. However, intravenous injection of bacteria for cancer treatment also has problems such as low bacterial survival rate and poor tumor accumulation. In order to improve the feasibility of treating tumors, more and more studies have modified target bacteria through genetic engineering to make them have the desired functions, such as releasing drugs and secreting immune stimulating molecules.

[0003] In tumors, limited blood supply makes tumor cells and immune cells compete for nutrients to meet their own needs. Tumors reprogram various metabolic pathways to take up a large amount of nutrients. As a kind of essential amino acid, tryptophan is an important nutrient component of T cell response and an important mediator of anti-tumor immune response. However, tumor cells can deplete tryptophan levels through the enzymatic activity of indoleamine 2,3-dioxygenase, leading to a lack of tryptophan and accumulation of metabolites such as kynurenine in the microenvironment, which inhibits effector T cells and establishes an immunosuppressive microenvironment that supports cancer cell proliferation and survival. Although IDO inhibitors show promise in preclinical models, there are still major challenges such as potential off-target effects. In addition, due to its chemical structure, traditional IDO inhibitors may exhibit toxicity by affecting multiple pathways. How to optimize the therapeutic potential of IDO inhibitors while reducing their limitations is crucial for the development of improved immunotherapy methods and programs.

[0004] Clostridium butyricum (CB) is a spore-forming probiotic bacteria, which is a strict anaerobe with good physiological and biochemical characteristics. Compared with non-spore probiotic agents, it has the advantages of stable storage, stable viable count, immune function and antioxidant property. It is a common microbial feed additive with the functions of regulating intestinal microecological balance and promoting growth and development. Clostridium butyricum can promote the fermentation of various carbohydrates to produce butyric acid. Butyric acid, as a short-chain fatty acid, can act on adaptive immune cells through various pathways such as immune cell recruitment, activation and promotion of antibody secretion, to regulate host immune response and maintain host immune homeostasis. With further research, it is found that butyric acid produced by intestinal flora can not only promote the immune response of CD8 + T cells, but also regulate intestinal homeostasis by reducing the level of STAT1 in colon epithelial cells and inhibiting the expression of IDO through HDAC inhibitor properties. In summary, it is a potential strategy to genetically modify Clostridium butyricum to produce tryptophan to target tumor microenvironment metabolism therapy to provide IDO inhibitors to meet the current needs of tumor immunotherapy. However, the microbial strains currently used to produce tryptophan basically exist in aerobic environment, and there is no report on the treatment of tumors by engineering strains with high tryptophan production and anaerobic characteristics.

[0005] SUMMARY

[0006] To solve the above technical problems, the present application provides an engineered Clostridium butyricum producing L-tryptophan and its application in preparing tumor treatment drugs. The present application uses thl promoter and pMTL82151 shuttle vector to overexpress tryptophan operon gene trpEDCBA in Clostridium butyricum, and constructs an engineered Clostridium butyricum with high yield of L-tryptophan. The strain inhibits the expression of IDO and regulates the metabolism of CD8 + T cells by secreting butyric acid and tryptophan metabolites, and changes the tumor immune microenvironment.

[0007] The first object of the present application is to provide an engineered Clostridium butyricum producing L-tryptophan, which overexpresses tryptophan operon gene trpEDCBA in Clostridium butyricum.

[0008] Further, the thl promoter is used to overexpress the tryptophan operon gene trpEDCBA.

[0009] Further, the shuttle vector pMTL82151 is used to express the tryptophan operon gene trpEDCBA.

[0010] The second object of the present application is to provide a construction method of an engineered Clostridium butyricum producing L-tryptophan, comprising the following steps:

[0011] S1, constructing plasmid pMTL82151-trpEDCBA;

[0012] S2, transfecting Escherichia coli with plasmid pMTL82151-trpEDCBA to obtain recombinant Escherichia coli;

[0013] S3, mixing the recombinant Escherichia coli with Clostridium butyricum to make pMTL82151-trpEDCBA conjugated to Clostridium butyricum, thereby obtaining the engineered Clostridium butyricum.

[0014] A third object of the present application is to provide a microbial inoculant comprising the engineered Clostridium butyricum described above.

[0015] Further, the engineered Clostridium butyricum described above is cultured in a thioethanolate medium, and the supernatant is collected to obtain a metabolite.

[0016] Further, the thioethanolate medium comprises tryptone 15 g / L, yeast extract powder 5 g / L, glucose 5 g / L, sodium thioethanolate 0.5 g / L, sodium chloride 2.5 g / L, L-cystine 0.5 g / L, agar 0.75 g / L, and resazurin 0.001 g / L.

[0017] Further, the culture time is 24 hours.

[0018] A fourth object of the present application is to provide the use of the engineered Clostridium butyricum described above, the microbial inoculant described above, or the metabolite described above in the preparation of a tumor treatment drug.

[0019] Further, the tumor comprises a colon tumor.

[0020] Further, the drug is administered by intravenous injection.

[0021] A fifth object of the present application is to provide the use of the engineered Clostridium butyricum described above or the microbial inoculant described above in the preparation of a drug for promoting T cell metabolism.

[0022] A sixth object of the present application is to provide the use of the engineered Clostridium butyricum described above or the microbial inoculant described above in the preparation of an IDO inhibitor.

[0023] Advantages of the present application:

[0024] The present application constructs an engineered Clostridium butyricum with high L-tryptophan yield through genetic engineering technology. The strain can specifically target and colonize in tumors in mice without additional treatment, inhibit IDO expression by secreting butyric acid and tryptophan metabolites, regulate CD8 + T cell metabolism, change the tumor immune microenvironment, thereby achieving the purpose of inhibiting tumor growth, and does not affect the physiological structure of major organs in the body, and has good safety. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the accompanying drawings, in which

[0026] FIG. 1 is a plasmid map of pMTL82151-thl and pMTL82151-trpEDCBA in embodiment 1 of the present application, in which A is the plasmid map of pMTL82151-thl and B is the plasmid map of pMTL82151-trpEDCBA;

[0027] FIG. 2 is an electrophoresis map of enzyme digestion identification of plasmid pMTL82151-trpEDCBA and a statistical diagram of RNA expression levels of trpE, trpD, trpC, trpB and trpA in embodiment 1 of the present application;

[0028] FIG. 3 is a schematic diagram of gene regulation of plasmid pMTL82151-trpEDCBA in Clostridium butyricum in embodiment 1 of the present application;

[0029] FIG. 4 is an electron microscope imaging diagram of engineered Clostridium butyricum L-Trp CB in embodiment 1 of the present application;

[0030] FIG. 5 is a statistical diagram of particle size and zeta potential of engineered Clostridium butyricum L-Trp CB in embodiment 1 of the present application;

[0031] FIG. 6 is a bacterial growth curve diagram of engineered Clostridium butyricum L-Trp CB in embodiment 1 of the present application;

[0032] FIG. 7 is a statistical diagram of contents of secreted tryptophan and butyric acid of engineered Clostridium butyricum L-Trp CB detected by HPLC in embodiment 1 of the present application;

[0033] FIG. 8 is a Western blot diagram and a statistical diagram of tumor cell IDO enzyme expression of metabolites of Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB in embodiment 2 of the present application;

[0034] FIG. 9 is a flow cytometry statistical diagram of T cell effector phenotype of lymph node obtained in flow cytometry experiment in embodiment 2 of the present application;

[0035] FIG. 10 is an OCR statistical diagram of lymph node T cells obtained in extracellular flux analyzer experiment in embodiment 2 of the present application;

[0036] FIG. 11 is an ECAR statistical diagram of lymph node T cells obtained in extracellular flux analyzer experiment in embodiment 2 of the present application;

[0037] FIG. 12 is a live fluorescence imaging diagram and a statistical diagram of ex vivo fluorescence imaging diagram of each organ of biodistribution of Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB in embodiment 3 of the present application;

[0038] Figure 13 is a statistical chart of intratumoral colony results for Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB in Example 3 of the present application;

[0039] Figure 14 is a statistical chart of IDO immunohistochemistry of tumor tissue and kynurenine, tryptophan and butyrate contents of tumor colonization by engineered Clostridium butyricum L-Trp CB detected by HPLC experiment in Example 3 of the present application;

[0040] Figure 15 is a statistical chart of tumor growth curve, mouse survival rate and body weight of tumor-bearing mice in Example 4 of the present application;

[0041] Figure 16 is a statistical chart of tumor representative graph and tumor growth curve of rabbits in Example 4 of the present application;

[0042] Figure 17 is a statistical chart of tumor T cell distribution map obtained by single cell sequencing analysis experiment in Example 4 of the present application;

[0043] Figure 18 is a statistical chart of tumor CD8 + T and CD4 + T cell representative graph obtained by flow cytometry experiment in Example 4 of the present application;

[0044] Figure 19 is a statistical chart of CD8 + T cell representative gene bubble chart and corresponding protein flow chart obtained by tumor single cell sequencing analysis and flow cytometry experiment in Example 4 of the present application;

[0045] Figure 20 is a CD8 + T cell other representative gene bubble chart of tumor single cell sequencing analysis in Example 4 of the present application;

[0046] Figure 21 is a gene enrichment analysis chart of CD8 + T cell of tumor single cell sequencing analysis in Example 4 of the present application;

[0047] Figure 22 is a KEGG enrichment analysis chart of CD8 + T cell of tumor single cell sequencing analysis in Example 4 of the present application;

[0048] Figure 23 is a statistical chart of CD8 + T cell representative related metabolic genes volcano plot and gene bubble chart obtained by tumor single cell sequencing analysis experiment in Example 5 of the present application;

[0049] Figure 24 is a statistical chart of CD8 + T cell related metabolic pathway gene enrichment analysis chart of tumor single cell sequencing analysis in Example 5 of the present application;

[0050] Figure 25 is a photograph of H&E sections of major organs and tissues obtained in the safety evaluation experiment in Example 6 of the present application. DETAILED DESCRIPTION

[0051] The present application will be further described with reference to the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it.

[0052] Example 1: Construction of L-Trp CB, a Clostridium butyricum strain producing L-tryptophan

[0053] (1) Construction of expression recombinant plasmid pMTL82151-thl

[0054] The strong promoter Pthl was cloned and inserted into the Clostridium-Escherichia coli shuttle plasmid pMTL82151, and the construction procedure included amplification using primers thl-Xba I-F / R (the nucleotide sequence of thl-Xba I-F is 5'-GCTCTAGAGACTATTCCTCCTAAATATT-3'; the nucleotide sequence of thl-Xba I-R is 5'-CCGGAGCTCTATATGATAAAAGCGACTTATA-3'), and then as a template, further amplification was performed using primers thl-F / R (the nucleotide sequence of thl-F is 5'-GACTATTCCTCCTAAATATT-3', and the nucleotide sequence of thl-R is 5'-TATATGATAAAAGCGACTTATA-3'), to obtain the amplification product of the thl gene. The PCR procedure was as follows: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, and elongation at 68°C for 20 seconds, for 30 cycles; and elongation at 65°C for 2 minutes. Subsequently, the amplification product of the thl gene was double-digested with pMTL82151 using Xba I and Xho I restriction endonucleases. The digested product was recovered to obtain the plasmid pMTL82151-thl (as shown in Figure 1A).

[0055] (2) Construction of expression recombinant plasmid pMTL82151-trpEDCBA

[0056] The structural gene sequence of the tryptophan biosynthesis enzyme (trpE, D, C, B, A) capable of expressing the tryptophan operon of E. coli obtained by gene synthesis (as shown in SEQ ID NO. 1-5, respectively) is amplified by polymerase chain reaction (PCR) using primers and flanking restriction enzyme recognition sites. According to the amplified tryptophan synthetase gene sequence, the tryptophan synthetase gene sequence is cloned into a plasmid vector. Then the pMTL82151-thl plasmid is digested with the corresponding restriction endonuclease, and the digestion product is recovered for ligation reaction. The amplified trpEDCBA gene fragment is inserted into the plasmid pMTL82151-thl to obtain the expression type recombinant plasmid pMTL82151-trpEDCBA (as shown in FIG. 1B). The primer sequences are shown in Table 1.

[0057] Table 1 trpEDCBA primer sequences

[0058] (3) Construction of engineered Clostridium butyricum L-Trp CB

[0059] The recombinant plasmid pMTL82151-trpEDCBA is heat-shocked and transformed into E. coli, and single colonies are picked and cultured in LB medium containing chloramphenicol for further culture. Clostridium butyricum is cultured in liquid thioacetate medium. E. coli and Clostridium butyricum BNCC337239 are mixed and plated on RCM plates and cultured anaerobically at 37°C to allow pMTL82151-trpEDCBA in E. coli to be transferred to Clostridium butyricum strain by conjugation. Subsequently, the bacterial cells on the plate are collected and plated on RCM plates containing thiamphenicol for antibiotic selection, and single colonies are picked and further cultured in liquid thioacetate medium to obtain engineered Clostridium butyricum strain L-Trp CB. The recombinant plasmid pMTL82151-trpEDCBA is digested with the plasmid pMTL82151-trpEDCBA and electrophoresis is performed to identify the success of insertion, and the positive engineered strain L-Trp CB is confirmed by quantitative reverse transcription PCR (RT-qPCR) to highly express the trpEDCBA gene. The schematic diagram of gene regulation of plasmid pMTL82151-trpEDCBA in Clostridium butyricum is shown in FIG. 3.

[0060] (4) Characterization of engineered Clostridium butyricum L-Trp CB

[0061] The morphology of the L-Trp-producing Clostridium butyricum strain was observed and its internal characterization was identified by transmission electron microscopy (TEM, model: FEI TF20) and Zeta potential instrument (model: Nano ZS90, manufacturer: MALVERN), as shown in FIGS. 4 and 5, L-Trp CB exhibited a similar rod-like morphology as the starting strain CB, and there was no significant difference in particle size or Zeta potential compared with CB, the average particle size was 2.5-3.5 mm, and the Zeta potential was -25 to -20 mV. The effect of L-Trp production on the activity of the bacteria itself was detected by bacterial growth state curve, CB and L-Trp CB were statically cultured in liquid thioethanolate medium at 37°C without or with chloramphenicol (15 pg / mL). At the designated time point, the absorbance value of different bacterial samples at 600 nm wavelength was measured by ultraviolet spectrophotometer (GENESYS10S, Thermo Fisher), as shown in FIG. 6, the growth curve OD 600 value of L-Trp CB was not different from that of ordinary Clostridium butyricum CB, indicating that the heterologous expression of the tryptophan operon in Clostridium butyricum did not affect the growth of the bacteria.

[0062] (5) Production performance

[0063] To determine the yield of tryptophan and butyric acid secreted by the engineered Clostridium butyricum L-Trp CB, the bacteria stored at -80°C were statically cultured in an anaerobic test tube of thioethanolate medium added with chloramphenicol (15 pg / mL) at 37°C for 24 hours. The fermentation medium composition was: tyroptone 15 g / L, yeast extract powder 5 g / L, glucose 5 g / L, sodium thioethanolate 0.5 g / L, sodium chloride 2.5 g / L, L-cystine 0.5 g / L, agar 0.75 g / L, resazurin 0.001 g / L. Then the bacterial culture was centrifuged at 12000 rpm for 10 minutes, and the bacterial supernatant was prepared by filtering through a 0.2 pm filter. The content of tryptophan and butyric acid in the bacterial supernatant was detected by high performance liquid chromatography (HPLC; Thermo, UltiMate 3000). The HPLC detection conditions for tryptophan were: 10% methanol and 90% KH2PO4 with a concentration of 0.03% were used for linear program to detect tryptophan from the supernatant, and the program time was 18 minutes. The solvent flow rate was 1.0 mL / min. The content of tryptophan (mg / mL) was analyzed by comparing the peak height (λ max = 278 nm) or peak area of the sample with the analysis standard. The HPLC detection conditions for butyric acid were: 20% acetonitrile and 80% phosphoric acid with a concentration of 0.1% were used for linear program to detect butyric acid in the supernatant, and the program time was 9 minutes, and the solvent flow rate was 0.8 mL / min. The content of butyric acid (mg / mL) was analyzed by comparing the peak height (λ max＝The peak area of butyrate was compared with the peak height and peak area of the analysis standard, and the concentration of butyrate (mg / mL) was calculated. The results are shown in Figure 7, after 24 hours of bacterial cultivation in thiolate medium, L-Trp CB still secreted more tryptophan, reaching about 1.25 mM, while CB secreted only 0.4 mM. Overexpression of trpEDCBA genes in engineered bacteria did not affect the production of butyric acid, and the content was basically maintained at 30 mM concentration.

[0064] Example 2: Effect of butyric acid and tryptophan metabolites secreted by engineered Clostridium butyricum on cell function

[0065] (1) Evaluation of tumor cell IDO enzyme expression

[0066] The inhibitory effect of butyric acid produced by Clostridium butyricum and engineered Clostridium butyricum on IDO enzyme was first evaluated. Tumor cells (CT26 mouse colon cancer cells) previously cultured in 6-well plates were incubated with the metabolites of Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB (1:10 ratio of supernatant to cell culture medium). After 24 hours of treatment, the expression of IDO enzyme in CT26 cells was detected by Western blotting method, as shown in Figure 8, the butyric acid metabolites of Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB can induce IDO down-regulation in CT26 cells. It is shown that butyric acid produced by Clostridium butyricum can be used as a potential IDO inhibitor to prevent cancer cells from taking up tryptophan.

[0067] (2) Effect on T cell function

[0068] First, T cells from the inguinal lymph nodes of BALB / c mice were extracted. Lymph node-derived T cells were activated with anti-CD3 and soluble anti-CD28 antibodies in the presence of 20 ng / ml IL-2 and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Then, T cells were treated with Clostridium butyricum CB and engineered Clostridium butyricum L-Trp CB metabolites (1:15 ratio with cell culture medium) for 48 hours. The proportion of CD8 + CD8 + T cells and the expression of IFN-γ+ on CD8 + CD3 + T and IFN-γ + CD8 +The frequency of T cells increased significantly. The effects of engineered Clostridium butyricum on T cell energy metabolism were then analyzed. ECAR and OCR were measured using a Seahorse Bioscience XF-96 extracellular flux analyzer to measure intracellular glycolysis and oxidative phosphorylation in T cells. A total of 1×10⁻⁶ cells were used. 4 Lymph node-derived T cells were seeded into 96-well plates supplemented with XF-96 growth medium and cultured overnight. They were then treated with *Clostridium butyricum* and engineered *Clostridium butyricum* metabolites, anti-CD3, anti-CD28, and IL-2 for 48 hours. Oligomycin (0.5 μM), 2.5 μM FCCP, and 5 μM rotinolone / antimycin A were automatically injected. As shown in Figures 10 and 11, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of T cells in the engineered *Clostridium butyricum* metabolite treatment group were higher than those in the control and *Clostridium butyricum* groups. This indicates that butyrate and tryptophan-containing metabolites of engineered *Clostridium butyricum* promote oxidative phosphorylation and glycolysis in T cells to enhance and maintain T cells in the effector phenotype.

[0069] Example 3: Tumor-targeting assessment of engineered Clostridium butyricum

[0070] (1) Distribution of mice in vivo

[0071] Bioluminescence was achieved by transforming engineered *Clostridium butyricum* L-Trp CB with the luminescent plasmid PMV306-G13-lux. The bioluminescent engineered *Clostridium butyricum* and *Clostridium butyricum* were delivered subcutaneously to colon tumor regions in mouse models. Observations were performed using a PE small animal in vivo imaging system at 0, 4, 12, 36, and 48 hours post-tail vein delivery, and at days 5 and 7. Simultaneously, mice were euthanized after 7 days, and their hearts, livers, spleens, lungs, kidneys, and tumors were collected for in vitro imaging. The tissues were then weighed and homogenized with sterile PBS. The homogenates were serially diluted and incubated on RCM plates at 37°C for 24–36 hours. The number of newly formed colonies on each culture dish was recorded using a colony counter (Czone 8), and the results were calculated as CFU / g tissue. As shown in Figures 12 and 13, engineered Clostridium butyricum L-Trp CB and Clostridium butyricum CB began to remain in the tumor area after 36 hours, but could not colonize other organs, indicating that the bacteria can specifically target and colonize tumors for a long time.

[0072] (2) Functional studies of tumor targeting

[0073] Firstly, the effect of engineered Clostridium butyricum L-Trp CB treatment on the expression of IDO enzyme in tumor tissue was evaluated. The tumors of mice injected with engineered Clostridium butyricum 7 days after tail vein injection were collected, and the expression of IDO in the tumor tissue section fixed with paraformaldehyde was detected by immunohistochemical method. Then, tumor homogenate was prepared from the excised tumor. The supernatant was collected by centrifugation (12000 rpm, 10 minutes) and membrane preparation was completed, and the concentrations of tryptophan, kynurenine and butyric acid were determined by high performance liquid chromatography (HPLC). As shown in Figure 14, the expression of IDO in the engineered Clostridium butyricum treatment group decreased significantly, and the tryptophan content in the engineered Clostridium butyricum group was significantly higher than that in the untreated group and the Clostridium butyricum CB group, and the kynurenine Kyn, a metabolite of tryptophan, was lower than that in the untreated group, while the butyric acid content was similar to that in the Clostridium butyricum CB group. It is shown that the engineered bacteria can exert its own butyric acid and genetically engineered tryptophan characteristics in the tumor.

[0074] Example 4: Anti-tumor effect study

[0075] (1) Effect on tumor growth

[0076] 2x10 6 CT26 tumor cells were subcutaneously injected into the right abdomen of mice to obtain CT26 tumor-bearing mice. Subsequently, when the tumor volume reached about 100mm 3 3, 5x10 7 CFU of engineered Clostridium butyricum L-Trp CB or Clostridium butyricum CB was injected into each tumor-bearing mouse through the tail vein. During this process, the tumor size and mouse body weight were measured every two days. As shown in Figure 15, engineered Clostridium butyricum can significantly inhibit tumor growth, prolong the survival time of tumor-bearing mice, and has no obvious effect on the body weight of mice. At the same time, a rabbit VX2 tumor model was constructed, and when the tumor grew to about 1000mm 3 3, each New Zealand rabbit was single intravenous injection of 1x10 8 CFU of engineered Clostridium butyricum L-Trp CB through the auricular vein, and the tumor volume of each rabbit was measured every other day and photographed every four days using the above mouse tumor experiment method. As shown in Figure 16, engineered Clostridium butyricum can also significantly inhibit the growth of rabbit tumors.

[0077] (2) Effect on tumor immune microenvironment

[0078] The above CT26 tumor tissues were collected, and the changes in tumor T cells were analyzed by single cell sequencing and flow cytometry. As shown in Figure 17, the T cell subpopulation analysis results show that the proportion of CD8 + T cell population increases after tail vein injection of engineered Clostridium butyricum, and the proportion of effector, memory and exhausted CD8 + T cell subpopulations increases, while the proportion of CD4+ T cell population. As shown in Figure 18, the results of flow cytometry experiments also confirmed that engineered Clostridia butyricum was able to significantly increase the proportion of CD8 + T cells and decrease the proportion of CD4 + T cells in the tumor region. Therefore, the proportion of CD8 + T cells in the tumor region was further analyzed by single-cell sequencing and flow cytometry experiments, and the results are shown in Figure 19, which showed that the levels of immune activation-related genes (such as Gran B + , IFN-γ + , Ki67 + , TNF-α + and PD-1 + ) and corresponding proteins were significantly increased. As shown in Figure 20, the gene bubble chart showed that the expression of other functional genes and signal regulation genes in CD8 + T cells of mice receiving engineered Clostridia butyricum L-Trp CB treatment was also up-regulated. The results are shown in Figures 21 and 22, and gene set enrichment analysis (GESA) showed that the pathways related to CD8 + T cells were significantly enriched, including significant enrichment of natural killer cell-mediated immunity, immune effect process, and MTORC1 signaling pathway. KEGG functional enrichment analysis also identified significantly enriched pathways in CD8 + T cells, including p53 signal transduction, T cell receptor signal transduction, and chemokine signal transduction pathways. The above results indicate that tail vein-engineered Clostridia butyricum can activate the anti-tumor effect of CD8 + T cells in the tumor.

[0079] Example 5: Study on the regulation of tumor CD8+T cell metabolism

[0080] The changes in energy metabolism of tumor CD8 + T cells were analyzed by single-cell sequencing. The results are shown in Figure 23, and the gene volcano plot showed that the most significantly up-regulated genes were related to glycolysis (such as Eno3, Ldh and Gapdh), tricarboxylic acid cycle (TCA) (such as Gpd2) and oxidative phosphorylation (OX PHOS) (such as Atp5a1, Ndufb4 and Cox5b). Gene bubble chart analysis further showed that the number and expression of related glycolysis, tricarboxylic acid cycle and oxidative phosphorylation metabolism genes were increased. As shown in Figure 24, gene set enrichment analysis again confirmed that engineered Clostridia butyricum L-Trp CB promoted the glycolysis and oxidative phosphorylation metabolic pathways of CD8 + T cells. These results indicate that the energy metabolism of CD8 + T cells in the tumor microenvironment is regulated by engineered Clostridia butyricum, which stimulates CD8 + T cell proliferation and immune response.

[0081] Example 6: Safety evaluation of major organs

[0082] To explore whether the engineered Clostridium butyricum would damage the major organs including heart, liver, spleen, lung and kidney, we collected the heart, liver, spleen, lung and kidney of the mice treated with engineered Clostridium butyricum L-Trp CB in the above examples and performed H&E section on them. As shown in Figure 25, the results of H&E section showed that the engineered Clostridium butyricum did not affect the normal physiological structure of the above organs. The above results showed that the engineered Clostridium butyricum had good safety.

[0083] In summary, the present application relates to the application of engineered Clostridium butyricum L-Trp-CB for specific colonization of tumor targeting, continuous expression of specific molecules to regulate tumor immune response. The engineered Clostridium butyricum administered through tail vein regulates the tumor immune microenvironment of mice, secretes butyric acid in the tumor to inhibit the expression of IDO enzyme of tumor, and secretes tryptophan to activate the function of CD8 + T cells, thereby achieving enhanced immunotherapy effect of tumor.

[0084] Obviously, the above examples are only examples for clearly illustrating, not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. An engineered butyric acid bacterium producing L-tryptophan, characterized in that: The tryptophan operon gene trpEDCBA was overexpressed in the engineered Clostridium butyricum.

2. The engineered Clostridium butyricum according to claim 1, characterized in that: The tryptophan operon gene trpEDCBA was overexpressed using the thl promoter.

3. The engineered Clostridium butyricum according to claim 1, characterized in that: The tryptophan operon gene trpEDCBA was expressed using the shuttle vector pMTL82151.

4. A method for constructing an engineered butyric acid-producing Clostridium, characterized in that, Includes the following steps: S1. Construct plasmid pMTL82151-trpEDCBA; S2. Use plasmid pMTL82151-trpEDCBA to transfect Escherichia coli and obtain recombinant Escherichia coli; S3. The recombinant Escherichia coli and Clostridium butyricum are mixed and cultured to allow the plasmid pMTL82151-trpEDCBA to be conjugated and transferred into Clostridium butyricum, thereby obtaining the engineered Clostridium butyricum.

5. A microbial agent comprising any one of the engineered Clostridium butyricum strains described in claims 1-3.

6. The use of the engineered Clostridium butyricum according to any one of claims 1-3 and the microbial agent according to claim 5 in the preparation of tumor therapeutic drugs.

7. The application according to claim 6, characterized in that: The tumors include colon tumors.

8. The application according to claim 6, characterized in that: The drug is administered via intravenous injection.

9. The use of the engineered Clostridium butyricum according to any one of claims 1-3 or the microbial agent according to claim 5 in the preparation of a drug that promotes T cell metabolism.

10. The use of the engineered Clostridium butyricum according to any one of claims 1-3 or the microbial agent according to claim 5 in the preparation of IDO inhibitors.

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