Engineered probiotics expressing D-lactate dehydrogenase, their construction methods and applications

Abstract

This invention discloses an engineered probiotic expressing D-lactate dehydrogenase, its construction method, and its applications, belonging to the field of biomedical technology. The engineered probiotic of this invention is constructed by exogenously introducing the D-lactate dehydrogenase encoding gene into *Escherichia coli* Nissle 1917. This invention utilizes synthetic biology techniques to construct a "living therapeutic factory" capable of in-situ clearing specific metabolites, realizing a paradigm shift from "inhibiting proteins" to "clearing metabolites" in treatment. Through lipid coating technology, oral delivery and intestinal targeted colonization of the engineered bacteria are achieved, improving the precision and efficiency of treatment. Based on the probiotic EcN, it exhibits high biosafety. The treatment process is oral administration, non-invasive, and avoids the systemic toxicity of traditional chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to engineered probiotics expressing D-lactate dehydrogenase, their construction methods, and applications. Background Technology

[0002] Gliomas are a type of tumor originating from glial cells in the central nervous system and are common primary brain tumors. Gliomas include diffuse astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, and mixed gliomas. Glioblastoma (GBM) is the most common and most malignant primary brain tumor of the central nervous system, with limited efficacy of current treatments and extremely poor patient prognosis. One of the typical characteristics of this tumor is its unique metabolic reprogramming phenomenon, where tumor cells, through abnormal metabolic activity, not only support their rapid proliferation and invasive behavior but also shape an immunosuppressive tumor microenvironment. Recent studies have shown that tumor-derived metabolites, in addition to serving as energy sources, act as key signaling molecules driving tumor progression and treatment resistance, making targeting key GBM metabolites a highly promising new therapeutic strategy.

[0003] Lactic acid plays a central role in tumor metabolic networks. High concentrations of lactic acid produced by tumors not only exert their effects by inhibiting the immune microenvironment, but their metabolic processes also directly regulate tumor prognosis, metastasis, and clinical outcomes. Notably, due to the presence of chiral carbon atoms, lactic acid exists in two enantiomers: L-lactate and D-lactate. Currently, most research focuses on the more abundant L-lactate, which plays a crucial role in glycolysis, energy transfer, and lactation modification of novel proteins, regulating metabolic processes and cell survival. However, increasing evidence suggests that the stereoisomer D-lactate has unique functions in solid tumors: its levels are significantly elevated in various cancers, promoting malignant progression through mechanisms such as regulating the acidic microenvironment, altering immune cell function, and influencing epigenetic modifications. D-lactate is not only closely associated with metabolic abnormalities, mitochondrial damage, and oxidative stress, but it can also induce D-lactoacidosis, characterized by metabolic acidosis and neurological symptoms, and plays an important role in tumor metabolic networks by regulating ferroptosis and macrophage function.

[0004] Although D-lactic acid has a clear harmful effect on tumor development and progression, targeted therapy against this small molecule metabolite faces significant technical bottlenecks: unlike proteins and other biomolecules, small molecule metabolites are difficult to intervene with through traditional "inhibition" strategies, while blocking their biosynthetic pathways can easily cause systemic toxic side effects. Therefore, developing tumor therapeutic drugs targeting D-lactic acid is quite challenging. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an engineered probiotic expressing D-lactate dehydrogenase, its construction method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an engineered probiotic expressing D-lactate dehydrogenase, wherein the engineered probiotic is based on Escherichia coli as the starting strain, and the encoding gene of D-lactate dehydrogenase is exogenously transferred into the starting strain.

[0008] The D-lactate dehydrogenase is any of the proteins shown below (A1)-(A4):

[0009] (A1) The MeLDHD2 protein, whose amino acid sequence is shown in SEQ ID NO.2;

[0010] (A2) The MeLDHD3 protein, whose amino acid sequence is shown in SEQ ID NO.3;

[0011] (A3) BfLDHD protein, the amino acid sequence of which is shown in SEQ ID NO.4;

[0012] (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1), (A2) or (A3).

[0013] Preferably, the Escherichia coli is Escherichia coli Nissle 1917 (EcN); Escherichia coli Nissle 1917 (EcN) has safety, probiotic properties and tumor colonization ability, making it an ideal biodelivery platform.

[0014] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0015] Based on codon encoding principles and the codon bias of the host bacterium, the nucleotide sequence of the encoding gene of D-lactate dehydrogenase can be obtained according to its amino acid sequence. The encoding gene is expressed in an expression vector or expressibly inserted into the *E. coli* genome.

[0016] A second aspect of the present invention provides a method for constructing engineered probiotics expressing D-lactate dehydrogenase, comprising the following steps:

[0017] (1) The gene encoding D-lactate dehydrogenase was ligated into a plasmid vector containing a promoter to obtain a recombinant expression vector;

[0018] (2) The recombinant expression vector was transferred into Escherichia coli Nissle 1917 (EcN) to construct an engineered probiotic expressing D-lactate dehydrogenase.

[0019] Preferably, in step (1), the promoter is selected from Ptac, Plac or Ptrc; the plasmid vector is selected from pTrc99a vector or pBBR1MCS-2 vector.

[0020] More preferably, the promoter is the Ptrc promoter; and the plasmid vector is the pTrc99a vector.

[0021] This invention, through screening of promoter and plasmid vector combinations, found that combining the Ptrc promoter with the pTrc99a vector can synergistically improve the expression level of D-lactate dehydrogenase and the degradation effect on D-lactate.

[0022] In some preferred embodiments of the present invention, the engineered probiotics constructed include: engineered probiotics expressing the MeLDHD2 protein (EcN:: pTrc99a-Ptrc-MeLDHD2), engineered probiotics expressing the MeLDHD3 protein (EcN:: pTrc99a-Ptrc-MeLDHD3), and engineered probiotics expressing the BfLDHD protein (EcN:: pTrc99a-Ptrc-BfLDHD).

[0023] A third aspect of the present invention provides the application of the above-described engineered probiotics in the preparation of products that degrade D-lactic acid.

[0024] In the above applications, the engineered probiotics expressing the MeLDHD2 protein (EcN:: pTrc99a-Ptrc-MeLDHD2), the engineered probiotics expressing the MeLDHD3 protein (EcN:: pTrc99a-Ptrc-MeLDHD3), and the engineered probiotics expressing the BfLDHD protein (EcN:: pTrc99a-Ptrc-BfLDHD) all showed good effects in degrading D-lactic acid.

[0025] Preferably, the product for degrading D-lactic acid is composed of engineered probiotics expressing the MeldHD2 protein, the MeldHD3 protein, and the BfLDHD protein; the live bacteria ratio of the three engineered probiotics is 1:1:1. Using these three engineered probiotics in combination, compared to using them individually, can synergistically improve the degradation effect on D-lactic acid, achieving rapid degradation of D-lactic acid.

[0026] In a fourth aspect, the present invention provides the use of the above-described engineered probiotics in the preparation of a medicament for treating glioma.

[0027] In the above applications, the drug contains a therapeutically effective amount of engineered probiotics. In this invention, "therapeutically effective amount" refers to the amount of engineered probiotics required for the treatment of glioblastoma. The effective amount level may depend on factors such as disease severity, drug sensitivity, timing of administration, route of administration, and duration of treatment.

[0028] In the above applications, the drug may further comprise a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes one or more of the following: coating agents, diluents, binders, disintegrants, lubricants, adsorbents, and humectants. For example, materials that can be used as coating agents include liposomes; materials that can be used as binders include gelatin, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, etc.; materials that can be used as disintegrants include corn starch, agar powder, bentonite, sodium alginate, calcium citrate, etc.; materials that can be used as lubricants include calcium stearate, magnesium stearate, talc, liquid paraffin, petrolatum, etc.

[0029] In the above applications, the drug can be formulated into various dosage forms according to conventional methods, such as granules, powders, capsules, tablets, sprays, inhalers, injections, etc.

[0030] In some preferred embodiments of the present invention, the medicament for treating glioma is prepared by the following method:

[0031] (1) Dissolve DOTAP, DOPE, cholesterol and DSPE-PEG2000 together in anhydrous ethanol to form a lipid mixture;

[0032] (2) Dissolve the above-mentioned engineered probiotics in citrate buffer, add them to the lipid mixture prepared in step (1), mix and incubate for 10-30 min; then dialyze after sonication.

[0033] To improve the survival rate and colonization ability of engineered bacteria in the gastrointestinal tract, this invention utilizes liposomes to coat engineered probiotics, forming a lipid coating layer. The lipid coating layer preferably contains DOTAP, DOPE, and cholesterol, and is formed into a thin film by vacuum evaporation. This effectively protects the engineered bacteria from gastric acid damage and enhances their stability and retention time in the intestine.

[0034] In the above applications, the glioma is preferably a glioblastoma.

[0035] The beneficial effects of this invention are:

[0036] (1) This invention investigates the catalytic efficiency of D-lactic acid dehydrogenases from different sources and selects the encoding genes of MeLDHD2, MeLDHD3, and BfLDHD proteins to construct engineered probiotics. During the construction of the engineered probiotics, the optimization of the expression vector and promoter significantly improved the degradation effect of the prepared engineered probiotics on D-lactic acid. Furthermore, combining the three engineered probiotics synergistically enhances the degradation effect of D-lactic acid, achieving rapid degradation of D-lactic acid.

[0037] (2) This invention achieves oral delivery and intestinal targeted colonization of engineered bacteria through lipid coating technology, thereby improving the precision and efficiency of treatment.

[0038] (3) The lipid-coated engineered probiotics prepared by the present invention can catalyze the conversion of D-lactic acid in the blood into pyruvate, thereby significantly reducing the D-lactic acid level in the whole body and tumor microenvironment. The reduction of D-lactic acid level can directly inhibit the proliferation and invasion of GBM tumor cells, and indirectly exert anti-tumor effects by reshaping the intestinal flora homeostasis and repairing the intestinal mucosal barrier. Attached Figure Description

[0039] Figure 1 SDS-PAGE analysis of D-lactic acid dehydrogenase (LDHD); In the figure, M: molecular weight standard (kDa); Lane 1: MeLDHD1; Lane 2: MeLDHD2; Lane 3: MeLDHD3; Lane 4: BuLDHD; Lane 5: BaLDHD; Lane 6: BfLDHD.

[0040] Figure 2 : Comparison of different LDHD enzyme activities; In the figure, A is the amount of NADH generated by D-lactate dehydrogenase at different pH values; B is the amount of NADH generated by D-lactate dehydrogenase at different temperatures; C is the amount of NADH consumed by D-lactate dehydrogenase in the reverse reaction.

[0041] Figure 3 : Schematic diagram of the strategy and results for optimizing LDHD expression in EcN; In the figure, A is a schematic diagram of plasmid construction; B is the whole-cell D-lactic acid degradation capacity of engineered EcN expressing different plasmids under different promoter regulation. Data are expressed as mean ± standard deviation (n=5). P < 0.001, P < 0.0001. Statistical significance was determined using two-way ANOVA.

[0042] Figure 4 : Graph showing the effect of engineered bacteria on D-lactic acid degradation in vivo. Whole-cell D-lactic acid degradation capacity of engineered EcN expressing different LDHDs. Data are expressed as mean ± standard deviation (n=5). P<0.001, P < 0.0001. Statistical significance was determined using two-way ANOVA.

[0043] Figure 5 : Schematic diagram of the preparation and characterization results of lipid-coated engineered bacteria (Lipo-EcN-LDHD). (A) Schematic diagram of the preparation of lipid-coated bacteria. (B) Representative transmission electron microscopy images of uncoated Escherichia coli (EcN) and lipid-coated Escherichia coli (Lipo-EcN) (scale bar: 1 μm and 2 μm).

[0044] Figure 6 Representative in vivo bioluminescence imaging of the tumor site at week 4 post-transplantation in Example 5.

[0045] Figure 7 Bioluminescence intensity values ​​for assessing tumor growth at week 4 post-transplantation in Example 5. Data are expressed as mean ± standard deviation (n=5). P<0.01, statistical significance was determined by one-way ANOVA.

[0046] Figure 8 Overall survival was assessed using Kaplan-Meier survival curves, and statistical significance was analyzed using the log-rank test (n=10).

[0047] Figure 9 Serum D-lactate levels in GBM-bearing mice and the treatment group at week 4 post-transplantation in Example 5. Data are expressed as mean ± standard deviation (n=5). P < 0.0001, statistical significance was determined by one-way ANOVA.

[0048] Figure 10 HE staining showing the extent of tumor invasion in the brain (scale bar: 1 mm). Detailed Implementation

[0049] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0050] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0051] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0052] Escherichia coli Nissle 1917 (EcN) was purchased from Baosai Biotechnology.

[0053] Example 1: Expression and catalytic efficiency of D-lactate dehydrogenase

[0054] Six D-lactate dehydrogenases—MeLDHD1 (GenBank: AVO73537.1; amino acid sequence as shown in SEQ ID NO.1), MeLDHD2 (AVO74211.1; amino acid sequence as shown in SEQ ID NO.2), MeLDHD3 (AVO74336.1; amino acid sequence as shown in SEQ ID NO.3), BfLDHD (AHK05341.1; amino acid sequence as shown in SEQ ID NO.4), BuLDHD (UWO07026.1; amino acid sequence as shown in SEQ ID NO.5), and BaLDHD (WP_175630329.1; amino acid sequence as shown in SEQ ID NO.6)—were expressed in prokaryotes to investigate the catalytic activity of different D-lactate dehydrogenases for D-lactate.

[0055] 1. Expression of D-lactate dehydrogenase:

[0056] (1) From respectively Megasphaera elsdenii DSM20460 Bacteroides fragilis NCTC 9343 Bacteroides uniformis ATCC 8492 and Bacteroides acidifaciens Genomic DNA was extracted from DSM 15896.

[0057] (2) Using genomic DNA as a template, amplify the primers shown in Table 1 respectively. MeLDHD1 Gene, MeLDHD2 Gene, MeLDHD3 Gene, BfLDHD Gene, BuLDHD Genes and BaLDHD Gene.

[0058] Table 1: Primers used for prokaryotic expression of D-lactate dehydrogenase

[0059]

[0060]

[0061] Note: The sequences marked with underline in the table are homologous arm sequences.

[0062] (3) Each MeLDHD1 Gene, MeLDHD2 Gene, MeLDHD3 Gene, BfLDHD Gene, BuLDHD Genes and BaLDHD Gene inserted into expression vector pET28a, transformed into E. coli DH5α, construction E. coli DH5α(pET28a-LDHD).

[0063] (4) Pick a single colony, perform colony PCR, and then perform sequencing analysis; incubate the correctly sequenced strains at 37℃ and 220rpm until OD. 600 =1, after plasmid extraction, transformed to E. coli BL21(DE3) yielded 6 protein expression strains. E. coli BL21(DE3)::pET28a-LDHD.

[0064] (5) The six protein expression strains obtained E. coli BL21(DE3)::pET28a-LDHD were cultured in LB medium containing kanamycin (50 μg / mL); when OD 600 When the concentration reaches 0.6-0.8, add 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) and lower the temperature to 16°C, then continue culturing for 20 hours.

[0065] (6) Collect bacterial cells by centrifugation and resuspend them in binding buffer (200 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole, pH=8.0). Cell disruption was performed by sonication at 4°C. After centrifugation to remove cell debris from the lysate, LDHD purification was performed using a nickel column, following the manufacturer's instructions. The eluted protein was then passed through a PD-10 desalting column to replace the buffer with sodium phosphate buffer (20 mM, pH 7.6).

[0066] The purified protein was analyzed by SDS-PAGE, and the results are as follows: Figure 1 As shown, the molecular weights of the purified MeLDHD1, MeLDHD2, MeLDHD3, BfLDHD, BuLDHD, and BaLDHD proteins are consistent with the target molecular weights. Sequencing verification confirmed the correct protein sequences, indicating successful expression of these six proteins.

[0067] 2. Catalytic efficiency evaluation:

[0068] The purified MeLDHD1, MeLDHD2, MeLDHD3, BfLDHD, BuLDHD, and BaLDHD proteins were mixed with D-lactic acid in PBS buffer (100 mM) to form an enzymatic reaction system. In the enzymatic reaction system, the concentration of each of the six LDHD proteins was 0.2 mg / mL, and the final concentration of D-lactic acid was 2 mM.

[0069] The enzymatic reaction system (pH=8) was reacted at different temperatures of 25℃, 30℃, and 37℃ for 5 min; and at different pH conditions of pH7, pH8, pH9, pH10, and pH11 for 5 min at 37℃. The amount of NADH produced under different temperature and pH conditions was quantitatively determined; the more NADH produced, the stronger the catalytic activity of the enzyme.

[0070] The amount of NADH consumed was quantitatively determined in the reverse reaction (37℃, pH 8); the more NADH consumed, the stronger the catalytic activity of the enzyme.

[0071] The results are as follows Figure 2 As shown, based on the catalytic efficiency measurements under different temperatures and pH conditions, and after comprehensive consideration, MeLDHD2 protein, MeLDHD3 protein, and BfLDHD protein were selected for the subsequent construction of engineered probiotics.

[0072] Example 2: Optimization of the construction strategy for engineered probiotics expressing D-lactate dehydrogenase

[0073] 1. Optimization of expression vector and promoter selection:

[0074] Using pBBR1MCS-2 and pTrc99a vectors as the vector backbones, and Ptac, Plac, and Ptrc as promoters, three types of vectors containing pBBR1MCS-2 and pTrc99a were constructed, respectively. BfLDHD The recombinant expression plasmids (pBBR1MCS2::Ptac-BfLDHD, pTrc99a::Ptrc-BfLDHD, and pTrc99a::Plac-BfLDHD) were constructed; the primer sequences used for constructing the recombinant expression plasmids are shown in Table 2; a schematic diagram of the construction of the recombinant expression plasmids is shown in the figure. Figure 3 As shown in Figure A.

[0075] Table 2: Primers used for constructing recombinant expression plasmids

[0076]

[0077] Note: The sequences marked with underline in the table are homologous arm sequences.

[0078] The recombinant expression plasmid containing BfLDHD was electroporated into Escherichia coli Nissle 1917 (EcN) to construct three engineered probiotics (EcN::pBBR1MCS2-Ptac-BfLDHD, EcN::pTrc99a-Ptrc-BfLDHD and EcN::pTrc99a-Plac-BfLDHD).

[0079] The three engineered probiotic strains (EcN::pBBR1MCS2-Ptac-BfLDHD, EcN::pTrc99a-Ptrc-BfLDHD, and EcN::pTrc99a-Plac-BfLDHD) were inoculated into LB liquid medium and activated by incubation at 37°C and 200 rpm for 12 hours. They were then transferred to shake flasks containing fresh LB liquid medium and cultured at 37°C and 200 rpm until OD (October Expiratory Time) was reached. 600 =1.5-2, centrifuge to collect bacteria, wash twice with PBS (10mM, pH 8), then resuspend in buffer and dilute to adjust OD. 600 =5, to obtain bacterial suspension.

[0080] The degradation capacity of the engineered probiotics for D-lactic acid was determined in PBS buffer:

[0081] 20 mL of bacterial suspension was directly transferred to a 50 mL centrifuge tube; D-lactic acid was added to a final concentration of 2 mM, and the tubes were incubated at 37°C in a shaker (200 rpm) for 30, 60, 90, and 120 minutes, respectively; the amount of D-lactic acid degradation was detected using a D-lactic acid assay kit (Beyotime Biotechnology, Shanghai, China). The bacterial suspension was replaced with an equal volume of buffer as a control.

[0082] The results are as follows Figure 3 As shown in Figure B, when LDHD expression was driven by the Ptrc promoter using the pTrc99a vector as the vector backbone, the whole-cell enzyme activity was the highest (2.3 times higher than that of Ptac).

[0083] 2. Optimization of cultivation conditions:

[0084] Using the engineered probiotic EcN::pTrc99a-Ptrc-BfLDHD as the test strain, a two-stage temperature control strategy was adopted: growth at 37℃ to OD 600 =0.6; then induce at 25℃ for 12 hours.

[0085] Continuous growth and induction at 37℃ served as a control.

[0086] The expression levels of soluble proteins and the inclusion body formation rate in the culture products were determined.

[0087] The results showed that, compared with the control, the two-stage temperature control strategy increased the expression level of soluble proteins by 4.1 times and reduced the inclusion body formation rate to below 15%.

[0088] 3. Construction of engineered probiotics expressing MeLDHD2 and MeLDHD3 proteins:

[0089] Based on the optimization results of the expression vector and promoter, the pTrc99a vector was selected as the vector backbone and Ptrc as the promoter. Using the primers in Table 2, expression vectors containing pTrc99a and Ptrc99a were constructed respectively. MeLDHD2 Recombinant expression plasmids and containing MeLDHD 3 recombinant expression plasmid.

[0090] The constructed containing MeLDHD2 Recombinant expression plasmids and containing MeLDHD The recombinant expression plasmids 3 were electroporated into Escherichia coli Nissle 1917 (EcN) to construct engineered probiotics expressing MeLDHD2 protein (EcN::pTrc99a-Ptrc-MeLDHD2) and MeLDHD3 protein (EcN::pTrc99a-Ptrc-MeLDHD3).

[0091] Example 3: Investigation on the degradation effect of engineered probiotics on D-lactic acid

[0092] The degradation effects of the engineered probiotics expressing MeLDHD2 protein (EcN::pTrc99a-Ptrc-MeLDHD2), MeLDHD3 protein (EcN::pTrc99a-Ptrc-MeLDHD3), and BfLDHD protein (EcN::pTrc99a-Ptrc-BfLDHD) constructed in Example 2 on D-lactic acid were determined in PBS buffer. The method for measuring D-lactic acid degradation was the same as in Example 2.

[0093] The results are as follows Figure 4 As shown, the results indicate that all three engineered probiotics exhibit superior D-lactic acid degradation performance.

[0094] OD 600 The three engineered probiotics (=5) were mixed at a volume ratio of 1:1:1 to obtain a bacterial suspension of the engineered probiotic combination. The degradation effect of the engineered probiotic combination on D-lactic acid was determined according to the method in Example 2. The results showed that treatment with the engineered probiotic combination for 30 minutes reduced the D-lactic acid content to 0.6 mM. Compared with treatment with equal amounts of the three engineered probiotics alone, it had a significant and synergistic effect, enabling rapid degradation of D-lactic acid.

[0095] Example 4: Preparation and characterization of lipid-coated engineered probiotics (LiPo-EcN)

[0096] 1. Dissolve 10 mg (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), 2.5 mg 1,2-dioleoyl-sn-glycerol-3-phosphorylethanolamine (DOPE), 1.25 mg cholesterol and 1.25 mg DSPE-PEG2000 together in 2 mL of anhydrous ethanol, transfer to a round-bottom flask and evaporate under reduced pressure to form a thin film, to obtain a lipid mixture.

[0097] 2. Dissolve 10 mg of the three engineered probiotics prepared in Example 2 in citrate buffer (50 mM citrate, pH 4) and slowly add them to the lipid mixture prepared in step 1. After thorough mixing, incubate for 20 minutes.

[0098] 3. After ultrasonic treatment, dialysis was performed using a nanodialysis device (polycarbonate membrane, 30 nm pore size) to collect the unintercepted components, and finally the volume was adjusted to 5 mL.

[0099] Transmission electron microscopy image of the prepared lipid-coated engineered probiotics is shown below. Figure 5 As shown in Figure B, coating engineered probiotics with a layer of lipids effectively protects them from stomach acid damage and enhances their stability and retention time in the intestines.

[0100] Example 5: Therapeutic effect analysis of lipid-coated engineered probiotics (LiPo-EcN) in GBM in situ model

[0101] 1. Construction of the GBM mouse model:

[0102] BALB / c-nu / nu nude mice (4-week-old males, purchased from Shanghai Slack Laboratory Animal Center) were housed for one week under specific pathogen-free conditions at 24°C with a 12-hour diurnal cycle. After anesthesia, human glioblastoma cells (GBM#P3) expressing luciferase (5 × 10⁻⁶ cells) were injected into the frontal lobe. 5 Cells were suspended in 10 μL PBS. The borehole was positioned 1 mm anterior to the anterior fontanelle and 2 mm lateral to it, with the injection depth adjusted to 2.5 mm. Tumor progression was continuously monitored using a bioluminescence imaging system (IVIS Spectrum, PerkinElmer, Massachusetts, USA), with fluorescence intensity within the ROI region used as a surrogate indicator of tumor volume.

[0103] 2. Experimental grouping and treatment:

[0104] GBM model mice were randomly divided into 5 groups of 15 mice each (10 mice were used for survival rate analysis, and 5 mice were used for other indicators). BALB / c-nu / nu nude mice that underwent sham surgery were used as controls. Specific treatments were as follows:

[0105] Control: PBS was administered by gavage starting on day 7, once every two days.

[0106] GBM: Start PBS by gavage from day 7, once every two days;

[0107] EcN: Starting from day 7, lipid-coated EcN was administered via gavage at a volume of 10 g / L. 9 CFU / ml / kg, administered via gavage every two days;

[0108] EcN-MeLDHD2: Starting from day 7, administer lipid-coated engineered probiotics (EcN:: pTrc99a-Ptrc-MeLDHD2) via gavage at a dose of 10 g / L. 9 CFU / ml / kg, administered via gavage every two days;

[0109] EcN-MeLDHD3: Starting from day 7, administer lipid-coated engineered probiotics (EcN:: pTrc99a-Ptrc-MeLDHD3) via gavage at a dose of 10 g / L. 9 CFU / ml / kg, administered via gavage every two days;

[0110] EcN-BfLDHD: Starting from day 7, administer lipid-coated engineered probiotics (EcN:: pTrc99a-Ptrc-BfLDHD) via gavage at a dose of 10 mg / L. 9 CFU / ml / kg, administered via gavage every two days.

[0111] Four weeks after tumor transplantation, in vivo bioluminescence imaging was performed on the tumor site to assess the bioluminescence intensity of tumor growth; D-lactate levels in mouse serum were measured; when mice began to show persistent symptoms of discomfort, they were euthanized, and brain tissue was collected and fixed in 4% paraformaldehyde for HE staining and immunohistochemical analysis.

[0112] 3. Test Results:

[0113] The results are as follows Figures 6-10 As shown, the results indicate that after oral administration, engineered probiotics can specifically colonize the intestine and continuously express LDHD enzyme. This enzyme can catalyze the conversion of D-lactic acid in the blood into pyruvate, thereby significantly reducing the level of D-lactic acid in the whole body and the tumor microenvironment. The reduction of D-lactic acid level can directly inhibit the proliferation and invasion of GBM tumor cells, and indirectly exert anti-tumor effects by reshaping the gut microbiota homeostasis and repairing the intestinal mucosal barrier.

[0114] In summary, addressing the technical bottlenecks in developing tumor therapeutics targeting D-lactic acid, this invention first investigated the catalytic efficiency of existing D-lactic acid dehydrogenases, selecting the MelDHD2, MelDHD3, and BfLDHD proteins, which exhibit superior overall catalytic performance, as experimental proteins. Then, the expression vectors and promoters for these three proteins were optimized, choosing the Ptrc promoter and pTrc99a vector as the optimal combination, which synergistically enhances protein expression levels and D-lactic acid degradation. The encoding genes of these three proteins were ligated into the pTrc99a vector containing the Ptrc promoter to construct recombinant expression vectors. These recombinant expression vectors were then transformed into *Escherichia coli* Nissle 1917 (EcN) to construct three engineered probiotic strains expressing D-lactic acid dehydrogenase. To improve the survival rate and colonization ability of engineered probiotics in the gastrointestinal tract, this invention further encapsulated the engineered probiotics with liposomes. The therapeutic effect of the liposome-encapsulated engineered probiotics on GBM model mice was then investigated. The results showed that oral administration of the engineered probiotics of this invention reduced D-lactate levels in mice and prolonged the survival time of GBM model mice. Furthermore, the activity of D-lactate dehydrogenase expressed by the engineered probiotics showed a positive correlation with the therapeutic effect on glioblastoma. By combining three engineered probiotics expressing D-lactate dehydrogenase to synergistically improve the degradation effect of D-lactate levels, the therapeutic effect on glioblastoma was also synergistically improved.

[0115] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An engineered probiotic expressing D-lactate dehydrogenase, characterized in that, The engineered probiotics are derived from Escherichia coli strains, into which the gene encoding D-lactate dehydrogenase is exogenously transferred. The D-lactate dehydrogenase is any of the proteins shown below (A1)-(A4): (A1) The MeLDHD2 protein, whose amino acid sequence is shown in SEQ ID NO.2; (A2) The MeLDHD3 protein, whose amino acid sequence is shown in SEQ ID NO.3; (A3) BfLDHD protein, the amino acid sequence of which is shown in SEQ ID NO.4; (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1), (A2) or (A3); The method for constructing the engineered probiotics is as follows: (1) The gene encoding D-lactate dehydrogenase was ligated into a plasmid vector containing a promoter to obtain a recombinant expression vector; (2) The recombinant expression vector was transferred into Escherichia coli to construct an engineered probiotic expressing D-lactate dehydrogenase; The promoter is the Ptrc promoter; the plasmid vector is the pTrc99a vector.

2. The engineered probiotic expressing D-lactate dehydrogenase according to claim 1, characterized in that, The Escherichia coli mentioned is Escherichia coli Nissle 1917.

3. The use of the engineered probiotic expressing D-lactic acid dehydrogenase as described in claim 1 or 2 in the preparation of products that degrade D-lactic acid.

4. The application according to claim 3, characterized in that, The product that degrades D-lactic acid consists of engineered probiotics expressing the MeLDHD2 protein, engineered probiotics expressing the MeLDHD3 protein, and engineered probiotics expressing the BfLDHD protein.

5. The use of the engineered probiotic expressing D-lactate dehydrogenase as described in claim 1 or 2 in the preparation of a medicament for treating glioma.

6. The application according to claim 5, characterized in that, The drug further comprises a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier is one or more of the following: coating agent, diluent, binder, disintegrant, lubricant, adsorbent, and humectant.

7. The application according to claim 6, characterized in that, The dosage form of the drug is granules, powders, capsules, tablets, sprays, inhalers, or injections.

8. The application according to claim 5, characterized in that, The drug for treating gliomas is prepared by the following method: (1) Dissolve DOTAP, DOPE, cholesterol and DSPE-PEG2000 together in anhydrous ethanol to form a lipid mixture; (2) Dissolve the engineered probiotics expressing D-lactate dehydrogenase in citrate buffer, add it to the lipid mixture prepared in step (1), and incubate for 10-30 min after mixing; Dialysis is performed after ultrasonic treatment.

Images