A coordination assembly modified microorganism, its preparation method and application

Abstract

This invention proposes a coordination assembly-modified microorganism, its preparation method, and its application, belonging to the field of biomedical materials technology. This invention obtains catechol lipid-modified microorganisms by reacting catechol lipid molecules with microbial culture via a thin-film hydration method. These microorganisms are then contacted with metal ions to form a catechol lipid-metal-modified microorganism through coordination. Further contact with polyphenolic compounds leads to the coordination assembly of a catechol lipid-metal-polyphenol complex, thus forming the coordination assembly-modified microorganism. This invention employs a novel bacterial surface coordination assembly method and, through the combined regulation of microorganisms and inflammatory mediators, achieves a combined treatment of periodontitis and neuroinflammatory conditions related to depression and cognitive impairment. The modification method of this invention is universal; it can be used to construct engineered bacteria by replacing different bacterial carriers and different metals and polyphenolic compounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and in particular relates to a coordination assembly modified microorganism, its preparation method and application. Background Technology

[0002] Existing methods for regulating microbial community imbalance include directly adjusting the microbial composition balance using probiotics, prebiotics, antibiotics, or bacteriophages, or directly regulating microbial function through DNA coupling, enzyme inhibitors, and engineered bacteria. Using nanomaterials for bacterial modification can achieve complementary properties between nanomaterials and bacteria, resulting in synergistic therapeutic effects. Currently, most bacterial modifications utilize the abundant reactive groups (such as free thiol groups, amino groups, hydroxyl groups, and carboxyl groups) provided by the rich components of the bacterial surface for chemical grafting, or modify the bacterial surface through electrostatic interactions using nanomaterials that utilize the negative charge of the bacterial surface. These modification methods are highly dependent on the bacterial surface structure, have poor universality, and easily damage the biological activity of the bacteria themselves, making them difficult to adapt to different types of Gram-positive and Gram-negative bacteria.

[0003] Current strategies for therapeutic biomaterials targeting inflammation control primarily involve clearing inflammatory mediators, blocking their contact with cells, or rescuing the inflammatory microenvironment through drug delivery. Microbial community regulation often lacks precision, potentially leading to further disruption of the microbial ecosystem. Furthermore, the number of genetically modified bacteria is limited, and many biomolecules (especially eukaryotic proteins) cannot be expressed efficiently and precisely by bacteria. Additionally, genetically engineered bacteria may face the potential risk of gene contamination through horizontal gene transfer, limiting the widespread application of engineered bacteria in inflammation treatment. Existing therapeutic materials for inflammation primarily focus on interventions at single sites and for single types of inflammation, failing to leverage microbial-cellular interactions to control the spread of inflammation from its source interface, and even less capable of cross-site joint regulation of local oral inflammation and secondary systemic distal neuroinflammation.

[0004] Meanwhile, existing catalytic systems for nano-modified bacteria mostly employ metal nanoparticles, which do not fully utilize the catalytic efficiency of metal atoms, resulting in limited clearance of inflammatory mediators. Furthermore, metal particle aggregation can easily lead to biotoxicity. Traditional anti-inflammatory materials also struggle to achieve organic integration and regulation of microorganisms, their metabolites, and inflammatory mediators, failing to achieve anti-inflammatory effects from multiple dimensions such as microbial community regulation, mediator clearance, and microenvironment improvement. Currently, few nanobiotechnology-driven engineered bacterial therapies can effectively integrate these functions to achieve combined treatment of oral and neuroinflammation. For clinical problems such as periodontitis, which are often associated with oral inflammation leading to depression and cognitive impairment-related neuroinflammation, there is a lack of effective cross-systemic treatment methods. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a coordination assembly-modified microorganism, its preparation method, and its application. This invention employs a novel bacterial surface coordination assembly method and, through the combined regulation of microorganisms and inflammatory mediators, achieves a combined treatment of periodontitis and neuroinflammatory conditions related to depression and cognitive impairment. The modification method of this invention is universally applicable; it can be used to construct engineered bacteria by replacing different bacterial carriers and various metals and polyphenolic compounds.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing coordination-assembled modified microorganisms, comprising the following steps: (1) Catechol lipid molecules are combined with microbial bacterial solution through a thin-film hydration method, so that the catechin lipid molecules are modified onto the surface of microorganisms by hydrophobic insertion, thereby obtaining microorganisms modified with catechin lipid molecules; (2) The catechol lipid molecules modified microorganisms obtained in step (1) are brought into contact with metal ions to form catechol lipid molecules-metal modified microorganisms through coordination. (3) The product obtained in step (2) is contacted with a polyphenol compound to form a catechol lipid molecule-metal-polyphenol complex through coordination assembly, thereby obtaining the coordination assembly modified microorganism (also known as coordination assembly engineered bacteria).

[0007] This invention achieves the joint regulation of microorganisms and inflammatory mediators through a novel engineered probiotic approach, thereby jointly regulating oral and neuroinflammation induced by bacteria. It also addresses the technical pain points of existing bacterial modifications, such as poor universality, high risk of gene modification, and low catalytic efficiency.

[0008] Further, in step (1), the thin film hydration method refers to the process in which catechol lipid molecules are dissolved in chloroform, completely dried with argon gas, and then microbial liquid is added to the surface of the thin film formed in the centrifuge tube by blowing and resuspending, so that the lipid molecules are re-dissolved in the bacterial liquid and mixed with bacteria.

[0009] Further, in step (1), the method for preparing the catechol lipid molecules includes the following steps: Diphenyl phosphite was reacted with n-decyl alcohol in the presence of pyridine to give bisdecyl phosphate. N,N-diethylethylenediamine was reacted with 3,4-dihydroxybenzaldehyde to generate an intermediate product, which was (E)-4-(((2-(diethylamino)ethyl)imino)methyl)benzene-1,2-diol; The intermediate product was mixed with the dicealkyl phosphate and reacted, and then purified to obtain the catechol lipid molecule.

[0010] Furthermore, the molar ratio of diphenyl phosphite to n-decyl alcohol is 1:2, the reaction temperature of diphenyl phosphite and n-decyl alcohol is 130-150℃, and the reaction time is 3-5h.

[0011] Furthermore, the molar ratio of N,N-diethylethylenediamine to 3,4-dihydroxybenzaldehyde is 1:1, and the reaction temperature of N,N-diethylethylenediamine and 3,4-dihydroxybenzaldehyde is 60-80℃, with a reaction time of 2-4 hours.

[0012] Furthermore, the molar ratio of N,N-diethylethylenediamine to the diecryl phosphate is 1:1, and the reaction temperature of the intermediate product with the diecryl phosphate is 90-110°C, and the reaction time is 1-2 h.

[0013] More specifically, in step (1), the method for preparing the catechol lipid molecules includes the following steps: A. Mix 5.0 mmol diphenyl phosphite and 10.0 mmol n-decyl alcohol (molar ratio 1:2), add 0.5-5 mL pyridine, heat at 130-150 °C for 3-5 h, and after the reaction is complete, remove pyridine and byproducts by vacuum distillation to obtain didecyl phosphate. B. Add 1.0 mmol N,N-diethylethylenediamine and 1.0 mmol 3,4-dihydroxybenzaldehyde (molar ratio 1:1) to 3 mL of methanol, and heat at 60-80 °C for 2-4 h under a nitrogen atmosphere. An intermediate product is generated during the reaction: (E)-4-(((2-(diethylamino)ethyl)imino)methyl)benzene-1,2-diol, with the following structural formula: ; C. Evaporate the intermediate product at 30°C to remove all solvent, then add 1.0 mmol of the dicealkyl phosphate ester prepared in step A, and react at 90-110°C for 1-2 h under a nitrogen atmosphere. After the reaction is complete, purify the crude product by silica gel column chromatography to obtain catechol lipid molecules.

[0014] Further, in step (1), the bacteria in the microbial culture are Gram-positive or Gram-negative bacteria, preferably Escherichia coli strain Nissell 1917 or Lactobacillus fermentum NB02; the amount of catechol lipid molecules and microbial culture is 1 mL for every 0.2 mg of catechol lipid molecules, and 10 mL for every 10 mg of catechol lipid molecules. 8 Microbial culture at CFU / mL.

[0015] Further, in step (2), the metal ions are derived from salts of cerium, iron, copper or zinc, preferably cerium ammonium nitrate, ferric chloride, copper sulfate or zinc sulfate; the molar ratio of the catechol lipid molecules to the metal ions is 1:1; Further, in step (3), the polyphenolic compound is one or more of tannic acid, ellagic acid, epigallocatechin gallate, and quercetin, and the molar ratio of the catechin lipid molecule to the polyphenolic compound is 1:1. More specifically, the preparation method of the coordination assembly modified microorganism of the present invention includes the following steps: (1) Dissolve 0.2 mg of catechol lipid molecules in 0.2 mL of chloroform to obtain a mother liquor; dry the chloroform in the mother liquor with argon gas, and then add 1 mL of 10 mL of chloroform. 8 CFU / mL bacterial suspension (using Escherichia coli Nissl 1917 or Lactobacillus fermentum NB02) was thoroughly vortexed (vortex speed 200-1000 rpm, vortex time 10-30 s) to obtain catechol lipid molecule modified microorganisms with lipid molecules whose hydrophobic tails were inserted into the bacterial membrane. (2) Add a metal element mother liquor (concentration of 10 mg / mL, molar ratio of catechol lipid molecules to metal elements (selected from cerium ammonium nitrate, ferric chloride hexahydrate, copper sulfate pentahydrate or zinc sulfate heptahydrate) to the catechol lipid molecule modified microorganisms obtained in step (1) and vortex thoroughly (vortex speed of 200-1000 rpm, vortex time of 10-30 s) to obtain catechol lipid molecule-metal modified microorganisms; (3) Add a polyphenolic compound mother liquor (concentration of 10 mg / mL, molar ratio of catechin lipid molecules to polyphenolic compounds (selected from tannic acid, ellagic acid, epigallocatechin gallate or quercetin) of 1:1) to the catechin lipid molecules-metal-polyphenol modified microorganisms obtained in step (2) and vortex thoroughly (vortex speed of 200-1000 rpm, vortex time of 10-30 s) to obtain catechin lipid molecules-metal-polyphenol modified microorganisms (i.e. coordination assembly modified microorganisms).

[0016] The present invention also provides a coordination assembly modified microorganism, which is prepared according to the above preparation method.

[0017] The present invention also provides the application of the above-mentioned coordination assembly modified microorganisms in the preparation of medicaments for treating oral inflammation and / or neuroinflammation.

[0018] Furthermore, the oral inflammation is periodontitis, and the neurological inflammation is neurological inflammation associated with depression or cognitive impairment.

[0019] The present invention also provides a pharmaceutical composition for the combined treatment of oral and neurological inflammation, comprising the above-mentioned coordination assembly modified microorganism and a pharmaceutically acceptable carrier.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects: This invention utilizes engineered bacteria constructed through coordination assembly to exert DNase-like activity, thereby disrupting inflammatory mediators (cell-free DNA, cfDNA). Furthermore, it possesses SOD and CAT-like enzyme activities, effectively regulating excessive reactive oxygen species (ROS) in the inflammatory microenvironment. This effectively clears inflammatory mediators from the local source of inflammation, preventing them from entering the bloodstream, circulating in the peripheral circulation, crossing the blood-brain barrier, and causing neuroinflammation. In addition, by modifying the characteristics of the bacteria themselves, such as probiotics, it can effectively regulate the local microbial ecological balance, preventing inflammatory responses caused by dysbiosis, and reducing the secretion of metabolites associated with increased vascular permeability, thus preventing inflammatory mediators from entering the bloodstream and disseminating to other parts of the body. The bacterial surface coordination assembly modification method of this invention is highly universal, allowing for flexible replacement of different Gram-positive / negative bacterial carriers, as well as different metal ions such as cerium, iron, copper, and zinc, and different polyphenolic compounds such as tannic acid and ellagic acid. All of these methods can efficiently construct engineered bacteria, providing flexible technical support for personalized treatment in different inflammatory scenarios. The specific principle is as follows: 1. Coordination assembly engineered bacteria can effectively destroy cfDNA that can trigger inflammation through DNase-like activity (cerium has DNase-like activity), while SOD / CAT-like activity can effectively clear high ROS in the inflammatory microenvironment (the valence state transition of metal elements has antioxidant effects, and polyphenolic compounds also have antioxidant effects). The combination of the two is conducive to synergistic anti-inflammatory effects, achieving precise intervention at the core mediator level of inflammation, and making the anti-inflammatory effect more direct and efficient.

[0021] 2. Coordination assemblies, due to the presence of catechol in lipid molecules and catechol in polyphenolic compounds, confine individual metal elements between them, thus forming single atoms. These single metal atoms can maximize catalytic efficacy, significantly enhancing the catalytic activity of DNase-like and SOD / CAT-like enzymes, thereby significantly improving the clearance efficiency of inflammatory mediators. Simultaneously, the presence of catechol in lipid molecules and catechol in polyphenolic compounds confines individual metal elements between them, thus forming single atoms, which can maximize catalytic efficacy.

[0022] 3. Specific Principle of Local Inflammation Regulation in the Oral Cavity: The coordination assembly-modified microorganisms of this invention utilize DNase-like and SOD / CAT-like enzyme activities to clear cfDNA and ROS that trigger inflammatory responses. By inhibiting inflammatory responses, the levels of inflammatory factors in the blood are reduced, thereby preventing these inflammatory factors from entering the brain and causing neuroinflammation. Therefore, the coordination assembly-modified microorganisms of this invention can achieve joint regulation of oral and neuroinflammation, breaking through the technical bottleneck of existing technologies that make it difficult to achieve cross-site joint treatment of oral and neuroinflammation. This provides a novel solution for the treatment of neuroinflammation such as depression and cognitive impairment secondary to periodontitis.

[0023] 4. This invention uses catechol lipid molecules to modify the surface of microorganisms through hydrophobic insertion. Combined with subsequent coordination assembly reactions, the entire modification process does not require genetic modification of bacteria, thus avoiding the risk of gene contamination caused by horizontal gene transfer in genetically engineered bacteria. It also solves the technical problems of the limited types of gene-modifiable bacteria and the difficulty in efficiently and accurately expressing biological macromolecules. At the same time, this modification method does not damage the biological activity of the bacteria themselves, ensuring that the modified bacteria can properly regulate the ecological balance of the microbial community.

[0024] 5. The coordination assembly modification process of the present invention is simple to operate, the reaction conditions are mild, and the reaction ratio, temperature, time and other parameters of each step have been precisely optimized, making it easy to prepare on a large scale. Moreover, the nano-assemblies on the surface of the modified engineered bacteria are uniformly distributed and structurally stable, and can maintain good catalytic activity and anti-inflammatory effects in vivo, thus having good prospects for industrial application. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the construction of Escherichia coli modified with cerium-tannins, a catechin lipid molecule, in Example 1; Figure 2 The image shows a comparison of the surface potential changes between Escherichia coli modified with catechol lipid molecules in Example 1 (lipid-modified bacteria) and Escherichia coli (unmodified bacteria) in Comparative Example 1, where a is an image of the bacterial surface potential distribution and b is a quantitative statistical graph of the surface potential distribution. Figure 3 TEM images of Escherichia coli (engineered bacteria) modified with cerium-tannin molecule-catechol lipid molecule obtained in Example 1 at different magnifications; Figure 4 The results of surface roughness (a) and Young's modulus (b) of Escherichia coli (engineered bacteria) modified with cerium-tannic acid molecule in Example 1 and Escherichia coli (unmodified bacteria) in Comparative Example 1 are shown. Figure 5 These are TEM images of microorganisms modified with different coordination assemblies under different bacterial species, different metals, or different polyphenolic compounds in the examples. Figure 6 The image shows an aberration-corrected transmission electron microscope image of the coordination assembly-modified microorganisms in Example 1. Figure 7The absorbance test results are as follows: Escherichia coli (engineered bacteria) modified with cerium-tannic acid in Example 1 and Escherichia coli (unmodified bacteria) in Comparative Example 1 were co-incubated with bis(4-nitrobenzene) phosphate (BNPP). Figure 8 The *E. coli* (engineered bacteria) modified with cerium-tannin cations of catechol lipid molecules in Example 1 or the *E. coli* (unmodified bacteria) in Comparative Example 1, for (a) H2O2 and (b) ·O2 - The results of the clearance rate comparison; Figure 9 The results of periodontal probing depth (a) and gingival bleeding index (b) tests in mice of different treatment groups; Figure 10 The results show the levels of inflammatory mediators in the brains of mice in different treatment groups, where a represents the IL-1β level and b represents the TNF-α level. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] This invention provides a method for preparing coordination-assembled modified microorganisms, comprising the following steps: (1) Catechol lipid molecules are combined with microbial bacterial solution through a thin-film hydration method, so that the catechin lipid molecules are modified on the surface of microorganisms by hydrophobic insertion, and microorganisms modified with catechin lipid molecules are obtained. (2) The catechol lipid molecules modified microorganisms obtained in step (1) are brought into contact with metal ions to form catechol lipid molecules-metal modified microorganisms through coordination. (3) The product obtained in step (2) is contacted with polyphenolic compounds to form a catechol lipid molecule-metal-polyphenol complex through coordination assembly, thereby obtaining coordination assembly modified microorganisms (also known as coordination assembly engineered bacteria).

[0032] This invention first utilizes hydrophobic insertion, where the hydrophobic tail of the catechol lipid molecule can insert into the phospholipid bilayer of the microbial cell membrane, achieving preliminary modification of the microbial surface and providing binding sites for subsequent coordination reactions. The catechol group has strong coordination ability and can form stable coordination bonds with metal ions, enabling the loading of metal elements on the microbial surface. Furthermore, using multi-level coordination assembly, the metal ions serve as bridging sites, further coordinating with the phenolic hydroxyl groups of polyphenolic compounds to form a catechol lipid molecule-metal-polyphenol ternary complex, completing the surface engineering modification of the microorganism and ultimately obtaining a coordination-assembled modified microorganism that possesses both microbial characteristics and catalytic anti-inflammatory activity.

[0033] Furthermore, the thin-film hydration method refers to the process in which catechol lipid molecules are dissolved in chloroform, completely dried with argon gas, and then microbial bacterial solution is added to the surface of the thin film formed by the dried lipid molecules in a centrifuge tube and resuspended by blowing, so that the lipid molecules are redissolved in the bacterial solution and mixed with bacteria.

[0034] In step (1) of the preferred embodiment of the present invention, the method for preparing catechol lipid molecules includes the following steps: A. Mix 5.0 mmol diphenyl phosphite and 10.0 mmol n-decyl alcohol (molar ratio 1:2), add 0.5-5 mL pyridine, heat at 130-150 °C for 3-5 h, and after the reaction is complete, remove pyridine and byproducts by vacuum distillation to obtain didecyl phosphate. B. Add 1.0 mmol N,N-diethylethylenediamine and 1.0 mmol 3,4-dihydroxybenzaldehyde to 3 mL of methanol, and heat at 60-80 °C for 2-4 h under a nitrogen (N2) protective atmosphere. An intermediate product is generated during the reaction: (E)-4-(((2-(diethylamino)ethyl)imino)methyl)benzene-1,2-diol, with the following structural formula: ; C. Evaporate the intermediate product at 30°C to remove all solvent, then add 1.0 mmol of the dicealkyl phosphate ester prepared in step A, and react at 90-110°C for 1-2 h under a nitrogen atmosphere. After the reaction is complete, purify the crude product by silica gel column chromatography to obtain catechol lipid molecules.

[0035] In the preparation process of the catechol lipid molecule of the present invention, an esterification reaction is first used. Diphenyl phosphite and n-decyl alcohol undergo esterification under pyridine catalysis to generate decyl phosphate, which provides a hydrophobic lipid chain for the catechol lipid molecule, ensuring its ability to subsequently insert into the microbial membrane. Then, the amino group of N,N-diethylethylenediamine condenses with the aldehyde group of 3,4-dihydroxybenzaldehyde to generate an intermediate product containing catechol groups and imino groups, which provides key phenolic hydroxyl coordination sites for subsequent coordination reactions. Finally, the intermediate product reacts with decyl phosphate to connect the hydrophobic lipid chain with the hydrophilic end containing the catechol group, synthesizing a catechol lipid molecule with both hydrophobic insertion and coordination capabilities, meeting the dual requirements of microbial surface modification.

[0036] In step (1) of the preferred embodiment of the present invention, the bacteria in the microbial culture are Gram-positive or Gram-negative bacteria, preferably Escherichia coli strain Nissell 1917 or Lactobacillus fermentum NB02; the amount of catechol lipid molecules and microbial culture is 1 mL for every 0.2 mg of catechol lipid molecules, and 10 mL for every 10 mg of catechol lipid molecules. 8 Microbial culture at CFU / mL.

[0037] In step (2) of the preferred embodiment of the present invention, the metal ions are derived from salts of cerium, iron, copper or zinc, preferably cerium ammonium nitrate, ferric chloride, copper sulfate or zinc sulfate; the molar ratio of catechol lipid molecules to metal ions is 1:1. In step (3) of the preferred embodiment of the present invention, the polyphenolic compound is one or more of tannic acid, ellagic acid, epigallocatechin gallate, and quercetin, and the molar ratio of catechin lipid molecules to polyphenolic compounds is 1:1. This invention also provides a coordination assembly modified microorganism, prepared according to the above preparation method.

[0038] This invention also provides the application of the above-mentioned coordination assembly modified microorganisms in the preparation of drugs for treating oral inflammation and / or neuroinflammation.

[0039] In a preferred embodiment of the present invention, oral inflammation is periodontitis, and neurological inflammation is neurological inflammation related to depression or cognitive impairment.

[0040] The microorganisms modified by this invention possess a dual-core anti-inflammatory mechanism. First, they destroy inflammatory mediators cfDNA through the DNA-like activity of metal single atoms and clear ROS through SOD / CAT-like enzyme activity, thereby eliminating key mediators of oral inflammation at its source and blocking the spread of inflammation. Second, the microorganisms themselves can regulate the local oral flora ecological balance, avoiding persistent inflammation caused by flora imbalance. At the same time, the effective removal of inflammatory mediators can prevent them from entering the bloodstream, crossing the blood-brain barrier, and causing neuroinflammation. Therefore, this product can effectively treat oral inflammation and / or neuroinflammation, providing a novel biomaterial solution for inflammation treatment.

[0041] This invention also provides a pharmaceutical composition for the combined treatment of oral and neurological inflammation, comprising the above-mentioned coordination-assembly modified microorganisms and a pharmaceutically acceptable carrier.

[0042] All raw materials used in the embodiments of this invention were purchased commercially. Escherichia coli Nissl 1917 was purchased from the Beina Biotechnology Culture Bank (Beina Chuanglian Biotechnology Co., Ltd.), Lactobacillus fermentum NB02 was isolated, screened and preserved by the School of Food and Pharmacy of Ningbo University, Porphyromonas gingivalis was purchased from the American Type Culture Collection, and the ELISA kit was purchased from Cybex (Shanghai) Biotechnology Co., Ltd.

[0043] The technical solution of the present invention will be further illustrated by the following embodiments.

[0044] Example 1 Preparation of catechol lipid molecules: A. 5.0 mmol of diphenyl phosphite and 10.0 mmol of n-decyl alcohol were mixed (molar ratio 1:2), and 0.5 mL of pyridine was added. The mixture was heated at 140 °C for 4 h. After the reaction was completed, pyridine and byproducts were removed by vacuum distillation to obtain didecyl phosphate with a yield of 94%. B. 1.0 mmol N,N-diethylethylenediamine and 1.0 mmol 3,4-dihydroxybenzaldehyde (molar ratio 1:1) were added to 3 mL of methanol and heated at 70 °C for 3 h under a nitrogen (N2) protective atmosphere. During the reaction, an intermediate product was generated, which was (E)-4-(((2-(diethylamino)ethyl)imino)methyl)benzene-1,2-diol; C. The intermediate product was evaporated at 30°C to remove all solvent, and then 1.0 mmol of the dicedyl phosphate prepared in step A was added. The mixture was heated to 100°C and reacted for 1.5 h under a nitrogen atmosphere. After the reaction was completed, the crude product was purified by silica gel column chromatography to obtain catechol lipid molecules with a yield of 50%, which were used for the preparation of coordination assembly modified microorganisms. The synthetic route for catechol lipid molecules in this embodiment is as follows: ; Preparation of coordination assembly modified microorganisms: (1) Dissolve 0.2 mg of catechol lipid molecules in 0.2 mL of chloroform to obtain a mother liquor; dry the chloroform in the mother liquor with argon gas, and then add 1 mL of 10 mL of chloroform. 8 CFU / mL of Escherichia coli Nissl 1917 bacterial suspension was thoroughly vortexed (vortex speed 300 rpm, vortex time 10 s, repeated 3 times) to obtain Escherichia coli modified with catechol lipid molecules whose hydrophobic tails were inserted into the bacterial membrane. (2) Add cerium ammonium nitrate mother liquor (cerium ammonium nitrate mother liquor concentration is 10 mg / mL, molar ratio of catechol lipid molecules to cerium ammonium nitrate is 1:1) to the catechol lipid molecules modified in step (1) and vortex thoroughly (vortex speed is 300 rpm, vortex time is 10 s, repeated 3 times) to obtain catechol lipid molecules-cerium modified Escherichia coli. (3) Add tannic acid stock solution (concentration of 10 mg / mL, molar ratio of catechol lipid molecules to tannic acid 1:1) to the cerium-modified Escherichia coli obtained in step (2) and vortex thoroughly (vortex speed of 200-1000 rpm, vortex time of 10 s, repeated 3 times) to obtain cerium-tannic acid-modified Escherichia coli, which is the coordination assembly modified microorganism.

[0045] Figure 1 This is a schematic diagram of the construction of Escherichia coli modified with cerium-tannins, a catechin lipid molecule, in Example 1.

[0046] Example 2 Same as Example 1, except that the Escherichia coli Nissler 1917 bacterial solution was replaced with an equal concentration of Lactobacillus fermentum NB02 bacterial solution, and the other steps remained unchanged. The product obtained was Lactobacillus fermentum modified with catechol lipid molecules - cerium-tannin.

[0047] Example 3 Same as Example 1, except that cerium ammonium nitrate is replaced with ferric chloride hexahydrate (the molar ratio of catechol lipid molecules to ferric chloride hexahydrate is 1:1), and the other steps remain unchanged. The product obtained is Escherichia coli modified with catechol lipid molecules-iron-tannic acid.

[0048] Example 4 Same as Example 1, except that ferric chloride hexahydrate is replaced with copper sulfate pentahydrate (the molar ratio of catechol lipid molecules to copper sulfate pentahydrate is 1:1), and the other steps remain unchanged. The product obtained is Escherichia coli modified with catechol lipid molecules-copper-tannic acid.

[0049] Example 5 Same as Example 1, except that ferric chloride hexahydrate is replaced with zinc sulfate heptahydrate (the molar ratio of catechol lipid molecules to zinc sulfate heptahydrate is 1:1), and the other steps remain unchanged. The product obtained is Escherichia coli modified with catechol lipid molecules-zinc-tannic acid.

[0050] Example 6 Same as Example 1, except that the tannic acid mother liquor is replaced with ellagic acid mother liquor (concentration of 10 mg / mL, molar ratio of catechol lipid molecules to ellagic acid 1:1), and the other steps remain unchanged, to obtain catechol lipid molecules-cerium-ellagic acid modified Escherichia coli.

[0051] Example 7 Same as Example 1, except that the tannic acid mother liquor is replaced with epigallocatechin gallate mother liquor (concentration of 10 mg / mL, molar ratio of catechin lipid molecules to epigallocatechin gallate ester 1:1), and the other steps remain unchanged, to obtain Escherichia coli modified with catechin lipid molecules-cerium-epigallocatechin gallate ester.

[0052] Example 8 Same as Example 1, except that the tannic acid mother liquor was replaced with quercetin mother liquor (concentration of 10 mg / mL, molar ratio of catechol lipid molecules to quercetin 1:1), and the other steps remained the same, to obtain catechol lipid molecules-cerium-quercetin modified Escherichia coli.

[0053] Comparative Example 1 Unmodified bacteria (i.e., Escherichia coli Nissl 1917 used in the examples).

[0054] Performance testing I. Surface Potential Test The catechol lipid-modified Escherichia coli (lipid-modified bacteria) obtained in step (1) of Example 1 and the Escherichia coli (unmodified bacteria) in Comparative Example 1 were respectively dropped onto a clean silicon wafer, and the surface potential of the bacteria was scanned using an atomic force microscope.

[0055] Figure 2The graph shows a comparison of the surface potential changes between *E. coli* modified with catechol lipid molecules in Example 1 (lipid-modified bacteria) and *E. coli* in Comparative Example 1 (unmodified bacteria), where a is an image of the bacterial surface potential distribution and b is a quantitative statistical graph of the surface potential distribution. It can be seen that the surface potential of the catechol lipid-modified bacteria significantly decreases due to the surface insertion of catechol lipid molecules, proving the success of the lipid molecule membrane insertion behavior.

[0056] II. TEM Test The *E. coli* modified with the cerium-tannin catechin lipid molecule obtained in Example 1 was fixed using electron microscopy fixative at 4°C, dehydrated using a gradient method, embedded in resin, and then ultra-thin sections were prepared and placed on a copper grid for TEM image capture. Results at different magnifications are shown in [Figure 1]. Figure 3 The results showed that uniformly sized nano-assemblies were visible on the surface of the coordinated assembly-engineered bacterial biofilm, proving that the modification was successful.

[0057] III. Roughness and Young's Modulus Testing The bacterial cultures of *E. coli* modified with cerium-tannin, a catechin lipid molecule, as described in Example 1 (engineered bacteria) and *E. coli* (unmodified bacteria), as described in Comparative Example 1, were respectively dropped onto a clean silicon wafer. The surface roughness (Ra) and Young's modulus of the bacteria were scanned using an atomic force microscope. The results are shown in [Figure 1]. Figure 4 In the figure, a represents the roughness (Ra) test result and b represents the Young's modulus test result. It can be seen that the average surface roughness of the coordinated assembly engineered bacteria is improved compared with the unmodified bacteria, and the Young's modulus value, which represents the stiffness of the bacterial cell, is also significantly improved, proving that the bacterial modification was successful.

[0058] IV. TEM images of engineered bacteria after exposure to different bacterial species, metals, and polyphenolic compounds. In this example, different coordination-assembly modified microorganisms were prepared by replacing different bacterial strains, metals, and polyphenolic compounds. The bacterial cultures of each coordination-assembly modified microorganism were dropped onto a copper grid, vacuum dried for 48 hours, and then TEM images were taken. The results are shown in [Figure number missing]. Figure 5 It can be seen that the engineered modification of the cell surface can be achieved under different bacterial species, different metals, or different polyphenolic compounds, proving the universality of the proposed modification strategy.

[0059] V. Aberration-corrected transmission electron microscope images The coordination-assembly modified microbial culture from Example 1 was dropped onto a microgrid copper grid, vacuum dried for 48 hours, and then subjected to aberration-sensor transmission electron microscopy. The results are shown in [Figure 1]. Figure 6 The results showed that the surface of the microbial cells modified by coordination assembly exhibited a large number of isolated, sharp bright spots of metal single atoms, proving that cerium exists in single-atom form and has more efficient catalytic activity.

[0060] VI. DNA-like enzyme performance testing 200 μL of 40 mM bis(4-nitrobenzene) phosphate (BNPP) was co-incubated with 200 μL of *E. coli* (engineered bacteria) modified with cerium-tannin molecule catechol lipid molecules from Example 1 or *E. coli* (unmodified bacteria) from Comparative Example 1, and the total volume was increased to 2 mL. The absorbance at 400 nm was measured using a UV spectrophotometer. The results are shown in [Figure 1]. Figure 7 The results showed that the engineered bacteria with coordinated assembly had a better ability to break the phosphate ester bonds in BNPP (DNPP-like ability) than the unmodified bacteria. That is, the content of p-nitrophenol produced after the phosphate ester bond was broken increased, and the absorbance value at 400 nm increased.

[0061] VII. H2O2 and ·O2 - Cleaning performance test (1) The Escherichia coli (engineered bacteria) modified with cerium-tannic acid in Example 1 or the Escherichia coli (unmodified bacteria) in Comparative Example 1 were incubated with an equal volume of H2O2 (2mM) and the residual H2O2 content was determined according to the instructions of the commercial H2O2 kit and the clearance rate was calculated.

[0062] (2) Take 50 μL of *E. coli* (engineered bacteria) modified with cerium-tannin molecule-catechin lipid molecules from Example 1 or *E. coli* (unmodified bacteria) from Comparative Example 1, and according to commercial inhibition of O2 - Perform the clearance rate determination according to the instructions of the proficiency test kit.

[0063] In Example 1, the cerium-tannin-modified Escherichia coli (engineered bacteria) or in Comparative Example 1, the unmodified Escherichia coli showed resistance to (a) H2O2 and (b) ·O2. - The results of the clearance rate comparison are shown in Figure 8 It can be seen that the engineered bacteria with coordination assembly exhibit superior scavenging ability for H2O2 and ·O2 compared to unmodified bacteria. - The ability refers to possessing SOD- and CAT-like enzyme activities.

[0064] 8. Periodontal probing depth and GBI index test in mice Use Porphyromonas gingivalis (10) 9A mouse periodontitis model was constructed by inoculating mice with CFU / mL every other day for 4 weeks. After 4 weeks, mice were treated with either Escherichia coli modified with cerium-tannins (engineered bacteria) from Example 1 or Escherichia coli (unmodified bacteria) from Comparative Example 1 (inoculated every other day for 4 weeks). The periodontal probing depth was then measured using a periodontal probe, and the gingival bleeding index (GBI) was recorded. A healthy group and an inflammation group were also set up. The healthy group consisted of healthy mice that did not have a mouse periodontitis model constructed, and the inflammation group consisted of mice that had a mouse periodontitis model constructed but did not receive treatment.

[0065] The results of periodontal probing depth (a) and gingival bleeding index (b) tests in mice of different treatment groups are shown in the figure. Figure 9 It can be seen that the mice treated with engineered bacteria assembled by coordination did not show significant periodontal tissue loss, that is, the periodontal probing depth was basically the same as that of healthy mice, and the gingival bleeding was significantly restored compared with the inflammation group.

[0066] IX. Test of Inflammatory Mediator Content in Mouse Brain After euthanizing mice in each of the eight test groups, brain tissue homogenates were collected. The levels of inflammatory factors in the tissue homogenate supernatant were detected using an ELISA kit. The results of the inflammatory mediator levels in the brains of mice in different treatment groups are shown below. Figure 10 Where a represents the IL-1β content and b represents the TNF-α content, it can be seen that the inflammatory factors detectable in the brain tissue of mice treated with engineered bacteria via coordination assembly also showed a significant decrease compared to the periodontitis group.

[0067] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing coordination-assembled modified microorganisms, characterized in that, Includes the following steps: (1) Catechol lipid molecules are combined with microbial bacterial solution through a thin-film hydration method, so that the catechin lipid molecules are modified onto the surface of microorganisms by hydrophobic insertion, thereby obtaining microorganisms modified with catechin lipid molecules; (2) The microorganisms modified with catechol lipid molecules obtained in step (1) are brought into contact with metal ions to form catechol lipid molecules-metal modified microorganisms through coordination. (3) The product obtained in step (2) is contacted with a polyphenol compound to form a catechol lipid molecule-metal-polyphenol complex through coordination assembly, thereby obtaining the coordination assembly modified microorganism.

2. The method for preparing coordination-assembled modified microorganisms according to claim 1, characterized in that, In step (1), the method for preparing the catechol lipid molecules includes the following steps: Diphenyl phosphite was reacted with n-decyl alcohol in the presence of pyridine to give bisdecyl phosphate. N,N-diethylethylenediamine was reacted with 3,4-dihydroxybenzaldehyde to generate an intermediate product, which was (E)-4-(((2-(diethylamino)ethyl)imino)methyl)benzene-1,2-diol; The intermediate product was mixed with the dicealkyl phosphate and reacted, and then purified to obtain the catechol lipid molecule.

3. The method for preparing coordination-assembled modified microorganisms according to claim 2, characterized in that, The molar ratio of diphenyl phosphite to n-decyl alcohol is 1:2, and the reaction temperature of diphenyl phosphite and n-decyl alcohol is 130-150℃, with a reaction time of 3-5h.

4. The method for preparing coordination-assembled modified microorganisms according to claim 2, characterized in that, The molar ratio of N,N-diethylethylenediamine to 3,4-dihydroxybenzaldehyde is 1:1, and the reaction temperature of N,N-diethylethylenediamine and 3,4-dihydroxybenzaldehyde is 60-80℃, with a reaction time of 2-4h.

5. The method for preparing coordination-assembled modified microorganisms according to claim 2, characterized in that, The molar ratio of N,N-diethylethylenediamine to the diecryl phosphate is 1:1, and the reaction temperature of the intermediate product with the diecryl phosphate is 90-110℃, and the reaction time is 1-2h.

6. The method for preparing coordination-assembled modified microorganisms according to claim 1, characterized in that, In step (1), the bacteria in the microbial culture are Gram-positive or Gram-negative bacteria, and the amount of catechol lipid molecules and microbial culture is 1 mL for every 0.2 mg of catechol lipid molecules. 8 Microbial culture at CFU / mL; In step (2), the metal ions are derived from salts of cerium, iron, copper or zinc, and the molar ratio of the catechol lipid molecules to the metal ions is 1:

1. In step (3), the polyphenolic compound is one or more of tannic acid, ellagic acid, epigallocatechin gallate and quercetin, and the molar ratio of the catechin lipid molecule to the polyphenolic compound is 1:

1.

7. A coordination assembly-modified microorganism, characterized in that, It is prepared according to any one of claims 1-6.

8. The use of a coordination assembly modified microorganism as described in claim 7 in the preparation of a medicament for treating oral inflammation and / or neuroinflammation.

9. The application according to claim 8, characterized in that, The oral inflammation is periodontitis, and the neurological inflammation is neurological inflammation associated with depression or cognitive impairment.

10. A pharmaceutical composition for the combined treatment of oral and neurological inflammation, characterized in that, It includes the coordination assembly modified microorganism and pharmaceutically acceptable carrier as described in claim 7.

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