Oral engineered microbe systems and uses thereof

Abstract

This invention pertains to genetic engineering and relates to an orally administered engineered microbial system and its applications. Through genetic manipulation techniques such as genome integration, a dominant mutant of the nicotine-degrading enzyme Nox is displayed on the surface of Bacillus subtilis spores. This mutant is then coupled with catalase or peroxidase, and genes related to spore germination and the system's own recombinant enzymes are knocked out to construct a strain with self-eliminating resistance genes. This invention utilizes orally administered engineered bacteria to colonize the intestines. The "molecular degradation machine and metabolic siphon" effect produced by the engineered bacteria continuously degrades nicotine diffused from the bloodstream, thereby significantly reducing the concentration of nicotine binding to central nicotine receptors. This fundamentally weakens the dopamine reward effect induced by smoking, effectively breaking the positive feedback loop of "smoking-pleasure," and providing a new method for treating nicotine dependence. As a "plug-and-play" platform technology, displaying different degradation enzymes on the surface of Bacillus subtilis spores can also degrade other harmful substances.

Description

Technical Field

[0001] This invention pertains to genetic engineering and relates to an orally administered engineered microbial system and its applications. Background Technology

[0002] According to the 2024 Global Burden of Disease data released by the IHME, smoking remains one of the top three causes of death globally, causing more than 6 million deaths annually. However, smoking is also the only preventable cause of death. As the material basis for smoking addiction, nicotine possesses an addiction reward mechanism similar to morphine (Nat Rev Dis Primers. 2022; 8(1):19.; ProcNatl Acad Sci U SA. 2022; 119(46):e2209870119.), making it difficult to quit smoking. In addition to the health problems associated with smoking, nicotine itself is also a highly toxic substance, with an oral LD50 in rats. 50 The LD50 for mice is 50 mg / kg. 50 The LD50 for adults is 3.3 mg / kg. 50 The dosage is 0.5–1 mg / kg. Studies have shown that nicotine can inhibit the killing of tumor cells by chemotherapy drugs such as paclitaxel, explaining why lung cancer patients who smoke are more difficult to treat (J Pharmacol ExpTher. 2018; 366(2):303-313.). In addition, existing evidence suggests that smoking affects both innate and adaptive immune responses, and the effects on adaptive immune responses persist for a long time after an individual quits smoking and are associated with epigenetic memory (Nature. 2024; 626(8000):827-835.). Although there are reports that long-term use of low-dose nicotine can prolong the lifespan of mice (Adv Sci; 2025 28:e15311), the concentration of nicotine in the blood and brain of smokers is usually significantly higher than the dose used in this experiment, and there are significant differences between humans and mice in terms of metabolism, stress response, sensitivity to toxic and harmful substances, and psychological effects. Therefore, based on the results of numerous clinical studies, it is shown that nicotine dependence is the material basis for human smoking addiction and the most important factor affecting health, and breaking nicotine dependence is the most important way to quit smoking addiction.

[0003] For a long time, the development of nicotine withdrawal or smoking cessation drugs has focused on nicotinic acetylcholine receptors (α4β2nAChRs), such as the nicotine withdrawal methods currently approved by the FDA: nicotine replacement therapy (NRT), varenicline, a partial agonist of nicotinic acetylcholine receptors, atypical antidepressants bupropion, and nicotine vaccines. However, these therapies have had little effect in clinical applications (J SuBSt Abuse. 1991; 3(4): 427-440.; Addiction. 2014; 109(8): 1268-1273.; Addiction. 2014; 109(8): 1252-1259.). According to data from Boyan Consulting's "2022–2028 In-depth Research and Market Prospect Analysis Report on Smoking Cessation Products Industry", the market size of smoking cessation products in my country reached 180 billion yuan in 2024, accounting for 35% of the global market, making it the world's largest smoking cessation product market. Among numerous smoking cessation drugs, Pfizer's Champix was once a blockbuster product that generated over $1 billion in sales for Pfizer. However, due to excessive levels of the impurity nitrosamine, sales of the product have been suspended. Several domestic pharmaceutical companies filed for generic drug approval after Champix's patent expired in 2020. However, according to reported research results, varenicline has a higher binding affinity to α4β2nAChRs (K... i =0.15nM vs 1.6nM [nicotine]), there is a risk of abuse, and there are side effects such as nausea, insomnia and headache. Patients with mental illness, commercial vehicle drivers and heavy machinery operators are high-risk groups for drug use. These three groups need to use varenicline with extreme caution (DrugHealthc Patient Saf.2010;2010(2):39-48.; Clin Ther.2009;31(3):463-491.). With the limited efficacy and large side effects of traditional smoking cessation drugs in clinical treatment, people have turned their attention to nicotine metabolism therapy, which reduces the concentration of nicotine reaching the brain by reducing the content of nicotine in the blood, thereby reducing the triggering of the reward mechanism. Based on the nicotine-degrading enzyme NicA2 isolated from the nicotine-degrading bacterium Pseudomonas putida S16, people have conducted a systematic study on its drug-likeness. Although its effectiveness has been validated in animals, it cannot be developed into a therapeutic product due to unavoidable immunogenicity from intravenous administration of exogenous proteins, short plasma half-life, and poor patient compliance. Furthermore, in animal studies, a dose of up to 70 mg / kg of NicA2-J1 is required to reduce blood nicotine concentrations (Biochemistry).

[0004] 2005; 44(50):16701-16709.; Diabetes Obes Metab. 2010; 12(3):179-185.), which also seriously affected further development.

[0005] In recent years, gut microbiota research has revealed that the gut bacterium *Bacteroides xylanisolvens* can effectively degrade nicotine by generating a novel nicotine metabolite, HPB, through the nicotine-degrading enzyme NicX. In smokers and mouse models, nicotine accumulates in the gut during smoking. Colonizing *B. xylanisolvens* in nicotine-exposed mice reduced intestinal nicotine concentration through NicX-catalyzed degradation, alleviating the progression of nicotine-induced non-alcoholic fatty liver disease (NAFLD) (Nature. 2022; 610(7932):562-568.). This work provides an example of nicotine degradation using gut microbiota, but the need for prior colonization of *B. xylanisolvens* and induction of NicX expression within the bacteria presents a challenge to its practical application.

[0006] Oral administration offers advantages such as convenience, low toxicity and side effects, and good patient compliance. However, proteins and peptides, when taken orally, are easily degraded by various proteolytic enzymes and the extremely low pH environment of the gastrointestinal tract, rendering them unable to function. Given these problems with nicotine-degrading enzymes, there is an urgent need to develop an oral formulation containing novel nicotine-degrading enzymes that overcomes gastrointestinal degradation issues, exhibits good acid and heat stability, and demonstrates high nicotine degradation efficiency. Summary of the Invention

[0007] Purpose of the invention

[0008] Unlike existing methods of nicotine degradation in vivo via injection based on NicA2, this invention develops engineered live microorganisms for nicotine degradation via oral administration. To achieve this, this invention identifies and modifies nicotine-degrading enzymes to obtain mutants with protease resistance and acid tolerance, and then uses gene editing techniques such as genome integration to display these mutants on the surface of Bacillus subtilis 168 spores.

[0009] One of the objectives of this invention is to construct a recombinant spore of a Bacillus subtilis display system, wherein the Bacillus subtilis is an engineered Bacillus subtilis with germination defects and self-elimination of resistance.

[0010] The second objective of this invention is to construct a superior mutant of the nicotine-degrading enzyme Nox derived from the strain *Stutzerimonasstutzeri*, which exhibits tolerance to a variety of protein-destructive factors.

[0011] The third objective of this invention is to demonstrate a mutant of the nicotine-degrading enzyme Nox in Bacillus subtilis.

[0012] Develop engineered live microorganisms for nicotine degradation via oral route on the surface of Bacillus subtilis 168 spores.

[0013] The fourth objective of this invention is to fully utilize the unique stress-resistant properties of spores, such as high temperature resistance, the ability to display exogenous proteins on their surface, non-growth, and the ability to germinate at any time, to effectively transform these advantages into reliable oral engineered live microorganisms for the prevention and treatment of various diseases or bodily discomforts.

[0014] The fifth objective of this invention is to utilize the degrading enzymes and their combinations or fusions displayed on the spore surface, through concentration gradient differences, to generate a "metabolic siphon effect" as the "molecular degradation machine" operates, thereby achieving a continuous reduction of substances (such as ethanol, glucose, urea, creatinine, uric acid, cholesterol, triglycerides, etc.) in the circulatory system, central nervous system, and other systems that are at concentrations higher than those in the human body's health.

[0015] The sixth objective of this invention is to improve upon the previously disclosed spore display system (ACS Sensors, 2025).

[0016] Based on 10(5):3589-3599.), significant conceptual innovations and technological advancements have been achieved: 1. Catalase and nicotine-degrading enzyme were efficiently and organically coupled in Bacillus subtilis spores to verify the feasibility of surface display of multiple proteins and to eliminate the inactivation of enzymes by hydrogen peroxide; 2. By optimizing the linker to ensure the activity of the two enzymes, catalase and nicotine-degrading enzyme were fused into a single protein for surface display; 3. Through resistance screening and preservation in the strain library and real-time resistance elimination of the working strain, the efficient production of oral-safe strains was achieved.

[0017] Technical solution

[0018] To achieve the above-mentioned objectives, this invention integrates protein engineering, metabolic engineering, and microbial engineering, and adopts synthetic biology concepts and technologies to construct living microorganisms (molecular degradation machines) that can be stationary in the intestines. Through the generation of a "metabolic siphon effect," these microorganisms continuously degrade nicotine and other human metabolites or exogenous substances that are toxic or harmful to human health and transported from the blood to the intestines.

[0019] An oral engineered microbial system is characterized in that engineered spores are obtained through a Bacillus subtilis display system; wherein Bacillus subtilis serves as the chassis, and the Bacillus subtilis is modified into a germination-deficient and resistance-self-eliminating recombinant Bacillus subtilis; one or more of the following spore capsid proteins—CgeA, CotB, CotE, CotG, CotY, and CotZ—are used as anchoring proteins for displaying the target protein; the integration site of the target protein or enzyme is the amyE, lacA, pyrD, gerK operon, or prpE.

[0020] The oral engineered microbial system is characterized by amplifying the capsid protein using the genome of Bacillus subtilis WB800n or B. subtilis 168 strains as a template, fusing the target protein and capsid protein with the Escherichia coli-Bacillus subtilis shuttle integration plasmid pDG364, constructing a Bacillus subtilis recombinant plasmid, wherein the amyE homologous arm is replaced with the upstream and downstream homologous arms of gerKA-gerKB-gerKC, and a gering defect is formed by integration and knockout of this site, and the resistance gene is seamlessly self-eliminated using the Xer / dif resistance self-elimination system in Bacillus subtilis, transforming into competent cells to form Bacillus subtilis recombinant bacteria, which form engineered spores under nutrient deprivation and conditional stress.

[0021] Seamless elimination of resistance genes is achieved by utilizing the Xer / dif resistance self-elimination system in Bacillus subtilis. Specifically, it is based on Bacillus subtilis' own Xer recombinase, which specifically recognizes the dif sequence (5'-ACTTCCTAGAATATATATTATGTAAACT-3') as shown in SEQ ID NO:2.

[0022] The oral engineered microbial system is characterized in that the nutrient deficiency is formed by DSM spore-forming medium; DSM spore-forming medium: 5g peptone, 3g yeast extract, 1g potassium chloride, diluted to 1L, and after moist heat sterilization, 1mL of filtered and sterilized 1M MgSO4, 1mL of 10mM MnCl4, 1mL of 1mM FeSO4, and 0.5mL of 1M CaCl2 solution are added.

[0023] The oral engineered microbial system is prepared as follows: Recombinant B. subtilis 168 is inoculated at 0.1% in sterile 2×YT culture medium at 37℃ and activated for 12–24 h. Then, it is inoculated at 1%–2% in DSM spore-forming medium. After spore-forming culture for 24–96 h, the spores are collected by centrifugation at 8000–12000 rpm and 4℃ for 10–15 min. The spores are then washed three times with sterile 1×PBS (pH 7.4) to obtain a clean spore precipitate.

[0024] The oral engineered microbial system described above produces spore precipitates which are then prepared into spore freeze-dried powder under the action of a freeze-drying protectant.

[0025] The spore freeze-dried powder contains recombinant spores and a freeze-drying protectant. The freeze-drying temperature is -80 to -50°C, and the time is 12 to 24 hours.

[0026] The freeze-drying protectant is characterized by using a compound or single protectant in a ratio of 6-3-3% (wt / vol) mannitol-lactose-sorbitol or using mannitol and whey protein as a single protectant.

[0027] The oral engineered microbial system has a "plug-and-play" characteristic, and its engineered spores can load and replace any target protein and its combination. In this invention, the target protein can be a degradation or destructive enzyme involved in the following application scenarios:

[0028] (1) Nervous system: Anxiety and depression caused by elevated serotonin (using monoamine oxidase, etc.); mental disorders caused by abnormal gamma-aminobutyric acid (using gamma-aminobutyric acid transaminase, etc.); mental disorders caused by psychotropic substances such as methamphetamine, ketamine, ecstasy, opium, morphine, marijuana, heroin, cocaine, synthetic cannabinoids, flunitrazepam, fentanyl, etc. (using catechin-O-methyltransferase and monoamine oxidase, etc.).

[0029] (2) Cardiovascular system: hypertension caused by elevated renin; atherosclerosis caused by elevated triglycerides (using lipoprotein lipase); thrombosis and atherosclerosis caused by high concentration of homocysteine ​​(using cystathionine β-synthase, methylenetetrahydrofolate reductase).

[0030] (3) Metabolic system: Diabetes and cataracts caused by elevated glucose levels (using enzymes such as hexokinase / glucokinase, phosphofructokinase, and glucose oxidase); organ damage caused by elevated urea concentration (using enzymes such as carbamoyl phosphate synthase I and ornithine carbamoyltransferase); organ damage caused by elevated creatinine concentration (using enzymes such as creatinine degrading enzymes); gout, hyperuricemia, and organ damage caused by elevated uric acid concentration (using enzymes such as xanthine oxidase).

[0031] (4) Endocrine system: Cushing's syndrome caused by elevated cortisol and glucocorticoids (using a drug such as type II 11β-hydroxysteroid dehydrogenase); refractory hypertension caused by elevated aldosterone (using aldosterone degrading enzyme).

[0032] (5) Digestive system: gallstones, hepatitis and pancreatic cancer caused by elevated bilirubin (using enzymes such as uridine diphosphate glucuronyl transferase).

[0033] (6) Reproductive system: female polycystic ovary syndrome, infertility and hirsutism caused by elevated androgen (testosterone) (using aromatase, etc.); male sexual dysfunction and breast development caused by elevated estrogen (using 17α-hydroxylase / 17,20-lyase, 3β-hydroxysteroid dehydrogenase, etc.).

[0034] (7) Exogenous substances: Acute gastritis, alcoholic liver disease (such as fatty liver, hepatitis and cirrhosis), pancreatitis, alcohol addiction, Alzheimer's disease, alcoholic heart failure, arrhythmia, hypertension, liver cancer, colorectal cancer, breast cancer, sexual dysfunction, infertility (using enzymes such as alcohol dehydrogenase and aldehyde dehydrogenase); liver cancer, liver failure, growth retardation in children, and immunosuppression caused by aflatoxin intake (using enzymes such as glutathione S-transferase).

[0035] Bacillus subtilis exists in a highly resilient spore state under nutrient deficiencies, and the latter can survive for extended periods in extreme environments. Therefore, this invention utilizes engineered spores (“molecular degradation machines”) to generate a “metabolic siphon” effect, continuously degrading nicotine diffused from the bloodstream. This significantly reduces the concentration of nicotine binding to central nicotine receptors, fundamentally weakening the dopamine reward effect induced by smoking in the brain. This effectively breaks the positive feedback loop of “smoking-pleasure,” providing a new method for treating nicotine dependence.

[0036] Furthermore, the oral engineered microbial system is characterized in that the target protein displayed by the spores is a nicotine-degrading enzyme or a mutant, or a fusion protein constructed by linking it with catalase.

[0037] Furthermore, the wild-type nicotine-degrading enzyme is Nox derived from the strain Stutzerimonasstutzeri.

[0038] Because wild-type Nox is completely intolerant to low concentrations of pepsin and trypsin, it easily loses its catalytic activity. Considering activity, tolerance to pepsin and trypsin hydrolysis, acid tolerance, and thermal stability (Tm), wild-type Nox was mutated to generate the nicotine-degrading enzyme mutant. The mutant sequence includes at least one substitution of an amino acid selected from positions 68, 86, 103, 126, 152, 184, 201, 217, 238, 245, 322, 347, 351, 364, 375, 383, 396, 398, 428, 433, 452, and 458 of SEQ ID NO:1; wherein the basic amino acids include Lys, His, and Arg; and the acidic amino acids include...

[0039] Asp and Glu; nonpolar hydrophobic amino acids include Phe, Leu, Ile, Trp, Pro, Val, Met, and Ala; polar and uncharged amino acids include Cys, Asn, Gly, Ser, Gln, Tyr, Thr, and Ser.

[0040] The sequences of the variants include the N-terminal truncated form Δ46 based on SEQ ID NO:1, and single-site or multi-site substitutions of K184H, K184F, K184V, K184S, K184T, K184Y, A103R, F351H, K322V, Q330I, Q330T, A103R&F351H, K322V&Q330I, K322V&Q330R201H, R201F, R201V, R201S, R201T, R201Y, R347H, R347F, R347V, R347S, R347T, R347Y, K398H, K398F, K398V, K398S, K398T, K398F, K398V, K398S, K398T, K398F, K398F, K398S, K398T, K398F, K398F, K398T ... 8Y, R433H, R433F, R433V, R433S, R433T, R433Y, C68L, G86D, G152W, E21 7N, Y238W, E245L, E245T, S375C, T383L, I396L, V428P, S452A, N458F, Q3 30I&R347S, R347S&N458F, Q330I&R347S&R433S, Q330I&R347S&N458F, Q330I&R347S&D126S, Q330I&R347S&H364R, G86D&Q330I&R347S&R433S.

[0041] The mutant is used in the preparation of oral formulations or smoking cessation drugs for nicotine dependence.

[0042] The recombinant proteins are linked together by linkers.

[0043] The genomic integration sites include amyE and gerKC, gerKB, gerKA, and prpE, which are related to germination.

[0044] The application of the oral engineered microbial system in the preparation of oral drug carriers.

[0045] Application of the nicotine-degrading enzyme and its mutants, and the oral engineered microbial system in the preparation of smoking cessation drugs.

[0046] Specifically: an oral engineered microbial system for treating nicotine addiction or assisting in smoking cessation and reducing the toxic effects of nicotine on those exposed.

[0047] In the application scenario described, the contactee is a human user.

[0048] The use of the oral engineered microbial system is characterized by its development into an oral formulation for the treatment of nicotine poisoning or diseases related to nicotine intake.

[0049] The use of the oral engineered microbial system is characterized by being both an oral engineered microbial system for pharmaceuticals and an oral engineered microbial system for food.

[0050] Overall design concept of this invention

[0051] Homologous proteins from the *Stutzerimonasstutzeri* strain were analyzed using bioinformatics methods.

[0052] Nox was used to mutate its protein sequence, increasing its tolerance to acid, pepsin, and trypsin, as well as its thermal stability and activity.

[0053] This invention utilizes genetic manipulation techniques such as genome integration to display a mutant of the wild-type nicotine-degrading enzyme Nox from the strain *Stutzerimonasstutzeri* on the surface of *Bacillus subtilis* 168 spores. It then couples this mutant with manganese catalase from the thermophilic bacterium *Anoxybacillaceae* or the spore's inherent hydrogen peroxide degradation activity, and knocks out genes related to spore germination to avoid effective dose reduction caused by spore germination. Finally, it leverages *Bacillus subtilis*' own recombinant enzyme system.

[0054] Xer / dif enables the construction of edible strains that achieve self-elimination of resistance genes.

[0055] Specifically as follows:

[0056] To address the aforementioned technical challenges, NicA2 from *P. putida* S16 was first used as the search sequence. The NCBI BLSATP tool was used for retrieval, and a phylogenetic tree was constructed from the top 500 homologous sequences from bacterial sources to obtain Nox from the *Stutzerimonas stutzeri* strain. However, wild-type Nox is intolerant to proteases and sensitive to acid. Therefore, the PeptideCutter tool (https: / / web.expasy.org / peptide_cutter / ) was first used to predict potential protease cleavage sites. Subsequently, the sites were further analyzed according to Keil rules, combined with I-TASSER...

[0057] (https: / / seq2fun.dcmb.med.umich.edu / I-TASSER / )NetSurfP-3.0

[0058] Protein surface solvent accessibility analysis and 3D structure modeling analysis using (https: / / services.healthtech.dtu.dk / services / NetSurfP-3.0 / ) ultimately identified K184, R201, R347, K398, and R433 as potential trypsin cleavage sites, and these sites were then mutated. In addition to the direct cleavage sites of the protease, improved protein thermostability can also enhance stability against protease hydrolysis to some extent. Using HotspotWizard (https: / / loschmidt.chemi.muni.cz / hotspotwizard / ) and

[0059] The FireProt 2.0 tool (https: / / loschmidt.chemi.muni.cz / fireprotweb / ) analyzed Nox, and the sites C68, G86, A103, G152, D162, E217, Y238, E245, K322, Q330, F351, H364, S375, T383, I396, V428, S452, and N458 were associated with thermal stability. Hydrophobic amino acids are embedded within proteins, while hydrophilic amino acids are distributed on the protein surface. Charged amino acids on the protein surface can form a protective layer through electrostatic interactions, stabilizing the protein structure. Acid-resistant proteins typically have a high proportion of acidic amino acids such as Asp and Glu. The surface charge of the protein was designed using the Rosetta Supercharge tool, and mutants were constructed and screened for potential sites that affect acid resistance. Subsequently, combined mutations with different properties were performed to obtain the superior mutant Nox (G86D&Q330I&R347S&R433S). This mutant showed significant improvements in activity, protease stability, thermal stability, and acid resistance.

[0060] Subsequently, a Bacillus subtilis spore display system was constructed. Using Bacillus subtilis WB800n as the host bacterium, the Nox sequence was fused with nine common spore capsid proteins (CgeA, CotB, CotC, CotE, CotG, CotX, CotY, CotZ, and OxdD) to screen for the optimal anchoring protein.

[0061] CotE. To meet the requirements for subsequent edible strains, CotE was used as the display molecule, and B. subtilis 168 was used as the host bacterium to construct a dominant mutant recombinant spore BS168::EN (where E in EN is the anchoring protein).

[0062] CotE (N is Nox) was used, and the gerKC, gerKB, and gerKA genes were further used as integration sites and knocked out to construct a recombinant spore with germination defects, BS168::ΔFrame-EN. Using Bacillus subtilis' own recombinase Xer, the integrated resistance marker was eliminated, resulting in a recombinant spore with both germination defects and eliminated resistance, BS168::ΔFrame-EN-Δ. Since Bacillus subtilis is a recognized probiotic and the resistance genes have been eliminated, the recombinant spores prepared by this method are suitable for oral application in pharmaceuticals or food.

[0063] According to the catalytic process of nicotine degradation by Nox, the use of Nox alone inevitably produces H2O2, and the additional H2O2 will damage the protein structure, leading to the inactivation of Nox. Therefore, this invention further conjugates catalase (CAT) with Nox, using manganese catalase (MnCAT, WP_015864320.1) from the thermophilic bacterium Anoxybacillaceae. First, the expression of MnCAT in B. subtilis 168 strain was verified using the free plasmid pMA5. Then, a fusion protein was constructed with Nox and displayed on the spore surface, and recombinant spore ENLs were constructed. 15 G, EGL 15 N and ENL6G, as well as the Nox double copy display of recombinant spore BS168::ΔFrame-ENL 15 G-YN-Δ and BS168::ΔFrame-EN-YN-Δ (where N stands for Nox and Y stands for the anchoring protein CotY). By replacing the natural promoter of CotE with the strong constitutive promoter p43 of Bacillus subtilis, the BS168::ΔFrame-p43-EN-Δ strain with further enhanced expression was obtained.

[0064] It should be noted that the embodiments of the present invention are merely examples for clearly illustrating the invention, and are not intended to limit the implementation of the invention. Those skilled in the art will be able to make various obvious changes, readjustments, and substitutions without departing from the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention can achieve the effects claimed in this invention. This includes other engineered microbial systems, such as lactic acid bacteria (e.g., *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, *Lactobacillus paracasei*, *Lactobacillus helveticus*, *Lactobacillus reuteri*, *Lactobacillus plantarum*, *Lactobacillus curvularis*, *Lactobacillus fermentum*, *Lactobacillus brevis*, *Lactobacillus brunelli*, *Lactobacillus cellobiose*, *Lactobacillus deuterans* subsp. bulgaricus*; bifidobacteria (e.g., *Bifidobacterium longum*, *Bifidobacterium bifidum*, *Bifidobacterium infantis*, *Bifidobacterium brevis*, *Bifidobacterium animalis* subsp. *lactobacterium*, *Bifidobacterium adolescentis*); Gram-positive cocci (e.g., *Enterococcus faecalis*, *Streptococcus thermophilus*); and spore-forming fungi (e.g., *Bacillus licheniformis*), yeasts (e.g., *Saccharomyces boulardii*, *Saccharomyces cerevisiae*), and butyric acid bacteria (e.g., *Clostridium butyricum*), as well as other edible strains such as *Escherichia coli*. Nissle 1917, *Lactobacillus thuringiensis*, *Lactobacillus sakei*, *Pediococcus lactis*, and *Pediococcus pentosaceus*. Furthermore, as this invention is a platform technology, it has broad applications, not limited to the degradation of nicotine, but also applicable to the degradation of other toxic substances or metabolites in the body, for the development into pharmaceuticals, functional foods, and liquid oral engineered microbial system preparations. For example, displaying the corresponding degrading enzymes or multi-enzyme degradation systems on the surface of the above-mentioned microorganisms and applying them to the human body can be used to treat neurological dysfunction caused by elevated levels of endogenous or exogenous substances such as serotonin, γ-aminobutyric acid, methamphetamine, and cocaine; displaying the corresponding degrading enzymes or multi-enzyme degradation systems on the surface of the above-mentioned microorganisms and applying them to the human body can be used to treat metabolic and cardiovascular diseases or discomfort caused by elevated levels of endogenous or exogenous substances such as glucose, urea, creatinine, uric acid, renin, cholesterol, triglycerides, and homocysteine; displaying the corresponding degrading enzymes or multi-enzyme degradation systems on the surface of the above-mentioned microorganisms and applying them to the human body can be used to treat cortical... This treatment addresses endocrine disorders or discomfort caused by elevated levels of endogenous or exogenous substances such as alcohol, thyroid hormones, growth hormone, prolactin, aldosterone, and glucocorticoids. By applying corresponding degrading enzymes or multi-enzyme degradation systems to the surfaces of these microorganisms in the human body, it can be used to treat gastric ulcers caused by abnormal gastric acid, bile reflux gastritis and bile diarrhea caused by abnormal bile acid, and gallstones, hepatitis, and pancreatic cancer caused by abnormal bilirubin levels. Furthermore, by applying corresponding degrading enzymes or multi-enzyme degradation systems to the surfaces of these microorganisms in the human body, it can be used to treat endocrine or reproductive system disorders or discomfort caused by elevated levels of endogenous substances such as female androgens (testosterone) and male estrogens.It is particularly important to emphasize that by displaying the corresponding degrading enzymes or multi-enzyme degradation systems on the surfaces of the aforementioned microorganisms and applying them to the human body, they can be used to degrade exogenously ingested substances such as ethanol and aflatoxin, thereby alleviating their harm to human health. In summary, the scope of application of this invention involves the engineering modification of various orally safe microbial strains and the treatment or prevention of human health problems caused by elevated levels of various endogenous or exogenous substances.

[0065] Beneficial effects

[0066] 1. This invention is innovative based on the physicochemical properties of nicotine molecules and their ability to easily travel through multiple tissues such as the brain, blood, and intestines. By leveraging the "molecular degradation machine" of the intestines, a "metabolic siphon" effect is generated to continuously degrade nicotine that diffuses from the blood, thereby significantly reducing the concentration of nicotine that binds to central nicotine receptors. This fundamentally weakens the dopamine reward effect induced by smoking in the brain, effectively breaking the positive feedback loop of "smoking-pleasure," and providing a new method for the treatment of nicotine dependence.

[0067] 2. The activity of nicotine-degrading enzymes has not been reported in this invention, and the mutant sequence is a completely new sequence, the function of which has not been reported.

[0068] 3. To avoid the deactivation of enzymes by hydrogen peroxide generated during nicotine degradation, this invention employs simultaneous display of catalase or a fusion protein of catalase and nicotine-degrading enzyme on the surface of spores, ensuring the efficient operation of the molecular degradation machine.

[0069] 4. This invention provides a new approach and strategy for the prevention and treatment of various human diseases or discomforts caused by in vivo metabolic abnormalities or exogenous intake through the engineering modification of living microorganisms and their oral administration to the intestines. It has important clinical significance and human health value. Attached Figure Description

[0070] Figure 1 It is a phylogenetic tree analysis of NicA2 homologous proteins from 500 bacterial species;

[0071] Figure 2 This is an analysis of the phylogenetic tree of NicA2 and Nox on the same evolutionary branch;

[0072] Figure 3 This is a multiple sequence alignment analysis of Nox with the highest homology to NicA2;

[0073] Figure 4 These are SDS-PAGE gel images of IPTG-induced expression at different final concentrations;

[0074] Figure 5 This is a Nox purification SDS-PAGE gel image;

[0075] Figure 6 This is the UV-Vis spectrum of NOx;

[0076] Figure 7 It uses LC-MS / MS to detect NIC, NMM, and PON;

[0077] Figure 8 This is the enzymatic kinetic curve of Nox degrading nicotine;

[0078] Figure 9 These are the Nox enzyme activity-pH curve (A) and the enzyme activity-temperature curve (B);

[0079] Figure 10 The results of the Nox intestinal sac inversion experiment;

[0080] Figure 11 It is the residual enzyme activity of Nox after protease treatment;

[0081] Figure 12 The relative activity of Nox and its mutants and T m value;

[0082] Figure 13 These are representative SDS-PAGE gel images of Nox and its mutants after trypsin treatment. A represents Q330I & R347S, and B represents Q330I & R347S & R433S.

[0083] Figure 14 It is the residual activity of Nox and its mutants after trypsin treatment;

[0084] Figure 15 It is the surface electrostatic potential of the proteins in Nox and its mutants;

[0085] Figure 16 This is a gel image showing the PCR verification of the recombinant Bacillus genome using different anchoring proteins;

[0086] Figure 17 This is a verification of the loss of recombinant Bacillus amylase activity by different anchoring proteins;

[0087] Figure 18 These are oil immersion images (10×100x oil immersion) of spore formation at different time points;

[0088] Figure 19 It represents the activity of spores in degrading nicotine at different time points;

[0089] Figure 20 Different anchoring proteins exhibit spore activity;

[0090] Figure 21 The results are Western blotting (A) and the standard curve (B).

[0091] Figure 22 The extraction amount of spore protein at different time points (A) and the statistical results (B);

[0092] Figure 23 The results are flow cytometry detection of recombinant spores (A) and transmission electron microscopy observation (B);

[0093] Figure 24 The results are genomic PCR verification (A) and germination defect verification (B) of BS168::ΔFrame-EN strain;

[0094] Figure 25 This is a verification of the self-elimination of resistance in the BS168::ΔFrame-EN-Δ strain; A. Electrophoresis results; B. Schematic diagram of the plasmid; C. Screening of resistance-eliminating strains by plate printing.

[0095] Figure 26 The images show the cell state during apoptosis observation under a microscope (10×4x); (a) 200 μL PBS, (b) nicotine at a final concentration of 3 ng / mL, (c) nicotine at a final concentration of 30 ng / mL, (d) nicotine at a final concentration of 300 ng / mL, (e) nicotine at a final concentration of 3 ng / mL and Nox enzyme at a final concentration of 0.1 mg / mL, (f) nicotine at a final concentration of 30 ng / mL and Nox enzyme at a final concentration of 0.1 mg / mL, (g) nicotine at a final concentration of 300 ng / mL and Nox enzyme at a final concentration of 0.1 mg / mL, and (h) nicotine at a final concentration of 300 ng / mL and Nox enzyme at a final concentration of 0.1 mg / mL, and CAT enzyme at a final concentration of 5 μg / mL (2000–5000 U / mg). (i) Nicotine and CAT pure enzyme with a final concentration of 300 ng / mL, (j) Nox pure enzyme with a final concentration of 0.1 mg / mL, (k) CAT pure enzyme with a final concentration of 5 μg / mL, and (l) H2O2 with a final concentration of 1.5%.

[0096] Figure 27 These are the results of flow cytometry apoptosis detection; among them, (a) 200 μL PBS, (b) final concentration 3 ng / mL nicotine, (c) final concentration 30 ng / mL nicotine, (d) final concentration 300 ng / mL nicotine, (e) final concentration 3 ng / mL nicotine and final concentration 0.1 mg / mL Nox purified enzyme, (f) final concentration 30 ng / mL nicotine and final concentration 0.1 mg / mL Nox purified enzyme, (g) final concentration 300 ng / mL nicotine and final concentration 0.1 mg / mL Nox purified enzyme, (h) final concentration 300 ng / mL nicotine and final concentration 0.1 mg / mL Nox purified enzyme and 5 μg / mL CAT purified enzyme (2000–5000 U / mg), (i) Nicotine and CAT pure enzyme with a final concentration of 300 ng / mL, (j) Nox pure enzyme with a final concentration of 0.1 mg / mL, (k) CAT pure enzyme with a final concentration of 5 μg / mL, and (l) H2O2 with a final concentration of 1.5%.

[0097] Figure 28 The results are: genomic PCR verification (A) and protein secretion expression results (B) of strain BS168::pMA5-MnCAT;

[0098] Figure 29 Here is a schematic diagram of the Nox-MnCAT spore co-display system (A) and the results of genomic PCR verification (B);

[0099] Figure 30 This is a result of the H2O2 degradation ability of recombinant spores;

[0100] Figure 31 The diagram shows the Ferritin-like superfamily domain of MnCAT (A), the construction of BS168::Δxpd C strain (B, C), and the results of catalase activity detection (D).

[0101] Figure 32 The results are H2O2 detection (A) and cell apoptosis detection (B) during the degradation of nicotine by spores.

[0102] Figure 33 This refers to the construction of double-copy recombinant spores. A is a schematic diagram of the construction of double-copy recombinant spores, and B is...

[0103] BS168::AFrame-ENL15-YN-Δ, where C represents the activity detection result;

[0104] Figure 34 This is the construction of promoter-substituted recombinant spores; where A is a schematic diagram of promoter-substituted recombinant spores, B is the electrophoresis result of pDG-AFrame-p43-EN-Δ(XhoI / NotI), and C is the electrophoresis result of BS168::AFrame-p43-EN-Δ.

[0105] Figure 35 These are growth curves for wild-type and recombinant spore strains;

[0106] Figure 36 These are the screening results for lyophilization protectants: A is a single lyophilization protectant, and B is a composite protectant.

[0107] Figure 37 The condition of recombinant spores after treatment with simulated body fluids (A) and residual activity (B);

[0108] Figure 38This is a growth curve determined by the generation time of recombinant bacteria.

[0109] Figure 39 H&E staining results of paraffin sections of mouse organs in each group;

[0110] Figure 40 Mouse weight change (A) and ALT (B) and AST levels (C) were measured. Detailed Implementation

[0111] The technical solution of the present invention will be further explained in detail below with reference to the embodiments and accompanying drawings. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available; experimental methods without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. The strains Bacillus subtilis 168 and Bacillus subtilis WB800n were purchased from Baosai Plasmid Strain Resource Co., Ltd., the Nox gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., the pDG364 plasmid was purchased from Hunan Fenghui Biotechnology Co., Ltd., the pMA5 and pET32a plasmids were purchased from Wuhan Miaoling Biotechnology Co., Ltd., the MnCAT gene sequence was synthesized by Wuhan Miaoling Biotechnology Co., Ltd., and the nicotine standard was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. All animal research procedures have been approved by the Animal Ethics Committee of China Pharmaceutical University.

[0112] The sequences of all gene amplification primers in this embodiment of the invention are shown in Table 1:

[0113] Table 1. Primer sequences used in the embodiments of the present invention

[0114]

[0115]

[0116]

[0117] Example 1: Mining of nicotine-degrading enzyme Nox

[0118] Nicotine-degrading enzyme NicA2 and its homologous protein Nox are enzymes that catalyze the first step of nicotine degradation in the pyrrole metabolic pathway, as shown in Equation 1. They catalyze the oxidative dehydrogenation of nicotine (NIC, I) to N-methylmasmin (NMM, II), which further undergoes spontaneous hydrolysis to generate pseudooxynicotine (PON, III). The spontaneous allosteric change and hydrolysis of NMM are pH-dependent. The dehydrogenated metabolite NMM has only a very low affinity for the α4β2 nicotinic acetylcholine receptor (nAChR), K iThe value was 3300 nM (vs 1.6 nM [nicotine]), indicating that degrading nicotine into NMM can significantly reduce the effects of nicotine on the central nervous system.

[0119]

[0120] Equation 1: Catalytic process of nicotine degradation by Nox oxidation

[0121] Using NicA2 (WP_013973880.1) from P. putida S16 as the search sequence, NCBI's BLSATP tool was used to retrieve sequences from a non-redundant database. Phylogenetic trees were constructed using TBtools-II and visualized using iTOL (https: / / itol.embl.de / ) for the top 500 homologous sequences from bacterial sources. Figure 1 As shown, Nox and NicA2 from different strains clustered on the same evolutionary branch, indicating the closest homology. Further analysis of nine proteins on the same branch (WP_013973880.1, WP_082457896.1, WP_181086758.1, WP_064613885.1, TDV98744.1, MCY4390314.1, MGB1882955.1, HJP37266.1, NKB39062.1) was performed using MAFFT.

[0122] (https: / / www.ebi.ac.uk / jdispatcher / msa / mafft?stype=protein) performs multiple sequence alignment and phylogenetic tree construction, and performs multiple sequence alignment and uses on the three proteins with the highest homology to NicA2.

[0123] Visualization is performed using ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi), as follows: Figure 2 and Figure 3 As shown, WP_082457896.1 from Pseudomonas sp. EGD-AK9, WP_064613885.1 (Nox) from Pseudomonas sp. JY-Q, and WP_181086758.1 from Stutzeri are most homologous to NicA2 (WP_013973880.1) and all belong to the flavin monoamine oxidase family protein.

[0124] Given that previous reports (Appl Microbiol Biotechnol. 2019; 103(11):4499-4510.; Appl Biochem Biotechnol. 2021; 193(9):2793-2805.) indicate that the genome of *Pseudomonas* sp. JY-Q simultaneously contains two nicotine homologous degradation clusters dominated by NicA2 and Nox (WP_064613885.1), respectively, and that this bacterium can degrade 5 g / L of nicotine within one day, and that the transcriptional level of Nox is...

[0125] It is 20 times more potent than NicA2. Studies have shown that while wild-type Nox has slightly lower activity than NicA2, it exhibits better stability, a wider pH range, a higher reaction temperature range, and better thermal and acid stability compared to NicA2. Furthermore, it has a higher substrate affinity than NicA2, i.e., K+. m It is lower (Protein Expr Purif. 2021; 178:105767.), and therefore has greater development and application potential than Nox.

[0126] Therefore, this invention selected WP_181086758.1 from Stutzerimonas stutzeri (as Nox), which has the highest homology with NicA2 and for which no activity has been reported yet, for further research.

[0127] Example 2: Enzymatic characterization of Nox derived from *Stutzerimonas stutzeri*

[0128] (1) Nox heterologous expression in BL21(DE3)

[0129] Using pET28a-sumo-Nox synthesized by Sangon Biotech as a template, the target fragment was amplified using the primers in Table 1 for Nox-F1 / Nox-R1. After double digestion with NcoI and XhoI, the fragment was ligated into the pET32a plasmid. This plasmid has a 6×His and TrxA tag at its N-terminus, which promotes lysing expression and facilitates affinity purification. The constructed pET32a-Nox plasmid was digested with enzymes and verified by sequencing by Qingke Biotechnology. It was then transformed into the BL21(DE3) strain to obtain the heterologous expression strain BL21::pET32a-Nox. Positive single clones or glycerol-containing bacteria were inoculated into 5 mL of LB or 2×YT liquid medium containing 100 μg / mL ampicillin. After overnight culture, 1% of the culture was inoculated into 200 mL of LB or 2×YT liquid medium and cultured at 37℃ and 200 rpm until OD. 600When the concentration is 0.6–1.0, the culture is placed in an ice-water mixture for 10–30 min, and then IPTG is added to a final concentration of 0.1–1.0 mM. Expression is induced at 18°C ​​for 16–20 h.

[0130] Result: As Figure 4 As shown, the BL21::pET32a-Nox strain exhibited some leakage expression even when cultured at 37°C without additional IPTG. The expression level significantly increased after induction with different IPTG concentrations at low temperatures for 16–20 h, but there was no significant difference in expression levels induced by different IPTG concentrations. Considering factors such as economy and expression stability, a final concentration of 0.2 mM IPTG was selected for subsequent experiments.

[0131] (2) Nox expression and purification

[0132] The induced bacterial cells were collected by centrifugation (5000 rpm, 10 min), washed once with 1×PBS, and resuspended in 10 times their weight volume of Lysis buffer (50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, 5% glycerol, and 20 mM imidazole). The suspension was then sonicated in an ice-water bath for 10 min (2 s working, 4 s interval) using an ultrasonic homogenizer set to 120 W. The homogenized sample was centrifuged at 15000 rpm for 30 min at 4 °C to obtain a bright yellow supernatant, which was the crude enzyme solution. The crude enzyme solution was then mixed with pre-treated Ni... 2+ Cytiva TM After incubation at 4°C for 2–3 hours, non-specifically bound proteins were eluted with buffers containing 20 and 50 mM imidazole, respectively, and the target protein was finally eluted with a buffer containing 300 mM imidazole. The target protein was then subjected to ultrafiltration in a 10 kDa ultrafiltration tube (Milipore). TM Concentrate to 1.8–2.5 mL, then pass through a PD-10 desalting column (Cytiva). TM (GE Life) To remove imidazole, the desalting solution was Buffer D (50 mM Na2HPO4-NaH2PO4, pH 7.5, containing 50 mM NaCl and 5% glycerol). The collected protein solution was processed using the BCA method. Protein content was determined and the purified protein was verified by 12% SDS-PAGE.

[0133] Result: As Figure 5 As shown, the final protein purity reached over 95%.

[0134] (3) Nox enzymatic characterization

[0135] Nox belongs to the flavin monoamine oxidase superfamily:

[0136] like Figure 6 As shown, Nox exhibits absorption peaks at 370 nm and 450 nm characteristic of flavoproteins when scanned with a Shimadzu UV1780 spectrophotometer.

[0137] LC-MS / MS (Shimadzu LC20A liquid chromatography, 8045MS, Luna 3μHILIC column) was used. The Nox enzyme activity was verified using a 15×0.46cm, 3μm, phenomenex column at 30℃ and a flow rate of 0.6mL / min, eluted for 10min in a mobile phase of 90-10 acetonitrile-methanol [containing 1‰ formic acid]. Figure 7 This demonstrates the effect of LC-MS on NIC (m / z 163 [M+H)). + ), NMM(m / z 161[M+H) + ) and PON(m / z179[M+H] + The test results.

[0138] In a system without the addition of any cofactors, 2 mg / mL wild-type Nox can convert 2 μM nicotine to NMM and PON within 30 min, with NMM being the predominant form and a small amount of spontaneous hydrolysis occurring. Using substrate concentrations of 1, 2.5, 5, 12.5, 25, 50, 100, 200, 400, and 800 μM, 0.2 mg / mL enzyme solution was incubated in 200 μL of 1×PBS at 37°C for 30 min. Michaelis-Menten curves (V0 vs. NIC concentration) were plotted using GraphPad Prism 8.0, and nonlinear fitting was performed using the Michaelis-Menten function. Figure 8 As shown, the K of wild-type Nox m =13.3μM, K cat =3.02×10 –1 / s.

[0139] In the study of optimal reaction temperature and pH, comparisons were made at 20, 30, 35, 40, 45, 50, 55, 60, 70, and 80℃ and with 0.1M citrate buffer (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5), 0.1M phosphate buffer (pH 6.0, 6.5, 7.0, 7.5), 0.1M Tris-HCl buffer (pH 8.0, 8.5, 9.0), and 0.1M glycine-NaOH buffer (pH 9.5, 10.0). The results showed that the optimal reaction temperature was approximately 55℃, and the optimal reaction pH was 7.5–8.0. Figure 9 ).

[0140] Example 3: Evaluation of the effect of Nox enzyme using an inverted intestinal sac model

[0141] To determine whether Nox enzymes can lower blood nicotine levels via enteral administration, [the following was taken]

[0142] An intestinal sac inversion experiment was performed on the intestines of SD rats. Specifically, a small intestinal segment from rats that had been fasting for one day was isolated and rinsed in Klebsiella pneumoniae buffer until no contents flowed out. The mesentery and fat on the surface of the intestinal segment were carefully peeled off, and the segment was inverted using a self-made glass tube. One end was ligated with surgical suture, and the other end was ligated to the glass tube to form a sac. 2 mL of blank Klebsiella pneumoniae buffer was then injected into the sac, and it was transferred to a glass trough containing Klebsiella pneumoniae buffer for equilibration for 10 min, while checking for leaks. After equilibration, the sac was transferred to 50 mL of Klebsiella pneumoniae buffer containing 10 μM nicotine. Klebsiella pneumoniae buffer containing 0.2 mg / mL Nox enzyme solution was added to the sac, and the temperature was maintained at 37°C and 5% CO2. 200 μL of parenteral fluid was collected at 0, 30, 60, 90, 120, and 180 min, and the samples were analyzed by MS. Another inverted intestinal sac model involved adding nicotine-containing K-buffered saline to the sac, with enzyme solution in the external fluid, and sampling the internal fluid. Western blotting was used to detect potential enzyme leakage, and the results showed no leakage; the solution volume within the sac remained stable.

[0143] Result: As Figure 10 As shown, the MS analysis results showed that, compared with the control group without enzyme solution, the sample showed the signal of nicotine degradation product NMM, and the NMM content increased with the extension of the sampling time point, while the nicotine content decreased accordingly, indicating that the intestinal administration model can reduce the nicotine content in body fluids.

[0144] Example 4: Construction and Characterization of Nox Mutants

[0145] Molecular modification can effectively improve enzyme stability. Factors affecting the acid resistance of enzyme proteins mainly include the pKa value of the active site, the pKa value of the substrate binding site, the protein surface charge, and the molecular forces within the protein. Through rationally designed site-directed mutagenesis, strategies such as homologous sequence alignment, surface charge optimization, and intermolecular interaction optimization can enhance the acid resistance of enzyme molecules. Furthermore, various proteases hydrolyze exogenous proteins and peptides at specific sites. For example, pepsin tends to cleave peptide bonds on the carboxyl side of F, L, E, and K residues, while trypsin acts more specifically on K and R residues. This invention modifies the Nox protein targeting the action sites of pepsin and trypsin.

[0146] (1) Nox protease hydrolysis tolerance

[0147] This invention first investigated the protease hydrolysis tolerance of Nox (WT). Pepsin (≥2500 U / mg, Shanghai Sangon Biotech) and trypsin (≥250 U / mg, Shanghai Sangon Biotech) were diluted with 0.1 M ly-HCl (pH 2.0) and 0.1 M Tris-HCl (pH 7.5) buffer, respectively. The diluted solution (1 mg / mL) was mixed with the pure protein solution at a mass ratio of 1:50 and treated at 37°C and 300 rpm for 4 h. After reacting at 37°C and pH 7.5 for 30 min, the residual enzyme activity was measured.

[0148] Result: As Figure 11 As shown, Nox is completely intolerant to low concentrations of pepsin and trypsin, and completely loses its catalytic activity after treatment.

[0149] (2) Construction of Nox mutant

[0150] Considering the application scenario of oral administration and the fact that the final site of function is in the intestine, firstly, based on the analysis of the Nox sequence, the potential trypsin action sites include 43 sites (amino acids at positions 4, 5, 11, 12, 43, 44, 46, 66, 74, 81, 83, 87, 118, 133, 161, 181, 183, 193, 201, 204, 221, 229, 249, 270, 286, 310, 322, 324, 333, 336, 342, 347, 380, 389, 394, and 397). Further analysis of the sites (Table 2) was conducted according to Keil rules. Combined with protein surface solvent accessibility analysis using I-TASSER (https: / / seq2fun.dcmb.med.umich.edu / I-TASSER / ) and NetSurfP-3.0 (https: / / services.healthtech.dtu.dk / services / NetSurfP-3.0 / ), K184, R201, R347, K398, and R433 were identified as potential protease cleavage sites, with pET32a-Nox as the target protein cleavage site. Using the template and the corresponding primers listed in Table 1, mutations were introduced using techniques such as overlap extension PCR to construct K184H, K184F, K184V, K184S, K184T, K184Y, R201H, R201F, R201V, R201S, R201T, R201Y, R347H, R347F, R347V, R347S, R347T, R347Y, K398H, K398F, K398V, K398S, K398T, K398Y, R433H, R433F, R433V, R433S, R433T, and R433Y. Pepsin typically acts on peptide bonds on the carboxyl side of F, L, E, and K residues on the protein surface. Acid-resistant proteins usually have a high proportion of acidic amino acids D and E on their surface, resulting in very low pI values ​​(pI = 2–3). This reduces the accumulation of positive charge under low pH conditions and improves acid stability (J Mol Biol. 1990; 214(1):199-222; J Mol Biol. 2002; 319(3):791-805). Furthermore, there is a correlation between acid resistance, resistance to protease hydrolysis, and thermal stability. Therefore, the Rosetta Supercharge tool (https: / / rosie.graylab.jhu.edu / supercharge) was used to calculate the surface charge of the protein, combined with the thermal stability mutation prediction tool HotspotWizard.

[0151] (https: / / loschmidt.chemi.muni.cz / hotspotwizard / ) and FireProt 2.0

[0152] (https: / / loschmidt.chemi.muni.cz / fireprotweb / ) A systematic analysis of the Nox enzyme was performed. Predictive results showed that single mutants A103R, D126S, F351H, K322V, Q330I, Q330T, C68L, G86D, G152W, E217N, E245L, E245T, S375C, T383L, V428P, Y238W, H364R, I396L, S452A, and N458F are potentially beneficial mutations that can improve protein stability. These mutants were constructed using overlap extension PCR. Furthermore, a free loop sequence exists in the N segment of Nox. Signal peptide online analysis tools (https: / / www.novopro.cn / tools / signalp) predicted the presence of a TAT (Tat / SPI) type signal peptide cleavage site at position 36 (A36) of the Nox amino acid sequence. This site would cause N-terminal breakage during purification and cryopreservation, resulting in protein size heterogeneity. Using a structural model established with AlphaFold Server (https: / / alphafoldserver.com / ), the N-terminal 46 amino acids were truncated to construct a Δ46WT protein. Then, based on the results of single-point mutation, double mutants K322V&Q330I, K322V&Q330T, Q330I&R347S and R347S&N458F, as well as triple mutants Q330I&R347S&R433S, Q330I&R347S&N458F, NoxQ330I&R347S&D126S, Q330I&R347S&H364R and quadruple mutants were constructed.

[0153] G86D&Q330I&R347S&R433S combined mutant.

[0154] The amino acid sequence of wild-type Nox is shown in SEQ ID NO.1.

[0155] MDEKRNNGLSRRSFIGGAAVVTAGAAGLGLIGSANATENGTSKRATGFDYDVIVVGGGFAGATAARECGHQGYKTLLLEARSRLGGRTFTSHFAGQEIEFGGAWVHWLQPHVWSEMQRY GLGVVEDPLTNLDKTLVMYNDGSVEDLPPEVFGTNIQVAFEKMCHDAWEAFPRPHEPMFTERARKLDKMSVLDRINQLELTRAQRAELNSYMALYGGETTDKYGLPGVLKLFACCGGWNYN AFMDTETHYRIEGGTIGLINAMLADSGAEVRLNMPVISVEQLNGGVRVETDDGETITAGTIIMTVPLNTYRHINFTPALSEGKQRFIQEGQLSKGAKLYVHVKENLGRVFAFADEQQPL NWVQTHDYGDELGTILSITIARAETIDVNDDRDAVTREIRKLFPGVEVLGIAAYDWTADPFSLGAWAAYGVGQLSRLTDLQQPEGRILFAGAETSNGWHANIDGAVESGLRAGREAKEILG

[0156] Table 2. Summary of Keil rules for trypsin cleavage sites (J Proteome Res, 2007; 6(1):399-408; J Proteome Res. 2014; 13(2):702-709.)

[0157]

[0158] * Different shearing patterns are represented in the form of regular expressions. A group of letters within square brackets is treated as a single symbol, equivalent to any single letter within the brackets; ^ indicates "except".

[0159] (3) High-throughput screening method and activity assay for Nox mutants

[0160] As shown in Equation 1, the Nox-catalyzed oxidation of nicotine involves the generation of O2 as a proton acceptor.

[0161] H2O2. Taking advantage of this property of H2O2 generation, a peroxidase detection method (Anal Biochem. 2005; 342(2):327-337.) was used as a high-throughput screening method for Nox mutant activity after appropriate modifications. Specifically, the generated H2O2, catalyzed by horseradish peroxidase (HRP), oxidizes the chromogenic substrate Amplex Red, producing a red product (N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine) with a maximum absorption wavelength of 570 nm. A standard curve was constructed using H2O2, and the H2O2 content in the samples was calculated to assess the activity differences between Nox and its mutants. 5 μL of 20 mM nicotine stock solution and 20 μL of purified protein solution (0.2 mg / mL) were added to 175 μL of 1×PBS (pH 7.5) and incubated at 37°C and 500 rpm for 10 min. Then, 50 μL of the reaction solution was added to a container containing 200 μL of chromogenic solution (1×HRP and 1×Amplex). The absorbance of the solution at 570 nm wavelength was measured using an ELISA reader after incubation at 25 °C for 30 min with a Red dye mixture in a 96-well plate, and the catalytic efficiency was calculated using the H2O2 standard curve.

[0162] Result: As Figure 11 As shown, compared to wild-type Nox, the constructed mutants Δ46WT, F351H, K322V, Q330I, Q330T, K322V&Q330I, Q330I&R347S, K184H, R201H, R347H, K398H, R433H, K184F, K184S, K184Y, R201T, R201V, R347S, R347T, R347Y, K398S, R433F, R433S, R433T, R433V, R433Y, and C68L... G152W, E217N, E245L, E245T, S375C, T383L, V428P, Y238W, I396L, S452A, N458F, R347S&N458F, Q330I&R347S&R433S, Q330I&R347S&N458F, Q330I&R347S&D126S, Q330I&R347S&H364R, G86D, G86D&Q330I&R347S&R433S showed varying degrees of influence on activity.

[0163] (4) Determination of thermal stability of Nox mutant

[0164] Protein melting temperature (T) mThe γ value reflects the thermal stability of a protein. Using SYPRO Orange dye (Merck) as a reference value... TM Differential scanning fluorescence (DSF) was used to detect Nox enzyme and the above mutant T. m The values ​​were determined. Specifically, 50 μL of 1×PBS containing 150 mM NaCl, 0.2 mg / mL protein solution, and SYPRO Orange dye to a final concentration of 5× was added to a qPCR-96-well plate and analyzed using Roche. 96. Determine the melting curve.

[0165] Result: As Figure 12 As shown, the T of wild-type Nox enzyme m The value was 67.9℃. The constructed mutants were Δ46WT, F351H, K322V, Q330I, Q330T, K322V&Q330I, Q330I&R347S, K184H, R201H, R347H, K398H, R433H, K184F, K184S, K184Y, R201T, R201V, R347S, R347T, and R347. Y, K398S, R433F, R433S, R433T, R433V, R433Y, C68L, G152W, E217N, E245L, E245T, S375C , T383L, V428P, Y238W, I396L, S452A, G86D, N458F, R347S&N458F, Q330I&R347S&R433S m The values ​​were 67.6℃, 70.7℃, 58.8℃, 72.5℃, 71.2℃, 70.3℃, 74.4℃, 68.7℃, 68.3℃, 71.2℃, 68.5℃, 63.5℃, 69.4℃, 69.2℃, 69.1℃, 69.5℃, 69.0℃, 70.2℃, 70.9℃, 72.0℃, and 6... 8.3℃, 63.1℃, 61.7℃, 60.6℃, 62.7℃, 63.6℃, ​​69.5℃, 64.9℃, 67.9℃, 66.1℃, 68.9℃, 67.2℃, 64.1℃, 67.2℃, 65.4℃, 70.7℃, 69.9℃, 67.1℃, 70.0℃, 64.3℃, 70.0℃. Among them, the mutants F351H, Q330I, Q330T, K322V&R347S, Q330I&R347S, R347H, R347S, R347T, R347V, I396L, Q330I&R347S&R433S, and G86D&Q330I&R347S&R433S showed a significant improvement in thermal stability (Tm≥70℃).

[0166] (5) Investigation of Nox mutant protease hydrolysis tolerance

[0167] The purified wild-type Nox enzyme solution and the mutant were mixed with simulated intestinal fluid at a trypsin activity-nicotine degrading enzyme mass ratio of 50-fold. After incubation at 37°C for 0–2 h, an equal volume of protease inhibitor (InStab) was added. TM Enzymatic digestion was terminated with a protease cocktail (EDTA-free). A portion was used for residual activity assay, and another portion was used for SDS-PAGE analysis. Thus, the superior mutants Q330I, R347S, and R433S, exhibiting enhanced activity, thermostability, and trypsin tolerance, were obtained. Their half-life (T5) under 50-fold trypsin activity-to-mass ratio treatment was [not specified in the original text]. 1 / 2) The activity retention time was 1054 min, which is far superior to the 26.98 ± min of wild-type Nox, with an activity retention of >80%. Figure 13 , Figure 14 Other mutants constructed include F351H, K322V, Q330I, Q330T, K322V&Q330I, Q330I&R347S, K184H, R201H, R347H, K398H, R433H, K184F, K184S, K184Y, R201T, R201V, R347S, R347T, and R347Y. K398S, R433F, R433S, R433T, R433V, R433Y, C68L, G152W, E217N, E245L, E245T, S375C, T383L, V428P, Y238W, I396L, S452A, N458F, and R347S & N458F also showed varying degrees of trypsin hydrolysis resistance.

[0168] (6) Investigation of acid tolerance in Nox mutants

[0169] Based on this mutant, a preferred combination of iterative mutations was performed with the G86D mutation site designed and optimized using the Rosetta Supercharge tool to obtain the dominant mutants G86D&Q330I&R347S&R433S. These mutants were incubated in simulated gastric fluid at pH 2.0 at a mass ratio of 1:50 (nicotine-degrading enzyme: pepsin) for 0–2 h, then the pH was adjusted to 7.5 to terminate the treatment, and enzyme activity was measured.

[0170] Results: Compared with wild-type Nox, this mutant showed significantly enhanced residual activity and gel electrophoresis results, indicating improved tolerance. Figure 15As shown, the surface charge distribution of wild-type Nox and mutants G86D, Q330I, R347S, and R433S was visualized using ChimeraX software. The mutants generally exhibited more negative potential, which is represented by red (white represents zero and blue represents positive potential). Moderate negative potential can reduce the accumulation of positive charge under low pH conditions and improve their acid stability.

[0171] Example 5: Construction of Bacillus subtilis display system and screening of display anchor proteins

[0172] Bacillus subtilis exists in a highly resistant spore state under nutrient deficiencies, capable of long-term survival in extreme environments. It is widely used as a human food and animal feed additive and is a Generally Recognized As Safe (GRAS) strain certified by the U.S. Food and Drug Administration (FDA). Its spores are encased in an outer capsid composed of more than 20 proteins. Recombinant gene technology and gene editing are used to fuse spore capsid proteins with exogenous proteins and display them on the spore surface. Recombinant spores displaying target proteins or enzymes can exhibit various biological activities and functions. For example, using CotC as a display vector, spores have successfully displayed heat-labile enterotoxins, alcohol dehydrogenase, β-galactosidase, proinsulin, enolase, and trehalose synthase. CotB is often used as a molecular carrier for displaying antigen fragments. However, recombinant spores prepared by conventional methods can germinate into vegetative cells relatively quickly in animal intestines, contaminated environments, or animal feed, leading to a reduction in the effective spore quantity. Furthermore, the resistance selection markers used in the construction of recombinant spores do not comply with the FDA's clinical guidelines for live edible microorganisms. Therefore, constructing recombinant spores with no residual resistance markers and spore germination defects is a necessary step. It is known that spore germination of *Bacillus subtilis* is controlled by operons such as gerA, gerB, and gerK, and regulated by the prpE-encoded protein. Deletion or mutation of these genes can reduce the germination rate of *Bacillus subtilis* spores. Furthermore, *Bacillus subtilis* possesses a site-specific recombinase Xer, which recognizes the dif sequence (5'-ACTTCCTAGAATATATATTATGTAAACT-3', SEQ ID NO:2) and mediates recombination between two repeated dif sequences, thereby eliminating the fragment between the two sites and achieving seamless elimination of resistance genes. Although the above methods for removing resistance genes exist, there are currently no reports of universal, residue-free recombinant *Bacillus* vectors that integrate resistance markers with spore germination defects, and the design and screening of genes causing spore germination defects and resistance elimination systems are still needed.

[0173] (1) Construction of Bacillus subtilis recombinant plasmid

[0174] To meet the needs of intestinal function, recombinant Bacillus subtilis was constructed using a probiotic Bacillus subtilis substrate. Bacillus subtilis spores are encased in multiple layers of capsids, which contain over twenty known proteins that serve as anchoring proteins to display exogenous proteins. Therefore, nine capsid proteins—CgeA, CotB, CotC, CotE, CotG, CotX, CotY, CotZ, and OxdD—were selected for screening suitable anchoring proteins. The sequence information of these proteins is shown in Table 3 below.

[0175] Specifically, using the genomes of Bacillus subtilis WB800n or B. subtilis 168 strains as templates, these nine capsid proteins were amplified using the corresponding primers listed in Table 1. The Escherichia coli-Bacillus subtilis shuttle-integrated plasmid pDG364 was amplified via BamHI / EcoRI. Linearization was achieved through double enzyme digestion. Nox was amplified using the corresponding primers in Table 1 with pET32a-Nox as a template, and a 6×His terminus was added to its C-terminus for Western blotting detection. Recombinant plasmids were constructed using a seamless cloning kit (Nanjing Novozymes Biotechnology), or by first performing overlap extension PCR followed by enzyme digestion and ligation. After transformation into DH5α competent cells (Sangon Biotech), enzyme digestion and sequencing verification yielded the following names: pDG364-CgeA-Nox-His, pDG364-CotB-Nox-His, pDG364-CotC-Nox-His, pDG364-CotE-Nox-His, pDG364-CotG-Nox-His, pDG364-CotX-Nox-His, pDG364-CotY-Nox-His, pDG364-CotZ-Nox-His, and pDG364-OxdD-Nox-His. The pDG364 plasmid contains a homologous arm to the Bacillus subtilis amylase amyE, enabling site-specific integration.

[0176] (2) Preparation of Bacillus subtilis competent cells

[0177] The preparation of competent Bacillus subtilis cells employed a two-step culture method. Specifically, single clones were picked from streaked plates or inoculated from glycerol tubes and incubated overnight in 5 mL of LB broth. Then, 200 μL of the inoculum was transferred to 10 mL of SPI medium and cultured at 37°C and 200 rpm until OD reached the target cell count. 600When the concentration is close to 1.0, add it to 10 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h. Then add 100 μL of 100×EGTA and continue culturing at 37 °C and 150 rpm for 10 min. Then add glycerol to the final concentration of 10% and finally dispense 500 μL into sterile tubes. After quick freezing in liquid nitrogen, store at -80 °C for later use. Each 20 mL SPI medium contains 9.8 mL SPI-A salt, 9.8 mL SPI-B salt, 200 μL 50% glucose solution, and 200 μL 100×CAYE; each 6 mL SPII medium contains 5.88 mL SPI medium, 60 μL 50 mM calcium chloride, and 60 μL 250 mM magnesium chloride; the 100×EGTA solution is at a final concentration of 10 mM, and the pH is adjusted to 8.0 with NaOH solution; SPI-A salt: 0.4% ammonium sulfate, 2.8% dipotassium hydrogen phosphate trihydrate, 1.2% potassium dihydrogen phosphate, and 0.2% trisodium citrate dihydrate; SPI-B salt: 0.04% magnesium sulfate heptahydrate; 100×CAYE: 2% casein acid hydrolyzed amino acids and 10% yeast extract.

[0178] (3) Bacillus subtilis plasmid transformation and genomic PCR verification

[0179] After linearizing the verified plasmid with a single enzyme digestion (linearization is not necessary if the plasmid has been completely transformed), the plasmid was added to the prepared competent cells. After recovery and culture at 37℃ and 200 rpm for 90 min, the cells were plated onto the corresponding antibiotic-resistant solid plates and incubated upside down at 37℃ for 12–16 h. Single clones were then picked and cultured in 5 mL of liquid culture medium for expansion, followed by extraction of genomic DNA for PCR amplification and verification. The single enzyme digestion linearization system consisted of: 2.0 μL of 10×QuickCutter Buffer, 1–2 μg of recombinant plasmid, 2 μL of XbaI rapid digestion enzyme, and ddH2O to a final volume of 20 μL. Digestion was performed at 37℃ for 0.5–3 h. Genomic DNA extraction was performed using the cell genomic DNA extraction kit from Hunan Aikerui Biotechnology Co., Ltd., following the manufacturer's instructions. Genomic PCR verification used the amyE-F / amyE-R primer pair listed in Table 1. The results are as follows: Figure 16As shown, wild-type Bacillus subtilis can amplify the complete amyE sequence, which is 2556 bp in size. The sizes of the recombinant Bacillus strains that successfully integrated the exogenous fragment are: B. subtilis::CgeA-Nox-His 6116 bp (AN), B. subtilis::CotB-Nox-His 6307 bp (BN), B. subtilis::CotC-Nox-His 5592 bp (CN), B. subtilis::CotE-Nox-His 6067 bp (EN), B. subtilis::CotG-Nox-His 6257 bp (GN), B. subtilis::CotX-Nox-His 6097 bp (XN), and B. subtilis::CotY-Nox-His... The strains were 6337bp (YN), 6455bp (ZN), and 6630bp (DN). B. subtilis strain WB800n was used as the host strain.

[0180] (4) Validation of integration site of recombinant Bacillus

[0181] The pDG364 plasmid used in the anchoring protein screening experiment of this invention carries the amyE integration site of the amylase gene. Successful integration will cause the recombinant Bacillus to lose its amylase activity. Therefore, a starch plate assay can be used to directly compare and verify the integration results. Specifically, 3 μL of PCR-verified positive bacterial suspension and wild-type Bacillus suspension are respectively added to a solid plate containing 1% soluble starch. After incubation at 37°C for 12–16 h, the plate is covered with prepared iodine solution, and the color change is observed.

[0182] like Figure 17 As shown, wild-type Bacillus has complete amylase activity and can decompose starch in agar plates; it does not change color upon contact with iodine solution. The integrated recombinant Bacillus loses its amylase activity, cannot decompose starch, and turns blue upon contact with iodine solution. Iodine solution: 2g potassium iodide, 2g iodine granules. First, dissolve the potassium iodide in a small amount of distilled water. After it is completely dissolved, add the iodine granules, shake to dissolve, and then bring the volume to 300mL. Store in a brown bottle.

[0183] (5) Conditional stress sporulation and activity verification of recombinant Bacillus

[0184] The recombinant Bacillus constructed in this invention requires the display of the target protein on the spore surface. Bacillus subtilis, under nutrient deprivation and conditional stress, will transform into a highly resistant spore state. This invention uses a modified DSM medium and culture conditions to achieve the transformation from bacillus to spores. Specifically, the activated bacterial solution is inoculated into the modified DSM medium at a 2% inoculum size and cultured at 37°C and 200 rpm for 24, 26, 36, 41, 48, and 72 hours. The spore formation status is observed under an oil immersion microscope, and 1 mL of the culture is used to determine nicotine degradation activity. To achieve stable and efficient spore formation, the modified DSM medium is prepared differently from the conventionally used DSM medium as follows: 5 g peptone, 3 g yeast extract, and 1 g potassium chloride are added to a final volume of 1 L. After moist heat sterilization, 1 mL of filtered and sterilized 1 M MgSO4, 1 mL of 10 mM MnCl4, 1 mL of 1 mM FeSO4, and 0.5 mL of 1 M CaCl2 solution are added.

[0185] like Figure 18 As shown, taking EN as an example, after modifying the preparation conditions of DSM medium, sporulation began to appear after 24 hours of culture and was complete after 72 hours. There was no significant effect on the catalytic activity of the sporulations at different time points. Figure 19 The activity of spores was demonstrated by comparing different anchoring proteins in spores that had sporulated for 72 hours. The results are as follows: Figure 20 As shown, under the same spore amount (0.1g wet spores), EN can achieve complete degradation of 40nM nicotine, YN has an 80% conversion rate, while CN, DN, and XN have conversion rates of <10%, indicating the importance of anchoring proteins in displaying exogenous proteins.

[0186] (6) Quantitative analysis of recombinant spore display protein content

[0187] Vetráková et al. (Comput Struct Biotechnol J. 2023; 21:1550-1556.) reported that as sporulation progresses, the displayed spore proteins integrate into the spore capsid, becoming part of its structure and thus resisting extraction by conventional methods. To verify the expression of the target protein and whether it integrates into the spore capsid over time, the target protein with different sporulation levels was extracted at different time points. Extraction method: 1 mL of LDSM culture was collected by centrifugation at 6000 rpm for 5 min, washed three times with 1×PBS, and then lysozyme was added to a final concentration of 2 mg / mL. After treatment at 37℃ for 2 h, the culture was washed three times with PBS, centrifuged, and the supernatant was discarded to obtain pure spores. Spore lysis buffer was then added, and the spores were dissolved by treatment at 70℃ for 30 min. The supernatant was collected by centrifugation at 12000 rpm for 15 min, which was the spore protein extract. Spore lysis buffer: 500 μL of 10% SDS and 1 mol / L LDTT were mixed with 8.5 mL of water. A Western blotting grayscale-concentration standard curve was plotted using purified protein expressed by BL21. Figure 21 After determining protein concentration using the BCA method and standardizing protein analysis amounts, the protein content of sporulated spores at 24, 26, 36, 41, 48, and 72 hours was compared. The results are as follows: Figure 22 As shown, a relatively large amount of protein could be extracted from the spores in the first 40 hours, but by the 48th hour, almost no more display protein could be extracted, indicating that the display protein had completely integrated into the spore capsid after 48 hours. Simultaneously, Western blotting was used to identify the target protein. The primary antibody used was a mouse-derived anti-His-tag mAb (1:2000 dilution, Wuhan Aibote Biotechnology Co., Ltd.), and the secondary antibody was HRP-conjugated anti-mouse IgG conjugated (1:10000 dilution, Cell Signaling Technology). Specifically, after separating the protein using 12% SDS-PAGE, a transfer apparatus was assembled using the "sandwich" wet transfer method, 1× transfer buffer was added, and the bands were transferred to a PVDF membrane (Millipore). TMThe transfer conditions were: a constant current of 300 mA in an ice-water bath for 2 hours. After the transfer, the membrane was immersed in 5% skim milk powder prepared with 1×TBST and sealed at room temperature for 2 hours. 1× transfer buffer: 200 mL methanol, 100 mL 10× transfer buffer, diluted to 1 L; 10× transfer buffer: 30.2 g Tris-base and 144 g glycine, adjusted to pH 8.3 and diluted to 1 L with deionized water; 10×TBS: 24.2 g Tris-base, 80 g sodium chloride, adjusted to pH 7.6 and diluted to 1 L with deionized water; 1×TBST: 100 mL 10×TBS, 1 mL Tween 20, diluted to 1 L. After blocking, the membrane was washed three times with 1×TBST for 5 min each time, then primary antibody was added and incubated overnight at 4°C. After overnight incubation, the membrane was removed and the primary antibody was recovered. The membrane was then washed three times with 1×TBST for 10 min each time, followed by incubation with secondary antibody at room temperature for 2 h. After incubation, the membrane was washed three times with 1×TBST for 10 min each time. Finally, the membrane was rinsed with ECL chemiluminescence buffer (solution A:solution B = 1:1, 1 mL each) from Yamei Biotechnology and exposed to light. Imaging was performed using a Tannon 5200 multifunctional imaging system (Tannon, Shanghai, China), and the bands were analyzed for grayscale using ImageJ.

[0188] (7) Flow cytometry and electron microscopy analysis of recombinant spore display proteins

[0189] To further confirm successful display and integration into the spore capsid, flow cytometry was used to analyze the displayed proteins. Purified wild-type and EN spores were fixed in 4% paraformaldehyde and then adjusted to a concentration of 10⁻¹⁰ with 1 mL of 1×PBS. 5 ~10 7 After spores / mL, the cells were blocked with 5% BSA solution at 37°C for 1 hour. Then, they were incubated with primary antibody (1:100 dilution) containing 5% BSA solution at room temperature for 2 hours. After washing three times with PBS, the cells were incubated with FITC-conjugated fluorescent secondary antibody IgG (Huaan Biotechnology) on ice in the dark for 2 hours (1:1000 dilution). After washing with PBS, the spores were resuspended in 0.5 mL of PBS, passed through a 300-mesh sieve, and transferred to flow cytometry tubes for analysis.

[0190] BD FACSCelesta flow cytometer (Becton, USA).

[0191] The results are as follows Figure 23As shown, 74% of EN spores were successfully labeled with fluorescence, while no fluorescence signal was detected in wild-type spores without a display system, further demonstrating the successful display of the target protein in the recombinant spores and its integration into the spore capsid. To determine whether the expression of the exogenous recombinant protein affected the spore structure, the recombinant spores were observed using transmission electron microscopy (TEM, Hitachi-7700, Hitachi, Japan). Compared with wild-type spores, the recombinant spores showed no morphological changes in the capsid, shape, and size; however, some fibrous material was present on the surface of the recombinant spores, which may be due to the displayed exogenous protein. Figure 23 ).

[0192] Table 3. Sequence information of spore cap protein in the embodiments of the present invention

[0193]

[0194]

[0195] Example 6: Construction of recombinant Bacillus subtilis with germination defects and self-eliminating resistance

[0196] (1) Construction of germination-deficient recombinant Bacillus subtilis

[0197] Example 5 demonstrates genome integration and display construction based on the conventional Bacillus subtilis integration site amyE, as well as other sites such as lacA and pyrD. Spores constructed using conventional methods will inevitably germinate into bacterial cells in the usage environment, reducing the effective spore quantity. The germination of Bacillus subtilis spores is regulated by the expression of a series of genes, including operons such as gerA, gerB, and gerK (e.g., gerKA-gerKB-gerKC), which are also regulated by the prpE-encoded protein. Knocking out these genes can reduce the spore germination rate, as described in patent CN201710338769.8. To demonstrate technological advancement, this invention modified the pDG364 plasmid by replacing the original amyE homologous arm with the upstream and downstream homologous arms of gerKA-gerKB-gerKC using a seamless cloning method, constructing the pDG-ΔFrame-EN plasmid. This enabled integration or knockout of the site, resulting in the construction of BS168::ΔFrame-EN recombinant Bacillus. The primers used are shown in Table 1. Genomic PCR verification results are shown in [Table 1]. Figure 24Wild-type Bacillus subtilis amplified a 3816 bp fragment, while the positive clone of the Nox-dominant mutant, which successfully achieved site insertion and genome integration, amplified a 5311 bp fragment. It should be noted that, unless otherwise specified, all subsequent construction strains used were germination-deficient integration strains, and the EN strains mentioned in the following examples are recombinant Bacillus subtilis, representing the Nox-dominant mutant. Unless otherwise specified, the following examples all used Bacillus subtilis 168 as the host strain.

[0198] Take 1 mL of LDSM culture (72 h), wash once with sterile water, treat with lysozyme at a final concentration of 2 mg / mL at 37°C for 2 h, centrifuge at 6000 rpm for 10 min to precipitate spores, wash once more with sterile water, resuspend in 1 mL of sterile water, and dilute to 10⁻⁶. -7 Take 100 μL and spread it onto a chloramphenicol-resistant plate containing 10 μg / mL of chloramphenicol. Incubate at 37°C for 12 hours and then observe the colony growth. Figure 24 As shown, compared with recombinant Bacillus with amyE as the integration site, the spore germination rate of recombinant Bacillus integrated into the germination-related gene site was significantly reduced, indicating that not only was the germination-deficient spore successfully constructed, but germination could also be effectively prevented.

[0199] (2) Construction of resistance-self-eliminating recombinant Bacillus subtilis

[0200] In the above construction, the chloramphenicol resistance gene was used as a selection marker. However, the introduction of the resistance gene does not comply with the relevant regulations of drug and food regulatory authorities regarding industrial host bacteria and live microbial therapy. Because Bacillus subtilis possesses the site-specific recombinase Xer, which can specifically act on the dif sequence (5'-ACTT CCTAGAATATATATTATGTAAACT-3', SEQ ID NO:2), mediating recombination between two repeated dif sequences, thereby eliminating the fragment between the two dots. Therefore, when constructing the recombinant plasmid, the action characteristics of the Xer enzyme can be utilized to perform PCR on the chloramphenicol resistance gene Cm... r DIF sequences were loaded on both sides of the plasmid, and after relaxation culture, chloramphenicol-sensitive strains were screened by plate printing. Using this method, the pDG-ΔFrame-EN plasmid was constructed using the pDG-ΔFrame-EN plasmid as a template (primers are shown in Table 1). Positive strains were easily obtained after introducing the recombinant plasmid into *B. subtilis* strain 168. After verification, 0.1% of preserved glycerol bacteria was inoculated into 5 mL of antibiotic-free LB or 2×YT liquid medium, cultured for 24–30 h, and then diluted to 10⁻⁶. -7Antibiotic-free plates were plated, and then plate-printed. Single clones were picked and streaked onto antibiotic-free plates and antibiotic-resistant plates containing 5 / 10 μg / mL chloramphenicol. Single clones that grew on antibiotic-free plates but not on antibiotic-resistant plates were identified as antibiotic-self-eliminating strains. These single clones were then selected for further culture and genomic analysis, followed by PCR amplification for verification.

[0201] like Figure 25 As shown, the amplified fragment of the strain that did not undergo resistance elimination was 5367 bp, while the amplified fragment of the strain that successfully achieved resistance elimination was 4415 bp, namely the target strain BS168::ΔFrame-EN-Δ.

[0202] Example 7: The effect of Nox degradation of nicotine on hydrogen peroxide production and the protective effect of Bacillus subtilis

[0203] (1) The production of H2O2 during the degradation of nicotine by Nox causes apoptosis in cells.

[0204] As described in Formula 1 and Example 4, H2O2 is inevitably generated during the degradation of nicotine using Nox alone. The additional H2O2 in vivo can disrupt the balance between oxidation and antioxidation, causing oxidative stress. To assess whether the H2O2 generated during this process affects cells, rat small intestinal crypt epithelial cells IEC-6 were used as the research subject to study the effect of H2O2 generated during the degradation of nicotine by Nox on IEC-6 cells. IEC-6 is a rat small intestinal crypt epithelial cell that retains the undifferentiated characteristics of small intestinal epithelial stem cells. It is not significantly different from proliferative crypt cells in terms of histology and immunology. Moreover, under suitable conditions, it can differentiate into mature intestinal cells, making it a good model for studying the effects of exogenous substances on the small intestine. Compared with colon cancer cell lines such as HT-29 and Caco-2, IEC-6 cells are more suitable for studying the effects on small intestinal function (Food Funct. 2019; 10(4): 2010-2021.). Specifically, IEC-6 cells were revived and passaged in DMEM medium (KGI Biotechnology) containing 10% FBS. After digestion with trypsin, they were seeded into six-well plates at a density of 1–5 × 10⁶ cells per well. 5Cells were cultured until they adhered to the culture medium and reached 60–70% healing on day 2. Then, 1.8 mL of fresh culture medium was added, followed by the following group treatments: (a) 200 μL PBS, (b) 3 ng / mL nicotine, (c) 30 ng / mL nicotine, (d) 300 ng / mL nicotine, (e) 3 ng / mL nicotine and 0.1 mg / mL Nox enzyme, (f) 30 ng / mL nicotine and 0.1 mg / mL Nox enzyme, (g) 300 ng / mL nicotine and 0.1 mg / mL Nox enzyme, (h) 300 ng / mL nicotine, 0.1 mg / mL Nox enzyme, and 5 μg / mL CAT enzyme (2000–5000 U / mg). (i) Final concentration of nicotine and CAT pure enzyme at 300 ng / mL, (j) Final concentration of Nox pure enzyme at 0.1 mg / mL, (k) Final concentration of CAT enzyme at 5 μg / mL, (l) Final concentration of H2O2 at 1.5%. Microscopic observation after 24 h of treatment revealed a significant number of floating cells in groups (f), (g), and (l), with fewer adherent cells, while the cells in other groups remained normal. This indicates that the H2O2 produced by the combined treatment of medium / high concentrations of nicotine and Nox leads to apoptosis or necrosis. Furthermore, similar to direct H2O2 treatment, the addition of catalase (CAT) protected cells from damage, while different concentrations of nicotine treatment did not affect the cells. Figure 26 Further flow cytometry was used to detect cell apoptosis and necrosis, and the results are as follows: Figure 27 As shown, >90% of cells in groups (f), (g), and (l) appeared in the early apoptosis (lower right) and late apoptosis (upper right) quadrants, consistent with microscopic observations.

[0205] (2) Construction of Nox-MnCAT spore co-display system

[0206] To avoid the effects of H2O2 generated during nicotine degradation on the body and to inactivate nicotine-degrading enzymes, a manganese catalase (MnCAT) gene derived from the thermophilic bacterium *Anoxybacillaceae* was synthesized. First, the MnCAT gene was inserted into the *E. coli*-*Bacillus subtilis* shuttle free plasmid pMA5 to construct the *Bacillus subtilis* secretory expression plasmid pMA5-MnCAT. MnCAT is transcribed, translated, and secreted under the control of the HpaII constitutive promoter and signal peptide. The BS168::pMA5-MnCAT strain was constructed according to the method described in Example 5. Genomic PCR results are shown below. Figure 28Simultaneously, its secretory expression effect and CAT activity were verified, indicating that this protein is suitable for expression in Bacillus subtilis. Subsequently, the coding sequence of this protein was linked with the coding sequence of the Nox dominant mutant through a linker, and both were displayed on the surface of the recombinant spores under the action of CotE (e.g., Figure 29 (As shown), successfully constructed

[0207] ENL 15 G, EGL 15 N and ENL6G recombinant spores. Using the modified peroxidase detection method described in Example 4, the H2O2 degradation capacity of the recombinant spores was determined after obtaining the spores. Figure 30 As shown, ENL 15 G, EGL 15 The H2O2 degradation capacity of N recombinant spores is comparable to that of 0.1 mg / mL CAT enzyme, but unexpectedly, wild-type spores and EN spores also showed a certain H2O2 degradation capacity. This result is consistent with the latest report by Yang et al. (ACS Synth Biol. 2025; 14(1):101-112), which is beneficial to the application of spores.

[0208] (3) Bacillus subtilis demonstrates the protective effect of spores on cells during the degradation of nicotine.

[0209] To investigate whether recombinant spores cause oxidative stress damage or damage repair function in cells during nicotine degradation, a comparative study was conducted. First, the H2O2 produced during nicotine degradation by the spores was measured. Figure 32 As shown, with Nox enzyme as a control, the H2O2 generated during the degradation of nicotine by recombinant spores was completely and immediately degraded, while the Nox enzyme-degraded nicotine group showed a significant color reaction. When wild-type spores were added to the Nox pure enzyme-degraded nicotine group, the generated H2O2 was completely degraded, and nicotine degradation and degradation products were detected by LC-MS / MS. Apoptosis experiments further verified the protective effect of the spores, with the following groups: (a) 200 μL PBS, (b) wild-type spores, (c) BS168::ΔFrame-EN spores, (d) ENL. 15 G spores, (e) wild-type spores with a final concentration of 300 ng / mL nicotine, (f) BS168::ΔFrame-EN spores with a final concentration of 300 ng / mL nicotine, (g) ENL 15(h) spores and nicotine at a final concentration of 300 ng / mL; (i) 5 μg / mL Nox enzyme and nicotine at a final concentration of 300 ng / mL; and wild-type spores. The results were consistent with the in vitro H2O2 detection experiment. Except for group (h), where apoptosis was clearly observed at >30%, the other groups showed no significant difference from PBS treatment. Figure 32 This result further highlights the safety advantage of using recombinant spores as a nicotine degradation tool compared to Nox enzymes.

[0210] Example 8: Multi-copy display system and starter optimization

[0211] (1) Construction of a multi-copy display system

[0212] Genomic integration of exogenous proteins onto the surface of *B. subtilis* spores results in single-copy display, leading to low expression levels. To increase expression, three main strategies exist: first, using different integration sites simultaneously, such as amyE, lacA, and pyrD, and integrating different sites by preparing competent cells, followed by screening with various resistance genes; second, inserting multiple exogenous proteins guided by different anchoring proteins at the same site; and third, replacing the natural promoter of the anchoring protein with a strong constitutive promoter such as p43 to increase protein expression. However, integration at different sites faces the challenge of using multiple antibiotics and their subsequent elimination, making it unsuitable for edible strains. Therefore, based on the aforementioned strains, recombinant strains displaying both CotE and CotY were constructed.

[0213] BS168::ΔFrame-ENL 15 G-YN-Δ( Figure 33 ).

[0214] Specifically, a promoter substitution strategy was employed, replacing the natural promoter of cotE with the strongly constitutive promoter p43 from Bacillus subtilis. To achieve efficient substitution, the corresponding primers listed in Table 1 were used to amplify the p43 promoter sequence with 15–30 bp nucleotides at both ends, using the Bacillus subtilis genome as a template. Simultaneously, the remaining sequence was amplified using reverse PCR with pDG-ΔFrame-EN-Δ as a template. Then, the two fragments were ligated using a seamless cloning method to construct the pDG-ΔFrame-p43-EN-Δ plasmid. After verification by enzyme digestion and third-generation nanopore sequencing (Shanghai Sangon Biotech), the plasmid was introduced into BS168 competent cells, and positive strains BS168::ΔFrame-p43-EN-Δ were obtained through screening. Figure 34Compared with the BS168::ΔFrame-EN-Δ recombinant spores controlled by the natural promoter, the expression level and catalytic activity were significantly improved after replacing the promoter with p43.

[0215] (2) Growth curve of recombinant Bacillus

[0216] After inoculating wild-type strains and recombinant strains into 2×YT or LB medium and culturing overnight, the cultures were transferred to fresh medium at a 1% inoculum size. OD600 values ​​were measured every 2 hours at 0, 2, 4, 6, 8, 10, and 12 hours to plot growth curves. A blank medium was used as a control. OD600 values ​​between 0.2 and 0.8 are considered meaningful. If the bacterial density is too high, the medium should be appropriately diluted, and the absorbance value multiplied by the dilution factor to determine the growth. A growth curve was plotted with incubation time on the x-axis and OD600 value on the y-axis. Figure 35 As shown, the growth trends of the recombinant strain and the wild-type strain are basically the same, indicating that the insertion of the exogenous gene did not affect the growth of the bacteria.

[0217] Example 9: Preparation of Recombinant Spore Lyophilized Powder

[0218] (1) Determination of the dry weight of recombinant spores

[0219] Spores were collected by centrifugation (8000 rpm, 10 min) after culturing in 100 mL LDSM medium at a 2% inoculum rate for 72 h. After washing twice with 1×PBS, the spores were dried in a low-temperature freeze dryer (Alpha 1-2LDplus, Martin Christ Company, Germany) for 12–24 h until constant weight was achieved. The spore powder was then weighed, and the results showed that 50 mg of dry spore powder could be obtained based on the inoculum rate and medium volume in the examples.

[0220] (2) Determination of recombinant spore enzyme activity

[0221] Recombinant spores with a dry weight of 3 mg were resuspended in 500 μL of 1×PBS (pH 7.4), and 10 μL of 400 μM nicotine was added. After mixing, the mixture was incubated at 37 °C. 100 μL samples were taken at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 min after incubation. The reaction was terminated by adding twice the volume of methanol, and 5 μL of internal standard d3-nicotine (500 μM, m / z = 166.01, Shanghai Zhenzhun Biotechnology) was added. 2 μL of the solution was injected for LC-MS / MS analysis. The spores were eluted with 95% methanol for 2 min, and the signal abundances at 163.15, 161.10, 166, 179.01, 161.10>119.10, 179.01>106.15, and 166.01>130.15 were recorded. The enzyme activity of the recombinant spores was then calculated based on the plotted standard curve. 1 U was defined as the amount of spores required to catalyze the degradation of 1 nM nicotine per minute in a 1×PBS system at 37°C. The recombinant spores completely degraded nicotine in the system within 20 min, and the specific activity of the nicotine-degrading enzyme was calculated to be 134 U / mg.

[0222] (3) Screening of freeze-drying protectants

[0223] To facilitate storage and use, the purified spores were further prepared into lyophilized powder. To avoid enzyme activity loss during vacuum freeze-drying, the effects of using dextran-40 (2, 4, 6, 8, 10%), glycerol (1, 2, 3, 4, 5%), mannitol (2, 4, 6, 8, 10%), glucose (2, 4, 6, 8, 10%), Tween-80 (1, 2, 3, 4, 5%), sucrose (1, 2, 3, 4, 5%), lactose (1, 2, 3, 4, 5%), and sorbitol (2, 4, 6, 8, 10%) as freeze-drying protectants were investigated. Glycerol and Tween-80, being oily, could not be freeze-dried, and their overall activity protection effect was less than 50%. Mannitol showed better excipient effect and better activity preservation, while other sugars resulted in higher viscosity after freeze-drying. Considering activity retention, excipient effect, and cost, mannitol at a concentration of 2% (20 mg / mL) was selected as the single lyophilization protectant. Furthermore, using Design-Expert 10.0.3 software for Plackett-Burman design and steepest climb experimental design, the combined protectant usage scheme is provided in Table 4. Detailed activity assay results are available in [link to table]. Figure 36 Meanwhile, the use of 10% whey protein (Shanghai Yuanye Bioreactor Co., Ltd.) was also investigated to improve the survival rate of recombinant bacteria in the acidic gastric environment.

[0224] Table 4. Experimental Design and Results for the Steepest Climb

[0225]

[0226] a: Spores that have not been freeze-dried

[0227] (4) Investigation of the tolerance of recombinant spores to trypsin and pepsin

[0228] Wild-type and recombinant spores were treated with simulated intestinal fluid (SIF-II, containing enzymes and bile salts) and simulated gastric fluid (SGF-II, containing enzymes), respectively. Enzyme activity was measured, and spore germination was observed by plating. Specifically, 3 mg dry weight spores were resuspended in 500 μL of SGF-II and SIF-II and treated for 2 h and 6 h, respectively. Then, 10 μL of an EDTA-free protease inhibitor (InStab) was added. TM After mixing the Protease Cocktail (10 mL / tablet) for 10 min, centrifuge at 5000 rpm for 5 min at room temperature to remove liquid. Wash three times with sterile water until the pH returns to neutral. Resuspend in 500 μL 1×PBS (pH 7.4), add 10 μL 400 μM nicotine, and react for 20 min. Take 100 μL of the reaction solution and terminate the reaction with an equal volume of methanol. Analyze by LC-MS / MS. Simultaneously, take the reaction solution, serially dilute it, and plate it on antibiotic resistance plates to determine the germination of spores treated with SIF-II and SGF-II. Use an equal amount of untreated spores as a control. Figure 37 As shown, the germination rate of recombinant spores was not affected after treatment with simulated body fluids. Even after extending the SIF-II treatment time by 24 hours, the degradation activity remained above 60%, indicating that the recombinant spores prepared in this invention are suitable for oral administration and the gastrointestinal environment.

[0229] Example 10: Genetic Stability

[0230] (1) Determination of the lag phase and generation time of the strain

[0231] Taking BS168::EN as an example, the generation time of the strain was determined. Specifically, glycerol bacteria frozen at -20℃ were inoculated at a 1% inoculum into resistant LB liquid or 2×YT medium and allowed to recover overnight. Then, a 1% inoculum was added to fresh liquid medium and cultured at 37℃ with shaking at 220 rpm. The inoculation time was set to 0 h. At 0, 2, 4, 6, 8, 10, 20, 22, 24, and 26 h after inoculation, 100 μL of spore suspension was collected and serially diluted to CFU = 10. -6 Spread 100 μL of the diluted solution onto a resistance plate and incubate at 37°C for 12 h until single colonies grow on the plate. According to the formula... Where ai is the plate colony count (CFU), v is the plating volume, d is the dilution factor, and m is the number of replicates, the bacterial count per unit volume (CFU / mL) is calculated. Then, a growth curve is plotted with the logarithm of the bacterial count as the ordinate and the incubation time as the abscissa. After determining the lag phase from the curve, a point is randomly selected from the logarithmic growth phase of the growth curve, and the generation time is determined using the formula G = (t - t1) * ln2 / (lnx - x0), where G is the generation time, t1 is the lag phase, t is the incubation time, x is the bacterial count at the incubation time point, and x0 is the initial bacterial count. Figure 38 As shown, the growth lag phase of Bacillus subtilis is 2 hours, and the calculated generation time is 0.75 hours, meaning that it takes 7.5 hours to grow 10 generations.

[0232] (2) Determination of genetic stability

[0233] After determining the generation time, the genetic stability of the recombinant bacteria was further investigated. Taking BS168::EN as an example, utilizing the characteristics of the chloramphenicol resistance gene, the activated bacterial culture was inoculated at 1% and cultured in fresh chloramphenicol-containing and chloramphenicol-free liquid media, respectively. The culture was transferred to fresh media midway through the process and passaged up to the 100th generation. Genetic stability was tested every 10 generations. Specifically, at generations 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100, 100 μL of the bacterial culture from both antibiotic-containing and antibiotic-free cultures was serially diluted to CFU = 10. -6 Subsequently, 100 μL was spread onto antibiotic-free solid culture dishes and incubated at 37°C for 12–16 h before plate printing. Approximately 30 single clones were randomly selected and streaked onto antibiotic-containing plates, and cultured for another 10–12 h before the growth of the strains on the antibiotic-resistant plates was recorded. The results are shown in Table 5. In the randomly selected single clones, even after 100 consecutive passages without chloramphenicol selection pressure, their genetic stability remained at 100%, indicating that the recombinant strains after genome integration have excellent genetic stability and are unlikely to lose the target gene during continuous passages.

[0234] Table 5. Results of genetic stability assay for recombinant spores

[0235]

[0236] Example 11: Nicotine toxicity and the detoxifying effect of recombinant spores

[0237] Although many studies suggest that organ damage caused by smoking is due to other harmful substances in inhaled tobacco, such as tar, the research by Jiang Changtao et al. (Nature. 2022; 610(7932): 562-568.) shows that nicotine accumulation during smoking exacerbates the progression of non-alcoholic fatty liver disease, and nicotine itself is considered a highly toxic substance. Therefore, in order to observe the toxicity of nicotine and the detoxification effect of recombinant spores in an animal model, a mouse nicotine injection model was used for verification. The groups were as follows: (a) 0.1% sodium saccharin aqueous solution group (blank control group), (b) wild spore lyophilized powder (10⁹ CFU / mL) and nicotine group, (c) recombinant spore lyophilized powder (10⁷ CFU / mL) and nicotine group (low concentration group), (d) recombinant spore lyophilized powder (10⁹ CFU / mL) and nicotine group (high concentration group), and (e) nicotine group. Six C57 mice were used in each group. The lyophilized spore powder was resuspended in 0.1% sodium saccharin solution and administered to each mouse by gavage (200 μL). Specifically, the experiment began after a week of acclimatization. For the first two days, each group was administered 200 μL of 0.1% sodium saccharin solution by gavage daily. Group (a) was administered 0.1% sodium saccharin solution by gavage daily. Groups (b), (c), and (d) were administered spore powder by gavage on the third day.

[0238] Thirty minutes later, a 0.5 mg / kg nicotine solution was injected intraperitoneally; group (d) received only the same amount of nicotine. The daily weight changes of each group were recorded for 5 consecutive days. On day 8, mice were euthanized after cardiac puncture and blood was collected. Serum was separated from the blood for biochemical analysis. After blood collection, mice were euthanized, and the heart, liver, spleen, lungs, kidneys, and brain tissues were separated, photographed, and the spleen, liver, and kidneys were weighed. The colon and small intestine were separated, photographed, and their lengths were measured. All tissues were immersed in a tissue fixative (4% paraformaldehyde). In the study, paraffin sections and H&E staining were performed. Sections were scanned using a slide scanner and pannoramic scanner software, and the scan results were observed using CaseViewer 2.4 viewing software.

[0239] Result: As Figure 39 and Figure 40As shown, compared with group (a), the pure nicotine treatment group (e) and the wild-type spore freeze-dried powder and nicotine group (b) showed significant weight loss during treatment, while the weight of mice in the recombinant spore gavage group remained basically normal, and the weight of mice in the low-concentration recombinant spore group (c) decreased slightly. Dissection of the euthanized mice revealed splenomegaly in groups (b) and (e), with pale livers and visible spots and fibrotic lesions on the surface. Further histological staining showed significant cell shrinkage and inflammatory cell infiltration in the liver and spleen of groups (b) and (e), with clusters of reduced cell nuclei in the spleen. Groups (c) and (d) showed protective effects; group (d) was essentially the same as the blank control group, while group (c) showed weak protective effects but no significant impact on other organs. Blood biochemistry tests also showed significantly elevated AST and ALT levels in groups (b) and (e). The above results indicate that long-term intake of high concentrations of nicotine causes irreversible damage to the liver and spleen. Oral administration of recombinant spores displaying nicotine-degrading enzymes can play a protective role in the body and eliminate the toxicity produced by nicotine. Moreover, the higher the dose of oral recombinant spores, the more obvious the effect, which is positively correlated with the amount of enzymes displayed.

Claims

1. An oral engineered microbe system, characterized in that, The engineered spores were obtained through a Bacillus subtilis display system; Bacillus subtilis was used as the chassis, and the Bacillus subtilis was modified into a germination-deficient and resistance-self-eliminating recombinant Bacillus subtilis; one or more spore capsid proteins among CgeA, CotB, CotE, CotG, CotY and CotZ were used as anchoring proteins to display the target protein and its combination or fusion; the integration site of the target protein or enzyme was amyE, lacA, pyrD, gerK operon or prpE.

2. The oral engineered microbial system according to claim 1, characterized in that, The capsid protein was amplified using the genome of Bacillus subtilis WB800n or B. subtilis 168 strains as templates. The target protein and capsid protein were fused together using the Escherichia coli-Bacillus subtilis shuttle integration plasmid pDG364 to construct a Bacillus subtilis recombinant plasmid. The amyE homologous arm was replaced with the upstream and downstream homologous arms of gerKA-gerKB-gerKC. A germination defect was formed by integration and knockout at this site. The resistance gene was seamlessly eliminated using the Xer / dif resistance self-elimination system in Bacillus subtilis. The recombinant Bacillus subtilis was transformed into competent cells to form Bacillus subtilis recombinant bacteria, which formed engineered spores under nutrient deprivation and conditional stress.

3. The oral engineered microbial system according to claim 2, characterized in that, Bacillus subtilis' own Xer recombinase specifically recognizes the dif sequence as described in SEQ ID NO:

2.

4. The oral engineered microbial system according to claim 2, characterized in that, Nutrient deficiency was caused by a modified DSM spore-forming medium, which was prepared as follows: 5g peptone, 3g yeast extract, 1g potassium chloride, and brought to a final volume of 1L; after moist heat sterilization, 1mL of filtered and sterilized 1M MgSO4, 1mL of 10mM MnCl4, 1mL of 1mM FeSO4, and 0.5mL of 1MCaCl2 solution were added.

5. The oral engineered microbial system according to claim 1, characterized in that, Recombinant B. subtilis168 was inoculated at 0.1% in sterile 2×YT medium at 37°C for 12–24 h to activate it. Then, it was inoculated at 1%–2% in DSM spore-forming medium. After spore-forming culture for 24–96 h, the spores were collected by centrifugation at 8000–12000 rpm and 4°C for 10–15 min. The spores were then washed three times with sterile 1×PBS, pH 7.4, to obtain the spore precipitate.

6. The oral engineered microbial system according to claim 5, characterized in that, The obtained spore precipitate was freeze-dried in the presence of a freeze-drying protectant to prepare spore freeze-dried powder.

7. The oral engineered microbial system according to claim 6, characterized in that, The freeze-drying temperature is -80 to -50℃, and the time is 12 to 24 hours.

8. The oral engineered microbial system according to claim 6, characterized in that, Use either a compound or a single lyophilization protectant; the compound lyophilization protectant is 6-3-3% wt / vol mannitol-lactose-sorbitol, or the single lyophilization protectant is mannitol and whey protein.

9. The oral engineered microbial system according to any one of claims 1-8, characterized in that, Engineered spores are loaded with target proteins and combinations or fusions thereof, wherein the target protein can be a degradation or destructive enzyme involved in the following application scenarios: (1) Nervous system: Anxiety and depression caused by elevated serotonin; cognitive and motor coordination dysfunction caused by abnormal γ-aminobutyric acid; mental abnormalities caused by substances of psychological dependence such as methamphetamine, ketamine, ecstasy, opium, morphine, marijuana, heroin, cocaine, cannabinoids, flunitrazepam, and fentanyl. (2) Cardiovascular system: hypertension caused by elevated renin; atherosclerosis caused by elevated cholesterol and triglycerides; thrombosis and atherosclerosis caused by high concentrations of homocysteine; (3) Metabolic system: diabetes and cataracts caused by elevated glucose levels; organ damage caused by elevated urea concentration; organ damage caused by elevated creatinine concentration; gout, hyperuricemia and organ damage caused by elevated uric acid concentration; (4) Endocrine system: Cushing's syndrome caused by elevated cortisol and glucocorticoids; refractory hypertension caused by elevated aldosterone; (5) Digestive system: Elevated bilirubin levels can lead to gallstones, hepatitis, and pancreatic cancer; (6) Reproductive system: Elevated androgen levels lead to polycystic ovary syndrome, infertility, and hirsutism in women; elevated estrogen levels lead to decreased sexual function and breast development in men. (7) Exogenous substances: Acute gastritis, alcoholic liver disease, pancreatitis, alcohol addiction, Alzheimer's disease, alcoholic heart failure, arrhythmia, hypertension, liver cancer, colorectal cancer, breast cancer, sexual dysfunction, and infertility caused by high ethanol intake; liver cancer, liver failure, growth retardation in children, and immunosuppression caused by aflatoxin intake.

10. A nicotine-degrading enzyme and its mutants, characterized in that, The target protein is a nicotine-degrading enzyme and its mutants; the nicotine-degrading enzyme is Nox derived from the strain Stutzerimonas stutzeri.

11. The nicotine-degrading enzyme and its mutant according to claim 10, characterized in that, The variant sequence comprises at least one substitution of an amino acid selected from positions 68, 86, 103, 126, 152, 184, 201, 217, 238, 245, 322, 347, 351, 364, 375, 383, 396, 398, 428, 433, 452, and 458 of SEQ ID NO:1; wherein basic amino acids include Lys, His, and Arg; acidic amino acids include Asp and Glu; nonpolar hydrophobic amino acids include Phe, Leu, Ile, Trp, Pro, Val, Met, and Ala; and polar and uncharged amino acids include Cys, Asn, Gly, Ser, Gln, Tyr, Thr, and Ser.

12. The nicotine-degrading enzyme and its mutants according to claim 11, characterized in that, The sequences of the variants include the N-terminal truncated form Δ46 based on SEQ ID NO:1, and single-site or multi-site substitutions of K184H, K184F, K184V, K184T, K184Y, F351H, Q330I, Q330T, K322V&Q330I, K322V&Q330T, R201H, R201F, R201V, R201S, R201T, R201Y, R347H, R347F, R347V, R347S, R347T, R347Y, K398H, K398F, K398V, K398S, K398T, K398Y, and R4. 33F, R433S, R433Y, C68L, G86D, G152W, E217N, Y238W, E245L, E245T, S375C, T383L, I396L, V428P, S452A, N458F, Q330I&R347S, R 347S&N458F, Q330I&R347S&R433S, Q330I&R347S&N458F, Q330I&R347S&D126S, Q330I&R347S&H364R, G86D&Q330I&R347S&R433S.

13. The oral engineered microbial system according to claim 1, characterized in that, The microorganisms mentioned include lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus curvilinearus, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus brusensis, Lactobacillus cellobiose, and Lactobacillus deutschlandiae subsp. bulgaricus; bifidobacteria such as Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium brevis, Bifidobacterium animalis subsp. lactis, and Bifidobacterium adolescentis; Gram-positive cocci such as Enterococcus faecalis and Streptococcus thermophilus; and Bacillus licheniformis, Saccharomyces boulardii, Saccharomyces cerevisiae, and Clostridium butyricum, as well as other edible strains such as Escherichia coli Nissle 1917, Lactobacillus styracifolius, Lactobacillus sakei, Pediococcus lactis, and Pediococcus pentosaceus.

14. The oral engineered microbial system according to claims 1-9, characterized in that, The target protein is the nicotine-degrading enzyme and its mutants as described in claims 10-12.

15. The oral engineered microbial system according to claim 13, characterized in that, The target protein is a Nox mutant and manganese catalase MnCA from the thermophilic bacterium Anoxybacillaceae, which are linked by a linker to form the Nox-MnCAT fusion protein.

16. The oral engineered microbial system according to claim 14, characterized in that, The natural promoter of the capsid protein was replaced with the strong constitutive promoter p43 used in Bacillus subtilis.

17. The use of the oral engineered microbial system according to any one of claims 1–8 in the preparation of oral drug carriers.

18. The use of the nicotine-degrading enzymes and their mutants according to claims 10-12, and the oral engineered microbial system according to claims 13-15 in the preparation of nicotine-dependent or smoking cessation drugs.

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