Application of engineered citrobacter freundii in fermentation synthesis of stabilizers

Abstract

The invention discloses an application of engineered citrobacter freundii in fermentation synthesis of a stable body. The engineered citrobacter freundii is citrobacter freundii overexpressing a polyphosphate kinase gene Ppk1. The stable body prepared by fermenting the engineered citrobacter freundii is rich in polyphosphates and polyamines, particularly, the content of the polyamines can be up to 21wt%, the polyphosphates in the stable body can store the polyamines in an ionic bond combination mode, the combined polyamines are insoluble in water, a natural anion coating is generated, and the stability of the stable body is improved. And the sustained-release tablet is not easily taken by intestinal epithelial cells, so that the sustained-release tablet has a certain sustained-release effect. The medicine prepared by taking the compound as a main active ingredient shows a remarkable curative effect in ulcerative colitis treatment, has the comprehensive effects of reducing oxidative stress, enhancing the anti-inflammatory ability of a body and regulating immunity, is suitable for patients of all ages, and has outstanding application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to the application of engineered Citrobacter freundii in the fermentation synthesis of stable compounds. Background Technology

[0002] Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease, primarily encompassing Crohn's Disease (CD) and ulcerative colitis (UC). A core characteristic of this disease is the dysregulation of immune cells, particularly the abnormal activation of macrophages in the lamina propria, leading to persistent inflammation of the colonic tissue. Normally, anti-inflammatory macrophages (M2) in the colon maintain immune homeostasis and suppress excessive immune responses by secreting anti-inflammatory factors such as IL-10 and TGF-β. However, in IBD patients, the differentiation and activity of colonic macrophages change, with an increase in pro-inflammatory macrophages (M1) that secrete large amounts of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, thereby exacerbating the inflammatory response. Furthermore, damage to the intestinal barrier further intensifies the immune response, allowing inflammation to persist within the colonic tissue, ultimately leading to ulcers, intestinal fibrosis, and other complications. Polyamines are considered key regulators of colonic immune homeostasis during the development of colonic inflammation. Spermine and spermine in the colon not only promote the differentiation of anti-inflammatory macrophages M2 but also inhibit the activation of pro-inflammatory macrophages M1. Studies have found that in a mouse model of colitis, exogenous spermmine supplementation significantly reduced the expression of pro-inflammatory cytokines TNF-α and IL-1β and increased IL-10 levels, thereby improving the inflammatory state. In addition to alleviating inflammation, spermmine also maintains intestinal barrier integrity by activating non-receptor protein tyrosine phosphatase 2 (PTPN2). In the absence of PTPN2, the anti-inflammatory effect of spermmine is significantly weakened, indicating that PTPN2 is an important mediator for the function of spermmine.

[0003] The primary source of polyamines in the colon relies on the symbiotic metabolism of gut microbes, which synthesize polyamines from amino acids such as ornithine and arginine. However, in colitis, the microbial capacity for polyamine synthesis is impaired, leading to a decrease in endogenous polyamine levels in the gut. For example, in a mouse model of colitis, colonization of... E. coli Germ-free mice lacking polyamine synthase (SK930) exhibited decreased colonic barrier function and exacerbated inflammatory responses. This suggests that alterations in the gut microbiota may lead to a reduction in endogenous polyamine supply, thereby exacerbating IBD symptoms.

[0004] Although polyamines play a crucial role in regulating colonic inflammation, oral supplementation of polyamines faces significant challenges in practical applications. Due to the complex environment of the gastrointestinal tract, orally administered polyamines may be rapidly absorbed in the small intestine, preventing them from effectively reaching the colon to exert their effects. Therefore, how to accurately deliver polyamines to the colon has become a key research issue. Most current research focuses on using specific probiotics or prebiotics to indirectly influence the synthesis and metabolism of polyamines within host cells by modulating the gut microbiota. This approach utilizes polyamine-producing microorganisms in the gut microbiota, thereby regulating the host's endogenous polyamine levels by improving the microbiota structure and activity. However, a treatment method that directly adds spermidine is still lacking. Direct supplementation of spermidine may face several challenges, such as the stability of spermidine in the gastrointestinal tract, absorption efficiency, and bioavailability in vivo, all of which require further investigation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing the application of engineered Citrobacter freundii in the fermentation synthesis of stable compounds.

[0006] Another technical problem to be solved by the present invention is to provide a method for synthesizing stable organisms by fermentation using engineered Citrobacter freundii.

[0007] The technical problem that this invention also aims to solve is to provide a method for preparing a stable body using engineered Citrobacter freundii fermentation.

[0008] Another technical problem to be solved by the present invention is to provide the use of the stabilizer in the preparation of a medicament for treating ulcerative colitis.

[0009] The final technical problem to be solved by this invention is to provide a drug for treating ulcerative colitis.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0011] The first aspect of this invention provides the application of engineered Citrobacter freundii in the fermentation synthesis of stable compounds, wherein the engineered Citrobacter freundii is an overexpression of the polyphosphate kinase gene. Ppk1 Citrobacter freundii.

[0012] A second aspect of this invention provides a method for synthesizing stable organisms using engineered Citrobacter freundii fermentation, wherein the engineered Citrobacter freundii is an overexpressing polyphosphate kinase gene. Ppk1 Citrobacter freundii.

[0013] Preferably, engineered Citrobacter freundii is inoculated into a fermentation medium for fermentation culture to synthesize a stable organism.

[0014] Preferably, the engineered Citrobacter freundii uses Citrobacter freundii as a host and introduces the host's own polyphosphate kinase gene. Ppk1 .

[0015] More preferably, the *Citrobacter freundii* is *Citrobacter freundii* ATCC 8090, and the polyphosphate kinase gene... Ppk1 The GenBank number is ANAVO1000007.1.

[0016] Most preferably, the method for constructing the engineered Citrobacter freundii is disclosed in patent CN 104531599 A.

[0017] The fermentation medium comprises tryptone, NaCl, MgSO4, NH4Cl, KH2PO4 and yeast extract.

[0018] Preferably, the fermentation medium comprises 50-150 mg / L tryptone, 20-70 mg / L NaCl, 200-250 mg / L MgSO4·7H2O, 150-210 mg / L NH4Cl, 50-150 mg / L KH2PO4·3H2O, and 1-5 mg / L yeast extract.

[0019] Most preferably, the fermentation medium comprises 100 mg / L tryptone, 50 mg / L NaCl, 226 mg / L MgSO4·7H2O, 180 mg / L NH4Cl, 102 mg / L KH2PO4·3H2O, 1 mg / L yeast extract and 10 mg / L kanamycin.

[0020] The fermentation culture is carried out at a temperature of 32-42℃, a rotation speed of 200-250 rpm, and a time of 150-240 min.

[0021] Preferably, the inoculum size of the engineered Citrobacter freundii in the fermentation medium is controlled such that the initial OD of the engineered Citrobacter freundii in the fermentation medium is such that... 600 The value is 0.15 to 0.2.

[0022] The application of the engineered Citrobacter freundii in the fermentation synthesis of stabilizers includes the step of extracting stabilizers from the fermentation broth obtained from the fermentation culture.

[0023] Preferably, the step of extracting the stabilizer includes: separating the fermentation broth into solid and liquid components, collecting the cells, resuspending and breaking them, centrifuging, collecting the supernatant, ultrafiltration centrifugation, collecting the retentate, and thus obtaining the stabilizer.

[0024] Preferably, the solid-liquid separation is centrifugation at 6000-8000 g for 7-10 min; the liquid used for resuspension is a buffer solution, preferably HEPES buffer; the disruption is ultrasonic disruption at 200-400 W for 10-20 min under ice bath conditions; the centrifugation is centrifugation at 6000-8000 g for 7-10 min; the molecular weight cutoff of the ultrafiltration centrifuge tube used for ultrafiltration centrifugation is 80-120 kDa; the centrifugal force of the ultrafiltration centrifugation is 2000-6000 g, and the centrifugation time is 7-10 min.

[0025] The third aspect of the present invention provides a method for preparing a stable body by fermentation synthesis of a stable body using engineered Citrobacter freundii.

[0026] The stabilizer is rich in polyphosphates and polyamines, wherein the polyphosphate content is 40-50 wt% and the polyamine content is 20-25 wt%.

[0027] A fourth aspect of the present invention provides the use of the stabilizer in the preparation of a medicament for treating ulcerative colitis.

[0028] A fifth aspect of the present invention provides a medicament for treating ulcerative colitis, comprising the stabilizer.

[0029] A stabilizer is a subcellular structural unit containing polyP n Electron-dense phosphates (Pi), metal cations, proteins, amino acids, lipids, and water are widely present in most prokaryotes and some eukaryotes. Under an optical microscope, they appear as dense, black granules, hence sometimes called electron-dense organelles. These granules have diverse transmembrane proteins distributed along their membranes. Different biologists have used different names for this subcellular structure containing granular polyphosphates. For example, this structure, initially isolated from trypanosoma, is called an acidocalcisome; in algae, it is sometimes called a polyphosphate granule; other scholars refer to it as metachromatic granules, volutin granules, and polyphosphate vacuoles. The terminology used to define this organelle has evolved gradually with advancements in scientific techniques, deeper research, and a more profound understanding of it; it is not static. Based on the core function of this organelle in maintaining cellular homeostasis and adapting to the biological environment, this invention uniformly names it as a stabilizer.

[0030] Beneficial effects:

[0031] (1) The stabilizer prepared by the engineered Citrobacter freundii fermentation of this invention is rich in polyphosphates and polyamines, especially polyamines, which can reach up to 21 wt%, playing an important role in regulating colonic inflammation. In contrast, the polyamine content of general natural stabilizers (such as the stabilizer extracted from cyanobacteria in patent CN 119193407 A) is only 0.001 wt%. Drugs prepared using the stabilizer prepared by this invention as the main active ingredient have shown significant efficacy in the treatment of ulcerative colitis, possessing comprehensive effects of reducing oxidative stress, enhancing the body's anti-inflammatory capacity, and regulating immunity. Furthermore, this stabilizer has high safety, good biocompatibility, and excellent hydrophilicity and interfacial compatibility, effectively reducing immune rejection and inflammatory responses, further promoting cell adhesion, growth, and tissue repair. It is suitable for patients of all ages and has outstanding application value and market prospects.

[0032] (2) The stabilizer prepared in this invention is a nanoparticle with a small particle size and controllable release characteristics. The structure of its polyphosphate component is highly similar to the inorganic components of human mineralized tissue, exhibiting good biocompatibility. Furthermore, its degradation products are non-toxic, metabolizable phosphate ions that can be safely absorbed and utilized by the body. The polyphosphate in the stabilizer stores polyamines via ionic bonds. These bound polyamines are poorly soluble in water, forming a natural anionic coating, and are not easily taken up by intestinal epithelial cells, thus providing a sustained-release effect. Therefore, this stabilizer not only effectively resists the damage caused by extreme pH and digestive enzymes in the gastrointestinal tract, achieving precise targeted delivery of the active ingredient polyamines, but also enhances its stability and bioavailability in vivo, ensuring that sufficient polyamines reach the colon after oral administration. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 This is a scanning electron microscope image of the stable organism extracted from the bacterial cells in Example 1.

[0035] Figure 2 The cells containing stable bodies were observed using Albert staining in Example 1.

[0036] Figure 3 The bacterial cells containing stable bodies were observed by TEM in Example 1.

[0037] Figure 4 The pathological changes and histological scores of mouse colon tissue were analyzed using hematoxylin-eosin (H&E) staining in Example 2. Detailed Implementation

[0038] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0039] The tryptone, NaCl, MgSO4·7H2O, NH4Cl, KH2PO4·3H2O and yeast extract used in the following examples were all domestically produced analytical grade or biological grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0040] The engineered Citrobacter freundii used in the following examples is a genetically engineered bacterium constructed using the method disclosed in patent CN 104531599 A.

[0041] Example 1

[0042] Engineered Citrobacter freundii fermentation synthesis of stable compounds:

[0043] Engineered Citrobacter freundii was inoculated into the fermentation medium to control the initial OD of the engineered Citrobacter freundii in the fermentation medium. 600 The concentration was 0.15. The fermentation system was incubated at 37℃ and 200 rpm for 240 min. After fermentation, the fermentation broth was centrifuged at 7000 g for 10 min, and the bacterial cells were collected and resuspended in 5 times the volume of HEPES buffer (10 mmol / L, pH 7.2) to obtain a bacterial suspension. The suspension was placed in an ice bath and ultrasonically disrupted at 300 W for 10 min. After disruption, the suspension was centrifuged at 7000 g for 10 min. The supernatant was ultrafiltered using a 100 kDa molecular weight cutoff ultrafiltration centrifuge tube at 4000 g for 10 min. The stable phase was found in the retentate phase and observed under a scanning electron microscope. Figure 1 As shown, a stable body in the form of round granules can be observed.

[0044] Analysis of the components of the stabilizer prepared in this embodiment showed that its main components were polyphosphate (44.9 wt%), polyamine (20.6 wt%), protein (16.2 wt%), lipid (9.9 wt%), and metal ions (5.3 wt%). This is significantly different from the stabilizer extracted from cyanobacteria in patent CN 119193407 A, where polyphosphate accounted for 21.2 wt%, polyamine accounted for only 0.001 wt%, and protein was the main component, accounting for 65.7 wt%.

[0045] The engineered *Citrobacter freundii* cells obtained during the above extraction process were stained using a modified Albert staining method and observed under an oil immersion microscope. Figure 2As shown, the rod-shaped bacteria exhibited extremely prominent growth of "huge", nearly round, blue-purple stable particles, giving the bacteria a very unique "dumbbell" shape.

[0046] In addition, the engineered Citrobacter freundii cells were observed under a transmission electron microscope, such as... Figure 3 As shown, engineered Citrobacter freundii cells were found to have white, nearly circular particles at their extreme points, which indicates that the cells are stable.

[0047] The fermentation medium described above consists of the following components: 100 mg / L tryptone, 50 mg / L NaCl, 226 mg / L MgSO4·7H2O, 180 mg / L NH4Cl, 1 mg / L yeast extract, 10 mg / L kanamycin, and 102 mg / L KH2PO4·3H2O (to control the concentration of inorganic phosphorus (Pi) in the fermentation medium to 20 mg / L).

[0048] Example 2

[0049] Stabilizers improve colitis symptoms in mice.

[0050] C57BL / 6 mice are highly sensitive to inflammatory responses, making them suitable for studying immune-mediated intestinal inflammation. Acute colitis in mice can be induced by administering a 3% (30 g / L) sodium dextran sulfate (DSS) aqueous solution to free access to water for 7 consecutive days. The 30 g / L DSS solution effectively disrupts the intestinal epithelial barrier in C57BL / 6 mice, inducing colonic tissue damage, inflammatory cell infiltration, and elevated pro-inflammatory factors, thus mimicking the pathological characteristics of ulcerative colitis (UC).

[0051] This experiment used C57BL / 6 mice as the experimental model, and four treatment groups were set up: CTL group, stabilizer group (PPGs) group, PA group, and PBS group, with 6 C57BL / 6 mice in each group. The CTL group was administered 200 μL of PBS buffer (0.2 M, pH 7) by gavage daily, and the drinking water did not contain sodium dextran sulfate. The other three groups had 30 g / L sodium dextran sulfate (DSS) added to their drinking water to induce colitis, and the test substances were administered by gavage daily starting from day 3 of culture. Specifically, the stabilizer group was administered 200 μL of a 3 mM suspension of stabilizers prepared in Example 1 by gavage daily, the PA group was administered 200 μL of a 3 mM spermidine aqueous solution by gavage daily, and the PBS group was administered 200 μL of 0.2 M PBS buffer (0.2 M, pH 7) by gavage daily. Ten days after culture, colon tissues from each mouse were collected for hematoxylin-eosin (H&E) staining. The pathological changes and histological scores of the mouse colon tissues were analyzed (colon injury scores were assessed according to the histological scoring system and statistically analyzed).

[0052] Experimental results are as follows Figure 4 As shown in the figure. After 10 days of culture, histological analysis of the colon of mice in the CTL group showed normal tissue structure, while the PBS group showed typical inflammatory symptoms, including goblet cell and epithelial cell depletion, crypt structure destruction, and inflammatory cell infiltration in the lamina propria. The histological score of the stabilizer group was significantly lower than that of the PA and PBS groups, and only slightly higher than that of the CTL group. These results indicate that gavage treatment with stabilizers significantly reduced colonic tissue damage induced by DSS stimulation.

[0053] This invention provides a concept and method for the application of engineered Citrobacter freundii in the fermentation synthesis of stable compounds. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of engineered Citrobacter freundii in the fermentation synthesis of stable compounds, characterized in that, The engineered Citrobacter freundii is an overexpression of the polyphosphate kinase gene. Ppk1 Citrobacter freundii.

2. A method for synthesizing stable compounds using engineered Citrobacter freundii fermentation, characterized in that, The engineered Citrobacter freundii is an overexpression of the polyphosphate kinase gene. Ppk1 Citrobacter freundii.

3. The method according to claim 2, characterized in that, Engineered Citrobacter freundii was inoculated into a fermentation medium for fermentation culture to synthesize a stable organism.

4. The method according to claim 3, characterized in that, The fermentation medium comprises tryptone, NaCl, MgSO4, NH4Cl, KH2PO4, and yeast extract; preferably, the fermentation medium comprises 50-150 mg / L tryptone, 20-70 mg / L NaCl, 200-250 mg / L MgSO4·7H2O, 150-210 mg / L NH4Cl, 50-150 mg / L KH2PO4·3H2O, and 1-5 mg / L yeast extract.

5. The method according to claim 3, characterized in that, The fermentation culture is carried out at a temperature of 32-42℃, a rotation speed of 200-250 rpm, and a time of 150-240 min.

6. The method according to claim 3, characterized in that, The method includes the step of extracting stabilizers from the fermentation broth obtained from the fermentation culture; preferably, the step of extracting stabilizers from the fermentation broth obtained from the fermentation culture includes: separating the solid and liquid components of the fermentation broth, collecting the cells, resuspending and breaking them, centrifuging, collecting the supernatant, ultrafiltration centrifugation, collecting the retentate, and thus obtaining the stabilizers.

7. The method according to claim 6, characterized in that, The solid-liquid separation is performed by centrifugation at 6000-8000 g for 7-10 min; the disruption is performed by ultrasonic disruption at 200-400 W for 10-20 min under ice bath conditions; the centrifugation is performed by centrifugation at 6000-8000 g for 7-10 min; the molecular weight cutoff of the ultrafiltration centrifuge tubes used in the ultrafiltration centrifugation is 80-120 kDa; the centrifugal force of the ultrafiltration centrifugation is 2000-6000 g, and the centrifugation time is 7-10 min.

8. A stable body obtained by the method according to any one of claims 2 to 7.

9. The use of the stabilizer according to claim 8 in the preparation of a medicament for treating ulcerative colitis.

10. A drug for treating ulcerative colitis, characterized in that, Includes the stabilizer as described in claim 8.

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