Application of engineered clostridium butyricum for producing tryptophan in delaying senescence and improving metabolism

Abstract

The invention relates to application of engineered clostridium butyricum for producing tryptophan in delaying senescence and improving metabolism, and belongs to the technical field of biomedical engineering. The invention discloses an application of engineered clostridium butyricum for expressing tryptophan biosynthetase trpEDCBA in preparation of drugs for delaying senescence, improving metabolism and increasing intestinal beneficial flora. An aging mouse model is constructed, it is found that after the aged mouse orally takes the engineered clostridium butyricum, the muscle strength, the blood glucose homeostasis, the cell metabolism, the cognitive ability and other aspects are remarkably improved, the improvement effect of the engineered clostridium butyricum is better than that of direct taking of butyric acid and tryptophan, the tryptophan-producing clostridium butyricum can be colonized in intestinal tracts for a long time, and the tryptophan-producing effect of the engineered clostridium butyricum is improved. The abundance of effective microbial communities in intestinal tracts is improved, a new tool and means are provided for anti-aging, and good application prospects are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to the application of engineered butyric acid bacteria that produce tryptophan in the preparation of anti-aging drugs. Background Technology

[0002] Aging is a complex physiological process that occurs in organisms over time, manifested as cellular functional decline, tissue and organ structural damage and dysfunction, leading to increased susceptibility to disease. Modern medicine views aging as the result of multiple factors, including oxidative stress damage, accumulation of gene mutations, protein homeostasis imbalance, telomere shortening, exacerbation of chronic inflammation, and cellular metabolic disorders. Currently, the world faces the challenge of an aging population, making delaying aging and improving the quality of life for the elderly a focus in the health field. Anti-aging research is gaining momentum, and many cutting-edge technologies such as cell reprogramming, gene editing, and mitochondrial function regulation are highly anticipated. The tryptophan metabolic pathway is related to the occurrence and development of various diseases, playing a key role in regulating the body's immunity, nerve signal transduction, and redox balance. Its tryptophan metabolites, such as kynurenine and serotonin, can participate in regulating multiple signaling pathways and physiological processes, such as affecting cellular energy metabolism and the aging process by regulating NAD+ levels, thus becoming an important target for aging intervention.

[0003] Probiotics hold immense potential in improving gut health and enhancing immune function, opening new avenues for anti-aging research. Some probiotics indirectly influence the host's aging process by regulating gut microbiota balance, promoting nutrient absorption, and secreting bioactive molecules. For example, Bifidobacteria can inhibit the growth of harmful bacteria and reduce the production of inflammatory factors; lactic acid bacteria, through fermentation, produce short-chain fatty acids, regulating intestinal epithelial cell function and enhancing intestinal barrier integrity. However, current probiotic applications in anti-aging still have limitations: firstly, most probiotics have low survival rates under the influence of gastric acid and bile, making it difficult to stably colonize the gut; secondly, the types and quantities of their secreted metabolites are limited, making it difficult to precisely meet the diverse needs of anti-aging; and thirdly, different individuals respond significantly differently to probiotics, constrained by multiple factors such as host genes, dietary habits, and the gut microenvironment, resulting in a lack of universality in the anti-aging effects of probiotics. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of engineered Clostridium butyricum expressing tryptophan biosynthetic enzyme trpEDCBA in the preparation of drugs for delaying aging, improving metabolism, and increasing beneficial gut flora. By constructing an aging mouse model, the significant effects of engineered Clostridium butyricum on muscle strength, blood glucose homeostasis, cell metabolism, and cognitive ability were verified.

[0005] The first objective of this invention is to provide the use of engineered butyric acid bacteria that produce tryptophan in the preparation of drugs for delaying aging and / or improving metabolism, characterized in that: the engineered butyric acid bacteria express tryptophan biosynthetic enzyme.

[0006] Clostridium butyricum (CB) is a Gram-positive, strictly anaerobic, spore-forming probiotic widely distributed in the intestines of humans and animals, and used in fermentation industries, livestock farming, food processing, and healthcare. Its unique anaerobic properties allow it to survive in the low-oxygen microenvironment of the intestine, and its spore structure ensures a high survival rate in gastric acid and bile, guaranteeing the successful arrival and function of the bacterial flora in the intestine. The short-chain fatty acid butyrate secreted by Clostridium butyricum is a preferred energy source for intestinal epithelial cells, and can repair the intestinal barrier, inhibit inflammatory factors, and regulate immune cell activity, thereby indirectly delaying aging.

[0007] Furthermore, the engineered Clostridium butyricum uses the strong promoter Pthl to express the tryptophan biosynthetic enzyme trpEDCBA.

[0008] Furthermore, the drug is administered orally.

[0009] In one embodiment of the present invention, the amount of Clostridium butyricum in the drug is 5 × 10⁻⁶. 8 CFU / 200μL PBS.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage form of the drug is tablets, capsules, granules, pills, suspensions, dispersants, or syrups.

[0012] A second objective of the present invention is to provide a medicament for delaying aging and / or improving metabolism, the medicament comprising engineered Clostridium butyricum expressing tryptophan biosynthetic enzyme trpEDCBA.

[0013] Furthermore, the drug is administered orally.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage form of the drug is tablets, capsules, granules, pills, suspensions, dispersants, or syrups.

[0016] Furthermore, the drug has at least one of the following effects:

[0017] (1) Improve blood oxygen saturation;

[0018] (2) Maintaining blood glucose homeostasis;

[0019] (3) Increase serum NAD+ levels;

[0020] (4) Increase muscle strength;

[0021] (5) Enhance cognitive abilities.

[0022] A third objective of this invention is to provide the use of engineered butyric acid bacteria that produce tryptophan in the preparation of drugs that increase the beneficial gut flora, wherein the engineered butyric acid bacteria express the tryptophan biosynthetic enzyme trpEDCBA.

[0023] A fourth object of the present invention is to provide a medicament for increasing beneficial gut flora, the medicament comprising engineered Clostridium butyricum, wherein the engineered Clostridium butyricum expresses the tryptophan biosynthetic enzyme trpEDCBA.

[0024] Furthermore, the beneficial bacteria include *Pseudomonas gondii*, *Prevotella*, butyrate-producing bacteria, or acetic acid-producing bacteria.

[0025] Furthermore, the drug is administered orally.

[0026] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0027] Furthermore, the dosage form of the drug is tablets, capsules, granules, pills, suspensions, dispersants, or syrups.

[0028] The beneficial effects of this invention are:

[0029] The engineered Clostridium butyricum provided by this invention significantly enhances the bacterial capacity to synthesize tryptophan by efficiently expressing trpEDCBA, a key enzyme in the tryptophan biosynthesis pathway. After oral administration, this engineered bacterium colonizes the intestine and continuously produces tryptophan and butyrate, exerting multiple beneficial physiological effects directly or indirectly: effectively delaying the aging process; significantly improving the host's metabolic state, specifically by increasing blood oxygen saturation, maintaining blood glucose homeostasis, increasing serum NAD+ levels, enhancing muscle strength, and improving cognitive abilities; simultaneously, the tryptophan and butyrate produced by this engineered bacterium selectively promote the growth and proliferation of beneficial bacteria in the intestine, such as *Pseudomonas gondii*, *Prevotella*, butyrate-producing bacteria, and acetic acid-producing bacteria, optimizing the intestinal microecological structure. This further enhances the overall efficacy of delaying aging and improving metabolism through the microbiota-host interaction mechanism, providing an effective tool for developing novel microbial therapies for related diseases. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0031] Figure 1 This is a schematic diagram of the engineered Clostridium butyricum strain that secretes tryptophan in Example 1 of the present invention;

[0032] Figure 2 This is a statistical chart showing the tryptophan and butyric acid content secreted by engineered Clostridium butyricum BT in Example 1 of the present invention;

[0033] Figure 3 These are representative images of aged mice and engineered Clostridium butyricum BT-treated mice in Example 2 of this invention;

[0034] Figure 4 This is a statistical chart of the frailty index scores of mice in each group in Example 2 of the present invention;

[0035] Figure 5 This is a statistical chart of blood oxygen saturation levels in each group of mice in Example 2 of the present invention;

[0036] Figure 6 This is a statistical chart showing the body weight, food intake, and water consumption of mice in each group in Example 2 of the present invention;

[0037] Figure 7 This is a statistical chart of blood glucose concentrations in mice of each group during weeks 0-4 in Example 2 of the present invention;

[0038] Figure 8 The graphs show the time curves of blood glucose concentration changes and the statistical graphs of the area under the AUC curves for each group of mice in Example 2 of this invention.

[0039] Figure 9 This is a statistical chart of serum NAD+ levels in mice in each group in Example 2 of the present invention;

[0040] Figure 10 This is a statistical chart of the suspension time of mice in each group in Example 3 of the present invention;

[0041] Figure 11 This is a statistical analysis chart showing representative images of mice in each group during the O-type maze experiment in Example 3 of the present invention and the time spent in the open area.

[0042] Figure 12 This is a statistical chart showing the spontaneous alternation rate of mice in each group during the Y-maze experiment in Example 3 of this invention.

[0043] Figure 13 This is a diagram showing the relative abundance of gut bacteria in phylum and class units in the gut microbiome analysis of Example 4 of the present invention;

[0044] Figure 14 This is a relative abundance diagram of gut microbiota analysis in Example 4 of the present invention;

[0045] Figure 15This is a statistical chart showing the relative abundance of specific bacterial strains in the intestinal microbial analysis of Example 4 of the present invention. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0047] C57BL / 6 mice aged 18-20 months were purchased from the Experimental Animal Center of Soochow University. All animal experiments were conducted in accordance with the animal experimental protocols approved by the Experimental Animal Center of Soochow University and met relevant ethical standards.

[0048] Example 1: Engineered Clostridium butyricum synthesizes and secretes tryptophan and butyric acid

[0049] First, the strong promoter Pthl was cloned and ligated into the Clostridium shuttle vector pMTL82151 to obtain the plasmid pMTL82151-thl. Then, the structural gene sequence encoding tryptophan biosynthetic enzymes (trpE, D, C, B, A) was cloned and amplified. The amplified product was double-digested with pMTL82151-thl, and the trpEDCBA gene was ligated into the plasmid pMTL82151-thl to prepare the expression plasmid pMTL82151-trpEDCBA. This plasmid was then heat-shocked into *E. coli*. Subsequently, the overexpressed recombinant plasmid, through conjugated transfer shuttle characteristics, yielded a *C. butyricum* strain carrying the trpEDCBA enzyme gene. Next, antibiotic screening culture was performed, and single colonies were selected for further culture to successfully prepare and preserve the engineered bacteria. The plasmid structure and strain preparation steps are as follows: Figure 1 As shown. Detailed construction and characterization methods of engineered Clostridium butyricum (BT) have been disclosed in Chinese Patent CN118792231A, "An engineered Clostridium butyricum producing L-tryptophan and its application in the preparation of tumor therapeutic drugs."

[0050] The yields of tryptophan and butyric acid secreted by engineered Clostridium butyricum were repeatedly determined using high-performance liquid chromatography (HPLC; Thermo, UltiMate 3000). Clostridium butyricum CB and engineered Clostridium butyricum BT were fermented in thioglycolate medium containing tryptophan (approximately 0.7 mM). The results are as follows: Figure 2 As shown, after 24 hours, engineered Clostridium butyricum BT still secreted more tryptophan, reaching approximately 1.10 mM, while Clostridium butyricum CB secreted only 0.6 mM of tryptophan. The butyric acid content secreted by Clostridium butyricum itself did not differ significantly, with yields generally around 30 mM.

[0051] Example 2: Evaluation of the effects of oral administration of engineered Clostridium butyricum on health and metabolic indicators in aged mice

[0052] (1) Assessment of the health status of aged mice

[0053] First, aged mice were given daily oral administration of engineered Clostridium butyricum (BT). The engineered bacteria were cultured to the early logarithmic growth phase before administration, with each mouse receiving 5 × 10⁻⁶ engineered bacteria. 8 The experiment was conducted using CFU / 200μL PBS over a period of 4 weeks. Results are as follows... Figure 3 As shown, aged mice treated with engineered Clostridium butyricum appeared healthier than age-matched control mice. The aged mice exhibited graying fur and hair loss, but oral administration of engineered Clostridium butyricum significantly reduced hair loss in the aged mice. This indicates that the continuously secreted tryptophan regulates the body's antioxidant capacity and improves overall physiological state; while the continuously secreted butyrate reduces the potential damage risk of inflammatory factors to hair follicles. These two anti-aging metabolites work synergistically to improve the color luster of the fur in aged mice. Based on previous clinical studies, a frailty index score was established to quantify the health status of aged mice (AClinical Frailty Index in Aging Mice: Comparinsons With Frailty Data in Humans, DOI:10.1093 / gerona / glt136). Using this objective index, we determined the frailty index score of aged mice treated with engineered Clostridium butyricum, and the results are as follows: Figure 4 As shown, using young mice as a control, the mean frailty index score of aged mice was significantly higher than that of young mice, and engineered Clostridium butyricum BT treatment significantly reduced the frailty index of aged mice. However, the CB (5×10) score in the oral Clostridium butyricum group was significantly lower. 8 Treatment with CFU / 200μL PBS or oral administration of tryptophan (10mg / kg, Sigma-Aldrich, USA) + butyric acid (500mg / kg, Beyotime, China) dissolved in 200μL PBS (volume ratio 1:1) did not result in a significant decrease.

[0054] The mice's blood oxygen saturation levels were then further measured using a small animal oxygenation monitor (mouse OX), and the results were as follows: Figure 5 As shown, the blood oxygen saturation level of the engineered Clostridium butyricum BT group was close to that of the young group, and significantly higher than that of the elderly group compared with the Clostridium butyricum group and the tryptophan + butyric acid group.

[0055] The body weight, food intake, and water consumption of mice in each treatment group were statistically analyzed, and the results are as follows: Figure 6As shown, there were no significant differences in body weight, food and water intake among the groups of aged mice, and the intake of food was increased slightly. This indicates that oral administration of engineered Clostridium butyricum BT does not affect the mice's eating or drinking, or cause a decrease in body weight.

[0056] (2) Assessment of metabolic indicators in aged mice

[0057] To investigate the assessment of metabolic indicators in aged mice, the changes in blood glucose concentration in the aged mice over four weeks were first detected using blood glucose test strips. Figure 7 As shown, blood glucose levels in aged mice were higher than in younger mice, while the blood glucose concentration in aged mice treated with engineered Clostridium butyricum (BT) at week 4 was significantly lower than that at week 1. Blood glucose concentrations in the CB group or the tryptophan + butyrate group did not show a significant decrease, indicating that the engineered bacteria have a unique advantage in regulating blood glucose through the continuous secretion of tryptophan and butyrate. The continuous secretion of these two metabolites can more efficiently maintain blood glucose homeostasis. Subsequently, a glucose tolerance test was used to assess glucose metabolism in aged mice, and the results are as follows... Figure 8 As shown, aged mice exhibited the highest peak blood glucose concentration with a slow decline, indicating the worst glucose tolerance. The AUC area of ​​the *Clostridium butyricum* CB group, the tryptophan + butyrate group, and the engineered *Clostridium butyricum* BT group was lower than that of the aged group and similar to that of the young group. This indicates that oral administration of engineered probiotics significantly improved glucose metabolism in aged mice, and oral administration of *Clostridium butyricum* or tryptophan + butyrate treatment also significantly enhanced glucose tolerance in aged mice.

[0058] NAD+ levels are crucial for maintaining cellular metabolism. Serum NAD+ levels in mice from different treatment groups were measured using an NAD+ detection kit. The results are as follows: Figure 9 As shown, compared to treatment with Clostridium butyricum alone or oral tryptophan + butyrate, the serum NAD+ level in mice in the engineered Clostridium butyricum group was significantly higher than that in the aged group, and similar to that in the young group. The synergistic effect of butyrate and tryptophan continuously secreted by engineered Clostridium butyricum significantly increased NAD+ levels, providing strong support for the maintenance of normal cellular metabolism and physiological functions. This indicates that the persistent secretion of butyrate and tryptophan by engineered Clostridium butyricum can significantly improve NAD+ levels in aged mice and has a positive effect on improving age-related metabolic functions.

[0059] Example 3: Evaluation of the effect of oral engineered Clostridium butyricum on the physical function of aged mice

[0060] (1) Assessment of muscle strength in aged mice

[0061] To assess the physical function of aged mice, muscle strength was evaluated by recording the duration of time the mice were suspended under a wire mesh. Figure 10The results showed that young mice had the longest suspension time, exceeding 120 seconds. The suspension time of aged mice was significantly shorter, indicating a decline in muscle strength. The suspension time of aged mice treated with engineered Clostridium butyricum was significantly longer than that of the Clostridium butyricum group and the tryptophan + butyrate group, demonstrating that oral administration of engineered Clostridium butyricum can improve muscle function in aged mice. This suggests that engineered bacteria that effectively secrete tryptophan and butyrate more comprehensively improve muscle physiology, thereby significantly enhancing muscle strength.

[0062] (2) Assessment of cognitive abilities in aged mice

[0063] To more systematically assess the physical function of aged mice, the Y-maze and O-maze tests were used to examine the short-term learning memory and autonomous exploration abilities of both young and aged mice. The O-maze is typically a circular open space, with half open and half closed areas. Mice were allowed to move around for 5 minutes, and the time spent in the open area was recorded to assess their exploratory behavior. The Y-maze consists of three arms of equal length, forming a Y shape. Mice were placed at the end of one arm and allowed to explore freely for 5 minutes. The order in which they entered the arms was recorded via video. A "spontaneous alternation" was counted when a mouse entered three different arms consecutively. The spontaneous alternation rate = number of alternations ÷ total number of arm entries × 100%, reflecting its short-term memory and spatial learning ability regarding the arm position just explored. Both tests were conducted under constant illumination and low noise conditions. The apparatus was cleaned with 75% ethanol before and after each round of experiments to eliminate odor interference and ensure that the obtained behavioral indicators reliably assess the mice's short-term learning memory and exploratory functions.

[0064] The results of the O maze test are as follows: Figure 11 As shown, older mice exhibited a significantly reduced time spent in the open area during the O maze test. Furthermore, the engineered Clostridium butyricum BT group significantly increased the time older mice spent in the open area during the O maze test compared to the Clostridium butyricum CB group and the tryptophan + butyric acid group.

[0065] The results of the Y-maze test are as follows: Figure 12 As shown, the autonomous selection ability of aged mice was significantly reduced, and the engineered Clostridium butyricum BT group enhanced the autonomous selection ability of aged mice more than the Clostridium butyricum CB group and the tryptophan + butyrate group. This may be because the engineered bacteria help maintain normal brain function and improve cognitive ability through the combined effect of continuously secreted tryptophan and butyrate. This indicates that oral administration of the engineered probiotic Clostridium butyricum significantly improves the cognitive function of aged mice.

[0066] Example 4: Oral administration of engineered Clostridium butyricum increases beneficial gut bacteria in aged mice

[0067] To investigate the gut microbiota of aged mice, fecal samples were collected from mice in each treatment group after 4 weeks of oral administration. These samples were then rapidly frozen in liquid nitrogen, followed by 16S rRNA gene sequencing analysis of the gut microbiota. Further analysis was performed comparing the gut bacteria before and after oral administration of engineered Clostridium butyricum. The relative abundance of the mouse gut microbiota is shown below. Figure 13 and 14 As shown, the abundance of Bacillus and Clostridium classes in Firmicutes increased, while the abundance of Bacteroidetes in Bacteroidetes decreased. Furthermore, oral administration of engineered Clostridium butyricum increased the abundance of gut microbiota.

[0068] Analysis of specific bacterial strains in the mouse gut yielded the following results: Figure 15 As shown, *Parabacteroides goldsteinii*, *Prevotella*, butyric acid-producing bacteria, and acetic acid-producing bacteria were significantly upregulated. These strains can produce short-chain fatty acids, reduce intestinal inflammation, resist bacterial infections, and have a positive effect on health. This indicates that engineered *Clostridium butyricum* creates an environment conducive to the growth of beneficial bacteria in the gut by secreting butyric acid, and its secreted tryptophan can further promote the proliferation of beneficial bacteria by regulating the redox state and nutrient composition of the intestinal microenvironment. The simultaneous and stable supply of both comprehensively meets the nutritional needs of beneficial bacteria in the gut, improving the intestinal flora structure.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of engineered butyric acid-producing Clostridium butyricum in the preparation of drugs for delaying aging and / or improving metabolism, characterized in that: The engineered Clostridium butyricum expresses tryptophan biosynthetic enzyme.

2. The application according to claim 1, characterized in that: The engineered Clostridium butyricum strain expresses the tryptophan biosynthetic enzyme trpEDCBA using the strong promoter Pthl.

3. The application according to claim 1, characterized in that: The drug is administered orally.

4. A drug for delaying aging and / or improving metabolism, characterized in that: The drug comprises engineered Clostridium butyricum that expresses tryptophan biosynthesis enzyme.

5. The drug according to claim 4, characterized in that: The drug is administered orally.

6. The drug according to claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 4, characterized in that, The drug has at least one of the following effects: (1) Improve blood oxygen saturation; (2) Maintaining blood glucose homeostasis; (3) Increase serum NAD+ levels; (4) Increase muscle strength; (5) Enhance cognitive abilities.

8. The application of engineered butyric acid-producing Clostridium butyricum in the preparation of drugs that increase beneficial intestinal flora, characterized in that: The engineered Clostridium butyricum expresses tryptophan biosynthetic enzyme.

9. A drug for increasing beneficial intestinal flora, characterized in that: The drug comprises engineered Clostridium butyricum, which expresses tryptophan biosynthetic enzyme.

10. The medicament according to claim 9, characterized in that: The beneficial bacteria include *Pseudomonas gossypii*, *Prevotella*, butyrate-producing bacteria, or acetic acid-producing bacteria.

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