Klebsiella pneumoniae and application thereof in production of indole-3-carboxylic acid

Abstract

The invention discloses klebsiella pneumoniae and application thereof in production of indole-3-carboxylic acid (I3CA), and belongs to the fields of microbial technology, biological catalysis and fermentation engineering. The preservation number of the klebsiella pneumoniae is CCTCC NO: M 20252762, the classification name of the klebsiella pneumoniae is Klebsiella pneumoniae I3CA-02, the strain can efficiently convert tryptophan into I3CA in a time-dependent mode through a one-step method, high sensitivity to various clinically common antibiotics is kept, and the klebsiella pneumoniae has good intestinal colonization ability and safety, and can be used for preparing I3CA. The method has the potential of being directly applied to oral micro-ecological preparations and industrial production. The technical bottlenecks that the existing I3CA biosynthesis efficiency is low and the safety of recombinant bacteria is limited are broken through, the microorganism sources of indole derivatives are widened, and a foundation is laid for green manufacturing of high-purity I3CA and development of intestinal-brain-immune axis related functional products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Klebsiella pneumoniae isolated from fresh fecal samples from healthy individuals, and its application in the biocatalytic conversion of tryptophan to indole-3-carboxylic acid (I3CA). Background Technology

[0002] Indole-3-carboxylic acid (I3CA) is the most stable indole derivative in the tryptophan metabolic pathway, with the molecular formula C9H7NO2. Recent studies have confirmed that I3CA, through weak agonist aryl hydrocarbon receptor (AhR) and activation of the PXR / CAR pathway, can inhibit the NLRP3 inflammasome, enhance the intestinal barrier, and alleviate neuroinflammation, and has been listed as a candidate functional molecule targeting the gut-brain-immune axis (Zelante et al., 2013; Rothhammer et al., 2018). However, the content of plant-derived I3CA is extremely low (<0.5 μg g). -1 DW), chemical synthesis requires high temperature and pressure and precious metal catalysis, which is costly and polluting, making it difficult to meet the urgent demand of pharmaceuticals and functional foods for high-purity, low-cost raw materials.

[0003] Microbial metabolism of tryptophan primarily occurs via three pathways: kynurenine, serotonin, or indole. The indole pathway begins with tryptophanase (TnaA) or aromatic transaminase, followed by deamination and oxidation to produce I3CA. Although *Escherichia coli*, lactic acid bacteria, and other microorganisms can produce indole-3-acetic acid (IAA), food-grade strains possessing "post-modified" dioxygenases or oxidases (ipdABC-like) are rare, resulting in I3CA yields generally below 5 mg / L. -1 A recent study increased I3CA levels to 78 mg / L by heterologous expression of the Pseudomonas ipd gene cluster in E. coli. -1 However, recombinant bacteria rely on IPTG and antibiotic labeling, which does not meet the safety standards for oral probiotics. Therefore, obtaining wild-type strains with natural I3CA synthesis capabilities, genetic stability, and tolerance to the gastrointestinal environment remains a technological gap in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Klebsiella pneumoniae and its application in the preparation of a strain capable of efficiently and in one step converting tryptophan to indole-3-carboxylic acid (I3CA).

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the present invention, a strain of Klebsiella pneumoniae is provided, with accession number CCTCC NO: M20252762.

[0006] In a second aspect of the invention, the use of the Klebsiella pneumoniae in the preparation of a product for converting tryptophan into I3CA is provided.

[0007] In a third aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising: The fermentation broth obtained by fermenting the aforementioned Klebsiella pneumoniae; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

[0008] In a fourth aspect of the invention, a product for converting tryptophan into I3CA is provided, the product comprising the aforementioned Klebsiella pneumoniae or the aforementioned fermentation agent.

[0009] Furthermore, the product for converting tryptophan into I3CA also includes pharmaceutically acceptable excipients.

[0010] The excipients include those required for preparation into oral dosage forms.

[0011] In a fifth aspect of the invention, a method for converting tryptophan to I3CA using the aforementioned Klebsiella pneumoniae is provided, the method comprising: I3CA was obtained by adding the Klebsiella pneumoniae or the fermentation agent to a culture medium containing tryptophan and fermenting it in vitro. Alternatively, I3CA can be obtained in vivo by gavage or oral administration of the aforementioned Klebsiella pneumoniae or the aforementioned fermentation agent.

[0012] Furthermore, the culture medium containing tryptophan is LB medium, with 400 mg / L-600 mg / L tryptophan added, and most preferably 500 mg / L tryptophan added.

[0013] Furthermore, the in vitro fermentation culture temperature is 36℃-38℃, the pH range is 6.5-7.5, and the optimal inoculum size is 5%-10%.

[0014] As a specific implementation method, the method for converting tryptophan into I3CA using the aforementioned Klebsiella pneumoniae specifically includes: The Klebsiella pneumoniae was inoculated into a test tube containing LB liquid medium and cultured overnight at 37 °C with shaking to activate it; Centrifuge the bacterial culture after overnight incubation, discard the supernatant, resuspend in sterile water, centrifuge again, discard the supernatant, and resuspend in sterile water; transfer the resuspended bacterial culture with OD600 to a triangular flask containing tryptophan-based LB medium and incubate on a shaker.

[0015] Furthermore, the gas chromatography-mass spectrometry (GC-MS) system is model G1540N, and the chromatographic column used is a TraceGOLDTG-5MS column (30m × 0.25μm). The gas flow rates are: high-purity nitrogen: 1.5 mL / min, hydrogen: 30 mL / min, and air: 300 mL / min. The injection port and detector temperatures are 280℃. The column temperature is: initial temperature 50℃ held for 2 min, increased to 230℃ at a rate of 40℃ / min, increased to 320℃ at a rate of 20℃ / min and held for 20 min. The injection volume is 1 μL. The detector temperature is 325℃.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The present invention provides a strain of Klebsiella pneumoniae and its application, which isolates a bacterium capable of converting tryptophan into I3CA from fresh fecal samples of healthy individuals. This strain has been identified as Klebsiella pneumoniae and is capable of converting tryptophan into I3CA in a time-dependent manner.

[0017] 2. The Klebsiella pneumoniae strain provided by this invention has the ability to produce I3CA, which broadens the production pathway and application field of I3CA.

[0018] The Klebsiella pneumoniae strain of this invention was deposited on December 3, 2025, with accession number CCTCC NO: M20252762. Its classification and naming... Klebsiella pneumoniae I3CA-02 ， The institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, China, 430072, China. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Image of Klebsiella pneumoniae I3CA-02 isolated in Example 1; Figure 2 This is the phylogenetic tree of Klebsiella pneumoniae I3CA-02; Figure 3 The detection results for I3CA production by Klebsiella pneumoniae I3CA-02 isolated in Example 3. Detailed Implementation

[0021] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0022] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0023] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0024] To solve the technical problem of this invention, the overall concept of this invention is as follows: Klebsiella pneumoniae is a facultative anaerobic, Gram-negative enterobacterium that can simultaneously utilize aromatic amino acids, oligosaccharides, and uronic acids. Approximately 42% of environmental / intestinal isolates carry the TnaA homolog in their genome, and some strains possess an ipdC-like fragment, suggesting a potential pathway for tryptophan-to-I3CA conversion. This bacterium is acid-tolerant (pH 4.0–9.5) and bile-tolerant (0.3% w / v), and its cell density can reach 150 g CDW L. -1 Furthermore, the Red / CRISPR-Cas12a editing system is mature, providing a feasible platform for food-grade modification. However, the function of K. pneumoniae in the targeted synthesis of I3CA from tryptophan has not been systematically reported.

[0025] This invention isolates strain KP-I3CA01 from fresh feces of healthy adults. This strain is free of multidrug resistance genes and classical virulence plasmids, and can directly synthesize I3CA from tryptophan without exogenous induction, with a shake-flask yield of 1.2 g / L. -1 A 5 L fermenter yielded 3.8 g / L. -1 With a molar conversion rate of >82%, significantly superior to existing wild-type strains, it provides a novel solution for developing gut-immune-nervous axis targeted microecological drugs and green I3CA production processes.

[0026] The following will provide a detailed description of a strain of Klebsiella pneumoniae and its application, in conjunction with embodiments and experimental data.

[0027] Example 1: Isolation, purification and identification of Klebsiella pneumoniae (1) Isolation of Klebsiella pneumoniae Based on the hypothesis that intestinal bacteria can convert tryptophan into I3CA through degradation, the inventors mixed 0.5 g of fresh fecal sample from healthy individuals with a selective culture medium (tryptophan as the sole carbon source). The medium was incubated in the dark on a rotary shaker at 37°C and 250 rpm. The medium was changed after three to four days of incubation. This process was repeated five times, and the culture suspension was then inoculated onto blood agar plates to isolate the bacterial strains. Figure 1 As shown.

[0028] (2) Identification of Klebsiella pneumoniae The isolated bacterial strains were inoculated into LB liquid medium containing 500 mg / L tryptophan and cultured at 37 °C, 200 rpm for 24 h on a shaker. The bacterial culture was then mixed with 30% glycerol at a 1:1 volume ratio and sent to MEGA Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of the obtained strain (as shown in SEQ ID NO.1) was analyzed by BLAST comparison in the NCBI database. Phylogenetic analysis of the strain and the type strain was performed using MEGA11 software, and a phylogenetic tree was constructed using the neighbor-linked method, as shown below. Figure 2 As shown; Based on physiological and biochemical characteristics, and identification by matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry, this bacterium was identified as *Klebsiella pneumoniae*, and we named it *Klebsiella pneumoniae*. It was deposited on December 3, 2025, at the China Center for Type Culture Collection (depository address: Wuhan University, Wuhan, China), and its classification name is... Klebsiella pneumoniae I3CA-02, accession number CCTCC M 20252762.

[0029] Example 2: Inoculum of Klebsiella pneumoniae I. Experimental Objective The study validated the practical application of this strain in the preparation of products that convert tryptophan to I3CA, covering both fermentation agents and oral formulations.

[0030] II. Experimental Materials and Methods 1. Strains and Fermentation Klebsiella pneumoniae I3CA-02 (accession number CCTCC M 20252762) was used as identified in Example 1.

[0031] 2. Preparation of fermentation broth The strain was inoculated into LB liquid medium (with 500 mg / L tryptophan added) and cultured at 37°C and 220 rpm for 72 hours to obtain the fermentation broth.

[0032] 3. Preparation of dry powder inoculant: The fermentation broth is spray-dried (inlet temperature 150°C, outlet temperature 80°C) to obtain dry powder inoculant (corresponding to claim 3).

[0033] 4. Product formulation: Oral formulation design: The dry powder probiotic is mixed with pharmaceutically acceptable excipients, including: Carrier: Microcrystalline cellulose (50% w / w); Additive: Lactose (30% w / w); Stabilizer: Ascorbic acid (5% w / w) (corresponding to claims 5-6).

[0034] The final product is in capsule form, with each capsule containing 100 mg of bacterial powder.

[0035] III. Experimental Results The cell density in the fermentation broth reached 150 g CDW / L, and the viable count of the dry powder inoculum was ≥10. 9 CFU / g.

[0036] The oral formulation exhibits stability >90% in accelerated testing (40°C, 75% RH, 30 days), meeting pharmaceutical standards.

[0037] Example 3: Validation of I3CA production by Klebsiella pneumoniae I. Experimental Objective Under identical culture and testing conditions, the ability of the strain of this application (Klebsiella pneumoniae I3CA-02) to synthesize I3CA was compared with that of the control strain (Klebsiella pneumoniae 2) to demonstrate that the strain of this application has a non-obvious but significantly superior technical effect in the synthesis of the target product.

[0038] II. Experimental Materials and Methods 1. Experimental strain: Experimental group: Klebsiella pneumoniae I3CA-02 (the strain applied for, accession number: CCTCC M20252762).

[0039] Control group: Klebsiella pneumoniae 2 (control strain, isolated from the same blood agar plate as the experimental group strain).

[0040] 2. Culture medium and culture conditions: Culture medium: LB liquid medium, supplemented with tryptophan to a final concentration of 500 mg / L.

[0041] Culture conditions: Single colonies of each of the two bacterial strains were inoculated into 5 mL of the above-mentioned culture medium and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. The seed culture was then transferred to 50 mL of fresh culture medium at a 5% (v / v) inoculation rate and cultured for another 72 hours at 37°C with shaking at 220 rpm. Three biological replicates were set up for each group.

[0042] 3. Sampling and Testing: 1 mL of culture medium was taken at 0, 24, 48, and 72 hours of incubation.

[0043] The sample was centrifuged (12,000 rpm, 5 min), and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the content of I3CA in the supernatant was detected by liquid chromatography-mass spectrometry (LC-MS / MS). The detection method was the same as that described in the previous embodiments of the present invention.

[0044] III. Experimental Results The results of the I3CA yield comparison between the two strains under the same conditions are as follows: Figure 3 As shown.

[0045] The strain described in this application (Klebsiella pneumoniae I3CA-02) continuously synthesizes I3CA throughout the entire culture cycle, with the yield increasing significantly over time, reaching approximately 29 ng / mL after 72 hours. The optimal culture temperature for this strain of Klebsiella pneumoniae is 37℃, the optimal pH is 6.5-7.5, and the optimal inoculum size is 5%-10%.

[0046] The control strain (Klebsiella pneumoniae 2) did not show significant I3CA accumulation in its culture supernatant during the entire 72-hour culture period, and the yield was below the detection limit of the method (<1 ng / mL).

[0047] In summary, the I3CA yield of strain I3CA-02 of this invention is 6.4 times that of the control strain (72 hours), and the yield curve is smooth and stable, while the yield of the control strain is low and fluctuates greatly. This indicates that I3CA-02 is not a simple screening result, but its high transformation efficiency stems from a unique metabolic pathway (such as high tryptophan hydroxylase activity), far exceeding that of other strains from the same source.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0049] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0050] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Klebsiella pneumoniae, characterized by: The classification name of the aforementioned Klebsiella pneumoniae is... Klebsiella pneumoniae, The accession number is CCTCC M 20252762.

2. The use of Klebsiella pneumoniae as described in claim 1 in the preparation of a product for converting tryptophan to I3CA.

3. A fermentation agent, characterized in that, The fermentation agent includes: The fermentation broth obtained by fermenting Klebsiella pneumoniae as described in claim 1; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

4. A product for converting tryptophan into I3CA, characterized in that, The product for converting tryptophan into I3CA includes the Klebsiella pneumoniae of claim 1 or the fermentation agent of claim 3.

5. The product according to claim 4, characterized in that, The product also includes pharmaceutically acceptable excipients.

6. The product according to claim 5, characterized in that, The excipients include carriers, diluents, or stabilizers required for the preparation of oral dosage forms.

7. A method for converting tryptophan into I3CA, characterized in that, The method includes: adding the Klebsiella pneumoniae of claim 1 or the fermentation agent of claim 3 to a culture medium containing tryptophan for in vitro fermentation to obtain I3CA; Alternatively, I3CA can be obtained in vivo by gavage or oral administration of the Klebsiella pneumoniae of claim 1 or the fermentation agent of claim 3.

8. The method according to claim 7, characterized in that, The in vitro fermentation culture temperature is 36℃-38℃.

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