Klebsiella aerogenes and application thereof in converting tryptophan into indole-3-carboxylic acid
By using Klebsiella pneumoniae to convert tryptophan into indole-3-carboxylic acid, the problem of low conversion efficiency in existing technologies has been solved, and efficient and stable I3CA production has been achieved, which is suitable for green agriculture and high-end pharmaceutical fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies struggle to efficiently and stably convert tryptophan into indole-3-carboxylic acid (I3CA), resulting in low I3CA production and high costs, making it difficult to meet the needs of green agriculture and high-end pharmaceutical fields.
A strain of Klebsiella aerogenes (I3CA-01) was used to convert tryptophan into I3CA through fermentation. By optimizing culture conditions and detection methods, efficient and stable I3CA production was achieved.
The efficient conversion of tryptophan to I3CA was achieved, with a yield of 116 ng/mL. The process exhibited good stability, broadened the production pathways of I3CA, and made it suitable for functional foods and microbial interventional therapy.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of microbial technology, biocatalysis and agricultural biotechnology, specifically to a strain of Klebsiella aerogenes and its application in the conversion of tryptophan into indole-3-carboxylic acid (I3CA). Background Technology
[0002] Indole-3-carboxylic acid (I3CA) is an important indole-based plant growth regulator, widely found in the metabolic networks of plants, microorganisms, and some animals. Its chemical formula is C9H7NO2, and its structure contains an indole ring and a carboxyl group. I3CA significantly improves crop resistance to drought, salinity, and pathogen stress through mechanisms such as regulating endogenous hormone balance, promoting root hair differentiation, and enhancing antioxidant enzyme activity. It is hailed as a core active ingredient of a new generation of "green biostimulants." Recent studies have further discovered that I3CA can activate the aryl hydrocarbon receptor (AhR) pathway in the mammalian gut, exerting potential functions such as anti-inflammation, maintaining the intestinal barrier, and regulating the neuro-immune axis. Therefore, it has attracted widespread attention in the fields of functional foods, special medical diets, and adjunctive interventions for neurodegenerative diseases.
[0003] Currently, the acquisition of I3CA mainly relies on chemical synthesis, typically involving a multi-step reaction of indole with pyruvate or oxalyl chloride. This method suffers from drawbacks such as long routes, low yields (<45%), the need for high temperatures and pressures, and organic solvent pollution, making it difficult to meet the urgent demand for "naturally derived I3CA" in green agriculture and high-end pharmaceutical fields. While plant extraction can yield natural I3CA, the content is low (≤0.02% DW), fluctuates greatly seasonally, and has high purification costs, resulting in poor industrial feasibility. Microbial transformation is considered the most promising alternative due to its mild conditions, high selectivity, and environmental friendliness. Reports indicate that some rhizosphere bacteria, enterococci, and pseudomonads can utilize tryptophan as a precursor to synthesize I3CA via a bypass route of "tryptophan-indole-pyruvate-I3CA" or "tryptophan-indole-3-acetaldehyde-I3CA," but bottlenecks such as low yields (<10 mg L⁻¹), unknown key genes, narrow substrate spectrum, and lack of fermentation technology have prevented large-scale production to date.
[0004] Therefore, developing a new strain that can efficiently, specifically, and stably convert tryptophan into I3CA can not only fill a technological gap, but also provide a sustainable and low-cost production solution for green agriculture (biostimulants), functional foods (natural I3CA raw materials) and high-value chemicals (pharmaceutical intermediates), which is of great significance for promoting the coordinated development of circular economy and precision agriculture. Summary of the Invention
[0005] The purpose of this invention is to provide an aerogenetic Klebsiella pneumoniae strain and its application in I3CA production, which can effectively convert tryptophan into indole-3-carboxylic acid (I3CA).
[0006] 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, the Klebsiella pneumoniae having the accession number: CCTCC NO: M 20252761.
[0007] In a second aspect of the invention, the use of the aforementioned Klebsiella pneumoniae in the preparation of a product for converting tryptophan into I3CA is provided.
[0008] 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.
[0009] 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.
[0010] Furthermore, the product for converting tryptophan into I3CA also includes pharmaceutically acceptable excipients.
[0011] The excipients include those required for preparation into oral dosage forms.
[0012] 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 gas-producing 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.
[0013] Furthermore, the culture medium containing tryptophan is LB medium, with 300 mg / L-800 mg / L tryptophan added.
[0014] Furthermore, the in vitro fermentation culture temperature is 35℃-37℃, the pH range is 6.5-7.5, and the optimal inoculum size is 5%-10%.
[0015] As a specific implementation method, the method for converting tryptophan into I3CA using the aforementioned Klebsiella pneumoniae specifically includes: The gas-producing Klebsiella pneumoniae was inoculated into a test tube containing LB liquid medium and cultured overnight at 35 °C with shaking to activate it. Centrifuge the bacterial culture after overnight incubation, discard the supernatant, resuspend in sterile physiological saline, centrifuge again, discard the supernatant, and resuspend in sterile physiological saline. The resuspended bacterial solution was categorized according to OD... 600 =0.05-0.1% was transferred to an Erlenmeyer flask containing tryptophan in LB medium and cultured on a shaker.
[0016] Furthermore, I3CA was detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): the chromatographic column was a Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0–2 min 5% B, 2–4 min 5%–95% B, 4–5 min 95% B, 5–5.1 min 95%–5% B, 5.1–7 min 5% B; flow rate 0.3 mL / min; column temperature 40℃; injection volume 5 μL; mass spectrometry was performed in electrospray ionization positive ion mode (ESI). + MRM monitoring of ion pairs m / z 176.0→130.0 (I3CA) and m / z 180.0→134.0 ( 13 C4-I3CA internal standard).
[0017] 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. A bacterium capable of converting tryptophan into I3CA was isolated from fresh fecal samples of healthy individuals. This strain was identified as Klebsiella pneumoniae, which can convert tryptophan into I3CA in a time-dependent manner, with a yield of 116 ng / mL in 72 hours. It is also sensitive to antibiotics and has high safety.
[0018] 2. The Klebsiella pneumoniae strain provided by this invention has the ability to produce I3CA, which broadens the production pathway of I3CA and its application in functional foods and microbial interventional therapy.
[0019] The *Klebsiella pneumoniae* strain of this invention was deposited on December 3, 2025, with accession number CCTCC NO: M20252761. Its classification and naming... Klebsiella aerogenes I3CA-01, deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, China, 430072, China. Attached Figure Description
[0020] Figure 1 This is a colony morphology diagram of the Klebsiella pneumoniae strain isolated in this invention. Figure 2 A phylogenetic tree diagram; Figure 3 The image shows the validation results of I3CA production by Klebsiella pneumoniae. 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] The following will provide a detailed description of an aerogenetic Klebsiella pneumoniae strain and its application in the conversion of tryptophan to indole-3-carboxylic acid, in conjunction with examples and experimental data.
[0025] Example 1: Isolation, purification and identification of Klebsiella pneumoniae (1) Isolation of Klebsiella pneumoniae 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 strain. Figure 1 As shown, two strains of Klebsiella pneumoniae were obtained.
[0026] (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 72 h on a shaker. The bacterial culture was then mixed with 30% glycerol at a 1:1 volume ratio and sent to Megvii Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of the obtained strain (as shown in SEQ ID NO.1) was subjected to BLAST alignment analysis 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 aerogenes*, and we named it *Klebsiella aerogenes* I3CA-01. It was deposited on December 3, 2025, at the China Center for Type Culture Collection (depository address: Wuhan University, Wuhan, China), and its taxonomic name is... Klebsiella aerogenes I3CA-01, accession number CCTCC M20252761.
[0027] Example 2: Preparation of Fermentation Agent The activated Klebsiella pneumoniae from Example 1 was inoculated into LB liquid medium and cultured at 35°C with shaking until the logarithmic phase. The cells were collected by centrifugation, resuspended in sterile physiological saline, and prepared into a fermentation broth.
[0028] Part of the fermentation broth was spray-dried to obtain a dry powder inoculum agent with a viable count ≥10⁻⁶. 9 CFU / g.
[0029] Example 3: Comparison and verification of the I3CA transformation ability of Klebsiella pneumoniae I3CA-01 and control strains. I. Experimental Objective In the screening process of Example 1, we isolated two strains of Klebsiella aerogenes from the same fecal sample: one was a high-efficiency strain I3CA-01 (the strain of this invention), and the other was a control strain "Klebsiella aerogenes 2" (not yet preserved, preliminarily identified as Klebsiella aerogenes, but with low expression levels of key genes for I3CA synthesis). To quantitatively compare the performance of the two strains, we designed a parallel fermentation experiment to detect the change in I3CA yield over time.
[0030] II. Experimental Methods 1. Preparation of bacterial strains: Experimental group: Klebsiella pneumoniae I3CA-01 (the strain of this invention).
[0031] Control group: Klebsiella pneumoniae 2 (control strain).
[0032] Both strains were isolated from the same blood agar plate. Figure 1 (The colonies showed similar morphology), and 16S rDNA sequencing confirmed that they belonged to the same genus, Klebsiella gas-producing.
[0033] 2. Fermentation culture The two strains were inoculated into LB liquid medium (containing 500 mg / L tryptophan), with three replicates for each group.
[0034] Culture conditions: 37℃, 220 rpm shaking culture, pH 7.0, inoculum size 5% (v / v).
[0035] Sampling was performed daily (0, 24, 48, and 72 hours) to detect I3CA concentration.
[0036] 3. I3CA testing: Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used under the same conditions as described in the original paper: the column was a Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution (0–2 min 5% B, 2–4 min 5%–95% B, 4–5 min 95% B, 5–5.1 min 95%–5% B, 5.1–7 min 5% B); flow rate 0.3 mL / min; column temperature 40℃; injection volume 5 μL.
[0037] Mass spectrometry was performed in positive electrospray ionization (ESI) mode. + MRM monitoring of ion pairs m / z 176.0→130.0 (I3CA) and m / z 180.0→134.0 (13C4-I3CA internal standard).
[0038] III. Experimental Results The results are as follows Figure 3 As shown, the statistics are presented in the table below.
[0039] Table 1
[0040] Depend on Figure 3 As shown in Table 1: The I3CA-01 strain of this invention showed a significant increase in I3CA yield over time, reaching 116 ng / mL after 72 hours, and the yield increase exhibited a linear trend (R0). 2>0.98), while the control group without tryptophan showed no I3CA production. This demonstrates that the strain I3CA-01 of this invention possesses highly efficient and stable transformation capabilities. 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%.
[0041] Control strain Klebsiella pneumoniae 2: Under the same conditions, the I3CA yield was only 18 ng / mL (average) after 72 hours, and the yield fluctuated greatly (standard deviation ±5 ng / mL), indicating that its transformation efficiency was low and unstable.
[0042] In summary, the I3CA yield of strain I3CA-01 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-01 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.
[0043] Example 4: Fermentation process optimization and stability test of control strains I. Experimental Objective To further verify the robustness of I3CA-01 under industrial conditions, and to compare it with the control strain.
[0044] II. Experimental Methods I3CA-01 and control strain 2 were inoculated into LB medium with different tryptophan concentrations (300, 500, and 800 mg / L), respectively, under the same culture conditions as in Example 3. The culture was extended to 96 hours, and samples were taken daily to detect I3CA yield and cell density (OD600).
[0045] III. Experimental Results I3CA-01: At a tryptophan concentration of 500 mg / L, the yield reached a peak of 116 ng / mL after 72 hours and remained at 110 ng / mL after 96 hours (a decrease of only 5%), demonstrating strong stability.
[0046] Control strain 2: At a tryptophan concentration of 500 mg / L, the yield was 18 ng / mL after 72 hours, and decreased to 10 ng / mL after 96 hours (a decrease of 44%), and the bacteria aged prematurely.
[0047] Optimal conditions: I3CA-01 maintained high yields in the range of 300-800 mg / L tryptophan, while the control strain only had slightly higher yields at 300 mg / L (but not exceeding 25 ng / mL).
[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 in that: The classification of Klebsiella pneumoniae is as follows: Klebsiella aerogenes I3CA-01, accession number CCTCC M 20252761.
2. The use of Klebsiella pneumoniae as described in claim 1 in the preparation of a product for converting tryptophan into indole-3-carboxylic acid.
3. A fermentation agent, characterized in that, The fermentation agent includes: The fermentation broth obtained by fermenting the gas-producing 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: I3CA was obtained by adding the Klebsiella pneumoniae of claim 1 or the fermentation agent of claim 3 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 gas-producing 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 was conducted at a temperature of 35℃-37℃, a pH range of 6.5-7.5, and an inoculum size of 5%-10% (v / v).