Lactobacillus rhamnosus for preparing rapamycin derivatives by transformation and application thereof

Abstract

The present application relates to the field of microbial medicine, and particularly relates to a lactobacillus rhamnosus for preparing rapamycin derivatives through transformation and application thereof.The lactobacillus rhamnosus is lactobacillus rhamnosus (Lactobacillus rhamnosus) Lacticaseibacillus rhamnosus ) SK20, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is No. 1, Yitian West Road, Chaoyang District, Beijing, the preservation date is May 9, 2026, and the preservation number is CGMCC NO. 38606.The lactobacillus rhamnosus is used for preparing rapamycin derivatives through microbial transformation.The present application proves that the lactobacillus rhamnosus SK20 can efficiently transform rapamycin to generate 7-O-demethyl rapamycin, lays a foundation for the development and application of rapamycin derivatives, and provides a feasible path for the research and development of structural modification and new derivatives.

Description

Technical Field

[0001] This invention relates to the field of microbial medicine, and more particularly to a strain of Lactobacillus rhamnosus that is transformed to prepare rapamycin derivatives and its applications. Background Technology

[0002] Rapamycin, as a specific inhibitor of the mTOR pathway, is currently widely used in many fields such as organ transplantation, tumor treatment, and autoimmune diseases.

[0003] Based on the broad pharmacological effects of rapamycin, researchers have further developed various rapamycin derivatives to optimize its drug performance. Everolimus, for example, can be used to prevent organ transplant rejection and plays an important role in the treatment of breast cancer, renal cell carcinoma, neuroendocrine tumors, and tuberous sclerosis-related subependymal giant cell astrocytoma. Tesirolimus has been used in the clinical treatment of advanced renal cell carcinoma and mantle cell lymphoma. However, rapamycin and its derivatives still have several drawbacks in certain applications, such as poor water solubility, rapid in vivo metabolism, short half-life, low bioavailability, and complex chemical structures. Furthermore, traditional chemical modifications suffer from stringent conditions, poor selectivity, and severe pollution, which greatly limit their clinical application and industrialization.

[0004] Microbial transformation is a structural modification method that utilizes enzyme systems produced by microbial metabolism to selectively catalyze substrates. It offers advantages such as mild reaction conditions, high regioselectivity, environmental friendliness, and low cost. In the pharmaceutical field, microbial fermentation is a key processing technology, releasing large amounts of existing active ingredients and synthesizing new substances. Research on the microbial transformation of rapamycin has made some progress. Bacillus, actinomycetes, and fungi can all modify rapamycin to obtain a series of derivatives with better activity and stability. For example, *Bacillus megaterium* 287 can efficiently transform rapamycin to produce products such as 29,42-O-bis(demethyl)rapamycin. *Micromonospora aridans* FIM03-712 can specifically deoxygenate rapamycin at position 14 through microbial transformation, yielding four derivatives including 14-deoxyrapamycin. *Bacillus subtilis* CGMCC 7764 can specifically glycosylate rapamycin at position 43, generating 43-O-(β-D-glucoside)-rapamycin in one step. However, research on the targeted biotransformation of rapamycin using lactic acid bacteria is still relatively lacking. The relevant bacterial strains, transformation patterns, product structures and activities need to be systematically explored, which has great research potential and application prospects.

[0005] Based on the above research background, this invention uses rapamycin as a substrate and screens efficient and safe lactic acid bacteria transformation strains in soil to provide experimental basis for rapamycin structural modification, new drug development, and the application of lactic acid bacteria in drug biotransformation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus rhamnosus that can be transformed to prepare rapamycin derivatives and its application.

[0007] This invention is implemented as follows: The present invention provides a strain of *Lactobacillus rhamnosus* that is transformed to prepare rapamycin derivatives. Lacticaseibacillus rhamnosus SK20, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on May 9, 2026, with accession number CGMCC NO.38606.

[0008] Furthermore, the rapamycin derivative includes 7-O-demethylrapamycin.

[0009] The present invention also provides the application of the Lactobacillus rhamnosus in the microbial transformation for the preparation of rapamycin derivatives.

[0010] Furthermore, the method for preparing rapamycin derivatives using the *Lactobacillus rhamnosus* microbial transformation includes the following steps: (1) Bacterial strain: Lactobacillus rhamnosus (Lactobacillus rhamnosus) Lacticaseibacillus rhamnosus SK20 is the starting strain; (2) Preservation of strains: The strains were inoculated onto MRS slant medium, cultured at 40°C for 24 h, and then stored in a refrigerator at 4°C for later use. (3) Seed culture: The preserved pure strain was inoculated into MRS liquid medium and cultured at 40℃ and 250 r / min for 18-24 h with shaking to obtain the seed culture of strain SK20; (4) Microbial transformation: The seed culture of strain SK20 was inoculated into MRS liquid medium and cultured at 40℃ and 250 r / min for 24 h. Rapamycin mother liquor was added to the fermentation broth to make the final concentration of rapamycin in the medium 250 μg / mL. The culture was continued for 48 hours to obtain the fermentation transformation broth containing rapamycin derivative.

[0011] Furthermore, the rapamycin stock solution was dissolved in DMSO to a concentration of 30 mg / mL.

[0012] Further, the fermentation broth was extracted twice with an equal volume of ethyl acetate, the extracts were combined, and concentrated under reduced pressure to obtain a crude extract. Anhydrous ethanol was added to the crude extract to dissolve it, and the extract was filtered through a 0.22 μm filter membrane to remove impurities. Then, it was purified using preparative liquid chromatography.

[0013] Furthermore, the chromatographic conditions of the prepared liquid chromatograph are as follows: the chromatographic column is a C18 column, the mobile phase is acetonitrile-water (52:48, v / v), the flow rate is 8 mL / min, the detection wavelength is 277 nm, and the column temperature is 30 ℃.

[0014] The present invention has the following advantages: This invention confirms that *Lactobacillus rhamnosus* SK20 can efficiently convert rapamycin to 7-O-demethylrapamycin, laying the foundation for the development and application of rapamycin derivatives and providing a feasible path for structural modification and the research and development of novel derivatives. Specifically: (1) This invention screened and obtained a strain SK20 with transformation ability, which was identified as Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ); (2) HPLC analysis showed that the strain could convert rapamycin into a variety of products. The main product JSY1 (7-O-demethylrapamycin) had a relative rapamycin retention time of 0.409. After preparative HPLC purification, JSY1 with a purity of over 95% was obtained. (3) This product has a certain inhibitory effect on Candida albicans and can inhibit the proliferation of three types of tumor cells, namely T47D, DU145 and ECA109, in a concentration-dependent manner. (4) The water solubility of the product after conversion is significantly improved, and its bioavailability is expected to be improved. Attached image description: Lactobacillus rhamnosus

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Figure 1 The HPLC chromatograms of strain SK20 after microbial transformation with rapamycin are shown below. Among them, (A) is the HPLC chromatogram of the extract of strain SK20 after culturing in transformation medium at 40°C for 72 hours; (B) is the HPLC chromatogram of the extract of strain SK20 after culturing in transformation medium containing rapamycin at 40°C for 72 hours; and (C) is the HPLC chromatogram of the extract after culturing in sterile transformation medium containing rapamycin for 72 hours.

[0017] Figure 2 Agarose gel electrophoresis image of PCR amplification of strain SK20.

[0018] Figure 3 Phylogenetic tree of strain SK20.

[0019] Figure 4 This is the HPLC chromatogram of JSY1, the conversion product of rapamycin.

[0020] Figure 5The structural formulas are those of rapamycin and 7-O-demethylrapamycin, where R=—OH for 7-O-demethylrapamycin and R=—OCH3 for rapamycin. Detailed Implementation

[0021] The technical solution of the present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0022] 1. Materials and Methods 1.1 Test strains Of the strains used in this experiment, strain SK20 was isolated from collected soil samples and identified as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is May 9, 2026, and the accession number is CGMCC NO.38606. The specific information of the other strains is as follows: Candida albicans (CPCC 360003), Candida albicans (CMCC 98001), Staphylococcus aureus (CMCC (B) 26003), Escherichia coli (CMCC (B) 44102), and Bacillus subtilis (CMCC (B) 63501).

[0023] 1.2 Materials, Reagents and Instruments 1.2.1 Soil Samples Soil samples used in the experiment were collected from the area near the institute. Strict aseptic techniques were followed during sampling; samples were collected using a sterile soil shovel and stored at 4 ℃ for subsequent bacterial isolation and screening experiments.

[0024] 1.2.2 Main Reagents and Consumables Reagents: Beef extract, yeast extract, peptone, agar, and other culture medium raw materials were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; Glucose, anhydrous sodium acetate, ammonium citrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, Tween 80, and other chemical reagents were all analytical grade (AR) and purchased from Sinopharm Chemical Reagent Co., Ltd.; PBS (Wuhan Sewell Biotechnology Co., Ltd.), RPMI-1640 medium, F12 medium, DMEM medium, trypsin (with EDTA, without EDTA) (Gibco, USA), FBS, Pen-Strep Solution (Shanghai Darthill Biotechnology Co., Ltd.), TCA (Guangdong Guanghua Technology Co., Ltd.), SRB (Sigma-Aldrich, USA), acetic acid, anhydrous ethanol (Xilong Scientific Co., Ltd.), DMSO, HCl (Sinopharm Chemical Reagent Co., Ltd.), Tris (Beijing Bio-Top Technology Co., Ltd.).

[0025] Experimental consumables: T25 culture flasks, cell cryopreservation tubes, 15 mL centrifuge tubes, 96-well plates (Wuxi Nice Life Technology Co., Ltd.).

[0026] Reference drug: Rapamycin (Fujian Kerui Pharmaceutical Co., Ltd., purity ≥98%, batch number: 20250312).

[0027] 1.3 Methods 1.3.1 Culture medium preparation 1.3.1.1 MRS culture medium (slant and liquid) 10 g tryptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 5 g anhydrous sodium acetate, 2 g ammonium citrate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.1 g manganese sulfate tetrahydrate, and 1 mL Tween 80 were added to distilled water to bring the volume to 1000 mL. The pH was adjusted to 6.2-6.6, and the mixture was autoclaved at 121 °C for 15 min. An additional 15 g / L agar was added to the slant culture medium for strain preservation. No agar was added to the liquid culture medium for strain cultivation.

[0028] 1.3.1.2 LB medium and LB agar medium Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, add distilled water to make up to 1 L, adjust pH to 7.0, and autoclave at 121 ℃ for 15 min; LB agar medium is prepared by adding 15 g / L agar to LB liquid medium and sterilizing under the same conditions.

[0029] 1.3.1.3 SDB and SDA media Add 10 g / L peptone and 40 g / L glucose to distilled water to a final volume of 1 L, adjust the pH to 5.4-5.8, and autoclave at 121 °C for 15 min. SDA medium is prepared by adding 15 g / L agar to SDB medium and sterilizing under the same conditions.

[0030] 1.3.2 Isolation and Screening of Lactic Acid Bacteria in Soil 1.3.2.1 Soil Sample Pretreatment Take 10 g of the preserved soil sample and, under aseptic conditions, add it to an Erlenmeyer flask containing 90 mL of sterile physiological saline (0.85% NaCl). Place the flask in a shaking incubator and shake at 40 ℃ and 250 r / min for 30 min to thoroughly mix the soil sample, obtaining a 10⁻¹ soil bacterial suspension. Use a serial dilution method to successively dilute the bacterial suspension to 10⁻¹. -2 10 -3 10 -4 10 -5 10 -6 Concentration gradients, with 3 replicates for each gradient.

[0031] 1.3.2.2 Isolation and purification of lactic acid bacteria Take 10 respectively -4 10 -5 10 -6 Three concentration gradients of bacterial suspension, 0.1 mL each, were evenly spread onto MRS agar plates. After spreading, the plates were inverted and incubated at 40 °C for 48 hours. After incubation, the colony morphology was observed. Colonies with different morphologies, neat edges, and smooth surfaces suspected to be lactic acid bacteria were selected and inoculated onto new MRS agar plates using the streak plating method. The streaking was repeated three times until pure cultures were obtained. The purified strains were inoculated onto MRS slant agar and incubated at 40 °C for 24 hours. Then, they were stored at 4 °C for later use. A total of 200 lactic acid bacteria strains were isolated and screened.

[0032] 1.3.3 Screening of rapamycin-transformed strains 1.3.3.1 Seed culture Two hundred preserved pure strains of lactic acid bacteria were inoculated into Erlenmeyer flasks containing 50 mL of MRS liquid medium and cultured at 40 ℃ and 250 r / min for 18-24 h with shaking to obtain lactic acid bacteria seed culture for later use.

[0033] 1.3.3.2 Transformation, Screening, and Cultivation Take 5 mL of the above seed culture and inoculate it into an Erlenmeyer flask containing 100 mL of MRS liquid medium. After 24 h, add rapamycin stock solution (dissolved in DMSO, concentration 30 mg / mL) to bring the final concentration of rapamycin in the medium to 250 μg / mL. Use MRS liquid medium without lactic acid bacteria and only with added rapamycin as a blank control, and use MRS liquid medium with lactic acid bacteria and no added rapamycin as a negative control. Set up 3 replicates for each strain. Place the Erlenmeyer flasks at 40 ℃ and shake at 250 r / min for 48 hours to complete the transformation culture.

[0034] 1.3.3.3 Screening of Transformed Strains After transformation culture, 2 ml of the transformation fermentation broth was placed in a centrifuge tube and extracted with 4 mL of ethyl acetate by shaking for 20 min. After centrifugation (4000 r / min, 5 min), the ethyl ester layer was collected, concentrated under reduced pressure to remove the ethyl ester, and the residue was dissolved in 1 mL of ethanol. After centrifugation (10000 r / min) for 10 min, the supernatant was collected for HPLC analysis. By comparing the blank control and negative control, strains capable of transforming rapamycin to produce new derivatives were screened, and the target transformant strain SK20 was obtained after further purification.

[0035] 1.3.4 Identification of the target transformed strain SK20 using 16S rRNA 1.3.4.1 Extraction of genomic DNA from the strain Strain SK20 was inoculated into MRS liquid medium and cultured at 40 °C with shaking for 24 h. 5 mL of the culture was taken and genomic DNA of strain SK20 was extracted according to the instructions of the bacterial genomic DNA extraction kit. The purity and integrity of the DNA were detected by 1% agarose gel electrophoresis and stored at -20 °C for later use.

[0036] 1.3.4.216S rRNA gene PCR amplification PCR amplification was performed using universal primers for bacterial 16S rRNA: upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction mixture (25 μL) consisted of: 12.5 μL Premix Taq, 1 μL upstream primer (10 μmol / L), 1 μL downstream primer (10 μmol / L), 1 μL genomic DNA template, and 9.5 μL sterile double-distilled water. PCR conditions were: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, and 72 ℃ extension for 1.5 min, for a total of 35 cycles; final extension at 72 ℃ for 10 min; and storage at 4 ℃. After confirming successful amplification by 1% agarose gel electrophoresis, the PCR products were sent to a sequencing company for sequencing.

[0037] 1.3.4.3 Strain Identification and Analysis The 16S rRNA gene sequence obtained from sequencing was BLAST-aligned using the NCBI database. Sequences with high homology were selected, and a phylogenetic tree was constructed using MEGA software. Combined with the morphological characteristics and culture properties of the strain, the species classification of strain SK20 was determined, and it was identified as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus ).

[0038] 1.3.5 Large-scale fermentation of strain SK20 and extraction and purification of transformation products 1.3.5.1 Large-scale fermentation culture The SK20 seed culture was inoculated into a 10 LMRS liquid medium at a seed volume of 10 % (v / v) and cultured at 40 ℃ and 250 r / min for 24 h. Rapamycin stock solution was then added to the fermentation broth to bring the final concentration to 250 μg / mL. The culture was continued for 48 hours to complete the large-scale transformation.

[0039] 1.3.5.2 Extraction of transformation products The large-scale fermentation broth was extracted twice with an equal volume of ethyl acetate. The extracts were combined and distilled under reduced pressure at 45 °C to dryness to obtain a crude extract. 10 mL of anhydrous ethanol was added to the crude extract to dissolve it, and the extract was filtered through a 0.22 μm filter membrane to remove impurities. The extract was then set aside for later use.

[0040] 1.3.5.3 Purification of transformation products The crude extract was purified using preparative liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (250 mm × 21.2 mm, 10 μm); mobile phase: acetonitrile-water (52:48, v / v); flow rate: 8 mL / min; detection wavelength: 277 nm; column temperature: 30 ℃. 2.5 mL of the ethanol solution was injected, and the eluent corresponding to the chromatographic peak was collected. The purification process was repeated three times. The eluents were combined and distilled under reduced pressure to dryness to obtain the pure main conversion product, which was stored at -20 ℃ for later use.

[0041] 1.3.6 Structural identification of major transformation products 1.3.6.1 High-resolution mass spectrometry (HR-MS) identification The conversion product was dissolved in methanol to prepare a sample solution with a concentration of 1 mg / mL. The solution was then detected using a high-resolution mass spectrometer with ESI as the ionization method. By determining the precise molecular weight of the sample and combining it with the molecular structure of rapamycin, the molecular composition of the conversion product was preliminarily inferred.

[0042] 1.3.6.2 Nuclear Magnetic Resonance (NMR) Identification The conversion product was dissolved in deuterated dimethyl sulfoxide (DMSO-d6), and relevant spectral detections such as ¹H-NMR and ¹³C-NMR were performed to determine data such as chemical shift and coupling constant. Combined with high-resolution mass spectrometry results and nuclear magnetic resonance data of rapamycin, the chemical structure of the conversion product was determined by comparative analysis.

[0043] 1.3.7 Solubility test of conversion products Take 20 μL each of 10 mmol / L rapamycin and the conversion product, add them to 4 mL of deionized water, shake for 20 min, centrifuge at 10000 rpm for 10 min, take the supernatant and analyze it by HPLC, and determine and compare the solubility of the two.

[0044] 2 Results 2.1 Results of the isolation of lactic acid bacteria from soil Two hundred strains of suspected lactic acid bacteria were isolated from collected soil samples using MRS medium through serial dilution and plating. The isolated strains exhibited mostly round, milky-white colonies with smooth, moist surfaces and regular edges on MRS agar plates. Microscopic examination revealed them to be Gram-positive bacilli without spores, consistent with the typical morphological characteristics of lactic acid bacteria.

[0045] 2.2 Screening results of rapamycin-transformed strains Two hundred soil lactic acid bacteria strains were used as experimental strains and transformed in MRS liquid medium supplemented with rapamycin. Preliminary HPLC analysis revealed that strain SK20 exhibited significant transformation activity to rapamycin, showing a new characteristic absorption peak in the liquid chromatogram. This indicated that this strain could catalyze the formation of a new transformation product from rapamycin, and was thus identified as the target transformation strain. Figure 1 ).

[0046] 2.3 Identification results of 16S rDNA of strain SK20 The genomic DNA of strain SK20 was clearly banded by agarose gel electrophoresis, with no obvious tailing, which meets the requirements for PCR amplification. Figure 2 A 16S rDNA gene fragment of approximately 1500 bp (as shown in SEQ NO. 1) was amplified using universal primers 27F / 1492R. This sequence was compared with the NCBI database using BLAST, and the results showed that strain SK20 was similar to several strains of *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus The homology reached over 99%. Based on phylogenetic tree analysis and strain morphology and culture characteristics, the transformed strain SK20 was ultimately identified as *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus () Figure 3 ).

[0047] 2.4 Large-scale fermentation and separation / purification of transformation products Lactobacillus rhamnosus SK20 was fermented on a 10 L scale. The fermentation broth was centrifuged, extracted with ethyl acetate, and concentrated under reduced pressure to obtain 1.87 g of crude extract. The crude extract was purified by preparative liquid chromatography (HPLC). Under optimized chromatographic conditions (acetonitrile:water = 52%:48% (v / v)), the main characteristic peak components were collected. After drying under reduced pressure, 27.54 mg of a white amorphous powder was obtained. HPLC analysis showed a purity of over 95%. Figure 4 ).

[0048] 2.5 Structural identification results of the transformation products 2.5.1 High-resolution mass spectrometry analysis High-resolution mass spectrometry results showed that the quasi-molecular ion peak of the conversion product JSY1 was 917.5769 [M+NH4]. + The corresponding molecular formula is C 50 H 77 NO 13 , structural formula as Figure 5 As shown, the molecular weight is reduced by 14 Da compared to rapamycin, suggesting that demethylation may occur in the rapamycin molecule.

[0049] 2.5.2 Nuclear Magnetic Resonance Analysis Analysis of ¹H-NMR, ¹³C-NMR, and two-dimensional NMR spectra, compared with standard NMR data for rapamycin, revealed that the proton and carbon signals corresponding to the methoxy group at the C-7 position of the transformed product JSY1 disappeared, while the hydroxyl signal appeared at the corresponding position. The distribution of proton and carbon signals in the remaining skeleton was highly consistent with that of rapamycin. Combined with the high-resolution mass spectrometry results, the transformed product was identified as 7-O-demethylrapamycin (as shown in Table 1).

[0050] Table 1. Chemical shifts of ¹³C and ¹H NMR for the conversion product JSY1 (solvent: DMSO)

[0051]

[0052] 2.5.3 Analysis of the difference in solubility between the transformation product JSY1 and rapamycin The solubility of rapamycin and its conversion product JSY1 in water was determined using the equilibrium method. Three parallel experiments were set up for each sample. The concentration of the two compounds in the supernatant was determined by HPLC, and then their solubility was determined. There was a significant difference in the solubility of the two compounds in water. The specific measurement results are shown in Table 2.

[0053] Table 2. Results of solubility determination of transformation product JSY1 and rapamycin

[0054] Table 2 shows that the solubility of rapamycin in water is 3.02 ± 0.12 μmol / mL, while the solubility of the transformed product JSY1 is 3.86 ± 0.18 μmol / mL, with the latter being approximately 1.28 times more soluble than the former. This indicates that the water solubility of rapamycin is significantly improved after demethylation at the 7-position, which may be related to the change in molecular polarity after modification. In summary, this invention screened a strain of *Lactobacillus rhamnosus* SK20 from soil, which can transform rapamycin into JSY1 (7-O-demethylrapamycin) under mild conditions. Experimental results show that the water solubility of JSY1 is significantly improved. Although its antibacterial activity (especially against *Saccharomyces cerevisiae*) is weakened, it still retains strong antitumor activity and inhibitory effect against *Candida albicans*.

[0055] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A strain of *Lactobacillus rhamnosus* transformed to prepare rapamycin derivatives, characterized in that: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus SK20, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on May 9, 2026, with accession number CGMCC NO.38606.

2. The Lactobacillus rhamnosus according to claim 1, characterized in that: The rapamycin derivatives include 7-O-demethylrapamycin.

3. The use of Lactobacillus rhamnosus as described in claim 1 or 2 in the microbial transformation for the preparation of rapamycin derivatives.

4. The application according to claim 3, characterized in that: The method for preparing rapamycin derivatives using *Lactobacillus rhamnosus* microbial transformation includes the following steps: (1) Bacterial strain: Lactobacillus rhamnosus (Lactobacillus rhamnos Lacticaseibacillus rhamnosus SK20 is the starting strain; (2) Preservation of strains: The strains were inoculated onto MRS slant medium, cultured at 40°C for 24 h, and then stored in a refrigerator at 4°C for later use. (3) Seed culture: The preserved pure strain was inoculated into MRS liquid medium and cultured at 40℃ and 250 r / min for 18-24 h with shaking to obtain the seed culture of strain SK20; (4) Microbial transformation: The seed culture of strain SK20 was inoculated into MRS liquid medium and cultured at 40℃ and 250 r / min for 24 h. Rapamycin mother liquor was added to the fermentation broth to make the final concentration of rapamycin in the medium 250 μg / mL. The culture was continued for 48 hours to obtain the fermentation transformation broth containing rapamycin derivative.

5. The application according to claim 4, characterized in that: The rapamycin stock solution described in step (4) is dissolved in DMSO to a concentration of 30 mg / mL.

6. The application according to claim 4, characterized in that: The fermentation broth was extracted twice with an equal volume of ethyl acetate. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. Anhydrous ethanol was added to the crude extract to dissolve it. The extract was filtered through a 0.22 μm filter membrane to remove impurities and then purified using preparative liquid chromatography.

7. The application according to claim 6, characterized in that: The chromatographic conditions for the prepared liquid chromatograph are as follows: C18 column; mobile phase: acetonitrile-water, volume ratio: 52:48; flow rate: 8 mL / min; detection wavelength: 277 nm; column temperature: 30 ℃.

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