Mass spectrum-assisted structure mining method for converting concephalosporium monosporum into spirolactone

Abstract

The invention relates to the technical field of microbial conversion, and discloses a mass spectrum-assisted structure mining method for converting conehead monospora into spirolactone, which comprises the following steps: S1, screening intestinal fungus strains with spirolactone bioconversion capability through high performance liquid chromatography; s2, carrying out in-vitro fermentation on spirolactone by utilizing the concephalosporium monosporum, and carrying out acetonitrile inactivation and ethyl acetate extraction to obtain fermentation liquor containing metabolites; s3, identifying the metabolites by adopting an HPLC-MS / MS technology; s4, performing separation and structure verification on the metabolite in S3 by combining preparative high performance liquid chromatography and nuclear magnetic resonance technology; and S5, evaluating the binding capacity of the metabolite and an aldosterone receptor through a molecular docking technology, and screening the metabolite with potential antagonistic activity. The concephalosporium monosporum is determined to be a strain for efficiently converting the spirolactone through HPLC (High Performance Liquid Chromatography) screening, and various metabolites including three unreported novel metabolites are generated in the conversion process, so that a candidate compound library is enriched.

Description

Technical Field

[0001] This invention relates to the field of microbial transformation technology, specifically to a mass spectrometry-assisted structure mining method for transforming spironolactone by a single-spore cocephalomycin. Background Technology

[0002] Microbial biotransformation technology, a key tool in drug development, utilizes microbial metabolic systems to modify the structure of substrate molecules, becoming an important pathway for discovering novel active compounds. Spironolactone, a commonly used aldosterone receptor antagonist in clinical practice, can undergo hydroxylation, deacetylation, and double bond isomerization reactions catalyzed by microbial enzyme systems to generate derivatives with potential pharmacological activity through its steroidal ring skeleton and thioacetyl groups.

[0003] In current technologies, microbial transformation studies of spironolactone mainly employ traditional strain screening methods, evaluating strain performance through single indicators and lacking systematic analysis of metabolite diversity. For example, some studies calculate conversion rates solely based on high-performance liquid chromatography (HPLC) peak areas, ignoring the potential for structural diversity in metabolites. Regarding metabolite identification, existing methods largely rely on HPLC-MS for molecular formula prediction; however, for common hydroxylation site isomers and stereoconfigurations in steroid compounds, mass spectrometry fragmentation analysis alone is insufficient for accurate differentiation.

[0004] Traditional screening methods rely on a single indicator and lack a systematic assessment of metabolite diversity, making it difficult to obtain strains that combine high-efficiency transformation capacity with abundant metabolites. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a mass spectrometry-assisted structure mining method for the transformation of spironolactone by monospora concephalomycin. This method solves the problem in existing technologies that lack a systematic assessment of metabolite diversity, making it difficult to obtain strains with both high transformation efficiency and abundant metabolites.

[0006] To achieve the above objectives, the present invention provides a mass spectrometry-assisted structure mining method for spironolactone transformed by a single-spore cocephalomycete, comprising the following steps: S1. Enteric fungal strains with spironolactone biotransformation ability were screened by high performance liquid chromatography, and *Syntrophus monospora* was selected as the target strain. S2. Spirolactone is fermented in vitro using the aforementioned monosporous cocephalomycin, and the fermentation broth containing metabolites is obtained by inactivation with acetonitrile and extraction with ethyl acetate. S3. The metabolites were preliminarily identified using HPLC-MS / MS technology to obtain mass spectrometry data and molecular formulas of each metabolite. S4. The metabolites in S3 were separated and their structures were verified by combining preparative high performance liquid chromatography and nuclear magnetic resonance techniques. S5. Evaluate the binding ability of the metabolites to aldosterone receptors using molecular docking technology, and screen for metabolites with potential antagonistic activity.

[0007] Using the above technical solution, a single-spore cocephalomyeloid with both high conversion efficiency and abundant metabolites was screened by high performance liquid chromatography (HPLC) based on both peak area ratio and diversity of metabolites. Metabolites were efficiently captured through in vitro fermentation, acetonitrile inactivation, and ethyl acetate extraction. Mass spectral profiles, including three previously unreported novel metabolites (C24H32O6S and C24H34O5S), were rapidly identified using alternating positive and negative ion modes and fragment ion matching by HPLC-MS / MS. Further analysis using preparative HPLC separation and multidimensional nuclear magnetic resonance (NMR) spectra (¹H NMR, ¹³CN MR, HMBC, NOESY, etc.) was conducted, and precise analysis was performed using hydrogen-hydrogen coupling constants, carbon chemical shifts, long-range correlations, and spatial correlations. The method identifies hydroxylation sites (e.g., C-6, C-7, C-12) and stereoconfigurations (e.g., C-12 β-hydroxyl, Δ¹,² olefinic bonds), and finally uses molecular docking technology to screen potential antagonistic metabolites based on the number of hydrogen bonds, binding energy (e.g., C8-2 binding energy < -7 kcal / mol) and RMSD value, targeting the aldosterone receptor. This method integrates multi-dimensional strain screening with efficient metabolite capture, a three-level structure resolution system of mass spectrometry screening-preparative separation-NMR confirmation, and molecular docking with biotransformation. It overcomes the limitations of traditional single-index screening and structure identification, significantly expands the candidate compound library, accurately resolves metabolite structures, and provides an efficient active ingredient screening paradigm for steroid drug development.

[0008] Preferably, the high-performance liquid chromatography screening conditions in S1 are as follows: the chromatographic column is a C18 reversed-phase column, the mobile phase is methanol-water gradient elution, the flow rate is 0.3-0.5 mL / min, the detection wavelength is 190-700 nm, the spironolactone retention time is 24.3-24.5 minutes, and the transformation ability of the strain is determined by the proportion of metabolite peak area and diversity.

[0009] Preferably, the HPLC-MS / MS analysis conditions in S3 include: alternating scanning in positive ion mode and negative ion mode, a collision energy of 35%, and precursor ions of M+HM+H⁺ or M+NaM+Na⁺. The mass spectrometry data are used to determine the metabolite structure through molecular formula deviation and fragment ion matching.

[0010] Preferably, the nuclear magnetic resonance technique in S4 includes: ¹H NMR, ¹³CNMR, HSQC, HMBC and NOESY spectral analysis, and the hydroxylation sites and stereoconfiguration of metabolites are determined by hydrogen-hydrogen coupling constant, carbon chemical shift and spatial correlation.

[0011] Preferably, the molecular docking technology in S5 includes: using the aldosterone receptor crystal structure as the target, CHARMM force field and LibDock algorithm for simulation, and evaluating the antagonistic activity of metabolites by the number of hydrogen bonds, binding energy and RMSD value.

[0012] Preferably, the fermentation conditions for the single-spore cocephalosporin are as follows: cultured in MTB medium at 32°C and 160 r / min for 5 days with shaking, and the supernatant is taken after centrifugation of the fermentation broth for metabolite extraction.

[0013] Preferably, the separation conditions for high performance liquid chromatography in step S4 are: C18 column, mobile phase is methanol-water gradient, flow rate is 5-10 mL / min, ultraviolet detection wavelength is 254 nm, and the fractions corresponding to each chromatographic peak are collected.

[0014] Preferably, the metabolite includes at least one of the following: C24H32O6S (m / z471.1810+Na); C24H34O5S (m / z457.2014 +Na); C22H28O3 (m / z363.1927+Na).

[0015] Preferably, the hydroxylation site of the metabolite is selected from the C-6, C-7, or C-12 position of the spironolactone nucleus, and verified by the long-range correlation of H-6 / C-8, H-7 / C-5, and H-12 / C-18 in the HMBC spectrum.

[0016] Preferably, the metabolite C8-2 forms five hydrogen bonds with the aldosterone receptor, with a binding energy of less than -7 kcal / mol, and its structure includes a C-12 β-hydroxyl group and a Δ1,2 olefin bond. The configuration is confirmed by the spatial correlation between H-12 and H-14 in the NOESY spectrum.

[0017] This invention provides a mass spectrometry-assisted structure mining method for spironolactone transformed from monosporous cocephalomycin. It has the following beneficial effects: 1. This invention identifies *Syntrophus monospora* as a strain that efficiently transforms spironolactone through HPLC screening. The transformation process produces a variety of metabolites, including three previously unreported novel metabolites, enriching the candidate compound library.

[0018] 2. This invention combines HPLC-MS / MS and NMR techniques to rapidly and accurately identify the structure of metabolites, overcoming the limitations of traditional separation methods in distinguishing isomers and detecting trace components.

[0019] 3. This invention utilizes molecular docking technology to evaluate the binding ability of metabolites to aldosterone receptors. Metabolites with binding energies below -5 kcal / mol show potential antagonistic activity, providing a screening basis for drug development.

[0020] 4. This invention integrates in vitro fermentation, mass spectrometry analysis, and molecular docking, with optimized operation procedures, making it suitable for high-throughput metabolite research and reducing interference from artificial products. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the qualitative analysis of the biotransformation capacity of intestinal fungi to spironolactone using the high-performance liquid chromatography (HPLC) method of this invention. Figure 2 This is a schematic diagram of the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) analysis of spironol biotransformation in this invention. Figure 3 This is a schematic diagram of the compounds isolated from spironolactone by intestinal fungi according to the present invention; Figure 4 This is a schematic diagram illustrating the main 1H-1HCOSY and HMBC correlations in C4, C5, and C8-1 of the present invention; Figure 5 This is a schematic diagram of the three-dimensional configuration of C4, C5, and C8-1 of the present invention; Figure 6 This is a schematic diagram illustrating the biotransformation mechanism of spironolactone by intestinal fungi according to the present invention. Figure 7 This is a schematic diagram illustrating the potential aldosterone receptor antagonistic effect of spironolactone metabolites discovered through molecular docking studies in this invention. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Please see the appendix Figure 1 -Appendix Figure 7 This invention provides a mass spectrometry-assisted structure mining method for spironolactone transformed by a single-spore cocephalomycin, comprising the following steps: S1. Enteric fungal strains with spironolactone biotransformation ability were screened by high performance liquid chromatography, and *Syntrophus monospora* was selected as the target strain. S2. Spironolactone was fermented in vitro using a single-spore cocephalosporin, and the fermentation broth containing metabolites was obtained by inactivation with acetonitrile and extraction with ethyl acetate. S3. Use HPLC-MS / MS technology to preliminarily identify metabolites and obtain mass spectrometry data and molecular formulas of each metabolite; S4. The metabolites in S3 were separated and their structures were verified by combining preparative high performance liquid chromatography and nuclear magnetic resonance techniques. S5. Evaluate the binding ability of metabolites to aldosterone receptors using molecular docking technology, and screen for metabolites with potential antagonistic activity.

[0024] Specifically, the mass spectrometry-assisted structure mining method for spironolactone transformation by *Cyclocarya monosporans* is implemented as follows: In step S1, nine enteric fungal strains, including *Penicillium spp.*, *Penicillium umbellatus*, *Fusarium oxysporum*, *Cyclocarya monosporans*, *Taraxacum odoratum*, *Smectia circinate*, *Rhizopus microsporum*, *Alfalfa*, and *Aspergillus niger*, were screened by high-performance liquid chromatography (HPLC) to evaluate their spironolactone biotransformation capacity. *Cyclocarya monosporans* was selected as the target strain due to its high transformation efficiency and strong metabolite diversity (confirmed by HPLC peak area analysis). In step S2, *Cyclocarya monosporans* was cultured in MTB medium at 32°C and 160 rpm for 5 days with shaking. The supernatant was collected by centrifugation, inactivated with acetonitrile, and extracted with ethyl acetate to obtain a fermentation broth containing metabolites. In step S3, HPLC-MS / MS technology was used to preliminarily identify the metabolites and obtain mass-to-charge ratio (m / z), molecular formula, and fragment ion mode mass spectrometry data. In step S4, eight major metabolites were separated by preparative HPLC, and their structures were verified by ¹H NMR, ¹³CNMR, HSQC, HMBC, and NOESY spectra, confirming the hydroxylation sites and stereoconfigurations. In step S5, molecular docking was performed targeting the aldosterone receptor (PDB: 2OAX) to analyze the hydrogen bond interactions and binding energies between the metabolites and the receptor, screening for metabolites with potential antagonistic activity.

[0025] The high-performance liquid chromatography (HPLC) screening conditions for S1 were as follows: C18 reversed-phase column, methanol-water gradient elution mobile phase, flow rate 0.3-0.5 mL / min, detection wavelength 190-700 nm, spironolactone retention time 24.3-24.5 min, and the transformation ability of the strain was determined by the proportion of metabolite peak area and diversity.

[0026] Specifically, in the HPLC screening process of step S1, a C18 reversed-phase column was used, with a methanol-water gradient elution system as the mobile phase. The flow rate was set to 0.3-0.5 mL / min, and the detection wavelength range was 190-700 nm. The retention time of spironolactone was approximately 24.3-24.5 minutes. The transformation capacity of each strain was evaluated by comparing the area ratio and diversity of metabolite peaks in the HPLC chromatograms. *Conopterus monospora* exhibited the highest transformation efficiency, producing multiple metabolite peaks with significant peak area ratios, indicating its strong biotransformation capacity and metabolite diversity.

[0027] The HPLC-MS / MS analysis conditions in S3 include: alternating scanning in positive and negative ion modes, a collision energy of 35%, and precursor ions of M+HM+H⁺ or M+NaM+Na⁺. The structure of metabolites was determined by molecular formula deviation (ppm < 10) and fragment ion matching based on the mass spectrometry data.

[0028] Specifically, in the HPLC-MS / MS analysis of step S3, alternating positive and negative ion modes were used, with the collision energy set to 35%, and [M+H]⁺ or [M+Na]⁺ selected as precursor ions. The metabolite structure was determined by molecular formula deviation (ppm) and fragment ion matching. For example, metabolites such as m / z 407.1881 [M+H]⁺ (C22H30O5S), m / z 471.1810 [M+Na]⁺ (C24H32O6S), and m / z 363.1927 [M+Na]⁺ (C22H28O3) were detected, with ppm values ​​ranging from -7.9 to 3.3, ensuring the accuracy of structural identification and reflecting metabolic processes such as hydroxylation, reduction, and deacetylation.

[0029] The nuclear magnetic resonance techniques used in S4 include ¹H NMR, ¹³CNMR, HSQC, HMBC, and NOESY spectral analysis. The hydroxylation sites and stereoconfigurations of metabolites are determined by hydrogen-hydrogen coupling constants, carbon chemical shifts, and spatial correlations.

[0030] Specifically, in step S4, nuclear magnetic resonance (NMR) techniques (including ¹H NMR, ¹³CNMR, HSQC, HMBC, and NOESY spectra) are used to verify the structure of the isolated metabolites. For example, metabolite C4 (C22H30O5) shows two oxymethyl groups (δH 3.92, 3.54) and two oxycarbon atoms (δC 76.4, 70.4) in its NMR spectrum, indicating that it is a deacetylated and dihydroxylated derivative. Long-range correlations from H-6 to C-8 and H-7 to C-5 in the HMBC spectrum confirm the hydroxylation sites at C-6 and C-7, while the correlation between H-6 and H-7 in the NOESY spectrum establishes the cis configurations of 6-OH and 7-OH. Similarly, NMR data for metabolite C5 show an additional double bond (δH 6.22, δC 129.3, 141.6) and a C-12 hydroxyl group, which are verified by HMBC and NOESY spectra.

[0031] The molecular docking technology in S5 includes: using the crystal structure of the aldosterone receptor as the target, CHARMM force field and LibDock algorithm are used for simulation, and the antagonistic activity of metabolites is evaluated by the number of hydrogen bonds, binding energy and RMSD value.

[0032] Specifically, in step S5, molecular docking targets the aldosterone receptor (PDB: 2OAX), and the CHARMM force field and LibDock algorithm are used to simulate the interaction between the metabolite and the receptor. The antagonistic activity of the metabolite is evaluated by the number of hydrogen bonds, binding energy, and RMSD value. For example, metabolite C8-2 forms five hydrogen bonds with key residues of the receptor (CYS, GLN, ARG, ASN, LEU), with a binding energy below -7 kcal / mol, showing strong antagonistic potential. The docking scheme is validated by re-docking the original configuration, and the RMSD value is below 2 Å, ensuring the reliability of the simulation results.

[0033] The fermentation conditions for *Syntrophus monospora* were as follows: cultured in MTB medium at 32°C and 160 r / min for 5 days with shaking. After centrifugation, the supernatant of the fermentation broth was used for metabolite extraction.

[0034] Specifically, in step S2, the fermentation of *Hypericytonne monospora* was carried out in MTB medium under conditions of 32°C and 160 rpm shaking for 5 days to optimize metabolite yield. After fermentation, the fermentation broth was centrifuged at 5,000 g for 10 minutes to separate the cells and supernatant. The supernatant was used to inactivate enzyme activity with acetonitrile, and then metabolites were extracted with ethyl acetate. This process ensured efficient extraction of multiple metabolites, and HPLC analysis showed multiple metabolite peaks, verifying the effectiveness of the fermentation conditions.

[0035] The separation conditions for high performance liquid chromatography in S4 are as follows: C18 column, mobile phase is methanol-water gradient, flow rate is 5-10 mL / min, UV detection wavelength is 254 nm, and the fractions corresponding to each chromatographic peak are collected.

[0036] Specifically, in step S4, the preparative HPLC used a C18 column, a methanol-water gradient elution system as the mobile phase, a flow rate of 5-10 mL / min, and a UV detection wavelength of 254 nm. Pure metabolite fractions were collected based on the chromatographic peaks, and their chemical structures were further confirmed by NMR and mass spectrometry analysis. This method successfully separated eight major metabolites, including C4, C5, and C8-1, ensuring the elucidation of the structures of high-purity metabolites.

[0037] Metabolites include at least one of the following: C24H32O6S (m / z471.1810+Na); C24H34O5S (m / z457.2014 +Na); C22H28O3 (m / z363.1927+Na).

[0038] Specifically, the metabolites identified in step S3 include at least one of the following: C24H32O6S (m / z 471.1810 [M+Na]⁺, ppm -0.4, UV area percentage 0.80%), C24H34O5S (m / z 457.2014 [M+Na]⁺, ppm -1.1, UV area percentage 2.88%), and C22H28O3 (m / z 363.1927 [M+Na]⁺, ppm -1.0, UV area percentage 0.81%). These metabolites were detected by HPLC-MS / MS analysis, and their molecular formulas and structures were confirmed by fragment ion mode and ppm deviation matching, reflecting the chemical changes of spironolactone during hydroxylation, reduction, and deacetylation.

[0039] The hydroxylation sites of the metabolites were selected from the C-6, C-7, or C-12 positions of the spironolactone nucleus and verified by the long-range correlations of H-6 / C-8, H-7 / C-5, and H-12 / C-18 in the HMBC spectrum.

[0040] Specifically, in step S4, the hydroxylation sites of the metabolites are located at C-6, C-7, or C-12 of the spironolactone core. For example, metabolite C4 undergoes hydroxylation at C-6 and C-7, confirmed by long-range correlations from H-6 to C-8 and H-7 to C-5 in the HMBC spectrum. Metabolite C5 undergoes hydroxylation at C-12, verified by correlations from H-12 to C-18 in the HMBC spectrum. Combined with NOESY spectral data, these long-range correlations further confirm the stereoconfiguration and precise location of the hydroxyl groups, ensuring the accuracy of structural characterization.

[0041] The metabolite C8-2 forms five hydrogen bonds with the aldosterone receptor with a binding energy of less than -7 kcal / mol. Its structure includes a C-12 β-hydroxyl group and a Δ1,2 olefin bond. The configuration was confirmed by the spatial correlation between H-12 and H-14 in the NOESY spectrum.

[0042] Specifically, in step S5, metabolite C8-2 forms five hydrogen bonds with the aldosterone receptor (with CYS, GLN, ARG, ASN, and LEU residues) via molecular docking, with a binding energy below -7 kcal / mol. Its structure includes a C-12 β-hydroxyl group and a Δ1,2 double bond, and its configuration is confirmed by the correlations of H-1 / C-3, H-1 / C-5, and H-19 / C-1 in the HMBC spectrum and the spatial correlations of H-12 and H-14 in the NOESY spectrum. This structural characteristic enhances the binding affinity of C8-2 to the receptor, exhibiting stronger antagonistic activity compared to spironolactone.

[0043] The following is a description with reference to specific embodiments: Example 1: Screening and Transformation Capacity Assessment of Gut Fungi Nine enteric fungal strains (Penicillium spp., Penicillium umbellatus, Fusarium oxysporum, *Cytocephalus monospora*, *Taraxacum acidophilus*, *Smectia circinate*, *Rhizopus microsporum*, *Alternaria solani*, and *Aspergillus niger*) were cultured in MTB medium at 32°C and 160 rpm for 5 days. Spironolactone (10 mg / mL) was then added for in vitro fermentation. The fermentation broth was centrifuged (5,000 g, 10 min), and the supernatant was collected. After inactivation with acetonitrile, the supernatant was analyzed by HPLC. The HPLC conditions were a C18 reversed-phase column, methanol-water gradient elution, flow rate of 0.3 mL / min, detection wavelength of 190-700 nm, and a spironolactone retention time of approximately 24.3 min. The results showed that *Cytocephalus monospora* exhibited the highest peak area and greatest diversity of metabolites, confirming it as the optimal transformation strain.

[0044] Example 2: Metabolite extraction and HPLC-MS / MS analysis After culturing *Cyclocarya monospora* in MTB medium for 5 days, spironolactone was added for fermentation. The supernatant was collected by centrifugation, inactivated with acetonitrile, and the metabolites were extracted with ethyl acetate. The extracts were analyzed by HPLC-MS / MS under alternating positive and negative ion modes, collision energy 35%, and precursor ions [M+H]⁺ or [M+Na]⁺. Eleven components were detected, including m / z 407.1881 [M+H]⁺ (C22H30O5S, ppm-1.4, UV area 0.46%) and m / z 471.1810 [M+Na]⁺ (C24H32O6S, ppm-0.4, UV area 0.80%), which preliminarily confirmed that the metabolites involved hydroxylation, reduction, and deacetylation processes.

[0045] Example 3: Metabolite Isolation and Structure Verification Preparative HPLC (C18 column, methanol-water gradient, flow rate 5 mL / min, detection wavelength 254 nm) was used to separate the fermentation broth extract, and eight major metabolite fractions were collected. The structures were verified using ¹H NMR, ¹³CNMR, HSQC, HMBC, and NOESY spectra. For example, metabolite C4 (C22H30O5) showed two oxymethyl groups (δH 3.92, 3.54) and an oxycarbon group (δC 76.4, 70.4), HMBC confirmed C-6 and C-7 hydroxylation, and NOESY established the cis configuration. Metabolites C5 and C8-1 were confirmed by similar methods with C-12 hydroxylation and Δ1,2 double bonds.

[0046] Example 4: Molecular docking and activity assessment Molecular docking was performed using the CHARMM force field and LibDock algorithm, targeting the aldosterone receptor (PDB: 2OAX). Metabolite C8-2 formed five hydrogen bonds with the receptor (CYS, GLN, ARG, ASN, LEU), with a binding energy below -7 kcal / mol, superior to the four hydrogen bonds of spironolactone. Metabolites C4, C9, and C11 formed four hydrogen bonds, showing antagonistic activity similar to spironolactone. The RMSD value was below 2 Å, validating the reliability of the docking.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mass spectrometry-assisted structure mining method for spironolactone transformed by a single-spore concephalomycete, characterized in that, Includes the following steps: S1. Enteric fungal strains with spironolactone biotransformation ability were screened by high performance liquid chromatography, and *Syntrophus monospora* was selected as the target strain. S2. Spirolactone is fermented in vitro using the aforementioned monosporous cocephalomycin, and the fermentation broth containing metabolites is obtained by inactivation with acetonitrile and extraction with ethyl acetate. S3. The metabolites were preliminarily identified using HPLC-MS / MS technology to obtain mass spectrometry data and molecular formulas of each metabolite. S4. Preparative high-performance liquid chromatography and nuclear magnetic resonance techniques were used to separate and verify the structure of metabolites in S3; S5. Evaluate the binding ability of the metabolites to aldosterone receptors using molecular docking technology, and screen for metabolites with potential antagonistic activity.

2. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalus according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) screening conditions in S1 are as follows: the chromatographic column is a C18 reversed-phase column, the mobile phase is methanol-water gradient elution, the flow rate is 0.3-0.5 mL / min, the detection wavelength is 190-700 nm, the spironolactone retention time is 24.3-24.5 minutes, and the transformation ability of the strain is determined by the proportion of metabolite peak area and diversity.

3. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalomycete according to claim 1, characterized in that, The HPLC-MS / MS analysis conditions in S3 include: alternating scanning in positive ion mode and negative ion mode, collision energy of 35%, precursor ions of M+HM+H⁺ or M+NaM+Na⁺, and determination of metabolite structure by molecular formula deviation and fragment ion matching of mass spectrometry data.

4. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalus according to claim 1, characterized in that, The nuclear magnetic resonance techniques in S4 include: ¹H NMR, ¹³CNMR, HSQC, HMBC and NOESY spectral analysis, and the hydroxylation sites and stereoconfigurations of metabolites are determined by hydrogen-hydrogen coupling constant, carbon chemical shift and spatial correlation.

5. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalus according to claim 1, characterized in that, The molecular docking technology in S5 includes: using the crystal structure of the aldosterone receptor as the target, CHARMM force field and LibDock algorithm are used for simulation, and the antagonistic activity of the metabolite is evaluated by the number of hydrogen bonds, binding energy and RMSD value.

6. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalus according to claim 1, characterized in that, The fermentation conditions for the single-spore cocephalosporin were as follows: cultured in MTB medium at 32°C and 160 r / min for 5 days with shaking. After centrifugation, the supernatant of the fermentation broth was used for metabolite extraction.

7. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalomycete according to claim 1, characterized in that, The separation conditions for high performance liquid chromatography in S4 are as follows: C18 column, mobile phase is methanol-water gradient, flow rate is 5-10 mL / min, ultraviolet detection wavelength is 254 nm, and the fractions corresponding to each chromatographic peak are collected.

8. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalomycete according to claim 3, characterized in that, The metabolite includes at least one of the following: C24H32O6S (m / z471.1810+Na); C24H34O5S (m / z457.2014 +Na); C22H28O3 (m / z363.1927+Na).

9. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalomycete according to claim 4, characterized in that, The hydroxylation sites of the metabolites are selected from the C-6, C-7, or C-12 positions of the spironolactone nucleus, and verified by the long-range correlations of H-6 / C-8, H-7 / C-5, and H-12 / C-18 in the HMBC spectrum.

10. The mass spectrometry-assisted structure mining method for spironolactone transformation by a single-spore cocephalomycete according to claim 5, characterized in that, The metabolite C8-2 forms five hydrogen bonds with the aldosterone receptor, with a binding energy of less than -7 kcal / mol. Its structure includes a C-12 β-hydroxyl group and a Δ1,2 olefin bond. The configuration was confirmed by the spatial correlation between H-12 and H-14 in the NOESY spectrum.

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