A meso-azurocidin-producing strain and a separation and purification process thereof

Abstract

This invention relates to the fields of biomedicine and microbial fermentation engineering technology, and discloses a strain of *Streptomyces citrus* YS-4 that produces mesocyclocarpine. The separation and purification process includes: solid-liquid separation of the fermentation broth; extraction of the supernatant with ethyl acetate and extraction of the bacterial cells by methanol soaking; combining and concentrating the extracts to obtain a crude extract; purification of the crude extract by silica gel column chromatography and Sephadex LH-20 gel column chromatography; and finally purification by C18 reversed-phase semi-preparative high-performance liquid chromatography to obtain mesocyclocarpine. This invention employs a dual extraction strategy to improve the yield and achieves efficient separation of the target product and its structural homologues through specific chromatographic conditions, effectively solving the problem of incomplete extraction caused by the coexistence of intracellular adsorption and extracellular secretion of the target product, resulting in a final product yield superior to that of single-solvent extraction methods.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbial fermentation engineering technology, specifically to a strain of *Mesenthalpyrrolizin* that produces erythrin and its isolation and purification process. Background Technology

[0002] Prodiginines are a class of microbial secondary metabolites with a pyrrole ring skeleton, attracting significant attention due to their unique chemical structure and various biological activities, including antibacterial, antitumor, and immunosuppressive effects. Among them, metacycloprodigiosin, as a member of this family with a special macrocyclic structure, has become a research hotspot in the biomedical field due to its excellent activity against drug-resistant strains and its potential medicinal value.

[0003] However, despite the broad application prospects of cyclophosphamide, its large-scale preparation and application still face many technical bottlenecks. Firstly, at the fermentation production source, the currently reported production strains are mostly *Serratia marcescens* (…). Serratia marcescens (or some wild-type strains of Streptomyces) These strains typically have metabolic networks that tend to synthesize linear prodigiosin. Metabolic prodigiosin often appears only as a trace byproduct in the metabolic pathway, resulting in extremely low potency of the target product in the fermentation broth. Furthermore, the fermentation product composition is complex, increasing the difficulty of subsequent separation and purification.

[0004] Secondly, in the product extraction stage, cyclophosphamide, as a fat-soluble pigment, exhibits a complex distribution during fermentation. Some is secreted into the extracellular fermentation broth, while a large amount is adsorbed onto the cell surface or retained intracellularly. Existing extraction processes often use a single solvent to extract from the entire fermentation broth, or only extract from the cells while discarding the fermentation supernatant. This extensive approach fails to capture both intracellular and extracellular products, resulting in low extraction yields of the target compound and significant resource waste.

[0005] More importantly, in terms of separation and purification techniques, metacyclophosphamide is extremely similar to linear cyclophosphamide and its derivatives, which are already present in high concentrations in the fermentation broth, in terms of molecular structure, polarity, and solubility. Conventional silica gel column chromatography or recrystallization methods are insufficient to effectively separate these structural homologues, often resulting in tailing and overlapping phenomena. To obtain high-purity samples, multiple repeated chromatography steps are often required. This not only consumes large amounts of organic solvents but also leads to degradation or loss of the target product during repeated processing, creating a dilemma where high purity and high yield are difficult to achieve simultaneously, thus limiting in-depth pharmacological research and industrial development of this compound.

[0006] Therefore, screening for specific high-yield strains and developing an efficient and precise integrated extraction and purification process are currently urgent technical challenges to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a cyclophosphamide erythrin-producing strain and its isolation and purification process, which solves the problems of poor metabolic specificity, low yield of single solvent extraction, and difficulty in obtaining high-purity products due to the similarity of structural homologous physicochemical properties in existing cyclophosphamide erythrin-producing strains.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain that produces mesocyclocarpine. This strain possesses a specific secondary metabolic network and, upon induction culture, can specifically synthesize mesocyclocarpine with a macrocyclic structure, reducing the proportion of linear homologues and increasing the abundance of the target product from the source.

[0009] A second aspect of this invention provides a process for isolating and purifying erythrin from *Mexicanthone* strains. This process, based on the amphiphilic characteristics of the target product and the stereochemical differences in its macrocyclic structure, constructs a combined technical route including fermentation regulation, biphasic extraction, polarity screening, and hydrophobic purification.

[0010] The specific technical solution is as follows: To address the distribution characteristics of cyclophosphamide erythrin during fermentation, this invention employs a phase-separation extraction strategy. First, fermentation is carried out for 6-8 days at 26-30℃, 160-200 r / min, and a specific culture medium to allow the cell biomass and secondary metabolite accumulation to reach equilibrium. Subsequently, solid-liquid separation is performed: the fermentation supernatant is extracted using a moderately polar solvent (ethyl acetate), while the wet cells are extracted by soaking in a highly polar solvent (methanol).

[0011] Mechanism Explanation: This dual extraction strategy overcomes the limitations of a single solvent. Ethyl acetate utilizes the principle of "like dissolves like" to recover extracellular products dissolved in the aqueous culture medium; methanol utilizes its permeability to disrupt cell walls and dissolve products adsorbed on the cell membrane or intracellularly. The combined two-phase extract is then concentrated to achieve full recovery of the target product.

[0012] To address impurities such as oils, proteins, and non-target pigments in the crude extract, this invention utilizes normal-phase silica gel media for polarity screening. The key technical approach lies in employing a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 3:1 as the elution system.

[0013] Mechanism explanation: The mixed solvent ratio creates a specific polar environment, ensuring that the adsorption-desorption equilibrium constant of metacyclophosphamide on the silica gel surface is within the optimal range, manifested as a stable Rf value between 0.2 and 0.3. This polar window effectively distinguishes between strongly lipophilic impurities (eluting with the solvent front) and strongly polar impurities (adsorbed at the column head), achieving preliminary enrichment and deimpurification of the target product.

[0014] Following normal phase chromatography, a Sephadex LH-20 gel column chromatography step was introduced, using methanol as the mobile phase.

[0015] Mechanism explanation: The network structure of the gel medium is used to remove impurities with large molecular weight differences based on the molecular size exclusion effect. At the same time, the specific adsorption of pigment molecules by the gel skeleton is used to further remove trace amounts of pigments remaining in the previous steps, reducing the load on subsequent high-precision separation.

[0016] For the most difficult-to-remove structural homologues (such as styraxin), this invention uses C18 reverse semi-preparative high performance liquid chromatography for final purification, the core of which is to control the mobile phase to a methanol-water volume ratio of 75:25.

[0017] Mechanism Explanation: Metacyclic thiazolinone possesses a unique metacyclic macrocyclic structure, exhibiting slight differences in steric hindrance and hydrophobic surface area compared to linear thiazolinone. In a 75:25 methanol-water system, the hydrophobic interaction between the C18 alkyl chain and the target molecule is precisely regulated, amplifying the retention time difference caused by this structural difference, thereby achieving complete separation of the target peak from the impurity peak in the chromatogram.

[0018] This invention provides a erythromycin-producing strain of *Ichthyophthirius multifiliis* and its isolation and purification process. It has the following beneficial effects: 1. The *Streptomyces citrus* YS-4 obtained by screening in this invention has a specific secondary metabolic pathway. Compared with conventional *Serratia marcescens*, its metabolic flow is more inclined to synthesize mesocytocin with a macrocyclic structure, reducing the generation of linear mesocytocin byproducts. Combined with the dual extraction strategy for fermentation supernatant and bacterial cells constructed in this invention, the problem of incomplete extraction caused by the coexistence of intracellular adsorption and extracellular secretion of the target product is effectively solved, resulting in a higher extraction yield of the final product than that of single solvent extraction method, and realizing the full recovery of fermentation products.

[0019] 2. This invention solves the technical challenge of separating mesoxadiazonin from its structural homologues through a chromatographic process with specific parameters. Specifically, a petroleum ether-ethyl acetate elution system (3:1 volume ratio) constructs a precise polarity screening window in normal-phase silica gel chromatography, effectively removing oils and highly polar impurities. A methanol-water mobile phase system (75:25 volume ratio) amplifies the subtle differences in steric hindrance and hydrophobicity between mesoxadiazonin and its linear derivatives in reversed-phase liquid chromatography, achieving baseline separation. The product prepared using this process exhibits good process reproducibility and is suitable for preparing high-purity samples.

[0020] 3. The cyclophosphamide prepared by the process of this invention maintains a complete bioactive framework and exhibits inhibitory effects on Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. In particular, the minimum inhibitory concentration against Staphylococcus aureus is better than that of the control drug sodium penicillin, thus providing a high-activity and high-quality raw material basis for the development of drugs against drug-resistant bacteria. Attached Figure Description

[0021] Figure 1 This is a morphological characteristic diagram of Streptomyces citrus YS-4 of the present invention; Figure 2 This invention presents a phylogenetic tree of Streptomyces citrus YS-4 constructed based on the whole genome. Figure 3 The UV-Vis full-wavelength scan spectrum of the crude extract of cymoxanil prepared in Example 1 of this invention; Figure 4 This is a thin-layer chromatography (TLC) image of the silica gel column chromatography eluted components in Example 1 of the present invention.

[0022] In the image, A shows the frontal morphology of YS-4 on Gao's No. 1 medium, B shows the back morphology, C shows the single colony morphology, D shows the electron micrograph of aerial hyphae, E shows the electron micrograph of spore chains, and F shows the electron micrograph of spore surface characteristics. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, examples, comparative examples, and test examples. 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.

[0024] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a method for preparing *Streptomyces citrus* fermentation broth for subsequent isolation and purification. First, a PDB liquid fermentation medium is prepared, consisting of: boiling 200g of peeled potatoes for 30min, filtering the filtrate through four layers of gauze, adding 20.0g of glucose, and adjusting the volume to 1L. The pH is adjusted to natural (approximately 7.2), and the mixture is autoclaved at 121℃ for 20min. *Streptomyces citrus* YS-4 is inoculated into a 250mL Erlenmeyer flask containing 50mL of the above medium and cultured at 28℃ and 180rpm for 24h to obtain a seed culture. The seed culture is then inoculated at a rate of 4% into a fermentation flask containing the above PDB medium, resulting in a total fermentation volume of 10L. The fermentation flask is placed on a shaker, and the culture temperature is controlled at 28℃, the rotation speed at 180rpm, and the culture period is 7 days. After fermentation, the fermentation broth is deep orange-red, which is used as the separation raw material in subsequent examples.

[0025] Preparation Example 2: This preparation example provides a method for preparing *Streptomyces citrus* fermentation broth for subsequent isolation and purification. First, a PDB liquid fermentation medium is prepared, consisting of 5.0 g potato starch and 15.0 g glucose, dissolved in 1 L of distilled water. The pH is adjusted to 7.0, and the medium is autoclaved at 121°C for 20 min. *Streptomyces citrus* YS-4 is inoculated into an Erlenmeyer flask containing the above medium and cultured at 26°C and 160 rpm for 36 h to obtain a seed culture. The seed culture is then inoculated into the fermentation medium at a rate of 2%, for a total fermentation volume of 10 L. The culture temperature is controlled at 26°C, the rotation speed at 160 rpm, and the culture period is 8 days. After fermentation, the fermentation broth is orange-red, which is used as the separation raw material in subsequent examples.

[0026] Preparation Example 3: This preparation example provides a method for preparing *Streptomyces citrus* fermentation broth for subsequent isolation and purification. First, a PDB liquid fermentation medium is prepared, consisting of: boiling and filtering 300g of peeled potatoes, adding 25.0g of glucose, bringing the volume to 1L, adjusting the pH to 7.4, and autoclaving at 121℃ for 20min. *Streptomyces citrus* YS-4 is inoculated into an Erlenmeyer flask containing the above medium and cultured at 30℃ and 200 rpm for 24h to obtain a seed culture. The seed culture is then inoculated into the fermentation medium at a rate of 5%, for a total fermentation volume of 10L. The culture temperature is controlled at 30℃, the rotation speed at 200 rpm, and the culture period is 6 days. After fermentation, the fermentation broth is dark red, which is used as the separation raw material in subsequent examples.

[0027] Examples 1-3: See attached document Figure 1 - Appendix Figure 4 Example 1: This embodiment provides a process for the isolation and purification of cyclophosphamide erythrin, using the fermentation broth obtained in Preparation Example 1 as raw material. The process includes the following steps: (1) The 10L fermentation broth obtained in Preparation Example 1 was filtered under reduced pressure through a Buchner funnel lined with two layers of filter paper to achieve solid-liquid separation, and the fermentation supernatant and wet cells were collected respectively.

[0028] (2) Transfer the fermentation supernatant to a separatory funnel, add ethyl acetate at a volume ratio of 1:1 to the supernatant for extraction, shake and allow to stand for separation, collect the ethyl acetate phase, and repeat the extraction 3 times; at the same time, place the wet cells in a beaker, add methanol at a volume of 2.5 times the volume of the wet cells, stir and soak for extraction 3 times, each extraction time is 45 min, filter and collect the methanol extract; combine the above ethyl acetate phase and methanol extract, and concentrate under reduced pressure using a rotary evaporator at 45℃ to remove the solvent, to obtain a dark red organic phase crude extract.

[0029] (3) Dissolve the crude organic extract in an appropriate amount of methanol, add 200-300 mesh silica gel for column chromatography and mix and grind, wherein the mass ratio of the silica gel to the crude organic extract is 1:1. Place it in a fume hood and wait for the methanol to evaporate completely before using it as the sample for loading. Take another 200-300 mesh silica gel for column chromatography and pack it into a column using the wet method. Add the sample to the top of the column and use a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 3:1 for isocratic elution. Collect the eluent in segments and detect it using thin-layer chromatography. Combine the eluents with Rf values ​​between 0.2 and 0.3 and showing a single main spot, concentrate and dry to obtain component 1.

[0030] (4) Weigh Sephadex LH-20 gel and soak it in methanol for 18 hours to swell. Pack it into a glass chromatography column and equilibrate the column with methanol at a flow rate of 2.0 mL / min for 7 hours. Dissolve and filter the fraction 1 obtained in step (3) and load it onto the column. Use methanol as the mobile phase for elution and control the elution flow rate at 1.0 mL / min. Collect the main fraction containing mesoxadiazine, concentrate and dry it.

[0031] (5) The components obtained in step (4) were separated by reverse semi-preparative high performance liquid chromatography. A C18 reverse semi-preparative column (10×250mm, 5μm) was selected. The mobile phase was a methanol-water system with a volume ratio of 75:25. The flow rate was set to 3.0mL / min, the column temperature was controlled at 28℃, and the detection wavelength was set to 531nm. The single main peak eluent at the retention time was collected, methanol was removed by rotary evaporation, and then the product was freeze-dried to obtain pure cyclophosphamide erythrin.

[0032] Example 2: This embodiment provides a process for the isolation and purification of cyclophosphamide erythrin, using the fermentation broth obtained in Preparation Example 2 as raw material, and includes the following steps: (1) The 10L fermentation broth obtained in Preparation Example 2 was filtered and separated, and the fermentation supernatant and wet cells were collected respectively.

[0033] (2) The fermentation supernatant was extracted three times with ethyl acetate at a volume ratio of 1:1 to the supernatant; the cells were extracted three times with methanol at a volume twice that of the wet cells, with each extraction lasting 30 min; all extracts were combined and concentrated under reduced pressure to obtain the crude organic extract.

[0034] (3) Dissolve the crude organic extract and mix it with 200-300 mesh column chromatography silica gel at a mass ratio of 0.8:1. Pack the column using wet method, and use petroleum ether-ethyl acetate at a volume ratio of 3:1 for isocratic elution. Collect the fractions with Rf values ​​between 0.2 and 0.3.

[0035] (4) The Sephadex LH-20 gel was soaked in methanol for 12 h to swell, and then packed into a column and equilibrated at a flow rate of 1.5 mL / min for 6 h. After loading the sample, it was eluted with methanol at a flow rate of 0.8 mL / min and the target component was collected.

[0036] (5) The obtained components were separated by reverse semi-preparative high performance liquid chromatography using a C18 column, a mobile phase of methanol-water with a volume ratio of 75:25, a flow rate of 2.0 mL / min, a column temperature of 25 °C, and a detection wavelength of 531 nm. The main peak eluent was collected and dried to obtain pure cyclophosphamide erythrin.

[0037] Example 3: This embodiment provides a process for the isolation and purification of cyclophosphamide erythrin, using the fermentation broth obtained in Preparation Example 3 as raw material, and includes the following steps: (1) The 10L fermentation broth obtained in Preparation Example 3 was filtered and separated, and the fermentation supernatant and wet cells were collected respectively.

[0038] (2) The fermentation supernatant was extracted three times with ethyl acetate at a volume ratio of 1:1 to the supernatant; the cells were extracted three times with methanol at a volume of 3 times that of the wet cells, with each extraction time being 60 min; all extracts were combined and concentrated under reduced pressure to obtain the crude organic extract.

[0039] (3) Dissolve the crude organic extract and mix it with 200-300 mesh column chromatography silica gel at a mass ratio of 1.2:1. Pack the column using wet method, use petroleum ether-ethyl acetate at a volume ratio of 3:1 as the eluent, elute isocratic, and collect the fractions with Rf values ​​between 0.2 and 0.3.

[0040] (4) The Sephadex LH-20 gel was soaked in methanol for 24 hours to swell. After packing the column, it was equilibrated at a flow rate of 2.5 mL / min for 8 hours. After loading the sample, it was eluted with methanol at a flow rate of 1.2 mL / min and the target component was collected.

[0041] (5) The obtained components were separated by reverse semi-preparative high performance liquid chromatography using a C18 column, a mobile phase of methanol-water with a volume ratio of 75:25, a flow rate of 4.0 mL / min, a column temperature of 30 °C, and a detection wavelength of 531 nm. The main peak eluent was collected and dried to obtain pure cyclophosphamide erythrin.

[0042] Comparative Examples 1-8: Comparative Example 1: Compared with Example 1, the difference lies in the extraction operation in step (2): only the fermentation supernatant is extracted with ethyl acetate, the wet cells are discarded, and methanol soaking extraction is not performed. The remaining steps and parameters are the same as in Example 1.

[0043] Comparative Example 2: Compared with Example 1, the difference lies in the extraction operation in step (2): only the wet bacterial cells are extracted by soaking in methanol, the fermentation supernatant is discarded, and the supernatant is not extracted with ethyl acetate. The remaining steps and parameters are the same as in Example 1.

[0044] Comparative Example 3: Compared with Example 1, the difference lies in the eluent system for silica gel column chromatography in step (3): the volume ratio of petroleum ether to ethyl acetate was adjusted to 10:1 (too low polarity). The remaining steps and parameters are the same as in Example 1.

[0045] Comparative Example 4: Compared with Example 1, the difference lies in the eluent system for silica gel column chromatography in step (3): the volume ratio of petroleum ether to ethyl acetate was adjusted to 1:1 (too polar). The remaining steps and parameters are the same as in Example 1.

[0046] Comparative Example 5: Compared with Example 1, the difference is that the Sephadex LH-20 gel column chromatography step (4) was omitted, and the component 1 obtained in step (3) was directly used for separation by reverse high performance liquid chromatography in step (5). The remaining steps and parameters are the same as in Example 1.

[0047] Comparative Example 6: Compared with Example 1, the difference lies in the mobile phase system of the reverse high performance liquid chromatography in step (5): the volume ratio of methanol to water is adjusted to 50:50. The remaining steps and parameters are the same as in Example 1.

[0048] Comparative Example 7: Compared with Example 1, the difference lies in the mobile phase system of reverse high performance liquid chromatography in step (5): the volume ratio of methanol to water is adjusted to 90:10. The remaining steps and parameters are the same as in Example 1.

[0049] Comparative Example 8: Compared with Example 1, the difference is that the production strain used in step (1) was replaced with commercially available Serratia marcescens, and the fermentation conditions were adjusted accordingly with reference to the optimal conditions of the commercially available strain. The extraction and purification process was exactly the same as in Example 1.

[0050] Test Examples 1-4: Test Example 1: Structural Confirmation of the Product Prepared by the Invention Experimental instructions and procedures: An appropriate amount of the deep yellow powder obtained in Example 1 was taken and its structure was identified by ultraviolet-visible absorption spectroscopy, high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

[0051] The specific operating parameters are as follows: Ultraviolet spectroscopy analysis: Weigh 1.2 mg of the sample, dissolve it in chromatographically pure methanol, and dilute to 100 mL to prepare the test solution. Using pure methanol as a blank control, perform a full wavelength scan in the wavelength range of 200 nm to 800 nm using a UV-Vis spectrophotometer, and record the maximum absorption wavelength.

[0052] Mass spectrometry analysis: A trace amount of sample was dissolved in methanol and injected into the high-resolution mass spectrometer. Electrospray ionization was used as the ionization method, and a full scan was performed in positive ion mode with a scan range of m / z 100-1000. The capillary voltage was set to 3.5 kV, the ion source temperature was 100℃, and the desolvation gas temperature was 350℃.

[0053] Nuclear magnetic resonance (NMR) analysis: Weigh 15 mg of the sample and dissolve it in 0.5 mL of deuterated methanol (CD3OD), then place the solution in a 5 mm NMR tube. Using a 600 MHz NMR spectrometer with tetramethylsilane (TMS) as an internal standard, determine the proton NMR (1H-NMR), carbon NMR (13C-NMR), and two-dimensional correlation spectra (HMBC, HSQC, COSY) at 25 °C.

[0054] Experimental data: Ultraviolet spectroscopy revealed a maximum characteristic absorption peak at 532 nm in acidic methanol solution. High-resolution mass spectrometry analysis showed a quasi-molecular ion peak [M+H]+ with a measured value of m / z 392.2691. Based on nitrogen rules and isotopic abundance ratios, its molecular formula was deduced to be C2. 25 H 33 N3O (theoretical value m / z 392.2696), with an unsaturation degree of 11.

[0055] The specific data attribution and analysis of the proton and carbon NMR spectra are shown in Table 1.

[0056] Table 1. NMR data of the products prepared in Example 1 (solvent: CD3OD)

[0057] Note: The "-" symbol in the table indicates no data or that the data is not applicable.

[0058] Based on the analysis of the above test data, the structure of the compound prepared by the method of the present invention is confirmed as follows: The characteristic absorption peak at 532 nm in the UV data indicates that this compound possesses a typical conjugated skeleton of sclerotinoids. The molecular formula, determined by HR-ESI-MS data, is C2. 25 H 33 N3O matches the target compound, cyclophosphamide.

[0059] The NMR data further revealed detailed structural features: Characteristics of methoxy groups: and The single-peak signal confirmed the presence of the methoxy group, and the HMBC spectrum showed that it was attached to the C-8 position of the pyrrole ring B.

[0060] Macrocyclic structure characteristics: A series of methylene signals appearing in the high-field region, combined with the correlation signals of C-12 and C-24, and C-2 and C-16 in the HMBC spectrum, confirmed that the macrocyclic structure was formed by the connection of pyrrole ring A and pyrrole ring C through aliphatic chains.

[0061] Ethyl side chain: The coupling signals of 1.06 (t) and 2.41 (q) clearly indicate the presence of the ethyl side chain, and the HMBC correlation point indicates that it is attached to the C-12 position of the pyrrole ring C.

[0062] In summary, the spectral data of the compound prepared in Example 1 are consistent with the standard data of mesocyclocarpine. This indicates that the fermentation culture of *Streptomyces citrus* YS-4, combined with a dual extraction and purification process using silica gel column, gel column, and reversed HPLC, can accurately separate structurally intact and purity-compliant mesocyclocarpine from complex metabolites. Specifically, the reversed HPLC step effectively removes structurally similar non-macrocyclic mesocyclocarpine impurities by utilizing polarity differences, ensuring the singleness of the final product.

[0063] Test Example 2: Effects of different process parameters on product yield and purity Experimental instructions and procedures: This experiment aims to compare the yield and purity of the products prepared in Examples 1-3 and Comparative Examples 1-7 to verify the necessity of key parameters in the extraction and purification process of this invention.

[0064] The specific steps are as follows: Sample preparation: Collect the dried solid powders obtained in the final step of Examples 1-3 and Comparative Examples 1-7 respectively.

[0065] Yield determination: The total mass of each group of samples was determined using a precision electronic balance (sensitivity 0.1 mg). Based on the initial fermentation volume (10 L for all samples), the product yield per unit volume of fermentation broth was calculated. The calculation formula is: Yield (mg / L) = Total mass of final product (mg) / 10 (L).

[0066] Purity testing: Take an appropriate amount of each sample and dissolve it in chromatographically pure methanol to prepare a test solution of 0.5 mg / mL. Detection was performed using high-performance liquid chromatography (HPLC). Chromatographic conditions were: C18 analytical column (4.6 × 250 mm, 5 μm), mobile phase methanol-water (75:25), flow rate 1.0 mL / min, detection wavelength 531 nm, and injection volume 10 μL. Chromatograms were recorded, and the percentage content of the main peak of cyclophosphamide was calculated using the peak area normalization method, which represents the product purity.

[0067] Experimental data: The yield and purity test data of each group of samples are shown in Table 2.

[0068] Table 2. Summary of yield and purity data of cyclophosphamide under different process conditions

[0069] Table 2 shows the influence of key parameters in the extraction and purification process on the final result, verifying the synergistic effect of the technical solution of this invention: The yield of Example 1 was higher than that of Comparative Examples 1 and 2 (9.12 mg / L). Data from Comparative Examples 1 and 2 showed that cyclophosphamide erythrin exhibited a distribution pattern of both extracellular secretion and intracellular accumulation in the fermentation system, with a slightly higher content in the cell fraction. Using only a single solvent for extraction of the supernatant or cells would result in approximately 50% product loss. This invention employs a dual extraction strategy combining ethyl acetate extraction of the supernatant and methanol soaking of the cells, achieving maximum product recovery.

[0070] Comparative Example 3 used a 10:1 low-polarity eluent, resulting in excessive adsorption of the target product on the column, making elution difficult and reducing the yield to 4.26 mg / L. Comparative Example 4 used a 1:1 high-polarity eluent, which yielded the highest total solids (215.8 mg), but the purity was only 68.5%, indicating that a large amount of polar impurities co-eluted with the target product, leading to a decrease in crude product quality and increasing the burden on subsequent purification. Example 1 used a 3:1 elution ratio, balancing elution capacity and separation selectivity, preliminarily removing impurities while ensuring yield.

[0071] In Comparative Example 5, omitting the gel column chromatography step resulted in a product purity of 86.3%, indicating the necessity of gel chromatography for removing pigments and impurities with significant molecular weight differences. In the HPLC purification step, Comparative Example 6 (50:50) suffered from prolonged retention time and peak broadening due to an excessively high aqueous phase ratio, affecting both yield and purity. Comparative Example 7 (90:10) exhibited reduced separation due to an excessively high organic phase ratio, failing to effectively separate structurally similar homologues, resulting in a purity of only 82.4%. Example 1, using a 75:25 mobile phase system, utilized the specific hydrophobic properties of metacyclophosphamide to achieve effective separation from structurally similar compounds, ultimately yielding a high-purity product with 98.6%.

[0072] In summary, this invention has developed a dedicated purification process for the metabolites of Streptomyces citrus YS-4 by systematically optimizing the extraction method, chromatography medium, and elution parameters, thus solving the technical problems of low yield and difficulty in removing impurities in traditional methods.

[0073] Test Example 3: Comparison Test of Yield of Production Strains Experimental instructions and procedures: This experiment aims to compare the yield of cypermethrin from the *Streptomyces citrus* YS-4 strain screened in this invention with that of conventional cypermethrin-producing strains (*Serratia marcescens*) in the prior art, in order to verify the high-yield characteristics of strain YS-4.

[0074] The specific steps are as follows: Fermentation grouping: Two experimental groups were set up. Experimental group: *Streptomyces citrus* YS-4 from Example 1. Control group: *Serratia marcescens* from Comparative Example 8.

[0075] Parallel experiments: To eliminate random errors, three parallel 10L fermentation batches were set up for each group. The two groups of strains were cultured according to their respective optimal fermentation conditions (the experimental group followed the conditions of Example 1, and the control group followed the conditions of Comparative Example 8).

[0076] Sampling and processing: After fermentation, the crude organic extract was prepared strictly according to the solid-liquid separation, dual extraction and concentration process of Example 1 of this invention.

[0077] Quantitative detection: The crude extracts from each batch were dissolved and diluted to a fixed volume, and quantified using high-performance liquid chromatography (HPLC) with external standard method. The chromatographic conditions were the same as in Test Example 2. A standard curve was plotted using the high-purity mesocyclophosphamide prepared in Example 1 as the standard, and the final potency (mg / L) of mesocyclophosphamide in each batch of fermentation broth was calculated.

[0078] Experimental data: The yield data of cyclophosphamide in each batch of fermentation broth are shown in Table 3.

[0079] Table 3. Comparison of yield data between Streptomyces citrus YS-4 and Serratia marcescens.

[0080] Note: The fermentation cycle of Serratia marcescens in the control group was shorter (4 days). Extending the fermentation time to 7 days would lead to cell autolysis and product degradation. Therefore, the data at the highest yield point (day 4) was used for comparison.

[0081] The comparative data in Table 3 show that the *Streptomyces citrus* YS-4 selected in this invention has an advantage in the synthesis ability of cyclophosphamide: The experimental group (YS-4) had an average titer of approximately 17.15 mg / L of cyclophosphamide, while the control group (Serratia marcescens) had an average titer of only 1.73 mg / L. The yield per unit area of ​​the strain of this invention is approximately 10 times that of conventional strains.

[0082] Although the control group *Serratia marcescens* also produced red pigments, HPLC analysis showed that its main product was linear cymoxanil, while mesocyclic cymoxanil existed only as a trace byproduct. In contrast, the secondary metabolic network of *Streptomyces citrus* YS-4 was more inclined to synthesize mesocyclic cymoxanil with a macrocyclic structure, and the target product accounted for a very high proportion of the total erythrocytes.

[0083] Based on the total crude extract data, the extract obtained after double extraction of the YS-4 strain fermentation broth showed a high abundance of the target product, which is beneficial for subsequent separation and purification. In contrast, although the control group also obtained a certain amount of crude extract, the content of the target component was extremely low, making separation difficult and not valuable for the industrial preparation of cyclophosphamide.

[0084] In summary, *Streptomyces citrus* YS-4 is an excellent strain that specifically produces high levels of cyclophosphamide. Combined with the extraction and purification process of this invention, this compound can be prepared efficiently.

[0085] Test Example 4: Bioactivity Assay Experimental instructions and procedures: This experiment used the micro-broth dilution method to determine the minimum inhibitory concentration of meclofenoxam prepared in Example 1 against common pathogenic bacteria, in order to evaluate its pharmacological activity and application potential.

[0086] The specific procedures are as follows: Test strains: Representative strains of Gram-positive bacteria: Staphylococcus aureus and Bacillus subtilis; Representative strains of Gram-negative bacteria: Escherichia coli and Pseudomonas aeruginosa.

[0087] Sample preparation: Accurately weigh the pure cyclophosphamide erythrin prepared in Example 1, and dissolve it in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1280 μg / mL. The positive control drug was penicillin sodium, which was also prepared into a stock solution.

[0088] MIC determination: Using sterile 96-well plates, the test samples and positive controls were serially diluted with Mueller-Hinton broth to obtain a concentration gradient from 64 μg / mL to 0.0625 μg / mL. Add 1 × 10⁻⁶ μg / mL to each well. 5 CFU / mL bacterial suspension. Growth control wells containing only bacterial suspension and culture medium, and blank control wells containing only culture medium.

[0089] Culture and observation: Place the 96-well plate in a 37℃ incubator and incubate statically for 18-24 hours. Observe with the naked eye and measure the OD using a microplate reader. 600 The MIC value is used to determine bacterial growth. The lowest drug concentration at which no bacterial growth occurs (clarity) is the MIC value for that drug on that bacterial strain. The assay should be repeated three times in parallel.

[0090] Experimental data: The MIC determination results for each strain are shown in Table 4.

[0091] Table 4. Results of determination of the minimum inhibitory concentration (MIC) of azoxystrobin against different indicator bacteria.

[0092] Note: >64 indicates that no significant antibacterial effect was observed at the maximum tested concentration of 64 μg / mL.

[0093] The bioactivity test data in Table 4 reveal that the mesoxadiazonin prepared in this invention has significant antibacterial selectivity, as detailed below: The MIC values ​​of the product in Example 1 against Staphylococcus aureus and Bacillus subtilis were 0.125 μg / mL and 0.25 μg / mL, respectively. Particularly against Staphylococcus aureus, its antibacterial activity was superior to the positive control drug sodium penicillin (0.5 μg / mL). This result confirms that the cyclophosphamide produced by the process of this invention retains the high biological activity of the natural product and can effectively disrupt the cell membrane integrity of Gram-positive bacteria or interfere with their metabolic processes.

[0094] The compound exhibits low activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), with MIC values ​​greater than 64 μg / mL. This difference is related to the hydrophobic macrocyclic backbone in its molecular structure. The macrocyclic structure makes it difficult to penetrate the dense outer lipopolysaccharide layer of Gram-negative bacteria, preventing the drug from reaching its target site.

[0095] Experimental data directly support the application potential of the product of this invention in the preparation of drugs against Gram-positive bacterial infections. Combined with the high yield and high purity advantages of the aforementioned embodiments, the technical solution provided by this invention offers a raw material foundation with industrial-scale prospects for the development of novel antibiotics.

Claims

1. A strain of *Ichthyophthirius multifiliis* that produces erythrin, characterized in that, The physiological and biochemical characteristics of the strain include: It can utilize glucose, sucrose, maltose, and mannitol as carbon sources, but cannot utilize fructose, xylose, and galactose; it can utilize L-leucine, L-serine, and potassium nitrate as nitrogen sources, but cannot utilize L-tyrosine and ammonium nitrate.

2. A process for isolating and purifying cyclophosphamide erythrin, characterized in that, The application of a cyclophosphamide erythrin-producing strain as described in any one of claims 1 includes the following steps: Step (1) Inoculate the production strain into PDB liquid fermentation medium for fermentation culture, and collect the fermentation supernatant and cell bodies after solid-liquid separation; Step (2) involves extracting the fermentation supernatant from step (1) with ethyl acetate and collecting the ethyl acetate phase; The bacterial cells described in step (1) were extracted by soaking in methanol, and the methanol extract was collected. The ethyl acetate phase and the methanol extract were combined and concentrated under reduced pressure to remove the solvent, yielding a crude organic extract. Step (3) Separate the crude organic extract from step (2) by silica gel column chromatography, and use a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 3:1 for isocratic elution, and collect the crude eluent containing mesoxadiazine. Step (4) After concentrating the crude eluent obtained in step (3), the crude eluent is subjected to Sephadex LH-20 gel column chromatography with methanol as the mobile phase to elute and collect the purified fraction containing mesoxane erythrin. Step (5) The purified component obtained in step (4) is separated by reverse semi-preparative high performance liquid chromatography. The elution is carried out using a methanol-water system with a volume ratio of 75:

25. The eluent of the target main peak is collected and dried to obtain mesoxadiazon red.

3. The process for isolating and purifying cyclophosphamide erythrin according to claim 2, characterized in that, In step (1), the fermentation conditions are as follows: The fermentation temperature is 26-30℃, the rotation speed is 160-200r / min, and the fermentation cycle is 6-8 days. The PDB liquid fermentation medium contains: Potato starch 5.0-8.0 g / L or potato boiled filtrate 200-300 g / L, glucose 15.0-25.0 g / L, balance distilled water, pH 7.0-7.

4.

4. The process for isolating and purifying cyclophosphamide erythrin according to claim 2, characterized in that, The specific operation of step (2) is as follows: The volume ratio of ethyl acetate to the fermentation supernatant is 1:1, and the extraction is performed 3 times. The volume of methanol is 2-3 times the wet volume of the bacterial cells, and the soaking and extraction are performed 3 times, with each extraction lasting 30-60 minutes.

5. The process for isolating and purifying cyclophosphamide erythrin according to claim 2, characterized in that, The sample loading method in step (3) is as follows: The crude organic extract was dissolved in methanol, then mixed and ground with column chromatography silica gel. The mass ratio of column chromatography silica gel to the crude organic extract was 0.8:1-1.2:

1. After the methanol had completely evaporated, the sample was loaded using a dry method.

6. The process for isolating and purifying metacyclophosphamide erythrin according to claim 2, characterized in that, The collection method in step (3) is as follows: The effluent was collected in segments and analyzed by thin-layer chromatography. Effluent with Rf values ​​between 0.2 and 0.3 and exhibiting a single main spot were combined.

7. The process for isolating and purifying mesoxadiazine according to claim 2, characterized in that, The elution flow rate in step (4) is 0.8-1.2 mL / min. Before loading the sample, the Sephadex LH-20 gel is soaked in methanol to swell for 12-24 h. After packing the column, the column is equilibrated with methanol at a flow rate of 1.5-2.5 mL / min for 6-8 h.

8. The process for isolating and purifying cyclophosphamide erythrin according to claim 2, characterized in that, The specific conditions for step (5) are as follows: The chromatographic column was a C18 reverse-phase semi-preparative column, the flow rate was 2.0-4.0 mL / min, the column temperature was 25℃-30℃, and the detection wavelength was 529-533 nm.

9. The process for isolating and purifying cyclophosphamide erythrin according to claim 2, characterized in that, In step (3), a wet packing method is used, and the silica gel column used has a mesh size of 200-300.

Images