A strain of streptomyces and its use for the production of pentostatin by fermentation

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to an antibiotic Streptomyces strain, and further disclosing its application in the fermentation production of pentostatin. This invention screened a high-yield pentostatin-producing antibiotic Streptomyces antibioticus FIM-PNT-23-66 using atmospheric pressure, room temperature plasma mutagenesis technology. This strain can ferment pentostatin to a high yield, with a pentostatin potency as high as 651 μg / mL, significantly increasing pentostatin production. Furthermore, it exhibits excellent genetic traits and is suitable for industrial fermentation production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to an antibiotic Streptomyces strain, and further disclosing its application in the fermentation production of pentostatin. Background Technology

[0002] Pentostatin (PNT) is a nucleoside antibiotic isolated in 1974 by Warner-Lambert from the fermentation broth of *Streptomyces antibioticus*. Pentostatin possesses broad-spectrum biological and pharmacological properties, including antibacterial, antitrypanogenic, anticancer, antiviral, herbicidal, insecticidal, and immunomodulatory effects, and is widely used clinically in the treatment of malignant tumors. As a highly effective adenosine deaminase inhibitor, pentostatin interferes with the metabolic processes of leukemia cells by inhibiting adenosine deaminase activity, preventing normal cell growth and proliferation, thereby exerting its therapeutic effect. It is a first-line treatment for acute T-cell, chronic lymphocytic, and hairy cell leukemia.

[0003]

[0004] Currently, pentostatin production mainly involves two routes: total chemical synthesis and microbial synthesis. However, chemical synthesis methods have many serious drawbacks, such as long synthetic routes, harsh reaction conditions, extremely low production efficiency, and high costs due to environmental requirements, making industrial-scale production difficult. While biosynthetic PNTs have been discovered in microorganisms such as Streptomyces, Actinomycetes, Aspergillus, and Cordyceps, their yields are low, fermentation processes are unstable, and production costs are high, resulting in persistently high prices and difficulty in meeting demand. In China, PNTs often experience supply shortages and lack market competitiveness.

[0005] Currently, research on the biosynthesis of pentostatin mainly focuses on strain selection and fermentation processes. Optimizing the external production environment of microbial strains, including fermentation parameters such as culture medium composition, temperature, and pH, aims to improve pentostatin synthesis efficiency. For example, in 2013, Zou Xin of the Fujian Institute of Microbiology optimized the fermentation conditions of antibiotic Streptomyces FIM06-063, achieving a pentostatin yield of 183 mg / L. In 2019, Zheng Xiaoxian of the Fujian Institute of Microbiology discovered that adding vegetable oil significantly improved the pentostatin production capacity of antibiotic Streptomyces. Through optimized fermentation processes, antibiotic Streptomyces FIM0426 achieved a shake-flask fermentation yield of 232 mg / L, and its pentostatin fermentation level in a 5-ton scale tank reached 210 mg / L, but this still falls short of industrial-scale production standards.

[0006] Atmospheric pressure room temperature plasma breeding technology (ARTP) is a rapid microbial genome mutation technology. The plasma generated is rich in various chemically active particles, causing multiple effects on the genetic material of the strain cells, including damage to cell membrane permeability and alterations in protein structure. This triggers the SOS repair mechanism in the cells, generating a wide variety of mismatch sites during the repair process, resulting in a high mutation rate. This technology has been successfully applied in the breeding of various industrial microorganisms. Therefore, the field seeks to obtain a strain suitable for industrial production and capable of efficiently producing pentostatin, which would be of positive significance for realizing the industrial production of pentostatin. Summary of the Invention

[0007] Therefore, the first technical problem to be solved by the present invention is to provide an antibiotic streptomycin strain that can efficiently ferment and produce pentostatin, so as to solve the problem of low fermentation capacity of pentostatin strains in the prior art.

[0008] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned antibiotic Streptomyces strain for the fermentation production of pentostatin.

[0009] The third technical problem to be solved by the present invention is to provide a method for producing pentostatin by fermentation based on the above-mentioned antibiotic Streptomyces strain.

[0010] To solve the above-mentioned technical problems, the present invention provides an antibiotic Streptomyces strain, strain number FIM-PNT-23-66, classified as Streptomyces antibioticus, which has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 67030, deposit date of September 25, 2025, and address of Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou.

[0011] The present invention also provides the application of the antibiotic Streptomyces strain in the fermentation production of pentostatin.

[0012] The present invention also provides a method for producing pentostatin by fermentation, comprising the step of inoculating the antibiotic Streptomyces strain into a suitable fermentation medium for fermentation culture.

[0013] Specifically, in the method for producing pentostatin by fermentation, the fermentation medium comprises the following components in the indicated mass percentages: dextrin 0.5-1.5 wt%, glucose 1.5-4.5 wt%, soybean flour 2.0-5.0 wt%, yeast powder 0.05-0.45 wt%, peptone 0.1-0.5 wt%, cottonseed oil 0.25-1.25 wt%, calcium carbonate 0.1-0.6 wt%, with a natural pH.

[0014] Preferably, the fermentation medium comprises the following components in the indicated mass percentages: 1.0 wt% dextrin, 3.0 wt% glucose, 3.5 wt% soybean flour, 0.25 wt% yeast powder, 0.3 wt% peptone, 0.75 wt% cottonseed oil, and 0.35 wt% calcium carbonate, with a natural pH.

[0015] Specifically, the fermentation production method for pentostatin includes the following conditions for the fermentation culture step: controlling the rotation speed at 100-280 rpm and fermenting at 25-30℃ for 96-192 hours.

[0016] Specifically, the method for producing pentostatin by fermentation further includes the step of inoculating the antibiotic Streptomyces strain into a seed culture medium for seed liquid culture.

[0017] The seed culture medium comprises the following components in the indicated mass percentages: glucose 0.5-3.5 wt%, soybean flour 0.5-3.5 wt%, sodium chloride 0.05-0.35 wt%, calcium carbonate 0.05-0.45 wt%, with a natural pH.

[0018] Preferably, the seed culture medium comprises the following components in the indicated mass percentages: 2.0 wt% glucose, 2.0 wt% soybean flour, 0.2 wt% sodium chloride, 0.25 wt% calcium carbonate, with a natural pH.

[0019] Specifically, in the method for producing pentostatin by fermentation, the conditions for the seed culture step include: controlling the rotation speed at 100-280 rpm and culturing the seed culture at 25-30℃ for 36-60 hours.

[0020] Specifically, the method for producing pentostatin by fermentation further includes the step of activating the antibiotic Streptomyces strain by inoculating it into an agar slant culture medium;

[0021] The slant culture medium comprises the following components in the indicated mass percentages: yeast extract 0.05-0.55 wt%, casein 0.05-0.55 wt%, glucose 8-12 wt%, agar 1.5-2.5 wt%, with a natural pH.

[0022] Preferably, the slant culture medium comprises the following components in the indicated mass percentages: 0.25 wt% yeast extract, 0.3 wt% casein, 10 wt% glucose, 2.0 wt% agar, with a natural pH.

[0023] Specifically, in the method for producing pentostatin by fermentation, the activation step includes the following conditions: constant temperature culture at 25-30℃ for 7-12 days.

[0024] This invention screened a high-yield pentostatin-producing antibiotic *Streptomyces antibioticus* FIM-PNT-23-66 using atmospheric pressure, room temperature plasma mutagenesis. This strain is capable of fermenting pentostatin to produce high yields. In fermentation experiments, the *Streptomyces antibioticus* FIM-PNT-23-66 produced pentostatin at a potency of 663 μg / mL, significantly increasing pentostatin production. Furthermore, its low impurity content makes it more suitable for industrial-scale fermentation production. The *Streptomyces antibioticus* FIM-PNT-23-66 strain screened in this invention exhibits good stability; its pentostatin potency remained relatively stable after five generations, maintaining a consistently high level, making it a suitable strain for further research and development. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0026] Figure 1 The curve showing the relationship between ARTP jetting time and lethality in the mutagenesis experiment;

[0027] Figure 2 This is the phylogenetic tree of the strain FIM-PNT-23-66 described in this invention. Detailed Implementation

[0028] This invention screened a high-producing pentostatin antibiotic Streptomyces FIM-PNT-23-66 using atmospheric pressure and room temperature plasma mutagenesis technology. The strain is classified as Streptomyces antibioticus and has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 67030, deposit date of September 25, 2025, and address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0029] In the following embodiments of the present invention, the culture medium involved includes:

[0030] The isolated plate and slant culture media consist of the following components by mass: yeast extract 0.25 wt%, casein 0.3 wt%, glucose 10 wt%, agar 2.0 wt%, natural pH, autoclaved at 121°C for 30 min;

[0031] The seed culture medium comprises the following components by weight: 2.0 wt% glucose, 2.0 wt% soybean flour, 0.2 wt% sodium chloride, and 0.25 wt% calcium carbonate, with natural pH and autoclaved at 121°C for 30 min.

[0032] The fermentation medium comprises the following components by mass: dextrin 1.0 wt%, glucose 3.0 wt%, soybean flour 3.5 wt%, yeast powder 0.25 wt%, peptone 0.3 wt%, cottonseed oil 0.75 wt%, and calcium carbonate 0.35 wt%. The pH is natural, and the medium is autoclaved at 121°C for 30 min.

[0033] In the following embodiments of the present invention, the pentostatin content was detected using high-performance liquid chromatography (HPLC). The method is as follows: An appropriate amount of fermentation broth was taken, centrifuged at 4500 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm membrane to obtain the fermentation test solution, which was then analyzed by HPLC. The chromatographic conditions included: a C18 column (4.6 mm × 250 mm, 5 μm), a detection wavelength of 280 nm, a flow rate of 1 mL / min, a column temperature of 40 °C, a mobile phase of methanol-ultrapure water (84:16), and an injection volume of 10 μL. Pentostatin standard was used as the reference standard, and the fermentation titer was indicated by the average of three detection results.

[0034] Example 1

[0035] Obtaining the starting strain FIM-PNT-23

[0036] The pentostatin-producing Streptomyces FIM 06-063 strain, preserved in the strain bank of Fujian Institute of Microbiology, was inoculated onto slant agar and cultured in a constant temperature incubator for 7-12 days at 30℃. The fresh mycelial growth was then isolated and purified. Single colonies of different morphologies were selected for fermentation screening to obtain a relatively stable pentostatin-producing Streptomyces strain, which was named Streptomyces FIM-PNT-23 and preserved in glycerol.

[0037] Obtaining the mutant strain FIM-PNT-23-66

[0038] Using the genetically stable antibiotic Streptomyces FIM-PNT-23 strain as the starting strain, this strain was transferred to slant agar and cultured in a constant temperature incubator for 7-12 days at 28℃. Afterwards, the spores on the slant agar were washed off with physiological saline, the glass beads were dispersed, and the mixture was filtered through gauze to prepare 10... 6 A spore suspension of 1 spore per mL.

[0039] 10 μL of the spore suspension prepared above was pipetted onto a circular iron plate with a diameter of 1 cm. The plate was placed in a room-temperature plasma mutagenesis system at atmospheric pressure using helium as the working gas, a power supply of 110 W, and a working gas flow rate of 10 L / min. The treatment distance was 2 mm. Treatment times were 15 s, 20 s, 25 s, 30 s, 40 s, 50 s, 60 s, 70 s, and 80 s. The treated spore suspension was then serially diluted and plated to create lethality curves. Figure 1 As shown, there is a clear dose-response relationship between the mutagenesis treatment dose and the lethality of the FIM-PNT-23 strain; the lethality gradually increases with the extension of treatment time.

[0040] Based on the lethality curve described above, an irradiation time of 40 seconds was selected to induce plasma mutagenesis in the spore suspension of strain FIM-PNT-23. The treated spore suspension was then placed in a test tube containing physiological saline and mixed thoroughly to obtain a mutagenic spore suspension for later use. The obtained spore suspension was spread onto agar plates containing streptomycin at concentrations of 2.5 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L. After incubation at 28°C for 10 days, colony growth on different plates was observed, and the results are shown in Table 1. The lowest effective concentration of streptomycin corresponding to the plates where no colonies grew was the minimum inhibitory concentration (MIC) of streptomycin. Therefore, based on Table 1, the MIC of streptomycin was determined to be 15 mg / L.

[0041] Table 1. Effect of streptomycin concentration on spore growth of strain FIM-PNT-23

[0042]

[0043] The obtained mutant spore suspension was serially diluted to 10⁻⁶ dilutions. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Select 10 -4 10 -5 10 -6 Three dilutions of spore suspension were spread onto resistance isolation plates containing 15 mg / L streptomycin and incubated at 28°C in the dark for 7–12 days.

[0044] After transferring single colonies grown on resistance plates to slant culture medium and culturing for 7-12 days, they are inoculated into seed culture medium at an inoculation rate of 0.2-0.5% and cultured at 28℃ and 230 r / min for 36-60 h to obtain seed liquid. Seed liquid is then inoculated into fermentation culture medium at an inoculation rate of 10% and cultured at 28℃ and 230 r / min for 4-7 days to obtain fermentation broth.

[0045] Take an appropriate amount of the obtained fermentation broth, centrifuge to discard the bacterial residue, filter through a membrane to obtain the fermentation supernatant, use pentostatin standard as a reference, and determine the yield of pentostatin using conventional high performance liquid chromatography to confirm that the structure of the product in the fermentation broth of this embodiment is correct.

[0046] In this embodiment, the strain with the highest pentostatin production was screened using this method. For ease of description, the screened strain was named strain FIM-PNT-23-66, and strain FIM-PNT-23-66 was stored in glycerol.

[0047] Example 2

[0048] This embodiment is based on biological identification of the screened strain FIM-PNT-23-66.

[0049] Physiological and biochemical characteristics identification

[0050] The obtained strain FIM-PNT-23-66 was streaked on an isolation medium plate and a coverslip was inserted. The culture was carried out at 28°C for 5-15 days. The morphological characteristics of single colonies and their hyphae were observed using an optical microscope, a transmission microscope, and a scanning electron microscope.

[0051] The main morphological and physiological-biochemical characteristics of strain FIM-PNT-23-66 are as follows: Colonies on agar plates are round, with a slightly raised center and wrinkled surface after maturity, producing a water-soluble brownish pigment. Microscopic observation revealed that most spore hyphae are curved and annular, occasionally straight or loosely spiral; the spore chain length is 10-18 strands; spores are spherical to elliptical, 1.0-1.3 × 0.9-1.0 μm in size, with a smooth exoskeleton. It can liquefy gelatin and peptone milk, is nitrate-reducing positive, and can readily utilize starch, dextrin, rhamnose, glycerol, glucose, raffinose, mannitol, sucrose, and inositol, but not cellulose, galactose, xylose, fructose, or arabinose. This strain FIM-PNT-23-66 is a highly aerobic bacterium. Dissolved oxygen has a significant effect on the production of pentostatin during fermentation. The optimal growth temperature is 25-30℃. The highest pentostatin yield is achieved when the shaking speed is 100-280 rpm and the culture time is 4-7 days.

[0052] Molecular biological identification

[0053] The 16S rDNA sequence of strain FIM-PNT-23-66 was sequenced, and the sequencing results are shown in SEQ ID No. 1.

[0054] SEQ ID NO.1:

[0055]

[0056] The 16S rDNA sequence of the tested strain was compared with existing sequences in the GenBank database, and homology analysis was performed. The 16S rRNA gene sequence of the corresponding type strain was selected from the LPSN website (http: / / www.bacterio.cict.fr), and phylogenetic analysis was performed using CLUSTAL-X software. The generated alignment files were then subjected to phylogenetic analysis using the neighbor-joining method in TREECON software. Topological analysis was performed using the results of 1000 replicates. The results are attached. Figure 2 16S rDNA sequence analysis showed that strain FIM-PNT-23-66 had 100% sequence homology with Streptomyces antibioticus.

[0057] Based on the above morphological, physiological and biochemical characteristics and molecular biological identification, strain FIM-PNT-23-66 was finally identified as *Streptomyces antibioticus*, and its taxonomic name was determined to be...

[0058] Streptomyces antibioticus is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 67030 and deposit date of September 25, 2025.

[0059] Example 3

[0060] This embodiment uses the starting strain FIM-PNT-23 to synthesize pentostatin through fermentation.

[0061] Activate the strain FIM-PNT-23: Transfer the strain FIM-PNT-23 preserved in glycerol to the slant medium and incubate in a constant temperature incubator for 7-12 days at 28℃.

[0062] Preparation of FIM-PNT-23 seed culture: Single colonies of the above-mentioned strain FIM-PNT-23 were inoculated into seed culture medium (100 mL of seed culture medium in a 500 mL Erlenmeyer flask) and cultured at 28 °C and 230 r / min for 48 h to obtain seed culture.

[0063] Fermentation culture: The prepared seed liquid was inoculated into fermentation medium (100 mL fermentation medium in a 500 mL Erlenmeyer flask) at a 10% (v / v) inoculation rate and fermented at 28℃ and 230 r / min for 168 h. The resulting fermentation broth was then tested.

[0064] The test results showed that the yields of pentostatin in the three batches of shake-flask fermentation were 183 μg / mL, 182 μg / mL, and 186 μg / mL, respectively. Further determination of the pentostatin purity in each batch of fermentation broth revealed values ​​of 13.5%, 12.8%, and 14.9%, respectively.

[0065] Example 4

[0066] This embodiment uses the mutant strain FIM-PNT-23-66 to synthesize pentostatin through fermentation.

[0067] Activate strain FIM-PNT-23-66: Transfer strain FIM-PNT-23-66 preserved in glycerol to slant culture medium and incubate in a constant temperature incubator for 7-12 days at a temperature of 28℃.

[0068] Preparation of FIM-PNT-23-66 seed culture: The approximately 0.5cm × 0.5cm mycelial growth obtained from the activation of the above strain FIM-PNT-23-66 was inoculated into seed culture medium (100mL of seed culture medium in a 500mL Erlenmeyer flask) and cultured at 28℃ and 230r / min for 46h to obtain the seed culture.

[0069] Fermentation culture: The prepared seed liquid was inoculated into fermentation medium (100 mL fermentation medium in a 500 mL Erlenmeyer flask) at a 10% (v / v) inoculation rate and fermented at 28℃ and 230 r / min for 168 h. The resulting fermentation broth was then tested.

[0070] The test results showed that the yields of pentostatin in the three shake-flask fermentations were 653 μg / mL, 651 μg / mL, and 656 μg / mL, respectively. Further determination of the pentostatin purity in the fermentation broth revealed values ​​of 45.5%, 45.8%, and 46.9%, respectively.

[0071] It is evident that the strains screened in this invention can not only efficiently ferment pentostatin, but also produce pentostatin with higher purity in the fermentation broth and lower impact from byproducts.

[0072] Example 5

[0073] This embodiment uses the mutant strain FIM-PNT-23-66 to produce pentostatin through fermentation.

[0074] Shake-flask seed culture: The above-mentioned strain FIM-PNT-23-66 bacterial moss was inoculated into seed culture medium (280 mL of seed culture medium in a 1000 mL Erlenmeyer flask) and cultured at 28℃ and 230 r / min for 46 h to obtain shake-flask seed solution.

[0075] Seed culture in seed tank: The seed culture in the shake flask was inoculated at a rate of 0.5% into the seed culture medium (70L of seed culture medium in a 100L tank), and cultured for 43h at a temperature of 28℃, a tank pressure of 0.05MPa, an air flow rate of 1:1vvm, and a stirring speed of 100-200r / min to obtain the seed culture in the seed tank.

[0076] Fermentation culture in a fermenter: The prepared seed liquid was inoculated into the fermentation medium (700L of fermentation medium in a 1-ton tank) at an inoculation rate of 10% (v / v). The fermentation was carried out at a culture temperature of 28℃, a tank pressure of 0.05MPa, an air flow rate of 1:0.8-1.8vvm, a stirring speed of 100-280r / min, and dissolved oxygen was controlled to be no less than 30%. The fermentation culture was carried out for 168h, and the fermentation broth was tested after being discharged from the tank.

[0077] The test results showed that the pentostatin yields from the three fermentation batches were 663 μg / mL, 658 μg / mL, and 660 μg / mL, respectively. Further analysis of the pentostatin purity in the fermentation broth revealed values ​​of 45.6%, 47.2%, and 48.1%, respectively.

[0078] It is evident that the strains screened in this invention can not only efficiently ferment and synthesize pentostatin, but also produce pentostatin with superior purity in the fermentation broth.

[0079] Example 6

[0080] This embodiment uses strain FIM-PNT-23-66 to verify genetic stability.

[0081] The high-yielding pentostatin strain FIM-PNT-23-66, which was screened and preserved above, was continuously cultured and passaged (F1, F2, F3, F4, F5, F6) according to the method in Example 4. After fermentation in 500 mL shake flasks, the fermentation titer was determined. The well-grown primary strain (F0) was used as a control. The results are shown in Table 2 below.

[0082] Table 2. Effects of passage on the production of pentostatin by strain FIM-PNT-23-66

[0083] strain generation F0 F1 F2 F3 F4 F5 F6 Relative valence (%) 100 100.7 100.2 98.9 96.9 95.3 88.6

[0084] As shown in Table 2 above, the fermentation level of strain FIM-PNT-23-66 screened in this invention was not significantly affected after five generations, and its potency of producing pentazocine remained basically stable at the same high level, thus indicating that strain FIM-PNT-23-66 has good genetic stability characteristics.

[0085] In summary, the target strain of this invention, FIM-PNT-23-66, produces pentostatin at a rate of no less than 651 μg / mL, and the purity of pentostatin in the fermentation broth is no less than 45%. This improves the content of the target product, effectively controls impurities, and facilitates the downstream purification and preparation of pentostatin. The mutant strain *Streptomyces violaceum* FIM-PNT-23-66 (*Fusarium solani*) screened in this invention can serve as a production strain for further research and development.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antibiotic Streptomyces strain, with strain number FIM-PNT-23-66 and classification name Streptomyces antibioticus, has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 67030 and deposit date of September 25, 2025.

2. The application of the antibiotic Streptomyces strain of claim 1 in the fermentation production of pentostatin.

3. A method for producing pentostatin by fermentation, characterized in that, The step includes inoculating the antibiotic Streptomyces strain of claim 1 into a suitable fermentation medium for fermentation culture.

4. The method for producing pentostatin by fermentation according to claim 3, characterized in that, The fermentation medium comprises the following components in the indicated mass percentages: dextrin 0.5-1.5 wt%, glucose 1.5-4.5 wt%, soybean flour 2.0-5.0 wt%, yeast powder 0.05-0.45 wt%, peptone 0.1-0.5 wt%, cottonseed oil 0.25-1.25 wt%, calcium carbonate 0.1-0.6 wt%, with a natural pH.

5. The method for producing pentostatin by fermentation according to claim 3 or 4, characterized in that, The conditions for the fermentation culture step include: controlling the rotation speed at 100-280 rpm and fermenting at 25-30℃ for 96-192 hours.

6. The method for producing pentostatin by fermentation according to any one of claims 3-5, characterized in that, It also includes the step of inoculating the antibiotic Streptomyces strain into a seed culture medium for seed culture.

7. The method for producing pentostatin by fermentation according to claim 6, characterized in that, The seed culture medium comprises the following components in the indicated mass percentages: glucose 0.5-3.5 wt%, soybean flour 0.5-3.5 wt%, sodium chloride 0.05-0.35 wt%, calcium carbonate 0.05-0.45 wt%, with a natural pH.

8. The method for producing pentostatin by fermentation according to claim 6 or 7, characterized in that, The conditions for the seed culture step include: controlling the rotation speed at 100-280 rpm and culturing the seed culture at 25-30℃ for 36-60 hours.

9. The method for producing pentostatin by fermentation according to any one of claims 3-8, characterized in that, It also includes the step of activating the antibiotic Streptomyces strain by inoculating it into an agar slant culture medium; The slant culture medium comprises the following components in the indicated mass percentages: yeast extract 0.05-0.55 wt%, casein 0.05-0.55 wt%, glucose 8-12 wt%, agar 1.5-2.5 wt%, with a natural pH.

10. The method for producing pentostatin by fermentation according to claim 9, characterized in that, The activation step is performed under the following conditions: constant temperature incubation at 25-30℃ for 7-12 days.

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