Strain for fermentation production of gentamicin B and application thereof

Abstract

The micromonospora echinospora HS-GT101-23 with high yield of gentamicin B is obtained by screening through a normal-pressure and room-temperature plasma mutagenesis technology, the fermentation process is simple, high yield of gentamicin B can be achieved through fermentation without material supplementation, and in a shake flask experiment, the titer of gentamicin B produced through fermentation of the strain reaches 4126 mu g / mL; the strain can reach 3881 mu g / mL on a fermentation tank, and is suitable for industrial fermentation production. The screened strain HS-GT101-23 is good in stability, and the titer of gentamicin B produced by the strain HS-GT101-23 is basically stabilized at the same high level after continuous passage for 5 generations, so that a foundation is laid for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a Micromonospora echinospora strain with high gentamicin B production and application of the strain in fermentation production of gentamicin B. Background Art

[0002] Gentamicin is an aminoglycoside antibiotic that plays an important role in clinical practice. It was first isolated from Micromonospora purpurea and Micromonospora echinospora by Schering Company in the United States in 1963 and put into use in the United States in 1969. Gentamicin has been widely used in clinical practice due to its broad spectrum antibacterial activity, rapid bactericidal activity, and low price, and once became the drug of choice for the treatment of Gram-negative bacterial infections.

[0003] The main technical bottleneck of producing gentamicin B by microbial fermentation is its low yield. Chinese patent application number 201710932451.2 discloses a method for producing gentamicin B using gentamicin B fermentation broth. 3 The fermentation unit of the fermentation tank is controlled at 800μg / mL, and its highest disclosed potency is 821μg / mL. Chinese patent application number 200810070844.8 discloses a production process of gentamicin B, and the final potency of gentamicin B is 1000μg / mL. Chinese patent application number 201510632886.6 discloses a method for constructing a gentamicin B production bacterium, by genetically engineering the gentamicin production bacterium, expressing the exogenous genes kanJ and kanK, catalyzing J1-20A to form gentamicin B, and increasing the shake flask fermentation unit of gentamicin B to 780μg / mL, which is 92 times higher than the original strain. Another example is the gentamicin B production bacteria and its preparation method and application disclosed in Chinese patent CN 109897862 A, which uses genetic engineering technology to replace the genK gene with genR and genS, strengthens the gentamicin B biosynthesis pathway to increase the yield of the target product, and ultimately increases the yield of gentamicin B to 2792 μg / mL. Although the above patents have improved the fermentation level of gentamicin B to varying degrees, overall, as a production strain, the fermentation level of gentamicin B is still low, the production cost is high, which is not conducive to its commercial production and application, so better fermentation capacity is still expected.

[0004] Atmospheric pressure room temperature plasma technology (ARTP) is a technology that uses active particles in plasma to cause damage to cellular genetic material, so that microorganisms can start the SOS repair mechanism, produce DNA base mismatches, and increase the mutation rate. Because this technology has the characteristics of broad spectrum, high positive mutation rate and yield increase rate, strong controllability, safe and simple operation process, and stable characteristics of induced mutants, it has become an emerging strain mutagenesis breeding technology in recent years and has been widely used. Therefore, by adopting ARTP mutagenesis technology, it is expected to selectively increase the positive mutation rate of strain product yield, thereby screening out efficient strains that meet industrial production requirements. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a strain of Micromonospora aculeatus that produces gentamicin B by fermentation, so as to solve the problem that the fermentation performance of gentamicin B in the prior art is not ideal;

[0006] The second technical problem to be solved by the present invention is to provide an application of gentamicin B produced by fermentation of the above-mentioned Micromonospora aculeatus strain.

[0007] In order to solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is to provide a Micromonospora echinospora HS-GT101-23, which was deposited on November 7, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 32537, and was registered to prove its survival.

[0008] The second technical solution provided by the present invention is to provide the use of Micromonospora aculeatus HS-GT101-23 in the fermentation production of gentamicin B or in the production of a pharmaceutical composition containing gentamicin B.

[0009] Furthermore, the present invention provides a method for producing gentamicin B by fermentation, which comprises the step of fermenting Micromonospora aculeatus HS-GT101-23 in a culture medium containing assimilable carbon source and nitrogen source and inorganic salt, wherein the assimilable carbon source is selected from one or more of corn starch, soluble starch, dextrin, raffinose, mannitol, sucrose, lactose, maltose, glucose, sorbitol, and glycerol; the assimilable nitrogen source is selected from one or more of corn flour, yeast extract, yeast extract, yeast powder, peptone, tryptone, gluten powder, cottonseed cake powder, soybean cake powder, peanut cake powder, corn steep liquor powder, ammonium salt, and nitrate; and the inorganic salt is selected from one or more of ammonium sulfate, cobalt chloride, potassium nitrate, ammonium nitrate, calcium carbonate, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0010] Furthermore, the present invention provides a method for producing gentamicin B by fermentation, which comprises the steps of inoculating Micromonospora aculeatus HS-GT101-23 into a fermentation medium for fermentation.

[0011] Preferably, the fermentation medium comprises the following components by mass: 4-6% corn starch, 2-4% soybean cake powder, 0.2-0.4% peptone, 0.01-0.03% potassium nitrate, 0.3-0.6% gluten powder, 0.2-0.6% calcium carbonate, 0.2-0.5% ammonium sulfate, 0.0008% cobalt chloride, and the rest is water; the pH of the fermentation medium is 6.8-7.5; more preferably, the fermentation medium comprises the following components by mass: 5% corn starch, 3.5% soybean cake powder, 0.3% peptone, 0.02% potassium nitrate, 0.5% gluten powder, 0.5% calcium carbonate, 0.3% ammonium sulfate, 0.0008% cobalt chloride, and the rest is water; the pH of the fermentation medium is 7.0.

[0012] Preferably, the fermentation culture conditions include: fermentation culture at 34-38° C. for 144-192 h.

[0013] Furthermore, the method for producing gentamicin B by fermentation provided by the present invention further comprises the step of inoculating Micromonospora aculeatus HS-GT101-23 into a seed culture medium for seed liquid culture;

[0014] The seed culture medium comprises the following components by mass: 2-4% corn starch, 0.5-1.5% soybean meal, 0.2-0.5% yeast extract, 0.2-0.4% peptone, 0.01-0.02% potassium nitrate, 0.2-0.6% calcium carbonate, and the remainder is water; the pH of the seed culture medium is 6.8-7.5;

[0015] Preferably, the seed culture medium comprises the following components by mass: 3.5% corn starch, 1% soybean cake powder, 0.4% yeast extract, 0.3% peptone, 0.01% potassium nitrate, 0.5% calcium carbonate, and the remainder is water; the pH of the seed culture medium is 7.0.

[0016] Preferably, the step of culturing the seed solution comprises: culturing at 34-38° C. for 22-48 hours.

[0017] Furthermore, the method for producing gentamicin B by fermentation provided by the present invention further comprises the step of inoculating Micromonospora aculeatus HS-GT101-23 on a slant culture medium for activation;

[0018] The slant culture medium comprises the following components by mass: 1-3% corn starch, 2-3% soybean meal, 0.1-0.3% glucose, 0.2-0.5% yeast extract, 0.1-0.3% sodium chloride, 0.01-0.03% potassium dihydrogen phosphate, 0.1-0.2% potassium nitrate, 0.1-02% calcium carbonate, and 1.8-2.2% agar; the pH of the slant culture medium is 6.8-7.5; preferably, the slant culture medium comprises the following components by mass: 2% corn starch, 2.5% soybean meal, 0.2% glucose, 0.3% yeast extract, 0.2% sodium chloride, 0.02% potassium dihydrogen phosphate, 0.1% potassium nitrate, 0.1% calcium carbonate, and 2.0% agar; the pH of the slant culture medium is 72.

[0019] Preferably, the activation step comprises: culturing at 34-38° C. for 7-9 days.

[0020] In the present invention, the “Micromonospora aculeatus HS-GT101-23”, “strain HS-GT101-23”, “mutant HS-GT101-23” and “HS-GT101-23 strain” have the same meaning.

[0021] In the present invention, the “Micromonospora aculeatus HS-1520-016-89”, “strain HS-1520-016-89” and “original strain HS-1520-016-89” have the same meanings.

[0022] The present invention obtains a strain of Micromonospora echinospora HS-GT101-23 with high gentamicin B production by screening through atmospheric pressure room temperature plasma mutagenesis (ARTP) technology, which can ferment high-yield gentamicin B. In a shake flask, the titer of gentamicin B produced by the Micromonospora echinospora HS-GT101-23 fermentation is as high as 4126 μg / mL (in a fermenter, it can also reach 3881 μg / mL), which greatly improves the yield of gentamicin B and has a simple fermentation process (no need for feeding), so that the fermentation cost of the product is further reduced, and it is suitable for industrial fermentation production. Moreover, the strain HS-GT101-23 screened by the present invention has good stability, and the titer of gentamicin B produced by the strain is basically stable after 5 generations of continuous passage, and is maintained at the same high level, meeting the fermentation needs of industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the relationship curve between the ARTP mutagenesis time and the lethality of strain HS-1520-016-89 in the mutagenesis experiment; DETAILED DESCRIPTION

[0024] In the following embodiments of the present invention, the culture medium involved includes:

[0025] The solid culture medium for plates and slant surfaces includes the following components by mass content: 2% corn starch, 2.5% soybean meal, 0.2% glucose, 0.3% yeast extract, 0.2% sodium chloride, 0.02% potassium dihydrogen phosphate, 0.1% potassium nitrate, 0.1% calcium carbonate, 2.0% agar, pH 72, 121°C high pressure steam sterilization for 30 minutes;

[0026] The seed culture medium includes the following components by mass: 3.5% corn starch, 1% soybean meal, 0.4% yeast extract, 0.3% peptone, 0.01% potassium nitrate, 0.5% calcium carbonate, and the rest is water, pH 7.0, and sterilized by high pressure steam at 121°C for 30 minutes:

[0027] The fermentation medium includes the following components by mass: 5% corn starch, 3.5% soybean meal, 0.3% peptone, 0.02% potassium nitrate, 0.5% gluten powder, 0.5% calcium carbonate, 0.3% ammonium sulfate, 0.0008% cobalt chloride, and the rest is water, pH 7.0, and high-pressure steam sterilization at 121°C for 30 minutes.

[0028] In the following examples of the present invention, the gentamicin B content is detected by liquid chromatography-evaporative light scattering (HPLC-ELSD), specifically: the fermentation broth is adjusted to pH 1.8-2.0 with 6N concentrated sulfuric acid, and then centrifuged to obtain the supernatant after oscillation for 30 minutes, diluted 5 times with deionized water, mixed and then centrifuged to obtain the supernatant and enter the HPLC-ELSD system (Thermo Scientific) for detection. Detection conditions: chromatographic column LP-C18 (welch, 250×4.6mm, 5μm); mobile phase is 1.5% trifluoroacetic acid aqueous solution (phase A) and 95% methanol (phase B); flow rate is 1mL / min; mobile phase adopts gradient method, as follows: 0-12.5min 100% phase A; 12.5-15.5min 50% phase A and 50% phase B; 15.5-23min 100% phase A; electrospray conditions: atomization temperature 110℃, gas flow rate 2.7L / min. Gentamycin B standard was used as reference, and the potency was calculated based on the positive correlation between peak area and concentration.

[0029] In the following examples, unless otherwise specified, the reagents and instruments used are commonly used reagents and instruments in the field and can be purchased from chemical or biological product / preparation companies; the methods used in the following examples are conventional methods in the field, and those skilled in the art can undoubtedly know the operating procedures of these experiments and obtain the corresponding results based on the existing technology or the operating manuals provided by the manufacturer.

[0030] Example 1 Obtaining the mutagenic strain HS-GT101-23

[0031] 1. Preparation of single spore suspension

[0032] A single colony obtained by natural separation from Micromonospora echinospora HS-1520-016-89 preserved in the laboratory of Hisun Pharmaceutical (which is deposited in the China Type Culture Collection, the deposit address is Wuhan University, China, and the deposit number of the collection center is CCTCC NO: M 2018898; the deposit time is December 17, 2018, see CN109897862A) was transferred to a slant culture medium and cultured in a constant temperature incubator at 36.5°C for 8 days. The spores on the slant were then washed with physiological saline, broken up by oscillation with glass beads, and diluted appropriately to prepare a monospore suspension (OD600=0.6-0.8) of the original strain HS-1520-016-89.

[0033] 2. Screening of ARTP mutagenesis conditions

[0034] Take 10 μl of the prepared monospore bacterial suspension and spread it evenly on the surface of the metal slide. Use sterile tweezers to place the slide in the corresponding hole of the plasma chamber. Set the power supply to 120 W, the working gas flow to 10 SLM, the irradiation distance to 2 mm, and the irradiation time to 0, 15 s, 30 s, 45 s, 60 s, 75 s and 90 s respectively. Use sterile tweezers to place the treated slide into an EP tube containing 1 ml of normal saline, place the EP tube on an oscillator and oscillate for 1 min to form a new bacterial suspension. Perform gradient dilution on the new bacterial suspension, spread 100 μL of the dilution on a plate, and culture at 36.5 ° C for 8 days. Use the unirradiated bacterial suspension to spread on the plate as a control, observe the growth of the colonies, and count the colonies. Investigate the relationship between irradiation time and lethality to prepare a lethality curve (such as Figure 1 shown).

[0035] 3. Tolerance of the original strain to gentamicin B

[0036] The single spore suspension of the original strain HS-1520-016-89 prepared above was gradiently diluted and then spread on solid culture media containing different concentrations of gentamicin B. The concentrations of gentamicin B in the solid culture media were 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, and 5.5 g / L, respectively. The culture was carried out at 36.5°C for 8 days, and the growth of the colonies was observed. The results are shown in Table 1. The minimum inhibitory concentration against gentamicin B was determined to be 5 g / L as shown in Table 1.

[0037] Table 1 Effect of gentamicin B concentration on spore production of strain HS-1520-016-89

[0038]

[0039] Note: + means colony growth, - means no colony growth.

[0040] IV. ARTP mutagenesis

[0041] according to Figure 1 The lethality curve was obtained by repeatedly inducing plasma mutagenesis on the spore suspension of strain HS-1520-016-89 with two different lethal doses of irradiation time of 45s and 60s. The induced bacterial suspension was gradiently diluted and spread on a solid culture medium containing 5g / L gentamicin B and cultured at 36.5℃ in the dark for 8 days.

[0042] 5. Screening of mutagenic strains

[0043] The single colonies grown on the above-mentioned resistance plates were transferred to the slant culture medium and cultured for 8 days, then transferred to the seed culture medium, and cultured at 36.5°C and 250rpm for 48h to obtain the seed liquid; the seed liquid was then inoculated into the fermentation medium with a liquid volume of 25ml / 250ml, an inoculation amount of 10%, a shaking speed of 250rpm, a culture temperature of 36.5°C, and a culture period of 7d to obtain the fermentation liquid.

[0044] The pH of the fermentation broth was adjusted to 1.8-2.0 with 6N concentrated sulfuric acid, and the supernatant was centrifuged after oscillation for 30 minutes, diluted 5 times with deionized water, mixed, and centrifuged again to obtain the supernatant for detection by HPLC-ELSD system (Thermo Scientific).

[0045] By this method, the mutagenic strain HS-GT101-23 with the largest gentamicin B production was screened out, and its titer was as high as 4126μg / mL. Under the same conditions, compared with the titer of 731μg / mL of the original strain HS-1520-016-89, the titer of the new strain HS-GT101-23 obtained by mutation increased by 464.4%, which is 5.64 times that of the original strain. The strain HS-GT101-23 was stored in a 20% glycerol tube in a -80℃ ultra-low temperature refrigerator.

[0046] Example 2 Morphological and cultural characteristics of strain HS-GT101-23

[0047] The experiment was conducted with reference to the relevant contents in books such as "Manual of Streptomyces Identification", "Classification and Identification of Actinomycetes", and "Manual of Identification of Common Bacterial Systems". The morphological and cultural characteristics of the strains were tested using 8 culture media, including ISP1, ISP2, ISP3, ISP4, ISP5, Gao's No. 1, nutrient agar, and calcium malate. After culturing at 36.5°C for 7 to 9 days, the growth status of the strains and the color of the mycelium were observed. The original strain HS-1520-016-89 was compared with the new strain HS-GT101-23, and the results are shown in Table 2.

[0048] Table 2 Comparison of culture characteristics of strains on 8 different media

[0049]

[0050] From the observation of the mycelium color of the strain, it can be seen that the growth status of the new strain HS-GT101-23 and the original strain HS-1520-016-89 is obviously different in several culture media.

[0051] Example 3 Physiological and biochemical characteristics of strain HS-GT101-23

[0052] (1) Utilization of carbon sources: ISP9 was used as the basic culture medium, and the final concentration of various carbon sources was 1.0% (mass percentage concentration). The results are shown in Table 3.

[0053] (2) Utilization of inorganic nitrogen source: ISP9 was used as the basic culture medium, and the concentrations of potassium nitrate and ammonium sulfate were both 0.1% (mass percentage concentration). The results are shown in Table 3.

[0054] Table 3 Utilization of carbon and nitrogen sources by strain HS-GT101-23

[0055]

[0056] (3) Physiological and biochemical tests, pH tests and temperature tests: The results of the physiological and biochemical tests are shown in Table 4; the pH test and temperature test were both conducted using ISP2 medium. The results showed that the optimal pH for the growth of this strain was 6.5-7.5; the strain could grow between 28°C and 45°C, and the optimal growth temperature was 37°C.

[0057] Table 4 Main physiological and biochemical characteristics of strain HS-GT101-23

[0058] Test Project result Test Project result Gelatin liquefaction + Nitrate reduction - Starch hydrolysis - Hydrogen sulfide production - Milk peptonization + Cellulose Utilization -

[0059] Note: The numbers and symbols in Table 3-4 represent: 0: no growth; 1: very weak growth; 2: able to grow; 3: good growth; 4: best growth; +: positive; -: negative.

[0060] Example 4 16S rDNA sequence analysis and bacterial species identification

[0061] The 16S rDNA sequence of strain HS-GT101-23 was sequenced, and the 16S rDNA sequence of the strain was compared with the sequences of related species and genera in the GenBank database by homologous sequence BLAST to determine the taxonomic status of the strain.

[0062] The 16S rDNA sequence of strain HS-GT101-23 was compared with the related sequences in GenBank by BLAST, and the results are shown in Table 5 (only strains with higher homology are listed in the table).

[0063] Table 5 Homology of strain HS-GT101-23 and related strains

[0064]

[0065]

[0066] The strain HS-GT101-23 was found to have 100% homology with Micromonospora echinospora through 16S rDNA region sequencing.

[0067] Based on the above morphological, culturological, physiological and biochemical characteristics and 16S rDNA sequence molecular biological identification, the strain HS-GT101-23 was finally determined to be of the genus Micromonospora echinospora, and its classification name was Micromonospora echinospora HS-GT101-23.

[0068] Example 5 Verification of genetic stability of strain HS-GT101-23

[0069] The gentamicin B high-producing strain HS-GT101-23 obtained by the above screening was transferred to the slant culture medium and cultured at a constant temperature of 36.5°C for 8 days to obtain the well-growing HS-GT101-23 primary slant (F0). The strain was continuously cultured and subcultured at a constant temperature of 36.5°C for 8 days to obtain the well-growing F1 to F5 slants respectively.

[0070] The F0 to F5 generations obtained from the above-mentioned strain HS-GT101-23 were cultured on plates respectively, and the single colonies grown were inoculated into seed culture medium, and cultured at 37°C and 250rpm for 48h to obtain seed liquid; the seed liquid was then inoculated into fermentation culture medium with a liquid volume of 25ml / 250ml, an inoculation amount of 10%, a shaking speed of 250rpm, a culture temperature of 36.5°C, and a culture period of 7d, and the fermentation liquid was obtained and tested.

[0071] The primary strain (F0) with good growth was used as the control, and its fermentation relative titer was set as 100%. The results are shown in Table 6.

[0072] Table 6 Effect of subculture on gentamicin B production by strain HS-GT101-23

[0073]

[0074] As shown in Table 6, the fermentation level of the strain HS-GT101-23 obtained by the present invention was not significantly affected by the 5th generation, and the titer of gentamicin B produced by the strain was basically stable and maintained at the same high level, indicating that the strain HS-GT101-23 has good genetic stability.

[0075] Example 6 Fermentation production of gentamicin B by mutagenic strain HS-GT101-23

[0076] After culturing the strain HS-GT101-23 on a slant at 36.5° C. for 8 days, the spores were scraped off and cultured in a seed culture medium with a loading volume of 20 mL / 250 mL, a culture temperature of 37° C., a rotation speed of 250 rpm, and a culture time of 48 h; then, the spores were inoculated into a primary seed tank (10 L seed culture medium in a 15 L tank) at a volume ratio of 0.2%, and the temperature was controlled at 36-38° C. and cultured for 44-48 h; then, the spores were inoculated into a secondary seed tank at a transplant volume ratio of 15%, and cultured at 37-38° C. for 22-26 h, completing the culture of the seed tank to obtain a secondary tank seed solution.

[0077] The prepared secondary tank seed liquid was inoculated into the fermentation medium (30L fermentation medium in a 50L tank) at a volume ratio of 20%, the culture temperature was 36-38°C, the tank pressure was 0.05Mpa, the ventilation volume was 1:0.5-1:1.5vvm, the stirring speed was 80-150r / min, the fermentation culture was 192h, and the tank was released for detection. The results showed that the yield of gentamicin B in this example was 3881μg / mL.

[0078] In summary, the present invention provides a Micromonospora echinospora HS-GT101-23, which has a simple fermentation process and can produce a high amount of gentamicin B without feeding, thereby greatly improving the yield of gentamicin B. Moreover, the strain has good stability, and the potency of gentamicin B produced by continuous passage for 5 generations is basically stable, so the strain can be applied to industrial fermentation production.

[0079] Although the specific embodiments of the present invention are described above, they are not intended to limit the present invention. Any modifications and changes in form and details made by any person skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A Micromonospora echinospora HS-GT101-23, whose deposit number is CGMCC No.32537.

2. Use of Micromonospora aculeatus HS-GT101-23 according to claim 1 in the fermentation production of gentamicin B or in the production of a pharmaceutical composition containing gentamicin B.

3. A method for producing gentamicin B by fermentation, characterized in that: The method comprises the step of fermenting the Micromonospora aculeatus HS-GT101-23 described in claim 1 in a culture medium containing assimilable carbon source and nitrogen source and inorganic salt, wherein the assimilable carbon source is selected from one or more of corn starch, soluble starch, dextrin, raffinose, mannitol, sucrose, lactose, maltose, glucose, sorbitol and glycerol; the assimilable nitrogen source is selected from one or more of corn flour, yeast extract, yeast extract, yeast powder, peptone, tryptone, gluten powder, cottonseed cake powder, soybean cake powder, peanut cake powder, corn steep liquor powder, ammonium salt and nitrate; and the inorganic salt is selected from one or more of ammonium sulfate, cobalt chloride, potassium nitrate, ammonium nitrate, calcium carbonate, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, magnesium sulfate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

4. A method for producing gentamicin B by fermentation, characterized in that: The method comprises the steps of inoculating the Micromonospora aculeatus HS-GT101-23 described in claim 1 into a fermentation medium for fermentation.

5. The method for producing gentamicin B by fermentation according to claim 4, characterized in that: The fermentation medium comprises components by weight: 4-6% corn starch, 2-4% soybean meal, 0.2-0.4% peptone, 0.01-0.03% potassium nitrate, 0.3-0.6% gluten powder, 0.2-0.6% calcium carbonate, 0.2-0.5% ammonium sulfate, 0.0008% cobalt chloride, and the rest is water; the pH of the fermentation medium is 6.8-7.5; Preferably, the fermentation medium comprises the following components by mass: 5% corn starch, 3.5% soybean cake powder, 0.3% peptone, 0.02% potassium nitrate, 0.5% gluten powder, 0.5% calcium carbonate, 0.3% ammonium sulfate, 0.0008% cobalt chloride, and the rest is water; the pH of the fermentation medium is 7.

0.

6. The method for producing gentamicin B by fermentation according to claim 4 or 5, characterized in that: The fermentation culture conditions include: fermentation culture at 34-38° C. for 144-192 hours.

7. The method for producing gentamicin B by fermentation according to any one of claims 4 to 6, characterized in that: The method further comprises the step of inoculating the Micromonospora aculeatus HS-GT101-23 of claim 1 into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components by mass: 2-4% corn starch, 0.5-1.5% soybean meal, 0.2-0.5% yeast extract, 0.2-0.4% peptone, 0.01-0.02% potassium nitrate, 0.2-0.6% calcium carbonate, and the remainder is water; the pH of the seed culture medium is 6.8-7.5; Preferably, the seed culture medium comprises the following components by mass: 3.5% corn starch, 1% soybean cake powder, 0.4% yeast extract, 0.3% peptone, 0.01% potassium nitrate, 0.5% calcium carbonate, and the remainder is water; the pH of the seed culture medium is 7.

0.

8. The method for producing gentamicin B by fermentation according to claim 7, characterized in that: The step of culturing the seed solution includes: culturing at 34-38° C. for 22-48 hours.

9. The method for producing gentamicin B by fermentation according to any one of claims 4 to 8, characterized in that: The invention also includes the step of inoculating the Micromonospora aculeatus HS-GT101-23 of claim 1 on a slant culture medium for activation; The slant culture medium comprises the following components by mass: 1-3% corn starch, 2-3% soybean meal, 0.1-0.3% glucose, 0.2-0.5% yeast extract, 0.1-0.3% sodium chloride, 0.01-0.03% potassium dihydrogen phosphate, 0.1-0.2% potassium nitrate, 0.1-0.2% calcium carbonate, and 1.8-2.2% agar; the pH of the slant culture medium is 6.8-7.5; preferably, the slant culture medium comprises the following components by mass: 2% corn starch, 2.5% soybean meal, 0.2% glucose, 0.3% yeast extract, 0.2% sodium chloride, 0.02% potassium dihydrogen phosphate, 0.1% potassium nitrate, 0.1% calcium carbonate, and 2.0% agar; the pH of the slant culture medium is 7.

2.

10. The method for producing gentamicin B by fermentation according to claim 9, characterized in that: The activation step includes: culturing at 34-38° C. for 7-9 days.

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