Strain for fermentation production of gentamicin b and use thereof

Abstract

Provided are a strain for fermentation production of gentamicin B and use thereof. A high-yield gentamicin B-producing strain, Micromonospora echinospora HS-GT101-23, is obtained through screening by means of atmospheric pressure and room temperature plasma mutagenesis technology. The fermentation process for the strain is simple and can achieve high-yield fermentation production of gentamicin B without the need for feeding. In a shake flask experiment, the titer of gentamicin B produced by the fermentation of the strain reaches 4126 μg / mL; in a fermentation tank, the titer can also reach 3881 μg / mL. Therefore, the strain is suitable for industrial fermentation production. The strain HS-GT101-23 obtained through screening has good stability, and the titer of gentamicin B produced by the strain over 5 consecutive passages remains basically stable at a consistently high level, which lays a foundation for industrial production.

Description

A strain for fermentation production of gentamicin B and its application Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of Micromonospora echinospora that produces gentamicin B at high yield and its application in the fermentation production of gentamicin B using this strain. Background Technology

[0002] Gentamicin is an aminoglycoside antibiotic that plays an important role in clinical practice. It was initially isolated by Schering in 1963 from *Micromonospora purpurea* and *Micromonospora echinospora*, and put into use in the United States in 1969. Due to its broad-spectrum antibacterial activity, rapid bactericidal action, and low cost, it has been widely used clinically and was once the first-line drug for treating Gram-negative bacterial infections.

[0003] The main technical bottleneck in the production of gentamicin B via microbial fermentation is its low yield. Chinese patent application number 201710932451.2 discloses a method for producing gentamicin B using gentamicin B fermentation broth, with a yield of 60m³. 3 The fermentation unit in the fermenter is controlled at 800 μg / mL, and its highest disclosed potency is 821 μg / mL. Chinese patent application number 200810070844.8 discloses a production process for gentamicin B, achieving a final potency of 1000 μg / mL. Chinese patent application number 201510632886.6 discloses a method for constructing a gentamicin B-producing strain. Through genetic engineering of the gentamicin-producing strain to express exogenous genes kanJ and kanK, it catalyzes the formation of gentamicin B from J1-20A, increasing the shake-flask fermentation unit of gentamicin B to 780 μg / mL, a 92-fold increase compared to the original strain. For example, Chinese patent CN 109897862 A discloses a gentamicin B producing strain, its preparation method, and its application. This strain uses genetic engineering to replace the genK gene with genR and genS, enhancing the gentamicin B biosynthetic pathway to increase the yield of the target product, ultimately raising the yield of gentamicin B to 2792 μg / mL. While these patents have improved the fermentation level of gentamicin B to varying degrees, overall, as a production strain, the fermentation level of gentamicin B remains relatively low, resulting in high production costs, which is detrimental to its commercial production and application. Therefore, a more efficient fermentation process is still anticipated.

[0004] Atmospheric pressure room temperature plasma (ARTP) is a technique that utilizes active particles in plasma to cause damage to the genetic material of cells, triggering the SOS repair mechanism in microorganisms and resulting in DNA base mismatches, thus increasing the mutation rate. Due to its broad applicability, high positive mutation rate and yield increase, strong controllability, safe and simple operation, and stable mutant traits, this technique has become a widely used emerging microbial mutagenesis and breeding technology in recent years. Therefore, by employing ARTP mutagenesis technology, it is hoped that the positive mutation rate of strain product yield can be selectively increased, thereby screening for highly efficient strains suitable for industrial production. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a strain of Micromonospora echinococcosis that ferments to produce gentamicin B, 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 echinococcosis strain.

[0007] To address the aforementioned technical problems, one of the technical solutions provided by this invention is to provide a strain of Micromonospora echinospora HS-GT101-23, which was deposited on November 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32537, and registered, proving its viability.

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

[0009] Furthermore, the present invention provides a method for fermenting and producing gentamicin B, the method comprising the step of fermenting Micromonas hydrophila HS-GT101-23 in a culture medium containing an assimilated carbon source, a nitrogen source, and inorganic salts, wherein the assimilated carbon source is selected from one or more of corn starch, soluble starch, dextrin, raffinose, mannitol, sucrose, lactose, maltose, glucose, sorbitol, and glycerol; the assimilated nitrogen source is selected from one or more of corn flour, yeast extract, yeast extract, yeast powder, peptone, tryptone, gluten flour, cottonseed meal, soybean meal, peanut meal, corn steep liquor powder, ammonium salts, and nitrates; the inorganic salts are 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, the method comprising the step of inoculating Micromonospora echinococcosis HS-GT101-23 into a fermentation medium for fermentation.

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

[0012] Preferably, the fermentation culture conditions include: fermentation culture at 34-38℃ for 144-192 hours.

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

[0014] The seed culture medium comprises the following components by weight: 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 being water; the pH of the seed culture medium is 6.8-7.5.

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

[0016] Preferably, the seed culture step includes: 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 includes the step of activating Micromonospora echinococcosis HS-GT101-23 by inoculating it onto an agar slant culture medium;

[0018] The slant culture medium comprises the following components by weight: 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 weight: 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.

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

[0020] Preferably, the cultivation process of Micromonospora echinococcosis HS-GT101-23 is as follows: inoculation onto slant culture medium for activation, seed culture in seed culture medium, and fermentation in fermentation culture medium. The strain is inoculated onto slant culture medium for activation, the activated strain is inoculated onto seed culture medium for seed culture, and the seed culture is inoculated onto fermentation culture medium for fermentation.

[0021] In this invention, "Micromonospora echinococcosis HS-GT101-23", "strain HS-GT101-23", "mutant HS-GT101-23", and "HS-GT101-23 strain" have the same meaning.

[0022] In this invention, "Micromonospora echinococcosis HS-1520-016-89", "strain HS-1520-016-89", and "original strain HS-1520-016-89" have the same meaning.

[0023] This invention utilizes ambient pressure, room temperature plasma mutagenesis (ARTP) technology to screen for a high-genapine B-producing strain of *Micromonospora echinospora* HS-GT101-23. This strain can ferment gentamicin B at high yields. In shake flasks, the gentamicin B potency produced by *Micromonospora echinospora* HS-GT101-23 reaches 4126 μg / mL (and 3881 μg / mL in fermenters), significantly increasing gentamicin B production. Furthermore, the fermentation process is simple (no feed required), further reducing fermentation costs and making it suitable for industrial-scale fermentation production. Moreover, the strain HS-GT101-23 exhibits good stability; its gentamicin B potency remains relatively stable at a high level after five consecutive passages, meeting the requirements for industrial-scale fermentation. Attached Figure Description

[0024] Figure 1 shows the relationship between ARTP mutagenesis time and the lethality of strain HS-1520-016-89 in the mutagenesis experiment. Detailed Implementation

[0025] In the following embodiments of the present invention, the culture medium involved includes:

[0026] Solid culture media, including plates and slant culture media, contain the following components by mass: corn starch 2%, soybean meal 2.5%, glucose 0.2%, yeast extract 0.3%, sodium chloride 0.2%, potassium dihydrogen phosphate 0.02%, potassium nitrate 0.1%, calcium carbonate 0.1%, agar 2.0%, pH 7.2, and are autoclaved at 121°C for 30 min.

[0027] The seed culture medium comprises the following components by weight: corn starch 3.5%, soybean meal 1%, yeast extract 0.4%, peptone 0.3%, potassium nitrate 0.01%, calcium carbonate 0.5%, with the remainder being water, pH 7.0, and autoclaved at 121°C for 30 min.

[0028] The fermentation medium comprises the following components by mass: 5% corn starch, 3.5% soybean meal, 0.3% peptone, 0.02% potassium nitrate, 0.5% gluten flour, 0.5% calcium carbonate, 0.3% ammonium sulfate, 0.0008% cobalt chloride, with the remainder being water. The pH is 7.0, and the medium is autoclaved at 121°C for 30 minutes.

[0029] In the following embodiments of the present invention, the gentamicin B content was detected using high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD). Specifically, the pH of the fermentation broth was adjusted to 1.8–2.0 with 3 mol / L concentrated sulfuric acid, shaken for 30 min, centrifuged, and the supernatant was collected. The supernatant was diluted 5 times with deionized water, mixed thoroughly, centrifuged again, and then fed into an HPLC-ELSD system (Thermo Scientific) for detection. Detection conditions: chromatographic column... LP-C18 (welch, 250 × 4.6 mm, 5 μm); mobile phase: 1.5% trifluoroacetic acid aqueous solution (phase A) and 95% methanol (phase B); flow rate: 1 mL / min; gradient method was used for the mobile phase, specifically as follows: 0–12.5 min 100% phase A; 12.5–15.5 min 50% phase A and 50% phase B; 15.5–23 min 100% phase A; electrospray conditions: atomization temperature 110 °C, gas flow rate 2.7 L / min. Gentamicin B standard was used as a reference, and the potency was calculated based on the positive correlation between peak area and concentration.

[0030] Unless otherwise specified, the reagents and instruments used in the following embodiments are commonly used in the art and can be purchased from chemical or biological product / preparation companies; the methods used in the following embodiments are conventional methods in the art, and those skilled in the art can undoubtedly understand the operation process of these experiments and obtain the corresponding results based on the prior art or the operation manual provided by the manufacturer.

[0031] The ARTP used in this embodiment is an ARTP-II device manufactured by Siqingyuan.

[0032] Example 1: Obtaining the mutagenic strain HS-GT101-23

[0033] I. Preparation of Single Spore Suspension

[0034] Single colonies of *Micromonospora echinospora* HS-1520-016-89 (deposited at the China Center for Type Culture Collection, Wuhan University, China, accession number CCTCC NO: M 2018898; deposited on December 17, 2018, see CN109897862A) were naturally isolated from the strain. These colonies were transferred to slant agar and incubated at 36.5℃ for 8 days. The spores were then washed off the slant with physiological saline, dispersed by shaking with glass beads, and diluted appropriately to prepare a single-spore suspension of the original strain HS-1520-016-89 (OD600 = 0.6–0.8).

[0035] II. Screening of ARTP Mutagenic Conditions

[0036] Take 10 μl of the prepared single-spore bacterial suspension and spread it evenly on the surface of a metal slide. Use sterile forceps to place the slide into the corresponding well in the plasma chamber. Set the power to 120 W, the working gas flow rate to 10 SLM, the irradiation distance to 2 mm, and the irradiation times to 0, 15 s, 30 s, 45 s, 60 s, 75 s, and 90 s. Use sterile forceps to place the prepared slide into an EP tube containing 1 ml of physiological saline. Place the EP tube on a shaker and shake for 1 min to form a new bacterial suspension. Serially dilute the new bacterial suspension, and take 100 μL of the diluted solution to spread on a plate. Incubate at 36.5 °C for 8 days. Use unirradiated bacterial suspension as a control. Observe the colony growth and count the colonies. Examine the relationship between irradiation time and lethality to construct a lethality curve (as shown in Figure 1).

[0037] III. Tolerance of the original strain to gentamicin B

[0038] The prepared single-spore suspension of the original strain HS-1520-016-89 was serially diluted and spread onto solid culture media containing different concentrations of gentamicin B: 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. The media were incubated at 36.5℃ for 8 days, and colony growth was observed. The results are shown in Table 1. Table 1 determined the minimum inhibitory concentration against gentamicin B to be 5 g / L.

[0039] Table 1. Effect of gentamicin B concentration on spore production of strain HS-1520-016-89

[0040] Note: + indicates colony growth, - indicates no colony growth.

[0041] IV. ARTP Mutagenesis

[0042] Based on the lethality curve in Figure 1, plasma mutagenesis was repeatedly performed on the spore suspension of strain HS-1520-016-89 at two different lethal dose irradiation times of 45s and 60s. The mutagenized bacterial suspension was serially diluted and spread on solid medium containing gentamicin B 5g / L, and incubated at 36.5℃ in the dark for 8 days.

[0043] V. Screening of Mutagenic Strains

[0044] The single colonies grown on the various resistance plates were transferred to slant culture medium and cultured for 8 days, then transferred to seed culture medium and cultured at 36.5℃ and 250rpm for 48 hours to obtain seed liquid; then the seed liquid was inoculated into fermentation medium with a volume of 25ml / 250ml, an inoculation amount of 10%, a shaking speed of 250rpm, a culture temperature of 36.5℃, and a culture period of 7 days to obtain fermentation broth.

[0045] The pH of the fermentation broth was adjusted to 1.8–2.0 with 3 mol / L concentrated sulfuric acid. After shaking for 30 min, the supernatant was collected by centrifugation, diluted 5 times with deionized water, mixed well, and then centrifuged again. The supernatant was then sent to an HPLC-ELSD system (Thermo Scientific) for analysis.

[0046] This method screened out the mutant strain HS-GT101-23, which produced the highest amount of gentamicin B, with a titer as high as 4126 μg / mL. Under the same conditions, compared with the original strain HS-1520-016-89 (731 μg / mL), the titer of the newly obtained mutant strain HS-GT101-23 was increased by 464.4%, which is 5.64 times that of the original strain. Strain HS-GT101-23 was stored in 20% glycerol tubes at -80°C.

[0047] Example 2: Morphological and cultural characteristics of strain HS-GT101-23

[0048] Experiments were conducted in accordance with relevant content from books such as *Streptomyces Identification Manual*, *Classification and Identification of Actinomycetes*, and *Manual of Systematic Identification of Common Bacteria*. Morphological and cultural characteristics of the strains were determined using eight different culture media: ISP1, ISP2, ISP3, ISP4, ISP5, Gao's No. 1, nutrient agar, and calcium malate. After incubation at 36.5℃ for 7–9 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 newly obtained strain HS-GT101-23; the results are shown in Table 2.

[0049] Table 2 Comparison of culture characteristics of strains on 8 different culture media

[0050] Observation of the mycelial color of the strain revealed that the growth status of the new strain HS-GT101-23 and the original strain HS-1520-016-89 differed significantly 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 basal culture medium, and the final concentration of each carbon source was 1.0% (mass percentage). The results are shown in Table 3.

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

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

[0055] (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 are as follows: the optimal pH for the growth of this strain is 6.5 to 7.5; the strain can grow between 28℃ and 45℃, and the optimal growth temperature is 37℃.

[0056] Table 4. Main physiological and biochemical characteristics of strain HS-GT101-23 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.

[0057] Example 4: 16S rDNA sequence analysis and strain identification

[0058] 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 using homology sequence BLAST to determine the taxonomic position of the strain.

[0059] The 16S rDNA sequence of strain HS-GT101-23 was compared with the relevant sequences in GenBank using BLAST. The results are shown in Table 5 (only strains with high homology are listed in the table).

[0060] Table 5. Homology between strain HS-GT101-23 and related strains

[0061] Strain HS-GT101-23 was found to have 100% homology with Micromonospora echinospora by 16S rDNA region sequencing.

[0062] Based on the above morphological and cultural characteristics, physiological and biochemical characteristics, and molecular biological identification of 16S rDNA sequence, strain HS-GT101-23 was finally identified as belonging to the genus Micromonospora echinospora, and its taxonomic name was Micromonospora echinospora HS-GT101-23.

[0063] Example 5: Validation of the genetic stability of strain HS-GT101-23

[0064] The high-yielding gentamicin B strain HS-GT101-23 obtained from the above screening was transferred to slant culture medium and cultured at 36.5℃ for 8 days to obtain well-grown primary slant HS-GT101-23 (F0). The strain was continuously cultured and passaged, and cultured at 36.5℃ for 8 days to obtain well-grown F1 to F5 slant ...

[0065] The F0 to F5 generations of the above-mentioned strain HS-GT101-23 were cultured on plates. Single colonies were picked and inoculated into seed culture medium and cultured at 37℃ and 250rpm for 48h to obtain seed liquid. The seed liquid was then inoculated into fermentation medium with a volume of 25ml / 250ml, an inoculation amount of 10%, a shaking speed of 250rpm, a culture temperature of 36.5℃, and a culture period of 7d. The fermentation broth was then tested.

[0066] Using the well-growing primary strain (F0) as a control, its relative fermentation titer was set to 100%, and the results are shown in Table 6.

[0067] Table 6. Effect of passage on gentamicin B production by strain HS-GT101-23

[0068] As shown in Table 6, the fermentation level of strain HS-GT101-23 obtained by screening in this invention was not significantly affected after 5 generations, and its gentamicin B titer remained basically stable at the same high level, thus indicating that strain HS-GT101-23 has good genetic stability.

[0069] Example 6: Gentamicin B production by fermentation of mutant strain HS-GT101-23

[0070] After culturing strain HS-GT101-23 on an agar slant at 36.5℃ for 8 days, spores were scraped off and cultured in seed culture medium at a volume of 20 mL / 250 mL. The culture temperature was 37℃, the rotation speed was 250 rpm, and the culture time was 48 h. Then, 0.2% by volume was inoculated into a primary seed tank (10 L of seed culture medium in a 15 L tank) and cultured at 36–38℃ for 44–48 h. Then, 15% by volume was inoculated into a secondary seed tank and cultured at 37–38℃ for 22–26 h to complete the seed tank culture and obtain the secondary seed solution.

[0071] The prepared secondary tank seed culture was inoculated into fermentation medium (30L of fermentation medium in a 50L tank) at a volume ratio of 20%. The culture temperature was 36–38℃, the tank pressure was 0.05 MPa, the aeration rate was 1:0.5–1:1.5 vvm, the stirring speed was 80–150 r / min, and the fermentation was carried out for 192 h. The tank was then discharged for testing. The results showed that the yield of gentamicin B in this example was 3881 μg / mL.

[0072] In summary, this invention provides a strain of Micromonospora echinospora HS-GT101-23, which has a simple fermentation process and can produce high yields of gentamicin B without the need for feedstock, thus significantly increasing the yield of gentamicin B. Furthermore, this strain has good stability, and its gentamicin B titer remains basically stable after five consecutive generations of subculturing, making it suitable for industrial fermentation production.

[0073] While specific embodiments of the present invention have been described above, they are not intended to limit the invention. Any modifications and variations in form and detail made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A Micromonospora echinospora HS-GT101-23, with accession number CGMCC No.32537.

2. The use of Micromonospora echinococcosis HS-GT101-23 according to claim 1 in the fermentation production of gentamicin B or in the production of pharmaceutical compositions containing gentamicin B.

3. A process for the fermentative production of gentamicin B, characterized in that, The method comprises fermenting the *Micromonas echinococcosis* HS-GT101-23 of claim 1 in a culture medium containing an assimilable carbon source, a nitrogen source, and inorganic salts, 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 flour, cottonseed meal, soybean meal, peanut meal, corn steep liquor powder, ammonium salts, and nitrates; and the inorganic salts are 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 the fermentative production of gentamicin B, characterized in that, The method includes the step of inoculating the Micromonospora echinococcosis HS-GT101-23 of claim 1 into a fermentation medium for fermentation.

5. The process for the fermentative production of gentamicin B according to claim 4, characterized in that, The fermentation medium comprises the following 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 meal, 0.2-0.6% calcium carbonate, 0.2-0.5% ammonium sulfate, 0.0008% cobalt chloride, with the remainder being water; the pH of the fermentation medium is 6.8-7.

5. Preferably, the fermentation medium comprises the following components by mass percentage: 5% corn starch, 3.5% soybean meal, 0.3% peptone, 0.02% potassium nitrate, 0.5% gluten flour, 0.5% calcium carbonate, 0.3% ammonium sulfate, 0.0008% cobalt chloride, with the remainder being water; the pH of the fermentation medium is 7.

0.

6. The method for the fermentative production of gentamicin B according to claim 4 or 5, characterized in that, The fermentation conditions include fermentation at 34–38°C for 144–192 hours.

7. The process for the fermentative production of gentamicin B according to any one of claims 4 to 6, characterized in that, The method also includes the step of inoculating the Micromonospora echinococcosis HS-GT101-23 of claim 1 into a seed culture medium for seed culture. The seed culture medium comprises the following components by weight: 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 being water; the pH of the seed culture medium is 6.8-7.

5. Preferably, the seed culture medium comprises the following components by weight: 3.5% corn starch, 1% soybean meal, 0.4% yeast extract, 0.3% peptone, 0.01% potassium nitrate, 0.5% calcium carbonate, and the remainder being water; the pH of the seed culture medium is 7.

0.

8. The process for the fermentative production of gentamicin B according to claim 7, characterized in that, The seed liquid culture step includes culturing at 34-38℃ for 22-48h.

9. The process for the fermentative production of gentamicin B according to any one of claims 4 to 8, characterized in that, The application also includes a step of inoculating the Microbispora rosea HS-GT101-23 of claim 1 on a slant medium for activation; The slant medium includes components with mass content of corn starch 1-3%, soybean meal 2-3%, glucose 0.1-0.3%, yeast extract 0.2-0.5%, sodium chloride 0.1-0.3%, potassium dihydrogen phosphate 0.01-0.03%, potassium nitrate 0.1-0.2%, calcium carbonate 0.1-0.2%, and agar 1.8-2.2%; the pH of the slant medium is 6.8-7.5; preferably, the slant medium includes components with mass content of corn starch 2%, soybean meal 2.5%, glucose 0.2%, yeast extract 0.3%, sodium chloride 0.2%, potassium dihydrogen phosphate 0.02%, potassium nitrate 0.1%, calcium carbonate 0.1%, and agar 2.0%; the pH of the slant medium is 7.

2.

10. The process for the fermentative production of gentamicin B according to claim 9, characterized in that, The activation step includes culturing at 34-38℃ for 7-9 days.

Images