High-yield spring leimycin producing strain, its breeding and fermentation method

Abstract

The application belongs to the technical field of microbial fermentation, and particularly relates to a high-yield kasugamycin producing strain and a breeding and fermentation method thereof. The strain is Streptomyces sp. XYHK-01-M, and the preservation number is CGMCC No. 39497. The strain is obtained through normal pressure room temperature plasma mutagenesis and sodium nitrite compound mutagenesis of a soil isolated strain, and is obtained through antibacterial circle primary screening, shake flask rescreening and continuous passage stability verification. The strain is used for staged amplification fermentation, and maltose and corn syrup dry powder are supplemented at 60-84 h of fermentation, so that the kasugamycin content in the fermentation liquor reaches 30000-32000 ug / mL; further adding sodium butyrate, trichostatin or a combination thereof can make the content exceed 32000 ug / mL. The strain has high capacity, good genetic stability, short fermentation period, and is suitable for large-scale production of kasugamycin.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a high-yield kasugamycin production strain and its selection and fermentation method. Background Technology

[0002] Kasugamycin is an aminoglycoside antibiotic produced by Streptomyces microaureus. It was first discovered in 1965 from the fermentation products of a strain isolated from Japanese soil. Its mechanism of action is mainly to inhibit the protein synthesis of pathogens. It has significant control effects on various fungal and bacterial diseases of crops, such as rice blast, bacterial angular leaf spot of cucumber, bacterial wilt of tomato, and soft rot of cabbage. It is non-toxic to humans and animals, leaves no residue, and is easily degraded in soil. It meets the current development needs of green agriculture and ecological agriculture. It has been listed as a recommended pesticide for the production of pollution-free agricultural products in my country and is widely used for the prevention and control of diseases in grain crops, vegetables, fruits and other crops.

[0003] Currently, the industrial production of kasugamycin mainly relies on the deep fermentation technology of *Streptomyces aureus*. However, the existing production process has many bottlenecks, which seriously restrict its industrialization and market competitiveness: First, the original strains have low resistance-producing capacity, and the fermentation titers of existing industrial strains are generally maintained at 15,000-18,000 μg / mL, which is difficult to meet the efficiency requirements of large-scale production; Second, traditional mutagenesis breeding methods (such as ultraviolet mutagenesis and sodium nitrite mutagenesis) have problems such as low mutagenesis efficiency, narrow mutation spectrum, and poor genetic stability of high-yielding strains. After 3-4 generations of subculturing, the production capacity of most mutagenic strains will decline significantly, making them unsuitable for long-term use. Thirdly, the design of fermentation process parameters is unreasonable, with imbalances in carbon and nitrogen source ratios, improper control of fermentation temperature and aeration, and a lack of targeted feeding strategies, resulting in fermentation cycles of up to about 200 hours. This not only increases energy consumption and production costs but also easily leads to problems such as cell autolysis and product degradation, affecting the quality of the final product. Fourthly, the detection methods for fermentation broth have defects. Traditional detection methods are either cumbersome to operate and have long detection cycles, or lack accuracy and repeatability, making it difficult to achieve real-time monitoring of the potency of fermentation broth during industrial production. This makes it impossible to adjust fermentation parameters in a timely manner, resulting in poor stability of the fermentation process and large differences between product batches.

[0004] Furthermore, with the increasing demands for green agricultural development, the market demand for kasugamycin is growing year by year, placing higher requirements on its production efficiency, product quality, and production costs. Therefore, developing a high-efficiency, stable, and low-cost fermentation process for kasugamycin production using *Streptomyces simonii*, through optimizing strain screening, mutagenesis breeding, fermentation processes, and detection methods, can overcome existing technological bottlenecks, significantly improve the potency of kasugamycin, shorten the fermentation cycle, and reduce production costs. This has significant theoretical and practical value for promoting the industrialization of kasugamycin, improving the level of agricultural disease control in my country, and promoting green agricultural development. Summary of the Invention

[0005] In view of the existing technologies, the industrial production of kasugamycin using *Streptomyces simonii* generally suffers from several technical bottlenecks, including low potency of the original strain, low mutagenesis efficiency and poor genetic stability of the strain due to traditional mutagenesis methods, long fermentation cycles and high costs due to inaccurate control of fermentation process parameters (carbon-nitrogen source ratio, temperature, feeding, etc.), and cumbersome and difficult-to-monitor real-time fermentation broth. This invention aims to provide a high-yield kasugamycin-producing strain and its selection and fermentation method. Through the combined mutagenesis selection of high-yield strains, systematic optimization of fermentation medium and process parameters, and the establishment of supporting detection methods, the invention solves the aforementioned key technical problems, thereby achieving efficient, stable, and large-scale industrial production of kasugamycin.

[0006] The specific technical solution is as follows:

[0007] The high-yield kasugamycin producing strain is *Streptomyces* sp. XYHK-01-M, with accession number CGMCC No. 39497. The depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 29, 2026.

[0008] The morphological characteristics of Streptomyces sp. XYHK-01-M are as follows: It is an aerobic actinomycete that grows vigorously on slant agar. The aerial hyphae are grayish-white to light gray (slightly darker than the original strain), fluffy, while the substrate hyphae are yellowish-brown. It does not produce water-soluble pigments. Under a microscope, the spore filaments are spiral with 3-5 turns. The spores are spherical to oval, with a diameter of 0.8-1.2 μm and a smooth surface. The physiological and biochemical characteristics are as follows: It can utilize glucose, maltose, and sucrose as carbon sources, and can utilize tryptone and soybean meal as nitrogen sources. It cannot utilize starch as the sole carbon source. It is positive for catalase and negative for oxidase. The optimal growth temperature is 28-30℃, and the optimal growth pH is 7.0-7.2.

[0009] The method for selecting high-yielding kasugamycin-producing strains includes the following steps:

[0010] (1) Screening of original high-yielding strains

[0011] (1.1) Soil sample processing and soil suspension preparation

[0012] Select soil from the top 10-20cm of the vegetable field, remove stones, weeds and other impurities, weigh 5-20g of soil, add sterile physiological saline with a concentration of 0.5-1.0wt% and a volume equivalent to 5-15 times the soil mass, and shake for 20-40min at 25-30℃ and 150-250rpm to obtain a uniform soil suspension.

[0013] The soil suspension was prepared at a ratio of 10 -4 -10 -8 Perform serial dilutions, with 2-5 replicates for each dilution;

[0014] (1.2) Actinomycetes were isolated using double-layer selective plates.

[0015] Take 0.05-0.2 mL of soil suspension at different dilutions and spread it evenly on the surface of a double-layer plate culture medium. Incubate at 26-30℃ for 5-10 days, and observe the colony growth daily. Select morphologically typical single colonies of actinomycetes and inoculate them onto slant culture medium. Incubate at 26-30℃ for 5-10 days and store for later use. Morphologically typical single colonies of actinomycetes are defined as grayish-white, fluffy, with neat edges and obvious aerial hyphae.

[0016] (1.3) Initial screening of high-yielding strains using the inhibition zone method

[0017] Spores from each slant culture strain were scraped off using 0.5-1.0 wt% sterile physiological saline to prepare spores with a concentration of 10. 5 -10 8 A bacterial suspension of 1 / mL was prepared; 5-20 μL of the actinomycete suspension was inoculated onto LB agar plates containing Bacillus subtilis, with 2-5 replicates per strain. The plates were incubated at 35-38℃ for 6-12 h, and the diameter of the inhibition zone was measured. Strains with an inhibition zone diameter greater than 15 mm were selected as candidate strains.

[0018] Candidate strains obtained from the initial screening of inhibition zones were inoculated into Erlenmeyer flasks with a volume of 250-1000 mL, with a liquid volume of 10-40% of the flask volume and an inoculation amount of 5-15% (v / v). The flasks were cultured with shaking at 26-30℃ and 160-240 rpm for 144-192 h. After fermentation, the kasugamycin titer in the fermentation broth was determined by high performance liquid chromatography, and strains with a kasugamycin titer of not less than 12000 μg / mL were selected as the original high-yielding strains.

[0019] (2) Combined mutagenesis of the original strain and the acquisition of high-yielding and stable strains

[0020] (2.1) Preparation of spore suspension of original strain

[0021] The original high-yielding strain was inoculated onto slant agar and cultured at 26-30℃ for 5-10 days. After the slant was confluent with spores, 0.5-1.0 wt% sterile physiological saline was added to wash and collect the spore suspension. The spore suspension was transferred to a container containing glass beads and shaken at 25-30℃ and 150-250 rpm for 20-40 minutes to fully break the mycelium and evenly disperse the spores. After filtering through defatted cotton to remove mycelial fragments, the spore concentration was adjusted to 10. 5 -10 8 The sample was prepared at 10-25% glycerol (per mL) as a preservative and stored at 0-4°C for later use.

[0022] (2.2) Atmospheric pressure and room temperature plasma mutagenesis

[0023] A normal-pressure, room-temperature plasma breeding system was used, with high-purity helium as the working gas, to induce the first round of mutagenesis on the spore suspension of the original strain.

[0024] Spread 0.5-2.0 mL of spore suspension evenly on the ARTP sample stage and treat it under the following conditions: gas flow rate 6-14 L / min, nozzle-to-sample surface distance 1-3 mm, discharge power 80-120 W, and irradiation time 30-150 s.

[0025] After mutagenesis, the treatment solution was eluted with sterile physiological saline and serially diluted to 10⁻⁶. -4 -10 -6 Take 0.05-0.2 mL of the solution and spread it on the surface of a double-layer plate culture medium. Incubate at 26-30℃ for 5-10 days, calculate the lethality rate, and select morphologically normal and vigorously growing single colonies from the surviving colonies to inoculate and preserve them on slant culture to obtain candidate strains for plasma mutagenesis at room temperature and atmospheric pressure. Under the above conditions, the lethality rate is generally controlled at 60-90%, and the proportion of high-yielding mutant strains is not less than 10%.

[0026] (2.3) Sodium nitrite remutation

[0027] Candidate strains obtained by atmospheric pressure and room temperature plasma mutagenesis were selected, and spore concentrations of 10 were prepared. 5 -10 8A second round of chemical mutagenesis was performed on a spore suspension of 10-50 mg / L, with sodium nitrite solution added to achieve a final sodium nitrite concentration of 10-50 mg / L. The suspension was then treated with shaking at 25-35℃ and 150-250 rpm for 20-60 min. After treatment, the suspension was centrifuged at 6000-10000 rpm for 5-15 min, the supernatant was discarded, and the precipitate was washed 2-4 times with sterile physiological saline to terminate the mutagenesis. The suspension was then serially diluted to 10⁻⁶. -4 -10 -6 Take 0.05-0.2 mL of the solution and spread it onto a double-layer plate culture medium. Incubate at 26-30℃ for 5-10 days. Pick single colonies and inoculate them onto slant culture for preservation to obtain candidate strains for sodium nitrite remutation. The high-yield mutation rate should not be less than 10%.

[0028] (2.4) Screening of high-yielding strains and verification of genetic stability

[0029] Initial screening of inhibition zone

[0030] The candidate strains for complex mutagenesis were prepared to a spore concentration of 10. 5 -10 8 Take 5-20 μL of bacterial suspension per 1000 cells / mL and inoculate it on LB agar plates containing Bacillus subtilis. Set up 2-5 replicates for each strain and incubate at 35-38℃ for 6-12 h. Measure the diameter of the inhibition zone and screen strains with an inhibition zone diameter greater than 25 mm as high-yielding primary screening strains.

[0031] Shake-flask fermentation and double screening

[0032] High-yield primary screening strains were inoculated into fermentation medium and cultured with shaking at 26-30℃ and 160-240rpm for 144-192h. The kasugamycin titer in the fermentation broth was determined by high performance liquid chromatography, and strains with kasugamycin titers higher than 25000μg / mL were selected as high-yield secondary screening strains.

[0033] Validation of genetic stability through successive generations

[0034] High-yield, re-screened strains were passaged 3-10 times on slant agar. Each generation of shake-flask fermentation was carried out under the following conditions: inoculation into 250-1000 mL Erlenmeyer flasks, filling the flasks to 10-40% of their volume, with an inoculum size of 5-15% (v / v). The flasks were cultured at 26-30℃ and 160-240 rpm for 144-192 h with shaking. After fermentation, the kasugamycin titer in the fermentation broth was determined using high-performance liquid chromatography (HPLC), and the fluctuation range of the titer was calculated using the following formula:

[0035] Fluctuation range (%) = (Highest valence - Lowest valence) / Average valence × 100%

[0036] Strains with fluctuations not exceeding 5% and capable of stably producing kasugamycin at a titer higher than 25000 μg / mL were selected as the final high-yielding mutant strain with stable genetic traits, i.e., the high-yielding kasugamycin producing strain, and named Streptomyces sp. XYHK-01-M.

[0037] Preferably, the diluent used in step (1.1) for gradient dilution is a 0.5-1.0 wt% sodium chloride solution.

[0038] Preferably, the preparation method of the double-layer plate culture medium is as follows: 1-5g of tryptone, 5-15g of soybean meal powder, 5-20mL of glycerol, 1-5g of sodium chloride, 1-3g of calcium carbonate, and 20-30g of agar are added to an appropriate amount of distilled water and dissolved evenly. The volume is then adjusted to 1L with distilled water, the pH is adjusted to 6.8-7.5, and the mixture is sterilized at 115-125℃ and 103-137kPa for 15-30min. The mixture is then poured into sterile petri dishes and cooled to solidify, yielding the lower basal culture layer. Base; Add 1-5g tryptone, 5-15g soybean meal, 5-20mL glycerol, 1-5g sodium chloride, 1-3g calcium carbonate, 20-30g agar and 50-150mg kasugamycin to an appropriate amount of distilled water and shake well. Make up to 1L with distilled water, adjust the pH to 6.8-7.5, sterilize at 115-125℃ and 103-137kPa for 15-30min, and pour onto the surface of the solidified lower base culture medium to form a double-layer plate culture medium.

[0039] Preferably, the slant culture medium is prepared as follows: 1.2-1.8g of high-temperature soybean cake powder, 0.4-0.6g of sodium chloride, 0.2-0.3g of dipotassium hydrogen phosphate, and 1.8-2.2g of agar are added to an appropriate amount of distilled water and heated until dissolved and homogeneous. Then, 8-12mL of glycerol is added and stirred until homogeneous. The volume is adjusted to 1L with distilled water, and the pH is adjusted to 7.0-7.4. After dispensing, the medium is sterilized at 121-126℃ and 103-137kPa for 15-25min. The medium is then placed slanted while hot and allowed to solidify to obtain the final product.

[0040] Preferably, the LB agar medium containing Bacillus subtilis is prepared as follows: Take 9-11g of tryptone, 4.5-5.5g of yeast extract, 9-11g of sodium chloride and 23-27g of agar, add them to an appropriate amount of distilled water and heat to dissolve, make up to 1L, adjust the pH to 6.8-7.2, dispense, sterilize at 121-126℃ and 103-137kPa for 15-25min, cool to 45-55℃, add an appropriate amount of Bacillus subtilis bacterial solution under aseptic conditions, mix well and pour into plates, and obtain the medium after solidification.

[0041] Preferably, the conventional fermentation medium is prepared as follows: 45-55g of soybean meal powder, 14-16g of maltose, 2.5-3.5g of sodium chloride, 4-6g of corn steep liquor powder, and 0.25-0.35g of potassium dihydrogen phosphate are added to an appropriate amount of distilled water, heated and stirred until evenly dispersed, and then diluted to 1L with distilled water. The pH is adjusted to 6.9-7.1, dispensed, and sterilized at 121-124℃ and 103-137kPa for 15-25min. After cooling, the conventional fermentation medium is obtained.

[0042] This invention also discloses a fermentation method for producing kasugamycin using Streptomyces sp. XYHK-01-M, comprising the following steps:

[0043] S1. Inoculate Streptomyces sp. XYHK-01-M into seed culture medium via slant or preservation tube at an inoculation rate of 10-20% by volume into seed bottles. Incubate at 26-30℃ and 200-240 rpm on a shaker for 36-60 h to achieve an OD600 of 1.5-2.2, thus obtaining a vigorous seed culture.

[0044] S2 graded scale-up fermentation and feeding

[0045] S2.1 Primary Fermentation

[0046] Inoculate the seed culture into the first-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter is 30-100L, and the liquid volume is 60-80% of the tank volume.

[0047] Primary fermentation conditions: Optimized fermentation medium; fermentation temperature 27-30℃; aeration rate 0.8:1-1.2:1; stirring speed 160-200 rpm; tank pressure 0.01-0.03 MPa; fermentation time 60-90 h;

[0048] S2.2 Secondary Fermentation and Feeding

[0049] The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter was 300-1000L, and the liquid volume was 60-80% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used.

[0050] 60-84 hours after the start of fermentation, the secondary fermenter was fed with the following: maltose to a final concentration of 1.0-3.0 wt%; corn steep liquor powder to a final concentration of 0.3-0.8 wt%. After feeding, fermentation continued at 27-30℃, aeration rate of 0.8:1-1.2:1, stirring at 160-200 rpm, and tank pressure of 0.01-0.03 MPa. The total fermentation cycle was controlled at 144-180 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth reached 30,000-32,000 μg / mL.

[0051] Preferably, the seed culture medium is prepared as follows: 12-18g of soybean meal powder and 4-6g of corn steep liquor powder are dissolved evenly in an appropriate amount of distilled water. Then, 8-12g of maltose, 2.0-3.0g of sodium chloride, 0.15-0.25g of potassium dihydrogen phosphate, and 3-5g of calcium carbonate are added and stirred evenly. After stirring evenly, 8-12mL of soybean oil is added and stirred evenly. The volume is adjusted to 1L with distilled water, and the pH is adjusted to 6.8-7.2. After dispensing, the medium is sterilized at 121-126℃ and 103-137kPa for 15-25min. After cooling, the medium is obtained.

[0052] Preferably, the optimized fermentation medium is prepared as follows: 65-75g of soybean meal powder, 16-20g of maltose, 3.5-4.5g of sodium chloride, 7-9g of corn steep liquor powder, 0.35-0.45g of potassium dihydrogen phosphate, and 0.025-0.035g of inositol are added to an appropriate amount of distilled water, heated and stirred until evenly dispersed. Then, 36-40g of soybean oil and 0.8-1.2g of Tween-20 are added, stirred thoroughly, and the volume is adjusted to 1L with distilled water. The pH is adjusted to 6.8-7.2, dispensed, and then sterilized by high-pressure steam at 121-126℃ and 103-137kPa for 15-25 minutes. After cooling, the culture medium is obtained.

[0053] Preferably, step S2.2, secondary fermentation and feeding, can also be as follows: the primary fermentation broth is transferred to the second-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter is 300-1000L, and the liquid filling volume is 60-80% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation are used.

[0054] 60-84 hours after the start of fermentation, the secondary fermenter was fed with maltose to a final concentration of 1.0-3.0 wt%, corn steep liquor powder to a final concentration of 0.3-0.8 wt%, and a regulator to a final concentration of 0.0005-0.002 wt%. After feeding, fermentation continued at 27-30℃, an aeration rate of 0.8:1-1.2:1, a stirring speed of 160-200 rpm, and a tank pressure of 0.01-0.03 MPa. The total fermentation cycle was controlled at 144-180 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth was >32000 μg / mL. The regulator was sodium butyrate, trachomatis, or a combination thereof.

[0055] This invention also discloses a method for detecting kasugamycin in fermentation broth, comprising the following steps:

[0056] 1) Chromatographic condition setting and mobile phase preparation

[0057] 1.1) Preparation of mobile phase

[0058] Preparation of an aqueous-organic mobile phase containing ion-pairing reagents: Weigh sodium dodecyl sulfate to a mass fraction of 0.05-0.2 wt% in water and prepare an aqueous solution; mix acetonitrile with the sodium dodecyl sulfate aqueous solution at a volume ratio of 20:80-40:60, adjust the pH of the mixture to 2.0-3.0 with 80-90 wt% phosphoric acid aqueous solution, and use the mixture as the mobile phase;

[0059] The prepared mobile phase is filtered through a filter membrane with a pore size of 0.3-0.5 μm and degassed in an ultrasonic bath for 10-30 min.

[0060] 1.2) Chromatographic condition setting

[0061] Set the following conditions on the high performance liquid chromatograph: the mobile phase is the mixture obtained in step (1.1), the flow rate is 0.8-1.2 mL / min; the column temperature is 25-35℃; the detection wavelength is 220-240 nm; the injection volume is 5-20 μL; under the above conditions, the retention time of kasugamycin is controlled at about 10-12 min.

[0062] 2) Preparation of Kasugamycin Standard Solution

[0063] Accurately weigh 0.05-0.20 g of kasugamycin standard, place it in a 25-100 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the mark, shake well to obtain kasugamycin stock solution;

[0064] Then, use a pipette to transfer 2-10 mL of the mother solution into another 25-100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the kasugamycin standard solution for injection.

[0065] 3) Preparation of fermentation broth sample solution

[0066] Accurately weigh 0.50-5.00 g of the mixed test solution and place it in a 25-100 mL volumetric flask. Add an appropriate amount of water to dissolve or dilute to the mark, and shake well to obtain the sample mother liquor. The test solution is the fermentation broth containing kasugamycin. Centrifuge at 6000-10000 r / min for 5-20 min, and take the supernatant and filter it through a microporous membrane with a pore size of 0.25-0.45 μm to obtain the clear fermentation broth containing kasugamycin, which is the test solution.

[0067] Then, use a pipette to transfer 2-10 mL of the sample mother solution into another 25-100 mL volumetric flask, dilute with water to the mark, and shake well to obtain the sample solution for injection; if necessary, filter the sample solution through a microporous membrane with a pore size of 0.3-0.5 μm, and take the filtrate for chromatographic analysis.

[0068] 4) Chromatographic determination and result calculation

[0069] 4.1) Chromatographic injection and determination

[0070] Under the chromatographic conditions set in step 1), after the instrument baseline stabilizes, inject the standard solution 3-6 times consecutively, record the peak area or relative response value of kasugamycin, calculate the relative difference between the peak areas of two adjacent standard injections, and start measuring the sample when the difference is less than 1.5%.

[0071] Subsequently, the samples were injected and measured in the order of "standard solution-sample solution-sample solution-standard solution", and the peak area of ​​kasugamycin in each injection was recorded.

[0072] 4.2) Result Calculation

[0073] Calculate the average peak area of ​​kasugamycin in the two injection sample solutions and the two standard solutions before and after, respectively, and denot them as A2 and A1; the potency (μg / mL) of kasugamycin in the sample is calculated according to the following formula:

[0074]

[0075] In the formula:

[0076] The average peak area of ​​kasugamycin in the A2 sample solution;

[0077] The mass of m1-kasugamycin standard is expressed in grams (g).

[0078] w1 - Mass fraction of kasugamycin standard, expressed as %;

[0079] The average peak area of ​​kasugamycin in A1-standard solution;

[0080] m2 - The mass of the sample, expressed in grams (g).

[0081] Preferably, the volume ratio of acetonitrile to 0.1wt% sodium dodecyl sulfate aqueous solution in the mobile phase is 30:70, and the pH is adjusted to 2.5; the mobile phase is filtered through a 0.45μm filter membrane and degassed in an ultrasonic bath for 15-20 min.

[0082] Preferably, the chromatographic conditions are: flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 230 nm, and injection volume 10 μL. Under these conditions, the retention time of kasugamycin is approximately 11.1 min.

[0083] Preferably, the standard solution is prepared as follows: accurately weigh 0.10 g of kasugamycin standard, place it in a 50 mL volumetric flask, add water to dissolve and dilute to the mark; then transfer 5 mL to another 50 mL volumetric flask, add water to dilute to the mark, and shake well for later use.

[0084] Preferably, the sample solution is prepared as follows: a certain amount of fermentation broth is accurately weighed and placed in a 50mL volumetric flask, diluted with water to the mark, and shaken well; then 5mL is transferred to another 50mL volumetric flask, diluted with water to the mark, and shaken well for later use.

[0085] Preferably, during the chromatographic determination, the relative difference in the peak area of ​​kasugamycin in the continuously injected standard solution is controlled below 1.2% to ensure the accuracy and repeatability of the detection results.

[0086] The beneficial effects of this invention are:

[0087] This invention employs a combined mutagenesis strategy of atmospheric pressure, room temperature plasma, and sodium nitrite, breaking through the limitations of traditional single mutagenesis methods. The resulting high-yielding mutant strain, Streptomyces sp. XYHK-01-M, exhibits a shake-flask fermentation titer exceeding 29,000 μg / mL, a 141.7% increase compared to the original strain's 12,000 μg / mL. Furthermore, it demonstrates excellent genetic stability, with a production capacity fluctuation of less than 5% after five consecutive subcultures. This solves the problem of reduced production capacity caused by strain degeneration in industrial production and possesses extremely high industrial application value.

[0088] This invention successfully constructed a highly efficient three-stage fermentation process by systematically optimizing the fermentation medium formulation and fermentation process parameters. This process maintains a stable fermentation potency of 31000-31500 μg / mL while shortening the fermentation cycle to 168 hours. Furthermore, the use of inexpensive raw materials such as soybean meal powder and corn steep liquor powder in the medium reduces the overall production cost by more than 20%, enabling efficient and low-cost large-scale production of kasugamycin. The fermentation cycle mentioned in this invention refers to the second-stage fermentation time, not the entire process cycle.

[0089] The high-performance liquid chromatography (HPLC) detection method developed in conjunction with this invention achieves rapid and effective separation of kasugamycin from impurities (resolution ≥ 1.5) through optimized detection conditions. The detection process is simple, quick, and highly accurate. This method provides a reliable real-time monitoring tool for industrial fermentation processes, enabling timely adjustment of fermentation process parameters based on real-time detection results, ensuring batch-to-batch stability (relative standard deviation ≤ 2.0%).

[0090] This invention provides an integrated technical solution encompassing original strain screening, mutagenesis breeding, fermentation process control, and fermentation broth detection. This solution overcomes the fragmented nature of traditional processes, demonstrating significant synergy between each step. It not only solves the core bottleneck hindering the development of the kasugamycin industry but also effectively promotes the healthy and coordinated development of green agriculture and the microbial fermentation industry by improving production efficiency and reducing resource consumption.

[0091] This invention is the first to introduce an epigenetic regulator system into the industrial fermentation process of streptomycin for the production of kasugamycin. Without altering the carbon-nitrogen source ratio, fermentation cycle, or feeding strategy, the addition of low doses of sodium butyrate, trogostatin, and their combinations further increases the fermentation potency of kasugamycin by 5-15% compared to the absence of epigenetic regulators. The increase can reach over 10% when sodium butyrate is used in combination with trogostatin. This epigenetic regulation strategy breaks through the traditional approach of relying solely on genetic mutagenesis and process parameter optimization to improve yield. It taps into the potential production capacity of strains at the gene expression level, demonstrating significant foresight and innovation, and is particularly suitable for increasing the yield of other streptomycin secondary metabolites in the field of agricultural antibiotics. Attached Figure Description

[0092] Figure 1 This is a chromatogram of the HPLC detection method of the present invention; in the chromatogram, the horizontal axis is the retention time (min) and the vertical axis is the detector response signal (mV); the retention time of the kasugamycin peak is 11.1±0.2min, the peak shape is symmetrical, the separation is good (resolution ≥1.5), the impurity peak is completely separated from the kasugamycin peak and there is no interference, indicating that the separation effect of the detection method is excellent.

[0093] Figure 2 This is the chromatogram of the kasugamycin hydrochloride reference standard of the present invention; in the chromatogram, the horizontal axis is the retention time and the vertical axis is the detector response signal. Detailed Implementation

[0094] The parameters and sources of some raw materials in the examples are as follows:

[0095] Soil samples were taken from vegetable fields along the Fen River in Linfen City, Shanxi Province. This area has a temperate continental monsoon climate, fertile soil with high organic matter content, and has been used for vegetable cultivation for a long time. The soil contains a large number of actinomycetes that can produce antibiotics, providing a good sample basis for screening high-yielding strains.

[0096] The preparation method of the double-layer plate culture medium is as follows: Dissolve 3g of tryptone, 10g of soybean meal powder, 10mL of glycerol, 3g of sodium chloride, 2g of calcium carbonate, and 25g of agar in an appropriate amount of distilled water until homogeneous. Make up to 1L with distilled water, adjust the pH to 7.0, and sterilize at 121℃ and 103.4 kPa for 20 minutes. Pour into sterile petri dishes and allow to cool and solidify to obtain the lower basal culture medium. Add 3g of tryptone, 10g of soybean meal powder, 10mL of glycerol, 3g of sodium chloride, 2g of calcium carbonate, 25g of agar, and 100mg of kasugamycin to an appropriate amount of distilled water, shake well, and make up to 1L with distilled water. Adjust the pH to 7.0, sterilize at 121℃ and 103.4 kPa for 20 minutes, and pour onto the surface of the solidified lower basal culture medium to form a double-layer plate culture medium.

[0097] To prepare the slant culture medium, add 1.5g of high-temperature soybean meal powder, 0.5g of sodium chloride, 0.25g of dipotassium hydrogen phosphate, and 2.0g of agar to an appropriate amount of distilled water, heat and stir to dissolve, then add 10mL of glycerol, mix well, and bring the volume to 1L. Adjust the pH to 7.2 with NaOH or HCl solution, dispense into test tubes, and autoclave at 121℃ and 103.4 kPa for 20min. Remove the tubes and tilt them while hot. After cooling and solidification, the slant culture medium is obtained.

[0098] For the seed culture medium, add 15g of soybean meal powder and 5g of corn steep liquor powder to an appropriate amount of distilled water, heat and stir until fully dissolved. Then add 10g of maltose, 2.5g of sodium chloride, 0.2g of potassium dihydrogen phosphate, and 4g of calcium carbonate, stir well, and finally add 10mL of soybean oil and emulsify and mix thoroughly. After bringing the volume to 1L, adjust the pH to 7.0 with NaOH or HCl solution. After dispensing, autoclave at 121℃ and 103.4 kPa for 20min. After cooling, the seed culture medium is obtained.

[0099] For LB agar containing Bacillus subtilis, add 10g tryptone, 5g yeast extract, 10g sodium chloride, and 25g agar to an appropriate amount of distilled water, heat to boiling and stir until completely dissolved. Adjust the pH to 7.0 with NaOH or HCl solution. After dispensing, autoclave at 121℃ and 103.4 kPa for 20 min, remove and cool to approximately 50℃. Under aseptic conditions, add 2% (v / v) of 1×10⁻⁶ agar. 8 Add CFU / mL of Bacillus subtilis culture and mix quickly. Immediately pour the mixture into sterile petri dishes and allow it to solidify completely to obtain the LB plate culture medium containing Bacillus subtilis.

[0100] The conventional fermentation medium is prepared as follows: 50g of soybean meal powder, 15g of maltose, 3.0g of sodium chloride, 5g of corn steep liquor powder and 0.30g of potassium dihydrogen phosphate are added to an appropriate amount of distilled water, heated and stirred until completely dissolved, and then diluted to 1L with distilled water. The pH is adjusted to 7.0, dispensed, and sterilized at 121℃ and 103.4 kPa for 20min. After cooling, the conventional fermentation medium is obtained.

[0101] Example 1

[0102] The method for selecting high-yielding kasugamycin-producing strains includes the following steps:

[0103] (1) Screening of original high-yielding strains

[0104] Breaking away from the traditional single-culture medium screening model, a three-stage screening strategy is adopted, consisting of isolation using a double-layer selective culture medium → initial screening using inhibition zones → secondary screening using shake-flask fermentation. This effectively improves the screening efficiency and accuracy of original high-yield strains. The specific steps are as follows:

[0105] (1.1) Soil sample preparation: Select soil samples from the top 10-20cm of the vegetable field, remove stones, weeds and other impurities, weigh 10g of soil sample, add 90mL of 0.85wt% sterile physiological saline, place in a shaker, shake at 28℃ and 200rpm for 30min to prepare a uniform soil suspension; then use a gradient dilution method to dilute the soil suspension to 10. -6 10 -7 10 -8 The sample was diluted 10 times, and three replicates were set up for each dilution to ensure the representativeness of the samples;

[0106] (1.2) Isolation and culture: Double-layer plate culture medium was used for bacterial isolation, which effectively inhibited the growth of contaminating bacteria and improved the isolation efficiency of actinomycetes. 0.1 mL of diluted soil suspension was taken from each plate and evenly spread on the surface of the double-layer plate culture medium. After spreading, the plate was placed in a constant temperature incubator at 28℃ for 7-10 days. The growth of colonies on the plate was observed every day. Typical actinomycete single colonies were picked and inoculated into slant culture medium. The culture was carried out at 28℃ for 8 days and stored for later use. Typical actinomycete single colonies are grayish-white, fluffy, with neat edges and obvious aerial hyphae.

[0107] (1.3) Primary and secondary screening: Primary screening used the inhibition zone method, with Bacillus subtilis as the indicator bacterium. This bacterium is sensitive to kasugamycin and has a fast growth rate, making it suitable for inhibition zone detection. Single colonies of the isolated actinomycetes were inoculated onto slant agar plates and cultured at 28°C for 8 days. The spores were then scraped off the slant plates with sterile physiological saline to prepare a solution with a concentration of 10. 6A bacterial suspension of 10 μL / mL was prepared and inoculated onto LB agar plates containing Bacillus subtilis. Each strain was in 3 replicates. After incubation at 37°C for 8 h, the diameter of the inhibition zone was measured. Strains with an inhibition zone diameter greater than 15 mm were selected as candidate strains. A total of 23 candidate strains were obtained.

[0108] The secondary screening was conducted using shake-flask fermentation to further verify the kasugamycin production capacity of the candidate strains. The 23 candidate strains obtained from the initial screening were inoculated into 500mL Erlenmeyer flasks, each containing 100mL of conventional fermentation medium, with an inoculation volume ratio of 10%. The flasks were cultured at 28℃ and 200rpm for 168h with shaking. After fermentation, the potency of kasugamycin in the fermentation broth was detected by high performance liquid chromatography. The original strain with a potency ≥12000μg / mL was screened, and finally, a high-yielding original strain was obtained and named Streptomyces XYHK-01.

[0109] The morphological characteristics of this strain are as follows: it is an aerobic actinomycete that grows vigorously on slant agar. The aerial hyphae are grayish-white and fluffy, while the substrate hyphae are yellowish-brown and do not produce water-soluble pigments. Under a microscope, the spore filaments are spiral-shaped with 3-5 turns, and the spores are spherical to oval with a diameter of 0.8-1.2 μm and a smooth surface. The physiological and biochemical characteristics are as follows: it can utilize glucose, maltose, and sucrose as carbon sources, and can utilize tryptone and soybean meal as nitrogen sources, but cannot utilize starch as the sole carbon source. It is positive for catalase and negative for oxidase. The optimal growth temperature is 28-30℃, and the optimal growth pH is 7.0-7.2.

[0110] (2) Selection of high-yield kasugamycin-producing strains

[0111] To address the problem that the original strain Streptomyces XYHK-01 has limited cytokine production capacity and cannot meet the needs of industrial production, we abandoned the shortcomings of traditional single mutagenesis methods and adopted a combined mutagenesis strategy of atmospheric pressure room temperature plasma mutagenesis and sodium nitrite mutagenesis. Combined with high-yield strain screening and genetic stability verification, we obtained a high-yield and stable mutant strain.

[0112] The advantages of this combined mutagenesis strategy are as follows: ambient pressure and room temperature plasma mutagenesis has the characteristics of high mutagenesis efficiency, broad mutation spectrum, and no toxic or harmful substance residues, and can rapidly induce gene mutations in strains; sodium nitrite, as a traditional chemical mutagen, can specifically induce DNA base substitution, and its synergistic effect with ambient pressure and room temperature plasma mutagenesis can significantly improve the mutation rate and the probability of screening high-yield mutant strains, while effectively solving the problem of poor genetic stability of strains caused by single mutagenesis. The specific steps are as follows:

[0113] (2.1) Preparation of spore suspension: Streptomyces XYHK-01 was inoculated onto agar slant culture medium and cultured at 28℃ for 8 days. After the slant was confluent with spores, it was rinsed with 0.85wt% sterile physiological saline and added to an Erlenmeyer flask containing glass beads. The spores were shaken at 28℃ and 200rpm for 30 minutes to break up the mycelium and ensure full dispersion. The spore suspension was then filtered through absorbent cotton to remove mycelial fragments and obtain a pure spore suspension. The concentration of the spore suspension was adjusted to 10 using a hemocytometer. 6 The cells were sampled at a concentration of 1 / mL, and 20 vol% glycerol was added as a protective agent. The cells were then stored at 4°C for later use in subsequent mutagenesis treatment.

[0114] (2.2) Optimization and treatment of atmospheric pressure and room temperature plasma mutagenesis conditions: An atmospheric pressure and room temperature plasma breeding system (model: ARTP-Ⅱ) was used with high-purity helium (purity ≥99.999%) as the working gas. The atmospheric pressure and room temperature plasma mutagenesis conditions were optimized through single-factor experiments. The effects of irradiation time, gas flow rate, irradiation distance and power on the mutagenesis effect of the strain were investigated. The lethality rate and high mutation rate of the mutagenized strain were used as evaluation indicators to determine the optimal mutagenesis conditions.

[0115] The single-factor experimental design was as follows: ① Irradiation time: five gradients were set at 30s, 60s, 90s, 120s, and 150s, with other conditions fixed at a gas flow rate of 10L / min, an irradiation distance of 2mm, and a power of 110W; ② Gas flow rate: five gradients were set at 6L / min, 8L / min, 10L / min, 12L / min, and 14L / min, with other conditions fixed at an irradiation time of 90s, an irradiation distance of 2mm, and a power of 110W. Each treatment was repeated in triplicate. After mutagenesis, the samples were diluted to an appropriate concentration, spread on double-layer plates, and incubated at 28℃ for 8 days. The lethality rate was calculated. At the same time, the potency of the surviving strains was detected by shake-flask fermentation, and the high-mutation rate (the proportion of surviving strains with a potency of more than 30% higher than that of the original strain) was calculated.

[0116] Experimental results showed that when the atmospheric pressure, room temperature plasma mutagenesis conditions were: gas flow rate 10 L / min, irradiation distance 2 mm, power 110 W, and irradiation time 90 s, the bacterial lethality rate was 78.5%, and the high-yield mutation rate reached 12.3%, indicating the optimal mutagenesis effect (see Table 1). Under these optimal conditions, 1 mL of spore suspension was placed on the sample stage of the atmospheric pressure, room temperature plasma mutagenesis instrument for mutagenesis treatment. After mutagenesis, the spore suspension was serially diluted with sterile physiological saline to 10⁻⁶. -5 0.1 mL of the culture medium was spread onto a double-layer plate and incubated at 28°C for 8 days. Single colonies with normal morphology and vigorous growth were picked, inoculated onto slant culture medium, and incubated at 28°C for 8 days. The culture was then stored for later use. A total of 86 candidate strains of plasma mutagenesis induced at room temperature under normal pressure were obtained.

[0117] Table 1. Experimental results of single-factor plasma mutagenesis induced by ambient pressure and room temperature.

[0118]

[0119] (2.3) Optimization and treatment of sodium nitrite remutation conditions: 86 candidate strains that had undergone atmospheric pressure and room temperature plasma mutagenesis were selected and remutated with sodium nitrite to further improve their catabolite production capacity. The sodium nitrite remutation conditions were optimized through single-factor experiments to investigate the effects of sodium nitrite concentration, treatment time, and treatment temperature on the mutagenesis effect. The optimal remutation conditions were determined with high mutation rate as the evaluation index.

[0120] The single-factor experimental design was as follows: ① Sodium nitrite concentration: five gradients were set at 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, with other conditions fixed at a treatment time of 40 min and a treatment temperature of 30℃; ② Treatment time: five gradients were set at 20 min, 30 min, 40 min, 50 min, and 60 min, with other conditions fixed at a sodium nitrite concentration of 20 mg / L and a treatment temperature of 30℃. Each treatment was repeated in triplicate. After treatment, the mutagenesis was terminated by centrifugation and washing. After plating and culturing, the titer of the product was detected by shake-flask fermentation, and the high-yield mutation rate was calculated.

[0121] Experimental results showed that when the sodium nitrite remutation conditions were a final concentration of 20 mg / L, a treatment time of 40 min, and a treatment temperature of 30 °C, the high-yield mutation rate reached 18.6%, indicating the optimal remutation effect (see Table 2). Under these optimal conditions, 86 candidate strains induced by atmospheric pressure and room temperature plasma mutagenesis were selected, and concentrations of 10 mg / L and 10 mg / L were prepared. 6 A spore suspension of 10 spores / mL was added to sodium nitrite solution to achieve a final concentration of 20 mg / L, and treated with shaking at 30℃ and 200 rpm for 40 min. After treatment, the suspension was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed three times with sterile physiological saline to terminate the mutagenesis. The treated spore suspension was then diluted to 10... -5 0.1 mL of the culture medium was spread onto a double-layer plate and incubated at 28°C for 8 days. Single colonies were picked and inoculated onto slant culture medium and incubated at 28°C for 8 days. The culture was then stored for later use. A total of 123 candidate strains for remutation were obtained.

[0122] Table 2 Results of Sodium Nitrite Complex Mutagenesis Single-Factor Experiment

[0123]

[0124] (2.4) Screening of high-yield strains and verification of genetic stability: The three-step method of primary screening, secondary screening and passage stability verification was used to screen 123 candidate strains obtained by secondary mutagenesis to ensure that high-yield and stable mutant strains were obtained.

[0125] Primary screening: Using the inhibition zone method, 123 candidate strains were inoculated onto slant culture medium, cultured, and then made into bacterial suspensions. These suspensions were then inoculated onto LB agar plates containing Bacillus subtilis and cultured at 37°C for 8 hours. The diameter of the inhibition zone was measured, and strains with an inhibition zone diameter greater than 25 mm were screened. A total of 37 high-yielding strains were obtained from the primary screening.

[0126] Secondary screening: Using the shake-flask fermentation method, 37 high-yielding strains from the initial screening were inoculated into conventional fermentation medium and cultured at 28℃ and 200rpm for 168h. The potency of kasugamycin in the fermentation broth was detected by the optimized HPLC method, and strains with potency higher than 25000μg / mL were screened. A total of 8 high-yielding strains were obtained from the secondary screening.

[0127] Stability verification by passage: Eight high-yielding strains were inoculated into slant culture medium and passaged five times. After each passage, the titer of the product was detected by shake-flask fermentation and the fluctuation range of the product yield was calculated: fluctuation range (%) = (highest titer - lowest titer) / average titer × 100%.

[0128] The experimental results showed that one strain exhibited excellent genetic stability with a production capacity fluctuation of less than 5% after five consecutive passages. This strain was named Streptomyces sp. XYHK-01-M.

[0129] The phytochemical production capacity of the mutant strain *Streptomyces* sp. XYHK-01-M was verified. The titer reached 29,000 μg / mL in shake-flask fermentation, a 141.7% increase compared to the original strain XYHK-01 (12,000 μg / mL). Compared to traditional single-mutant strains, its phytochemical production titer increased by more than 40%, and its genetic stability was significantly improved (see Table 3). Simultaneously, the morphological and physiological-biochemical characteristics of this mutant strain were examined. The results showed that its morphological characteristics were basically consistent with the original strain, with only a slightly darker aerial mycelium color (grayish-white to light gray). There were no significant changes in physiological and biochemical characteristics, indicating that the mutagenesis treatment only altered the phytochemical production capacity of the strain without changing its basic biological characteristics, making it suitable for industrial fermentation production.

[0130] Table 3 Comparison of glycogen production capacity and genetic stability of different strains

[0131]

[0132] Example 2

[0133] Fermentation process optimization of high-yield kasugamycin production strains

[0134] Fermentation process is a key factor affecting the potency and production efficiency of kasugamycin. In this embodiment, the mutant strain Streptomyces sp. XYHK-01-M was used as the production strain. Through single-factor experiments and orthogonal experiments, the fermentation medium formulation and fermentation process parameters were systematically optimized to solve the problems of long fermentation cycle, low potency, and high cost of existing processes, and to establish an efficient and stable three-stage fermentation process, as detailed below:

[0135] Fermentation medium optimization: The components (carbon source, nitrogen source, inorganic salts, growth factors, etc.) and their ratios in the fermentation medium directly affect the growth and factor production capacity of the strain. In this embodiment, based on the initial fermentation medium, suitable carbon source, nitrogen source, inorganic salts and growth factors were screened through single-factor experiments, and the ratios of each component were optimized through orthogonal experiments to finally obtain the optimal fermentation medium formula.

[0136] (1) Screening of culture medium components by single-factor experiment: The effects of carbon source type and concentration, nitrogen source type and concentration, inorganic salt type and concentration, and growth factor on the cytokine production capacity of Streptomyces sp. XYHK-01-M were investigated. Five gradients were set for each factor. Other conditions were fixed as follows: fermentation temperature 28℃, shaking speed 200rpm, inoculum size 10%, and fermentation period 168h. Three replicates were set for each treatment. After fermentation, the kasugamycin titer was detected to screen the optimal component and concentration range.

[0137] (1.1) Carbon source screening: Carbon source is the main energy source for the growth and production of the strain. Five common carbon sources, glucose, raffinose, maltose, sucrose and glycerol, were investigated. The concentration of each was set at 15 g / L. The results showed that when maltose was used as the carbon source, the strain produced the highest potency of the product, reaching 25600 μg / mL. Raffinose was the second highest (23200 μg / mL). The potency of glucose, sucrose and glycerol was relatively low (18500 μg / mL, 17800 μg / mL and 19200 μg / mL, respectively). Therefore, maltose was selected as the optimal carbon source, and its concentration (12 g / L, 15 g / L, 18 g / L, 21 g / L, 24 g / L) was further investigated on the effect of the product on the production of the product. The results showed that the product potency was highest when the maltose concentration was 18 g / L (27800 μg / mL). Both excessively high and low concentrations led to a decrease in the product potency. This may be because excessively high carbon source concentrations can lead to excessive cell growth, consuming a large amount of nutrients and inhibiting product synthesis; while excessively low concentrations cannot meet the energy requirements for cell growth and product production.

[0138] (1.2) Nitrogen source screening: Nitrogen source is an important raw material for the synthesis of proteins, nucleic acids and antibiotics by strains. Five nitrogen sources were investigated: soybean meal powder, soybean cake powder, tryptone, corn steep liquor powder and yeast extract. The concentration of each was set at 5.0% (mass-volume ratio). The experimental results showed that soybean meal powder had the highest yield (26300 μg / mL), followed by corn steep liquor powder (24100 μg / mL). The yields of tryptone, soybean cake powder and yeast extract were relatively low (20100 μg / mL, 21500 μg / mL and 22300 μg / mL, respectively). Soybean meal powder, as an inexpensive plant-based nitrogen source, is not only rich in nutrients, but also provides a variety of amino acids and growth factors. Moreover, its low cost makes it suitable for industrial production. Further investigation was conducted on the effects of soybean meal concentration (5.0%, 6.0%, 7.0%, 8.0%, 9.0%) on the production of the product. The results showed that the product potency was highest when the soybean meal concentration was 7.0% (28200 μg / mL). Excessive concentration would lead to an imbalance in the carbon-nitrogen ratio and inhibit product synthesis; while excessively low concentration would not meet the growth requirements of the microorganisms.

[0139] (1.3) Screening of Inorganic Salts: Inorganic salts can regulate the osmotic pressure and pH of the culture medium and participate in the metabolic process of the strain. Four common inorganic salts, sodium chloride, potassium dihydrogen phosphate, calcium carbonate (CaCO3), and magnesium sulfate, were investigated at concentrations of 0.3%, 0.03%, 1.0%, and 0.05% (mass-volume ratio), respectively. Experimental results showed that the addition of potassium dihydrogen phosphate and sodium chloride significantly increased the product potency. Potassium dihydrogen phosphate promoted the metabolism and product synthesis of the strain, while sodium chloride regulated the osmotic pressure of the culture medium and maintained normal bacterial growth. Calcium carbonate and magnesium sulfate had no significant effect on product potency. Further optimization of the potassium dihydrogen phosphate concentration (0.02%, 0.03%, 0.04%, 0.05%, 0.06%) and sodium chloride concentration (0.2%, 0.3%, 0.4%, 0.5%, 0.6%) revealed that the highest product potency (28500 μg / mL) was achieved when the potassium dihydrogen phosphate concentration was 0.04% and the sodium chloride concentration was 0.4%.

[0140] (1.4) Screening of growth factors: Growth factors can promote the growth and product production of the strain. Four growth factors—inositol, vitamin B1, vitamin B6, and biotin—were investigated, all at a concentration of 0.03 g / L. The results showed that the addition of inositol resulted in the highest product potency (29100 μg / mL), an increase of 8.2% compared to the absence of growth factors. Other growth factors had no significant effect on product production. Inositol was extracted from spring corn soaking solution using sodium phytate hydrolysis, which is low-cost and synergistically works with other components in the culture medium to promote cell growth and product release. Further investigation was conducted on the effect of inositol concentrations (0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, and 0.05 g / L) on product production. The results showed that the highest product potency was achieved at an inositol concentration of 0.03 g / L; excessively high or low concentrations led to a decrease in product potency.

[0141] (1.5) Surfactant Screening: Surfactants can improve cell membrane permeability, promote the release of intracellular products, and increase fermentation potency. Three surfactants, Tween-20, Tween-80, and sodium dodecyl sulfate (SDS), were investigated, all at a concentration of 1 g / L. Experimental results showed that the addition of Tween-20 resulted in the highest product potency (29800 μg / mL), a 10.5% increase compared to the absence of surfactant. Tween-80 was the second most effective (28700 μg / mL), while SDS showed some toxicity to the strain and a lower product potency (25300 μg / mL). Therefore, Tween-20 was selected as the surfactant, with a concentration of 1 g / L.

[0142] (2) Orthogonal experiment to optimize the culture medium ratio: Based on the single factor experiment, four key factors affecting the efficacy of the product were selected: maltose concentration (A), soybean meal powder concentration (B), potassium dihydrogen phosphate concentration (C), and inositol concentration (D). Three levels were set for each factor. The L9 (34) orthogonal experimental design was adopted to optimize the culture medium ratio. The experimental factors and levels are shown in Table 4, and the results of the orthogonal experiment are shown in Table 5.

[0143] Table 4. Factors and Levels in Orthogonal Experiments

[0144]

[0145] Table 5 L9 (3) 4 Orthogonal experimental results

[0146]

[0147] Analysis of the orthogonal experimental results showed that the order of influence of each factor on the potency of kasugamycin production was: soybean meal concentration (B) > inositol concentration (D) > potassium dihydrogen phosphate concentration (C) > maltose concentration (A). Based on the K-value and range analysis of the orthogonal experiment, the optimal fermentation medium formula (mass-volume ratio) was determined to be: 7.0 wt% soybean meal, 18 g / L maltose, 0.4 wt% sodium chloride, 0.8 wt% corn steep liquor powder, 3.8 wt% soybean oil, 0.04 wt% potassium dihydrogen phosphate, 0.03 g / L inositol, 1 g / L Tween-20, pH 7.0. Specifically, the optimized fermentation medium is prepared by sequentially adding 70g of soybean meal powder, 18g of maltose, 4g of sodium chloride, 8g of corn steep liquor powder, 0.4g of potassium dihydrogen phosphate, and 0.03g of inositol to an appropriate amount of distilled water, heating and stirring until completely dissolved. Then, 38g of soybean oil and 1.0g of Tween-20 are added, and the mixture is thoroughly emulsified and mixed. The volume is adjusted to 1L, and the pH is adjusted to 7.0 with NaOH or HCl solution. After dispensing, the mixture is sterilized by autoclaving at 121℃ and 103.4 kPa for 20 minutes. After cooling, the optimized fermentation medium is obtained.

[0148] (3) Culture medium optimization verification experiment: The optimal fermentation culture medium formula was used, and the mutant strain Streptomyces sp. XYHK-01-M was inoculated for shake flask fermentation verification experiment. Three replicates were set up, and the fermentation conditions were 28℃, 200rpm, inoculum size of 10%, and fermentation period of 168h. After fermentation, the kasugamycin titer was measured. The experimental results showed that the average kasugamycin titer in the optimized fermentation broth was 29500μg / mL, which was 15.2% higher than that before optimization (25600μg / mL), and the repeatability was good (relative standard deviation ≤2.0%), indicating that the optimized culture medium formula was reasonable and could significantly improve the kasugamycin production capacity of the strain. At the same time, all components in this culture medium formula are inexpensive and readily available industrial raw materials. The use of corn steep liquor powder and soybean meal powder can further reduce production costs and is suitable for large-scale industrial production.

[0149] Fermentation process parameter optimization: Based on the optimized fermentation medium formula, a three-stage fermentation mode (seed culture → primary fermentation → secondary fermentation) was adopted. Through single-factor experiments, fermentation process parameters were optimized, including seed culture parameters, fermentation temperature, aeration rate, stirring speed, tank pressure, feeding strategy, and fermentation cycle, to further improve the yield of nutrients and shorten the fermentation cycle.

[0150] (3.1) Optimization of seed culture parameters: The quality of the seed culture directly affects the subsequent fermentation effect. Optimizing the seed culture parameters to obtain a vigorous and sufficient seed culture is a prerequisite for improving the fermentation potency. The effects of seed culture medium inoculum size, culture temperature, shaker speed, and culture time on the quality of the seed culture were investigated. The optimal seed culture parameters were determined using the OD600 value (cell concentration) and fermentation potency of the seed culture as evaluation indicators.

[0151] Experimental results showed that when the seed culture medium inoculum was 15% (v / v), the culture temperature was 28℃, the shaker speed was 220 rpm, and the culture time was 48 h, the OD600 value of the seed culture reached 1.8-2.0, indicating suitable cell concentration and vigorous activity. After inoculating the seed culture into the fermentation medium, the fermentation titer reached 29800 μg / mL, which was more than 3.5% higher than that under other parameters. Therefore, the optimal seed culture parameters were determined to be: inoculum size 15%, 28℃, 220 rpm shaking culture for 48 h.

[0152] (3.2) Fermentation Temperature Optimization: Fermentation temperature is a crucial environmental factor affecting strain growth and product synthesis. Excessively high temperatures can lead to metabolic disorders, decreased enzyme activity, and even cell autolysis; conversely, excessively low temperatures inhibit cell growth and toxin production. The effects of fermentation temperatures (26℃, 27℃, 28℃, 29℃, and 30℃) on toxin potency were investigated. Other conditions were fixed as follows: seed culture inoculum 12%, aeration rate 1:1 (v / v), stirring speed 180 rpm, tank pressure 0.02 MPa, and fermentation period 168 h. Each treatment was replicated three times. The results showed that the highest toxin potency (30200 μg / mL) was observed at a fermentation temperature of 29℃. Below 29℃, cell growth was slow, and the toxin potency increased with increasing temperature. Above 29℃, cell metabolism accelerated, nutrient consumption was excessive, and the toxin potency decreased. Therefore, the optimal fermentation temperature was determined to be 29℃.

[0153] (3.3) Aeration Optimization: *Streptomyces simonii* is an aerobic microorganism, requiring sufficient oxygen during fermentation to meet the needs of cell growth and product synthesis. Insufficient aeration leads to cell hypoxia, accumulation of metabolites, and inhibition of phytochemical production; excessive aeration increases energy consumption and may cause rapid evaporation of the culture medium, affecting the stability of the fermentation system. The effect of aeration rates (0.6:1, 0.8:1, 1:1, 1.2:1, 1.4:1, v / v, i.e., the ratio of air volume introduced into the fermenter per minute to fermentation liquid volume) on phytochemical potency was investigated. With other conditions fixed, the experimental results showed that the phytochemical potency was highest (30500 μg / mL) when the aeration rate was 1:1 (v / v); when the aeration rate was lower than 1:1, cell hypoxia occurred, and the phytochemical potency was lower; when the aeration rate was higher than 1:1, the phytochemical potency did not increase significantly, but energy consumption increased. Therefore, the optimal aeration rate was determined to be 1:1 (v / v).

[0154] (3.4) Optimization of stirring speed: The stirring speed and aeration rate work synergistically to affect the oxygen solubility and cell dispersion in the fermentation broth. Too low a stirring speed results in insufficient oxygen solubility, cell aggregation, and impaired metabolism; too high a stirring speed leads to cell damage and increased energy consumption. The effect of stirring speed (140 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm) on the potency of the product was investigated. With other conditions fixed, the experimental results showed that the potency was highest (30800 μg / mL) at a stirring speed of 180 rpm. At this speed, the oxygen solubility in the fermentation broth was suitable, the cells were evenly dispersed, and metabolism was vigorous. Both excessively high and low stirring speeds led to a decrease in the potency of the product. Therefore, the optimal stirring speed was determined to be 180 rpm.

[0155] (3.5) Tank Pressure Optimization: Tank pressure can affect the dissolved oxygen in the fermentation broth. Appropriately increasing the tank pressure can increase oxygen solubility, meeting the needs of cell growth and nutrient production. However, excessively high tank pressure will increase the equipment burden and may inhibit cell metabolism. The effect of tank pressure (0.01MPa, 0.02MPa, 0.03MPa, 0.04MPa, 0.05MPa) on nutrient potency was investigated. With other conditions fixed, the experimental results showed that the nutrient potency was highest (31000μg / mL) when the tank pressure was 0.02MPa. When the tank pressure was below 0.02MPa, the dissolved oxygen was insufficient. When the tank pressure was above 0.02MPa, the nutrient potency did not increase significantly, and the equipment energy consumption increased. Therefore, the optimal tank pressure was determined to be 0.02MPa.

[0156] (3.6) Optimization of feeding strategy: During fermentation, the carbon and nitrogen sources are consumed rapidly. If they are not replenished in time, cell growth and nutrient production will be hindered, affecting fermentation potency. This invention adopts a batch feeding strategy to investigate the effects of feeding time, type of feed, and amount on nutrient potency. The optimal feeding strategy is determined through single-factor experiments.

[0157] Experimental results showed that the optimal results were achieved by feeding the bacteria after 72 hours of fermentation, adding 2% (w / v) maltose and 0.5% (w / v) corn steep liquor powder. At this stage, the bacteria were in the late logarithmic growth phase and began to synthesize kasugamycin in large quantities. Adding maltose provided a carbon source to meet the energy requirements for product synthesis; adding corn steep liquor powder provided a nitrogen source and growth factors, maintaining a carbon-nitrogen balance and promoting product synthesis. After feeding, the kasugamycin titer in the fermentation broth reached 31500 μg / mL, an increase of 5.2% compared to the unfed state. Feeding too early would lead to excessive bacterial growth and consumption of large amounts of nutrients; feeding too late would result in insufficient nutrient supply and inhibited kasugamycin production; both excessive and insufficient feeding would affect the kasugamycin titer.

[0158] (3.7) Fermentation cycle optimization: The effects of fermentation cycles (144h, 156h, 168h, 180h, 192h) on the potency of the microbial agent were investigated. With other conditions fixed, the experimental results showed that the potency of the microbial agent was highest (31500μg / mL) when the fermentation cycle was 168h. When the fermentation cycle was less than 168h, the microbial agent production did not reach its peak and the potency was low. When the fermentation cycle was more than 168h, the potency of the microbial agent did not increase significantly and it led to excessive consumption of nutrients and microbial autolysis, affecting product quality and increasing energy consumption and production costs. Therefore, the optimal fermentation cycle was determined to be 168h, which is 16% shorter than the traditional process (200h).

[0159] (3.8) Validation of the three-stage fermentation process: The optimized fermentation medium formula and process parameters were used to conduct a three-stage fermentation validation experiment. The specific process was as follows: ① Seed culture: The mutant strain Streptomyces sp. XYHK-01-M was inoculated into the seed medium at an inoculation amount of 15%, and cultured at 28℃ and 220rpm for 48h to obtain the seed liquid; ② Primary fermentation: The seed liquid was inoculated into a 50L fermenter at an inoculation amount of 12%, the fermentation temperature was 29℃, the aeration rate was 1:1 (v / v), the stirring speed was 180rpm, the tank pressure was 0.02MPa, and the fermentation was carried out for 72h; ③ Secondary fermentation: The primary fermentation liquid was inoculated into a 500L fermenter at an inoculation amount of 12%, 2% maltose and 0.5% corn steep liquor powder were added, and the fermentation continued, maintaining the same process parameters as the primary fermentation, and fermentation was carried out for 168h to end the fermentation.

[0160] The verification experiment was conducted in triplicate. The results showed that the kasugamycin potency in the secondary fermentation broth was stable at 31,000-31,500 μg / mL, with an average of 31,200 μg / mL, which is 73.3% higher than that of the traditional process (18,000 μg / mL). The fermentation cycle was shortened by 16%, the culture medium utilization rate reached over 88%, and the production cost was reduced by 20%. Moreover, the fermentation process was stable, and the batch-to-batch differences in the product were small (relative standard deviation ≤2.0%). This indicates that the optimized three-stage fermentation process is feasible and suitable for large-scale industrial production.

[0161] Example 3

[0162] The fermentation process of Streptomyces sp. XYHK-01-M, a high-yield strain of kasugamycin, includes the following steps:

[0163] S1. Streptomyces sp. XYHK-01-M was inoculated into the seed culture medium at a volume ratio of 15% into the seed bottle and cultured at 28℃ and 220rpm on a shaker for 48h to achieve an OD600 of 1.8-2.0, thus obtaining a vigorous seed culture.

[0164] S2 graded scale-up fermentation and feeding

[0165] S2.1 Primary Fermentation

[0166] The seed culture was inoculated into the first-stage fermenter at a volume ratio of 12%. The effective volume of the fermenter was 50L, and the liquid volume was 60% of the tank volume.

[0167] Primary fermentation conditions: Optimized fermentation medium was used; fermentation temperature was 29℃; aeration rate was 1:1; stirring speed was 180 rpm; tank pressure was 0.02 MPa; fermentation time was 72 h.

[0168] S2.2 Secondary Fermentation and Feeding

[0169] The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 12% by volume. The effective volume of the fermenter was 500L, and the liquid volume was 60% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used.

[0170] 72 hours after the start of fermentation, the secondary fermenter was fed with maltose to a final concentration of 2.0 wt%, corn steep liquor powder to a final concentration of 0.5 wt%, and sodium butyrate to a final concentration of 0.001 wt%. Fermentation continued at 29°C, with an aeration rate of 1:1, stirring at 200 rpm, and a tank pressure of 0.02 MPa. The total fermentation period was controlled to 168 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth reached 33453 μg / mL (average of three tests). The total fermentation period of 168 hours refers to the total fermentation time of the secondary fermentation being 168 hours. Based on the optimized fermentation process in Example 2, low-dose sodium butyrate, without significantly altering cell growth, can activate the kasugamycin biosynthetic gene cluster through epigenetic regulation pathways, increasing secondary metabolic flux and thus significantly improving the fermentation yield of kasugamycin.

[0171] The optimized fermentation medium was prepared as follows: 70g of soybean meal powder, 18g of maltose, 4g of sodium chloride, 8g of corn steep liquor powder, 0.4g of potassium dihydrogen phosphate, and 0.03g of inositol were added to an appropriate amount of distilled water and heated and stirred until completely dissolved. Then, 38g of soybean oil and 1.0g of Tween-20 were added and stirred thoroughly. The volume was adjusted to 1L with distilled water, the pH was adjusted to 7.0, and the medium was dispensed and sterilized by autoclaving at 121℃ and 103.4 kPa for 20 minutes. After cooling, the medium was obtained.

[0172] Example 4

[0173] The fermentation process of Streptomyces sp. XYHK-01-M, a high-yield strain of kasugamycin, includes the following steps:

[0174] S1. Streptomyces sp. XYHK-01-M was inoculated into the seed culture medium at a volume ratio of 15% into the seed bottle and cultured at 28℃ and 220rpm on a shaker for 48h to achieve an OD600 of 1.8-2.0, thus obtaining a vigorous seed culture.

[0175] S2 graded scale-up fermentation and feeding

[0176] S2.1 Primary Fermentation

[0177] The seed culture was inoculated into the first-stage fermenter at a volume ratio of 12%. The effective volume of the fermenter was 50L, and the liquid volume was 60% of the tank volume.

[0178] Primary fermentation conditions: Optimized fermentation medium was used; fermentation temperature was 29℃; aeration rate was 1:1; stirring speed was 180 rpm; tank pressure was 0.02 MPa; fermentation time was 72 h.

[0179] S2.2 Secondary Fermentation and Feeding

[0180] The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 12% by volume. The effective volume of the fermenter was 500L, and the liquid volume was 60% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used.

[0181] 72 hours after the start of fermentation, the secondary fermenter was fed with maltose to a final concentration of 2.0 wt%, corn steep liquor powder to a final concentration of 0.5 wt%, and kasugamycin to a final concentration of 0.001 wt%. Fermentation continued at 29°C, with an aeration rate of 1:1, stirring at 200 rpm, and a tank pressure of 0.02 MPa. The total fermentation period was controlled to 168 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth reached 34117 μg / mL (average of three tests). The total fermentation period of 168 hours refers to the total fermentation time of the secondary fermentation being 168 hours. As a potent HDAC inhibitor, kasugamycin, under the optimized fermentation conditions of this invention, can more effectively activate the kasugamycin biosynthetic gene cluster through epigenetic regulators, thereby improving the synthesis level of the target agricultural antibiotic.

[0182] The optimized fermentation medium was prepared as follows: 70g of soybean meal powder, 18g of maltose, 4g of sodium chloride, 8g of corn steep liquor powder, 0.4g of potassium dihydrogen phosphate, and 0.03g of inositol were added to an appropriate amount of distilled water and heated and stirred until completely dissolved. Then, 38g of soybean oil and 1.0g of Tween-20 were added and stirred thoroughly. The volume was adjusted to 1L with distilled water, the pH was adjusted to 7.0, and the medium was dispensed and sterilized by autoclaving at 121℃ and 103.4 kPa for 20 minutes. After cooling, the medium was obtained.

[0183] Example 5

[0184] The fermentation process of Streptomyces sp. XYHK-01-M, a high-yield strain of kasugamycin, includes the following steps:

[0185] S1. Streptomyces sp. XYHK-01-M was inoculated into the seed culture medium at a volume ratio of 15% into the seed bottle and cultured at 28℃ and 220rpm on a shaker for 48h to achieve an OD600 of 1.8-2.0, thus obtaining a vigorous seed culture.

[0186] S2 graded scale-up fermentation and feeding

[0187] S2.1 Primary Fermentation

[0188] The seed culture was inoculated into the first-stage fermenter at a volume ratio of 12%. The effective volume of the fermenter was 50L, and the liquid volume was 60% of the tank volume.

[0189] Primary fermentation conditions: Optimized fermentation medium was used; fermentation temperature was 29℃; aeration rate was 1:1; stirring speed was 180 rpm; tank pressure was 0.02 MPa; fermentation time was 72 h.

[0190] S2.2 Secondary Fermentation and Feeding

[0191] The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 12% by volume. The effective volume of the fermenter was 500L, and the liquid volume was 60% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used.

[0192] 72 hours after the start of fermentation, the secondary fermenter was fed with maltose to a final concentration of 2.0 wt%, corn steep liquor powder to a final concentration of 0.5 wt%, sodium butyrate to a final concentration of 0.0005 wt%, and kasugamycin to a final concentration of 0.0005 wt%. Fermentation continued at 29°C, aeration rate of 1:1, stirring at 200 rpm, and a tank pressure of 0.02 MPa, with a total fermentation period controlled at 168 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth reached 35137 μg / mL (average of three tests). The total fermentation period of 168 hours refers to the total fermentation time of the secondary fermentation being 168 hours. Compared to Example 2 (31200 μg / mL) without epigenetic regulators, the kasugamycin titer increased by 12.6%. It also showed a significant improvement compared to Example 3 (with only sodium butyrate) and Example 4 (with only kasugamycin), demonstrating a significant synergistic effect when the two HDAC inhibitors are used in combination.

[0193] The optimized fermentation medium was prepared as follows: 70g of soybean meal powder, 18g of maltose, 4g of sodium chloride, 8g of corn steep liquor powder, 0.4g of potassium dihydrogen phosphate, and 0.03g of inositol were added to an appropriate amount of distilled water and heated and stirred until completely dissolved. Then, 38g of soybean oil and 1.0g of Tween-20 were added and stirred thoroughly. The volume was adjusted to 1L with distilled water, the pH was adjusted to 7.0, and the medium was dispensed and sterilized by autoclaving at 121℃ and 103.4 kPa for 20 minutes. After cooling, the medium was obtained.

[0194] Example 6

[0195] Detection methods for fermentation broth

[0196] This embodiment optimizes the high-performance liquid chromatography (HPLC) detection method, solving the problems of cumbersome operation, long detection cycle, and insufficient accuracy and repeatability of traditional detection methods. It establishes a rapid, accurate and stable method for detecting kasugamycin in fermentation broth, which can realize real-time monitoring of fermentation broth potency during industrial production.

[0197] The high-performance liquid chromatography (HPLC) injection method is as follows:

[0198] Mobile phase: (acetonitrile: 0.1wt% sodium dodecyl sulfate aqueous solution) = 30:70 (V / V); (adjust pH to 2.5 with 85wt% phosphoric acid aqueous solution), the mobile phase is filtered through a 0.45μm pore size filter membrane and degassed in an ultrasonic bath.

[0199] Flow rate: 1.0 mL / min.

[0200] Column temperature: 30℃.

[0201] Detection wavelength: 230nm.

[0202] Injection volume: 10 μL.

[0203] Retention time: Kasugamycin approximately 11.1 min.

[0204] Measurement steps:

[0205] Preparation of standard solution: Accurately weigh 0.1 g of kasugamycin standard (accurate to 0.0001 g), place it in a 50 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the mark, and shake well; use a pipette to transfer 5 mL of the above solution into a 50 mL volumetric flask, dissolve and dilute to the mark with water, shake well, and set aside.

[0206] Preparation of sample solution: Accurately weigh a certain amount (accurate to 0.0001 g) of the kasugamycin-containing fermentation broth sample and place it in a 50 mL volumetric flask. Add an appropriate amount of water to dissolve and dilute to the mark, and shake well. Transfer 5 mL of the above solution to a 50 mL volumetric flask using a pipette, dissolve and dilute to the mark with water, and shake well. Centrifuge the kasugamycin-containing fermentation broth at 6000 r / min for 10 min. Take the supernatant and filter it through a microporous membrane with a pore size of 0.45 μm to obtain a clear kasugamycin-containing fermentation broth, which is the test solution.

[0207] Measurement: Under the above operating conditions, after the instrument baseline stabilizes, inject several standard solutions consecutively, calculate the repeatability of the relative response values ​​of each injection, and when the relative response value of two adjacent injections of kasugamycin is less than 1.2%, perform the measurement in the order of standard solution, sample solution, sample solution, and standard solution.

[0208] The peak areas of kasugamycin in the two sample solutions and the two standard solutions were averaged. The potency (μg / mL) of kasugamycin in the sample was calculated using the following formula:

[0209]

[0210] In the formula:

[0211] The average peak area of ​​kasugamycin in the A2 sample solution;

[0212] The mass of m1-kasugamycin standard is expressed in grams (g).

[0213] w1 - Mass fraction of kasugamycin standard, expressed as %;

[0214] The average peak area of ​​kasugamycin in A1-standard solution;

[0215] m2 - The mass of the sample, expressed in grams (g).

[0216] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A high-yield kasugamycin-producing strain, characterized in that: The high-yield kasugamycin producing strain is *Streptomyces* sp. XYHK-01-M, with accession number CGMCC No. 39497. The depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 29, 2026.

2. A method for breeding a high-yield kasugamycin-producing strain, characterized in that, Includes the following steps: (1) Screening of original high-yielding strains (1.1) Soil sample processing and soil suspension preparation Select soil from the top 10-20cm of the vegetable field, remove stones, weeds and other impurities, weigh 5-20g of soil, add sterile physiological saline with a concentration of 0.5-1.0wt% and a volume equivalent to 5-15 times the soil mass, and shake for 20-40min at 25-30℃ and 150-250rpm to obtain a uniform soil suspension. The soil suspension was prepared at a ratio of 10 -4 -10 -8 Perform serial dilutions, with 2-5 replicates for each dilution; (1.2) Actinomycetes were isolated using double-layer selective plates. Take 0.05-0.2 mL of soil suspension at different dilutions and spread it evenly on the surface of a double-layer plate culture medium. Incubate at 26-30℃ for 5-10 days, and observe the colony growth daily. Select morphologically typical single colonies of actinomycetes and inoculate them onto slant culture medium. Incubate at 26-30℃ for 5-10 days and store for later use. Morphologically typical single colonies of actinomycetes are defined as grayish-white, fluffy, with neat edges and obvious aerial hyphae. (1.3) Initial screening of high-yielding strains using the inhibition zone method Spores from each slant culture strain were scraped off using 0.5-1.0 wt% sterile physiological saline to prepare spores with a concentration of 10. 5 -10 8 A bacterial suspension of 1 / mL was prepared; 5-20 μL of the actinomycete suspension was inoculated onto LB agar plates containing Bacillus subtilis, with 2-5 replicates per strain. The plates were incubated at 35-38℃ for 6-12 h, and the diameter of the inhibition zone was measured. Strains with an inhibition zone diameter greater than 15 mm were selected as candidate strains. Candidate strains obtained from the initial screening of inhibition zones were inoculated into Erlenmeyer flasks with a volume of 250-1000 mL. The fermentation medium was a conventional fermentation medium, with the liquid volume being 10-40% of the flask volume and the inoculation amount being 5-15% by volume. The flasks were cultured at 26-30℃ and 160-240 rpm with shaking for 144-192 h. After fermentation, the kasugamycin titer in the fermentation broth was determined by high performance liquid chromatography, and strains with a kasugamycin titer of not less than 12000 μg / mL were screened as the original high-yielding strains. (2) Combined mutagenesis of the original strain and the acquisition of high-yielding and stable strains (2.1) Preparation of spore suspension of original strain The original high-yielding strain was inoculated onto an agar slant and cultured at 26-30℃ for 5-10 days. After the slant was covered with spores, 0.5-1.0wt% sterile physiological saline was added to wash and collect the spore suspension. The spore suspension was then transferred into a container containing glass beads and shaken at 25-30℃ and 150-250rpm for 20-40 minutes to fully break the mycelium and evenly disperse the spores. After filtering with absorbent cotton to remove mycelial debris, the spore concentration was adjusted to 10. 5 -10 8 The sample was prepared at 0-4℃ and stored at 0-4℃ for later use, with 10-25% glycerol added as a protective agent. (2.2) Atmospheric pressure and room temperature plasma mutagenesis A normal-pressure, room-temperature plasma breeding system was used, with high-purity helium as the working gas, to induce the first round of mutagenesis on the spore suspension of the original strain. Spread 0.5-2.0 mL of spore suspension evenly on the ARTP sample stage and treat it under the following conditions: gas flow rate 6-14 L / min, nozzle-to-sample surface distance 1-3 mm, discharge power 80-120 W, and irradiation time 30-150 s. After mutagenesis, the treatment solution was eluted with sterile physiological saline and serially diluted to 10⁻⁶. -4 -10 -6 Take 0.05-0.2 mL of the culture medium and spread it on a double-layer plate. Incubate at 26-30℃ for 5-10 days, calculate the lethality, and select morphologically normal and vigorously growing single colonies from the surviving colonies to inoculate and preserve them on slant culture to obtain candidate strains for plasma mutagenesis at room temperature and atmospheric pressure. Under the above conditions, the lethality is generally 60-90%, and the proportion of high-yielding mutant strains is not less than 10%. (2.3) Sodium nitrite remutation Candidate strains obtained by atmospheric pressure and room temperature plasma mutagenesis were selected, and spore concentrations of 10 were prepared. 5 -10 8 A second round of chemical mutagenesis was performed on a spore suspension of 10-50 mg / L, with sodium nitrite solution added to achieve a final sodium nitrite concentration of 10-50 mg / L. The suspension was then treated with shaking at 25-35℃ and 150-250 rpm for 20-60 min. After treatment, the suspension was centrifuged at 6000-10000 rpm for 5-15 min, the supernatant was discarded, and the precipitate was washed 2-4 times with sterile physiological saline to terminate the mutagenesis. The suspension was then serially diluted to 10⁻⁶. -4 -10 -6 Take 0.05-0.2 mL of the solution and spread it onto a double-layer plate culture medium. Incubate at 26-30℃ for 5-10 days. Pick single colonies and inoculate them onto slant culture for preservation to obtain candidate strains for sodium nitrite remutation. The high-yield mutation rate should not be less than 10%. (2.4) Screening of high-yielding strains and verification of genetic stability Initial screening of inhibition zone The candidate strains for complex mutagenesis were prepared to a spore concentration of 10. 5 -10 8 Take 5-20 μL of bacterial suspension per 1000 cells / mL and inoculate it on LB agar plates containing Bacillus subtilis. Set up 2-5 replicates for each strain and incubate at 35-38℃ for 6-12 h. Measure the diameter of the inhibition zone and screen strains with an inhibition zone diameter greater than 25 mm as high-yielding primary screening strains. Shake-flask fermentation and double screening High-yield primary screening strains were inoculated into conventional fermentation medium and cultured with shaking at 26-30℃ and 160-240rpm for 144-192h. The kasugamycin titer in the fermentation broth was determined by high performance liquid chromatography, and strains with kasugamycin titers higher than 25000μg / mL were selected as high-yield secondary screening strains. Validation of genetic stability through successive generations High-yield, re-screened strains were passaged 3-10 times on slant agar. Each generation of shake-flask fermentation was carried out under the following conditions: inoculation into 250-1000 mL Erlenmeyer flasks, filling the flasks to 10-40% of their volume, with an inoculum size of 5-15% (v / v). The flasks were cultured at 26-30℃ and 160-240 rpm for 144-192 h with shaking. After fermentation, the kasugamycin titer in the fermentation broth was determined using high-performance liquid chromatography (HPLC), and the fluctuation range of the titer was calculated using the following formula: Fluctuation range (%) = (Highest valence - Lowest valence) / Average valence × 100% Strains with fluctuations not exceeding 5% and capable of stably producing kasugamycin at a titer higher than 25000 μg / mL were selected as the final high-yielding mutant strains with stable genetic traits, i.e., high-yielding kasugamycin-producing strains.

3. The method for breeding high-yield kasugamycin-producing strains as described in claim 2, characterized in that, The diluent used in step (1.1) for gradient dilution is a 0.5-1.0 wt% sodium chloride solution.

4. The method for breeding high-yield kasugamycin-producing strains as described in claim 2, characterized in that, The method for preparing the double-layer plate culture medium is as follows: 1-5g of tryptone, 5-15g of soybean meal powder, 5-20mL of glycerol, 1-5g of sodium chloride, 1-3g of calcium carbonate, and 20-30g of agar are added to an appropriate amount of distilled water and dissolved evenly. The volume is then adjusted to 1L with distilled water, the pH is adjusted to 6.8-7.5, and the medium is sterilized at 115-125℃ and 103-137kPa for 15-30min. The mixture is then poured into sterile petri dishes and cooled to solidify, yielding the lower basal culture medium. Add 1-5g of tryptone, 5-15g of soybean meal, 5-20mL of glycerol, 1-5g of sodium chloride, 1-3g of calcium carbonate, 20-30g of agar, and 50-150mg of kasugamycin to an appropriate amount of distilled water and shake well. Make up to 1L with distilled water, adjust the pH to 6.8-7.5, sterilize at 115-125℃ and 103-137kPa for 15-30min, and pour onto the surface of the solidified lower basal culture medium to form a double-layer plate culture medium.

5. The method for breeding high-yield kasugamycin-producing strains as described in claim 2, characterized in that, The preparation method of the slant culture medium described in steps (1.2), (2.1), and (2.4) is as follows: 1.2-1.8g of high-temperature soybean cake powder, 0.4-0.6g of sodium chloride, 0.2-0.3g of dipotassium hydrogen phosphate, and 1.8-2.2g of agar are added to an appropriate amount of distilled water and heated until dissolved evenly. Then, 8-12mL of glycerol is added and stirred evenly. The volume is adjusted to 1L with distilled water, and the pH is adjusted to 7.0-7.

4. After dispensing, the medium is sterilized at 121-126℃ and 103-137kPa for 15-25min. The medium is then placed slanted while hot and allowed to solidify to obtain the final product.

6. The method for breeding high-yield kasugamycin-producing strains as described in claim 2, characterized in that, The preparation method of LB plate culture medium containing Bacillus subtilis described in steps (1.3) and (2.4) is as follows: Take 9-11g of tryptone, 4.5-5.5g of yeast extract, 9-11g of sodium chloride and 23-27g of agar, add them to an appropriate amount of distilled water and heat to dissolve, make up to 1L, adjust the pH to 6.8-7.2, dispense and sterilize at 121-126℃ and 103-137kPa for 15-25min, cool to 45-55℃, add an appropriate amount of Bacillus subtilis bacterial solution under aseptic conditions, mix well and pour into plates, and obtain the culture medium after solidification.

7. The method for breeding high-yield kasugamycin-producing strains as described in claim 2, characterized in that, The preparation method of the conventional fermentation medium in steps (1.3) and (2.4) is as follows: 45-55g of soybean meal powder, 14-16g of maltose, 2.5-3.5g of sodium chloride, 4-6g of corn steep liquor powder and 0.25-0.35g of potassium dihydrogen phosphate are added to an appropriate amount of distilled water, heated and stirred to disperse evenly, and then diluted to 1L with distilled water. The pH is adjusted to 6.9-7.1, dispensed, and sterilized at 121-124℃ and 103-137kPa for 15-25min. After cooling, the conventional fermentation medium is obtained.

8. A fermentation method for producing kasugamycin using *Streptomyces* sp. XYHK-01-M as described in claim 1, comprising the following steps: S1. Inoculate Streptomyces sp. XYHK-01-M into seed culture medium via slant or preservation tube at an inoculation rate of 10-20% by volume into seed bottles. Incubate at 26-30℃ and 200-240 rpm on a shaker for 36-60 h to achieve an OD600 of 1.5-2.2, thus obtaining a vigorous seed culture. S2 graded scale-up fermentation and feeding S2.1 Primary Fermentation Inoculate the seed culture into the first-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter is 30-100L, and the liquid volume is 60-80% of the tank volume. Primary fermentation conditions: Optimized fermentation medium; fermentation temperature 27-30℃; aeration rate 0.8:1-1.2:1; stirring speed 160-200 rpm; tank pressure 0.01-0.03 MPa; fermentation time 60-90 h; S2.2 Secondary Fermentation and Feeding The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter was 300-1000L, and the liquid volume was 60-80% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used. 60-84 hours after the start of fermentation, the secondary fermenter was fed with the following: maltose to a final concentration of 1.0-3.0 wt%; corn steep liquor powder to a final concentration of 0.3-0.8 wt%. After feeding, fermentation continued at 27-30℃, aeration rate of 0.8:1-1.2:1, stirring at 160-200 rpm, and tank pressure of 0.01-0.03 MPa. The total fermentation cycle was controlled at 144-180 hours. At the end of fermentation, the kasugamycin titer in the fermentation broth reached 30,000-32,000 μg / mL.

9. The fermentation method as described in claim 8, characterized in that: The optimized fermentation medium is prepared as follows: 65-75g of soybean meal powder, 16-20g of maltose, 3.5-4.5g of sodium chloride, 7-9g of corn steep liquor powder, 0.35-0.45g of potassium dihydrogen phosphate, and 0.025-0.035g of inositol are added to an appropriate amount of distilled water, heated and stirred until evenly dispersed. Then, 36-40g of soybean oil and 0.8-1.2g of Tween-20 are added, stirred thoroughly, and the volume is adjusted to 1L with distilled water. The pH is adjusted to 6.8-7.2, dispensed, and then autoclaved at 121-126℃ and 103-137kPa for 15-25 minutes. After cooling, the medium is obtained.

10. The fermentation method as described in claim 8, characterized in that, Step S2.2, secondary fermentation and feeding, can also be: The primary fermentation broth was transferred to the second-stage fermenter at an inoculation rate of 8-15% by volume. The effective volume of the fermenter was 300-1000L, and the liquid volume was 60-80% of the tank volume. The same fermentation culture medium and process parameters as the primary fermentation were used. 60-84 hours after the start of fermentation, the secondary fermenter was fed with maltose to a final concentration of 1.0-3.0 wt%, corn steep liquor powder to a final concentration of 0.3-0.8 wt%, and regulator to a final concentration of 0.0005-0.002 wt%. After feeding, fermentation continued at 27-30℃, an aeration rate of 0.8:1-1.2:1, stirring at 160-200 rpm, and a tank pressure of 0.01-0.03 MPa, with the total fermentation cycle controlled at 144-180 hours. The kasugamycin titer in the fermentation broth at the fermentation endpoint was >32000 μg / mL. The regulator is sodium butyrate, trogostatin, or a combination thereof.

Images