Streptomyces parvus xhssm-001 and application thereof in fermentation production process of spring thunder mycin

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a Streptomyces microflavus XHSSM-001 and application thereof in fermentation production process of springamycin. The strain is obtained through normal pressure room temperature plasma mutagenesis, ultraviolet complex mutagenesis, photorepair and step-by-step screening, and has a preservation number of CGMCC No.38870. Fermentation culture is carried out by using the strain, scandium chloride hexahydrate is supplemented at 18-24 h of fermentation, 1,4-butyrolactone is supplemented at 48-60 h of fermentation, and maltose and corn syrup dry powder are supplemented in the subsequent fermentation stage. The present application combines the high-yield stable strain with the phase metabolic regulation, shortens the time required to reach the target yield, improves the springamycin synthesis efficiency and fermentation yield, and has good fermentation repeatability.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a small golden streptomyces XHSSM-001 and its application in the fermentation production process of kasugamycin. Background Technology

[0002] Kasugamycin, also known as kasugamycin or kasugamycin, is an aminoglycoside agricultural fungicide produced by the metabolism of *Streptomyces kasugaensis* or *Streptomyces microaureus*. The earliest kasugamycin-producing organism was *Streptomyces kasugaensis*, isolated from Japanese soil in April 1963. In my country, the earliest kasugamycin-producing organism was *Streptomyces microaureus*, isolated from Jiangxi soil by the Institute of Microbiology, Chinese Academy of Sciences in 1964. Although the two organisms are different, their products are the same. Currently, *Streptomyces microaureus* is the main kasugamycin-producing organism in China. Kasugamycin exerts its antibacterial mechanism by inhibiting 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, leaf mold of tomato, soft rot of Chinese cabbage, and early blight of celery. It also has relatively low toxicity and is easily degraded in the environment, which 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 in the prevention and control of diseases in grain crops, vegetables, fruits and other crops.

[0003] Currently, the industrial production of kasugamycin in China mainly relies on the deep fermentation technology of *Streptomyces simonii*. However, the existing production process has many bottlenecks, which seriously restrict its industrial development and market competitiveness: First, the original strains have low kasugamycin production capacity, and the fermentation content of existing industrial strains generally has low fermentation titers, making it 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 kasugamycin production capacity of most mutagenic strains will decline significantly, making them unsuitable for long-term industrial use. Thirdly, unreasonable design of fermentation process parameters, such as imbalance of carbon and nitrogen source ratio, improper control of fermentation temperature and aeration rate, and lack of targeted feeding strategies, result in a fermentation cycle 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. It is difficult to achieve real-time monitoring of the potency of fermentation broth during industrial production and 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 highly efficient, 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 kasugamycin production capacity, shorten the fermentation cycle, and reduce production costs. This has significant theoretical and practical value for promoting the industrialization of kasugamycin, improving agricultural disease control in my country, and fostering green agriculture. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a small golden streptomyces XHSSM-001 and its application in the fermentation production process of kasugamycin.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for producing kasugamycin by fermentation includes the following steps:

[0008] The seed culture of Streptomyces microaureus XHSSM-001 was inoculated into a fermentation medium for liquid fermentation. The preservation number of Streptomyces microaureus XHSSM-001 is CGMCC No.38870.

[0009] Based on the initial fermentation medium volume, 3.3-9.8 mg / L scandium chloride hexahydrate was added to the fermentation system at 18-24 h of fermentation, and 0.22-0.65 g / L 1,4-butyrolactone was added to the fermentation system at 48-60 h of fermentation, with the addition of scandium chloride hexahydrate preceding the addition of 1,4-butyrolactone.

[0010] After the addition was completed, fermentation continued to obtain a fermentation broth containing kasugamycin.

[0011] Preferably, based on the initial fermentation medium volume, the amount of scandium chloride hexahydrate added is 4.5-8.5 mg / L, and the amount of 1,4-butyrolactone added is 0.30-0.60 g / L; the scandium chloride hexahydrate is added after 20-24 h of fermentation, and the 1,4-butyrolactone is added after 50-58 h of fermentation.

[0012] Preferably, scandium chloride hexahydrate is added at 24 h of fermentation, with an addition amount of 6.5 mg / L; and 1,4-butyrolactone is added at 54 h of fermentation, with an addition amount of 0.43 g / L.

[0013] The application of the aforementioned *Streptomyces simonii* XHSSM-001 in the fermentation production process of kasugamycin involves using *Streptomyces simonii* XHSSM-001 as the production strain for fermentation culture, and supplementing metabolic regulators, carbon sources, and nitrogen sources in stages during the fermentation process; the metabolic regulators include scandium chloride hexahydrate and 1,4-butyrolactone.

[0014] To further enhance the kasugamycin synthesis capacity of *Streptomyces simonii* XHSSM-001, this invention introduces 1,4-butyrolactone and scandium chloride hexahydrate for staged metabolic regulation during fermentation. These two substances are not used as ordinary carbon or nitrogen sources, but rather act on the signal regulation and secondary metabolic processes of *Streptomyces simonii*, respectively, making it easier for the cells to enter the kasugamycin synthesis stage after completing normal growth, thereby improving the conversion efficiency of nutrients to the kasugamycin synthesis pathway.

[0015] The role of scandium chloride hexahydrate: Scandium chloride hexahydrate provides trace amounts of Sc. 3+ It is speculated that trace amounts of Sc 3+ It helps regulate the secondary metabolic state of bacteria, enhances the metabolic activity of the kasugamycin synthesis pathway, and improves the strain's ability to produce toxins.

[0016] The role of 1,4-butyrolactone: 1,4-butyrolactone can induce the 1,4-butyrolactone signaling regulation process in Streptomyces, which is beneficial to promoting the transformation of bacterial cells from growth metabolism to secondary metabolism. The addition of both in stages can improve the conversion efficiency of nutrients to the kasugamycin synthesis pathway.

[0017] Using *Streptomyces simonii* XHSSM-001 (CGMCC No. 38870) as the fermentation strain, trace amounts of scandium chloride hexahydrate were added during the cell growth stage. 1,4-Butyrolactone was added after 48-60 h of fermentation during the transition from growth metabolism to secondary metabolism. Through the phased synergistic effect of rare earth ion regulation and *Streptomyces* signal regulation, it is speculated that the phased addition of these two regulators is beneficial for regulating the secondary metabolic state of the cells and improving the conversion efficiency of nutrients to the kasugamycin synthesis pathway.

[0018] Preferably, after adding scandium chloride hexahydrate and 1,4-butyrolactone, a carbon source and a nitrogen source are added after 68-76 h of fermentation; the amount of carbon source added is 18-22 g / L and the amount of nitrogen source added is 4-6 g / L based on the initial fermentation medium volume; the carbon source includes maltose and the nitrogen source includes corn steep liquor powder.

[0019] Preferably, the carbon source is maltose and the nitrogen source is corn steep liquor powder; the maltose and corn steep liquor powder are added during fermentation for 72 hours, with the amount of maltose added being 20 g / L and the amount of corn steep liquor powder added being 5 g / L.

[0020] Preferably, the fermentation medium comprises the following components per liter:

[0021] 50-90 g of soybean meal powder, 12-24 g of corn starch, 2-6 g of sodium chloride, 0.2-0.6 g of potassium dihydrogen phosphate, 0.01-0.05 g of inositol, 5-10 g of corn steep liquor powder, 35-40 g of fish oil and 0.8-1.2 g of Tween-20;

[0022] The initial pH of the fermentation medium is 6.8-7.2.

[0023] Preferably, the conditions for liquid fermentation include: fermentation temperature 27-30 ℃, aeration rate 0.8-1.2 vvm, dissolved oxygen controlled at 30%-60%, fermentation tank gauge pressure 0.05MPa, and total fermentation time 156-180 h.

[0024] Preferably, the fermentation employs a three-stage process consisting of shake flask seed culture, seed tank culture, and production tank fermentation.

[0025] The culture system is as follows: the temperature for seed culture in shake flasks is 26-30 ℃, the shaking speed is 180-240 rpm, and the culture time is 36-54 h; the temperature for seed tank culture is 26-30 ℃, the aeration rate is 0.8-1.2 vvm, and the culture time is 36-54 h; the inoculation amount of seed liquid into the seed tank and the inoculation amount of seed tank culture into the production tank are 10-15% of the corresponding initial culture medium volume.

[0026] A small golden Streptomyces microaureus, XHSSM-001, was deposited on May 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38870.

[0027] The application of the aforementioned *Streptomyces simonii* XHSSM-001 in the fermentation production of kasugamycin.

[0028] This invention first treats the parental bacterial strain using a combination of ARTP mutagenesis, UV remutation, and photoremediation to increase the probability of beneficial genetic mutations. Then, through initial antibacterial screening, high-performance liquid chromatography (HPLC) screening, and continuous subculturing verification, the genetically stable and high-yielding *Streptomyces simonii* strain XHSSM-001 was obtained. Mutagenesis primarily improved the strain's metabolic capabilities related to kasugamycin synthesis. Stepwise screening eliminated mutants with abnormal growth, low yield, or genetic instability, ensuring that the final XHSSM-001 maintained normal growth, sporulation, and fermentation performance while enhancing kasugamycin synthesis. Furthermore, by optimizing the culture medium composition, oxygen supply, and feeding methods, suitable carbon, nitrogen, and dissolved oxygen conditions were provided to the strain, thereby improving the conversion efficiency of nutrients to the kasugamycin synthesis pathway, ultimately increasing kasugamycin yield and shortening the fermentation cycle.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention employs a combined mutagenesis method integrating ambient pressure room temperature plasma mutagenesis, ultraviolet remutation, and photoremediation. Through initial screening for antibacterial activity, secondary screening by high-performance liquid chromatography, and continuous subculturing verification, a genetically stable *Streptomyces simonii* strain, XHSSM-001, with strong kasugamycin synthesis capacity was obtained. Compared to the parental strain and single-mutated strains, this strain maintains normal growth, sporulation, and continuous subculturing performance while enhancing its kasugamycin production capacity.

[0031] 2. In this invention, scandium chloride hexahydrate and 1,4-butyrolactone are added in stages during the fermentation process. First, a trace amount of Scandium chloride is used… 3+ The process regulates the early secondary metabolic state of the cells, and then uses 1,4-butyrolactone to promote the transition of the cells from growth metabolism to the kasugamycin synthesis stage. This allows the two regulatory substances to play their roles in their respective fermentation stages, avoiding the adverse effects on cell metabolism caused by simultaneous addition, reversed order, or excessive addition.

[0032] 3. This invention combines phased metabolic regulation with subsequent carbon and nitrogen source feeding of maltose and corn steep liquor powder, so that cell growth, secondary metabolism initiation and continuous synthesis of kasugamycin are continuously regulated and nutritionally supported, thereby promoting the accumulation of kasugamycin, improving the synthesis efficiency of kasugamycin, and shortening the fermentation time required to reach the target kasugamycin content.

[0033] 4. This invention establishes a three-stage fermentation system that combines shake flask seed culture, seed tank culture, and production tank fermentation by optimizing seed age, culture medium composition, fermentation temperature, aeration rate, stirring speed, tank pressure, and feeding time. The fermentation process has good repeatability and is suitable for large-scale fermentation production of kasugamycin. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The high-performance liquid chromatogram of kasugamycin in the fermentation broth of Example 2 is shown below.

[0036] Figure 2 This is a standard curve of kasugamycin hydrochloride reference standard.

[0037] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0038] The raw materials described in this application are partially described; all other raw materials not described are commercially available.

[0039] Bacillus subtilis was purchased from the China Medical Bacterial Culture Collection Center, strain number: 63556. Detailed Implementation

[0040] 1. Isolation, screening and identification of the original strain

[0041] This invention isolates actinomycetes from soil in vegetable growing areas of Shouguang, Weifang, Shandong Province, and uses a combination of selective isolation, initial screening with antibacterial fermentation supernatant, and secondary screening with high-performance liquid chromatography to screen parental strains capable of producing kasugamycin. The specific steps are as follows:

[0042] 1.1 Soil Sample Collection and Processing: Multiple sampling points were selected in the Shouguang vegetable planting area of ​​Weifang City, Shandong Province. Dead branches, weeds, and stones on the soil surface were removed, and soil samples from the top 10-20 cm of the vegetable fields were collected. Soil samples from different sampling points were mixed thoroughly and passed through an 80-mesh sieve to obtain the soil samples to be separated.

[0043] Weigh 10g of the soil sample to be separated and add it to a 90mL Erlenmeyer flask containing 0.9% sodium chloride in sterile physiological saline. Place the Erlenmeyer flask in a shaker at 28℃ and 200rpm for 30min to fully disperse the microorganisms in the soil and obtain a soil suspension.

[0044] The original soil solution was serially diluted tenfold using sterile physiological saline to prepare solutions with dilution ratios of 10. -6 10 -7 10 -8 For each dilution of soil solution, three parallel samples were prepared.

[0045] 1.2 Selective isolation and purification of actinomycetes: The isolation medium consisted of the following components per liter: 3g tryptone, 10g soybean meal, 10mL glycerol, 3g sodium chloride, 2g calcium carbonate, 25g agar, and the balance water. The pH was adjusted to 7.0-7.2. The medium was then autoclaved at 121℃ and 0.1 MPa for 20 min, cooled to approximately 50℃, poured into sterile petri dishes, and allowed to solidify for later use.

[0046] Take 0.1 mL of each of the following solutions, diluted 10 times: -6 10 -7 and 10 -8 The diluted soil solution was evenly spread on the surface of the above-mentioned isolation medium and incubated at 28 °C for 6-8 days. During the incubation period, the colony morphology was observed, and single colonies with dry or wrinkled surfaces, obvious aerial hyphae, and typical actinomycete morphology were selected and inoculated onto slant agar for further purification.

[0047] Single colonies were selected and cultured on slant agar at 28 °C for 7 days. Purification was performed three times via streak plating or slant transfer until the colony morphology and microstructure of each culture were essentially consistent, yielding actinomycete isolates. The strain's tolerance to kasugamycin was not used as a criterion for determining its kasugamycin-producing ability during the isolation stage; the ability of each isolate to produce kasugamycin was determined through initial antibacterial screening of the subsequent fermentation supernatant and secondary screening by high-performance liquid chromatography.

[0048] 1.3 Fermentation culture of actinomycete isolates: Each actinomycete isolate was inoculated onto agar slant culture medium and cultured at 28℃ for 7 days. After mature spores formed on the surface of the slant, sterile physiological saline was added to each slant, the spores were gently scraped off, and dispersed by shaking to obtain a spore suspension. The concentration of the spore suspensions for each strain was uniformly adjusted to 1×10⁻⁶ using a hemocytometer. 6 per mL.

[0049] The initial fermentation culture consisted of the following components per liter: 50 g / L soybean meal powder, 15 g / L corn starch, 3 g / L sodium chloride, 5 g / L corn steep liquor powder, and 0.3 g / L potassium dihydrogen phosphate, with a total water volume of 1 L. The pH was adjusted to 7.0, and the culture was sterilized at 121℃ for 20 min. Spore suspensions of each strain were inoculated at 10% of the fermentation medium volume into 500 mL Erlenmeyer flasks containing 100 mL of initial fermentation medium. The flasks were incubated at 28℃ and 200 rpm with shaking for 168 h. Each strain was used in three parallel fermentation flasks.

[0050] After fermentation, the fermentation broth was centrifuged at 8000×g for 10 min, the supernatant was collected, and then filtered through a 0.22 μm filter membrane to obtain sterile fermentation supernatant, which was used for primary antibacterial screening and secondary screening of kasugamycin content.

[0051] 1.4 Initial screening using inhibition zones: The initial screening method was adopted, 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.

[0052] Preparation of test plates: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 200 rpm until the logarithmic growth phase. The LB medium contained 10 g peptone, 5 g yeast extract, and 10 g sodium chloride per liter. LB solid medium was prepared by adding 25 g / L agar to the LB liquid medium. After autoclaving at 121°C and 0.1 MPa for 20 min, the medium was cooled to approximately 50°C, and Bacillus subtilis culture was added to a volume of 1% of the LB solid medium. The mixture was thoroughly mixed and poured into sterile petri dishes to prepare indicator bacteria test plates.

[0053] Four sterile Oxford cups were placed on LB plates containing Bacillus subtilis. 150 μL of sterile fermentation supernatant was added to each Oxford cup. The supernatant of the blank fermentation medium without inoculated strains was centrifuged and filtered in the same way as a negative control. After incubation at 37 ℃ for 16 h, the diameter of the inhibition zone was measured. Three replicates were set up for each strain. Strains with an inhibition zone diameter greater than 15 mm were screened as candidate strains. A total of 17 candidate strains were obtained.

[0054] 1.5 High performance liquid chromatography rescreening: The 17 candidate strains obtained from the initial screening were subjected to three separate shake-flask fermentations under the conditions described in Section 1.3 of this section.

[0055] After fermentation, the kasugamycin content in each fermentation broth was determined using the high performance liquid chromatography method described in this instruction manual. The average value of three parallel fermentation flasks was taken as the kasugamycin yield of the corresponding strain.

[0056] Based on the high-performance liquid chromatography (HPLC) results, the strain with the highest average kasugamycin content and not less than 12.0 g / L was selected. Finally, a parent strain with an average kasugamycin content of 12.0 g / L was obtained and named *Streptomyces simonii* XHSCL-001.

[0057] 1.6 Biological characteristics of parent strain XHSCL-001: Actinomycete morphology, vigorous growth on slant culture medium, aerial hyphae are grayish-white, spores are slightly green and fluffy, and substrate hyphae are yellowish-brown and do not produce obvious water-soluble pigments.

[0058] Microscopic observation of XHSCL-001 revealed that the hyphae were curved or arc-shaped, and the spores were arranged in chains. When dispersed, they were spherical to oval in shape, with a diameter of 0.8-1.2 μm and a smooth surface.

[0059] Physiological and biochemical test results show that XHSCL-001 can utilize glucose, maltose, and sucrose as carbon sources, and peptone and soybean meal as nitrogen sources. It cannot utilize starch as the sole carbon source. The catalase test is positive, the oxidase test is negative, the optimal growth temperature is 28-30℃, and the optimal growth pH is 7.0-7.2.

[0060] Based on colony morphology, microscopic morphology, and physiological and biochemical characteristics, the parent strain XHSCL-001 was preliminarily identified as a strain related to the genus Streptomyces genus, and was used as the starting strain for subsequent compound mutagenesis breeding.

[0061] 2. Strain mutagenesis and the acquisition of Streptomyces simonii XHSSM-001

[0062] The original strain XHSCL-001 had limited catalytic activity (12 g / L in shake-flask fermentation), which was insufficient to meet the needs of industrial production. To improve the catalytic activity of the strain, this invention abandons the shortcomings of traditional single mutagenesis methods. This invention sequentially employs ambient pressure room temperature plasma (ARTP) mutagenesis, ultraviolet remutation, and photoremediation treatment, combined with high-yield strain screening and genetic stability verification, to obtain a high-yield and stable mutant strain.

[0063] The advantages of this combined mutagenesis strategy are as follows: ARTP 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; kasugamycin-producing bacteria are too sensitive to ultraviolet light, and even extremely short-term ultraviolet irradiation shows an excessively high lethality rate of over 99.9%. However, photoremediation can reduce the lethality rate and increase the positive mutation rate. Synergistically with ARTP mutagenesis, it can significantly improve the mutation rate and the probability of screening high-yielding mutant strains, while effectively solving the problem of poor genetic stability of strains caused by single mutagenesis. The specific steps are as follows:

[0064] 2.1 Preparation of spore suspension: The original strain XHSCL-001 was inoculated onto an agar slant and cultured at 28℃ for 7 days until mature spores formed on the surface of the slant. The slant was rinsed with sterile physiological saline (0.9% NaCl), and the spores and aerial hyphae on the surface of the slant were gently scraped off using a sterile inoculation loop. The resulting suspension was transferred to an Erlenmeyer flask containing sterile glass beads and shaken at 28℃ and 200 rpm for 30 min to ensure complete spore dispersion. Subsequently, the suspension was filtered through sterile rapid filter paper to remove hyphal fragments and culture medium debris, obtaining a pure spore suspension. The spore concentration was adjusted to 10 using a hemocytometer. 6The spores were prepared at a concentration of 1 spore per mL to obtain a spore suspension for mutagenesis. No glycerol was added to the spore suspension for mutagenesis. It should be used within 4 hours of preparation and temporarily stored at 4 °C before use. The suspension for preservation was prepared with 20% glycerol and stored at −80 °C. The glycerol was removed by centrifugation before mutagenesis.

[0065] 2.2 Screening of ARTP Mutagenesis Conditions: The first stage of mutagenesis was conducted using the ARTP-II ambient pressure room temperature plasma breeding system, with helium gas of no less than 99.999% purity as the working gas. Preliminary experiments determined the output power of the ARTP device to be 110 W and the distance between the plasma nozzle and the sample slide to be 2 mm. Based on this, the effects of treatment time and helium flow rate on the lethality and high-mutation rate of the strain were investigated. Single-factor experiments were used to optimize the ARTP mutagenesis conditions, examining the effects of irradiation time and gas flow rate on the mutagenesis effect. The lethality and high-mutation rate of the mutagenized strain were used as evaluation indicators to determine the optimal mutagenesis conditions.

[0066] The single-factor experimental design is as follows: ① Irradiation time: five gradients were set at 30s, 60s, 90s, 120s, and 150s, while other conditions were 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, while other conditions were fixed at an irradiation time of 90s, an irradiation distance of 2mm, and a power of 110W; each treatment was set up with 3 replicates.

[0067] Each ARTP treatment condition was set up in 3 independent replicates. For each replicate, 10 μL of a 1×10⁻⁶ concentration was aspirated. 6 A spore suspension of 1 spore / mL was dropped into the center of a sterile stainless steel sample slide (6 mm in diameter) used with the ARTP-II equipment. Using a sterile pipette tip, the spore suspension was gently spread onto the slide surface to form a uniform thin layer. ARTP treatment was performed immediately after sample addition.

[0068] Immediately after ARTP treatment, each sample slide was transferred to a sterile centrifuge tube containing 1.0 mL of sterile physiological saline and vortexed for 60 s to fully elute the spores from the slide surface, yielding the mutagenic spore eluent. The three independent replicates of the mutagenic spore eluent were used for subsequent processing separately and were not combined.

[0069] Each mutagenic spore eluent was serially diluted tenfold, and 100 μL of the appropriately diluted bacterial suspension was evenly spread on the surface of the isolation medium and incubated at 28 ℃ for 8 days. The control group without ARTP treatment used the same sample slides, the same concentration and volume of spore suspension, and the same elution, dilution, and incubation procedures, except that the ARTP treatment time was set to 0 s.

[0070] For each treatment condition, lethality was calculated based on the colony count in three independent replicates. Morphologically normal and vigorous single colonies were selected from the surviving colonies for subsequent shake-flask fermentation and screening of high-yielding mutant strains. A control group consisting of parental strains that were not treated with ARTP and underwent the same procedures as before was also included.

[0071] Calculate the mortality rate; the mortality rate of ARTP treatment is calculated according to the following formula: Mortality rate (%) = (N0 - N) t ) / N0×100%;

[0072] In the formula, N0 is the number of viable bacteria in the control group that was not treated with ARTP, and N t This represents the number of viable bacteria after treatment with the corresponding ARTP conditions.

[0073] Single colonies with normal morphology and vigorous growth were selected from the surviving colonies of each treatment group for shake-flask fermentation. A high-yielding mutant strain was defined as one with a kasugamycin content that was more than 30% higher than that of the parent strain XHSCL-001, i.e., a kasugamycin content of not less than 15.6 g / L.

[0074] The high-yield mutation rate is calculated using the following formula:

[0075] High-yield mutation rate (%) = number of high-yield mutant strains / total number of surviving strains that completed shake-flask fermentation testing × 100%. The results of ARTP mutagenesis screening are shown in Table 1.

[0076] Table 1. Screening results of ARTP mutagenic conditions

[0077]

[0078] Table 1 shows that when the ARTP mutagenesis conditions are: helium flow rate of 10 L / min, irradiation distance of 2 mm, power of 110 W, and irradiation time of 90 s, the bacterial lethality rate is 87.2%, and the high-yield mutation rate reaches 12.3%, indicating the optimal mutagenesis effect. Under these conditions, a certain number of surviving colonies can be maintained while obtaining a relatively high mutation rate; therefore, this condition has been determined as the subsequent ARTP mutagenesis condition.

[0079] ARTP mutagenesis was performed on XHSCL-001 under the above conditions. After serial dilution and spread culture, single colonies with normal morphology and vigorous growth were selected from the treated spore suspension, and a total of 93 ARTP mutagenesis candidate strains were obtained.

[0080] 2.3 UV remutation and photoremediation treatment: Candidate strains obtained after ARTP mutagenesis were inoculated onto slant culture medium and cultured at 28 °C for 7 days. A concentration of 1×10⁻⁶ was prepared according to the method described in section 2.1. 6 A spore suspension of 1 spore per mL.

[0081] Re-mutation treatment was performed using a low-pressure mercury UV lamp with a main wavelength of 254 nm and a rated power of 15 W. The UV lamp was preheated for 15 min before use to stabilize the output. For each treatment group, 10.0 mL of spore suspension was placed in a sterile petri dish with a diameter of 90 mm. The dish lid was removed to allow the spore suspension to form a uniform liquid layer at the bottom. The petri dish was placed horizontally directly below the UV lamp, with a distance of 20 cm between the spore suspension surface and the lamp. During irradiation, the petri dish was slowly rotated horizontally to ensure uniform UV irradiation of the spores.

[0082] The UV treatment times were set to 10 s, 30 s, 60 s, 90 s, and 120 s, with three independent replicates for each treatment. Photoremediation was performed immediately after treatment. When evaluating the UV treatment time, the photoremediation time was fixed at 90 min.

[0083] Photoremediation was performed using a white fluorescent lamp as the visible light source at 30 ℃, with the illuminance at the sample surface controlled at 2000±200 lx. The photoremediation times were set to 30 min, 60 min, 90 min, 120 min, and 180 min, respectively. When examining the photoremediation time, the UV treatment time was fixed at 30 s.

[0084] Immediately after photoremediation, the spore suspension was serially diluted tenfold under dark conditions. 100 μL of the appropriately diluted bacterial solution was evenly spread onto the surface of the isolation medium and incubated at 28 ℃ in the dark for 8 days. Morphologically normal surviving single colonies were then selected for subsequent fermentation screening. The results of UV mutagenesis and photoremediation screening are shown in Table 2.

[0085] Table 2 Screening results of UV-induced mutagenesis photoremediation

[0086]

[0087] Table 2 shows that when the UV mutagenesis conditions are wavelength 254 nm, treatment time 30 s, treatment temperature 30 °C, and photorepair time 90 min, the high-yield mutation rate reaches 12.8%, which is the highest. Therefore, the UV treatment time of 30 s and the photorepair time of 90 min are determined as the conditions for complex mutagenesis.

[0088] Following the above conditions, 93 ARTP mutagenesis candidate strains were subjected to UV remutation and photoremediation treatments. After serial dilution, spreading, and culturing, 76 morphologically normal remutation candidate strains were obtained from the treated spore suspensions.

[0089] 2.4 Initial screening of candidate strains for repeated mutagenesis

[0090] Using the inhibition zone method, 76 candidate bacterial strains were inoculated onto slant agar and cultured at 28°C for 7 days to prepare spore suspensions. These suspensions were then subjected to shake-flask fermentation under the conditions described in Section 1.3. After fermentation, the fermentation broth was centrifuged and filtered through a 0.22 μm filter membrane. The sterile fermentation supernatant was collected, and the diameter of the inhibition zone was determined using the Oxford cup method described in Section 1.4.

[0091] Strains with inhibition zone diameter greater than 15 mm were screened as candidate strains, and a total of 32 candidate strains were obtained.

[0092] This step also uses fermentation supernatant for inhibition zone detection, instead of directly adding the spore suspension of the candidate strain to the Oxford cup, to avoid interference with the inhibition results due to bacterial proliferation or nutrient competition in the culture medium.

[0093] 2.5 High-performance liquid chromatography (HPLC) secondary screening: Thirty-two high-yielding strains from the initial screening were inoculated into fermentation medium using a shake-flask fermentation method. Fermentation was carried out under the following conditions: 100 mL of liquid was added to a 500 mL Erlenmeyer flask, and the spore suspension inoculum was 10%. The culture was carried out at 28℃ and 200 rpm for 168 h with shaking. Each strain was used in triplicate. After fermentation, the potency of kasugamycin in the fermentation broth was determined using an optimized HPLC method. Strains with potency higher than 20 g / L were screened as high-yielding candidate strains, resulting in seven high-yielding strains obtained from the secondary screening.

[0094] 2.6 Validation of stability through continuous subculturing: Seven high-yielding strains selected through secondary screening were inoculated onto slant agar and cultured at 28°C for 7 days to serve as the first generation. For each generation, samples were taken from normally growing and morphologically consistent cultures and transferred to new slant agar, then cultured under the same conditions for five consecutive subculturings.

[0095] After each generation of culture was completed, shake-flask fermentation was carried out with the same inoculum size, culture medium composition, liquid volume, culture temperature, shaker speed and fermentation time, and the content of kasugamycin in the fermentation broth was determined by high performance liquid chromatography.

[0096] The yield fluctuation of each strain over 5 consecutive generations is calculated using the following formula: Calculate the fluctuation range of the production capacity (fluctuation range = (highest kasugamycin content over 5 consecutive generations - lowest kasugamycin content over 5 consecutive generations) / average kasugamycin content over 5 consecutive generations × 100%).

[0097] Using high kasugamycin content and a yield fluctuation of no more than 5% over 5 consecutive generations as screening criteria, a strain with a kasugamycin content of 24.5 g / L in shake-flask fermentation and a yield fluctuation of 4.5% over 5 consecutive generations was finally obtained. This strain was named Streptomyces simonii XHSSM-001.

[0098] 2.7 Comparison of factor production capacity and genetic stability

[0099] The parental strain XHSCL-001, the single ARTP mutant strain ARTP-01, the single UV mutant strain UV-01, and the composite mutant strain XHSSM-001 were subjected to shake-flask fermentation and continuous subculture experiments under the same conditions. The results are shown in Table 3.

[0100] Table 3. Kasugamycin yield and genetic stability of different strains

[0101]

[0102] As shown in Table 3, the kasugamycin content of the mutant strain XHSSM-001 was 104.17% higher than that of the parent strain XHSCL-001, 28.95% higher than that of the single ARTP mutant strain ARTP-01, and 36.11% higher than that of the single UV mutant strain UV-01.

[0103] The yield fluctuation of XHSSM-001 after five consecutive passages was 4.5%, which is close to the 4.2% of the parental strain XHSCL-001, but significantly lower than the 9.2% of ARTP-01 and the 11.7% of UV-01. Therefore, XHSSM-001 significantly increased the yield of kasugamycin while maintaining passage stability comparable to that of the parental strain, and its passage stability was superior to that of a single ARTP mutant and a single UV mutant.

[0104] 2.8 Preservation of bacterial strains

[0105] After identification, the obtained strain XHSSM-001 was classified and named *Streptomyces microaureus*. This strain was deposited on May 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38870.

[0106] 3. Fermentation process development

[0107] This invention uses the mutant strain *Streptomyces simonii* XHSSM-001 as the production strain. Transplantation indices were determined through inoculum age experiments. Through single-factor experiments, orthogonal experiments, and scale-up experiments, the fermentation medium formulation and fermentation process parameters were systematically optimized. This addresses the problems of long fermentation cycles, low yields, and high costs associated with existing processes, establishing a cultivation process suitable for the fermentation production of kasugamycin from *Streptomyces simonii* XHSSM-001, as detailed below:

[0108] 3.1 Preparation of seed solution and determination of seed age

[0109] The seed culture medium comprises the following components per liter: 50 g / L soybean meal powder, 15 g / L corn starch, 3 g / L sodium chloride, 5 g / L corn steep liquor powder, and 0.3 g / L potassium dihydrogen phosphate. The total volume of water added is 1 L. The above components are mixed evenly, the pH is adjusted to 7.0, sterilized at 121℃ for 20 min, and cooled to room temperature to obtain the seed culture medium.

[0110] 3.2 Seed liquid preparation

[0111] Streptomyces simonii XHSSM-001 was inoculated onto an agar slant and cultured at 28°C for 7 days. After mature spores formed on the slant, sterile physiological saline was added, and the spores were gently scraped off and dispersed by shaking to obtain a spore suspension.

[0112] Spore concentration was determined using a hemocytometer, and the spore suspension concentration was adjusted to 1×10⁻⁶ using sterile physiological saline. 6 per mL.

[0113] 100 mL of seed culture medium was placed in a 500 mL Erlenmeyer flask, and the spore suspension was inoculated at 2% of the volume of the seed culture medium. The flask was then shaken and cultured at 28 °C and 220 rpm to obtain the seed solution. The spore inoculation amount is the volume percentage of the spore suspension inoculated into the seed culture medium in the shake flask.

[0114] 3.3 Seed Age Trial

[0115] Because *Streptomyces simonii* XHSSM-001 easily forms hyphal clumps during liquid seed culture, a uniform dispersion treatment was performed on the seed culture before measurement to minimize the impact of hyphal aggregation on the OD600 results. Specifically, 5.0 mL of seed culture was taken from a shake flask and placed into a 15 mL sterile centrifuge tube containing 10 sterile glass beads with a diameter of 3-4 mm. The tube was vortexed at 2500 rpm for 60 s to ensure thorough dispersion of the hyphal clumps. After standing for 30 s, the uniformly dispersed suspension was taken and diluted 5 times with 0.9% sterile physiological saline. The absorbance was measured at 600 nm. An equivalent volume of seed culture medium treated in the same manner served as a blank control. The measured absorbance multiplied by the dilution factor was used as the OD600 of the seed culture. Each sample was measured in triplicate, and the average value was taken.

[0116] OD600 was measured at 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, and 60 h of seed culture using the method described above, and pH was measured simultaneously. The optimal seed age was determined based on the kasugamycin content observed during subsequent shake-flask fermentation. A qualified seed culture was defined as one where the culture time was 48 h, the seed culture pH was not lower than 7.0, the OD600 measured using the standardized dispersion method described above was 1.8–2.0, and microscopic observation showed normal mycelial growth and the absence of contaminating microorganisms.

[0117] Seed culture solutions from different incubation times were inoculated into 500 mL Erlenmeyer flasks containing 100 mL of fermentation medium at 10% of the fermentation medium volume. Fermentation was carried out at 28°C and 200 rpm for 168 h with shaking. After fermentation, the kasugamycin content in the fermentation broth was determined using the high-performance liquid chromatography method described in 1.3 of Test Example 1.

[0118] The results showed that the pH generally increased and then decreased during seed culture, reaching a maximum value of 7.39 after 42 h of culture. The cell concentration growth rate gradually decreased after 24 h of culture. When fermenting seed culture with a culture period of 24-48 h, the kasugamycin content increased with the extension of seed age; when the seed age was 48 h, the kasugamycin content reached 25.0 g / L; with further extension of seed age, the kasugamycin content decreased.

[0119] Therefore, the optimal seed culture age for *Streptomyces simonii* XHSSM-001 was determined to be 48 h. In actual transplanting, the transplanting criteria were a culture time of 48 h, a seed culture pH of not less than 7.0, and an OD600 of 1.8-2.0 after dispersion treatment.

[0120] 3.4 Optimization of Fermentation Medium

[0121] 3.4.1 Initial fermentation medium and experimental conditions

[0122] The initial fermentation medium consisted of 50 g of soybean meal powder, 15 g of corn starch, 3 g of sodium chloride, 5 g of corn steep liquor powder, 0.3 g of potassium dihydrogen phosphate, and the remainder water per liter, with an initial pH of 7.0 ± 0.1.

[0123] When conducting single-factor experiments on culture media, all other components of the culture media and fermentation conditions, except for the factor under investigation, were kept consistent. Three replicates were set up for each treatment.

[0124] The culture medium was screened using 500 mL Erlenmeyer flasks, each containing 100 mL of liquid. The seed culture inoculum was 10%, the culture temperature was 28℃, the shaker speed was 200 rpm, and the fermentation time was 168 h. After fermentation, the kasugamycin content was determined by high performance liquid chromatography.

[0125] 3.4.2 Raw material screening

[0126] (1) Carbon source screening: Carbon source is the main energy source for the growth and production of the strain. Five common carbon sources, namely glucose, corn starch, maltose, sucrose and glycerol, were investigated, and the concentration of each was set to 15 g / L.

[0127] Experimental results showed that the highest kasugamycin content (25.6 g / L) was observed in the fermentation broth when corn starch was used as the carbon source; the highest was 22 g / L when maltose was used; and the highest were 18.5 g / L, 17.8 g / L, and 19.2 g / L when glucose, sucrose, and glycerol were used. Therefore, corn starch was selected as the optimal carbon source, and the effect of corn starch concentration on kasugamycin yield was further investigated at concentrations of 12 g / L, 15 g / L, 18 g / L, 21 g / L, and 24 g / L. The results showed that the highest kasugamycin content (27.8 g / L) was observed at a corn starch concentration of 18 g / L. Therefore, the optimal corn starch concentration was determined to be 18 g / L. Both excessively high and low concentrations led to a decrease in kasugamycin production capacity. This may be because excessively high carbon source concentrations lead to excessive cell growth, consuming large amounts of nutrients and inhibiting product synthesis; while excessively low concentrations cannot meet the energy requirements for cell growth and kasugamycin production.

[0128] (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, peptone, corn steep liquor powder and yeast extract. The concentration of each was set to 50 g / L.

[0129] The results showed that when soybean meal powder was used as the nitrogen source, the kasugamycin content was the highest at 26.3 g / L, followed by corn steep liquor powder as the main nitrogen source at 24.1 g / L, and peptone, soybean meal powder, and yeast extract as the main nitrogen sources at 20.1 g / L, 21.5 g / L, and 22.3 g / L, respectively.

[0130] Therefore, soybean meal was chosen as the main nitrogen source for the fermentation medium. As an inexpensive plant-based nitrogen source, soybean meal is not only rich in nutrients but also provides various amino acids and metabolic regulators, and its low cost makes it suitable for industrial production. The effect of soybean meal addition on kasugamycin yield was further investigated. The addition amounts were 50 g / L, 60 g / L, 70 g / L, 80 g / L, and 90 g / L. The results showed that the highest kasugamycin content (28.2 g / L) was achieved at a soybean meal concentration of 70 g / L. Therefore, the optimal addition amount of soybean meal was determined to be 70 g / L. Excessive concentration leads to an imbalance in the carbon-nitrogen ratio, inhibiting product synthesis; insufficient concentration fails to meet the growth requirements of the microorganisms.

[0131] (3) Screening of inorganic salts: Inorganic salts can regulate the osmotic pressure and pH value of the culture medium and participate in the metabolic process of the strain. The effects of sodium chloride, potassium dihydrogen phosphate, calcium carbonate and magnesium sulfate on the fermentation of Streptomyces simonii XHSSM-001 to produce kasugamycin were investigated.

[0132] In the preliminary experiment, the amounts of sodium chloride, potassium dihydrogen phosphate, calcium carbonate, and magnesium sulfate added were 3.0 g / L, 0.3 g / L, 10.0 g / L, and 0.5 g / L, respectively. The experimental results showed that the addition of KH₂PO₄ and NaCl significantly promoted the synthesis of kasugamycin, while calcium carbonate and magnesium sulfate did not show significant promoting effects under the conditions of this experiment.

[0133] KH₂PO₄ promoted the metabolism and product synthesis of the strain, while NaCl regulated the osmotic pressure of the culture medium and maintained normal bacterial growth. CaCO₃ and MgSO₄ had no significant effect on the production of the product. Further optimization of the KH₂PO₄ concentration (0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, and 0.6 g / L) and NaCl concentration (2.0 g / L, 3.0 g / L, 4.0 g / L, 5.0 g / L, and 6.0 g / L) revealed that the highest kasugamycin content (28.5 g / L) was achieved when the KH₂PO₄ concentration was 0.4 g / L and the sodium chloride concentration was 4.0 g / L. Therefore, the optimal concentrations of potassium dihydrogen phosphate and sodium chloride are 0.4 g / L and 4.0 g / L, respectively.

[0134] (4) Screening of metabolic regulators: Metabolic regulators can promote the growth and production of the strain. Four metabolic regulators, namely inositol, vitamin B1, vitamin B6 and biotin, were investigated. The concentration of each component was set at 0.03 g / L, and a blank control without the addition of growth-promoting components was set.

[0135] The results showed that the addition of inositol resulted in the highest kasugamycin content in the fermentation broth (29.1 g / L), an increase of 8.2% compared to the absence of metabolic regulators. Other metabolic regulators had no significant effect on the production of the product. Inositol was selected as the sodium phytate hydrolysis extract from spring corn soaking solution, 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 at inositol concentrations of 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, and 0.05 g / L. The results indicated that the highest kasugamycin content was observed at an inositol concentration of 0.03 g / L. Therefore, the optimal inositol concentration was determined to be 0.03 g / L.

[0136] (5) Surfactant screening: Surfactants can improve cell membrane permeability, promote the release of intracellular products, and increase fermentation content. Three surfactants, Tween-20, Tween-80, and sodium dodecyl sulfate (SDS), were investigated. The concentration of each component was set to 1 g / L, and a blank control without surfactant was set up.

[0137] The results showed that the kasugamycin content was 29.8 g / L with the addition of Tween-20, which was 10.5% higher than that without surfactant; the content was 28.7 g / L with Tween-80. SDS had some toxicity to the strain and a low kasugamycin production capacity of only 25.3 g / L. Therefore, Tween-20 was selected as the surfactant, and its concentration was determined to be 1 g / L.

[0138] 3.4.3 Orthogonal Experiment to Optimize Culture Medium Ratio: Based on the single-factor experiments, four key factors affecting phytochemical production capacity were selected: corn starch addition amount (factor A), soybean meal addition amount (factor B), KH2PO4 addition amount (factor C), and inositol addition amount (factor D). Each factor was set with three levels, using an L9(3) orthogonal experimental design. 4 Orthogonal experimental design was used, and the culture medium ratio was optimized. The experimental factors and levels are shown in Table 4, and the results of the orthogonal experiment are shown in Table 5.

[0139] In the orthogonal experiment, sodium chloride was fixed at 4.0 g / L, corn steep liquor powder at 8.0 g / L, fish oil at 38 g / L, Tween-20 at 1.0 g / L, and the initial pH of the culture medium was 7.0 ± 0.1.

[0140] Table 4. Factors and Levels in Orthogonal Experiments

[0141]

[0142] Table 5 L9(3) 4 Orthogonal experimental results

[0143]

[0144] Where k1, k2, and k3 represent the average values ​​of kasugamycin content at levels 1, 2, and 3, respectively; R represents the difference between the maximum and minimum values ​​of the average values ​​at each level for the same factor.

[0145] Table 5 shows that the order of influence of each factor on the kasugamycin content is: soybean meal powder addition (B) > inositol addition (D) > KH2PO4 addition (C) > corn starch addition (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: soybean meal powder 70 g / L, corn starch 18 g / L, sodium chloride 4.0 g / L, corn steep liquor powder 8.0 g / L, fish oil 38 g / L, KH2PO4 0.4 g / L, inositol 0.03 g / L, Tween-20 1 g / L, pH 7.0.

[0146] 3.4.4. Culture medium optimization validation experiment

[0147] Mix 70 g / L soybean meal powder, 18 g / L corn starch, 4.0 g / L sodium chloride, 8.0 g / L corn steep liquor powder, 38 g / L fish oil, 0.4 g / L KH2PO4, 0.03 g / L inositol, and 1 g / L Tween-20 with water to a final volume of 1 L. Sterilize at 121°C for 20 min at pH 7.0.

[0148] The optimal fermentation medium formulation was used to conduct a shake-flask fermentation verification experiment. XHSSM-001 seed culture for 48 h was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of optimized fermentation medium at a volume ratio of 10%. Three parallel experiments were set up, and the fermentation was carried out at 28℃ and 200 rpm for 168 h with shaking. After the fermentation was completed, the kasugamycin content was detected.

[0149] Experimental results showed that the average kasugamycin content in the optimized fermentation broth was 29.5 g / L, a 20.4% increase compared to 24.5 g / L using the unoptimized medium, with good repeatability (relative standard deviation ≤ 2.0%). This indicates that the optimized medium formulation is reasonable and can significantly improve the kasugamycin production capacity of the strain. Furthermore, all components of this medium formulation are inexpensive and readily available industrial raw materials. The use of corn steep liquor powder and soybean meal powder can further reduce production costs, making it suitable for large-scale industrial production.

[0150] 3.4.5 Optimization of Fermentation Process Parameters

[0151] Fermentation process parameters were optimized in a 5 L mechanically stirred fermenter with an initial liquid volume of 3 L, a seed culture inoculation volume of 12% of the fermentation medium volume, and an initial pH of 7.0 ± 0.1. The fermentation inoculation volume is the volume percentage of the mature seed culture inoculated into 5 L of fermentation medium.

[0152] Except for the factor under investigation, all other conditions were determined to be suitable conditions in the previous step. Three parallel fermentation batches were set up for each experimental condition, and the kasugamycin content was measured after fermentation.

[0153] Based on the optimized fermentation medium formulation, a three-stage fermentation model (seed culture → primary fermentation → secondary fermentation) was adopted. The fermentation process was optimized through single-factor experiments, orthogonal experiments, and scale-up fermentation verification. The fermentation process parameters, including seed culture parameters, fermentation temperature, aeration rate, stirring speed, tank pressure, feeding strategy, and fermentation cycle, were optimized to further improve the nutrient production capacity and shorten the fermentation cycle.

[0154] 1. Seed Culture Parameter Optimization: The quality of the seed culture directly affects the subsequent fermentation effect. Optimizing seed culture parameters to obtain a vigorous and abundant seed culture is a prerequisite for improving the fermentation content. The effects of seed culture 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 content of the seed culture as evaluation indicators.

[0155] 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 medium with the seed culture medium, the fermentation content reached 29.8 g / L, which was more than 3.5% higher than that under other parameter conditions. Therefore, the optimal seed culture parameters were determined to be: inoculum size 15%, 28℃, 220 rpm shaking culture for 48 h.

[0156] 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 of 26℃, 27℃, 28℃, 29℃, and 30℃ on toxin production were investigated. Other conditions were kept constant: seed culture inoculum 12%, aeration rate 1:1 (v / v), stirring speed 180 rpm, tank pressure 0.05 MPa, and fermentation time 168 h. Three replicates were set up for each treatment.

[0157] The results showed that the highest kasugamycin content (30.2 g / L) in the fermentation broth was observed at a fermentation temperature of 29℃. Below 29℃, cell growth was slow, and the kasugamycin production capacity increased with increasing temperature. Above 29℃, cell metabolism accelerated, nutrients were consumed too quickly, and the kasugamycin production capacity decreased. Therefore, the optimal fermentation temperature was determined to be 29℃.

[0158] 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 metabolic products, and inhibition of toxin production; excessive aeration increases energy consumption and may cause rapid evaporation of the culture medium, affecting the stability of the fermentation system. Aeration rates of 0.6 vvm, 0.8 vvm, 1.0 vvm, 1.2 vvm, and 1.4 vvm were investigated; where 1.0 vvm indicates that the volume of air introduced into the fermenter per minute is equal to the volume of the fermentation broth.

[0159] The results showed that the highest kasugamycin content (30.5 g / L) was achieved at an aeration rate of 1.0 vvm. When the aeration rate was below 1.0 vvm, the bacteria were hypoxic, resulting in lower kasugamycin production. When the aeration rate was above 1.0 vvm, kasugamycin production did not significantly improve, but energy consumption increased. Therefore, the optimal aeration rate was determined to be 1.0 vvm.

[0160] 4. Dissolved Oxygen Control Optimization: The stirring speed and aeration rate work synergistically to affect the dissolved oxygen in the fermentation broth and the dispersibility of the microorganisms. Under a constant aeration rate, if dissolved oxygen is controlled too low, the stirring speed will be low, resulting in insufficient dissolved oxygen, microorganism aggregation, and impaired metabolism. Conversely, if dissolved oxygen is controlled too high, the stirring speed will be too high, leading to microorganism damage and increased energy consumption. Under fermentation conditions of 29℃ and an aeration rate of 1.0 vvm, the effects of dissolved oxygen control at 10%-30%, 30%-60%, 60%-80%, and above 80% on factor production capacity were investigated.

[0161] The results showed that the highest kasugamycin content (30.8 g / L) was achieved when dissolved oxygen was controlled between 30% and 60%. At this range, the oxygen content in the fermentation broth was suitable, the cells were evenly dispersed, and metabolism was vigorous. Both excessively high and low dissolved oxygen levels led to a decrease in kasugamycin production. Therefore, the optimal dissolved oxygen control range was determined to be 30%-60%.

[0162] 5. Optimization of Feeding Method: 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 the fermentation content. This invention adopts a batch feeding strategy to investigate the effects of feeding time, type of feed, and amount on nutrient production capacity, and determines the optimal feeding strategy through single-factor experiments.

[0163] The results showed that adding feed once 72 hours after the start of fermentation, and adding 20 g / L maltose and 5 g / L corn steep liquor powder according to the initial fermentation medium volume, resulted in the highest kasugamycin content (31.5 g / L) and the best effect.

[0164] At this stage, the bacteria are in the late logarithmic growth phase and begin to synthesize large amounts of kasugamycin. Adding maltose provides a carbon source to meet the energy requirements for product synthesis; adding corn steep liquor powder provides a nitrogen source and metabolic regulators to maintain carbon-nitrogen balance and promote product synthesis. After feeding, the kasugamycin titer in the fermentation broth reached 31.5 g / L, an increase of 5.2% compared to before feeding. Feeding too early can lead to excessive bacterial growth and consumption of large amounts of nutrients; feeding too late can result in insufficient nutrient intake and inhibited kasugamycin production; both excessive and insufficient feeding will affect kasugamycin production capacity.

[0165] The feed solution is prepared according to the following method:

[0166] (1) Prepare a maltose aqueous solution of 500 g / L and filter it through a 0.22 μm sterile filter membrane for sterilization;

[0167] (2) Prepare a 200 g / L corn steep liquor suspension and sterilize it at 121℃ for 20 min;

[0168] (3) Cool the two feed solutions to room temperature and add them aseptically to the fermenter after 72 h of fermentation, so that the amount of maltose and corn steep liquor powder added relative to the initial culture medium volume is 20 g / L and 5 g / L, respectively.

[0169] After feeding, maintain the fermentation temperature at 29℃, the aeration rate at 1.0 vvm, the stirring speed at 180 rpm, and the internal pressure at 0.05 MPa.

[0170] 6. Fermentation time optimization: The effects of fermentation times of 144h, 156h, 168h, 180h, and 192h on the kasugamycin content were investigated, while other conditions were kept constant.

[0171] The results showed that when the fermentation time was 168 h, the kasugamycin content reached 31.5 g / L. When the fermentation period was less than 168 h, the kasugamycin production did not reach its peak and the potency was low. When the fermentation period was more than 168 h, the kasugamycin production capacity did not improve significantly, and it led to excessive consumption of nutrients and cell autolysis, affecting product quality and increasing energy consumption and production costs. Therefore, the optimal fermentation period was determined to be 168 h, which is 16% shorter than the traditional process (200 h).

[0172] Based on the above results, the suitable fermentation conditions for the production of kasugamycin by *Streptomyces simonii* XHSSM-001 are as follows:

[0173] Fermentation temperature 29℃;

[0174] Ventilation rate 1.0 vvm;

[0175] Dissolved oxygen controlled at 50%;

[0176] The gauge pressure of the fermenter is 0.05 MPa;

[0177] Add 20 g / L maltose and 5 g / L corn steep liquor powder after 72 hours of fermentation;

[0178] Total fermentation time: 168 hours.

[0179] Based on the above optimization results of fermentation parameters, in a 5 L mechanically stirred fermenter, with a fermentation temperature of 29 ℃, an initial aeration rate of 1.0 vvm, dissolved oxygen controlled within a suitable range during fermentation, and a tank gauge pressure of 0.05 MPa, and with the addition of 20 g / L maltose and 5 g / L corn steep liquor powder after 72 h of fermentation, the kasugamycin content reached approximately 31.5 g / L after 168 h of fermentation. These results represent the results obtained during the optimization phase of the fermentation process parameters. To further verify the reproducibility of the determined basic fermentation process, three independent 5 L fermentation experiments were conducted in subsequent Example 1 under the same basic fermentation conditions. The kasugamycin content in each batch showed normal inter-batch fluctuations, and the average value was basically consistent with the results of the above optimization experiments.

[0180] 3.4.6 Validation of the three-stage fermentation process

[0181] To verify the feasibility of using the optimized basic fermentation process for the fermentation of Streptomyces simonii XHSSM-001 in production tanks, a three-stage culture system was constructed, consisting of shake flask seed culture, 50 L seed tank culture, and 500 L production tank fermentation.

[0182] (1) Shake flask seed culture

[0183] Streptomyces simonii XHSSM-001 was inoculated onto agar slant culture medium and cultured at 28 °C for 7 days. After mature spores formed on the slant, a concentration of 1×10⁻⁶ was prepared according to the aforementioned method. 6 A spore suspension of 1 spore per mL.

[0184] Add 100 mL of seed culture medium to a 500 mL Erlenmeyer flask, and inoculate with spore suspension at 2% of the seed culture medium volume, i.e., add 2.0 mL of spore suspension to each flask. Incubate at 28 ℃ and 220 rpm with shaking for 48 h. Prepare multiple shake-flask seed solutions using the same method. Aseptically combine the qualified shake-flask seed solutions to obtain at least 4.2 L of shake-flask seed solution.

[0185] (2) Culture in a 50 L seed tank

[0186] Add 35.0 L of seed culture medium to a 50 L seed tank, sterilize at 121 °C for 20 min, cool to 28 °C, and aseptically inoculate with 4.2 L of shake flask seed solution. The amount of shake flask seed solution added is equivalent to 12% of the initial seed culture medium volume.

[0187] The seed culture was conducted in a 50 L seed tank at a temperature of 28 ℃, an initial aeration rate of 1.0 vvm, an initial stirring speed of 180 rpm, an internal gauge pressure of 0.05 MPa, and a culture time of 48 h. During the culture process, the aeration rate and stirring speed were adjusted to maintain normal aerobic growth of the seed culture.

[0188] After cultivation, the pH of the seed culture was measured, and the OD600 was measured after the aforementioned uniform dispersion treatment. A qualified production seed culture is defined as one where the pH is not lower than 7.0, the OD600 is 1.8-2.0, and microscopic observation shows normal mycelial growth, uniform morphology, and the absence of contaminating microorganisms. At least 36.0 L of qualified seed culture should be available for aseptic transfer after cultivation.

[0189] (3) Fermentation in a 500 L production tank

[0190] Add 300 L of optimized fermentation medium to a 500 L production tank, sterilize at 121 °C for 20 min, cool to 29 °C, and aseptically transfer 36.0 L of qualified seed culture from a 50 L seed tank to a 500 L production tank. The inoculation volume of the seed culture is 12% of the initial fermentation medium volume in the production tank.

[0191] The fermentation temperature in the production tank was 29 ℃, the initial aeration rate was 1.0 vvm, the initial stirring speed was 180 rpm, and the gauge pressure inside the tank was 0.05 MPa. During fermentation, the stirring speed and aeration rate were adjusted according to changes in dissolved oxygen to maintain the fermentation broth in an aerobic state.

[0192] At 72 h of fermentation, 6.0 kg of maltose and 1.5 kg of corn steep liquor powder were added, which were equivalent to 20 g / L and 5 g / L of the initial fermentation medium volume of 300 L, respectively. After the addition of materials, fermentation continued until the total fermentation time reached 168 h.

[0193] In the aforementioned 500 L three-stage fermentation basic process, no additional scandium chloride hexahydrate and 1,4-butyrolactone were added. Three independent batches of fermentation validation were conducted in production tanks, and the kasugamycin content was determined using the method described in Test Example 1. This experiment was used to verify the adaptability of *Streptomyces aureus* XHSSM-001 and the basic fermentation process in the 500 L production tank, and was not used to evaluate the yield increase effect of the staged addition of scandium chloride hexahydrate and 1,4-butyrolactone.

[0194] Three independent scale-up fermentation validations were conducted. The results showed that the kasugamycin content in the fermentation broth of the three independent 500 L production tanks ranged from 31.0 to 31.4 g / L, with an average content of 31.2 g / L. Compared with the results of the basic process validation in the 5 L fermenter, the average kasugamycin content decreased slightly after scale-up in the 500 L production tank, but remained at a similar level overall, with minimal fluctuations between different production batches. This indicates that *Streptomyces aureus* XHSSM-001 and the established basic fermentation process have good adaptability to scale-up and fermentation reproducibility.

[0195] 4. Examples and Comparative Examples

[0196] 4.1 Common Test Conditions

[0197] Unless otherwise stated, Examples 1-4 and Comparative Examples 1-5 all used *Streptomyces simonii* XHSSM-001 with accession number CGMCC No. 38870 as the fermentation strain and were carried out in a 5 L mechanically stirred fermenter.

[0198] Add 3.0 L of optimized fermentation medium to each 5 L fermenter. The optimized fermentation medium consists of 70 g soybean meal powder, 18 g corn starch, 4.0 g sodium chloride, 8.0 g corn steep liquor powder, 38 g fish oil, 0.4 g potassium dihydrogen phosphate, 0.03 g inositol, 1.0 g Tween-20, and the balance water per liter, with an initial pH of 7.0 ± 0.1.

[0199] XHSSM-001 seed culture, which had been cultured for 48 h and met quality standards, was inoculated into the fermenters at 12% of the initial fermentation medium volume, i.e., 360 mL of seed culture per fermenter. The fermentation temperature was 29 ℃, the initial aeration rate was 1.0 vvm, the initial stirring speed was 180 rpm, and the gauge pressure inside the fermenter was 0.05 MPa. During fermentation, the dissolved oxygen level was controlled between 30-60% by adjusting the aeration rate and stirring speed. The total fermentation time was 168 h.

[0200] At 72 h of fermentation, 60 g of maltose and 15 g of corn steep liquor powder were added to all examples and comparative examples, making the added amounts of maltose and corn steep liquor powder relative to the initial fermentation medium volume 20 g / L and 5 g / L, respectively. Except for the amount, time, and order of addition of scandium chloride hexahydrate and 1,4-butyrolactone, the composition of the culture medium, inoculum size, nutrient feeding method, and fermentation conditions remained consistent. Three independent fermentation experiments were conducted for each group.

[0201] 4.2 Preparation of Feed Solution

[0202] The amount of scandium chloride hexahydrate added is based on the mass of ScCl3·6H2O. Weigh 6.5 g of scandium chloride hexahydrate with a purity of not less than 99.0%, dissolve it in water, and make up to 1 L to obtain a scandium chloride hexahydrate feed solution with a concentration of 6.5 g / L. Filter the feed solution through a sterile filter membrane with a pore size of 0.22 μm for sterilization, and prepare it immediately before use.

[0203] Weigh 100 g of 1,4-butyrolactone with a purity of not less than 99.0%, add water, mix and make up to 1 L to obtain a 1,4-butyrolactone feed solution with a concentration of 100 g / L; filter the feed solution through a sterile filter membrane with a pore size of 0.22 μm for sterilization, and prepare and use immediately.

[0204] Maltose was prepared into a 500 g / L aqueous solution and sterilized by filtration through a 0.22 μm sterile filter membrane; corn steep liquor powder was prepared into a 200 g / L suspension and sterilized at 121 ℃ for 20 min. Based on an initial fermentation medium volume of 3.0 L, 120 mL of maltose feed solution and 75 mL of corn steep liquor powder feed solution were added after 72 h of fermentation.

[0205] The amounts of scandium chloride hexahydrate and 1,4-butyrolactone added as described below are calculated based on the initial fermentation medium volume and are not presented as the final concentration of the actual fermentation broth after inoculation.

[0206] Example 1

[0207] This embodiment uses a 5 L mechanically stirred fermenter for basic fermentation.

[0208] No scandium chloride hexahydrate or 1,4-butyrolactone was added during the fermentation process; only 60 g of maltose and 15 g of corn steep liquor powder were added at 72 h of fermentation, and fermentation continued until the total fermentation time reached 168 h.

[0209] The composition of the remaining culture media, the amount of seed culture inoculated, and the fermentation conditions were all carried out in accordance with the common experimental conditions described in Section 4.1.

[0210] Example 2

[0211] At 24 h of fermentation, 3.0 mL of scandium chloride hexahydrate feed solution was added through a sterile feed line, which is equivalent to adding 19.5 mg of scandium chloride hexahydrate; based on the initial fermentation medium volume of 3.0 L, the amount of scandium chloride hexahydrate added was 6.5 mg / L.

[0212] At 54 h of fermentation, 12.9 mL of 1,4-butyrolactone feed solution was added through a sterile feed line, which is equivalent to adding 1.29 g of 1,4-butyrolactone; based on an initial fermentation medium volume of 3.0 L, the amount of 1,4-butyrolactone added was 0.43 g / L.

[0213] Add 60 g of maltose and 15 g of corn steep liquor powder after 72 h of fermentation, and continue fermentation for 168 h.

[0214] The composition of the remaining culture medium, the amount of seed culture inoculation, and the fermentation conditions were the same as in Example 1.

[0215] Example 3

[0216] The method is basically the same as in Example 2, except that: 9.9 mg of scandium chloride hexahydrate was added after 18 h of fermentation, and the amount of scandium chloride hexahydrate added was 3.3 mg / L based on the initial fermentation medium volume of 3.0 L.

[0217] 0.66 g of 1,4-butyrolactone was added after 48 h of fermentation. Based on an initial fermentation medium volume of 3.0 L, the amount of 1,4-butyrolactone added was 0.22 g / L.

[0218] The composition of the remaining culture medium, the amount of seed liquid inoculated, the method of feeding carbon and nitrogen sources during fermentation for 72 h, and the fermentation conditions were all the same as in Example 2.

[0219] Example 4

[0220] The method is basically the same as in Example 2, except that 29.4 mg of scandium chloride hexahydrate was added after 24 h of fermentation. Based on an initial fermentation medium volume of 3.0 L, the amount of scandium chloride hexahydrate added was 9.8 mg / L.

[0221] 1.95 g of 1,4-butyrolactone was added after 60 h of fermentation. Based on an initial fermentation medium volume of 3.0 L, the amount of 1,4-butyrolactone added was 0.65 g / L.

[0222] The composition of the remaining culture medium, the amount of seed liquid inoculated, the method of feeding carbon and nitrogen sources during fermentation for 72 h, and the fermentation conditions were all the same as in Example 2.

[0223] Comparative Example 1

[0224] The method is basically the same as Example 2, except that: only 19.5 mg of scandium chloride hexahydrate is added at 24 h of fermentation, and the amount of scandium chloride hexahydrate added is 6.5 mg / L based on the initial fermentation medium volume of 3.0 L; 1,4-butyrolactone is not added at 54 h of fermentation.

[0225] The composition of the remaining culture medium, the amount of seed liquid inoculated, the method of feeding carbon and nitrogen sources during fermentation for 72 h, and the fermentation conditions were all the same as in Example 2.

[0226] Comparative Example 2

[0227] The method is basically the same as Example 2, except that scandium chloride hexahydrate is not added during fermentation for 24 h; 1,4-butyrolactone 1.29 g is added only during fermentation for 54 h. Based on the initial fermentation medium volume of 3.0 L, the amount of 1,4-butyrolactone added is 0.43 g / L.

[0228] The composition of the remaining culture medium, the amount of seed liquid inoculated, the method of feeding carbon and nitrogen sources during fermentation for 72 h, and the fermentation conditions were all the same as in Example 2.

[0229] Comparative Example 3

[0230] The method is basically the same as in Example 2, except that scandium chloride hexahydrate and 1,4-butyrolactone are not added in stages, but are added simultaneously during 24 hours of fermentation.

[0231] The amount of scandium chloride hexahydrate added was 19.5 mg, which is 6.5 mg / L based on the initial fermentation medium volume of 3.0 L; the amount of 1,4-butyrolactone added was 1.29 g, which is 0.43 g / L based on the initial fermentation medium volume of 3.0 L. No more 1,4-butyrolactone was added after 54 h of fermentation.

[0232] The composition of the remaining culture medium, the total amount of the two regulatory substances added, the seed liquid inoculation amount, the carbon and nitrogen source feeding method and fermentation conditions at 72 h of fermentation were the same as in Example 2.

[0233] Comparative Example 4

[0234] It is basically the same as Example 2, except that the order of addition of scandium chloride hexahydrate and 1,4-butyrolactone is reversed compared to Example 2.

[0235] 1.29 g of 1,4-butyrolactone was added at 24 h of fermentation, which was 0.43 g / L based on the initial fermentation medium volume of 3.0 L; 19.5 mg of scandium chloride hexahydrate was added at 54 h of fermentation, which was 6.5 mg / L based on the initial fermentation medium volume of 3.0 L.

[0236] The composition of the remaining culture medium, the total amount of the two regulatory substances added, the seed liquid inoculation amount, the carbon and nitrogen source feeding method and fermentation conditions at 72 h of fermentation were the same as in Example 2.

[0237] Comparative Example 5

[0238] The method is basically the same as in Example 2, except that: 39.0 mg of scandium chloride hexahydrate was added after 24 h of fermentation, and the amount of scandium chloride hexahydrate added was 13.0 mg / L based on the initial fermentation medium volume of 3.0 L.

[0239] 2.58 g of 1,4-butyrolactone was added after 54 h of fermentation. Based on an initial fermentation medium volume of 3.0 L, the amount of 1,4-butyrolactone added was 0.86 g / L.

[0240] The composition of the remaining culture medium, the amount of seed liquid inoculated, the method of feeding carbon and nitrogen sources during fermentation for 72 h, and the fermentation conditions were all the same as in Example 2.

[0241] Test case

[0242] 1. Test method for kasugamycin content

[0243] 1.1 Instruments and Chromatographic Conditions

[0244] The content of kasugamycin in the fermentation broth was determined using a high-performance liquid chromatography (HPLC) system equipped with an ultraviolet detector. An ODS reversed-phase C18 column with dimensions of 250 mm × 4.6 mm and a packing particle size of 5 μm was used.

[0245] The mobile phase consisted of acetonitrile and an aqueous solution of sodium dodecyl sulfate. The sodium dodecyl sulfate aqueous solution had a mass concentration of 1.0%, and its pH was adjusted to 2.5 ± 0.2 using phosphoric acid. The volume ratio of acetonitrile to sodium dodecyl sulfate aqueous solution was 20:80. After mixing, the mobile phase was filtered through a 0.45 μm filter membrane and degassed before use.

[0246] The column temperature was 30 ℃, the flow rate was 1.2 mL / min, the UV detection wavelength was 215 nm, the injection volume was 5 μL, isocratic elution was used, and the run time per injection was 25 min. Measurements were performed after the instrument baseline stabilized.

[0247] 1.2 Preparation of reference solution

[0248] Kasugamycin hydrochloride reference standard with an assigned active ingredient value was used. The purity or active ingredient content of the reference standard was determined according to its test report or standard certificate, and the concentration of all standard solutions was calculated based on the active ingredient content of kasugamycin indicated in the certificate.

[0249] Accurately weigh an appropriate amount of kasugamycin hydrochloride reference standard, dissolve it in ultrapure water and dilute to volume to prepare a reference standard stock solution with a mass concentration of 4.0 g / L based on kasugamycin. Dilute with ultrapure water to obtain a series of reference standard working solutions with concentrations of 0.25 g / L, 0.50 g / L, 1.00 g / L, 2.00 g / L and 4.00 g / L, respectively.

[0250] Separate samples were injected and analyzed. A standard curve was established by linear regression with the mass concentration of kasugamycin as the abscissa and the corresponding chromatographic peak area as the ordinate.

[0251] 1.3 Fermentation broth sample processing

[0252] Take 10.0 mL of fermentation broth aseptically from the fermenter, centrifuge at 8000×g for 10 min, and collect the supernatant.

[0253] Select an appropriate quantitative dilution factor based on the expected kasugamycin content in different fermentation samples to ensure that the final kasugamycin concentration in the test solution falls within the standard curve range of 0.25-4.00 g / L. For the fermentation endpoint samples of Examples 1-4 and comparative examples, a 50-fold dilution can be used; for screening samples of strains with low kasugamycin content, a 20-40-fold dilution can be used.

[0254] The diluted sample was filtered through a hydrophilic membrane with a pore size of 0.22 μm. The first 1 mL of filtrate was discarded, and the subsequent filtrate was collected as the sample to be tested.

[0255] 1.4 Determination and Qualitative Analysis

[0256] The reference solution and the test sample were determined under the above chromatographic conditions. The retention time of the target peak in the sample was consistent with that of the kasugamycin reference standard under the same chromatographic conditions as the qualitative basis. The change in the area of ​​the target peak before and after spiked sample was used to further confirm the kasugamycin peak.

[0257] Each sample was injected twice consecutively, and the average value of the two measurements was taken as the result of the sample if the relative deviation between the two measurements was no greater than 2.0%.

[0258] 1.5 Content Calculation

[0259] The mass concentration of kasugamycin in the test sample was calculated based on the standard curve, and then converted to the kasugamycin content in the original fermentation broth according to the total dilution factor of the sample.

[0260] Kasugamycin content (g / L) = C × D;

[0261] In the formula, C is the mass concentration of kasugamycin in the test solution obtained according to the standard curve, in g / L; D is the total dilution factor from the original fermentation broth to the final test solution.

[0262] The concentration of the reference standard was already converted to kasugamycin according to the active ingredient content in the standard certificate when establishing the standard curve; therefore, a separate conversion between hydrochloride and free kasugamycin was not performed. The test results are shown in Tables 7 and 8.

[0263] Test Example 2

[0264] Method validation of high performance liquid chromatography detection methods

[0265] To verify the applicability of the high performance liquid chromatography method described in Test Example 1 for the determination of kasugamycin content in the fermentation broth of Streptomyces simonii, the specificity, linearity, injection precision, repeatability, spiked recovery rate and sample solution stability of the method were verified.

[0266] (1) Specificity: Blank fermentation medium without inoculated strain, kasugamycin reference solution, fermentation broth sample to be tested in Example 2, and spiked fermentation broth sample in Example 2 were prepared separately and determined according to the chromatographic conditions described in Test Example 1. No obvious interfering peaks affecting quantification should appear in the blank medium near the retention time of the kasugamycin target peak; the retention time of the target peak in the sample should be consistent with that of the reference standard, and the resolution between the target peak and adjacent impurity peaks should not be less than 1.5.

[0267] (2) Linearity: Prepare kasugamycin solutions with concentrations of 0.20 g / L, 0.40 g / L, 0.60 g / L, 0.8 g / L, and 1.0 g / L respectively. Use the kasugamycin concentration as the abscissa and the peak area as the ordinate to establish a linear regression equation. The correlation coefficient r should not be less than 0.999.

[0268] (3) Injection precision: Inject 1.00 g / L kasugamycin reference solution 6 times consecutively, calculate the relative standard deviation of the peak area of ​​kasugamycin, and the RSD should not be greater than 2.0%.

[0269] (4) Repeatability: Take 6 portions of the same batch of fermentation broth, each 10.0 mL, and centrifuge, dilute and filter them independently according to the method described in Test Example 1. Calculate the RSD of the kasugamycin content in the 6 samples. The RSD should not be greater than 3.0%.

[0270] (5) Spiking recovery rate: Take the same batch of fermentation broth with known kasugamycin content, and add known amounts of kasugamycin reference standard at three spiking levels: low, medium, and high. Prepare three parallel samples for each level and determine the spiking rate according to the method described in Test Example 1. Calculate the spiking recovery rate as follows: Recovery rate = (Measured amount of spiked sample - Measured amount of original sample) / Theoretical spiking amount × 100%. The average recovery rate for each level should be between 90% and 110%, and the RSD should not exceed 5.0%.

[0271] (6) Sample solution stability: The same portion of fermentation broth was used as the test sample and injected at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h after preparation. The RSD of the peak area of ​​kasugamycin at different time points was calculated. When the RSD is not greater than 3.0%, the test sample is considered to be stable within 24 h under the corresponding storage conditions.

[0272] Table 7. Changes in kasugamycin content in different groups with fermentation time

[0273]

[0274] Table 8. Kasugamycin content and batch repeatability in different groups at 168 h

[0275]

[0276] Based on the kasugamycin production capacity and continuous subculturing experiments, this invention utilizes a combination of atmospheric pressure room temperature plasma mutagenesis, ultraviolet remutation, and photoremediation to expand the range of genetic variation in the strain. Further screening using antibacterial activity, high-performance liquid chromatography (HPLC) rescreening, and continuous subculturing validation eliminates mutant strains with low yield, abnormal growth, and poor genetic stability. The resulting *Streptomyces simonii* XHSSM-001 exhibits stronger kasugamycin synthesis capacity compared to the parent strain and single-mutated strains, while maintaining similar continuous subculturing stability as the parent strain. This demonstrates that combining combined mutagenesis with stepwise screening can balance kasugamycin production capacity and genetic stability.

[0277] As shown in Tables 7 and 8, Example 1, using *Streptomyces simonii* XHSSM-001, an optimized fermentation medium, and maltose and corn steep liquor powder as feed, can provide suitable nutrition, temperature, and dissolved oxygen conditions for cell growth and continuous synthesis of kasugamycin. However, it does not specifically regulate the process of cell transition from growth metabolism to secondary metabolism. Therefore, its kasugamycin synthesis rate and final fermentation content are lower than those of Examples 2-4.

[0278] Example 2, based on Example 1, adds scandium chloride hexahydrate during the cell growth stage and then adds 1,4-butyrolactone during the transition from growth metabolism to secondary metabolism, allowing the two regulatory substances to function at appropriate fermentation stages. Scandium chloride hexahydrate helps to establish a suitable metabolic state for subsequent secondary metabolism, while 1,4-butyrolactone promotes the timely entry of cells into the kasugamycin synthesis stage, thus connecting the early metabolic regulation with the initiation of subsequent secondary metabolism.

[0279] In Example 3, the amounts of scandium chloride hexahydrate and 1,4-butyrolactone added were both at low levels, and the addition time of 1,4-butyrolactone was relatively early. Although both substances could still promote kasugamycin synthesis to some extent, the regulatory intensity was relatively insufficient; at the same time, the bacterial cells may not have fully completed the growth stage when 1,4-butyrolactone was added, making it difficult to fully exert its regulatory effect on the conversion of secondary metabolic stages. Therefore, its kasugamycin synthesis rate and final content were lower than those in Example 2.

[0280] In Example 4, the amounts of both regulatory substances were increased, and the addition time of 1,4-butyrolactone was delayed. This scheme could still promote the synthesis of kasugamycin, but the higher amount of scandium chloride hexahydrate added may increase the ion regulation and metabolic burden of the cells, and the late addition of 1,4-butyrolactone could not sufficiently prolong the effective synthesis stage of kasugamycin. Therefore, although its technical effect was better than that of Example 1, it was still lower than that of Example 2.

[0281] Comparative Example 1, with the addition of only scandium chloride hexahydrate, can regulate the secondary metabolic state of the bacteria to some extent, but lacks the further promoting effect of 1,4-butyrolactone on the transition of bacterial metabolic stages, thus the improvement in kasugamycin synthesis efficiency is limited. Comparative Example 2, with the addition of only 1,4-butyrolactone, although beneficial to promoting the transition of bacteria to the secondary metabolic stage, cannot form a sequential staged regulatory process due to the lack of regulation of the early metabolic state by scandium chloride hexahydrate, and its kasugamycin synthesis effect is also lower than that of Example 2.

[0282] In Comparative Example 3, scandium chloride hexahydrate and 1,4-butyrolactone were added simultaneously in the early stage of fermentation. Although the total amount of the two substances added was the same as in Example 2, the 1,4-butyrolactone was added too early and failed to match the stage of cell transition from growth metabolism to secondary metabolism, so that the two regulatory substances could not play their full role in the corresponding stages.

[0283] Comparative Example 4 reversed the order of addition of the two regulatory substances, adding 1,4-butyrolactone first, followed by scandium chloride hexahydrate. This order of addition prevented the bacteria from first forming a suitable secondary metabolic basis before completing the metabolic transition, which does not conform to the process logic of connecting early metabolic regulation with late signal regulation established in this invention. As a result, the accumulation rate and final content of kasugamycin were lower than in Example 2, indicating that the order of addition of the two regulatory substances has practical technical significance.

[0284] In Comparative Example 5, the amounts of scandium chloride hexahydrate and 1,4-butyrolactone added were significantly higher than those used in the embodiments of this invention. Consequently, the synthesis effect of kasugamycin decreased, and the target content was not reached at the end of fermentation. This may be because excessive Sc³⁺ interferes with the ion balance and normal enzymatic reactions of the cells, and excessive 1,4-butyrolactone may also lead to an imbalance in cell signal regulation, thus adversely affecting normal cell growth and the secondary metabolism of kasugamycin.

[0285] Furthermore, the three independent fermentation results of each example and comparative example in Table 8 all showed small batch fluctuations, indicating good reproducibility of the experiment. A summary of Tables 7 and 8 shows that the combined use, dosage, timing, and order of addition of scandium chloride hexahydrate and 1,4-butyrolactone in this invention are closely related. Adding scandium chloride hexahydrate first during the cell growth stage, then adding 1,4-butyrolactone during the cell transition to secondary metabolism, and supplementing with carbon and nitrogen sources during the subsequent continuous synthesis of kasugamycin, allows for seamless integration of early cell growth, mid-stage metabolic conversion, and late-stage product synthesis, thereby improving the synthesis rate of kasugamycin, the final fermentation content, and the reproducibility of the fermentation process.

Claims

1. A method for producing kasugamycin by fermentation, characterized in that, Includes the following steps: The seed culture of Streptomyces microaureus XHSSM-001 was inoculated into a fermentation medium for liquid fermentation. The preservation number of Streptomyces microaureus XHSSM-001 is CGMCC No.38870. Based on the initial fermentation medium volume, 3.3-9.8 mg / L scandium chloride hexahydrate was added to the fermentation system at 18-24 h of fermentation, and 0.22-0.65 g / L 1,4-butyrolactone was added to the fermentation system at 48-60 h of fermentation, with the addition of scandium chloride hexahydrate preceding the addition of 1,4-butyrolactone; after the addition was completed, fermentation continued to obtain a fermentation broth containing kasugamycin.

2. The method for producing kasugamycin by fermentation according to claim 1, characterized in that, Based on the initial fermentation medium volume, the amount of scandium chloride hexahydrate added is 4.5-8.5 mg / L, and the amount of 1,4-butyrolactone added is 0.30-0.60 g / L; the scandium chloride hexahydrate is added after 20-24 h of fermentation, and the 1,4-butyrolactone is added after 50-58 h of fermentation.

3. The method for producing kasugamycin by fermentation according to claim 1 or 2, characterized in that, Scandium chloride hexahydrate was added at 24 h of fermentation at a dosage of 6.5 mg / L; 1,4-butyrolactone was added at 54 h of fermentation at a dosage of 0.43 g / L.

4. The method for producing kasugamycin by fermentation according to any one of claims 1-3, characterized in that, After adding scandium chloride hexahydrate and 1,4-butyrolactone, a carbon source and a nitrogen source are added after 68-76 h of fermentation. The amount of carbon source added is 18-22 g / L and the amount of nitrogen source added is 4-6 g / L, based on the initial fermentation medium volume. The carbon source includes maltose and the nitrogen source includes corn steep liquor powder.

5. The method for producing kasugamycin by fermentation according to claim 4, characterized in that, The carbon source is maltose, and the nitrogen source is corn steep liquor powder. The maltose and corn steep liquor powder are added during fermentation for 72 hours, with the amount of maltose added being 20 g / L and the amount of corn steep liquor powder added being 5 g / L.

6. The method for producing kasugamycin by fermentation according to any one of claims 1-5, characterized in that, The fermentation medium comprises the following components per liter: 50-90 g soybean meal powder, 12-24 g corn starch, 2-6 g sodium chloride, 0.2-0.6 g potassium dihydrogen phosphate, 0.01-0.05 g inositol, 5-10 g corn steep liquor powder, 35-40 g fish oil, and 0.8-1.2 g Tween-20, with water added to a final volume of 1 L; the initial pH of the fermentation medium is 6.8-7.

2.

7. The method for producing kasugamycin by fermentation according to any one of claims 1-6, characterized in that, The conditions for liquid fermentation include: fermentation temperature 27-30 ℃, aeration rate 0.8-1.2 vvm, dissolved oxygen controlled at 30%-60%, fermentation tank gauge pressure 0.05MPa, and total fermentation time 156-180 h.

8. The method for producing kasugamycin by fermentation according to any one of claims 1-7, characterized in that, The fermentation employs a three-stage culture system consisting of shake flask seed culture, seed tank culture, and production tank fermentation. The temperature for shake flask seed culture is 26-30 ℃, the shaker speed is 180-240 rpm, and the culture time is 36-54 h. The temperature for seed tank culture is 26-30 ℃, the aeration rate is 0.8-1.2 vvm, and the culture time is 36-54 h. The inoculum volume of the seed liquid in the seed tank and the inoculum volume of the seed tank culture in the production tank are 10-15% of the corresponding initial culture medium volume, respectively.

9. A small golden Streptomyces microaureus XHSSM-001, characterized in that, The *Streptomyces simonii* XHSSM-001 was deposited on May 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 38870.

10. The application of the *Streptomyces simonii* XHSSM-001 as described in claim 9 in the fermentation production of kasugamycin.

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