A mycelium or bacterial cell and its application, and a method for breeding strains using the same

Abstract

The present invention discloses a mycelium or bacterial cell and its application, and a method for breeding strains using the same. The mycelium or bacterial cell is a mycelium or bacterial cell obtained from the fermentation broth at the end of the rapid growth and accumulation stage of secondary metabolite production in the fermentation production process of the strain. When the mycelium or bacterial cell obtained by the present invention is used to breed Streptomyces lilacinus strains resistant to its own metabolites, a high-yield strain of sinoprotinin is screened out, and the industrial fermentation production level of the high-yield strain can be greatly improved, which greatly reduces the industrial production cost of sinoprotinin, lays a foundation for the market demand for sinoprotinin in biological pesticides, and meets the expansion of use in fields such as large-scale aerial spraying of green ecological agriculture.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to a mycelium or mycelium and its application, and a method for selecting strains tolerant to their own metabolites. Background Technology

[0002] The vigorous development of safe, low-toxicity and non-toxic, low-accumulation and non-accumulation, and environmentally friendly biological pesticides has become an inevitable trend and a global consensus.

[0003] Casserole (chemical name: 1-N-glycosidic-2-amino-L-lysine-2-deoxygulosamine; molecular formula: C) 19 H 34 O8N8 (molecular weight: 502.0) is a novel agricultural antibiotic-type biological pesticide, classified as a fungicide for the control of crop diseases. Its producing organism, *Streptomyces lavendulae* var. *hainanensis*, was isolated from soil in Hainan Island and is protected under the CGMCC No. 1026. As an agricultural antibiotic-type biological pesticide, it exhibits strong inhibitory or bactericidal effects against Gram-positive bacteria, Gram-negative bacteria, and filamentous fungi. It inhibits the formation of peptide bonds in pathogenic bacterial proteins, ultimately leading to bacterial death. Against fungi, it inhibits mycelial growth and spore germination, thus controlling fungal diseases. It shows good control efficacy against rice bacterial blight, cabbage soft rot, apple ring rot, and apple leaf spot, while being low in toxicity to humans and livestock and environmentally friendly.

[0004] Therefore, improving the fermentation level of the traditional Chinese medicine industry, achieving energy conservation and consumption reduction in the industrialization of traditional Chinese medicine, expanding the production capacity of traditional Chinese medicine raw materials, and increasing the market share of traditional Chinese medicine as a green food production material will contribute to the cause of human food health and have good social and economic benefits.

[0005] The industrial fermentation production of kasugamycin has been underway for 20 years. However, both laboratory research and industrial fermentation production are hampered by numerous single and multi-factor inhibition and feedback regulation from the kasugamycin-producing bacteria's own metabolites, such as the glucose effect and its secondary metabolite kasugamycin. This has prevented further improvement in the fermentation production level of kasugamycin, with the biopotency hovering between 8000 μ / ml and 12000 μ / ml. Consequently, the content of kasugamycin technical material cannot be further increased, limiting the market supply and demand for kasugamycin technical material and its formulations, as well as its application in large-scale, comprehensive aerial spraying and other fields.

[0006] Based on this, it is hoped that by starting with the kasugamycin-producing bacteria, high-yield kasugamycin-producing strains that are resistant to their own secondary metabolites kasugamycin and other self-metabolites can be screened and applied to industrial fermentation production. This will improve the level of industrial fermentation production of kasugamycin as a green production material, obtain a large amount of fully water-soluble kasugamycin with high content, and meet the needs of rapidly developing green ecological agriculture. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides mycelium or mycelium, its applications, and a method for selecting strains using these mycelium. The technical solution of this invention starts with streptomycin-producing bacteria, screening for high-yield streptomycin-producing strains tolerant to their own secondary metabolites (such as streptomycin) and other self-metabolites, and applying them to industrial-scale fermentation production. This improves the level of industrial-scale fermentation production of streptomycin as a green production material, obtaining large quantities of fully water-soluble streptomycin with high content, thus meeting the needs of rapidly developing green ecological agriculture.

[0008] To achieve the above-mentioned objectives, the present invention is accomplished through the following aspects:

[0009] In a first aspect, the present invention provides a mycelium or mycelium obtained from the fermentation broth at the end of the rapid growth and accumulation stage of secondary metabolite production during the fermentation process of the strain.

[0010] In some preferred embodiments, the strain is *Streptomyces lavendulae*, preferably *Streptomyces lavendulae* var. *hainanensis*; more preferably, *Streptomyces lavendulae* var. *hainanensis* with accession numbers CGMCC NO. 1026 or CGMCC 61906.

[0011] Preferably, the fermentation satisfies one or more of the following conditions:

[0012] Fermentation temperature is 26℃-35℃;

[0013] Air ventilation rate: 0.5V / VM - 1.5V / VM;

[0014] The stirring speed of the fermentation tank is 50 rpm-200 rpm;

[0015] The fermentation culture cycle is 3-10 days;

[0016] The fermentation medium used for fermentation comprises: 4.0%-15.0% glucose, 1.0%-6.0% corn starch, 0.5%-7.5% corn flour, 3.0%-12.0% soybean flour, 0.2%-1.6% sodium chloride, 0.1%-1.8% ammonium chloride, 0.2%-1.6% calcium carbonate, 0.01%-0.13% potassium dihydrogen phosphate, 0.01%-0.16% magnesium sulfate, and pH 6.0-7.0, where % refers to mass-volume percentage.

[0017] Secondly, the present invention provides a method for selecting strains tolerant to their own metabolites, the method comprising the following steps:

[0018] Step S1: Ferment the selected starting strain to obtain the fermentation broth at the end of the rapid growth and accumulation stage of secondary metabolite production and prepare cell filtrate.

[0019] Step S2: Obtain the fermentation broth at the end of the stagnation stage of the production growth of the same batch of secondary metabolites, filter it to obtain filtrate, and then concentrate the filtrate, preferably by vacuum concentration, to obtain concentrated fermentation broth containing substrate factors.

[0020] Step S3: Prepare a screening culture medium containing concentrated fermentation broth;

[0021] Step S4: Spread and culture the bacterial cell filtrate on the screening medium to screen for strains that are tolerant to their own metabolites.

[0022] Preferably, the screening medium is a gradient concentration medium of substrate factors, wherein the maximum biopotency of the serotonin in the gradient concentration medium is 20,000 to 50,000 μ / ml; for example, 30,000 μ / ml.

[0023] And / or, in the final stage of the rapid growth and accumulation phase of production, the reducing sugar content is 15-20 g / L, preferably 18-19 g / L; the amino nitrogen content is 1.2-1.5 mg / L, preferably 1.3-1.4 mg / L; the total phosphorus content is 25-30 mg / L, preferably 28-29 mg / L; the bacterial concentration is 27-30%, preferably 28-29%; and the pH is 6.5-7.0.

[0024] And / or, in the final stage of the rapid growth and accumulation phase of production, the potency of kasugamycin is 6000-10000, preferably 7000-8000 μ / ml.

[0025] And / or, at the end of the production growth stagnation phase, the reducing sugar content is 5-7 g / L, the amino nitrogen content is 0.6-0.9 mg / L, the total phosphorus content is 8-12 mg / L, the bacterial concentration is 25-27%, and the pH is 6.8-7.0.

[0026] And / or, the potency of sclerosin is 10,000 to 12,000 μ / ml at the end of the production growth stagnation phase.

[0027] More preferably, in step S1, the starting strain is *Streptomyces lavendulae*, more preferably *Streptomyces lavendulae* var. *hainanensis*, and even more preferably, *Streptomyces lavendulae* var. *hainanensis* with accession numbers CGMCC NO. 1026 or CGMCC 61906.

[0028] In some preferred embodiments, in step S1, the fermentation satisfies one or more of the following conditions:

[0029] Fermentation temperature is 26℃-35℃;

[0030] Air ventilation rate: 0.5V / VM - 1.5V / VM;

[0031] The stirring speed of the fermentation tank is 50 rpm-200 rpm;

[0032] The fermentation culture cycle is 3-10 days;

[0033] The fermentation medium used for fermentation comprises: 4.0%-15.0% glucose, 1.0%-6.0% corn starch, 0.5%-7.5% corn flour, 3.0%-12.0% soybean flour, 0.2%-1.6% sodium chloride, 0.1%-1.8% ammonium chloride, 0.2%-1.6% calcium carbonate, 0.01%-0.13% potassium dihydrogen phosphate, 0.01%-0.16% magnesium sulfate, and pH 6.0-7.0, where % refers to mass-volume percentage.

[0034] In some preferred embodiments, in step S2, the bacterial filtrate is obtained under aseptic conditions by: fully separating and dispersing the mycelium of the fermentation broth from the culture medium of the fermentation broth, and then naturally filtering it at room temperature using a Buchner funnel with sterile cotton (preferably sterile cotton) as the filter medium to obtain the bacterial filtrate.

[0035] And / or, the concentrated fermentation broth containing substrate factors is obtained under aseptic conditions by the following method: the mycelium of the fermentation broth is fully separated and dispersed from the culture medium of the fermentation broth, and natural filtration is performed at room temperature using a Buchner funnel with sterile cotton (preferably sterile cotton) as the filter medium. The obtained filtrate is then filtered through a sterile bacterial funnel to obtain the concentrated fermentation broth containing substrate factors; the concentration is preferably vacuum concentration.

[0036] Preferably, the vacuum concentration includes the following steps: placing the filtrate in a rotary evaporator equipped with a filter, and concentrating it under reduced pressure at a water bath temperature <45℃, a vacuum degree of -0.098 MPa, and a cooling water temperature of 5℃-7℃ to obtain a concentrated fermentation broth containing substrate factors; the volume ratio of the concentrated fermentation broth to the fermentation broth is preferably 1:(5-10), for example, 1:5.

[0037] In some preferred embodiments, in step S2,

[0038] Before filtration, glass beads are placed in the fermentation broth and shaken to fully separate the mycelium from the culture medium.

[0039] Preferably, 20 to 50, preferably 30 to 40, glass beads are placed in every 500 ml of fermentation broth; and / or, the oscillation speed is 220 rpm and the oscillation time is 60 min.

[0040] In some preferred embodiments, in step S3, the screening medium is a gradient concentration medium.

[0041] In some preferred embodiments, a step of mutagenesis of the bacterial cell filtrate is included before step S4.

[0042] In some preferred embodiments, the mutagenesis employs a cumulative decreasing ultraviolet-photoreactivation composite mutagenesis, wherein ultraviolet light and sunlight are alternately irradiated 2 to 10 times, and the irradiation time decreases sequentially.

[0043] Preferably, the ultraviolet light comes from a 30W ultraviolet lamp, with an effective wavelength range of [wavelength range missing]. The sunlight comes from a 30W fluorescent lamp.

[0044] More preferably, the alternation between ultraviolet light and sunlight occurs five times, among which,

[0045] The first treatment consisted of: turning on the UV lamp for 30 seconds, then turning it off for 20 seconds; turning on the fluorescent lamp for 20 seconds; and turning it off for 20 seconds.

[0046] The second step was: turn on the UV lamp for 25 seconds, then turn it off for 20 seconds; turn on the fluorescent lamp for 20 seconds; turn off the fluorescent lamp for 20 seconds.

[0047] The third step is as follows: turn on the ultraviolet lamp for 20 seconds, then turn it off for 10 seconds; turn on the fluorescent lamp for 10 seconds; turn off the fluorescent lamp for 10 seconds.

[0048] The fourth step is as follows: turn on the ultraviolet lamp for 15 seconds, then turn off the ultraviolet lamp for 10 seconds; turn on the fluorescent lamp for 10 seconds; turn off the fluorescent lamp for 10 seconds.

[0049] The fifth step is as follows: turn on the ultraviolet lamp for 10 seconds, then turn it off for 5 seconds; turn on the fluorescent lamp for 5 seconds; turn off the fluorescent lamp for 5 seconds.

[0050] Thirdly, the present invention proposes the application of the above-mentioned mycelium or mycelium in the selection of strains resistant to their own metabolites.

[0051] In the above-mentioned technical solution, the inventors of this case start from the source of the kasugamycin-producing bacteria, rationally select and breed high-yielding kasugamycin strains that are tolerant to their own metabolites, and apply them to the industrial fermentation production of kasugamycin, thereby improving the level of industrial fermentation production of kasugamycin.

[0052] The specific process is as follows: Using the mutagenized strain (UV-3) of *Streptomyces lilacinus* Hainan variant (CGMCC No. 1026) to industrially produce streptomycin, fermentation batches with a fermentation level ≥10000 μ / ml were selected. The mycelium in the fermentation broth at the end of the rapid growth and accumulation phase of streptomycin production during this batch was used as the target starting strain for screening for tolerance to its own metabolites. The target starting strain was mutated through a cumulative decreasing UV-photoreactivation combined mutagenesis method. The concentrated filtrate from the end of the stagnant phase of streptomycin production in the same batch was used as the substrate source for screening streptomycin-producing bacteria resistant to their own metabolites. Gradient concentration culture media were prepared using the concentrated substrate as the source for rational selection of strains with higher concentrations of streptomycin-producing bacteria's own metabolites. By combining the aforementioned target starter strains, combined mutagenesis techniques, and screening substrate factors, single-colony culture was conducted to select target strains that tolerated high concentrations of their own metabolites. Following primary screening, secondary screening, and stability tests, a high-yield streptomycin-producing strain was obtained. During large-scale industrial fermentation, this high-yield streptomycin-producing strain exhibited high fermentation yield and stable characteristics.

[0053] The method for rationally selecting high-yielding streptavidin strains tolerant to their own metabolites addresses the problem that streptavidin-producing bacteria's own metabolites, such as streptavidin, during the entire fermentation and growth process can exert feedback inhibition or repression on streptavidin production, causing stagnation in the industrial-scale fermentation of streptavidin and preventing further improvement. This method addresses the issue by starting with the selection of target strains, screening substrates, mutagenesis methods, and targeted screening media for streptavidin production. It rationally integrates these screening factors to design a method for mutagenesis, screening, and optimization of streptavidin-producing bacteria to obtain high-yielding streptavidin strains.

[0054] The metabolites of kasugamycin and other kasugamycin-producing bacteria are produced during the fermentation process by which kasugamycin-producing bacteria utilize inorganic salts such as carbon sources, nitrogen sources, and phosphates in the fermentation medium. These inorganic salts are decomposed and converted into energy sources such as ATP, amino acids, oligopeptides, polypeptides, monosaccharides, disaccharides, and polysaccharides, as well as bioactive enzymes and other vitamins and minerals necessary for the growth and reproduction of kasugamycin-producing bacteria. This ensures the normal growth and reproduction of kasugamycin-producing bacteria, enabling them to transition from the reproductive growth phase to the antibiotic production phase. This completes the primary metabolic process of bacterial growth and increased reproduction during fermentation, followed by the secondary metabolic process of kasugamycin production accumulation. The process encompasses various metabolic products of kasugamycin-producing bacteria and decomposition products generated during the decomposition and utilization of the culture medium by the kasugamycin-producing bacteria.

[0055] The selection of the target starting strain for the rational selection of high-yielding streptomycin-producing strains that are tolerant to their own metabolites is based on the fermentation, growth, and production metabolic cycle of streptomycin-producing bacteria. This cycle involves the growth and reproduction of the bacteria during primary metabolism, followed by induction and conversion into secondary metabolism to produce streptomycin. The target starting strain is the mycelium at the end of the rapid growth and accumulation stage of streptomycin production, which occurs during different stages of the growth and production process: the bacterial growth and reproduction stage, the rapid growth and accumulation stage of streptomycin production, the stagnation stage of streptomycin production, and the bacterial death stage.

[0056] The mycelium in the final stage of the rapid growth and accumulation phase of kasugamycin production, a secondary metabolite of the kasugamycin-producing bacteria (the final stage of the rapid growth and accumulation phase refers to the last two-thirds of the time period from the initial increase in the biopotency or concentration of the target secondary metabolite in the fermentation broth to the point where the growth rate reaches its maximum value, which can be determined by those skilled in the art through real-time measurement or derived by plotting a curve), represents the mycelium of the kasugamycin-producing bacteria during its fermentation and growth process. This stage represents the period when the kasugamycin-producing bacteria have transitioned from primary metabolism to secondary metabolism and exhibit the most vigorous growth and production capacity, enabling the industrial-scale production of kasugamycin. The mycelium from the late stage of the rapid growth and accumulation phase of kasugamycin production, a secondary metabolite in the fermentation production environment, was used as the target starting strain for screening. The selected strains are more adapted to the primary and secondary metabolites in the growth and production process of kasugamycin industrial fermentation. The cell wall permeability of the mycelium from the rapid growth and accumulation phase of kasugamycin production is better than that of the cell wall of colony spores, making it more susceptible to combined mutagenesis induced by ultraviolet light and photoreactivation. On rationally selected culture media, it is easier to screen out target strains that are tolerant to their own metabolites. The selected target strains have a stronger ability to adapt to the growth and production environment of industrial fermentation.

[0057] The selection of screening substrate factors for rationally selecting high-yielding streptavidin-producing strains tolerant to their own metabolites is based on the fermentation, growth, and production process of streptavidin-producing bacteria. This process involves the growth and reproduction of primary metabolites, followed by induction and conversion into secondary metabolites that produce streptavidin. The concentrated filtrate of the fermentation broth, rich in streptavidin and other metabolic substances produced during the growth and production of streptavidin, as well as decomposition products of the culture medium, is used as the source of screening substrate factors for rationally selecting high-yielding streptavidin-producing strains. This filtrate is rich in streptavidin and other metabolic substances produced during the growth and production process of streptavidin-producing bacteria, as well as decomposition products of the culture medium, at the end of the stagnant stage of streptavidin production.

[0058] The fermentation of kasugamycin reaches the final stage of stagnant growth in secondary metabolites (the period during which fermentation is discharged from the tank). At this stage, the fermentation broth is rich in kasugamycin, other metabolites from primary and secondary metabolism, and decomposition products of the culture medium. These substances inhibit or feedback-inhibit kasugamycin production, resulting in slow or no further kasugamycin growth. Continued fermentation leads to the cell death stage, where some active enzymes and other substances in the kasugamycin's own metabolites decompose the already formed kasugamycin into smaller molecules, causing the fermentation level to stop increasing or even decrease. This results in wasted fermentation time and energy consumption, failing to achieve the uneconomical goal of increasing kasugamycin fermentation levels.

[0059] In the selection of strains resistant to antibiotic self-products, traditional methods are basically to select strains that are resistant to single factors such as feedback inhibition of their own secondary metabolites, relief of glucose effect, and phosphate inhibition, so the effect is relatively weak.

[0060] Unlike existing technologies, the fermentation broth of the streptomycin-producing bacteria involved in this case, at the end of the stagnant stage of streptomycin production growth in its secondary metabolite production process, is not only rich in streptomycin itself, but also rich in various other complex self-metabolites from the streptomycin growth and fermentation process. The concentrated filtrate of fermentation broth from the final stage of the stagnant growth phase of kasugamycin production in this secondary metabolite was selected as a screening substrate for kasugamycin-resistant strains. This not only relieves the feedback inhibition of kasugamycin in its own secondary metabolite, but also relieves the inhibition or feedback inhibition of various carbon sources produced or remaining in the fermentation medium during the fermentation of kasugamycin-producing bacteria, such as starch, polysaccharides, disaccharides, glucose, pentose, tetrose, triose, α-ketoglutarate, oxaloacetic acid, etc.; various nitrogen sources, such as β-amino acids like β-lysine, oligopeptides, short peptides, polypeptides, as well as kasugamycin precursors, N-acetylglucosamine and N-acetylmuramic acid from the cell wall peptidoglycan of the producing bacteria that can be converted into the basic structural units of aminoglycoside antibiotics, and intracellular and extracellular enzymes with various biological activities such as oxidoreductases and dehydrogenases. Relieving the inhibition and feedback inhibition of endoblastin and its growth and production metabolites by the endoblastin-producing bacteria during fermentation, and achieving tolerance to various single or multiple factors of inhibition or feedback inhibition, as well as multiple synergistic factors, is the fundamental way to improve the level of industrialized fermentation production of endoblastin.

[0061] The method for selecting high-yielding streptavidin-producing strains resistant to their own metabolites involves a cumulative decreasing UV-photoreactivation combined mutagenesis method. This method transforms the single-factor physical mutagenesis caused by simple UV irradiation of the streptavidin-producing strain into a combined application of UV irradiation followed by photoreactivation and repair of the mutation sites produced by the streptavidin-producing strain using natural sunlight or fluorescent lamps. This results in a UV-photoreactivation dual-factor combined mutagenesis. Furthermore, this UV-photoreactivation combined mutagenesis is cumulatively decreased through multiple dose reductions. This ensures that the mutation sites of thymine dimers between single-stranded or double-stranded DNA resulting from UV irradiation mutagenesis undergo multiple cycles of dimer formation → dimer depolymerization → dimer formation → dimer depolymerization → dimer formation, preventing easy reversion and repair.

[0062] In the aforementioned ultraviolet-photoreactivation combined mutagenesis method, the ultraviolet light is selected from the wavelength range that is most effective in inducing mutagenesis in microbial strains. The bactericidal and mutagenic effects of ultraviolet (UV) light are best within this wavelength range. The biological effects of UV light can cause biological damage to DNA and dimerization of thymine base pairs. Therefore, the formation of thymine dimers is a major pathway by which UV light alters DNA biological activity, causing bacterial death or mutation, and plays a significant role in inducing bacterial mutation. During DNA replication, the double helix first becomes a single helix, and then each helix forms a complementary strand with nearby bases. However, due to the formation of thymine dimers between the two strands, the two DNA strands cross-link, thus hindering the separation of the double helix and replication. The formation of dimers between adjacent thymine nucleotides on the same strand also hinders normal base pairing. Under normal circumstances, thymine pairs with adenine. If two adjacent thymine nucleotides form dimers, the original pairing may be altered, disrupting the normal incorporation of adenine. Replication then abruptly stops or proceeds incorrectly at this point. If replication occurs incorrectly, and a modified base sequence appears on the newly formed strand, the altered DNA strand will continue to replicate in the subsequent replication process, resulting in a molecule with incorrect base sequences on both strands, thus causing a mutation. However, the damage to DNA genetic activity caused by ultraviolet (UV) radiation can be reversed by visible light, a phenomenon known as photoreactivation. Since the substances damaged by UV radiation are mainly dimers, under the action of photoreactivating enzymes, the dimers are broken down back into monomers, restoring the DNA to normal. Therefore, strains treated with UV-induced mutagenesis should avoid exposure to visible light. The photoreactivation effect of UV radiation indicates that UV-induced mutagenesis is a complex process. The initial effect of UV radiation is to induce various types of metastable genetic material. The transition from metastable to stable mutant states requires a certain amount of time. Therefore, a time-cumulative UV-photoreactivation combined mutagenesis method, using UV radiation and visible light (bright white light) to repeatedly treat the mycelium, makes it difficult for the mutated sites in the mycelium to revert to their initial normal base sequences, thus increasing the mutation rate and expanding the mutation range of the mycelium.

[0063] The selection of a targeted screening medium for the rational selection of high-yielding streptomycin-producing strains tolerant to their own metabolites involves using a gradient concentration medium that allows streptomycin-producing bacteria to tolerate their own growth and metabolite production. This medium utilizes a concentrated filtrate of fermentation broth rich in streptomycin and its metabolites from the final stage of streptomycin production stagnation during the industrial-scale fermentation process. The concentrated broth, rich in streptomycin and other primary, secondary, and culture medium decomposition products generated during the fermentation process, serves as a major component of the screening medium. This creates a gradual increase in the concentration of metabolites on the screening plates, enabling targeted and rapid selection of high-yielding strains tolerant to their own metabolites. Mycelia mutated by the cumulative diminishing UV-photoreactivation mutagenesis method can be rapidly expressed and manifested on the rationally selected gradient substrate medium, significantly reducing workload.

[0064] The gradient concentration culture medium for kasugamycin-producing bacteria tolerating their own metabolites is prepared by placing a layer of screening medium on the bottom of a sterilized 90mm diameter double-dish petri dish, tilting it, and allowing the bottom screening medium to solidify. After the bottom screening medium is placed horizontally, a screening medium rich in the concentrated filtrate of the fermentation broth containing kasugamycin and other primary metabolites, secondary metabolites, and culture medium decomposition products produced during the fermentation and growth of kasugamycin-producing bacteria is added to the petri dish and allowed to solidify. This forms a screening medium containing various substances of kasugamycin and its own metabolites, with the concentration of screening substrate factors gradually increasing from low to high, for the rational selection of kasugamycin-producing bacteria.

[0065] The method for rationally selecting high-yielding streptavidin-producing strains tolerant to their own metabolites involves selecting mycelia with high streptavidin-producing capacity at the end of the rapid growth and accumulation phase of streptavidin production during the fermentation process of streptavidin-producing bacteria as the target starting strain; selecting high-unit streptavidin, large amounts of carbon sources, nitrogen sources, phosphates, precursor substances, and bioactive enzymes produced during primary and secondary metabolism in the fermentation broth at the end of the stagnant phase of streptavidin production as tolerance screening substrates; using a combined mutagenesis method of cumulatively decreasing ultraviolet light-photoreactivation over time as the mutagenic factor; and using culture dishes with a gradient concentration of streptavidin-representing metabolites from low to high as the screening tool to detect the target strains that have undergone mutagenesis.

[0066] The above-mentioned combined screening method was used to select high-yielding kasugamycin-producing strains resistant to their own metabolites. This method involved using mycelia or cells from the final stage of rapid growth and accumulation of kasugamycin production (a secondary metabolite) in the same batch of industrial-scale kasugamycin fermentation tanks as the target starting strain; using concentrated filtrate from the final stage of stagnant kasugamycin production as the screening substrate; employing a cumulative decreasing UV-photoreactivation combined mutagenesis as the mutagenesis method; and using culture dishes with progressively increasing concentrations of their own metabolites as the detection tool. A kasugamycin-producing strain was screened and, after stability verification, applied to a 40m... 3 The fermenter was used for large-scale industrial fermentation production. Under the same fermentation process and cycle conditions as the control strain, the fermentation level of the selected strain of streptomycin was increased by 52.5%, and the fermentation unit could reach 18050 μ / ml. This shows that the fermentation level of the streptomycin-producing strain obtained in this case has been greatly improved compared with the existing technology.

[0067] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0068] The reagents and raw materials used in this invention are all commercially available.

[0069] The positive and progressive effects of this invention are as follows:

[0070] When the mycelia or mycelia obtained by this invention are used in the selection of *Streptomyces lilacinus* strains resistant to their own metabolites, high-yielding strains of kasugamycin are screened. These high-yielding strains can significantly increase the fermentation production level of kasugamycin by more than 50% during industrial-scale production, reaching a maximum of over 18,000 μ / ml. This results in a large quantity of fully water-soluble kasugamycin technical powder products with a kasugamycin content of over 500,000 μ / g, greatly reducing the industrial-scale production cost of kasugamycin. This lays the foundation for the market demand for kasugamycin as a biopesticide and meets the expanded application needs in areas such as large-scale aerial spraying in green ecological agriculture.

[0071] Information on the preservation of biological materials

[0072] The *Streptomyces lavendulae* var. *hainanensis* UV-11 of this invention was deposited on August 31, 2021, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC). The depository address is: Strain Laboratory, Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Microbial Analysis and Testing Center), Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The accession number is GDMCC No. 61906, the culture name is UV-11, and the classification name is *Streptomyces sp.*. Attached Figure Description

[0073] Figure 1 The results are HPLC test results for the control strain UV-3.

[0074] Figure 2 The results are HPLC test results of the UV-11 strain of the present invention;

[0075] Figure 3 The structure of the screening culture medium of the present invention is illustrated. Detailed Implementation

[0076] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0077] The culture medium formulation and fermentation conditions involved in the fermentation are as follows:

[0078] Seed culture medium formula (components are by mass / volume percentage): glucose 1.0%, corn starch 1.5%, corn flour 1.0%, soybean flour 2.0%, sodium chloride 0.3%, ammonium chloride 0.3%, calcium carbonate 0.3%, adjusted to pH 6.6.

[0079] Fermentation medium formula (components are by mass / volume percentage): glucose 5.0%, corn starch 2.0%, corn flour 2.0%, soybean flour 4.0%, sodium chloride 0.6%, ammonium chloride 0.8%, calcium carbonate 0.7%, potassium dihydrogen phosphate 0.02%, magnesium sulfate 0.05%, adjusted to pH 6.6.

[0080] Fermentation conditions were as follows: fermentation temperature 26℃-35℃; air flow rate 0.5V / VM-1.5V / VM; fermentation tank stirring speed 50rpm-200rpm.

[0081] The formulations for the slant and plate culture media involved are as follows:

[0082] Slant and plate culture media (components by mass / volume percentage): soluble starch 1.05%, peptone 0.06%, magnesium sulfate 0.07%, sodium nitrate 0.25%, potassium chloride 0.05%, ferrous sulfate 0.02%, potassium dihydrogen phosphate 0.12%, agar 2.05%, adjusted to pH 6.6.

[0083] Example 1: A mutant strain (UV-3) of *Streptomyces lilacinus* Hainan variant (accession number CGMCC No. 1026) was used as the industrial production strain for the industrial fermentation production of streptomycin.

[0084] Strain UV-3 is a mutant strain of Streptomyces lilacinus Hainan variety obtained by the applicant through mutagenesis of Streptomyces lilacinus Hainan variety strain with accession number CGMCC No.1026, which has been applied to the industrial fermentation production of kasugamycin.

[0085] UV-3 at 40m 3 Industrial-scale fermentation was carried out in a fermenter (fermentation culture medium formula and fermentation culture conditions as described above, with a fermentation culture cycle of 5 days) and fermentation broth from different stages of the fermentation process was collected.

[0086] The above-mentioned industrial-scale fermentation broth samples were collected starting at "0" hour after inoculation of the fermenter, following aseptic sampling requirements, with samples collected every 12 hours. The specific aseptic sampling method was as follows: The sampling port of the bifidobacteria fermenter was sterilized with ≥0.3 MPa live steam for 30 minutes. Under flame protection, the steam valve at the sampling port was quickly closed, and the discharge valve was opened, allowing the fermentation broth to flow out. A sterilized glass triangular flask with a cotton plug was placed under flame protection. The cotton plug was quickly removed from the sampling port (while the removed plug remained under flame protection). The flask opening was aligned with the flowing fermentation broth to quickly collect the broth. The cotton plug was then quickly replaced, the discharge valve was closed, and the steam valve was opened to sterilize the sampling port for 10 minutes before closing it. The collected fermentation broth samples were immediately placed in a 4°C refrigerator for refrigeration after each collection until the batch was discharged from the industrial-scale fermentation tank. Simultaneously, after sampling, aseptic tests were immediately performed on the slant and broth, and mycelial morphology was observed under a microscope. Only fermentation broth samples that showed no contamination by other microorganisms during aseptic testing and microscopic observation could be used as samples to determine relevant parameters. The values ​​for the final stage of rapid growth accumulation and the final stage of stagnant growth are shown in Table 1. It should be noted that the biological potency growth rate v can be calculated using the following formula, where u1 refers to the biological potency at the previous time t1, u2 refers to the biological potency at the next time t2, and the interval between u1 and u2 can be limited according to the specific circumstances of each implementation method. In this implementation method, the interval between u1 and u2 is 12 hours.

[0087]

[0088] Table 1.

[0089]

[0090] It should be noted that the test methods mentioned in Table 1 were tested using the following methods:

[0091] 1.1 Determination of biological potency

[0092] The determination of the biopotency was performed using the antibiotic microbial assay method in the Chinese Pharmacopoeia (2015 edition). The reagents and solutions used were: phosphate buffer (pH 7.8); culture medium I; Bacillus subtilis suspension (Bacillus subtilis number: CMCC63501); volumetric flasks (100ml and 50ml).

[0093] 1.2 Determination of reducing sugars in microbial metabolism

[0094] The determination of reducing sugars in microbial metabolism was performed using the Fehling's reagent colorimetric method. The specific preparation method for Fehling's reagent is as follows:

[0095] 1. Preparation of Fehling's reagent A: Accurately weigh 120 g of copper sulfate hydrate (CuSO4·5H2O, analytical grade, content greater than 99.0%) and dissolve it completely in 2000 ml of distilled water.

[0096] 2. Preparation of Fehling's reagent B: Pour 2000 ml of distilled water into an open glass container. Accurately weigh 375 g of sodium potassium tartrate (C4H4KNaO6·4H2O, analytical grade, purity greater than 99.0%) and add it to the solution, stirring thoroughly until dissolved. Then accurately weigh 250 g of sodium hydroxide (analytical grade) and slowly add it to the solution in portions while stirring continuously.

[0097] 3. Preparation of Fehling's reagent C solution: Accurately weigh 300 g of potassium iodide (analytical grade, content greater than 98.0%), dissolve and dilute with distilled water to 1000 ml.

[0098] 4. Preparation of Fehling's reagent: Transfer Fehling's reagent solutions A, B, and C sequentially through a large funnel into a 10,000 ml brown reagent bottle, and shake carefully and vigorously until well mixed to prepare 5,000 ml of Fehling's reagent.

[0099] 5. Preparation of 0.1 mol / L sodium thiosulfate solution: Weigh 26 g of sodium thiosulfate, add 0.2 g of anhydrous sodium carbonate, dissolve in 1000 ml of water, slowly boil for 10 min, and cool. Filter after standing for two weeks. Standardize according to GB / T601-2002 after filtration.

[0100] Preparation of 6.1% starch indicator: Weigh 1 gram of starch, add a small amount of water to make a starch solution, add boiling distilled water to 100 ml, and boil for 2 minutes to complete the preparation.

[0101] Operating procedures

[0102] The predicted range of total residual sugar concentration is determined by comprehensively considering the grade of the microbial culture medium and the culture time. For culture medium filtrate or supernatant with a concentration exceeding 10 g / 100 ml, take 1 ml, dilute with 4 ml of distilled water, mix well, and then take 1 ml for measurement. For filtrate or supernatant with a total residual sugar concentration less than or equal to 10 g / 100 ml but greater than 5 g / 100 ml, take 0.5 ml for measurement. For filtrate or supernatant with a total sugar concentration less than or equal to 5 g / 100 ml, take 1 ml for measurement.

[0103] Accurately pipette 1 ml of filtrate or supernatant into a 150 ml Erlenmeyer flask, add 20 ml of Fehling's reagent directly, and heat evenly on a hot plate until boiling. Start timing after two minutes. Remove the Erlenmeyer flask and place it on a surgical tray containing cooling water to cool. The exhaust fan should be on throughout the procedure.

[0104] After cooling, accurately add 15 ml of 2 mol / L sulfuric acid solution, and quickly titrate with 0.0998 mol / L Na₂S₂SO₃ solution until a pale yellow color appears. Add approximately 1 ml of starch indicator during titration and continue titrating until the blue color disappears. Record the volume of sodium thiosulfate solution consumed: V 样 .

[0105] Blank experiment: Accurately measure 20 ml of Fehling's reagent into a 150 ml Erlenmeyer flask, without adding the test solution, and add 1 ml of distilled water to replace the sample. Follow the same procedure as for total sugar determination. The blank flask should be heated to boiling simultaneously with the sample flask to minimize random errors. After titration, record the volume of sodium thiosulfate solution consumed as V. 空 .

[0106] ΔV=V 空 -V 样 Look up the sugar value in the sugar table according to the ΔV value, and multiply the sugar value found by the dilution factor to get the total sugar or reducing sugar concentration of the sample, in g / 100ml.

[0107] 1.3 Determination of amino nitrogen in microbial metabolism

[0108] Reagent preparation

[0109] Preparation of 0.1% methyl red indicator: Accurately weigh 0.1g of methyl red into a 250ml beaker, add 100ml of 75% ethanol, and stir thoroughly with a clean glass rod until the methyl red is completely dissolved. Once the reagent is prepared, fill approximately 2 / 3 of a dropper bottle for use.

[0110] Preparation of 0.015 mol / L sulfuric acid solution: Accurately measure 75 ml of distilled water into a 1000 ml beaker. Accurately measure 2 mol / L sulfuric acid solution using a 100 ml graduated cylinder, and slowly add it to the beaker using a tilted glass rod, stirring constantly until homogeneous.

[0111] Preparation of 0.02858 mol / L sodium hydroxide solution: Dissolve 1.1432 g of sodium hydroxide (analytical grade) in distilled water and dilute to a volumetric flask of 1000 ml. Perform standardization.

[0112] Preparation of 18% neutral formaldehyde solution: Mix 50ml of 37%-40% formaldehyde with 50ml of distilled water to obtain an 18% formaldehyde solution.

[0113] Preparation of 1% phenolphthalein indicator: Accurately weigh 1g of phenolphthalein into a 250ml beaker, add 100ml of 75% alcohol, and stir thoroughly with a clean glass rod until the phenolphthalein is completely dissolved.

[0114] Operating procedures

[0115] Accurately pipette 2 ml of the culture medium filtrate or the supernatant after centrifugation into a 200 ml Erlenmeyer flask, and add 50 ml of distilled water.

[0116] Add 1 drop of 1% methyl red indicator and titrate with 0.015 mol / L sulfuric acid solution until faintly red.

[0117] Drop the solution of 0.02858 mol / L sodium hydroxide solution until it turns orange-yellow.

[0118] Add 2 ml of 18% neutral formaldehyde solution, shake gently until well mixed, and let stand for 10 minutes.

[0119] Add 2-8 drops of 1% phenolphthalein indicator, and titrate with 0.02858 mol / L sodium hydroxide solution until a faint red color appears. Record the volume of 0.02858 mol / L standard sodium hydroxide solution titrated in this step.

[0120] 1.4 Microbial concentration determination

[0121] First, shake the fermentation liquid well;

[0122] Weigh the centrifuge tube m1 using an electronic scale, pour about 10ml of fermentation broth into the centrifuge tube, and weigh the centrifuge tube m2 again; place it in a centrifuge and centrifuge at 3000r / min for 15 minutes.

[0123] Pour out the supernatant and weigh the remaining portion (m3).

[0124] Result: Bacterial concentration = (m3-m1) / (m2-m1)×100%.

[0125] 1.5 Microbial Total Phosphorus Detection

[0126] Total phosphorus was determined using an instrumental method.

[0127] The instrument used for total phosphorus was a multi-parameter water quality analyzer (5B-3B(V11)) manufactured by Beijing Lianhua Yongxing Technology Development Co., Ltd., and all reagents used were produced by the manufacturer.

[0128] Example 2: Preparation of the target starting strain

[0129] 1. Based on the determination results of the biopotency of the fermentation broth samples, the fermentation broth sample collected after 48 hours of fermentation in Example 1 (the middle and late stage of rapid growth and accumulation) was selected.

[0130] 2. Take out the Erlenmeyer flask containing the fermentation broth sample of kasugamycin that has not been contaminated by other microorganisms and store it in a 4℃ refrigerator. After the outer surface of the Erlenmeyer flask is disinfected by ultraviolet irradiation and the outer wall of the Erlenmeyer flask is wiped and disinfected by 75% alcohol swabs, it is transferred to the ultra-clean workbench in the sterile operating room for a completely sterile operation.

[0131] 3. In a sterile room, shake the above-mentioned kasugamycin fermentation broth evenly at room temperature, and take 100ml into a 250ml Erlenmeyer flask containing 20 glass beads (the Erlenmeyer flask and its contents have been sterilized). Place it on a shaker at 220rpm for 60 minutes at room temperature to fully separate and disperse the mycelium of the fermentation broth from the culture medium.

[0132] 4. In a sterile room under aseptic conditions, the fermentation broth, which has been thoroughly shaken to ensure complete separation and dispersion of mycelia from the culture medium, is filtered naturally at room temperature using a Buchner funnel with sterile cotton as the filter medium. The resulting filtrate of the target bacterial strain is stored at 4°C. Simultaneously, other microbial tests (referred to as sterility tests) are performed. Only filtrate with a negative sterility test result can be used as the starting strain filtrate.

[0133] Example 3 Preparation of tolerance substrate factor

[0134] 1. Based on the determination results of the biopotency of the fermentation broth samples, the fermentation broth sample collected after 120 hours of fermentation in Example 1 (the end of the growth stagnation stage) was selected.

[0135] 2. Take out the triangular flask containing the fermentation broth sample of streptomycin that has not been contaminated by other microorganisms and store it in a 4℃ refrigerator. After disinfecting the outer surface of the triangular flask and wiping the outer wall of the flask with 75% alcohol swabs, transfer it into the ultra-clean workbench of the sterile operating room for a completely sterile operation.

[0136] 3. Shake the above-mentioned kasugamycin fermentation broth evenly, and take 500ml into a 1000ml Erlenmeyer flask containing 50 glass beads (the Erlenmeyer flask and its contents have been sterilized). Place it on a shaker at 220rpm for 60min to allow the mycelium of the fermentation broth to be fully separated and dispersed from the culture medium of the fermentation broth.

[0137] 4. The sterile fermentation broth that has been thoroughly shaken to fully separate and disperse the mycelium from the culture medium is naturally filtered through a Buchner funnel using sterile cotton as the filter medium. The obtained filtrate is then filtered through a sterile bacterial funnel to obtain 400ml of the target filtrate with mycelium removed. Store at 4°C.

[0138] 5. In a sterile room, using aseptic techniques, 400 ml of the target filtrate obtained above (without mycelium) was placed in a sterilized rotary evaporator and concentrated under low-temperature reduced pressure at a temperature <45℃, a vacuum of -0.098 MPa, and a cooling water temperature of 5℃-7℃ until 80 ml remained. This yielded a sterile filtrate concentrate containing mycotoxin and other metabolites, but free of mycotoxin mycelium (this sterile filtrate concentrate is the concentrate containing the substrate factor). The data for this concentrate are shown in Table 2 below.

[0139] Table 2.

[0140]

[0141] Example 4: Preparation of screening culture medium

[0142] 1. Preparation of plate culture medium for concentrated solution without target filtrate: Prepare the culture medium according to the above-mentioned "slant and plate culture medium formula", sterilize the culture medium by steam heat at 121°C for 30 minutes, and store it in a water bath at 48°C-50°C for later use.

[0143] 2. Preparation of plate culture medium containing the concentrated target filtrate: The amount of concentrated target filtrate used in Example 3 was calculated as 3.0% based on a kasugamycin biopotency of 30,000 μ / ml; other components were the same as described in the "Slant and Plate Culture Medium Formulation" above. The culture medium was sterilized by steam heat at 121°C for 30 minutes, cooled, and then stored in a water bath at 48°C-50°C until use.

[0144] The specific steps are as follows:

[0145] 2.1. Based on the target filtrate sterile concentrate of Example 3 having a biopotency of 53286 μ / ml, the volume of the target filtrate sterile concentrate required for a 3% kasugamycin content is 56.6 ml. 56.6 ml of this sterile concentrate should be taken for aseptic operation.

[0146] 2.2. Preparation of other components of the target filtrate concentrate plate culture medium: A total of 43.4 ml of other culture medium components, excluding the target filtrate sterile concentrate, were prepared. The culture medium was weighed according to the aforementioned "Slant and Plate Culture Medium Formulas," with a total culture medium volume of 100 ml. This 43.3 ml of culture medium was sterilized by steam heat sterilization at 121℃ for 30 min, then cooled to 48℃-50℃ and stored in a 48℃-50℃ water bath for later use.

[0147] 2.3. Take 56.6 ml of the aseptic concentrate of the target filtrate with a biopotency of 53286 μ / ml as described above, heat it to 45°C aseptically, and store it in a 45°C water bath for later use. The shorter the 45°C holding time, the better, and it should not exceed 10 minutes, as this will reduce the inactivation of the bioactivity of the self-metabolites in the concentrate.

[0148] 2.4. Quickly mix 43.4 ml of culture medium (excluding the sterile concentrate of the target filtrate, which has been sterilized and stored in a water bath at 48℃-50℃) with 56.6 ml of the sterile concentrate of the target filtrate, which has been stored in a water bath at 45℃, to obtain a plate culture medium containing 3% streptomycin and other metabolites but without streptomycin cells. After the culture medium containing 3% streptomycin and other metabolites is mixed evenly, immediately prepare gradient concentration culture media.

[0149] 3. The preparation of gradient concentration culture media is as follows:

[0150] 3.1. Under aseptic conditions in a sterile room, take 10 ml of agar plate culture medium stored at 48℃-50℃ in a water bath and place it in a 90 mm petri dish. Quickly lean the petri dish against a horizontal bar so that the bottom of the petri dish at the higher position of the bar just touches the culture medium, forming a petri dish with a thinner top and thicker bottom. After the culture medium solidifies, place the petri dish flat. The petri dish will form a slant of culture medium that gradually thickens from one end to the other. Mark the back of the petri dish with an arrow indicating the thinnest part of the culture medium, with the "↑" arrow head indicating the thinnest part of the culture medium.

[0151] 3.2. Under aseptic conditions in a sterile room, after marking the thinnest part of the plate culture medium with the arrows (as indicated by the arrows), place the plate culture medium with solid slant culture medium horizontally. Take 10 ml of the freshly prepared culture medium containing 3% kasugamycin and other self-metabolites, and quickly place it into the plate culture medium with solid slant culture medium. Shake well and spread horizontally. Let the culture medium containing kasugamycin and other self-metabolites solidify to obtain a plate culture medium with a gradient concentration of kasugamycin and other self-metabolites. The content of kasugamycin and other self-metabolites in the plate culture medium gradually increases in the direction of the arrows. The content of kasugamycin at the head of the arrow "↑" is 3%. Similarly, the concentration of other self-metabolites in the culture medium is also the highest at the head of the arrow "↑".

[0152] 3.3. Under aseptic conditions in a sterile room, prepare six culture dishes containing gradient concentrations of their own metabolites, such as streptomycin, according to the method described above. The streptomycin content at the tip of the arrow at the bottom of the culture dish is 3%, and similarly, the concentrations of other endogenous products in the culture medium are highest at the tip of the arrow "↑".

[0153] The resulting gradient concentration culture medium is the screening medium used for rational screening. The structure of this screening medium is described in [reference needed]. Figure 3 .

[0154] like Figure 3 As shown, the side where the arrow "↑" is located is 0mm, and the concentration of the self-metabolite gradient gradually increases along the direction of the arrow. Figure 3 Plate I shows a plate culture medium containing the target filtrate but not prepared as a concentrate. Plate II shows a plate culture medium containing the target filtrate but prepared as a concentrate.

[0155] Example 5: Preparation of a composite mutagenic solution for target bacterial cell filtrate

[0156] 1. Take out the bacterial cell filtrate of the target starting strain obtained in Example 2 and stored in a refrigerator at 4°C, and place it together with the culture dish containing the gradient concentration of its own metabolites such as streptomycin prepared in Example 4 in a sterile dark room with a red light.

[0157] 2. Cumulative decreasing ultraviolet-photoreactivation complex mutagenesis:

[0158] 2.1. In a sterile darkroom equipped with a red light, a 30W fluorescent lamp and a 30W wavelength lamp will be installed. Both the fluorescent lamp and the ultraviolet lamp in the mutation chamber were turned on simultaneously for preheating and stabilization for 15 minutes, and then the ultraviolet lamp and the fluorescent lamp were turned off (the mutation chamber was covered with a red and black cloth).

[0159] 2.2. Take 10 ml of the target strain cell filtrate into a 90 mm double-dish sterile culture dish. In a sterile dark room with a red light, remove the top cover of the culture dish and place it on a rotating plate 20 cm below the two lamps in a mutagenesis chamber equipped with a 30 W fluorescent lamp and a 30 W ultraviolet lamp. Rotate it slowly.

[0160] 2.3. Perform five consecutive ultraviolet-photoreactivation mutagenesis cycles:

[0161] 2.3.1. First, turn on the ultraviolet lamp for 30 seconds, then turn it off for 20 seconds; turn on the fluorescent lamp for 20 seconds; turn it off for 20 seconds.

[0162] 2.3.2. Then turn on the UV lamp for 25 seconds, and then turn off the UV lamp for 20 seconds; turn on the fluorescent lamp for 20 seconds; turn off the fluorescent lamp for 20 seconds.

[0163] 2.3.3. Then turn on the UV lamp for 20 seconds, then turn off the UV lamp for 10 seconds; turn on the fluorescent lamp for 10 seconds; turn off the fluorescent lamp for 10 seconds.

[0164] 2.3.4. Then turn on the UV lamp for 15 seconds, then turn off the UV lamp for 10 seconds; turn on the fluorescent lamp for 10 seconds; turn off the fluorescent lamp for 10 seconds.

[0165] 2.3.5. Then turn on the UV lamp for 10 seconds, then turn off the UV lamp for 5 seconds; turn on the fluorescent lamp for 5 seconds; turn off the fluorescent lamp for 5 seconds.

[0166] 2.4. After five dose-decreasing combined mutagenesis cycles of ultraviolet-photoreactivation, a combined mutagenesis solution of the target starting strain cell filtrate was obtained.

[0167] Example 6 Rational Screening

[0168] 1. The composite mutagenesis solution of the target starting strain cell filtrate obtained through the above-mentioned dose-decreasing composite mutagenesis of 5 cycles of ultraviolet-photoreactivation was spread, cultured, and screened, specifically including the following steps:

[0169] 1.1. Spreading: Place six culture dishes containing gradient concentrations of kasugamycin and other self-metabolites in a red-light sterile dark room. Under aseptic conditions, perform the same operation on five culture dishes: take 1 ml of the composite mutagenic solution of the target starting strain cell filtrate obtained in Example 5 and spread it evenly on a 90 mm double-dish culture dish containing gradient concentrations of kasugamycin and other self-metabolites; spread the remaining target starting strain cell filtrate that has not been subjected to ultraviolet-photoreactivation mutagenesis on the remaining culture dish as a control culture dish.

[0170] 1.2. Incubation: In a sterile dark room, adjust the temperature and humidity to be constant: temperature 28.5℃-29.5℃, relative humidity 55%-65%; cover the control culture dish and 5 culture dishes coated with the same bacterial solution after compound mutagenesis, with the lids facing up, and incubate for one day. Then, invert the entire set of culture dishes with the lids facing down and incubate for 7-10 days to avoid the culture medium drying out.

[0171] 1.3. Selecting single-colony strains tolerant to high concentrations of their own metabolites:

[0172] 1.3.1. On the control culture dish, sparse colonies grew at the end of the arrow. The colonies became sparser as they extended in the direction of the arrow, with only one colony at a maximum of 22 mm. All colonies were pink. According to the streptomycin gradient concentration, the streptomycin concentration tolerated by this 22 mm colony was approximately 7300 μ / ml. This colony was labeled d22.

[0173] 1.3.2. Sparse colonies grew at the ends of the arrows on the five culture dishes used in the experiment. Colonies almost disappeared as the distance extended towards the arrows, but three culture dishes had one colony each at a maximum extension of 52mm, 58mm, and 63mm, respectively. Based on the streptomycin gradient concentration, the streptomycin concentrations tolerated by these 52mm, 58mm, and 63mm colonies were approximately 17000μ / ml, 19000μ / ml, and 21000μ / ml, respectively. The 52mm and 63mm colonies were small and dense, appearing as flat, dome-shaped colonies covered with pink spores. The 58mm colony was also small and dense, appearing as a dome-shaped colony with a higher central bulge, covered with spores, but the spores were mostly white with less pronounced pink. The 52mm, 58mm, and 63mm colonies on these three culture dishes were labeled n52, n58, and n63.

[0174] 1.3.3. Select colonies of d22, n52, n58, and n63 for inoculation slant culture. Incubate at 29℃ for 7 days. The d22 slant has a thicker, pinkish bacterial growth, and the back of the slant is dark pink. The n52 and n63 slants have a thick, pinkish bacterial growth, and the back of the slant is dark pink. The n58 slant has a thick, grayish-white bacterial growth with a slight pale pink tinge, and the back of the slant is pale pink.

[0175] 1.3.4 Fermentation Experiment: The screened resistant strains n52, n58, and n63, which are resistant to streptomycin and its metabolites, were subjected to a 5L fermenter fermentation experiment together with the control strain d22 and the strain UV-3 used in industrial production (fermentation culture was conducted according to the aforementioned "fermentation culture medium formula and fermentation culture conditions", with a fermentation cycle of 7 days). The experiment was repeated 3 times to examine the streptomycin fermentation level. The relevant results are shown in Table 3 below:

[0176] Table 3.

[0177]

[0178] As shown in Table 3, the streptomycin titers obtained by n52, n58, and n63 were all significantly higher than the 11389 μ / ml of the starting strain UV-3, indicating that the screening method of this invention can obtain a large number of high-yielding strains. The strain with the highest yield, n58, was preserved as Streptomyces lilacinus Hainan varietal UV-11, with the preservation number GDMCC No. 61906.

[0179] Example 7: Strain preservation and stability verification

[0180] 1. After sealing the five slant surfaces of UV-3, d22, n52, n58, and n63 involved in Example 6 above with sterile liquid paraffin, they were frozen and stored at -80°C for 3 months.

[0181] 2. Take out the UV-3, d22, n52, n58, and n63 slant strains that were sealed and frozen in sterile liquid paraffin, perform single colony isolation on plates, and observe the growth of single colonies after 7-10 days. The observation results are shown in Table 4 below:

[0182] Table 4.

[0183]

[0184] 3. Select characteristic single colonies from single-colony isolation plates, transfer the slant agar to a sterile room at 28.5℃-29.5℃ and 55%-65% relative humidity, and incubate for 7-10 days. Further observe the morphology of the bacterial growth on the slant: UV-3 slant has a thicker, pinkish bacterial growth; d22 slant has a thicker, pinkish bacterial growth, with a dark pink underside; n52 slant has a thicker, pinkish bacterial growth, with a dark pink underside; n58 slant has a thicker, pinkish bacterial growth, with a dark pink underside; n63 slant has a thicker, pinkish bacterial growth, with a dark pink underside.

[0185] 4. Fermentation test: The above-mentioned liquid paraffin slant strains, which were frozen at -80℃, were isolated by single colony isolation and then transferred to slant strains UV-3, d22, n52, n58, and n63 for 5L fermentation test (fermentation culture medium formula and fermentation culture conditions as described above, fermentation culture cycle 7 days), and the test was repeated 3 times.

[0186] The fermentation level of streptomycin was investigated, and the results are shown in Table 5 below:

[0187] Table 5.

[0188] Name\Group 1 (μ / ml) 2 (μ / ml) 3 (μ / ml) Average (μ / ml) increase(%) UV-3 10287 11032 11926 11081 100 d22 11389 10426 11386 11067 99.87 n52 14543 16235 15768 15515 140 n58 16899 17365 18256 17506 158 n63 15436 15228 15698 15453 139

[0189] As shown in Table 5, among the three strains (n52, n58, and n63) that tolerated higher doses of their own metabolites such as kasugamycin, the kasugamycin fermentation level was not only higher than that of the existing strains, but also significantly higher than that of the control group (UV-3 and d22), especially n58, which showed the highest increase of more than 55%.

[0190] Example 8: Validation of Industrial-Scale Fermentation Production

[0191] Applying UV-3 and UV-11 to 40m 3 The fermenter was used for industrial-scale fermentation production verification (fermentation culture medium formulation and fermentation conditions as described above, fermentation cycle 5 days), and the parameters of the UV-11 fermentation broth were obtained. The parameters of the UV-11 fermentation broth are as follows:

[0192] Total sugar: 1.306 g / 100 ml, reducing sugar: 0.6359 g / 100 ml, amino nitrogen: 88.58 mg / 100 ml, total phosphorus: 10.25 mg / L, bacterial concentration: 27.2%, pH: 6.95, bioavailability: 18050 μ / ml.

[0193] Under the same fermentation conditions and cycle, the kasugamycin fermentation level of the UV-3 strain in the tank batch was only 11936 μ / ml, while that of the UV-11 strain reached 18050 μ / ml. This means that the industrial-scale fermentation level of the UV-11 strain was 52.5% higher than that of the control strain UV-3. Further analysis of the fermentation broth components in the UV-11 fermentation tank broth using high-performance liquid chromatography (HPLC) is shown in the attached table. Figure 1 and Figure 2 .in, Figure 1 The results are HPLC test results for the control strain UV-3. Figure 2 The results are from the HPLC test of the UV-11 strain.

[0194] Combination Figure 1 and Figure 2 It can be seen that the retention times of each component in the fermentation broth of UV-11 are within the same range as those of the control strain UV-3. The specific data are shown in Table 6 below.

[0195] Table 6.

[0196]

[0197] It should be noted that the conditions used in HPLC are as follows:

[0198] Model: Waters E2695

[0199] Mobile phase: Gradient elution conditions are shown in Table 7 below:

[0200] Table 7.

[0201] Time / min Acetonitrile / % (V / V) Mixed salt solution / % (v / v) 0 15 85 2 15 85 10 22 78 20 22 78 22 15 85 30 15 85

[0202] Flow rate: 1.0 mL / min.

[0203] Column temperature: 30℃±2℃.

[0204] Detection wavelength: 200nm.

[0205] Injection volume: 10 μL.

[0206] Pillar: Water SymmetryShield TM RP18 5μm

[0207] Example 9: Preparation of a product containing kasugamycin

[0208] 1. Fermentation broth acidification: The fermentation broth with a biopotency of 18050 μ / ml obtained in Example 8 above was adjusted to pH 3.5 to form an acidified solution;

[0209] 2. Solid-liquid separation of acidified fermentation broth: The acidified fermentation broth is passed through a ceramic membrane with a pore size of 0.20 μm for solid-liquid separation to obtain permeate. This permeate is then passed through a ceramic membrane with a pore size of 0.05 μm for liquid-solid separation to obtain secondary permeate of fermentation broth.

[0210] 3. Concentration of secondary permeate from fermentation broth: The secondary permeate is concentrated through a nanofiltration membrane with a molecular weight of 400 Da to obtain a concentrated solution;

[0211] 4. Spray drying: The concentrate was directly spray dried to obtain the fully water-soluble raw material powder of kasugamycin. The biopotency of the raw material powder was determined to be 510025 μ / g.

[0212] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, any changes or modifications made in accordance with the claims and specification of the present invention should fall within the scope of the present invention.

Claims

1. A mycelium or a bacterial cell, characterized in that: The mycelium or bacterial cell is the mycelium or bacterial cell obtained from the fermentation liquid at the end of the rapid growth and accumulation stage of secondary metabolite production in the fermentation production process of the strain; The strain is Streptomyces lavendulae var. hainanensis, and its deposit number is GDMCC No.61906.

2. The mycelium or bacterial cell according to claim 1, characterized in that: The fermentation satisfies one or more of the following conditions: The fermentation temperature is 26℃-35℃; Air ventilation volume 0.5V / VM-1.5V / VM; The stirring speed of the fermentation tank is 50rpm-200rpm; The fermentation culture cycle is 3 to 10 days; The fermentation medium formula used in the fermentation includes: 4.0%-15.0% glucose, 1.0%-6.0% corn starch, 0.5%-7.5% corn flour, 3.0%-12.0% soybean flour, 0.2%-1.6% sodium chloride, 0.1%-1.8% ammonium chloride, 0.2%-1.6% calcium carbonate, 0.01%-0.13% potassium dihydrogen phosphate, 0.01%-0.16% magnesium sulfate, pH 6.0-7.0, wherein the % is the mass volume percentage.

3. Use of the mycelium or thalline as claimed in claim 1 or 2 in breeding strains resistant to their own metabolites.

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