A method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance

Abstract

This application relates to the technical field of microbial breeding, specifically disclosing a method for targeted screening of high-yielding chlortetracycline strains based on streptomycin resistance. The method includes the following steps: taking mycelium as starting material and subjecting it to mutagenesis to obtain a mutagenic bacterial solution; spreading the mutagenic bacterial solution onto agar plates containing different concentrations of streptomycin, culturing, counting colonies, and calculating the survival rate of the mutagenic bacterial solution at different streptomycin concentrations based on the colony count; determining the streptomycin resistance screening pressure based on the survival rate results from the statistical step; diluting the mutagenic bacterial solution and spreading it onto a screening plate containing the streptomycin resistance screening pressure, culturing, and picking streptomycin-resistant single colonies; subjecting the streptomycin-resistant single colonies to shake-flask fermentation to obtain a fermentation broth; screening: using high-performance liquid chromatography (HPLC) for screening to obtain high-yielding chlortetracycline strains.

Description

Technical Field

[0001] This application relates to the technical field of microbial breeding, and in particular to a method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance. Background Technology

[0002] Chlortetracycline, also known as tetracycline, is a tetracycline antibiotic produced by the fermentation of *Streptomyces aureus*. It is widely used in feed additives and related fermentation industries. Chlortetracycline is a typical secondary metabolite, and its biosynthesis is influenced by multiple genes, regulatory factors, and fermentation metabolic states. The potency of the producing strain directly determines fermentation efficiency and production costs. Therefore, obtaining high-yielding and stable *Streptomyces aureus* strains is a key step in improving the industrial production level of chlortetracycline.

[0003] Existing methods for breeding high-yielding chlortetracycline strains typically involve constructing a mutant library using methods such as UV mutagenesis, ARTP mutagenesis, chemical mutagenesis, and combined mutagenesis. Screening is then performed using techniques such as plate isolation, well plate culture, shake-flask fermentation, colorimetric detection, or high-performance liquid chromatography (HPLC). While these methods can yield a certain number of mutant strains, the mutagenesis process is highly random, resulting in a high proportion of negative mutants, low-yielding strains, and ineffective mutants in the mutant population. This leads to a large workload for subsequent rescreening, a long screening cycle, and a low positive mutation rate.

[0004] Other techniques have attempted to improve the screening efficiency of high-yielding chlortetracycline strains through flow cytometry, high-throughput plate staining, product tolerance screening, or salt stress screening. However, flow cytometry is mainly used to improve the sorting efficiency of viable spores, staining or plate detection is mainly used to increase detection throughput, and product tolerance or salt stress screening is more about selecting tolerant or adaptive strains. These methods do not address the issue of ribosome-mediated regulation of secondary metabolism to selectively enrich high-yielding chlortetracycline mutants of *Streptomyces aureus*. Therefore, there is an urgent need to establish a targeted screening method suitable for chlortetracycline-producing strains of *Streptomyces aureus*, thereby increasing the probability of obtaining high-yielding chlortetracycline strains, reducing the workload of subsequent shake-flask rescreening, and obtaining high-yielding strains with good genetic stability. Summary of the Invention

[0005] To increase the probability of obtaining high-yield chlortetracycline strains, this application provides a method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance.

[0006] This application provides a method for targeted screening of high-yield chlortetracycline-producing strains based on streptomycin resistance, which adopts the following technical solution: A method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance includes the following steps: Preparation of mutagenic bacterial solution: Take the spores or mycelium from the fermentation broth of Streptomyces aureus as the starting material, and perform mutagenic treatment on the starting material to obtain the mutagenic bacterial solution; Statistics: The mutant bacterial solution was spread on agar plates containing different concentrations of streptomycin, and the colony count was counted after incubation. The survival rate of the mutant bacterial solution at different streptomycin concentrations was calculated based on the colony count. Determine the screening pressure: Based on the survival rate results of the statistical steps, determine the streptomycin concentration that will result in a survival rate of 5%-15% for the mutant bacterial culture as the streptomycin resistance screening pressure; Culture and selection: Dilute the mutant bacterial solution and spread it on a selection plate containing the streptomycin resistance selection pressure, culture and select streptomycin resistant single colonies; Fermentation: The streptomycin-resistant single colony was subjected to shake-flask fermentation to obtain a fermentation broth; Screening: The chlortetracycline titer in the fermentation broth was detected by high performance liquid chromatography, and high-yielding chlortetracycline strains with higher chlortetracycline titers than the starting strain were screened.

[0007] By adopting the above technical solution, and adding a streptomycin resistance-oriented screening step after mutagenesis, the method no longer relies entirely on blind screening after random mutagenesis. Instead, it utilizes the correlation between streptomycin resistance and streptomycin ribosome regulation and secondary metabolic activation to pre-enrich mutant strains with high chlortetracycline production potential. At the same time, by limiting the screening pressure to a streptomycin concentration that results in a 5%-15% survival rate of the mutagenic bacterial solution, a certain number of effective mutant strains can be retained while eliminating a large number of low-yield, ineffective, or negative mutant strains. This increases the probability of obtaining high-yield strains in subsequent shake-flask rescreening, reduces the workload of rescreening, and improves the screening efficiency and positive mutation rate of high chlortetracycline-producing strains.

[0008] In one specific implementation, in the step of preparing the mutagenic bacterial solution, the mutagenesis treatment is ARTP mutagenesis or ultraviolet mutagenesis; the ARTP mutagenesis uses a suspension of Streptomyces aureus spores or mycelium as the treatment object, and the ultraviolet mutagenesis uses a suspension of Streptomyces aureus spores or mycelium as the treatment object.

[0009] By adopting the above technical solutions, ARTP mutagenesis can produce high frequency of genetic variations in Streptomyces aureus under normal pressure and room temperature conditions, while ultraviolet mutagenesis has the characteristics of simple operation, low cost and suitability for conventional microbial breeding. Both can be used to expand the genetic diversity of Streptomyces aureus mutant populations.

[0010] In one specific implementation, the different concentrations of streptomycin in the statistical step include 0 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 7.5 μg / mL, and 10.0 μg / mL.

[0011] In one specific implementation, in the step of determining the screening pressure, the streptomycin resistance screening pressure is the streptomycin concentration that enables the survival rate of the mutant bacterial culture to be 8%-12%; the streptomycin resistance screening pressure is 1.0 μg / mL.

[0012] By adopting the above technical solution, the streptomycin screening pressure is further limited to a streptomycin concentration that results in a survival rate of 8%-12% for the mutant bacterial culture. This screening intensity can effectively eliminate most of the mutant strains that are sensitive to streptomycin and whose metabolic regulation has not undergone beneficial changes, while also preventing the number of surviving strains from being too small to affect subsequent screening. Preferably, 1.0 μg / mL streptomycin is used as the screening pressure, which can make the screening conditions more specific, facilitate repeated implementation, and is conducive to enriching mutant strains that are resistant to streptomycin and have high chlortetracycline titers.

[0013] In one specific implementation, in the culture and selection step, the screening plate includes flour, agar powder, and streptomycin; wherein the mass percentage of flour in the screening plate is 2.8%, the mass percentage of agar powder is 1.7%, the concentration of streptomycin is 1.0 μg / mL, and the volume of each culture medium is 25 mL.

[0014] By adopting the above technical solutions, flour can provide a relatively mild and slow-release carbon source and nutrients for the growth of Streptomyces aureus, which is conducive to the recovery and growth of damaged spores or mycelia after mutagenesis; agar powder provides a stable solid culture medium structure, which is conducive to the isolation of single colonies; streptomycin serves as a directional screening pressure to screen for streptomycin-resistant mutant strains; by limiting the culture medium components and the volume of culture medium per plate, the batch consistency of screening plates can be improved, making the screening pressure of streptomycin in the plates more stable, thereby improving the accuracy and repeatability of the screening results of resistant single colonies.

[0015] In one specific implementation, during the culture and selection step, the culture temperature is 29-34℃; after the culture has progressed to the point where separable single colonies are formed on the screening plate, streptomycin-resistant single colonies are selected for purification culture, and then the purified streptomycin-resistant single colonies are used for shake-flask fermentation for re-screening.

[0016] By adopting the above technical solution, 29-34℃ is suitable for the growth and single colony formation of Streptomyces aureus on plates. This avoids slow colony growth due to excessively low temperatures, and also avoids difficulties in the recovery of mutagenic strains or abnormal colony morphology due to excessively high temperatures. Purification culture of streptomycin-resistant single colonies can eliminate interference from mixed colonies or mixed growth of multiple strains, ensuring that the strains used for subsequent shake-flask rescreening are of a single source and have a relatively stable genetic background, thereby improving the reliability of chlortetracycline titer test results.

[0017] In one specific implementation, the fermentation culture medium used in the shake-flask fermentation step comprises the following components at the following concentrations: corn starch 60–90 g / L, peanut meal powder 15–20 g / L, soybean meal powder 10–15 g / L, corn steep liquor powder 12–16 g / L, yeast powder 6–9 g / L, ammonium sulfate 5–8 g / L, CaCO3 6–8 g / L, NaCl 2–3 g / L, CaCl2 1–6 g / L, MgSO4·7H2O 0.3–1.6 g / L, KH2PO4 0.2–0.5 g / L, amylase 0.1–0.2 g / L, and defoamer 0.1–0.3 g / L; the pH of the fermentation culture medium is 6.6–7.2.

[0018] By adopting the above technical solution, the culture medium can provide a stable carbon source, nitrogen source, inorganic salts and buffer system for the fermentation of Streptomyces aureus to produce chlortetracycline.

[0019] In one specific implementation, in the fermentation step, the liquid volume for the shake flask fermentation is 25 mL in a 250 mL Erlenmeyer flask, the fermentation temperature is 29–31°C, and the shaker speed is 100–150 rpm.

[0020] By adopting the above technical solution, 29-31℃ is suitable for the accumulation of fermentation products of Streptomyces aureus; a shaking speed of 100-150 rpm can take into account cell growth, dissolved oxygen supply and shear intensity, and avoid the impact of insufficient dissolved oxygen or excessive shear on the yield of chlortetracycline. This condition is conducive to improving the stability and comparability of the shake flask screening results.

[0021] In one specific implementation scheme, the detection conditions of the high-performance liquid chromatography (HPLC) in the screening step include: a C8 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a packing particle size of 5 μm; a mobile phase of a mixture of 0.01 mol / L oxalic acid, acetonitrile, and methanol, wherein the volume ratio of 0.01 mol / L oxalic acid, acetonitrile, and methanol is 71:16:13; a flow rate of 1.2 mL / min; an ultraviolet detector; a detection wavelength of 360 nm; a column temperature of 30 °C; and an injection volume of 20 μL.

[0022] By adopting the above technical solution, this detection method can improve the accuracy of comparing the titers of chlortetracycline in different mutant strains, and provide a reliable basis for screening high-yield strains.

[0023] In one specific feasible implementation, after the obtained high-yield chlortetracycline strain is continuously passaged for 4 generations, the chlortetracycline potency of each generation in shake-flask fermentation is maintained at no less than 98% relative to the chlortetracycline potency of the first generation in shake-flask fermentation.

[0024] By adopting the above technical solution, it can be seen that the strains obtained by screening are not just random high-yield strains or short-term metabolic fluctuations, but have relatively stable genetic and fermentation production performance; after continuous subculturing, they still maintain a high chlortetracycline titer, indicating that the screening method can not only improve the acquisition rate of high-yield strains, but also obtain stable production strains suitable for subsequent industrial scale-up and long-term preservation applications.

[0025] In summary, this application includes at least one of the following beneficial technical effects: The method in this application adds a streptomycin resistance-directed screening step after mutagenesis, no longer relying entirely on blind screening after random mutagenesis. Instead, it utilizes the correlation between streptomycin resistance and streptomycin ribosome regulation and secondary metabolic activation to pre-enrich mutant strains with high chlortetracycline production potential. At the same time, by limiting the screening pressure to a streptomycin concentration that results in a 5%-15% survival rate of the mutagenic bacterial culture, it is possible to exclude a large number of low-yield, ineffective, or negative mutant strains while retaining a certain number of effective mutant strains. This increases the probability of obtaining high-yield strains in subsequent shake-flask rescreening, reduces the workload of rescreening, and improves the screening efficiency and positive mutation rate of high chlortetracycline-producing strains. The method in this application provides a relatively mild and slow-release carbon source and nutrients for the growth of *Streptomyces aureus*, which is beneficial for the recovery of damaged spores or mycelia after mutagenesis; agar powder provides a stable solid culture medium structure, which is beneficial for the isolation of single colonies; streptomycin acts as a directional screening pressure to screen for streptomycin-resistant mutants; by limiting the culture medium components and the volume of culture medium per plate, the batch-to-batch consistency of screening plates can be improved, making the screening pressure of streptomycin in the plates more stable, thereby improving the accuracy and repeatability of the screening results for resistant single colonies. The method in this application uses a fermentation temperature of 29–31°C, which is suitable for the accumulation of fermentation products of Streptomyces aureus; and a shaking speed of 100–150 rpm, which can balance cell growth, dissolved oxygen supply and shear intensity, and avoid the impact of insufficient dissolved oxygen or excessive shear on the yield of chlortetracycline. This condition is conducive to improving the stability and comparability of the shake flask screening results. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the embodiments.

[0027] All raw materials used in the embodiments are commercially available. The defoamer includes, but is not limited to, silicone oil or defoaming agents; defoaming agents are preferred in this application. The *Streptomyces aureus* strain is designated DSM 40127. Preparation Example

[0028] Preparation Example 1 Preparation Example 1 provides a method for preparing fermentation broth of Streptomyces aureus, comprising the following steps: The *Streptomyces aureus* strain was activated and cultured, then transferred to a seed culture medium to obtain a seed solution. This seed solution was inoculated into a fermentation medium at an inoculation rate of 17 v / v%, and fermented at 30°C. Samples were taken between 30 and 50 hours of fermentation to obtain the fermentation broth of *Streptomyces aureus*. The seed culture medium contained the following components: corn starch 38 g / L, peanut meal 17 g / L, soybean meal 14 g / L, yeast powder 10.5 g / L, ammonium sulfate 4.5 g / L, CaCO3 4 g / L, NaCl 1.2 g / L, KH2PO4 0.3 g / L, MgSO4·7H2O 0.3 g / L, soybean oil 5 g / L, and pH 7.0. Example

[0029] Example 1 Example 1 provides a method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance, comprising the following steps: Preparation of mutagenic bacterial solution: Mycelia from the fermentation broth of Streptomyces aureus in Preparation Example 1 were used as starting material. The starting material was subjected to ARTP mutagenesis treatment to obtain mutagenic bacterial solution. The ARTP mutagenesis conditions were: helium as working gas, treatment distance 2 mm, gas flow rate 10 SLM, radio frequency power 120 W, and treatment time 15 s. Statistics: The mutant bacterial suspension was serially diluted and spread onto agar plates containing different concentrations of streptomycin. After incubation, the number of colonies was counted, and the survival rate and lethality rate at each streptomycin concentration were calculated. The streptomycin concentration gradient and test results are shown in Table 1. Table 1. Effect of streptomycin concentration on the survival rate of mutant bacterial cultures

[0030] Determine the screening pressure: Based on the survival rate results of the statistical steps, determine the streptomycin concentration that results in a 10% survival rate of the mutant bacterial culture as the streptomycin resistance screening pressure; Culture and selection: The mutagenic bacterial solution was diluted and spread onto screening plates containing streptomycin resistance screening pressure. The plates were incubated at 32°C until isolated single colonies formed on the screening plates. Streptomycin-resistant single colonies were then picked and purified. The purified streptomycin-resistant single colonies were then used for shake-flask fermentation and re-screening. The screening plates consisted of flour, agar powder, and streptomycin. The mass percentage of flour in the screening plates was 2.8%, the mass percentage of agar powder was 1.7%, the concentration of streptomycin was 1.0 μg / mL, and the volume of each culture medium was 25 mL. Fermentation: Streptomycin-resistant single colonies were subjected to shake-flask fermentation to obtain a fermentation broth. The fermentation medium used for shake-flask fermentation included the following components at the following concentrations: corn starch 75 g / L, peanut meal powder 18 g / L, soybean meal powder 12 g / L, corn steep liquor powder 14 g / L, yeast powder 7 g / L, ammonium sulfate 6 g / L, CaCO3 7 g / L, NaCl 2.5 g / L, CaCl2 3 g / L, MgSO4·7H2O 1 g / L, KH2PO4 0.3 g / L, amylase 0.15 g / L, and antifoaming agent 0.2 g / L. The pH of the fermentation medium was 6.8. The volume of liquid for shake-flask fermentation was 25 mL in a 250 mL Erlenmeyer flask, the fermentation temperature was 30℃, and the shaking speed was 120 rpm. Screening: After fermentation, the fermentation broth was taken, diluted, and centrifuged. The chlortetracycline titer was then determined using high-performance liquid chromatography (HPLC). High-yielding chlortetracycline strains with higher titers than the starting strain were screened. The detection conditions were as follows: a C8 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a packing particle size of 5 μm; a mobile phase of a mixture of 0.01 mol / L oxalic acid, acetonitrile, and methanol in a volume ratio of 71:16:13; a flow rate of 1.2 mL / min; an ultraviolet detector; a detection wavelength of 360 nm; a column temperature of 30 °C; and an injection volume of 20 μL.

[0031] Example 2 The difference between Example 2 and Example 1 lies in the preparation of the mutagenic bacterial solution: *Streptomyces aureus* spore suspension was used as the starting material, and ultraviolet mutagenesis was performed on the starting material to obtain the mutagenic bacterial solution. The *Streptomyces aureus* spore suspension was prepared by scraping and crushing mature *Streptomyces aureus* spores using a spore scraper, washing the spores with sterile water, filtering through a funnel, shaking well, and then dispensing. The ultraviolet mutagenesis treatment was performed in the dark under a 15W ultraviolet lamp at a distance of 30cm for 50 seconds. The remaining steps were consistent with Example 1.

[0032] Example 3 The difference between Example 3 and Example 1 is that the high-yield chlortetracycline strain obtained in Example 1 was subjected to continuous subculture to obtain F1, F2, F3 and F4 generation strains respectively. The continuous subculture method was to ferment each generation strain under the same shake-flask fermentation conditions as in Example 1, and to detect the chlortetracycline titer under the same HPLC conditions as in Example 1. Comparative Example

[0033] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the obtained mutagenic bacterial culture was not subjected to streptomycin concentration gradient tolerance test, nor was resistance screening performed on streptomycin-containing screening plates. Instead, single colonies were randomly selected directly from the mutagenic colonies for shake-flask fermentation and re-screening. The shake-flask fermentation medium, fermentation conditions, and HPLC detection conditions were the same as those in Example 1.

[0034] Comparative Example 2 The difference between Comparative Example 2 and Example 1 lies in the culture and selection: the mutagenic bacterial solution was diluted and spread onto a screening plate containing streptomycin resistance screening pressure, and cultured at 32°C until a single colony that could be isolated formed on the screening plate. Then, streptomycin-resistant single colonies were picked and purified, and the purified streptomycin-resistant single colonies were used for shake-flask fermentation for re-screening. The screening plate consisted of flour, agar powder, and streptomycin. The mass percentage of flour in the screening plate was 2.8%, the mass percentage of agar powder was 1.7%, the concentration of streptomycin was 1.5 μg / mL, and the volume of culture medium per plate was 25 mL. The remaining steps were the same as in Example 1. Performance testing experiment

[0035] A *Streptomyces aureus* strain that had not undergone mutagenesis or streptomycin resistance screening was used as the control strain. This strain was fermented under the same shake-flask fermentation medium and conditions as in Example 1, and the chlortetracycline titer was determined using the same HPLC detection conditions as in Example 1. Then, the chlortetracycline titers in the examples and comparative examples were sequentially determined by HPLC. Twenty streptomycin-resistant mutant strains were randomly selected from the examples or comparative examples for re-screening in shake flasks. Finally, in Example 3, strain number 1 from screening in Example 1 was selected for passage stability testing.

[0036] Table 2 Results of shake-flask rescreening of strain 1 in Example 1

[0037] Table 3. Passage Stability

[0038] Table 4. Results of shake-flask rescreening of Comparative Example 1 strain

[0039] Table 5. Effects of different screening methods on the screening effect of high-yielding chlortetracycline strains

[0040] As can be seen from Tables 1, 2 and 5, by controlling the streptomycin screening pressure to a level that results in a 10% survival rate of the strain, it is possible to effectively enrich high-yield mutant strains of chlortetracycline, thereby increasing the positive mutation rate and the probability of obtaining high-yield strains in subsequent shake-flask rescreening.

[0041] As shown in Table 3, after continuous subculturing, the chlortetracycline titer of the high-yield strain obtained in this application was maintained at no less than 98% in the F2, F3 and F4 generations. This indicates that the high-yield strain has good genetic stability and fermentation production stability, and is suitable for further use as a chlortetracycline production strain.

[0042] As can be seen from Table 4, compared with Example 1, Comparative Example 1 did not set streptomycin resistance screening pressure, and could not effectively enrich mutants with high production potential. Therefore, the positive mutation rate, average titer and highest titer were all lower than those of Example 1.

[0043] As shown in Table 5, Example 1, using ARTP mutagenesis combined with 1.0 μg / mL streptomycin resistance screening, achieved a mean rescreening titer of 13608 u / mL and a positive mutation rate of 65%. After passage, the mean titer was 13389 u / mL and the positive mutation rate was 60%, both significantly higher than Comparative Example 1, which used single-mutation blind screening. This indicates that streptomycin resistance screening can effectively enrich mutants with high chlortetracycline production potential. Example 2, using UV mutagenesis combined with 1.0 μg / mL streptomycin resistance screening, achieved a mean rescreening titer of 13452 u / mL and a positive mutation rate of 55%. After passage, the mean titer was 13291 u / mL. The streptomycin resistance screening in this application does not rely on a single mutagenesis method. Whether ARTP mutagenesis or UV mutagenesis is used, the screening probability of high-yield chlortetracycline mutant strains can be improved. In contrast, the streptomycin screening pressure in Comparative Example 2 was increased to 1.5 μg / mL. The average titer of the rescreening was 12942 u / mL, and the positive mutation rate after rescreening was 25%. The average titer after subculturing was 12796 u / mL, and the positive mutation rate after subculturing was 20%. These values ​​were significantly lower than those in Examples 1 and 2. Combined with the results in Table 1 showing that the survival rate of the mutagenic bacterial solution under the streptomycin 1.5 μg / mL condition was only 1%, it can be seen that when the streptomycin screening pressure is too high, the mutant strains will be excessively killed, resulting in an insufficient number of effective mutant populations, which is not conducive to obtaining high-yield chlortetracycline strains.

[0044] This demonstrates that this application does not simply use streptomycin as a screening antibiotic, but rather uses streptomycin concentration gradient statistics to determine that the survival rate of the mutant bacterial solution is 5%-15%, with a preferred streptomycin concentration of 10% as the screening pressure. This screening pressure can eliminate a large number of low-yield, ineffective, or negative mutant strains while retaining effective mutant strains with high-yield potential, thereby increasing the positive mutation rate, average titer, and probability of obtaining high-yield strains in subsequent shake-flask rescreening.

[0045] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for targeted screening of high-yield chlortetracycline-producing strains based on streptomycin resistance, characterized in that: Includes the following steps: Preparation of mutagenic bacterial solution: Take the spores or mycelium from the fermentation broth of Streptomyces aureus as the starting material, and perform mutagenic treatment on the starting material to obtain the mutagenic bacterial solution; Statistics: The mutant bacterial solution was spread on agar plates containing different concentrations of streptomycin, and the colony count was counted after incubation. The survival rate of the mutant bacterial solution at different streptomycin concentrations was calculated based on the colony count. Determine the screening pressure: Based on the survival rate results of the statistical steps, determine the streptomycin concentration that will result in a survival rate of 5%-15% for the mutant bacterial culture as the streptomycin resistance screening pressure; Culture and selection: Dilute the mutant bacterial solution and spread it on a selection plate containing the streptomycin resistance selection pressure, culture and select streptomycin resistant single colonies; Fermentation: The streptomycin-resistant single colony was subjected to shake-flask fermentation to obtain a fermentation broth; Screening: The chlortetracycline titer in the fermentation broth was detected by high performance liquid chromatography, and high-yielding chlortetracycline strains with higher chlortetracycline titers than the starting strain were screened.

2. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 1, characterized in that: In the preparation step of the mutagenic bacterial solution, the mutagenesis treatment is ARTP mutagenesis or ultraviolet mutagenesis; the ARTP mutagenesis uses Streptomyces aureus spore suspension or mycelial suspension as the treatment object, and the ultraviolet mutagenesis uses Streptomyces aureus spore suspension or mycelial suspension as the treatment object.

3. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 1, characterized in that: In the statistical steps, the different concentrations of streptomycin include 0 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 7.5 μg / mL, and 10.0 μg / mL.

4. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 3, characterized in that: In the step of determining the screening pressure, the streptomycin resistance screening pressure is the streptomycin concentration that makes the survival rate of the mutant bacterial solution 8%-12%; the streptomycin resistance screening pressure is 1.0 μg / mL.

5. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 4, characterized in that: In the culture and selection step, the screening plate includes flour, agar powder, and streptomycin; wherein the mass percentage of flour in the screening plate is 2.8%, the mass percentage of agar powder is 1.7%, the concentration of streptomycin is 1.0 μg / mL, and the volume of each culture medium plate is 25 mL.

6. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 5, characterized in that: In the culture and selection step, the culture temperature is 29-34℃; after the culture is carried out until a single colony can be isolated on the screening plate, streptomycin-resistant single colonies are selected for purification culture, and then the purified streptomycin-resistant single colonies are used for shake-flask fermentation for re-screening.

7. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 1, characterized in that: In the fermentation step, the fermentation medium used for the shake-flask fermentation comprises the following components at the following concentrations: corn starch 60–90 g / L, peanut meal powder 15–20 g / L, soybean meal powder 10–15 g / L, corn steep liquor powder 12–16 g / L, yeast powder 6–9 g / L, ammonium sulfate 5–8 g / L, CaCO3 6–8 g / L, NaCl 2–3 g / L, CaCl2 1–6 g / L, MgSO4·7H2O 0.3–1.6 g / L, KH2PO4 0.2–0.5 g / L, amylase 0.1–0.2 g / L, and defoamer 0.1–0.3 g / L; the pH of the fermentation medium is 6.6–7.

2.

8. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 7, characterized in that: In the fermentation step, the liquid volume for the shake flask fermentation is 25 mL in a 250 mL Erlenmeyer flask, the fermentation temperature is 29–31°C, and the shaker speed is 100–150 rpm.

9. The method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to claim 1, characterized in that: In the screening step, the detection conditions of the high-performance liquid chromatography (HPLC) include: a C8 column with an inner diameter of 4.6 mm, a column length of 250 mm, and a packing particle size of 5 μm; a mobile phase of a mixture of 0.01 mol / L oxalic acid, acetonitrile, and methanol, with a volume ratio of 71:16:13; a flow rate of 1.2 mL / min; an ultraviolet detector; a detection wavelength of 360 nm; a column temperature of 30 °C; and an injection volume of 20 μL.

10. A method for targeted screening of high-yield chlortetracycline strains based on streptomycin resistance according to any one of claims 1-9, characterized in that: After the obtained high-yield chlortetracycline strain was passaged for four generations, the chlortetracycline potency of each generation in shake-flask fermentation was maintained at no less than 98% compared with the potency of the first generation in shake-flask fermentation.

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