Aureusidin-producing strain of streptomyces aureofaciens and its use

Abstract

The present application belongs to the field of microorganisms, and particularly relates to a kind of aureofaciens strain of fermentation aureofacin, and further discloses the application of fermentation aureofacin thereof. 60 Co and ARTP complex mutagenesis technology are screened to obtain a high aureofacin-producing kitasatospora aureofaciens FIM-CTC-Z23-94, which can ferment high aureofacin, in 1 ton tank fermentation experiment, the aureofacin-producing kitasatospora aureofaciens FIM-CTC-Z23-94 has a titer as high as 30259 μg / mL, and a fermentation cycle is only 90h, and is more suitable for industrial fermentation production.Moreover, the strain FIM-CTC-Z23-94 screened by the present application has good stability, and the aureofacin-producing titer is basically stable when continuously transmitted for five generations, is maintained at the same high level, and can be used as a production strain for further research and development.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and in particular relates to a Streptomyces aureus strain capable of producing chlortetracycline by fermentation, and further discloses an application of the strain in producing chlortetracycline by fermentation. Background Art

[0002] Chlortetracyclin is a broad-spectrum tetracycline antibiotic, typically produced by fermentation of Streptomyces aureofaciens. Its antimicrobial spectrum is similar to that of tetracycline, with antibacterial activity against Gram-negative and Gram-positive bacteria, spirochetes, chlamydiae, mycoplasmas, and rickettsiae. Due to its antibacterial properties, growth promotion, high feed utilization, and low residual levels in the body, it is widely used as a medicated feed additive in the livestock industry and is currently the most widely used medicated feed additive. In recent years, with the expansion of livestock farming, the increasing intensification and confinement of livestock, demand for medicated feed additives has increased both domestically and internationally. my country has become the world's largest producer of chlortetracycline, with the current fermentation level of chlortetracycline in domestic industrial production remaining at approximately 22,000 μg / mL.

[0003] For the improvement of chlortetracycline strains, the current main breeding methods are the traditional selection method of ultraviolet mutagenesis combined with different mutagens and other breeding methods, while the fermentation process optimization is combined to further improve the fermentation level of chlortetracycline, and have achieved certain results. 60 Co mutagenesis combined with self-resistance screening and optimization of fermentation medium has enabled the fermentation level of chlortetracycline to reach 23,000 μg / mL, with a fermentation cycle of 96 hours; Xuan Hailian obtained the high-yield strain M14-2 through ultraviolet light, lithium chloride and chlortetracycline resistance experiments, and used the optimized fermentation medium in 60T tank production, with a titer of 23,200 μg / mL, a 15% increase over the original strain, and a fermentation cycle of 166 hours; Sun Fei et al. disclosed in Chinese patent CN107177661A a chlortetracycline fermentation medium that uses rice residue to replace part of the corn steep liquor powder, with a chlortetracycline fermentation titer of up to 25,768 μg / mL and a fermentation cycle of 120 hours to 144 hours; Xie Changxian et al. disclosed in Chinese patent CN113388556A a method for producing chlortetracycline using Streptomyces aureus, with a chlortetracycline yield of 26.88 g / L after 6 days of shake flask fermentation. It can be seen that traditional composite mutagenesis breeding combined with fermentation process optimization has achieved certain results in improving the strain's ability to produce chloramphenicol. However, the domestic chloramphenicol potency is lower than that of foreign manufacturers, resulting in low overall fermentation production efficiency and affecting market competitiveness. Therefore, improving the fermentation level of chloramphenicol plays an important role in improving the production efficiency of this product.

[0004] Atmospheric pressure room temperature plasma (ARTP) breeding technology is a rapid mutation technology for microbial genomes. The plasma it produces is rich in various chemically active particles, which damage the genetic material of the strain cells, cause changes in cell membrane permeability and protein structure, and other multiple effects. The cells activate the SOS repair mechanism, and during the repair process, a rich variety of mismatch sites are generated with a high mutation rate. In addition, the resistance genes of microorganisms are closely linked to the product synthesis genes and can be expressed through induction, which largely overcomes the blindness and non-directionality of random screening of mutagenesis. It has the characteristics of broad spectrum, high positive mutation rate and high yield increase rate. Microorganisms have been 60 The combined mutagenesis of Co and ARTP can make the strain's mutagenic damage more diverse and obtain more mutation types than single mutagenesis, thereby enriching the diversity of the mutagenized strain and greatly improving the probability of obtaining high-yield mutant strains.

[0005] Therefore, by adopting 60 Co+ARTP mutagenesis combined with resistance mutations against its own products is expected to selectively increase the positive mutation rate of strain product yield, thereby screening out efficient strains suitable for industrial production of chloramphenicol. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a Streptomyces aureus strain that can ferment and produce chlortetracycline, so as to solve the problem that the performance of chlortetracycline fermentation strains in the prior art is not ideal;

[0007] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned Streptomyces aureus strain in fermentation production of chlortetracycline.

[0008] In order to solve the above technical problems, the golden Streptomyces strain described in the present invention is classified and named as golden Streptomyces (Kitasatospora aureofaciens) FIM-CTC-Z23-94, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center, located on the 5th Floor, Dayuanyuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No. 63321 and a deposit date of April 4, 2023.

[0009] The invention also discloses the application of the Streptomyces aureus strain in the fermentation production of chlortetracycline.

[0010] The present invention also discloses a method for producing chlortetracycline by fermentation, which comprises the steps of inoculating the Streptomyces aureus strain into a suitable fermentation medium for fermentation culture.

[0011] Specifically, the fermentation medium includes the following components by mass content: 5.5-10.5wt% of corn flour, 3.5-7.0wt% of yeast powder, 0.5-2.0wt% of glucose, 0.3-1.5wt% of peptone, 0.15-0.6wt% of sodium chloride, 0.015-0.08wt% of magnesium sulfate, 0.005-0.05wt% of potassium dihydrogen phosphate, 0.2-0.5wt% of calcium carbonate, 0.01-0.05wt% of amylase, and the balance is water, with a pH of 5.8-7.5.

[0012] Preferably, the fermentation medium comprises the following components by mass: 8.5wt% corn flour, 4.5wt% yeast powder, 1.0wt% glucose, 0.8wt% peptone, 0.3wt% sodium chloride, 0.03wt% magnesium sulfate, 0.01wt% potassium dihydrogen phosphate, 0.6wt% calcium carbonate, 0.02wt% amylase, and the balance is water, with a pH of 5.8-7.5.

[0013] Specifically, the conditions of the fermentation culture step include: controlling the rotation speed to 180-280 rpm and performing fermentation culture at 29-35° C. for 84-144 hours.

[0014] As a preferred embodiment, in the fermentation process of the present invention, the fermentation tank scale culture adopts variable temperature culture, that is, the fermentation is controlled for 0-48 hours at a temperature of 32-35°C, and after 48 hours, the temperature is controlled at 29-32°C, the tank pressure is 0.05MPa, the air flow rate is 1:0.8-1.5vvm, the stirring speed is 180-250r / min, the dissolved oxygen is controlled to be not less than 30% during the process, and the fermentation culture is 84-144h.

[0015] Specifically, the method for producing chlortetracycline by fermentation further comprises the step of inoculating the Streptomyces aureus strain into a seed culture medium for seed liquid culture;

[0016] The seed culture medium comprises the following components by weight: 2.0-6.5 wt% of corn flour, 1.5-4.0 wt% of yeast powder, 0.3-1.5 wt% of glucose, 0.3-1.5 wt% of peptone, 0.1-0.5 wt% of sodium chloride, 0.01-0.05 wt% of potassium dihydrogen phosphate, 0.01-0.05 wt% of magnesium sulfate, 0.005-0.05 wt% of amylase, 0.2-0.5 wt% of calcium carbonate, and the balance is water, with a pH value of 5.8-7.5.

[0017] Preferably, the seed culture medium comprises the following components by mass: 4.5wt% corn flour, 2.0wt% yeast powder, 0.5wt% glucose, 0.5wt% peptone, 0.2wt% sodium chloride, 0.025wt% potassium dihydrogen phosphate, 0.025wt% magnesium sulfate, 0.01wt% amylase, 0.4wt% calcium carbonate, and the balance is water, with a pH of 5.8-7.5.

[0018] Specifically, the conditions of the seed liquid culture step include: controlling the rotation speed to 180-280 rpm, preferably 200-250 rpm, and culturing the seed liquid at 29-35° C. for 16-24 hours.

[0019] Specifically, the method for producing chlortetracycline by fermentation further comprises the step of inoculating the Streptomyces aureus strain into a slant culture medium for activation;

[0020] The slant culture medium comprises the following components by weight: 4.0-6.5 wt% of bran, 0.01-0.05 wt% of potassium dihydrogen phosphate, 0.01-0.08 wt% of magnesium sulfate, 0.01-0.05 wt% of diammonium hydrogen phosphate, 1.8-2.0 wt% of agar, and the balance is water, with a pH of 5.8-7.5.

[0021] Preferably, the slant culture medium comprises the following components by mass: 5.0 wt% bran, 0.02 wt% potassium dihydrogen phosphate, 0.03 wt% magnesium sulfate, 0.02 wt% diammonium hydrogen phosphate, 1.8-2.0 wt% agar, and the balance is water, with a pH of 5.8-7.5.

[0022] Specifically, the conditions of the slant culture medium activation step include: constant temperature culture at 29-35° C. for 7-10 days.

[0023] The present invention 60 Co and ARTP composite mutagenesis technology was used to screen a strain of golden Streptomyces (Kitasatospora aureofaciens) FIM-CTC-Z23-94 with high chlortetracycline production, which can ferment high-yield chlortetracycline. In a 1T tank fermentation experiment, the titer of chlortetracycline produced by the golden Streptomyces FIM-CTC-Z23-94 fermentation was as high as 30259μg / mL, and the fermentation cycle was only 90h, which is more suitable for industrial fermentation production. Moreover, the strain FIM-CTC-Z23-94 screened by the present invention has good stability, and its chlortetracycline production titer is basically stable after five generations of continuous passage, maintained at the same high level, and can be used as a production strain for further research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 This is the relationship curve between ARTP efflux time and lethality in the mutagenesis experiment;

[0026] Figure 2 Phylogenetic tree of the strain FIM-CTC-Z23-94 described in the present invention. DETAILED DESCRIPTION

[0027] In the following embodiments of the present invention, the culture medium involved includes:

[0028] The separation plate culture medium and the slant culture medium comprise the following components by weight: 5.0 wt% of bran, 0.02 wt% of potassium dihydrogen phosphate, 0.03 wt% of magnesium sulfate, 0.02 wt% of diammonium hydrogen phosphate, 1.8-2.0 wt% of agar, and the balance being water; the pH value is 5.8-7.5; and the medium is sterilized by high-pressure steam at 121° C. for 30 min.

[0029] The seed culture medium comprises the following components by weight: 4.5 wt% corn flour, 2.0 wt% yeast powder, 0.5 wt% glucose, 0.5 wt% peptone, 0.2 wt% sodium chloride, 0.025 wt% potassium dihydrogen phosphate, 0.025 wt% magnesium sulfate, 0.01 wt% amylase, 0.4 wt% calcium carbonate, and the balance is water, with a pH of 5.8-7.5, and is sterilized by high-pressure steam at 121° C. for 30 min.

[0030] The fermentation medium includes the following components by weight: 8.5wt% corn flour, 4.5wt% yeast powder, 1.0wt% glucose, 0.8wt% peptone, 0.3wt% sodium chloride, 0.03wt% magnesium sulfate, 0.01wt% potassium dihydrogen phosphate, 0.6wt% calcium carbonate, 0.02wt% amylase, and the balance is water. The pH value is 5.8-7.5 and the medium is sterilized by high-pressure steam at 121°C for 30 min.

[0031] In the following embodiments of the present invention, the content of chlortetracycline and impurity components is detected by high performance liquid chromatography (HPLC). The chromatographic separation conditions are: C8 separation column (250*4.6mm); detection wavelength: 360nm; mobile phase: 0.01mol / L oxalic acid / acetonitrile / methanol (71 / 16 / 13, V / V / V) solution; flow rate: 1.0mL / min; column temperature: 30°C. The fermentation broth is acidified with oxalic acid to pH1.5-2.0, centrifuged at 4000r / min, 0.1mL of the supernatant is taken to a 100ml volumetric flask and fixed to volume with distilled water. The fixed solution is filtered with a 0.22μm microporous filter membrane, and the filtrate is tested by HPLC to determine the content of the target product chlortetracycline (CTC) and the content of impurities tetracycline (TC) and demethylchlortetracycline (DMCTC).

[0032] Example 1 Obtaining the mutagenic strain FIM-F-Z23-94

[0033] A single colony of Streptomyces aureus FIM-CTC-Z23 with relatively stable genetic traits was naturally isolated and transferred to a slant culture medium. The culture was cultured in a constant temperature incubator at 32°C for 8 days. The spores on the slant culture medium were then washed with physiological saline and transferred to a sterilized test tube containing 5 Φ5 mm glass beads and shaken. The suspension was filtered through gauze to prepare a single spore suspension (OD 600 value 0.6-0.8).

[0034] Use a pipette to draw 10 μL of spore suspension onto a round iron sheet with a diameter of 1 cm, and place it in a normal pressure and room temperature plasma mutagenesis system with helium as the working gas, a power supply of 110 W, and a working gas flow rate of 10 L / min. The treatment distance is 2 mm, and the treatment is carried out for 10 s, 20 s, 30 s, 45 s, 60 s, and 75 s respectively. The treated spore suspension is gradiently diluted and plated to prepare a lethality curve, as shown in FIG. Figure 1 As shown. Figure 1 It can be seen that there is an obvious dose-effect relationship between the mutagenesis treatment dose and the lethality of the strain FIM-CTC-Z23. As the treatment time increases, the lethality gradually increases.

[0035] The spore suspensions obtained above were respectively spread on plate culture media containing chlortetracycline, wherein the chlortetracycline concentrations in the plate culture media were 0.25 g / L, 0.5 g / L, 1.0 g / L, 1.25 g / L, 1.5 g / L, and 2.0 g / L, respectively; after culturing at 32°C for 8-10 days, the colony growth on different plates was observed. The results are shown in Table 1. The lowest effective concentration of chlortetracycline in the plate culture medium where no colonies grew was the minimum inhibitory concentration of chlortetracycline against its own metabolite, and the minimum inhibitory concentration against chlortetracycline was determined to be 2.0 g / L according to Table 1.

[0036] Table 1 Effect of chlortetracycline concentration on spore growth of strain FIM-CTC-Z23

[0037] Chlortetracycline (μg / mL) 0 250 500 1000 1250 1500 2000 Colony growth +++ +++ ++ + + +- -

[0038] Note: + means colony growth, - means no colony growth.

[0039] The FIM-CTC-Z23 spore suspension obtained above was subjected to a 500 Gy dose and a dose rate of 900 rad / min. 60 Co radiation; According to the above lethality curve, two different lethal dose irradiation times of 30s and 45s were selected. 60 The Co-treated strain FIM-CTC-Z23 spore suspension was subjected to ARTP mutagenesis, and the two spore suspensions with different ARTP treatment times were mixed in a test tube filled with physiological saline and mixed evenly to obtain a FIM-CTC-Z23 composite mutagenized spore suspension for later use.

[0040] The FIM-CTC-Z23 composite mutagenesis spore suspension obtained above was diluted in a gradient manner, with dilutions of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , select 10 -4 , 10 -5 , 10 -6 Three dilutions of spore suspension were spread on resistance separation plates containing 2.0 g / L of its own metabolite chlortetracycline and cultured in the dark at 32°C for 6-10 days.

[0041] The single colony grown on the resistance plate was transferred to the slant culture medium and cultured for 8 days, then inoculated into the seed culture medium at an inoculum size of 0.5-1%, and cultured at 32°C and 230r / min for 16-24h to obtain the seed liquid; the seed liquid was inoculated into the fermentation medium at an inoculum size of 10%, and cultured at 32°C and 230r / min for 84-144h to obtain the fermentation liquid, and the chloramphenicol content in the fermentation liquid was detected by chemical determination method.

[0042] By this method, the strain with the largest chlortetracycline production was screened out. For the convenience of description, the screened strain was named strain FIM-CTC-Z23-94, and strain FIM-CTC-Z23-94 was preserved in glycerol.

[0043] Example 2 Identification of strain FIM-CTC-Z23-94

[0044] Identification of physiological and biochemical characteristics

[0045] The obtained strain FIM-CTC-Z23-94 was streaked on a separation medium plate and inserted into a coverslip. It was cultured at 32°C for 5-15 days. The morphological characteristics of single colonies and their hyphae were observed using optical microscopy, transmission electron microscopy, and scanning electron microscopy.

[0046] The main morphological and physiological and biochemical characteristics of the strain FIM-CTC-Z23-94 are as follows: spore threads are flexible, initially spiraling to a loose, open spiral. Spores are oval to cylindrical with a smooth surface. Colonies are round on separation plates, aerial hyphae are gray-brown, and golden pigment is produced on glucose agar and potato tubers. The strain can utilize D-glucose, L-arabinose, D-xylose, D-fructose, and sucrose; it does not utilize rhamnose, raffinose, inositol, or D-mannitol. This strain is highly aerobic, and dissolved oxygen has a significant effect on the production of chlortetracycline during fermentation. The optimal growth temperature is 28-36°C, and the optimal growth pH is 5.7-8.2. The highest chlortetracycline production is achieved when the shaker speed is 180-250 r / min and the culture time is 60-144 h.

[0047] Molecular biology identification

[0048] The 16S rDNA sequence of strain FIM-CTC-Z23-94 was sequenced, and the sequencing result is shown in SEQ ID No. 1. The 16S rDNA sequence of the strain was compared with the existing sequences in the GenBank database, and homology analysis was performed. The corresponding model strain 16S rRNA gene sequence was selected from the LPSN website (http: / / www.bacterio.cict.fr), and phylogenetic analysis was performed using CLUSTAL-X software. The generated alignment file was used for phylogenetic analysis using the neighbor-joining method of TREECON software. The topological analysis was the result of 1000 repeated samplings. The results are shown in the Appendix. Figure 2 ; 16SrDNA sequence analysis showed that the sequence homology between strain FIM-CTC-Z23-94 and Kitasatospora aureofaciens was 99.78%.

[0049] SEQ ID NO.1:

[0050]

[0051] Based on the above morphological, physiological and biochemical characteristics and molecular biological identification, the strain FIM-CTC-Z23-94 was finally determined to be the genus Kitasatospora aureofaciens, and its classification name was Kitasatospora aureofaciens FIM-CTC-Z23-94. It has been deposited in the Guangdong Provincial Microbiological Culture Collection, located at 5th Floor, Dayuanyuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number GDMCC No. 63321 and the deposit date April 4, 2023.

[0052] Example 3 Verification of genetic stability of the mutagenized strain FIM-F-Z23-94

[0053] Strain subculture: The high-yield chlortetracycline strain FIM-CTC-Z23-94 selected above was transferred to the slant culture medium and cultured at 32°C under a humidity of 30-50% for 8 days to obtain the well-growing FIM-CTC-Z23-94 primary slant (F0). The strain was continuously cultured and subcultured, and cultured at 32°C for 8 days to obtain well-growing F1, F2, F3, F4, F5, and F6 slants, respectively.

[0054] Preparation of FIM-CTC-Z23-94 seed liquid: Wash the fresh moss obtained from the above-mentioned strain FIM-CTC-Z23-94 with physiological saline to prepare a spore suspension, inoculate it into a seed culture medium (60 mL seed culture medium in a 500 mL Erlenmeyer flask) at a 0.5% inoculum amount, and culture it at 32°C and 230 r / min for 20 h to obtain FIM-CTC-Z23-94 seed liquid.

[0055] Fermentation culture: The prepared seed liquid was inoculated into the fermentation medium (60 mL fermentation medium in a 500 mL Erlenmeyer flask) at a 10% (v / v) inoculation rate, and fermented at 32°C and 230 r / min for 120 h. The obtained fermentation liquid was then tested.

[0056] The primary strain (F0) with good growth was used as the control, and its fermentation relative titer was 100%. The results are shown in Table 2.

[0057] Table 2 Effect of subculture on chlortetracycline production by strain FIM-CTC-Z23-94

[0058] Strain generations F1 F2 F3 F4 F5 F6 Relative potency (%) 100.9 101.3 99.3 98.0 96.7 94.7

[0059] As can be seen from Table 2, the fifth generation of the strain FIM-CTC-Z23-94 screened by the present invention has no obvious effect on the fermentation level, and its chlortetracycline production titer is basically stable and maintained at the same high level, indicating that the strain FIM-CTC-Z23-94 has good genetic stability.

[0060] Example 4 Shake flask fermentation of starting strain FIM-CTC-Z23

[0061] Preparation of FIM-CTC-Z23 slants: Transfer the starting strain FIM-CTC-Z23 to a slant culture medium and culture at 32°C under a humidity of 30-50% for 8 days to obtain fresh FIM-CTC-Z23 slants with good growth.

[0062] Preparation of FIM-CTC-Z23 seed liquid: Wash the fresh moss obtained from the above-mentioned strain FIM-CTC-Z23 with physiological saline to prepare a spore suspension, inoculate it into the seed culture medium (60 mL seed culture medium in a 500 mL Erlenmeyer flask) at a 0.5% inoculum amount, and culture it at 32°C and 230 r / min for 20 h to obtain the FIM-CTC-Z23 seed liquid.

[0063] Fermentation culture: The prepared seed liquid was inoculated into the fermentation medium (60 mL fermentation medium in a 500 mL Erlenmeyer flask) at a 10% (v / v) inoculation rate, and fermented at 32°C and 230 r / min for 120 h. The obtained fermentation liquid was then tested.

[0064] The test results showed that the CTC yield in this example was 22162 μg / mL, with a daily yield of 4432 μg / mL. The main impurity TC content was 2216 μg / mL, and the DMCTC content was 266 μg / mL.

[0065] Example 5: Fermentation and production of chlortetracycline by mutagenic strain FIM-CTC-Z23-94

[0066] Shake flask seed culture: Wash the fresh bacterial moss of the above-mentioned strain FIM-CTC-Z23-94 with physiological saline to prepare a spore suspension, inoculate it into a seed culture medium (150 mL seed culture medium in a 1000 mL Erlenmeyer flask) at a 0.5% inoculum size, and culture it at 32°C and 230 r / min for 20 h to obtain the shake flask seed liquid.

[0067] Seed culture in seed tanks: inoculate 0.5% of the shake flask seed liquid into the seed culture medium (60L seed culture medium in a 100L tank), and culture for 18 hours at a culture temperature of 32°C, a tank pressure of 0.05MPa, an air flow rate of 1:1vvm, and a stirring speed of 100-200r / min to obtain the seed liquid in the seed tank.

[0068] Fermentation culture in a fermenter: inoculate the prepared seed liquid into the fermentation medium (600L fermentation medium in a 1-ton tank) at a 10% (v / v) inoculation rate, adopt variable temperature culture, the fermentation temperature is 33.5°C for 0-48h, and the temperature is controlled at 30.5°C after 48h, the tank pressure is 0.05MPa, the air flow rate is 1:0.8-1.5vvm, the stirring speed is 180-250r / min, the dissolved oxygen is controlled to be not less than 30% during the process, and the fermentation culture is carried out for 90h. Then, the fermentation liquid is placed in the tank for testing.

[0069] The test results showed that the CTC yield in this example was 30259 μg / mL, with a daily yield of 7894 μg / mL.d, which was a 78.1% increase in daily yield compared to the starting strain. The main impurity TC content was 1915 μg / mL, a 13.6% decrease compared to the starting strain, and the DMCTC content was 60.5 μg / mL, a 77.3% decrease compared to the starting strain.

[0070] Example 6: Fermentation production of chlortetracycline by mutagenic strain FIM-CTC-Z23-94

[0071] Shake flask seed culture: Wash the fresh bacterial moss of the above-mentioned strain FIM-CTC-Z23-94 with physiological saline to prepare a spore suspension, inoculate it into a seed culture medium (150 mL seed culture medium in a 1000 mL Erlenmeyer flask) at a 0.5% spore amount, and culture it at 32°C and 230 r / min for 20 h to obtain the shake flask seed liquid.

[0072] Seed culture in seed tanks: inoculate 0.5% of the shake flask seed liquid into the seed culture medium (60L seed culture medium in a 100L tank), and culture for 18 hours at a culture temperature of 32°C, a tank pressure of 0.05MPa, an air flow rate of 1:1vvm, and a stirring speed of 100-200r / min to obtain the seed liquid in the seed tank.

[0073] Fermentation culture in a fermenter: inoculate the prepared seed liquid into a fermentation medium (600L fermentation medium in a 1-ton tank) at a 10% (v / v) inoculation rate, adopt variable temperature culture, the fermentation temperature is 33.5°C for 0-48h, the temperature is controlled at 30.5°C after 48h, the tank pressure is 0.01-0.05MPa, the air flow rate is 1:0.8-1.5vvm, the stirring speed is 180-250r / min, the dissolved oxygen is controlled to be not less than 30% during the process, the fermentation culture is carried out for 90h, and the obtained fermentation liquid is placed in the tank for detection.

[0074] The test results showed that the CTC yield in this example was 30303 μg / mL, with a daily yield of 7905 μg / mL.d, a daily yield increase of 78.4% compared to the starting strain. The main impurity TC content was 1818 μg / mL, a decrease of 18.0% compared to the starting strain. The DMCTC content was 69.7 μg / mL, a decrease of 73.8% compared to the starting strain.

[0075] Example 7: Fermentation production of chlortetracycline by mutagenic strain FIM-CTC-Z23-94

[0076] Shake flask seed culture: Wash the fresh bacterial moss of the above-mentioned strain FIM-CTC-Z23-94 with physiological saline to prepare a spore suspension, inoculate it into a seed culture medium (150 mL seed culture medium in a 1000 mL Erlenmeyer flask) at a 0.5% inoculum size, and culture it at 32°C and 230 r / min for 20 h to obtain the shake flask seed liquid.

[0077] Seed culture in seed tanks: inoculate 0.5% of the shake flask seed liquid into the seed culture medium (60L seed culture medium in a 100L tank), and culture for 18 hours at a culture temperature of 32°C, a tank pressure of 0.01-0.05MPa, an air flow rate of 1:1vvm, and a stirring speed of 100-200r / min to obtain the seed liquid in the seed tank.

[0078] Fermentation culture in a fermenter: inoculate the prepared seed liquid into a fermentation medium (600L fermentation medium in a 1-ton tank) at a 10% (v / v) inoculation rate, adopt variable temperature culture, the fermentation temperature is 33.5°C for 0-48h, the temperature is controlled at 30.5°C after 48h, the tank pressure is 0.01-0.05MPa, the air flow rate is 1:0.8-1.5vvm, the stirring speed is 180-250r / min, the dissolved oxygen is controlled to be not less than 30% during the process, the fermentation culture is carried out for 90h, and the obtained fermentation liquid is placed in the tank for detection.

[0079] The test results showed that the CTC yield in this example was 30267 μg / mL, with a daily yield of 7896 μg / mL.d, which was a 78.2% increase in daily yield compared to the starting strain. The main impurity TC content was 1877 μg / mL, a 15.3% decrease compared to the starting strain. The DMCTC content was 63.6 μg / mL, a 76.1% decrease compared to the starting strain.

[0080] In summary, the mutant strain FIM-CTC-Z23-94 of the present invention produced no less than 30,259 μg / mL of chlortetracycline in a 1-ton fermentation tank, with a fermentation cycle of only 90 hours. This suggests that the mutant strain FIM-CTC-Z23-94 (Kitasatospora aureofaciens) screened in the present invention can be used as a production strain for further research and development.

[0081] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A strain of Streptomyces aureus, which is classified as Kitasatospora aureofaciens The strain number is FIM-CTC-Z23-94, and it has been deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No. 63321 and the deposit date is April 4, 2023.

2. Use of the Streptomyces aureus strain according to claim 1 in the fermentation production of chlortetracycline.

3. A method for producing chlortetracycline by fermentation, characterized in that: The method comprises the steps of inoculating the Streptomyces aureus strain according to claim 1 into a suitable fermentation medium for fermentation culture.

4. The method for producing chlortetracycline by fermentation according to claim 3, characterized in that: The fermentation medium includes the following components by weight: 5.5-10.5wt% corn flour, 3.5-7.0wt% yeast powder, 0.5-2.0wt% glucose, 0.3-1.5wt% peptone, 0.15-0.6wt% sodium chloride, 0.015-0.08wt% magnesium sulfate, 0.005-0.05wt% potassium dihydrogen phosphate, 0.2-0.5wt% calcium carbonate, 0.01-0.05wt% amylase, and the balance is water, with a pH of 5.8-7.

5.

5. The method for producing chlortetracycline by fermentation according to claim 3, characterized in that: The conditions of the fermentation culture step include: controlling the rotation speed to 180-280 rpm and performing fermentation culture at 29-35° C. for 84-144 hours.

6. The method for producing chlortetracycline by fermentation according to any one of claims 3 to 5, characterized in that: The method further comprises the step of inoculating the Streptomyces aureus strain according to claim 1 into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components by weight: 2.0-6.5wt% corn flour, 1.5-4.0wt% yeast powder, 0.3-1.5wt% glucose, 0.3-1.5wt% peptone, 0.1-0.5wt% sodium chloride, 0.01-0.05wt% potassium dihydrogen phosphate, 0.01-0.05wt% magnesium sulfate, 0.005-0.05wt% amylase, 0.2-0.5wt% calcium carbonate, and the balance is water, with a pH of 5.8-7.

5.

7. The method for producing chlortetracycline by fermentation according to claim 6, characterized in that: The conditions of the seed liquid culture step include: controlling the rotation speed to 180-280 rpm and culturing the seed liquid at 29-35° C. for 16-24 hours.

8. The method for producing chlortetracycline by fermentation according to any one of claims 3 to 5, characterized in that: The method further comprises the step of inoculating the Streptomyces aureus strain according to claim 1 into a slant culture medium for activation; The slant culture medium comprises the following components by weight: 4.0-6.5 wt% bran, 0.01-0.05 wt% potassium dihydrogen phosphate, 0.01-0.08 wt% magnesium sulfate, 0.01-0.05 wt% diammonium hydrogen phosphate, 1.8-2.0 wt% agar, and the balance is water, with a pH of 5.8-7.

5.

9. The method for producing chlortetracycline by fermentation according to claim 8, characterized in that: The conditions of the slant culture medium activation step include: constant temperature culture at 29-35° C. for 6-10 days.

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