阅读说明：本技术 一种金霉素产量高、杂质比例低的金色链霉菌突变菌株及其应用 (Streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof ) 是由 谢昌贤 王月清 蔡玉凤 梁玲 黄钦耿 赵燕玉 翁雪清 陈健 孙岩宁 陈红梅 王鹏 于 2021-08-02 设计创作，主要内容包括：本发明公开了金色链霉菌突变菌株及其选育方法以及优化的培养基。本发明的突变株能高效合成和积累金霉素、杂质四环素和去甲基金霉素含量显著降低,遗传稳定性良好,连续传代五代其形态、颜色、产金霉素水平以及主要杂质的含量基本稳定,能够应用于工业化发酵生产及作为进一步研究开发的生产菌株。本发明优化的培养基,有利于促进金霉素的合成与积累,进一步减少杂质四环素和去甲基金霉素的比例。本发明基于多重复合诱变和前体/产物耐受构建了高产菌株选育模型,不仅突变库的多样性更高,而且简便快捷,提高了筛选效率并减少了筛选工作量,对金霉素及其他链霉菌来源抗生素的生产成本降低和产能提高都具有一定的应用价值和指导意义。(The invention discloses a streptomyces aureofaciens mutant strain, a breeding method thereof and an optimized culture medium. The mutant strain can efficiently synthesize and accumulate aureomycin, the impurity tetracycline and the demethyl aureomycin, the content is obviously reduced, the genetic stability is good, the form, the color, the aureomycin production level and the content of main impurities are basically stable after five generations of continuous passage, and the mutant strain can be applied to industrial fermentation production and can be used as a production strain for further research and development. The optimized culture medium is favorable for promoting the synthesis and accumulation of aureomycin and further reducing the proportion of impurities tetracycline and demethylaureomycin. The high-yield strain breeding model is constructed based on multiple compound mutagenesis and precursor/product tolerance, the diversity of a mutation library is higher, the method is simple, convenient and quick, the screening efficiency is improved, the screening workload is reduced, and the method has certain application value and guiding significance for reducing the production cost and improving the productivity of aureomycin and other streptomycete-derived antibiotics.)

1. The Streptomyces aureofaciens is characterized by being Streptomyces aureofaciens, the strain number of the Streptomyces aureofaciens is FJC-505, and the preservation number of the Streptomyces aureofaciens in China center for type culture collection is CCTCC NO: m2021499.

2. A culture for the production of aureomycin, characterized in that it contains Streptomyces aureofaciens according to claim 1.

3. A method of producing a culture of streptomyces aureofaciens, the method comprising: culturing the Streptomyces aureofaciens of claim 1 in a medium to obtain a culture of Streptomyces aureofaciens.

4. The method of claim 3, wherein the medium comprises a seed medium and/or a fermentation medium, and wherein the seed medium and the fermentation medium comprise ammonium chloride, calcium chloride, and magnesium chloride.

5. The method of claim 4, wherein the composition of the seed medium is: 30.0g/L of corn starch, 15.0g/L of soybean cake powder, 10.0g/L of peanut cake powder, 3.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 1.2g/L of calcium chloride, 1.8g/L of calcium carbonate, 5.0g/L of peptone, 5.0g/L of yeast powder, 0.05g/L of magnesium chloride, 0.05g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of soybean oil and the balance of water.

6. The method according to claim 4 or 5, wherein the composition of the fermentation medium is: 60.0g/L of corn starch, 25.0g/L of peanut cake powder, 20.0g/L of corn flour, 10.0g/L of soybean cake powder, 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of corn steep liquor, 2.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 2.0g/L of calcium chloride, 3.0g/L of calcium carbonate, 0.1g/L of magnesium chloride, 0.1g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of amylase, 20.0g/L of soybean oil and the balance of water.

7. The method according to any one of claims 3 to 6, characterized in that said culture medium further comprises a spore culture medium consisting of an equal mass of bran and a nutrient solution consisting of: 30.0g/L of corn starch, 2.0g/L of peanut cake powder, 5.0g/L of yeast powder, 3.0g/L of peptone, 2.0g/L of ammonium chloride, 2.0g/L of sodium chloride, 0.1g/L of monopotassium phosphate, 0.15g/L of magnesium chloride, 0.15g/L of magnesium sulfate and the balance of water.

8. Use of Streptomyces aureofaciens according to claim 1 for the preparation of chlortetracycline.

9. Use of the culture of claim 2 for the preparation of chlortetracycline.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof.

Background

Aureomycin (chlorotetracycline) is a tetracycline broad-spectrum antibiotic generated by fermenting Streptomyces aureofaciens, and becomes a bacteriostatic growth promoter with the largest dosage in the feed industry due to the characteristics of bacteriostasis, growth promotion, high feed utilization rate, low residual quantity in organisms and the advantages of mature production technology and low production cost. Streptomyces aureofaciens is taken as an important medicinal microorganism and belongs to actinomycetes with high aerobic and gram-positive staining. However, the titer of the existing strain for producing aureomycin by fermentation is not high, and the production stability is not good, thus the fermentation index and the production benefit of aureomycin are seriously influenced. In order to establish an optimized, economical and competitive aureomycin bioproduction process, a series of problems such as production level, fermentation mode, post-treatment refining and the like must be fully considered. Among them, obtaining a desired characteristic and an appropriate high-efficiency production strain is essential for fermentation production of aureomycin and is the core of the entire aureomycin fermentation industry. Therefore, breeding high-yield and stable aureomycin strains is always a hotspot of technical development and fermentation engineering research of aureomycin fermentation industry, and will also always accompany the development of aureomycin fermentation industry.

The Streptomyces aureofaciens actinomycetes has difficult genetic operation and complex genetic background. In addition, aureomycin is used as a secondary metabolite, the biosynthesis process is complex, and the regulation and control means are various, so that the application of the metabolic engineering means in the breeding aspect of aureomycin strains is greatly limited. The traditional mutation breeding is not limited to the change of a certain gene or a certain number of metabolic flows, can form global interference on cell membranes and genomes, is convenient to form rich mutation types, and is widely applied to the breeding of high-yield strains in the fields of scientific research and production. However, the streptomyces aureofaciens industrial strain is generally subjected to multiple traditional mutagenesis, the phenomena of low mutagenesis efficiency, high negative rate, easy back mutation and the like often occur, and the repeated mutagenesis often generates a mutagenesis saturation effect, so that the yield is difficult to improve. Compared with the traditional conventional mutagenesis method, the atmospheric-pressure room-temperature plasma (ARTP) mutagenesis system is a novel mutagenesis method for carrying out microorganism mutagenesis breeding by utilizing radio-frequency atmospheric-pressure glow discharge (RF APGD) plasma jet, has the characteristics of simple and convenient operation, high safety, rapid mutagenesis, large mutation-generating diversity and the like, is successfully applied to strain breeding of various microorganisms such as bacteria, algae, fungi, actinomycetes and the like, and becomes an effective method for obtaining specific and high-yield strains in the fermentation industry. The physical and chemical compound mutagenesis method combining the normal-pressure room-temperature plasma, the ultraviolet rays and the lithium chloride can enable mutagenesis damage to be more diversified, and more mutation types than single mutagenesis can be obtained, so that the diversity of an mutagenesis library is increased, the probability of obtaining high-yield mutant strains is greatly improved, high-yield and high-quality strains meeting the industrial production can be screened and obtained, the enterprise cost is further reduced, the enterprise profit is increased, and the method has wide application value and important economic significance for the industrial production of the aureomycin.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion, a method for breeding the mutant strain and a method for preparing high-yield aureomycin by using the mutant strain. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the technical problems, the invention firstly provides Streptomyces aureofaciens, the Streptomyces aureofaciens is Streptomyces aureofaciens, the strain number is FJC-505, and the preservation number of the Streptomyces aureofaciens in China center for type culture preservation is CCTCC NO: m2021499. Hereinafter referred to as Streptomyces aureofaciens FJC-505.

The invention also provides a culture for preparing aureomycin, which contains the streptomyces aureofaciens FJC-505.

The culture consists of Streptomyces aureofaciens (Streptomyces aureofaciens) strain FJC-505CCTCC NO: m2021499 was obtained by culturing in a medium.

Further, the culture has a higher aureomycin content and/or a lower impurity content relative to Streptomyces aureofaciens D29 (starting strain, supplied by Jinhe Biotech Co., Ltd.).

The term "culture" refers to a general term for liquid or solid products (all substances within a culture vessel) on which a population of microorganisms has grown after artificial inoculation and culture. I.e. a product obtained by growing and/or amplifying a microorganism, which may be a biologically pure culture of the microorganism, or which may contain a certain amount of a culture medium, metabolite or other component produced during the cultivation. The term "culture" also includes subcultures obtained by passaging the microorganism, which may be a generation of culture or a mixture of generations. The ability of the (subculture) culture to produce aureomycin is essentially the same as that of the S.aureofaciens strain FJC-505 of the present invention.

The invention also provides a method for preparing a culture of streptomyces aureofaciens, comprising: culturing the streptomyces aureofaciens FJC-505 in a culture medium to obtain a streptomyces aureofaciens culture.

The culture is to culture the streptomyces aureofaciens FJC-505 under the condition suitable for culturing the streptomyces aureofaciens to produce aureomycin. It is well known to those skilled in the art that various methods can be used to culture the strain of the present invention, batch method or continuous method such as fed-batch method or repeated fed-batch method, etc., but the present invention is not limited thereto.

In the above method, the culture medium comprises a seed culture medium and/or a fermentation culture medium, and the seed culture medium and the fermentation culture medium contain ammonium chloride, calcium chloride and magnesium chloride.

In the above method, the seed culture medium may have the following composition: 30.0g/L of corn starch, 15.0g/L of soybean cake powder, 10.0g/L of peanut cake powder, 3.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 1.2g/L of calcium chloride, 1.8g/L of calcium carbonate, 5.0g/L of peptone, 5.0g/L of yeast powder, 0.05g/L of magnesium chloride, 0.05g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of soybean oil and the balance of water.

In the above method, the composition of the fermentation medium may be: 60.0g/L of corn starch, 25.0g/L of peanut cake powder, 20.0g/L of corn flour, 10.0g/L of soybean cake powder, 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of corn steep liquor, 2.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 2.0g/L of calcium chloride, 3.0g/L of calcium carbonate, 0.1g/L of magnesium chloride, 0.1g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of amylase, 20.0g/L of soybean oil and the balance of water.

In the above method, the culture medium further comprises a spore culture medium, the spore culture medium is composed of bran and a nutrient solution with equal mass, and the nutrient solution may be composed of: 30.0g/L of corn starch, 2.0g/L of peanut cake powder, 5.0g/L of yeast powder, 3.0g/L of peptone, 2.0g/L of ammonium chloride, 2.0g/L of sodium chloride, 0.1g/L of monopotassium phosphate, 0.15g/L of magnesium chloride, 0.15g/L of magnesium sulfate and the balance of water.

The invention also provides application of the Streptomyces aureofaciens strain FJC-505 in preparation of aureomycin.

Further, the application can be the production of aureomycin by a fermentation method.

The invention also provides application of the culture in preparation of aureomycin.

The Streptomyces aureofaciens strain FJC-505CCTCC NO: m2021499 can efficiently synthesize and accumulate aureomycin in the liquid fermentation process, and the impurity tetracycline and demethyl aureomycin content is obviously reduced, and the genetic stability is good.

The method comprises the steps of preparing a streptomyces aureofaciens strain into a protoplast, carrying out ARTP mutagenesis treatment, then carrying out ultraviolet mutagenesis, coating a mutagenesis suspension on a regeneration culture medium plate containing aureomycin hydrochloride, LiCl and NaCl with a certain concentration for directional primary screening, selecting mutant strains with the characteristics of high strain growth speed, obvious spore color difference, regular colony morphology and the like for secondary screening by shake flask fermentation, and detecting aureomycin yield and main impurity content by HPLC (high performance liquid chromatography), thereby finally obtaining the excellent mutant strains with high yield and stable heredity.

The starting strain is Streptomyces aureofaciens D29 provided by Jinhe Biotechnology GmbH.

The preparation of the protoplast comprises the steps of firstly preparing spore suspension, then carrying out liquid culture to collect mycelium, and finally carrying out enzymolysis wall breaking under certain conditions to prepare the protoplast.

The spore suspension is obtained by solid slant culture, and the glycerol strain of original strain Streptomyces aureofaciens D29 is inoculated into slant culture medium (18X 180mm test tube slantThe preparation method of the slant and the solid plate culture medium comprises the following steps: 40.0g of bran, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, distilled water with constant volume of 1000mL, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30min), culturing the inoculated strain for 4 days under the conditions of 34 ℃ and 45% of humidity, eluting spores in a solid inclined plane by using 5mL of sterile physiological saline to prepare spore eluent, and filtering the spore eluent by using sterile filter paper to obtain spore suspension, wherein the concentration of the spores in the spore suspension is 2.3 multiplied by 10⁹one/mL.

The mycelium is obtained by liquid culture, 1mL of spore suspension is sucked into a mycelium culture medium S1 in a 30mL/250mL triangular flask (with a spring) (the preparation method of the mycelium culture medium S1 is as follows: 5.0g of peptone, 3.0g of yeast extract, 1.5g of beef extract, 1.0g of glucose, 3.5g of sodium chloride, 3.68g of dipotassium hydrogen phosphate, 1.32g of potassium dihydrogen phosphate, pH 7.2, distilled water constant volume to 1000mL,121 ℃ high-pressure steam sterilization for 20-30min), the mycelium culture medium S2 is placed in a 30 ℃ and 270r/min constant temperature oscillator for culturing for 48h, the mycelium culture medium is transferred into a mycelium culture medium S2 in a 1 mL/30 mL/250mL triangular flask (with a spring placed at the bottom) (the preparation method of the constant volume culture medium S2 is as follows: 6.0g of peptone, 3.0g of yeast extract, 1.5g of beef extract, 1.0g of glucose, 15.0g of glycine, 1000mL of distilled water, 6.8 to 1000, 6.20 min constant volume of pH steam sterilization at 121 ℃) at 121 ℃, and continuously culturing for 24h at 30 ℃ under the condition of 270r/min, centrifuging the culture solution at 4000r/min for 15min after the culture is finished, collecting mycelium, carrying out heavy suspension and cleaning twice by using 10.3% of sucrose solution, centrifuging, and removing supernatant to finally obtain the collected mycelium of the starting strain streptomyces aureofaciens D29.

The enzymolysis wall breaking under the certain conditions is prepared by adopting mixed enzymolysis liquid of muramidase (purchased from Guangdong microorganism strain preservation center), lysozyme and cellulase (all purchased from biological engineering (Shanghai) corporation), wherein the using concentration of the muramidase enzymolysis liquid is 0.5-2.5%, the optimal enzymolysis concentration is 0.5-1.0%, the concentration of the lysozyme enzymolysis liquid is 0.5-2.5%, the optimal enzymolysis concentration is 0.5-1.0%, the optimal concentration of the cellulase is 0.2-1.5%, the optimal concentration of the cellulase is 0.5-1.0%, the enzymolysis temperature range is 24-35 ℃, the optimal enzymolysis temperature is 26-30 ℃, the enzymolysis time is 2-8h, and the optimal enzymolysis temperature is 2-30 DEG CThe enzymolysis time is 3-5h, the enzymolysis pH is 5.0-7.0, and the optimum enzymolysis pH is 6.2-6.8. The prepared protoplast was finally resuspended in 1mL of 10.3% sucrose solution to obtain a protoplast suspension, and the number of the prepared protoplasts reached 2.2X 10⁷The regeneration rate reaches 23.5 percent per mL.

The ultraviolet mutagenesis is carried out after the ARTP treatment, which means that an ARTP mutagenesis instrument (purchased from No-Sn source Qingtian Wood Biotech Co., Ltd.) is firstly adopted to carry out mutagenesis treatment on the protoplast, and the conditions are as follows: taking 900 mu L of the protoplast suspension, adding 100 mu L of 50% glycerol, fully and uniformly mixing, sucking 10 mu L of the protoplast suspension, uniformly coating the protoplast suspension on a slide, uniformly covering the surface of the slide, drying, and transferring the slide with the protoplast suspension to an objective table by using sterile forceps. Adopting high-purity helium as working gas of the plasma, setting power supply power of 80W, setting irradiation distance of 4mm, temperature of the plasma of 26-30 ℃, gas flow of 10L/min, treating the bacterial slide for 30s, closing the carrier gas after treatment, opening the ultraviolet lamp, and continuing to treat for 25 s. After treatment, the slide glass was transferred to an EP tube containing 1mL of a hypertonic solution (wherein the hypertonic solution is prepared by adding 103.0g of sucrose, 0.25g of potassium sulfate, 2.0g of magnesium chloride, 2.0mL of a trace element solution and 800.0mL of distilled water to 80.0mL of the hypertonic solution, and after sterilization, 1.0mL of filter sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris were added to each 80.0mL of the solution, and the composition of the trace element (mg/L) was ZnCl₂ 40.0，FeCl₃·6H₂O 200.0，CuCl₂·2H₂O10.0，MnCl₂·4H₂O 10.0，Na₂B₄O₇·10H₂O 10.0，(NH₄)6Mo₇O₂₄·4H₂O10.0 and the balance of water), and shaking and eluting to form the protoplast suspension after mutagenesis treatment.

The directional primary screening is to apply the treated protoplast suspension to a regeneration culture medium plate containing lithium chloride with the final concentration of 1.5%, sodium chloride with the final concentration of 2.5% and aureomycin hydrochloride with the final concentration of 5.0g/L after gradient dilution (the preparation method of the regeneration culture medium comprises the following steps of 40.0g of bran, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 103.0g of sucrose, 20.0g of agar, constant volume of distilled water to 1000mL, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30min), and culture at 30 ℃ in a dark place until monoclone grows out, namely the mutant strain after plate selection.

The shake flask fermentation re-screening is to select different colonies according to the colony size, color and morphological characteristics of a selective plate for shake flask fermentation verification, firstly, a mutant strain is transferred to a slant culture medium (the preparation method comprises the steps of 40.0g of bran, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, distilled water with the constant volume of 1000mL, natural pH, and high-pressure steam sterilization at 121 ℃ for 20-30min) at 34 ℃ and the relative humidity of 45%, slant activation culture is carried out for 3 days, 5mL of sterile water is added to the slant of the cultured mutant strain to prepare a spore suspension, 1mL of the spore suspension is inoculated into 25mL of a seed culture medium (the preparation method comprises the steps of 30.0g of corn starch, 15.0g of soybean cake powder, 10.0g of peanut cake powder, 3.0g of sodium chloride, 4.0g of ammonium sulfate, 3.0g of calcium carbonate, 5.0g of peptone, 5.0g of mother yeast powder, 0g of magnesium sulfate, 0.1g of monopotassium phosphate, 1.0g of soybean oil, and the like, wherein the volume of tap water is fixed to 1000mL, the pH is natural, the materials are sterilized by high-pressure steam at the temperature of 121 ℃ for 20-30min), and the materials are cultured for 22h at the temperature of 30 ℃ at 270 r/min; after the seeds are cultured, the seeds are transferred into a fermentation medium by 10 percent (volume ratio) of seed transfer amount (the preparation method comprises the following steps of 60.0g of corn starch, 25.0g of peanut cake powder, 20.0g of corn flour, 10.0g of soybean cake powder, 10.0g of peptone, 5.0g of yeast powder, 10.0g of corn steep liquor, 2.0g of sodium chloride, 4.0g of ammonium sulfate, 5.0g of calcium carbonate, 0.2g of magnesium sulfate, 0.1g of potassium dihydrogen phosphate, 1.0g of amylase, 20.0g of soybean oil, and carrying out fermentation culture for 6 days at 30 ℃ and 270r/min, wherein the volume of tap water is up to 1000mL, the pH is natural, the sterilization is carried out at 121 ℃ for 25min, and the fermentation culture is carried out for 6 days.

The HPLC detection refers to that after fermentation culture is finished, oxalic acid is added into fermentation liquor and stirred to be completely dissolved and uniformly mixed, the pH value of the fermentation liquor is adjusted to 1.5-2.0, the fermentation liquor is centrifuged for 15min at 4000rpm, supernatant is taken, 0.1mL of supernatant is taken, water is added into the supernatant to fix the volume to a 100mL volumetric flask according to estimated titer, and the volumetric flask is filtered by a 0.22 mu m microporous filter membrane and then HPLC is carried out to detect aureomycin content, tetracycline and demethyl aureomycin content (the detection method is liquid chromatography-mass spectrum/mass spectrum method and high performance liquid chromatography according to the detection method of the residual quantity of the tetracycline veterinary drugs in the animal-derived food GB/T21317-.

The genetic stability refers to that excellent positive mutant strains obtained by screening in a shake flask are subjected to continuous passage for five times through a slope, strains with stable forms and colors are selected, and shake flask fermentation is carried out to verify the yield and the main impurity content of the aureomycin. Finally, selecting a strain with stable aureomycin content in the passage process, namely an excellent mutant with stable heredity.

In the method, one of the obtained excellent strains is numbered as Streptomyces aureus auriculafaciens FJC-505, spores are light green and have obvious difference compared with the gray brown of the original strain, and the yield of aureomycin obtained by shake flask fermentation reaches 25.8g/L and is improved by 12.1 percent compared with the original strain, wherein the proportion of tetracycline content in the aureomycin content is 7.5 percent, the proportion of noraureomycin content in the aureomycin content is reduced by 11.8 percent compared with the original strain, the proportion of noraureomycin content in the aureomycin content is 0.28 percent, and the proportion of noraureomycin content in the aureomycin content is reduced by 50.0 percent compared with the original strain. Streptomyces aureofaciens FJC-505, which has been preserved in China Center for Type Culture Collection (CCTCC for short; address: China, Wuhan university China Center for Type Culture Collection, postal code 430072) at 5 months and 7 days 2021, with the preservation number of CCTCC NO: m2021499, categorically named Streptomyces aureus species FJC-505.

The second of the two strains is named as Streptomyces aureus FJW-507, spores are light yellow, and have obvious difference compared with the gray brown of the original strain, the yield of aureomycin obtained by shake flask fermentation reaches 25.5g/L and is improved by 10.9 percent compared with the original strain, wherein the proportion of tetracycline content in aureomycin content is 7.1 percent, the proportion of noraureomycin content in aureomycin content is reduced by 16.5 percent compared with the original strain, the proportion of noraureomycin content in aureomycin content is 0.25 percent, and the proportion of noraureomycin content in aureomycin content is reduced by 55.4 percent compared with the original strain. Streptomyces aureofaciens FJW-507, which has been preserved in China Center for Type Culture Collection (CCTCC for short; address: China, Wuhan university China Center for Type Culture Collection, postal code 430072) at 5 months and 7 days 2021, with the preservation number being CCTCC NO: m2021500, categorically named Streptomyces aureus species FJW-507.

Another purpose of the invention is to provide an optimized culture medium and a liquid fermentation culture method which are beneficial to the accumulation of high-content aureomycin by the excellent aureomycin mutant strain.

The activity of a key rate-limiting enzyme in aureomycin biosynthesis, namely, the chloroase and the affinity with chloride ions have important influence on aureomycin biosynthesis, and the chloride ions are used as precursors for aureomycin synthesis and have higher possibility of obtaining high-yield aureomycin for chloride ion-tolerant mutant strains. Therefore, the method takes high-concentration lithium chloride, sodium chloride and a product chlortetracycline as a first-stage screening method in screening, and contributes to the biosynthetic metabolic flux of chlortetracycline. In addition, the streptomyces aureofaciens is easy to generate an aging phenomenon in the liquid fermentation process, so that the synchronization of the growth of the strain is reduced, the biosynthesis capacity of aureomycin is weakened, the synthesis of other homologues is increased, and the synthesis and accumulation of aureomycin in a fermentation tank by the excellent strain are influenced finally. The seed culture is strengthened, the growth cycle of the seeds of the strain is greatly reduced, and the synthesis cycle of the aureomycin is enhanced.

The method has the obvious characteristics that the promotion of chloride ions on aureomycin biosynthesis is enhanced through an optimized formula, the seed culture is enhanced, the strain activity is greatly improved, the fermentation period is shortened, the cost is saved, the aureomycin synthesis is effectively promoted, and the content of homologous compound impurities is reduced.

In one embodiment of the present invention, the optimized formula mainly comprises a seed formula and a fermentation formula, and specifically, the optimized seed culture medium obtained by replacing ammonium sulfate with equal ammonium chloride, calcium chloride with calcium carbonate added in an amount of 40% and magnesium chloride with magnesium sulfate added in an amount of 50% respectively comprises: 30.0g/L of corn starch, 15.0g/L of soybean cake powder, 10.0g/L of peanut cake powder, 3.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 1.2g/L of calcium chloride, 1.8g/L of calcium carbonate, 5.0g/L of peptone, 5.0g/L of yeast powder, 0.05g/L of magnesium chloride, 0.05g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of soybean oil, the balance of water and natural pH. The optimized fermentation medium obtained consists of: 60.0g/L of corn starch, 25.0g/L of peanut cake powder, 20.0g/L of corn flour, 10.0g/L of soybean cake powder, 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of corn steep liquor, 2.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 2.0g/L of calcium chloride, 3.0g/L of calcium carbonate, 0.1g/L of magnesium chloride, 0.1g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of amylase, 20.0g/L of soybean oil, the balance of water and natural pH.

The intensified seed culture is characterized in that activated culture slant strains (slant of a test tube with the diameter of 18 multiplied by 180 mm) are washed by 5.0ml of sterile physiological saline to be removed to prepare spore suspension, the spore suspension is transferred to an eggplant bottle containing the slant of a seed intensified culture medium to prepare a culture medium which has large surface area, good ventilation capacity and easy spore propagation, and the culture medium is cultured in an incubator at the temperature of 34 ℃ for 3 days to further improve the germination number of spores and intensify the activity of hyphae, thereby achieving the aim of the intensified culture of strain seeds.

The preparation method of the seed strengthening culture medium comprises the following steps: weighing 30g of bran, uniformly mixing with 30mL of nutrient solution, steaming for 30min in a water-proof way, weighing 30g of bran nutrient solution culture medium in an eggplant bottle after steaming, and sterilizing at 121 ℃ for 20-30min under high temperature and high pressure according to a conventional method. The obtained bran kernel reinforced culture medium (also called spore culture medium) is prepared.

The nutrient solution comprises the following components: 30.0g/L of corn starch, 2.0g/L of peanut cake powder, 5.0g/L of yeast powder, 3.0g/L of peptone, 2.0g/L of ammonium chloride, 2.0g/L of sodium chloride, 0.1g/L of monopotassium phosphate, 0.15g/L of magnesium chloride, 0.15g/L of magnesium sulfate and the balance of water.

The fermentation culture refers to strain culture by adopting an enhanced seed culture medium, and fermentation by adopting optimized seed culture medium and fermentation culture medium components. The yield of the aureomycin produced by shake flask fermentation of the strain streptomyces aureofaciens FJC-505 is 26.88g/L, which is improved by 4.2 percent compared with that before optimization, wherein the tetracycline accounts for 6.8 percent of the aureomycin content, which is reduced by 9.3 percent compared with that before optimization, the demethyl aureomycin accounts for 0.22 percent compared with that before optimization, which is reduced by 21.4 percent. The yield of aureomycin produced by shake flask fermentation of the streptomyces aureofaciens FJW-507 is 26.52g/L, which is improved by 4.0 percent compared with that before optimization, wherein the tetracycline accounts for 6.3 percent of aureomycin content, which is reduced by 11.3 percent compared with that before optimization, the demethyl aureomycin accounts for 0.20 percent compared with that before optimization, which is reduced by 20.0 percent.

Experiments prove that compared with the prior art, the invention has the following advantages:

the invention screens and obtains the excellent streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion based on the normal-pressure room-temperature plasma and ultraviolet composite mutagenesis technology, and the yield of the aureomycin generated by the mutant strain is higher than that of the original strain. The streptomyces aureofaciens mutant strain can efficiently synthesize and accumulate aureomycin in the liquid fermentation process, has obviously reduced contents of tetracycline and demethylaureomycin impurities, can be applied to industrial fermentation production, has good genetic stability, has basically stable form, color, aureomycin production level and main impurity content after five generations of continuous passage, maintains aureomycin content at the same higher level, and can be used as a production strain for further research and development.

The yield of aureomycin obtained by shake flask fermentation of the bred streptomyces aureofaciens FJC-505 strain can reach 26.88g/L, and is increased by 16.9 percent compared with the original strain, wherein the tetracycline content accounts for 6.8 percent of the aureomycin content, the tetracycline content is reduced by 20.0 percent compared with the original strain, the demethyl aureomycin content accounts for 0.22 percent of the aureomycin content, and the demethyl aureomycin content is reduced by 60.7 percent compared with the original strain.

The yield of aureomycin obtained by shake flask fermentation of the bred streptomyces aureofaciens FJW-507 strain can reach 26.52g/L, and is improved by 15.3 percent compared with the original strain, wherein the tetracycline content accounts for 6.3 percent of the aureomycin content, the tetracycline content is reduced by 25.9 percent compared with the original strain, the demethyl aureomycin content accounts for 0.20 percent of the aureomycin content, and the demethyl aureomycin content is reduced by 64.3 percent compared with the original strain.

Experiments prove that the bred streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507 strains have better chloride ion tolerance and transformation capability, the capability of synthesizing and accumulating aureomycin is obviously enhanced, the contents of main impurities tetracycline and demethyl aureomycin are obviously reduced, the post-treatment cost of aureomycin products can be greatly reduced, the product quality is improved, the excellent capability of producing aureomycin premix and hydrochloride thereof is realized, the strains accord with the high yield and high quality of industrial production, the enterprise cost can be reduced, the enterprise profit is increased, and the strain has wide application value and important economic significance for the industrial production of aureomycin.

In addition, the invention strengthens the promotion of chloride ions to the biosynthesis of aureomycin through an optimized formula, simultaneously strengthens seed culture, greatly improves the activity of strains, shortens the fermentation period, saves the cost, not only effectively promotes the synthesis of aureomycin, but also is beneficial to reducing the content of homologous compound impurities.

Deposit description

The strain name is as follows: streptomyces aureofaciens

Latin name: streptomyces aureofaciens

The strain number is as follows: FJC-505

The preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

Address: china center for type culture Collection, zip code 430072, university of Wuhan, China

The preservation date is as follows: 2021, 5 months and 7 days

Registration number of the preservation center: CCTCC NO: m2021499

The strain name is as follows: streptomyces aureofaciens

Latin name: streptomyces aureofaciens

The strain number is as follows: FJW-507

The preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

Address: china center for type culture Collection, zip code 430072, university of Wuhan, China

The preservation date is as follows: 2021, 5 months and 7 days

Registration number of the preservation center: CCTCC NO: m2021500

Drawings

FIG. 1 shows a comparison of the colonies of the mutagenized strains (S.aureofaciens FJC-505 and S.aureofaciens FJW-507) with the starting strain (S.aureofaciens D29).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged.

The Streptomyces aureofaciens D29 strain and the Streptomyces aureofaciens F3 strain (Rongpenfei. optimization of the seed production of aureomycin and fermentation process research [ D ]. university of inner Mongolia, 2013.) in the following examples were publicly available from the applicant as the biomaterial, which was used only for repeating the experiment of the present invention and was not used for other purposes.

Example 1 Breeding of Streptomyces aureofaciens FJC-505

On the basis of an original strain streptomyces aureofaciens D29 strain, a protoplast composite mutagenesis method is adopted to improve the yield of aureomycin, reduce impurities and breed excellent strains with stable heredity, and the specific method is as follows:

1. collection of mycelia of the original Strain

Inoculating glycerol strain of original strain Streptomyces aureofaciens D29 to solid slant culture medium (slant of 18 × 180mm test tube, wherein the slant culture medium and solid plate culture medium are prepared by adding bran 40.0g, potassium dihydrogen phosphate 0.2g, magnesium sulfate 0.2g, diammonium hydrogen phosphate 0.5g, agar 20.0g, distilled water to a constant volume of 1000mL, sterilizing at 121 deg.C under high pressure steam for 20-30min), culturing the inoculated strain at 34 deg.C and humidity of 45% for 4 days, eluting spore in the solid slant with 5mL of sterile physiological saline to prepare spore eluate, and filtering the spore eluate with sterile filter paper to obtain spore suspension containing spore concentration of 2.3 × 10⁹one/mL.

Sucking 1mL of spore suspension into a 30mL/250mL triangular flask (with spring) mycelium culture medium S1 (the preparation method of the mycelium culture medium S1 is that peptone 5.0g, yeast extract 3.0g, beef extract 1.5g, glucose 1.0g, sodium chloride 3.5g, dipotassium hydrogen phosphate 3.68g, potassium dihydrogen phosphate 1.32g, pH 7.2, distilled water with constant volume of 1000mL, high-pressure steam sterilization at 121 ℃ for 20-30min), placing in a 30 ℃ and 270r/min constant-temperature oscillator for culturing for 48h, transferring into the mycelium culture medium S2 (the mycelium culture medium S2 is prepared by placing a spring at the bottom of the 1mL to 30mL/250mL triangular flask (with constant volume of the bottom) and placing the spring at the following steps of peptone 6.0g, yeast extract 3.0g, beef extract 1.5g, glucose 1.0g, glycine 15.0g, distilled water with constant volume of 1000mL, high-pressure steam sterilization at 121 ℃ for 20min, continuing culturing at 270r/min, and after the culture is finished, centrifuging the culture solution at 4000r/min for 15min to collect mycelium, resuspending and cleaning twice by using 10.3% of sucrose solution, centrifuging and removing supernatant, and finally obtaining the mycelium of the collected starting strain streptomyces aureofaciens D29 strain.

2. Preparation of starting Strain protoplast suspension

Taking the mycelium of the original strain streptomyces aureofaciens D29 strain prepared in the step 1 as a raw material, adopting a mixed enzymolysis liquid of muramidase (purchased from Guangdong microbial strain preservation center), lysozyme and cellulase (purchased from Biotechnology engineering (Shanghai) GmbH), wherein the use concentration of the muramidase enzymolysis liquid is 0.5-2.5%, the optimal enzymolysis concentration is 0.5-1.0%, the concentration of the lysozyme enzymolysis liquid is 0.5-2.5%, the optimal enzymolysis concentration is 0.5-1.0%, the use concentration of the cellulase is 0.2-1.5%, the optimal concentration is 0.5-1.0%, the enzymolysis temperature range is 24-35 ℃, the optimal enzymolysis temperature is 26-30 ℃, the enzymolysis time is 2-8h, the optimal enzymolysis time is 3-5h, the enzymolysis pH is 5.0-7.0, and the optimal enzymolysis pH is 6.2-6.8, this is the best condition for the preparation of protoplasts of streptomyces aureofaciens D29, and the efficiency of protoplast preparation is the highest (microscopic observation shows that the mycelium forms protoplasts almost entirely), and the regeneration rate of protoplasts is also better (regeneration rate ═ number of regenerated colonies/total number of protoplasts × 100%). The prepared protoplast is finally used for resuspending the cells by 1mL of 10.3 percent sucrose solution to obtain the protoplast suspension of the streptomyces aureofaciens D29 strain, and the number of the prepared protoplast reaches 2.2 multiplied by 10⁷The regeneration rate reaches 23.5 percent per mL.

3. Complex mutagenesis treatment of starting strain protoplast

3-1 ARTP (atmospheric pressure room temperature plasma) mutagenic instrument treatment for preliminary experiment

The protoplast of the S.aureofaciens D29 strain prepared in step 2 was first mutagenized by an ARTP mutagenizer (purchased from Qingtian Wood Biotech Co., Ltd., No. Sn source), subjected to preliminary experiments, and the lethality was calculated. The pre-experimental steps before compound mutagenesis and the method for calculating the lethality are as follows:

10-20. mu.L of the protoplast suspension of the S.aureus D29 strain prepared in step 2 was uniformly coated on the upper surface of a metal slide, and after drying, the slide was transferred to a stage with tweezers. High-purity helium gas is adopted as working gas of plasma, the power supply power is set to be 80W, the irradiation distance is 4mm, the temperature of the plasma is 26-30 ℃, the gas flow is 10L/min, the bacterial slide is treated, different treatment groups are set, and the treatment time of each treatment group is 0s (comparison), 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s and 55 s. After treatment, the slide glass was transferred to an EP tube containing 1mL of a hypertonic solution (wherein the hypertonic solution is prepared by adding 103.0g of sucrose, 0.25g of potassium sulfate, 2.0g of magnesium chloride, 2.0mL of a trace element solution and 800.0mL of distilled water to 80.0mL of the hypertonic solution, and after sterilization, 1.0mL of filter sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris were added to each 80.0mL of the solution, and the composition of the trace element (mg/L) was ZnCl₂ 40.0，FeCl₃·6H2O 200.0，CuCl₂·2H2O 10.0，MnCl₂·4H₂O 10.0，Na₂B₄O₇·10H₂O10.0，(NH₄)6Mo₇O₂₄·4H₂O10.0 and the balance of water), shaking and eluting to form a new bacterial suspension, and coating the new bacterial suspension on a regeneration plate (the preparation method of the regeneration medium is as follows: 40.0g of bran, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 103.0g of sucrose, 20.0g of agar, distilled water with constant volume of 1000mL, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30min), placing in an incubator at 34 ℃ for culturing for 4 days, counting plate colonies, and calculating the lethality:

percent lethality ═ number of colonies not subjected to mutagenesis treatment-number of colonies subjected to mutagenesis treatment)/number of colonies not subjected to mutagenesis × 100%

Three replicates of each treatment group were irradiated for a period of 70-75% lethality for a formal complex mutagenesis experiment, and the treatment time was determined to be 30s by computational statistics.

3-2 Complex mutagenesis

The specific compound mutagenesis mode of the protoplast of the original strain streptomyces aureofaciens D29 is as follows: and (3) adding 100 mu L of 50% glycerol into 900 mu L of the streptomyces aureofaciens D29 strain protoplast suspension prepared in the step (2), fully and uniformly mixing, absorbing 10 mu L of the mixture, uniformly coating the mixture on a metal slide, uniformly covering the surface of the metal slide with the protoplast suspension, drying, and transferring the slide attached with the protoplast suspension to a carrying table by using sterile forceps. Adopting high-purity helium as working gas of the plasma, setting power supply power of 80W, setting irradiation distance of 4mm, temperature of the plasma of 26-30 ℃, gas flow of 10L/min, treating the bacterial slide for 30s, closing the carrier gas after treatment, opening the ultraviolet lamp, and continuing to treat for 25 s. Transferring the slide glass into an EP tube containing 1mL of hypertonic solution after ultraviolet treatment (wherein the preparation method of the hypertonic solution comprises the following steps of 103.0g of sucrose, 0.25g of potassium sulfate, 2.0g of magnesium chloride, 2.0mL of trace element solution and 800.0mL of distilled water, and adding 1.0mL of filter sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris into 80.0mL of the solution after sterilization, wherein the composition of the trace elements is (mg/L): ZnCl)₂ 40.0，FeCl₃·6H2O 200.0，CuCl₂·2H2O10.0，MnCl₂·4H₂O 10.0，Na₂B₄O₇·10H₂O 10.0，(NH₄)6Mo₇O₂₄·4H₂O10.0 and the balance of water), shaking and eluting to form a protoplast suspension of the streptomyces aureofaciens D29 strain after compound mutagenesis treatment.

4. Screening of mutant strains

Diluting the protoplast suspension of the streptomyces aureofaciens D29 strain subjected to the compound mutagenesis treatment in the step 3 by 1000 times, and coating 100. mu.L of diluent on a selective plate, namely a regeneration culture medium plate containing 1.5 percent of lithium chloride, 2.5 percent of sodium chloride and 5.0g/L of aureomycin hydrochloride (the preparation method of the regeneration culture medium comprises the following steps of 40.0g of bran, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium phosphate, 103.0g of sucrose, 20.0g of agar, constant volume of distilled water to 1000mL, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30min), and gently shaking up in the sampling and coating processes to avoid bottom deposition. The selective plates were incubated at 30 ℃ in the dark until single colonies grew out.

And selecting single colonies according to the size, color and morphological characteristics of the colonies on the selective plate, and selecting 360 colonies with large colonies, regular edges and different colony colors for shake flask fermentation verification.

Firstly, respectively transferring 360 mutant strains to a slant culture medium (the preparation method comprises the steps of 40.0g of bran, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, constant volume of distilled water to 1000mL, high-pressure steam sterilization at 121 ℃ for 20-30min), carrying out slant activation culture at 34 ℃ and a relative humidity of 45% for 3 days, adding 5mL of sterile water into the slant of the cultured mutant strain to prepare a spore suspension, inoculating 1mL of the spore suspension into 25mL of seed culture medium (the preparation method comprises the steps of 30.0g of corn starch, 15.0g of soybean cake powder, 10.0g of peanut cake powder, 3.0g of sodium chloride, 4.0g of ammonium sulfate, 3.0g of calcium carbonate, 5.0g of peptone, 5.0g of yeast powder, 0.1g of magnesium sulfate, 0.1g of potassium dihydrogen phosphate, 1.0g of soybean oil, constant volume of tap water to 1000mL, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30min), culturing at 30 deg.C and 270r/min for 22 h; after the seeds are cultured, the seeds are transferred into a fermentation medium by 10 percent (volume ratio) of seed transfer amount (the preparation method comprises the following steps of 60.0g of corn starch, 25.0g of peanut cake powder, 20.0g of corn flour, 10.0g of soybean cake powder, 10.0g of peptone, 5.0g of yeast powder, 10.0g of corn steep liquor, 2.0g of sodium chloride, 4.0g of ammonium sulfate, 5.0g of calcium carbonate, 0.2g of magnesium sulfate, 0.1g of potassium dihydrogen phosphate, 1.0g of amylase, 20.0g of soybean oil, and carrying out fermentation culture for 6 days at 30 ℃ and 270r/min, wherein the volume of tap water is up to 1000mL, the pH is natural, the sterilization is carried out at 121 ℃ for 25min, and the fermentation culture is carried out for 6 days.

After the fermentation culture is finished, adding oxalic acid into the fermentation liquor, stirring to completely dissolve and uniformly mix the oxalic acid, adjusting the pH of the fermentation liquor to 1.5-2.0, centrifuging for 15min at 4000rpm, taking the supernatant, adding water into 0.1mL of the supernatant according to the estimated titer to fix the volume to a 100mL volumetric flask, filtering the solution through a 0.22 mu m microporous filter membrane, and detecting the aureomycin content, the tetracycline and the demethyl aureomycin content of the filtrate by High Performance Liquid Chromatography (HPLC) (the detection method is liquid chromatography-mass spectrum/mass spectrometry and high performance liquid chromatography according to the detection method of the residue quantity of the tetracycline veterinary drugs in the GB/T21317-2007 animal-derived food).

5. Acquisition of Excellent mutant Strain Streptomyces aureofaciens FJC-505

And (4) performing shake flask fermentation detection on 360 mutant strains selected in the step (4), obtaining 10 positive mutant strains with obviously improved aureomycin content (the aureomycin content is improved by more than 5%) by taking the original strain streptomyces aureofaciens D29 strain as a reference, performing shake flask fermentation re-screening on the 10 obtained mutant strains, and detecting the contents of aureomycin, tetracycline and demethylaureomycin according to the method in the step (4). The mutant strain with the number of FJC-505 is obtained from 10 positive mutant strains, spores of the mutant strain are light green, the difference of the spores in gray brown is obvious compared with the original strain D29, the yield of aureomycin obtained by shake flask fermentation reaches 25.8g/L and is improved by 12.1 percent compared with the original strain, wherein the tetracycline content accounts for 7.5 percent of the aureomycin content, the tetracycline content is reduced by 11.8 percent compared with the original strain, the demethyl aureomycin content accounts for 0.28 percent and is reduced by 50.0 percent compared with the original strain.

6. Verification of genetic stability

Subculturing the screened high-quality strain streptomyces aureofaciens FJC-505 to examine the genetic stability, carrying out passage once every three days and five times, carrying out shake flask fermentation to determine the contents of aureomycin, tetracycline and demethyl aureomycin of the strain, wherein the experimental result of the strain streptomyces aureofaciens FJC-505 subculture is as follows:

TABLE 1 Effect of passages on S.aureofaciens FJC-505 aureomycin content and impurities

Number of passages	Generation 1	Generation 2	Generation 3	Generation 4	5 generation
						Aureomycin content (g/L)	25.81	25.80	25.78	25.83	25.86
Tetracycline content (g/L)	1.94	1.93	1.93	1.94	1.94
						Demethylated aureomycin content (g/L)	0.072	0.072	0.068	0.072	0.073

The result shows that the five-generation fermentation level of the streptomyces aureofaciens FJC-505 has no obvious influence, the contents of the aureomycin, the tetracycline and the demethyl aureomycin in the fermentation liquor have no obvious change in the passage process, the aureomycin content is maintained at the same higher level, and the excellent genetic stability is realized.

The obtained Streptomyces aureofaciens (Streptomyces aureus) FJC-505 with stable heredity, high aureofaciens yield and low content of main impurities is preserved in China center for type culture Collection (CCTCC for short; address: China, Wuhan university China center for type culture Collection, postal code 430072) in 2021, 5 months and 7 days, and the preservation number is CCTCC NO: m2021499, hereinafter abbreviated as Streptomyces aureofaciens FJC-505.

The morphological characteristics of the streptomyces aureofaciens FJC-505 in a bran agar culture medium are as follows: culturing for 48h, wherein the colony is in straw hat shape, the whole is green, the edge has white bacteria halo, the spore is spherical or oval, and the surface is smooth.

Example 2: breeding of streptomyces aureofaciens FJW-507

On the basis of an original strain streptomyces aureofaciens D29 strain, a protoplast composite mutagenesis method is adopted to improve the yield of aureomycin, reduce impurities and breed excellent strains with stable heredity, and the specific method is as follows:

steps 1, 2, 3, 4, 5 and 6 are the same as the steps 1, 2, 3, 4, 5 and 6 for breeding the Streptomyces aureofaciens FJC-505 in example 1.

Wherein, the strain streptomyces aureofaciens FJW-507 passage fermentation experiment result is as follows:

TABLE 2 Effect of passages on aureomycin content and impurities from Streptomyces aureofaciens FJW-507

The result shows that the five-generation fermentation level of the streptomyces aureofaciens FJW-507 has no obvious influence, the contents of the aureomycin, the tetracycline and the demethyl aureomycin in the fermentation liquor have no obvious change in the passage process, the aureomycin content is maintained at the same higher level, and the excellent genetic stability is realized.

The mutant strain with the number of FJW-507 is obtained by breeding, spores are light yellow, the difference of gray brown compared with the original strain is obvious, the yield of aureomycin obtained by shake flask fermentation reaches 25.5g/L and is improved by 10.9 percent compared with the original strain, wherein the proportion of tetracycline content in aureomycin content is 7.1 percent, the proportion of noraureomycin content in aureomycin content is reduced by 16.5 percent compared with the original strain, and the proportion of noraureomycin content in aureomycin content is 0.25 percent and is reduced by 55.4 percent compared with the original strain.

Streptomyces aureofaciens FJW-507, which has stable heredity, high aureomycin yield and low content of main impurities, has been preserved in China center for type culture Collection (CCTCC for short; address: China, Wuhan university China center for type culture Collection, postal code 430072) at 5 months and 7 days 2021, with the preservation number of CCTCC NO: m2021500, hereinafter referred to as Streptomyces aureofaciens FJW-507.

Example 3: fermentation strengthening method for streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507

First, a seed enrichment medium (spore medium) was prepared: weighing 30g of bran, uniformly mixing with 30ml of nutrient solution, and steaming for 30min in a water-proof manner to obtain the bran nutrient solution culture medium. Weighing 30g of bran nutrient solution culture medium in an eggplant bottle, and sterilizing at 121 ℃ for 20-30min under high temperature and high pressure according to a conventional method. The preparation method of the nutrient solution comprises the following steps: 30.0g of corn starch, 2.0g of peanut cake powder, 5.0g of yeast powder, 3.0g of peptone, 2.0g of ammonium chloride, 2.0g of sodium chloride, 0.1g of monopotassium phosphate, 0.15g of magnesium chloride, 0.15g of magnesium sulfate and distilled water to reach the constant volume of 1000 ml. This is the bran seed enriched medium (spore culture medium).

Then, preparing streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507 spore suspensions, respectively taking 2.5mL of the spore suspensions to transfer into eggplant bottles containing bran seed enrichment culture medium (spore culture medium) (namely transferring into the culture medium which has large surface area, good ventilation capacity and easy spore propagation) to culture for 3 days at 34 ℃ in an incubator, further improving the germination number of spores, simultaneously strengthening the activity of hyphae and achieving the purpose of strain seed enrichment culture. Then, optimizing a seed formula and a fermentation formula according to the tolerance characteristics of chloride ions of the mutant strain, wherein the specific steps are respectively to replace ammonium sulfate with equal ammonium chloride in a seed culture medium and a fermentation culture medium, replace calcium chloride for 40% of the addition amount of calcium carbonate, replace magnesium chloride for 50% of the addition amount of magnesium sulfate, and obtain the optimized seed culture medium with the following composition: 30.0g/L of corn starch, 15.0g/L of soybean cake powder, 10.0g/L of peanut cake powder, 3.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 1.2g/L of calcium chloride, 1.8g/L of calcium carbonate, 5.0g/L of peptone, 5.0g/L of yeast powder, 0.05g/L of magnesium chloride, 0.05g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of soybean oil, the balance of water and natural pH. The composition of the optimized fermentation medium obtained was: 60.0g/L of corn starch, 25.0g/L of peanut cake powder, 20.0g/L of corn flour, 10.0g/L of soybean cake powder, 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of corn steep liquor, 2.0g/L of sodium chloride, 4.0g/L of ammonium chloride, 2.0g/L of calcium chloride, 3.0g/L of calcium carbonate, 0.1g/L of magnesium chloride, 0.1g/L of magnesium sulfate, 0.1g/L of potassium dihydrogen phosphate, 1.0g/L of amylase, 20.0g/L of soybean oil, the balance of water and natural pH.

The optimization method of the specific seed culture medium and the fermentation culture medium comprises the following steps:

according to the chlorine-resistant characteristic of the bred mutant strain, ammonium sulfate is used as an inorganic nitrogen source, magnesium sulfate and calcium carbonate are used as providers of magnesium metal and calcium metal in a fermentation formula of an original strain, and chloride ions are used as precursors for aureomycin synthesis, so that the chloride ions with a certain concentration are beneficial to conversion from tetracycline to aureomycin, the yield of the aureomycin is improved, and the content of the tetracycline in a product is reduced. However, too high a concentration of chloride ions may also have an adverse effect on the growth of the strain. The possibility of using ammonium chloride instead of ammonium sulphate, calcium chloride instead of calcium carbonate and magnesium chloride instead of magnesium sulphate is therefore considered. Firstly, the significant difference between the influence of one, two or three formula components of ammonium sulfate, calcium carbonate and magnesium sulfate on aureomycin synthesis and the influence of each chlorine source on aureomycin fermentation is respectively carried out. And then aiming at the significant factors, designing gradient level by taking the original addition amount as a reference, carrying out multi-factor uniform optimization experimental design of six-factor nine-level by adopting a uniform design method, carrying out chloride ion optimization on the whole fermentation culture medium formula, taking the contents of aureomycin, tetracycline and demethyl aureomycin as indexes, repeating three times in each level, and repeating three times in each experiment, wherein the experimental result is subjected to regression analysis and fitting verification by using a DPS18.10 data processing system, so as to finally obtain optimized seeds and a fermentation formula.

And finally, performing strain culture by adopting an enhanced seed culture medium, and performing fermentation verification on the streptomyces aureofaciens D29 strain, the streptomyces aureofaciens F3 strain, the streptomyces aureofaciens FJW-507 and the streptomyces aureofaciens FJC-505 by utilizing the optimized seed culture medium and the fermentation culture medium.

The specific method comprises the following steps: the experiment was carried out using 4 treatments, i.e., S.aureofaciens D29 strain treatment, S.aureofaciens F3 strain treatment, S.aureofaciens FJW-507 treatment and S.aureofaciens FJC-505 treatment.

Treating streptomyces aureofaciens FJC-505 (abbreviating FJC-505): streptomyces aureofaciens FJC-505 was inoculated into an eggplant flask containing the above-mentioned seed-strengthening medium, cultured in an incubator at 34 ℃ for 3 days, the spores were eluted with 5mL of sterile physiological saline to prepare a spore eluate, and the spore eluate was filtered using sterile filter paper to obtain a spore suspension. Inoculating the spore suspension into the optimized seed culture medium, and culturing at 30 ℃ and 270r/min for 22h to obtain a seed solution. Inoculating the seed solution into a 250mL triangular flask containing the optimized fermentation medium, wherein the inoculation amount is 20% (volume ratio), so that the thallus content in the optimized fermentation medium is 2.0 × 10⁸cfu/mL. Fermenting and culturing for 6 days at the temperature of 30 ℃ and at the speed of 270r/min, collecting fermentation liquor, adding oxalic acid into the fermentation liquor, stirring to completely dissolve the oxalic acid and uniformly mixing, adjusting the pH of the fermentation liquor to 1.5-2.0, centrifuging at 4000rpm for 15min, taking supernatant, taking 0.1mL of supernatant, adding water, fixing the volume to a 100mL volumetric flask, filtering by a 0.22-micron microporous filter membrane, and performing High Performance Liquid Chromatography (HPLC) on the filtrate to detect the content of aureomycin and the content of tetracycline and demethyl aureomycin (the detection method is liquid chromatography-mass spectrometry/mass spectrometry and high performance liquid chromatography in GB/T21317-2007 animal-derived food residual quantity detection methods of tetracycline veterinary drugs). Three replicates were set and 3 flasks were inoculated per replicate.

Treating streptomyces aureofaciens FJW-507 (FJW-507 for short): the same procedure was followed except that S.aureofaciens FJC-505 in the S.aureofaciens FJC-505 treatment was replaced with S.aureofaciens FJW-507.

Treating a streptomyces aureofaciens D29 strain (short for D29): the same procedure was followed except that S.aureofaciens FJC-505 in the S.aureofaciens FJC-505 treatment was replaced with S.aureofaciens D29 strain.

Treating a streptomyces aureofaciens F3 strain (F3 for short): the same procedure was followed except that S.aureofaciens FJC-505 in the S.aureofaciens FJC-505 treatment was replaced with S.aureofaciens F3 strain.

The results show that the original strains D29 and F3 can grow and metabolize in an optimized culture medium, but compared with the original culture medium, the aureomycin yield is respectively reduced by 8.7 percent and 7.8 percent, the impurity tetracycline is respectively increased by 13.2 percent and 7.2 percent, and the content of the demethylated aureomycin is not greatly changed. But has better chloride ion tolerance for mutant strains of streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507, and shows better aureomycin fermentation level, wherein:

the yield of the aureomycin produced by shake flask fermentation of the strain streptomyces aureofaciens FJC-505 is 26.88g/L, which is improved by 4.2 percent compared with that before optimization, wherein the tetracycline accounts for 6.8 percent of the aureomycin content, which is reduced by 9.3 percent compared with that before optimization, the demethyl aureomycin accounts for 0.22 percent compared with that before optimization, which is reduced by 21.4 percent. The yield of aureomycin produced by shake flask fermentation of the streptomyces aureofaciens FJW-507 is 26.52g/L, which is improved by 4.0 percent compared with that before optimization, wherein the tetracycline accounts for 6.3 percent of aureomycin content, which is reduced by 11.3 percent compared with that before optimization, the demethyl aureomycin accounts for 0.20 percent compared with that before optimization, which is reduced by 20.0 percent. The results of the yield and impurity measurement of the fermentation production of aureomycin by the mutant strains of Streptomyces aureofaciens FJC-505 and Streptomyces aureofaciens FJW-507 are shown in Table 3:

TABLE 3 yield and impurities of fermentation production of aureomycin by Streptomyces aureofaciens FJC-505 and Streptomyces aureofaciens FJW-507

Treatment of	Aureomycin (g/L)	Tetracycline (g/L)	Demethyl aureobasidium (g/L)
				D29	21.00±0.11	2.58±0.02	0.150±0.001
F3	23.50±0.16	2.78±0.05	0.161±0.001
				FJC-505	26.88±0.14	1.83±0.01	0.059±0.0003
FJW-507	26.52±0.12	1.67±0.01	0.053±0.0002

The results show that: the bred streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507 bacterial strains have better chloride ion tolerance and transformation capability on the basis of the reinforced culture of the invention, the capability of synthesizing and accumulating aureomycin is obviously enhanced, the contents of main impurities tetracycline and demethyl aureomycin are obviously reduced, the post-treatment cost of aureomycin products is reduced, the product quality is improved, and the excellent capability of producing aureomycin premix and hydrochloride thereof is provided.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.