CN113388556B - Method for producing aureomycin by using streptomyces aureofaciens - Google Patents
Method for producing aureomycin by using streptomyces aureofaciens Download PDFInfo
- Publication number
- CN113388556B CN113388556B CN202110901176.4A CN202110901176A CN113388556B CN 113388556 B CN113388556 B CN 113388556B CN 202110901176 A CN202110901176 A CN 202110901176A CN 113388556 B CN113388556 B CN 113388556B
- Authority
- CN
- China
- Prior art keywords
- aureomycin
- culture
- streptomyces aureofaciens
- strain
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing aureomycin by using streptomyces aureofaciens. The invention promotes the synthesis and accumulation of aureomycin and further reduces the proportion of impurities tetracycline and demethylaureomycin by combining and optimizing the culture medium and the culture method. The invention constructs a high-yield strain breeding model based on multiple compound mutagenesis and precursor/product tolerance, not only has higher diversity of a mutation library, but also is simple, convenient and quick, improves the screening efficiency, reduces the screening workload, and has certain application value and guiding significance for reducing the production cost and improving the productivity of aureomycin and other streptomyces source antibiotics. 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.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing aureomycin by using Streptomyces aureofaciens.
Background
Aureomycin (chlorotetracycline) is a broad-spectrum antibiotic of the tetracycline class produced by fermentation of Streptomyces aureofaciens. Streptomyces aureofaciens belongs to actinomycetes with high aerobic and gram-positive color as an important medicinal microorganism. If the culture conditions are not proper, hyphae are aged, so that the yield of aureomycin is influenced to a great extent. 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.
Microbial fermentation is a complex biological process and is influenced by various factors, wherein the composition and the proportion of a culture medium are one of key factors. The requirements of different microorganisms for the nutrient composition of the medium are different, as are the raw materials required for different fermentative productions. Whether the composition and the proportion of the culture medium are proper or not can greatly influence the growth and the propagation of the production strains, the fermentation titer of target products, the subsequent refining process of the fermentation products and the quality and the purity of final products. The optimized formula is researched, so that the fermentation titer is improved, the cost is reduced, the energy consumption is reduced, and the interference on the ecological environment is reduced through the optimization of the culture medium, therefore, the selection of a proper culture medium and the design of a reasonable proportion have very important significance on fermentation production.
Disclosure of Invention
The invention aims to solve the technical problem of how to promote the aureomycin biosynthesis of the streptomyces aureofaciens by optimizing the culture medium, improve the strain activity, shorten the fermentation period and save the cost. 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 a method for producing aureomycin by using streptomyces aureofaciens, which comprises the following steps:
a1 Culturing streptomyces aureofaciens in an optimized culture medium to obtain a culture;
a2 Collecting chlortetracycline from the culture;
the optimized 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.
The collection can be an extraction separation, processing and refining of the target component, eventually into a product.
Further, the collection may include steps of culture (or fermentation broth) pretreatment and solid-liquid separation, extraction (primary purification), refining (high purification), or finished product processing.
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.
In the above method, the culturing comprises the steps of:
b1 Spore culture; b2 Seed culture; b3 ) fermentation culture.
In the method, the spore culture is to inoculate the streptomyces aureofaciens to the spore culture medium and culture the streptomyces aureofaciens under the condition of 34 ℃ and 45 percent of relative humidity.
In the method, the seed culture is to prepare the spores into suspension, inoculate the suspension in the seed culture medium, and culture at 30 ℃ and 270 r/min.
In the method, the fermentation culture is to transfer the seed culture solution obtained by the method to the fermentation culture medium according to the seed transfer amount of 10% of the volume ratio, and culture the seed culture solution at the temperature of 30 ℃ and at the speed of 270 r/min.
In the method, the Streptomyces aureofaciens is Streptomyces aureofaciens (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 preservation is CCTCC NO: m2021499.
In the method, the Streptomyces aureofaciens is Streptomyces aureofaciens (Streptomyces aureofaciens), the strain number of the Streptomyces aureofaciens is FJW-507, and the preservation number of the Streptomyces aureofaciens in China center for type culture preservation is CCTCC NO: m2021500.
The invention also provides application of the method in preparation of aureomycin.
Any of the media described herein are also within the scope of the invention. The culture medium can be a spore culture medium, a seed culture medium or a fermentation culture medium.
The invention also provides a culture for preparing aureomycin, which contains the streptomyces aureofaciens FJC-505 and/or FJW-507.
The culture is prepared from Streptomyces aureofaciens strain FJC-505 CCTCC NO: m2021499 and/or Streptomyces aureofaciens (Streptomyces aureofaciens) strain FJW-507 CCTCC NO: and (3) culturing by using M2021500.
Further, the culture has a higher aureomycin content and/or a lower impurity content relative to Streptomyces aureofaciens D29.
The term "culture" refers to a general term for a liquid or solid product (all substances in a culture vessel) on which a microorganism population grows 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 invention also provides application of the Streptomyces aureofaciens strain FJC-505 and/or FJW-507.
In the above application, the application may be an application in preparing 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-505 CCTCC NO: m2021499 and/or Streptomyces aureofaciens (Streptomyces aureofaciens) strain FJW-507 CCTCC NO: m2021500 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, then carrying out ARTP mutagenesis treatment, then carrying out ultraviolet mutagenesis, coating mutagenesis suspension on a regeneration culture medium flat plate containing aureomycin hydrochloride, liCl and NaCl with certain concentration to carry out directional prescreening, prescreening to select mutant strains with the characteristics of high strain growth speed, obvious spore color difference, regular colony morphology and the like to carry out shake flask fermentation rescreening, and detecting aureomycin yield and main impurity content by HPLC (high performance liquid chromatography), thus 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, inoculating glycerol strain of original strain Streptomyces aureofaciens D29 into slant culture medium (slant of test tube of 18 × 180mm, wherein the slant culture medium and solid plate culture medium are prepared by inoculating 40.0g of testa Tritici, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, diluting with distilled water to 1000mL, adjusting pH to natural, sterilizing with high pressure steam at 121 deg.C for 20-30 min), culturing the inoculated strain at 34 deg.C and 45% humidity for 4 days, eluting spore in solid slant with 5mL of sterile physiological saline to obtain spore eluate, and making the spore eluate be obtained by subjecting the spore to elution with 5mL of sterile physiological salineFiltering the spore eluate with sterile filter paper to obtain spore suspension containing spore with spore concentration of 2.3 × 10 9 One per mL.
The mycelium was obtained by liquid culture, and 1mL of spore suspension was pipetted into a 30mL/250mL triangular flask (with spring) of mycelium culture medium S1 (the preparation method of mycelium culture medium S1 was 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 phosphate, 1.32g of potassium dihydrogen phosphate, pH 7.2, distilled water with a constant volume of 1000mL, and high-pressure steam sterilization at 121 ℃ for 20-30min, culturing in a 30-270-r/min constant-temperature oscillator for 48h, transferring into a mycelium culture medium S2 in a 1-30-mL/250-mL triangular flask (with a spring placed at the bottom) (the preparation method of the mycelium culture medium S2 comprises the steps of culturing for 6.0g of peptone, 3.0g of yeast extract, 1.5g of beef extract, 1.0g of glucose, 15.0g of glycine, fixing the volume of distilled water with a constant volume of 1000mL, pH 6.8, high-pressure steam sterilization at 121 ℃ for 20-30 min), culturing for 24h under the condition of 270r/min, collecting the culture solution 4000r/min, collecting sucrose, washing with a 10.3% gold mycelium solution, centrifuging, and removing the supernatant of streptomyces strain D to obtain the finally, and centrifuging and collecting the streptomyces strain D.
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 (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 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. The prepared protoplast is finally re-suspended in 1mL of 10.3% sucrose solution to obtain protoplast suspension, and the number of prepared protoplasts reaches 2.2 × 10 7 The regeneration rate reaches 23.5 percent per mL.
The ARTP treatment is followed by ultraviolet mutagenesis, which means that an ARTP mutagenesis instrument (purchased from ARTP)In qingtian wood biotechnology limited without tin source) to mutagenize protoplasts under the conditions: 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 25s. After treatment, the slide glass was transferred to an EP tube containing 1mL of a hypertonic solution (wherein the hypertonic solution was prepared by mixing 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, and 1.0mL of filter sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris per 80.0mL of the above solution, wherein the composition of the trace element was (mg/L): znCl) 2 40.0，FeCl 3 ·6H 2 O 200.0，CuCl 2 ·2H 2 O 10.0，MnCl 2 ·4H 2 O 10.0，Na 2 B 4 O 7 ·10H 2 O 10.0，(NH 4 )6Mo 7 O 24 ·4H 2 O10.0 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 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 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 20-30 min), and culturing at 30 ℃ in the 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 to transfer a mutant strain to a slant culture medium (the preparation method is as follows: 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 constant volume to 1000mL, pH natural, and high-pressure steam sterilization at 121 ℃ for 20-30 min), to perform slant activation culture for 3 days, to add 5mL of sterile water to the slant of the cultured mutant strain to prepare a spore suspension, to inoculate 1mL of spore suspension to 25mL of seed culture medium (the preparation method is as follows: 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 calcium carbonate, 3.0g of calcium carbonate, 5.0g of peptone, 5.0g of mother powder, 5.0g of yeast powder, 0g of 10000.1 g of potassium dihydrogen phosphate, 1.1 g of soybean oil with constant volume to 20min, pH high-pressure sterilization at 121 ℃ for 30-30 mL of tap water, to 20 h, natural steam sterilization for 30 h/30/20 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, fixing the volume of tap water to 1000mL, naturally adjusting the pH value, sterilizing at 121 ℃ for 25min, and carrying out fermentation culture at 30 ℃ and 270r/min 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 the aureomycin content, the tetracycline and the demethyl aureomycin content (the detection method is according to the liquid chromatography-mass spectrum/mass spectrum method and the high performance liquid chromatography of the residue detection method of the tetracycline veterinary drugs in the GB/T21317-2007 animal-derived food).
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. The streptomyces aureofaciens FJC-505 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, classified and named as 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 has been preserved in China Center for Type Culture Collection (CCTCC for short) in 2021, 5 months and 7 days, and the address: china, wuhan university Center for Type Culture Collection, postal code 430072), the preservation number is CCTCC NO: m2021500, categorically named Streptomyces aureofaciens 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 aureomycin superior mutant strain.
The activity of a key rate-limiting enzyme in aureomycin biosynthesis, namely, the activity of 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 slant strains (slant of a test tube with the diameter of 18 multiplied by 180 mm) subjected to activated culture are eluted by 5.0ml of sterile physiological saline to prepare spore suspension, the spore suspension is transferred into an eggplant bottle containing the slant of a seed intensified culture medium to prepare a culture medium which is large in surface area, good in ventilation capacity and easy for spore propagation, the culture medium is cultured in an incubator at the temperature of 34 ℃ for 3 days, the germination number of spores is further increased, the hypha activity is intensified, and the aim of strain seed intensified culture is fulfilled.
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 performing high-temperature and high-pressure sterilization according to a conventional method at 121 ℃ for 20-30min. The culture medium is the prepared bran seed strengthening culture medium (also called spore culture medium).
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 medium components. The yield of the shake flask fermentation aureomycin 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 in shake flask fermentation of 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 strengthens the promotion of chloride ions to the biosynthesis of aureomycin by 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.
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, the content of tetracycline and demethylaureomycin in impurities is obviously reduced, the streptomyces aureofaciens mutant strain can be applied to industrial fermentation production, the streptomyces aureofaciens mutant strain has good genetic stability, 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 aureomycin content is maintained at the same higher level, so that the streptomyces aureofaciens mutant strain can be used as a production strain for further research and development.
By the optimized culture medium culture and culture method, the yield of aureomycin in the shake flask fermentation of the bred Streptomyces aureofaciens FJC-505 strain can reach 26.88g/L, and is improved 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 aureomycin content is reduced by 60.7 percent compared with the original strain.
By the optimized culture medium culture and culture method, the yield of the streptomyces aureofaciens FJW-507 strain bred by the invention through shake flask fermentation 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, is reduced by 25.9 percent compared with the original strain, and the demethylated aureomycin content accounts for 0.20 percent of the aureomycin content, and 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.
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 CC)
Address: wuhan, wuhan university, postcode 430072
The preservation date is as follows: 2021, 5 months and 7 days
The registration number of the collection 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: wuhan, wuhan university, postcode 430072
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 specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. 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 carried out in triplicate and the results averaged.
The Streptomyces aureofaciens D29 strain and Streptomyces aureofaciens F3 strain (Rongpeng. University of inner Mongolia, 2013. For the study of the selection of a producing strain of aureomycin and optimization of fermentation process) in the following examples were publicly available from the applicant as the biomaterial, which was used only for repeating the experiments 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 strain to solid slant culture medium (slant of 18 × 180mm test tube, wherein the slant culture medium and solid plate culture medium are prepared by preparing slant culture medium from 40.0g of testa Tritici, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, distilled water to 1000mL, sterilizing with high pressure steam at 121 deg.C for 20-30min, culturing inoculated strain at 34 deg.C and 45% humidity for 4 days, eluting spore in solid slant with 5mL of sterile physiological saline to obtain spore eluate, and filtering the eluate with sterile filter paper to obtain spore suspension containing spore with spore concentration of 2.3 × 10 9 One per mL.
Sucking 1mL of spore suspension into a 30mL/250mL triangular flask (with a spring) mycelium culture medium S1 (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 and 1.32g of potassium dihydrogen phosphate are pH 7.2, distilled water is subjected to constant volume to 1000mL, and high-pressure steam sterilization at 121 ℃ is carried out for 20-30 min), placing the mixture into a 30 ℃ and 270r/min constant-temperature oscillator for culturing for 48h, transferring the mixture into a 30mL/250mL triangular flask (the bottom of which is placed with a spring) (the preparation method of the mycelium 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, constant volume to 1000mL of distilled water, carrying out high-pressure steam sterilization at 121 ℃ for 20min, carrying out golden steam sterilization for 1.5 min, continuously carrying out centrifugal culture on streptomycete strain culture for two times, and collecting 15.0g of mycelium, and finally, and centrifuging culture solution for 15.10% of streptomycete strain D after finishing centrifugation, and obtaining strain suspension.
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, and adopting a mixed enzymolysis solution of lywallzyme (purchased from Guangdong culture collection center of microorganisms), lysozyme and cellulase (all purchased from bio-engineering (Shanghai) limited), wherein the use concentration of the lywallzyme enzymolysis solution is 0.5% -2.5%, the optimal enzymolysis concentration is 0.5% -1.0%, the concentration of the lysozyme enzymolysis solution 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%, the optimal enzymolysis pH is 6.2-6.8%, and the optimal condition for preparing the streptomyces D29 protoplast is obtained, the preparation efficiency is the highest (the basic formation of the protoplast is observed), and the regeneration rate of the mycelium is better than the total protoplast/regeneration rate of the protoplast is 100%). The prepared protoplast is finally used for resuspending 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 10 7 The regeneration rate reaches 23.5 percent per mL.
3. Composite mutagenesis treatment of starting strain protoplast
3-1ARTP (atmospheric pressure room temperature plasma) mutagenic instrument treatment for preliminary experiment
The protoplast of the Streptomyces aureofaciens D29 strain prepared in the step 2 was first subjected to mutagenesis treatment using an ARTP mutagenesis apparatus (purchased from Tokyo biosciences, 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:
and (3) taking 10-20 mu L of the streptomyces aureofaciens D29 strain protoplast suspension prepared in the step (2), uniformly coating the protoplast suspension on the upper surface of a metal slide, drying the suspension, and transferring the slide to a stage by using 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 55s. After treatment, the slide glass was transferred to an EP tube containing 1mL of a hypertonic solution (wherein the hypertonic solution was 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, and 1.0mL of filter-sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris to 80.0mL of the above solution after sterilization, wherein the composition of the trace element was (mg/L): znCl) 2 40.0，FeCl 3 ·6H2O 200.0，CuCl 2 ·2H2O 10.0，MnCl 2 ·4H 2 O 10.0，Na 2 B 4 O 7 ·10H 2 O 10.0，(NH 4 )6Mo 7 O 24 ·4H 2 O10.0 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 20-30 min), placing in an incubator at 34 ℃ for culturing for 4 days, counting plate colonies, and calculating the lethality:
mortality% = (number of colonies not mutagenized-number of colonies mutagenized)/number of colonies not mutagenized × 100%
Three replicates per treatment group were selected for a full-scale complex mutagenesis experiment with an exposure time of 70-75% lethality, which was determined by computational statistics to ultimately determine a treatment time of 30s.
3-2 Complex mutagenesis
Specific composition of protoplast of original strain streptomyces aureofaciens D29 strainThe mutagenesis mode 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 so that the surface of the metal slide is uniformly covered with the protoplast suspension, drying, and transferring the slide attached with the protoplast suspension to a carrying platform 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 25s. After the UV treatment, the slide glass was transferred to an EP tube containing 1mL of a hypertonic solution (wherein the hypertonic solution was prepared by mixing 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, and 1.0mL of filter sterilized 0.5% monopotassium phosphate, 10.0mL of 3.7% calcium chloride and 10.0mL of 2.0% Tris per 80.0mL of the solution after sterilization, wherein the composition of the trace element was (mg/L): znCl) 2 40.0，FeCl 3 ·6H2O 200.0，CuCl 2 ·2H2O 10.0，MnCl 2 ·4H 2 O 10.0，Na 2 B 4 O 7 ·10H 2 O 10.0，(NH 4 )6Mo 7 O 24 ·4H 2 O10.0 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, coating 100-300 mu L of diluent on a selective plate, namely 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 (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 hydrogen phosphate, 103.0g of sucrose, 20.0g of agar, fixing the volume of distilled water to 1000mL, naturally adjusting the pH value, sterilizing by high-pressure steam at 121 ℃ for 20-30 min), 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 following steps of 40.0g of bran, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.5g of diammonium hydrogen phosphate, 20.0g of agar, constant volume of distilled water to 1000mL, natural pH and high-pressure steam sterilization at 121 ℃ for 20-30 min), carrying out slant activation culture at 34 ℃ and a relative humidity of 45%, adding 5mL of sterile water to the slant of the cultured mutant strains to prepare a spore suspension, and inoculating 1mL of the spore suspension into 25mL of seed culture medium (the preparation method comprises the following 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 monopotassium phosphate, 1.0g of soybean oil, constant volume to 1000mL of tap water, natural pH, high-pressure steam sterilization at 121 ℃ for 20-30 min), and carrying out culture at a temperature of 270 r/270 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, fixing the volume of tap water to 1000mL, naturally adjusting the pH value, sterilizing at 121 ℃ for 25min, and carrying out fermentation culture at 30 ℃ and 270r/min 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) carrying out 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, carrying out 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 a gray brown color is obvious compared with that of an original strain D29, the yield of aureomycin in shake flask fermentation reaches 25.8g/L and is improved by 12.1 percent compared with the original strain, wherein the content of tetracycline accounts for 7.5 percent of the content of aureomycin, the content of noraureomycin accounts for 11.8 percent of the yield of the original strain, the content of noraureomycin accounts for 0.28 percent of the content of aureomycin, and the content of noraureomycin accounts for 50.0 percent of the yield of the original strain.
6. Verification of genetic stability
Subculturing the screened high-quality strain Streptomyces aureofaciens FJC-505 to examine the genetic stability of the strain, 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 subculturing of the strain Streptomyces aureofaciens FJC-505 is as follows:
TABLE 1 Effect of passages on Streptomyces aureofaciens FJC-505 aureomycin content and impurities
| Passage number | 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 liquid 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 aureofaciens) FJC-505 with stable heredity, high yield of aureomycin 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 referred to as Streptomyces aureofaciens FJC-505.
Morphological characteristics of streptomyces aureofaciens FJC-505 in a bran agar medium: 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, 6 are the same as the breeding steps 1, 2, 3, 4, 5, 6 of Streptomyces chromogenes 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 Streptomyces aureofaciens FJW-507 aureomycin content and impurities
The result shows that the five-generation fermentation level of the streptomyces aureofaciens FJW-507 has no obvious influence, the contents of the fermentation liquor aureomycin, tetracycline and demethyl aureomycin are not obviously changed in the passage process, the aureomycin content is maintained at the same higher level, and the excellent genetic stability is realized.
The mutant strain numbered 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 the aureomycin content is 7.1 percent, the proportion of noraureomycin content in the original strain is reduced by 16.5 percent, the proportion of noraureomycin content in the aureomycin content is 0.25 percent, and the proportion of noraureomycin content in the original strain is reduced by 55.4 percent.
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) in 2021, 5 months and 7 days, and the preservation number is 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 1000ml. 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 2.5 mL of the spore suspensions to transfer into eggplant bottles containing bran seed enrichment culture media (spore culture media) (namely, transferring into culture media which have large surface areas, 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, chloride ions are used as precursors for aureomycin synthesis, and the chloride ions with a certain concentration are beneficial to conversion from tetracycline to aureomycin, so that the yield of aureomycin is improved, and meanwhile, the content of tetracycline in a product can be reduced. However, too high a concentration of chloride ions may also adversely affect 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 (short for FJC-505): streptomyces aureofaciens FJC-505 is inoculated into an eggplant bottle filled with the seed strengthening medium, cultured in an incubator at 34 ℃ for 3 days, spore eluent is prepared by eluting the spores with 5mL of sterile normal saline, and the spore eluent is filtered by using sterile filter paper to obtain 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 × 10 8 cfu/mL. Performing fermentation culture at 30 ℃ at 270r/min for 6 days, collecting fermentation liquor, adding oxalic acid into the fermentation liquor, stirring to completely dissolve the oxalic acid and uniformly mix the oxalic acid, adjusting the pH of the fermentation liquor to 1.5-2.0, centrifuging at 4000rpm for 15min, taking supernate, taking 0.1mL supernate, adding water into the supernate, fixing the volume to a 100mL volumetric flask, filtering the supernate by a 0.22 mu m 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 demethylaureomycin (the detection method is according to the detection method of the residue quantity of the tetracyclic veterinary drug in GB/T21317-2007 animal-derived food, namely liquid chromatography-mass spectrometry/mass spectrometry and high performance liquid chromatography). Three replicates were set and 3 flasks were inoculated per replicate.
FJW-507 (FJW-507 for short): the operation is the same except that the Streptomyces aureofaciens FJC-505 in the treatment of the Streptomyces aureofaciens FJC-505 is replaced by the Streptomyces aureofaciens FJW-507.
Treating a streptomyces aureofaciens D29 strain (D29 for short): the operation is the same except that the streptomyces aureofaciens FJC-505 in the treatment of the streptomyces aureofaciens FJC-505 is replaced by the streptomyces 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 the S.aureofaciens F3 strain.
The results show that the starting 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 capability and shows better aureomycin fermentation level for mutant strains of streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507, wherein:
the yield of aureomycin in the strain streptomyces aureofaciens by shake flask fermentation is 26.88g/L, which is improved by 4.2 percent compared with that before optimization, wherein, the content proportion of tetracycline in aureomycin is 6.8 percent, which is reduced by 9.3 percent compared with that before optimization, the content proportion of demethylaureomycin in aureomycin is 0.22 percent, which is reduced by 21.4 percent compared with that before optimization. The yield of aureomycin in shake flask fermentation of 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 mutant strain Streptomyces aureofaciens FJC-505 and Streptomyces aureofaciens FJW-507 fermentation production aureomycin are shown in Table 3:
TABLE 3 yield and impurities of fermentation of Streptomyces aureofaciens FJC-505 and Streptomyces aureofaciens FJW-507 for aureomycin production
| Treatment of | Aureomycin (g/L) | Tetracycline (g/L) | Demethylated 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 streptomyces aureofaciens FJC-505 and streptomyces aureofaciens FJW-507 bacterial strains bred by the invention 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 of tetracycline and demethyl aureomycin are obviously reduced, the post-treatment cost of aureomycin products is reduced, the product quality is improved, and the invention has excellent capability of producing aureomycin premix and hydrochloride thereof.
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 within 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.
Claims (7)
1. A method for producing aureomycin by utilizing streptomyces aureofaciens, which is characterized by comprising the following steps:
a1 Culturing streptomyces aureofaciens in an optimized culture medium to obtain a culture;
a2 Collecting chlortetracycline from the culture;
the optimized 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;
the Streptomyces aureofaciens is Streptomyces aureofaciens, the strain number of the Streptomyces aureofaciens is FJC-505, and the preservation number of the Streptomyces aureofaciens in the China center for type culture preservation is CCTCC NO: m2021499; or the Streptomyces aureofaciens is Streptomyces aureofaciens, the strain number of the Streptomyces aureofaciens is FJW-507, and the preservation number of the Streptomyces aureofaciens in China center for type culture collection is CCTCC NO: m2021500.
2. The method of claim 1, wherein the seed medium has a composition of: 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.
3. The method according to claim 1 or 2, 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.
4. The method according to claim 3, 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.
5. The method of claim 1, wherein said culturing comprises the steps of:
b1 Spore culture; b2 Seed culture; b3 ) fermentation culture.
6. The method according to claim 5, wherein the seed culture is carried out by preparing the spore of claim 5 into a suspension, inoculating the suspension into the seed culture medium of claim 2, and culturing at 30 ℃ and 270 r/min.
7. The method according to claim 5 or 6, wherein the fermentation culture is carried out by transferring a seed culture solution obtained by the method according to claim 6 to the fermentation medium according to claim 3 at a transfer amount of 10% by volume and culturing at 30 ℃ at 270 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110901176.4A CN113388556B (en) | 2021-08-02 | 2021-08-02 | Method for producing aureomycin by using streptomyces aureofaciens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110901176.4A CN113388556B (en) | 2021-08-02 | 2021-08-02 | Method for producing aureomycin by using streptomyces aureofaciens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113388556A CN113388556A (en) | 2021-09-14 |
| CN113388556B true CN113388556B (en) | 2022-12-02 |
Family
ID=77622485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110901176.4A Active CN113388556B (en) | 2021-08-02 | 2021-08-02 | Method for producing aureomycin by using streptomyces aureofaciens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113388556B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113549580B (en) * | 2021-08-02 | 2022-08-16 | 金河生物科技股份有限公司 | Streptomyces aureofaciens and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2734018A (en) * | 1953-09-28 | 1956-02-07 | Ircso | |
| CN101851648A (en) * | 2010-04-09 | 2010-10-06 | 福建省微生物研究所 | Method for preparing demethyl rapamycin by utilizing bacillus megaterium |
| CN103214409A (en) * | 2004-05-21 | 2013-07-24 | 哈佛大学校长及研究员协会 | Synthesis of tetracyclines and analogues thereof |
| CN111763667A (en) * | 2020-05-28 | 2020-10-13 | 河北圣雪大成制药有限责任公司 | Method for increasing yield of nortetracycline |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609491A (en) * | 2018-12-27 | 2019-04-12 | 陕西麦可罗生物科技有限公司 | It is a kind of it is efficient produce kasugarnycin streptomyces microaureus mutagenesis and screening technique |
-
2021
- 2021-08-02 CN CN202110901176.4A patent/CN113388556B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2734018A (en) * | 1953-09-28 | 1956-02-07 | Ircso | |
| CN103214409A (en) * | 2004-05-21 | 2013-07-24 | 哈佛大学校长及研究员协会 | Synthesis of tetracyclines and analogues thereof |
| CN101851648A (en) * | 2010-04-09 | 2010-10-06 | 福建省微生物研究所 | Method for preparing demethyl rapamycin by utilizing bacillus megaterium |
| CN111763667A (en) * | 2020-05-28 | 2020-10-13 | 河北圣雪大成制药有限责任公司 | Method for increasing yield of nortetracycline |
Non-Patent Citations (5)
| Title |
|---|
| "Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens";Zhu, T等;《METABOLIC ENGINEERING》;20130930;第19卷;第69-78页 * |
| "Optimization of composition of media for the production of extracellular glucoamylase by Candida guilliermendii";Mohamed, Lagzouli等;《Pakistan journal of biological sciences : PJBS》;20071001;第10卷(第19期);第3322-3329页 * |
| "多重复合诱变选育金霉素高产菌株";蔡玉凤;《福建农业科技》;20200630(第6期);第3页左栏倒数第3段-右栏第3段，第4页左栏第1段、右栏第1段 * |
| "脱H2S菌株的筛选及降解特性的研究";戴芬;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20130115(第1期);摘要 * |
| "金霉素高产菌的选育及发酵工艺优化";梁玲;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20211215(第12期);B018-40 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113388556A (en) | 2021-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108624524B (en) | Bacterial strain for producing bacterial cellulose and separation and screening method thereof | |
| CN106190921A (en) | A kind of corynebacterium glutamicum and application | |
| CN106282044B (en) | A kind of preparation method of Hyphomicrobium and pyrroloquinoline quinone | |
| CN111349588B (en) | Bacillus belgii and application thereof | |
| CN111100827B (en) | Bacillus capable of producing high-activity alginate lyase and application thereof | |
| CN111073839B (en) | Siam bacillus, microbial inoculum and application thereof | |
| CN113444659B (en) | Saccharopolyspora spinosa for high yield of spinosad | |
| CN112831421B (en) | Cephalosporin compound production strain and application thereof | |
| CN113388556B (en) | Method for producing aureomycin by using streptomyces aureofaciens | |
| CN110564580B (en) | Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation | |
| CN113549579B (en) | Streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof | |
| CN108823110B (en) | Strain for producing griseofulvin and application thereof | |
| CN108841889B (en) | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation | |
| CN111849807B (en) | Saccharopolyspora spinosa DS190375 and fermentation product, microbial inoculum, fermentation and screening method and application thereof | |
| CN113549580B (en) | Streptomyces aureofaciens and application thereof | |
| CN112300953A (en) | Bacillus subtilis and application thereof in fermentation production of adenosine deaminase | |
| CN112661807B (en) | Application of antibacterial lipopeptide Fengycin in inhibition of growth of aspergillus flavus and synthesis of toxin | |
| CN113502282A (en) | Method for producing pectinase preparation by solid-state fermentation of penicillium | |
| CN111548967B (en) | Pseudomonas putida X14 and application method thereof | |
| CN107641602A (en) | One plant of candida utili and its fermentation lay eggs it is white in application | |
| CN113604390A (en) | Corynebacterium glutamicum and application thereof in fermentation production of L-ornithine | |
| CN108774626B (en) | Method and strain for producing nemadectin | |
| CN109234194B (en) | Doramectin producing strain and application thereof | |
| CN110846300B (en) | Method for producing trypsin | |
| CN115322918B (en) | Strain for high-yield ansamitocin P-3 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |