CN112300968B - Arthrobacter for producing adenosine cyclophosphate and application thereof - Google Patents
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- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 title claims abstract description 29
- 241000186063 Arthrobacter Species 0.000 title abstract description 6
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 23
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 239000004202 carbamide Substances 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Natural products O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims description 4
- 244000061456 Solanum tuberosum Species 0.000 claims description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 235000015278 beef Nutrition 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 241000185994 Pseudarthrobacter oxydans Species 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 2
- -1 acetyl hypoxanthine Chemical compound 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- FRMOIGUYDRLNFT-UHFFFAOYSA-N 2-acetyl-3,7-dihydropurin-6-one Chemical compound N1C(C(=O)C)=NC(=O)C2=C1N=CN2 FRMOIGUYDRLNFT-UHFFFAOYSA-N 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 241000186073 Arthrobacter sp. Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/06—Arthrobacter
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- 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
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a Arthrobacter for producing adenosine cyclophosphate by fermentation, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) in the year 03 and 23. The invention also discloses a method for producing the adenosine cyclophosphate by fermenting the arthrobacter, which comprises the steps of slant, seed and culture medium and culture conditions required by fermentation culture. The bacterium has high adenosine cyclophosphate yield and good industrialization prospect.
Description
Technical Field
The invention relates to arthrobacter for producing adenosine cyclophosphate by fermentation and application thereof.
Technical Field
Adenosine cyclophosphate, a second messenger substance involved in regulating cellular functions, plays an important role in regulating the metabolism and synthesis of saccharides, fats, nucleic acids, and proteins. The clinical adenosine cyclophosphate is mainly used for the adjuvant therapy of angina pectoris, acute myocardial infarction and the like, and has the effects of improving myocardial anoxia, expanding coronary artery, enhancing myocardial contractility, increasing heart blood discharge and the like.
At present, the related drugs of the clinical adenosine cyclophosphate are mostly synthesized by adopting a chemical method, and mainly comprise an alkaline hydrolysis method, an active ester method, a DCC dehydration method and a phosphorus oxychloride method. However, the chemical synthesis method has the defects of toxic raw materials, high cost, large amount of organic solvents used, environmental pollution and the like. Thus, the microbial fermentation synthesis of adenosine cyclophosphate has important value.
Adenosine cyclophosphate is widely present in plants, animals and microorganisms but is not normally accumulated, for example, the amount of adenosine cyclophosphate per gram of wet cells in mammals is only 10 -12 To 10 -9 Gram (g).
Disclosure of Invention
The invention aims to provide a strain for high-yield adenosine cyclophosphate. In order to achieve the purpose, the invention adopts the following technical mode:
the high-yield strain JL-cAMP is preserved by China general microbiological culture Collection center (CGMCC No. 13923) and the preservation date is 2017.03.23. The strain is obtained by carrying out mutagenesis on a strain producing adenosine cyclophosphate obtained by soil screening. The bacterial colony is yellow, round, moist in surface and neat in edge, gram staining is positive, and the bacterial colony is short rod-shaped when observed by a microscope. The primary identification was made as Arthrobacter sp by 16S rDNA alignment. The strain has the advantages of high adenosine cyclophosphate yield, stable yield and the like.
Another technical problem to be solved by the present invention is to provide a fermentation method for producing adenosine cyclophosphate, and in order to achieve the purpose, the present invention adopts a technical scheme as follows:
first, a strain liquid-cryopreserved at-80℃is subjected to activation culture on a slant medium comprising: potato 200g/L MgSO 4 0.01g/L, 0.03g/L guanine, 20g/L agar, pH 7.2. The slant culture process comprises the following steps: 31 ℃. Subsequently, inoculated into a seed tank, the seed tank medium comprising: 25g/L glucose, 5g/L beef extract, 20g/L peptone, 10g/L yeast extract, 6g/L urea and NaCl1g/L. The seed tank culture process comprises the following steps: the pH value is 7.0, the temperature is 31 ℃, and the dissolved oxygen is less than 30-40%. Finally, inoculating into a fermentation tank, wherein the fermentation tank culture medium comprises the following components: glucose 50g/L, peptone 1g/L, KH 2 PO 4 2g/L, coCl 0.01g/L, biotin 0.02g/L, urea 2g/L, hypoxanthine (acetyl) 3g/L, naF 0.06g/L, feSO 4 0.1g/L,MgSO 4 3g/L, and 3g/L of yeast powder. The fermentation tank culture process comprises the following steps: the pH value is 7.4-7.5, the temperature is 30-31 ℃, and the dissolved oxygen is 30-45%.
The bacteria with the preservation number of CGMCC No.13923 have the preservation address of Beijing, chaoyang area North Chen West Lu No.1 and No. 3, and are classified and named as Arthrobacter oxydans (Pseudarthrobacter oxydans).
Detailed Description
The following summary of the preparation and advantages of the high-yield cyclic adenosine monophosphate strain JL-cAMP:
taking a strain which is preserved in a laboratory and produces the adenosine cyclophosphate as an initial strain, carrying out compound mutagenesis, picking single colony on a flat plate into a seed culture medium, culturing to the middle and later stages of logarithm, transferring into a fermentation culture medium, fermenting, and measuring the adenosine cyclophosphate content in fermentation broth. The strain with higher yield is selected and subjected to the next round of mutagenesis. Through 3 rounds of mutagenesis, one strain shows good adenosine cyclophosphate production performance, and the yield is improved by more than 2 times compared with the original strain, and the number is JL-cAMP. After 20 passages, the cyclic adenosine monophosphate yield of the JL-cAMP of the bacterium is basically kept stable.
Determination method of adenosine cyclophosphate used in the examples (high performance liquid chromatography):
chromatographic conditions:
chromatographic column: c18 4.6 mm. Times.150 cm. Times.5 μm, detection wavelength: 258nm, flow rate: 1.0ml/min, sample injection amount: 20 μl, mobile phase: phosphate buffer: acetonitrile = 85:15 phosphate buffer: 6.8g of monopotassium phosphate and 3.2g of tetrabutylammonium bromide are precisely weighed, dissolved by adding water and fixed to 1000ml of volume, and the pH value is regulated to 4.3 by phosphoric acid. Preparing a solution:
system adaptation solution: precisely weighing adenosine cyclophosphate reference substance 20mg, adding 5ml of water for dissolution, adding 1ml of 1mol/L hydrochloric acid solution, heating in water bath for 30min, cooling, adjusting to neutrality with sodium hydroxide test solution, diluting with water to obtain solution containing about 0.2mg of adenosine cyclophosphate per 1ml, and injecting 20uL into liquid chromatograph.
Sample solution: accurately weighing 10mg of the sample, placing in a 100ml volumetric flask, dissolving with water, diluting to scale, and shaking.
System applicability test: the separation degree R of the adenosine cyclophosphate peak and the adjacent peak is not less than 1.5, the tailing factor (T) is not more than 1.4, the relative standard deviation RSD of the response factor is not more than 2.0%, and the theoretical plate number is not less than 2000.
Sample injection sequence:
blank solution (n=6) →system adaptive solution (n=3) →test solution (n=1) …
The content is calculated according to an external standard method:
examples: production of adenosine cyclophosphate by fermentation of Arthrobacter JL-cAMP
The strain frozen and preserved by the liquid at the temperature of minus 80 ℃ is activated and cultured on a slant culture medium, wherein the slant culture medium comprises the following components: potato 200g/L MgSO 4 0.01g/L, 0.03g/L guanine, 20g/L agar, pH 7.2. The slant culture process comprises the following steps: 31 ℃. Subsequently, inoculated into a seed tank, the seed tank medium comprising: 25g/L of glucose, 5g/L of beef extract, 20g/L of peptone, 10g/L of yeast extract, 6g/L of urea and 1g/L of NaCl. The seed tank culture process comprises the following steps: the pH value is 7.0, the temperature is 31 ℃, and the dissolved oxygen is less than 30-40%. Finally, inoculating into a fermentation tank, wherein the fermentation tank culture medium comprises the following components: glucose 50g/L, peptone 1g/L, KH 2 PO 4 2g/L, coCl 0.01g/L, biotin 0.02g/L, urea 2g/L, hypoxanthine (acetyl) 3g/L, naF 0.06g/L, feSO 4 0.1g/L,MgSO 4 3g/L, and 3g/L of yeast powder. The fermentation tank culture process comprises the following steps: the pH is 7.5, the temperature is 30 ℃ and the dissolved oxygen is 45 percent. After fermentation, the yield of adenosine cyclophosphate was 15g/L as measured by the above method.
Claims (10)
1. Arthrobacter oxydans with preservation number of CGMCC No.13923Pseudarthrobacter oxydans)。
2. A method for producing adenosine cyclophosphate, which comprises producing adenosine cyclophosphate by fermentation using the pseudoarthrobacter oxydans of claim 1.
3. The method of claim 2, wherein the fermentation is a liquid fermentation.
4. A method according to claim 2 or 3, wherein the fermentation medium is selected from at least one of the following:
medium a, comprising the following components: potato, mgSO 4 Guanine and agar;
medium b, comprising the following components: glucose, beef extract, peptone, yeast extract, urea and NaCl;
medium c, comprising the following components: glucose, peptone, KH 2 PO 4 ,CoCl 2 Biotin, urea, acetyl hypoxanthine, naF, feSO 4 ,MgSO 4 And yeast powder.
5. The method according to claim 4, wherein the pH of the culture medium a is 7.0-7.4, and the composition comprises: potato 200g/L MgSO 4 0.01g/L, guanine 0.02-0.04 g/L, agar 20 g/L.
6. The method of claim 4, wherein the pH of the medium b is 6.5-7.5, and the medium b comprises the following components: glucose 25/g/L, beef extract 5/g/L, peptone 20/g/L, yeast extract 10/g/L, urea 6g/L, naCl 1g/L.
7. The method of claim 4, wherein the medium c composition comprises: glucose 50g/L, peptone 1g/L, KH 2 PO 4 2 g/L,CoCl 2 0.01g/L, biotin 0.02g/L, urea 2g/L, acetyl hypoxanthine 2-4 g/L, naF 0.06g/L, feSO 4 0.1 g/L,MgSO 4 3-g/L and 2-4 g/L of yeast powder.
8. The method of claim 7, wherein the concentration of acetylhypoxanthine or yeast powder is independently selected from 2g/L, 3g/L, or 4g/L.
9. The method of claim 4, wherein the pH of the fermentation of the medium c is 7.4 to 7.5.
10. The method of claim 4, wherein the process conditions for fermentation of medium c comprise: the temperature is 30-31 ℃ and/or the dissolved oxygen is 30-45%.
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