CN113278545B - Streptomyces mutant and application thereof - Google Patents
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- CN113278545B CN113278545B CN202110517764.8A CN202110517764A CN113278545B CN 113278545 B CN113278545 B CN 113278545B CN 202110517764 A CN202110517764 A CN 202110517764A CN 113278545 B CN113278545 B CN 113278545B
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- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 claims abstract description 55
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Abstract
The invention discloses a streptomyces mutant strain and application thereof. A streptomycete mutant is named as streptomycete sp with the strain number of 891-B6 and the preservation number of CGMCC No. 21775. The mutant strain 891-B6 is obtained by ultraviolet mutagenesis and morphological screening with streptomycete 891 as the original strain. The content of the aureomycin A in the fermentation product of the mutant strain 891-B6 is 17.44% higher than that of the aureomycin A in the fermentation product of the original strain, the aureomycin A in the aureomycin produced by the mutant strain 891-B6 provided by the invention is high in active component content, the content of inactive component aureomycin B is obviously reduced, and the content of inactive component aureomycin C is stable and unchanged. Meanwhile, the mutant strain 891-B6 provided by the invention has good genetic stability, and the level of the produced aureomycin A is equivalent to that of the first generation when the mutant strain is transferred to the third generation; can be applied to the industrial production of the golden yellow mycin.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a streptomycete mutant strain and application thereof.
Background
Chryseomycin (chrysomycin) is a glycoside compound with a structure of benzonaphthopyrone, which was first discovered from Streptomyces A-419 strain in 1955 and later isolated from other strains of Streptomyces. Biological experiments show that the compounds have remarkable activities of resisting phage, bacteria and tumors.
In recent years, research shows that the aureomycin A (with a structural formula shown as formula I) has a strong inhibiting effect on drug-resistant pathogens including mycobacterium tuberculosis, and early pharmacological research shows that the aureomycin A has a strong inhibiting effect on drug-resistant G+Bacteria such as methicillin-resistant staphylococcus aureus (MRSA) have a significant antibacterial effect. Therefore, the lead compound has great potential to be developed into a novel therapeutic drug for treating infectious diseases caused by drug-resistant pathogens. Therefore, golden yellow A, one of the drug leads, has great potential for application in the pharmaceutical industry, and more researchers such as chemists, biologists and pharmacologists are beginning to pay attention to the golden yellow class of substances. However, the fermentation process of the aureomycin producing strain has the problems of low titer, more components and the like, which causes expensive cost and separation difficulty for scale preparation in the later period.
The invention with the publication number of CN112574185A discloses a large-scale separation and purification method of aureomycin A. Filtering a golden yellow mycin fermentation liquor plate frame, drying to obtain dry fungus residues, crushing, performing ultrasonic extraction by using an organic solvent, concentrating and drying an extracting solution, adding a solvent to clean water-soluble impurities, adding dichloromethane to extract, decoloring a dichloromethane extracting solution, performing suction filtration, concentrating and drying a filtrate to obtain a golden yellow mycin A crude product, adding the golden yellow mycin A crude product into the solvent to dissolve the golden yellow mycin A crude product, and performing preparative chromatographic separation to obtain a golden yellow mycin A pure product. The method is simple to operate, the using amount of the organic solvent is small, a large amount of wastewater and the organic solvent generated by using macroporous resin, silica gel or organic solvent for crystallization are avoided, the yield of the aureomycin A finally reaches more than 95%, the content of the aureomycin A in the obtained pure product of the aureomycin A is more than 96%, the purity is more than 98%, and the method is more suitable for large-scale production, low in cost and high in benefit.
In the prior art, streptomyces 891 is used for producing aureomycin by fermentation, but the produced aureomycin is a mixture containing A, B, C three components, and aureomycin A plays a role in inhibiting staphylococcus aureus, so that the content of aureomycin A needs to be increased in order to improve the antibacterial effect of aureomycin on staphylococcus aureus.
Disclosure of Invention
The invention aims to solve the problems of more original strain components, high later-stage separation cost, low aureomycin A yield and the like, and provides a streptomyces mutant strain and application thereof.
A Streptomyces mutant strain is named as Streptomyces sp, has the strain number of 891-B6, is preserved in the China general microbiological culture Collection center (CGMCC) in the microbiological research institute of China academy of sciences No. 3 of the West Lu No. 1 Hopkin, Chaoyang, 1 month and 25 days in 2021, and has the preservation number of CGMCC No. 21775.
The streptomycete mutant strain is obtained by screening the original strain streptomycete 891 separated from mangrove forest sediment in south China sea in the laboratory through ultraviolet irradiation mutagenesis.
The invention also provides application of the streptomyces mutant strain in producing aureomycin.
The invention also provides a method for producing the aureomycin, which comprises the steps of carrying out fermentation culture by using the streptomyces mutant bacteria, and purifying to obtain the aureomycin.
Preferably, the fermentation culture process comprises the following steps:
and 2, inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
Preferably, said ISP in step 12The agar medium had the composition: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, 20.0g/L agar powder, pH 7, sterilizing at 121 deg.C for 20 min;
ISP2the composition of the liquid medium was: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, pH 7.0, sterilizing at 121 deg.C for 20min。
Preferably, the temperature of the seed culture in the step 1 is 28 ℃, the rotation speed of the seed culture is 200rpm, and the time of the seed culture is 48-72 h.
Preferably, the inoculation amount of the seed liquid in the step 2 is 5-10% by volume ratio.
Preferably, the composition of the fermentation medium in step 2 is: 5.0g/L starch, 20.0g/L glucose, 10.0g/L soybean powder, 2.0g/L calcium carbonate, pH 7.0, and sterilizing at 121 deg.C for 20 min.
Preferably, the temperature of the culture in the step 2 is 28 ℃, the rotation speed of the culture is 200rpm, and the time of the culture is 168-192 h.
The invention has the beneficial effects that: the invention provides a streptomycete mutant strain 891-B6 for producing aureomycin, with the preservation number of CGMCC No. 21775; the mutant strain 891-B6 is obtained by taking streptomycete 891 as an original strain, performing ultraviolet mutagenesis and performing morphological screening. The content of the aureomycin A in the fermentation product of the mutant strain 891-B6 is 17.44% higher than that of the aureomycin A in the fermentation product of the original strain, the aureomycin A in the aureomycin produced by the mutant strain 891-B6 provided by the invention is high in active component content, the content of inactive component aureomycin B is obviously reduced, and the content of inactive component aureomycin C is stable and unchanged. Meanwhile, the mutant strain 891-B6 provided by the invention has good genetic stability, and the level of the produced aureomycin A is equivalent to that of the first generation when the mutant strain is transferred to the third generation; can be applied to the industrial production of the golden yellow mycin.
Drawings
FIG. 1 is a graph showing the relationship between the UV irradiation time and the lethality of the strain.
FIG. 2 is a diagram showing morphological differences between the original strain and the mutant strain.
FIG. 3 is HPLC analysis spectrum of fermentation product of original strain Streptomyces 891.
FIG. 4 is HPLC analysis spectrum of fermentation product of high-producing strain of aureomycin A (Streptomyces 891-B6).
Detailed Description
Example 1
The streptomycete mutant strain is obtained by ultraviolet irradiation mutagenesis of streptomycete 891 which is separated from mangrove forest sediment in south China sea in the laboratory and is an original strain, and the mutagenesis method comprises the following steps:
And 4, carrying out constant-temperature shaking culture on the strains subjected to ultraviolet irradiation for different time periods in the step 3 at 28 ℃ under the condition of 200rpm for 168-.
and 6, extracting the thalli collected in the step 6 by using a proper amount of methanol, performing HPLC analysis and detection, and screening the mutant strain with high-yield aureomycin A compared with the original strain.
And 7, selecting the high-yield aureomycin A mutant strain obtained in the step 6, carrying out third-generation culture fermentation, carrying out HPLC analysis, and screening the stable high-yield aureomycin A mutant strain.
ISP (Internet service provider) in steps 1 and 32The agar medium had the composition: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, 20.0g/L agar powder, pH 7, and sterilizing at 121 deg.C for 20 min.
The lethality calculation formula in step 3 is:
survival rate (number of colonies surviving after mutagenesis/number of colonies surviving before mutagenesis) × 100%
Lethality rate is 1-survival rate
The positive mutation rate calculation formula in the step 4 is as follows:
positive mutation rate (number of positive mutant strains after mutagenesis/total number of strains after mutagenesis) × 100%
The relationship between the UV irradiation time and the lethality of the strain and the positive mutation of the strain is shown in FIG. 1.
And 3, selecting 120s with the highest positive mutation rate as the irradiation time under the ultraviolet irradiation condition of 16-20W of ultraviolet lamp power and the irradiation distance of about 15 cm.
The fermentation medium in the step 5 comprises the following components: 5.0g/L starch, 20.0g/L glucose, 10.0g/L soybean powder, 2.0g/L calcium carbonate, pH 7.0, and sterilizing at 121 deg.C for 20 min.
Analysis of fermentation product aureomycin A:
detecting the change of the flavomycin component in the fermentation product by HPLC (high performance liquid chromatography), and screening the bacterial strain with the difference from the secondary metabolic component of the original bacterial strain 891 by the increase and decrease of the peak shape and the change of the relative peak area.
Sample treatment: centrifuging 10mL fermentation liquid at 4000rpm for 10min, discarding supernatant, adding 40mL methanol into precipitate, ultrasonic extracting for 20min, centrifuging for 10min, and collecting supernatant 100 μ L for HPLC analysis.
HPLC conditions: the column used was Phenomenex (Luna, 250X 4.6mm) C18 column. HPLC mobile phase is 50% acetonitrile water solution, detection wavelength is 254nm, and injection volume is 20 μ L.
The result shows that a Streptomyces mutant strain derived from the China south China sea mangrove forest sediment for producing the aureomycin is screened, is classified and named as Streptomyces sp, has the strain number of 891-B6, is preserved in the China general microbiological culture Collection center (CGMCC) in the institute of microbiology of China academy of sciences No. 3 of the West Lu 1 of the northwest sunward area of Beijing city within 1 month and 25 days of 2021 year, and has the preservation number of CGMCC No. 21775.
FIG. 2 is a morphological diagram of the original strain and the mutant strain, which were streaked on ISP respectively2In agar medium, it can be observed that the original strain thallus is white, the aerial hyphae are abundant, the spore grows vigorously: the mutant strain has yellow-green thallus, abundant aerial hyphae and vigorous spore growth.
FIG. 3 is HPLC analysis spectrum of fermentation product of original strain Streptomyces 891. FIG. 4 is HPLC analysis map of fermentation product of screened high-producing strain of aureomycin A (Streptomyces 891-B6). FIG. 4 is a comparison with FIG. 3, in which the proportion of other components is reduced, and it can be seen that the secondary metabolites of the selected strains are changed.
Quantitative analysis of the aureomycin A is carried out by adopting an external standard method, the aureomycin A in the fermentation product of the high-yield strain of the aureomycin A (streptomycete 891-B6) accounts for the main component, and part of other components are reduced to a lower level. As a result of detection, the content of the aureomycin A in the control product accounts for 62% (438 +/-12.37 mg/L) of the total components, the content of the aureomycin A in the mutagenized strain accounts for 79.52% (486.20 +/-15.68 mg/L) of the total components, and the content of the aureomycin A produced by fermentation of the mutagenized strain 891-B1 is obviously increased.
Example 2
Stability analysis of aureomycin production by mutant strain 891-B6, comprising the following steps:
and 2, inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
The ISP in the step 12The agar medium had the composition: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, 20.0g/L agar powder, pH 7.0, and sterilizing at 121 deg.C for 20 min.
The temperature of the seed culture in the step 1 is 28 ℃, the rotation speed of the seed culture is 200rpm, and the time of the seed culture is 48-72 h.
The inoculation amount of the seed liquid in the step 2 is 5-10% (V/V).
The fermentation medium in the step 2 comprises the following components: 5.0g/L starch, 20.0g/L glucose, 10.0g/L soybean powder, 2.0g/L calcium carbonate, pH 7, and sterilizing at 121 deg.C for 20 min.
And 2, the fermentation culture temperature is 28 ℃, the rotation speed of the fermentation culture is 200rpm, and the fermentation culture time is 168-192 h.
Sample treatment: 10mL of fermentation liquid is centrifuged at 4000rpm for 10min, the supernatant is discarded, 40mL of methanol is added into the precipitate for ultrasonic extraction for 20min, the centrifugation is carried out for 10min, 100 mu L of supernatant is taken for HPLC analysis, the third generation of fermentation products 891-B6 is analyzed, and the stability result of the aureomycin produced by 891-B6 is shown in Table 1.
Table 1: 891-B6 strain golden yellow mycin A content
| Strain numbering | Golden yellow mycin A (%) |
| 891-B6 | 79.52 |
| 891 | 62.08 |
The data in Table 1 show that the fermentation product produced by the 891 original strain had an average content of chlorotetracycline 62.08%, the fermentation broth produced by the 891-B6 strain had an average content of chlorotetracycline 79.52%, the chlorotetracycline A content of the 891-B6 strain was increased by 17.44% over that of the 891 original strain, and the mutant strain 891-B6 was genetically stable as shown in Table 1.
As can be seen from the above examples, the aureomycin produced by the mutant strain 891-B6 provided by the invention has high content of aureomycin A as an active ingredient, remarkably reduced content of aureomycin B as an inactive ingredient and stable and unchanged content of aureomycin C as an inactive ingredient, and can be applied to industrial production of aureomycin.
Claims (9)
1. A streptomycete mutant is named as streptomycete sp with the strain number of 891-B6 and the preservation number of CGMCC No. 21775.
2. The use of the Streptomyces mutant as defined in claim 1 for producing aureomycin.
3. A method for producing aureochrysin, characterized in that the streptomyces mutant strain of claim 1 is used for fermentation culture, and then the aureochrysin is obtained after purification.
4. The method of claim 3, wherein the fermentation process comprises the steps of:
step 1, the streptomycete mutant is accessed into ISP2Activating the agar culture medium for 6-7 days, and then inoculating the agar culture medium to ISP2Obtaining seed liquid after seed culture of the liquid culture medium;
and 2, inoculating the seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
5. The method of claim 4, wherein the ISP in step 12The agar medium had the composition: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, 20.0g/L agar powder, pH 7, sterilizing at 121 deg.C for 20 min;
ISP2the composition of the liquid medium was: 4.0g/L glucose, 4.0g/L yeast extract, 10.0g/L malt extract, pH 7.0, sterilizing at 121 deg.C for 20 min.
6. The method according to claim 4, wherein the temperature of the seed culture in the step 1 is 28 ℃, the rotation speed of the seed culture is 200rpm, and the time of the seed culture is 48-72 h.
7. The method of claim 4, wherein the seed solution is inoculated in an amount of 5 to 10% by volume in step 2.
8. The method of claim 4, wherein the fermentation medium of step 2 has a composition of: 5.0g/L starch, 20.0g/L glucose, 10.0g/L soybean powder, 2.0g/L calcium carbonate, pH 7.0, and sterilizing at 121 deg.C for 20 min.
9. The method according to claim 4, wherein the temperature of the culture in step 2 is 28 ℃, the rotation speed of the culture is 200rpm, and the culture time is 168-192 h.
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| CN109867663A (en) * | 2018-12-29 | 2019-06-11 | 浙江工业大学 | A method of with preparation chromatographic isolation chrysomycin A and chrysomycin B |
| CN112280812A (en) * | 2020-09-30 | 2021-01-29 | 浙江工业大学 | Method for improving fermentation yield of aureomycin A and ratio of aureomycin A to aureomycin B |
| CN112574185A (en) * | 2020-09-30 | 2021-03-30 | 浙江工业大学 | Large-scale preparation method of aureomycin A |
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| CN109867663A (en) * | 2018-12-29 | 2019-06-11 | 浙江工业大学 | A method of with preparation chromatographic isolation chrysomycin A and chrysomycin B |
| CN112280812A (en) * | 2020-09-30 | 2021-01-29 | 浙江工业大学 | Method for improving fermentation yield of aureomycin A and ratio of aureomycin A to aureomycin B |
| CN112574185A (en) * | 2020-09-30 | 2021-03-30 | 浙江工业大学 | Large-scale preparation method of aureomycin A |
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| Optimization of fermentation conditions and medium compositions for the production of chrysomycin a by a marine-derived strain Streptomyces sp. 891;Hong-Jin Ni等;《Prep Biochem Biotechnol》;20210218;第51卷(第10期);第998-1003页，参见全文 * |
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