CN105400853A - Method for effectively increasing yield of tetraene macrolide antibiotics - Google Patents

Method for effectively increasing yield of tetraene macrolide antibiotics Download PDF

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Publication number
CN105400853A
CN105400853A CN201510966000.1A CN201510966000A CN105400853A CN 105400853 A CN105400853 A CN 105400853A CN 201510966000 A CN201510966000 A CN 201510966000A CN 105400853 A CN105400853 A CN 105400853A
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strain
antibiotic
mutant strain
tetraene
tetraene macrolide
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申屠旭萍
俞晓平
许益鹏
汤谷
刘楠楠
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China Jiliang University
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China Jiliang University
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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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Abstract

The invention discloses a method for effectively increasing the yield of tetraene macrolide antibiotics, and belongs to the field of microbial technologies. According to the method, by screening joint drug-resistant mutant strains, mutant strains of streptomyces diastatochromogenes D with drug resistance to rifampicin, low-concentration streptomycin and high-concentration streptomycin are obtained, and compared with streptomyces diastatochromogenes D, the capability of the obtained mutant strains for synthesizing tetraene macrolide antibiotics is significantly improved.

Description

A kind of method of effective raising tetraene macrolide antibiotic yield
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of method of effective raising tetraene macrolide antibiotic yield.
Background technology
The novel agricultural microbiotic of current many independent developments just rests on laboratory stage, and wherein crucial reason is exactly that the product element level of producing bacterium does not reach industrial requirement.So when forward swing, in research worker face, previous main problem is exactly improve the product element level that bacterium produced by agricultural antibiotic that is existing and that developing, accelerates the industrialization process of agricultural antibiotic.Conventional microorganism mutation breeding is wasted time and energy, randomness is high, can be realized the orthogenesis of the enzyme of single-gene coding by genetic engineering means and carry out overexpression, but then difficult to the output increased of the natural products such as the microbiotic needing genes in cluster to encode.Research shows that associating drug-resistant mutation screening method is a kind of novel method of optimization antibiotics generated bacterium of novelty, associating drug-resistant mutation can strengthen the antibiotic level of Microbe synthesis, and the novel method of this optimization bacterial strain can be applied to Multiple Classes of Antibiotics produces the optimization of bacterial strain and can improve the antibiotic yield of wild type strain.
Tetraene macrolide microbiotic can suppress the growth of various plants pathogenic fungi, and biological control has broad application prospects.Streptomyces diastatochromogenes ( streptomycesdiastatochromogenes) D can synthesize 3 kinds of tetraene macrolide microbiotic: tetramycin P, Tetrin B and tetramycin A, utilizes associating drug-resistant mutation screening method modified starch enzyme streptomyces chromogenes D to make its research improving tetraene macrolide microbiotic synthesis capability there are no report.
Summary of the invention
The present invention is by the screening of associating drug-resistant mutation strain, obtain the mutant strain to the streptomyces diastatochromogenes D that Rifampin, lower concentration Streptomycin sulphate and high density Streptomycin sulphate all develop immunity to drugs, the mutant strain synthesis tetraene macrolide of acquisition is antibiotic can be significantly improved by force rate streptomyces diastatochromogenes D.This invention provides a kind of method reliably for improving the antibiotic output of tetraene macrolide.
The object of the invention is to produce the low deficiency of tetraene macrolide antibiotic levels for wild type strain streptomyces diastatochromogenes D, provide a kind of and improve the method that tetraene macrolide microbiotic produces plain level; Another object of the present invention there is provided a plant height and produces tetraene macrolide antibiotic streptomyces diastatochromogenes D mutant strain.
The object of the invention is achieved through the following technical solutions:
A kind of method of effective raising tetraene macrolide antibiotic yield is carried out according to the following steps:
(1) acquisition (first round drug-resistant mutation strain screening) of rifampicin resistance mutant strain
Streptomyces diastatochromogenes ( streptomycesdiastatochromogenes), name as streptomyces diastatochromogenes D.This bacterial strain in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation address, preservation date on May 25th, 2007, preservation registration number CGMCCNo.2060.
Preparing the GYM solid plate that Rifampin final concentration is 20 μ g/mL, is also that in GYM solid plate, Concentration of Rifampicin is 20 μ g/mL.GYM solid medium component and concentration are glucose 4g/L, Driedyeastextract4g/L, DriedMaltextract-S10g/L, N-Z-AmineA1g/L, NaCl2g/L, OB solution 600 μ L, pH to 7.3, agar 20g/L is regulated with 2mol/LNaCl solution after constant volume; Wherein OB solution allocation method: take MgSO47H 2o25g, CuSO 45H 2o2.5g, FeSO 47H 2o3.75g, MnSO 45H 2o1.8g, CaCl 22H 2o17.5g, ZnSO 47H 2o4.5g, adds 500mL water, with front shaking up; 100 μ L streptomyces diastatochromogenes D spore suspensions (1 × 10 are drawn with liquid-transfering gun 6individual/mL) on the GYM solid plate containing 20 μ g/mL Rifampins, even with aseptic spreading rod coating, put 28 DEG C of incubators and cultivate 7 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 20 μ g/mL Rifampins respectively, put 28 DEG C of incubators and cultivate 5 days, the bacterial strain that can again grow on the GYM solid plate that this is new is rifampicin resistance mutant strain.
(2) acquisition (second takes turns drug-resistant mutation strain screening) of lower concentration streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 30 μ g/mL, also namely GYM solid plate streptomycin concentration is 30 μ g/mL.GYM solid medium component and the same step of concentration (1); 100 μ L rifampicin resistance mutant strains (mutant strain of also i.e. first round resistance screening acquisition) spore suspension (1 × 10 is drawn with liquid-transfering gun 6individual/mL) on the GYM solid plate containing 30 μ g/mL Streptomycin sulphates, even with aseptic spreading rod coating, put 28 DEG C of incubators and cultivate 7 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 30 μ g/mL Streptomycin sulphates respectively, put 28 DEG C of incubators and cultivate 5 days, the bacterial strain that can again grow on the GYM solid plate that this is new is the strain of lower concentration streptomycin resistant mutation.
(3) acquisition (third round drug-resistant mutation strain screening) of high density streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 300 μ g/mL, also namely GYM solid plate streptomycin concentration is 300 μ g/mL.GYM solid medium component and the same step of concentration (1); The mutant strain spore suspension (1 × 10 that 1000 μ L second take turns screening acquisition is drawn with liquid-transfering gun 12individual/mL) on the GYM solid plate containing 300 μ g/mL Streptomycin sulphates, even with aseptic spreading rod coating, put 28 DEG C of incubators to cultivate 10 ~ 15 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 300 μ g/mL Streptomycin sulphates respectively, put 28 DEG C of incubators to cultivate 7 ~ 10 days, the bacterial strain that can again grow on the GYM solid plate that this is new is the strain of high density streptomycin resistant mutation.
(4) streptomyces diastatochromogenes mutant strain liquid fermenting
Fermentation culture: fermentation culture is GYM liquid nutrient medium, component and concentration are glucose 4g/L, Driedyeastextract4g/L, DriedMaltextract-S10g/L, N-Z-AmineA1g/L, NaCl2g/L, OB solution 600 μ L, regulates pH to 7.3 with 2mol/LNaCl solution after constant volume; Wherein OB solution allocation method: take MgSO47H 2o25g, CuSO 45H 2o2.5g, FeSO 47H 2o3.75g, MnSO 45H 2o1.8g, CaCl 22H 2o17.5g, ZnSO 47H 2o4.5g, adds 500mL water, with front shaking up; The bottled 60mL fermentation culture of every 300mL triangle, prepares rear 121 DEG C of sterilizing 20min, to be cooled to 50 DEG C, difference picking one platinum ring streptomyces diastatochromogenes D and mutant strain spore inoculating under aseptic condition, 28 DEG C ± 1 DEG C, fermentation culture 6 days; The centrifugal 10min of fermentation liquor 5000r/min, supernatant liquor is used for the mensuration of tetraene macrolide antibiotic content;
(5) the antibiotic detection of tetraene macrolide
Method: adopt SHIMADZUC18 chromatographic column (150mm × 4.6mm, 5 μm), methyl alcohol is mobile phase A, and water is Mobile phase B, gradient elution, and elution program is 0-30min, 65%-80%A; 30-50min, 80%-100%A; 50-60min, 100%-65%A; Flow velocity 1.0mL/min, determined wavelength 305nm, column temperature 30 DEG C.
Beneficial effect of the present invention:
One is the screening that the present invention passes through the strain of associating drug-resistant mutation, the mutant strain synthesis tetraene macrolide obtained is antibiotic can be significantly improved by force rate streptomyces diastatochromogenes D, and this provides a kind of method reliably for improving the antibiotic output of tetraene macrolide;
Two is the invention provides a plant height to produce the antibiotic streptomyces diastatochromogenes mutant strain of tetraene macrolide, for tetraene macrolide Antibiotic Industryization production from now on lays the foundation.
Embodiment
Be described further below in conjunction with the method for embodiment to a kind of effective raising tetraene macrolide antibiotic yield of the present invention.
Embodiment 1: the acquisition (first round drug-resistant mutation strain screening) of rifampicin resistance mutant strain
Preparing the GYM solid plate that Rifampin final concentration is 20 μ g/mL, is also that in GYM solid plate, Concentration of Rifampicin is 20 μ g/mL.100 μ L streptomyces diastatochromogenes D spore suspensions (1 × 10 are drawn with liquid-transfering gun 6individual/mL) on GYM solid plate, even with aseptic spreading rod coating, put 28 DEG C of incubators and cultivate 7 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 20 μ g/mL Rifampins respectively, put 28 DEG C of incubators and cultivate 5 days, the bacterial strain that can again grow on the GYM solid plate that this is new is rifampicin resistance mutant strain.Obtain the strain of rifampicin resistance mutant strain 2 altogether, numbering is respectively f31 and f32, carry out liquid fermentation and culture to this 2 plant mutant strain according to the step (4) in technical scheme, fermentation liquor HPLC detects, and finds that wherein mutant strain f31 produces tetraene macrolide microbiotic ability comparatively strong (see table 1);
Embodiment 2: the acquisition (second takes turns drug-resistant mutation strain screening) of lower concentration streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 30 μ g/mL, also namely GYM solid plate streptomycin concentration is 30 μ g/mL.100 μ L rifampicin resistance mutant strain f31 spore suspensions (1 × 10 are drawn with liquid-transfering gun 6individual/mL) on GYM solid plate, even with aseptic spreading rod coating, put 28 DEG C of incubators and cultivate 7 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 30 μ g/mL Streptomycin sulphates respectively, put 28 DEG C of incubators and cultivate 5 days, the bacterial strain that can again grow on the GYM solid plate that this is new is the strain of lower concentration streptomycin resistant mutation.Obtain lower concentration streptomycin resistant mutation strain 2 strain altogether, numbering is respectively G2-13 and G2-18, according to the step (4) in technical scheme, liquid fermentation and culture is carried out to this 2 plant mutant strain, fermentation liquor HPLC detects, and finds that wherein mutant strain G2-13 produces tetraene macrolide microbiotic ability comparatively strong (see table 1);
Embodiment 3: the acquisition (third round drug-resistant mutation strain screening) of high density streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 300 μ g/mL, also namely GYM solid plate streptomycin concentration is 300 μ g/mL.1000 μ L lower concentration streptomycin resistant mutation strain G2-13 spore suspensions (1 × 10 are drawn with liquid-transfering gun 12individual/mL) on GYM solid plate, even with aseptic spreading rod coating, put 28 DEG C of incubators to cultivate 10 ~ 15 days, picking list bacterium colony is to the new GYM solid plate (standardized of a single bacterium colony is dull and stereotyped) containing 300 μ g/mL Streptomycin sulphates respectively, put 28 DEG C of incubators to cultivate 7 ~ 10 days, the bacterial strain that can again grow on the GYM solid plate that this is new is the strain of high density streptomycin resistant mutation.Obtain high density streptomycin resistant mutation strain 2 strain altogether, numbering is respectively G3-13 and G3-15;
Embodiment 4: mutant strain liquid fermenting
Fermentation culture is GYM liquid nutrient medium, and component and concentration are glucose 4g/L, Driedyeastextract4g/L, DriedMaltextract-S10g/L, N-Z-AmineA1g/L, NaCl2g/L, OB solution 600 μ L, regulates pH to 7.3 with 2mol/LNaCl solution after constant volume; Wherein OB solution allocation method: take MgSO47H 2o25g, CuSO 45H 2o2.5g, FeSO 47H 2o3.75g, MnSO 45H 2o1.8g, CaCl 22H 2o17.5g, ZnSO 47H 2o4.5g, adds 500mL water, with front shaking up; The bottled 60mL fermentation culture of every 300mL triangle, prepares rear 121 DEG C of sterilizing 20min, to be cooled to 50 DEG C, difference picking one platinum ring streptomyces diastatochromogenes D and mutant strain spore inoculating under aseptic condition, 28 DEG C ± 1 DEG C, fermentation culture 6 days; The centrifugal 10min of fermentation liquor 5000r/min, supernatant liquor is used for the mensuration of tetraene macrolide antibiotic content;
Embodiment 5: the antibiotic detection of tetraene macrolide
Method: adopt SHIMADZUC18 chromatographic column (150mm × 4.6mm, 5 μm), methyl alcohol is mobile phase A, and water is Mobile phase B, gradient elution, and elution program is 0-15min, 5%-37%A; 15-30min, 37%-100%A; 30-40min, 100%-5%A; Flow velocity 1.0mL/min, determined wavelength 279nm, column temperature 30 DEG C.
Result shows: in streptomyces diastatochromogenes mutant strain G3-13 and G3-15 fermented liquid, the antibiotic content of tetraene macrolide is all high than contrast streptomyces diastatochromogenes D, wherein the antibiotic ability of mutant strain G3-13 synthesis tetraene macrolide is the strongest, tetramycin P, the fermentation titer of Tetrin B and tetramycin A is up to 1065.18mg/L, 912.47mg/L, 885.24mg/L, is respectively 19.13 times of contrast, 17.19 times and 21.45 times.
table 1 streptomyces diastatochromogenes D and the antibiotic ability of mutant strain synthesis tetraene macrolide

Claims (2)

1. one kind is effectively improved the method for tetraene macrolide antibiotic yield, it is characterized in that the screening by associating drug-resistant mutation strain, obtain Rifampin 20 μ g/mL, lower concentration Streptomycin sulphate 30 μ g/mL and high density Streptomycin sulphate 300 μ g/mL are developed immunity to drugs streptomyces diastatochromogenes ( streptomycesdiastatochromogenes) mutant strain of D, the mutant strain synthesis tetraene macrolide obtained is antibiotic can be significantly improved by force rate streptomyces diastatochromogenes D, described streptomyces diastatochromogenes D is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation address, preservation date on May 25th, 2007, preservation registration number CGMCCNo.2060.
2. the antibiotic method of mutant strain synthesis tetraene macrolide according to claim 1, it is characterized in that fermentation culture is GYM liquid nutrient medium, component and concentration are glucose 4g/L, Driedyeastextract4g/L, DriedMaltextract-S10g/L, N-Z-AmineA1g/L, NaCl2g/L, OB solution 600 μ L, regulates pH to 7.3 with 2mol/LNaCl solution after constant volume; Wherein OB solution allocation method: take MgSO47H 2o25g, CuSO 45H 2o2.5g, FeSO 47H 2o3.75g, MnSO 45H 2o1.8g, CaCl 22H 2o17.5g, ZnSO 47H 2o4.5g, adds 500mL water, with front shaking up; The bottled 60mL fermentation culture of every 300mL triangle, prepares rear 121 DEG C of sterilizing 20min, to be cooled to 50 DEG C, difference picking one platinum ring streptomyces diastatochromogenes D and mutant strain spore inoculating under aseptic condition, 28 DEG C ± 1 DEG C, fermentation culture 6 days; The centrifugal 10min of fermentation liquor 5000r/min, supernatant liquor is used for the mensuration of tetraene macrolide antibiotic content.
CN201510966000.1A 2015-12-22 2015-12-22 Method for effectively increasing yield of tetraene macrolide antibiotics Pending CN105400853A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109142589A (en) * 2018-10-25 2019-01-04 湖南农业大学 A method of utilizing tetramycin residual quantity in high performance liquid chromatograph measurement soil

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104430407A (en) * 2014-12-18 2015-03-25 中国计量学院 Application of tetramycin P in inhibition of rhizoctonia solani kuhn of cucumbers and botrytis cinerea
CN104450835A (en) * 2014-12-18 2015-03-25 中国计量学院 Preparation method of new compound
CN104531798A (en) * 2014-12-18 2015-04-22 中国计量学院 Method for improving output of tetraene macrolide antibiotics

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104430407A (en) * 2014-12-18 2015-03-25 中国计量学院 Application of tetramycin P in inhibition of rhizoctonia solani kuhn of cucumbers and botrytis cinerea
CN104450835A (en) * 2014-12-18 2015-03-25 中国计量学院 Preparation method of new compound
CN104531798A (en) * 2014-12-18 2015-04-22 中国计量学院 Method for improving output of tetraene macrolide antibiotics

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109142589A (en) * 2018-10-25 2019-01-04 湖南农业大学 A method of utilizing tetramycin residual quantity in high performance liquid chromatograph measurement soil
CN109142589B (en) * 2018-10-25 2022-03-01 湖南农业大学 Method for determining tetramycin residual quantity in soil by using high performance liquid chromatograph

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