CN104342379A - Method utilizing fosfomycin to carry out breeding of daptomycin high-yield bacterial strains - Google Patents

Method utilizing fosfomycin to carry out breeding of daptomycin high-yield bacterial strains Download PDF

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Publication number
CN104342379A
CN104342379A CN201310311308.3A CN201310311308A CN104342379A CN 104342379 A CN104342379 A CN 104342379A CN 201310311308 A CN201310311308 A CN 201310311308A CN 104342379 A CN104342379 A CN 104342379A
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phosphonomycin
daptomycin
colonies
fosfomycin
culture medium
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沈依群
张晓来
陆茂林
钱国芬
许春燕
司竑飞
杨晓伟
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JIANGSU PROV MICROBILOGY INST CO Ltd
Jiangsu Institute of Microbiology
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JIANGSU PROV MICROBILOGY INST CO Ltd
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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
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

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Abstract

The invention discloses a method for obtaining daptomycin high-yield bacterial strains through screening streptomyces roseosporus, which is sensitive to fosfomycin, and belongs to the technology of bacterial strain breeding. The method comprises the following steps: dissolving spores cultured in a slope in sterile water to prepare a spore suspension, subjecting the spore suspension to a UV treatment, painting the suspension on a separated culture medium, picking the colonies growing on the culture medium by a sterile bamboo stick, then inoculating the colonies on solid screening culture mediums with different fosfomycin concentrations, selecting colonies that are sensitive to fosfomycin in different concentrations, inoculating the selected colonies on a slope culture medium to culture the colonies, then transferring the colonies to a liquid culture medium to carry out liquid culture in the end, and finally picking out the high-yield bacterial strains with stable genetic characters.

Description

A kind of method utilizing phosphonomycin seed selection daptomycin superior strain
Technical field
The invention belongs to the technical field of microorganism strains filtration, particularly utilize phosphonomycin to screen the technology of daptomycin superior strain.
Background technology
Along with antibiotic medicine widespread use clinically, the drug resistance problems of bacterium is more and more serious, causes the extensive concern of people.Vancomycin is for a long time always as last line of defense that the many serious Gram-positive bacterium of defence infect, but along with clinical application frequently, its Resistant strain also gets more and more, and the current clinical application of vancomycin is non-very good.Therefore research and develop novel antimicrobial agent microbiotic just to have very important significance.
Daptomycin (daptomycin/Cubicin) carrys out (Lily) company original research by gift, a kind of Cyclic lipopeptide antibiotic of Cubist drugmaker exploitation.In September, 2003, as a kind of microbiotic of brand new classification in U.S.'s Initial Public Offering.This microbiotic is from Streptomyces roseosporus (Streptomyces roseosporus) fermented liquid, extract the cyclic lipopeptide material obtained, it not only has novel chemical structure, and its binding mode is also all different from any one granted microbiotic, it is by upsetting cytolemma to amino acid whose transhipment, thus hinder the biosynthesizing of bacteria cell wall peptidoglycan, change the character of cytoplasmic membrane; In addition, it also by destroying the cytolemma of bacterium, makes its content leak and reach the object of sterilization.
Due to antimicrobial characteristic and the good market outlook of its uniqueness, in recent years, domestic many units began one's study successively.2006, University Of Tianjin disclosed a kind of method of selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete: within 2007, disclose a kind of synthetic method of daptomycin by Shanghai Organic Chemistry Institute, Chinese Academy of Sciences.Domestic also have people doing bacteria selection and genetic breeding work, but be all just started, also enough could not reach the requirement of suitability for industrialized production, so, the output how improving the daptomycin in roseosporus streptomycete will be produced its industrialization, and the sales and marketing possession share of product all has great importance.
Phosphonomycin is the reaction by UDP-GlcNAc-pyruvic acid transferring enzyme in the synthesis of block cell wall, thus the synthesis of T suppression cell wall.It is contemplated that, if be " sieve " with phosphonomycin (FOM), every bacterial strain high to FOM susceptibility, this point then in its Cell wall synthesis is possibility defectiveness just, making it can not synthetic cell wall well, and turns to synthetic antibiotic, reaches the object of breeding high-yield bacterial strain.Fan Mingqi etc. utilize and obtain successfully in phosphonomycin screening aminoglycoside antibiotics GMC1a superior strain, and tiring compared with parental plant improves 75 ~ 101%.And phosphonomycin application and the seed selection aspect of daptomycin producing strains, then have no relevant patent and the report of document.
Summary of the invention
The object of the invention is to propose a kind of utilization to screen the Streptomyces roseosporus of phosphonomycin sensitivity thus the method for acquisition daptomycin superior strain, Using such method effectively can improve the output of daptomycin.
The object of the invention is to realize by the following technical solutions: a kind of method utilizing phosphonomycin seed selection daptomycin superior strain, is characterized in that comprising following process: the slant pore sterilized water of Streptomyces roseosporus (Streptomyces roseosporus) parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 1-3ml, under 15 watts of ultraviolet lamps, process 0.5-1.5 minute, then the spore suspension sterilized water of radiotreatment is diluted to 10 -3-10 -5after, coat 26-28 DEG C of cultivation 7-8 days in culture plate, with aseptic bamboo let, the bacterium colony grown is chosen on the screening flat board of the phosphonomycin be inoculated in containing 200-1000 μ g/ml, picking is to the bacterium colony of respective concentration phosphonomycin sensitivity, after slant medium cultivates 6-7 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed, in 28-30 DEG C, rotating speed is cultivate 120-160h in the shaking table of 200-220rpm, pass through HPLC, detect daptomycin output, thus obtain the daptomycin producing strains of high yield.
In above-mentioned screening flat board, the content of phosphonomycin is: 200 μ g/ml or 400 μ g/ml or 600 μ g/ml or 800 μ g/ml or 1000 μ g/ml.
Above-mentioned liquid fermentation medium composition and mass content are: Zulkovsky starch 6-10g, yeast powder 4-6g, dextrin 10-20g, casein food grade 3-5g, Tryptones 4-10g, K 2sO 43-5g, altheine 1-3g, be dissolved in 1L tap water, PH7.5.
What the present invention adopted obtains daptomycin superior strain by screening phosphonomycin sensitive strain, and equipment is simple, method should be gone, respond well, and the bacterial strain positive mutation rate selected is 1-25%, and fermentation unit improves 0.3-2.9 doubly than starting strain.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but the non-scope being only limitted to these embodiments of scope of the present invention should be understood.
The slant pore sterilized water of embodiment 1 parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 2ml, processes 0.5 minute, then the spore suspension sterilized water of radiotreatment is diluted to 10 under 15 watts of ultraviolet lamps -3-10 -5after, to coat in culture plate 26 DEG C to cultivate 7 days, the screening flat board with aseptic bamboo let the bacterium colony grown being chosen the phosphonomycin be inoculated in containing 1000 μ g/ml (consists of, yeast powder 3g, Fructus Hordei Germinatus leaches powder 9g, glucose 3g, phosphonomycin 1g, agar 20g, deionized water 1L, PH nature), picking is to the bacterium colony of this phosphorus concentration mycin sensitivity, after slant medium cultivates 6 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed and (consist of Zulkovsky starch 8g, yeast powder 5g, dextrin 10g, casein food grade 5g, Tryptones 6g, K 2sO 43g, altheine 1g, be dissolved in 1L tap water, PH7.5), in 28 DEG C, rotating speed is cultivate 120h in the shaking table of 200rpm, and by HPLC, detect daptomycin output, positive mutation rate is 1-2%.Fermentation unit improves 0.3-0.4 doubly than parental plant.
Embodiment 2
The slant pore sterilized water of parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 2ml, processes 0.6 minute, then the spore suspension sterilized water of radiotreatment is diluted to 10 under 15 watts of ultraviolet lamps -3-10 -5after, to coat in culture plate 26 DEG C to cultivate 7 days, the screening flat board with aseptic bamboo let the bacterium colony grown being chosen the phosphonomycin be inoculated in containing 800 μ g/ml (consists of, yeast powder 3g, Fructus Hordei Germinatus leaches powder 9g, glucose 3g, phosphonomycin 0.8g, agar 20g, deionized water 1L, PH nature), picking is to the bacterium colony of this phosphorus concentration mycin sensitivity, after slant medium cultivates 6 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed and (consist of Zulkovsky starch 8g, yeast powder 5g, dextrin 10g, casein food grade 5g, Tryptones 6g, K 2sO 43g, altheine 1g, be dissolved in 1L tap water, PH7.5), in 28 DEG C, rotating speed is cultivate 120h in the shaking table of 200rpm, and by HPLC, detect daptomycin output, positive mutation rate is 4-5%.Fermentation unit improves 0.8-1.0 doubly than parental plant.
Embodiment 3
The slant pore sterilized water of parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 2ml, processes 1.0 minutes, then the spore suspension sterilized water of radiotreatment is diluted to 10 under 15 watts of ultraviolet lamps -3-10 -5after, to coat in culture plate 26 DEG C to cultivate 7 days, the screening flat board with aseptic bamboo let the bacterium colony grown being chosen the phosphonomycin be inoculated in containing 600 μ g/ml (consists of, yeast powder 3g, Fructus Hordei Germinatus leaches powder 9g, glucose 3g, phosphonomycin 0.6g, agar 20g, deionized water 1L, PH nature), picking is to the bacterium colony of this phosphorus concentration mycin sensitivity, after slant medium cultivates 6 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed and (consist of Zulkovsky starch 8g, yeast powder 5g, dextrin 10g, casein food grade 5g, Tryptones 6g, K 2sO 43g, altheine 1g, be dissolved in 1L tap water, PH7.5), in 28 DEG C, rotating speed is cultivate 120h in the shaking table of 200rpm, and by HPLC, detect daptomycin output, positive mutation rate is 12-13%.Fermentation unit improves 1.4-1.6 doubly than parental plant.
Embodiment 4
The slant pore sterilized water of parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 2ml, processes 1.0 minutes, then the spore suspension sterilized water of radiotreatment is diluted to 10 under 15 watts of ultraviolet lamps -3-10 -5after, to coat in culture plate 26 DEG C to cultivate 7 days, the screening flat board with aseptic bamboo let the bacterium colony grown being chosen the phosphonomycin be inoculated in containing 400 μ g/ml (consists of, yeast powder 3g, Fructus Hordei Germinatus leaches powder 9g, glucose 3g, phosphonomycin 0.4g, agar 20g, deionized water 1L, PH nature), picking is to the bacterium colony of this phosphorus concentration mycin sensitivity, after slant medium cultivates 6 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed and (consist of Zulkovsky starch 8g, yeast powder 5g, dextrin 10g, casein food grade 5g, Tryptones 6g, K 2sO 43g, altheine 1g, be dissolved in 1L tap water, PH7.5), in 28 DEG C, rotating speed is cultivate 120h in the shaking table of 200rpm, and by HPLC, detect daptomycin output, positive mutation rate is 15-16%.Fermentation unit improves 2.0-2.3 doubly than parental plant.
Embodiment 5
The slant pore sterilized water of parental plant makes 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 2ml, processes 1.0 minutes, then the spore suspension sterilized water of radiotreatment is diluted to 10 under 15 watts of ultraviolet lamps -3-10 -5after, to coat in culture plate 26 DEG C to cultivate 7 days, the screening flat board with aseptic bamboo let the bacterium colony grown being chosen the phosphonomycin be inoculated in containing 200 μ g/ml (consists of, yeast powder 3g, Fructus Hordei Germinatus leaches powder 9g, glucose 3g, phosphonomycin 0.4g, agar 20g, deionized water 1L, PH nature), picking is to the bacterium colony of this phosphorus concentration mycin sensitivity, after slant medium cultivates 6 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed and (consist of Zulkovsky starch 8g, yeast powder 5g, dextrin 10g, casein food grade 5g, Tryptones 6g, K 2sO 43g, altheine 1g, be dissolved in 1L tap water, PH7.5), in 28 DEG C, rotating speed is cultivate 120h in the shaking table of 200rpm, and by HPLC, detect daptomycin output, positive mutation rate is 20-21%.Fermentation unit improves 2.8-2.9 doubly than parental plant.

Claims (4)

1. utilize a method for phosphonomycin seed selection daptomycin superior strain, it is characterized in that comprising following process: the slant pore sterilized water of Streptomyces roseosporus (Streptomyces roseosporus) parental plant is made 10 5-10 7the monospore suspension of individual spore/ml, is sub-packed in glass dish by the spore suspension of 1-3ml, under 15 watts of ultraviolet lamps, process 0.5-1.5 minute, then the spore suspension sterilized water of radiotreatment is diluted to 10 -3-10 -5after, coat 26-28 DEG C of cultivation 7-8 days in culture plate, with aseptic bamboo let, the bacterium colony grown is chosen on the screening flat board of the phosphonomycin be inoculated in containing 200-1000 μ g/ml, picking is to the bacterium colony of respective concentration phosphonomycin sensitivity, after slant medium cultivates 6-7 days, be inoculated in the 250ml shaking flask that 30ml liquid fermentation medium is housed, in 28-30 DEG C, rotating speed is cultivate 120-160h in the shaking table of 200-220rpm, pass through HPLC, detect daptomycin output, thus obtain the daptomycin producing strains of high yield.
2. by method according to claim 1, it is characterized in that, screening culture medium composition and mass content are: yeast powder 2-5g, and Fructus Hordei Germinatus leaches powder 8-12g, glucose 3-5g, phosphonomycin 0.2-1g, agar 18-22g, deionized water 1L, PH nature.
3. by the method described in claim 1 and 2, it is characterized in that, in screening flat board, the content of phosphonomycin is: 200 μ g/ml or 400 μ g/ml or 600 μ g/ml or 800 μ g/ml or 1000 μ g/ml.
4. will go the method described in 1 by right, it is characterized in that, liquid fermentation medium composition and mass content are: Zulkovsky starch 6-10g, yeast powder 4-6g, dextrin 10-20g, casein food grade 3-5g, Tryptones 4-10g, K 2sO 43-5g, altheine 1-3g, be dissolved in 1L tap water, PH7.5.
CN201310311308.3A 2013-07-23 2013-07-23 Method utilizing fosfomycin to carry out breeding of daptomycin high-yield bacterial strains Pending CN104342379A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104774834A (en) * 2015-04-14 2015-07-15 中国医药集团总公司四川抗菌素工业研究所 Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model
CN105543212A (en) * 2016-01-15 2016-05-04 江西师范大学 Selective breeding method for eukaryotic microorganism anti-benomyl marked mutant strains

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104774834A (en) * 2015-04-14 2015-07-15 中国医药集团总公司四川抗菌素工业研究所 Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model
CN105543212A (en) * 2016-01-15 2016-05-04 江西师范大学 Selective breeding method for eukaryotic microorganism anti-benomyl marked mutant strains

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Application publication date: 20150211