CN105349476A - Method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains - Google Patents

Method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains Download PDF

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CN105349476A
CN105349476A CN201510966008.8A CN201510966008A CN105349476A CN 105349476 A CN105349476 A CN 105349476A CN 201510966008 A CN201510966008 A CN 201510966008A CN 105349476 A CN105349476 A CN 105349476A
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toyokamycin
screening
drug
resistant mutation
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申屠旭萍
许益鹏
俞晓平
汤谷
刘楠楠
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China Jiliang University
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    • 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
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    • C12R2001/525—Streptomyces diastatochromogenes
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides

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Abstract

The invention discloses a method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains and belongs to the field of microbial technologies. According to the method, Streptomyces diastatochromogenes D mutant strains generating drug resistance to low-concentration streptomycin, high-concentration streptomycin and paromomycin are obtained through screening of the united drug-resistant mutant strains, and the toyocamycin productivity of the obtained mutant strains is remarkably improved when compared with that of Streptomyces diastatochromogenes D.

Description

Associating drug-resistant mutation strain screening is utilized to improve the method for toyokamycin output
Technical field
The present invention relates to microbial technology field, particularly relate to the method utilizing associating drug-resistant mutation strain screening to improve toyokamycin output.
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.
Toyokamycin can suppress the growth of multiple pathogenic fungi, has wide biological and ecological methods to prevent plant disease, pests, and erosion application prospect.Streptomyces diastatochromogenes ( streptomycesdiastatochromogenes) D can synthesize toyokamycin, utilizes associating drug-resistant mutation screening method modified starch enzyme streptomyces chromogenes to make its research improving toyokamycin 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 lower concentration Streptomycin sulphate, high density Streptomycin sulphate and paromycin all develop immunity to drugs, the energy force rate streptomyces diastatochromogenes D of the mutant strain product toyokamycin of acquisition significantly improves.This invention is that the product element level improving toyokamycin provides a kind of method reliably.
The object of the invention is to produce the low deficiency of toyokamycin level for wild type strain streptomyces diastatochromogenes D, provide a kind of and improve the method that toyokamycin produces plain level; Another object of the present invention there is provided the streptomyces diastatochromogenes D mutant strain that a plant height produces toyokamycin.
The object of the invention is achieved through the following technical solutions:
The method utilizing associating drug-resistant mutation strain screening to improve toyokamycin output is carried out according to the following steps:
(1) acquisition (first round drug-resistant mutation strain screening) of lower concentration streptomycin resistant mutation 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.Early-stage Study shows to cultivate with GYM solid medium, and Streptomycin sulphate suppresses the MIC value of streptomyces diastatochromogenes D to be 20 μ g/mL.
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 100 μ g/mL, also namely GYM solid plate streptomycin concentration is 100 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; 1000 μ L streptomyces diastatochromogenes D spore suspensions (1 × 10 are drawn with liquid-transfering gun 6individual/mL) on the GYM solid plate containing 100 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 100 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.
(2) acquisition (second takes turns drug-resistant mutation strain screening) of high density streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 2000 μ g/mL, also namely GYM solid plate streptomycin concentration is 2000 μ g/mL.GYM solid medium component and the same step of concentration (1); 1000 μ L lower concentration streptomycin resistant mutation strains (mutant strain of also i.e. first round resistance screening acquisition) spore suspension (1 × 10 is drawn with liquid-transfering gun 12individual/mL) on the GYM solid plate containing 2000 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 2000 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.
(3) acquisition (third round drug-resistant mutation strain screening) of paromycin resistant mutant strain
Preparing the GYM solid plate that paromycin final concentration is 50 μ g/mL, is also that in GYM solid plate, paromycin concentration is 50 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 50 μ g/mL paromycin, 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 50 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 paromycin resistant mutant strain.
(4) mutant strain fermentation culture
Fermentation culture: fermentation culture is containing Zulkovsky starch 20g in every 1000mL, analysis for soybean powder 40g, NH 4cl3g, CaCO 35g, MgSO 42g, surplus is water; 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 drug-resistant mutation 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 toyokamycin content.
(5) detection of toyokamycin
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.
Beneficial effect of the present invention:
One is the screening that the present invention passes through the strain of associating drug-resistant mutation, and the energy force rate streptomyces diastatochromogenes D of the mutant strain synthesis toyokamycin of acquisition significantly improves, and this is that the output improving toyokamycin provides a kind of method reliably;
Two is the invention provides the streptomyces diastatochromogenes D mutant strain that a plant height produces toyokamycin, produces lay the foundation for toyokamycin industrialization from now on.
Embodiment
Utilize the strain of associating drug-resistant mutation to screen the method improving toyokamycin output below in conjunction with embodiment to the present invention to be described further.
Embodiment 1: the acquisition (first round drug-resistant mutation strain screening) of lower concentration streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 100 μ g/mL, also namely GYM solid plate streptomycin concentration is 100 μ g/mL.1000 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 100 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 8 strain altogether, numbering is respectively f11, f12, f13, f14, f15, f16, f17 and f18, carry out liquid fermentation and culture to this 8 plant mutant strain according to the step (4) in technical scheme, fermentation liquor HPLC detects, and finds that wherein mutant strain f11 product toyokamycin ability is higher, for 483.58mg/L, fermentation level is control strain streptomyces diastatochromogenes D(151.12mg/L) 3.2 times;
Embodiment 2: the acquisition (second takes turns drug-resistant mutation strain screening) of high density streptomycin resistant mutation strain
Prepare the GYM solid plate that Streptomycin sulphate final concentration is 2000 μ g/mL, also namely GYM solid plate streptomycin concentration is 2000 μ g/mL.1000 μ L lower concentration streptomycin resistant mutation strain f11 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 2000 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 3 strain altogether, numbering is respectively s223, s225, s228, carry out liquid fermentation and culture to this 3 plant mutant strain according to the step (4) in technical scheme, fermentation liquor HPLC detects, and finds that wherein mutant strain s228 product toyokamycin ability is higher, for 925.63mg/L, fermentation level is control strain streptomyces diastatochromogenes D(153.01mg/L) 6.05 times;
Embodiment 3: the acquisition (third round drug-resistant mutation strain screening) of paromycin resistant mutant strain
Preparing the GYM solid plate that paromycin final concentration is 50 μ g/mL, is also that in GYM solid plate, paromycin concentration is 50 μ g/mL.1000 μ L high density streptomycin resistant mutation strain s228 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 50 μ g/mL paromycin 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 paromycin resistant mutant strain.Obtain the strain of paromycin resistant mutant strain 2 altogether, numbering is respectively t3141 and t3145;
Embodiment 4: mutant strain liquid fermenting
Fermentation culture: fermentation culture is containing Zulkovsky starch 20g in every 1000mL, analysis for soybean powder 40g, NH 4cl3g, CaCO 35g, MgSO 42g, surplus is water; The bottled 60mL fermentation culture of every 300mL triangle, prepare rear 121 DEG C of sterilizing 20min, to be cooled to 50 DEG C, difference picking one platinum ring streptomyces diastatochromogenes D, mutant strain t3141 and mutant strain t3145 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 toyokamycin content;
Embodiment 5: the detection of toyokamycin
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 t3145 and t3141 fermented liquid, the content of toyokamycin is respectively 2294.95mg/L and 1985.15mg/L, improve 15.26 and 13.20 times respectively compared with contrast 150.39mg/L, wherein streptomyces diastatochromogenes mutant strain t3145 synthesizes the ability of toyokamycin the most by force, and fermentation titer reaches 2294.95mg/L.

Claims (2)

1. utilize associating drug-resistant mutation strain screening to improve the method for toyokamycin output, it is characterized in that the screening by associating drug-resistant mutation strain, obtain lower concentration Streptomycin sulphate 100 μ g/mL, high density Streptomycin sulphate 2000 μ g/mL and paromycin 50 μ g/mL are developed immunity to drugs streptomyces diastatochromogenes ( streptomycesdiastatochromogenes) mutant strain of D, the energy force rate streptomyces diastatochromogenes D that the mutant strain obtained produces toyokamycin significantly improves, 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 method for mutant strain synthesis toyokamycin according to claim 1, is characterized in that fermentation culture is containing Zulkovsky starch 20g in every 1000mL, analysis for soybean powder 40g, NH 4cl3g, CaCO 35g, MgSO 42g, surplus is water, the bottled 60mL fermentation culture of every 300mL triangle, prepare rear 121 DEG C of sterilizing 20min, to be cooled to 50 DEG C, difference picking one platinum ring streptomyces diastatochromogenes D and drug-resistant mutation 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 toyokamycin content.
CN201510966008.8A 2015-12-22 2015-12-22 Method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains Pending CN105349476A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103243062A (en) * 2013-05-08 2013-08-14 中国计量学院 Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof
CN104404111A (en) * 2014-12-18 2015-03-11 中国计量学院 Method for improving capacity of Streptomyces diastatochromogenes for synthesizing toyocamycin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103243062A (en) * 2013-05-08 2013-08-14 中国计量学院 Streptomyces diastatochromogenes engineering strain for producing toyocamycin, as well as construction method and application thereof
CN104404111A (en) * 2014-12-18 2015-03-11 中国计量学院 Method for improving capacity of Streptomyces diastatochromogenes for synthesizing toyocamycin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李丹婷等: "淀粉酶产色链霉菌1628发酵培养基及发酵条件的优化", 《中国计量院学报》 *

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Application publication date: 20160224