CN103882005B - The mutagenesis screening method of streptomycete bacterial strain SPC6-50-1 and application - Google Patents

The mutagenesis screening method of streptomycete bacterial strain SPC6-50-1 and application Download PDF

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CN103882005B
CN103882005B CN201410057958.4A CN201410057958A CN103882005B CN 103882005 B CN103882005 B CN 103882005B CN 201410057958 A CN201410057958 A CN 201410057958A CN 103882005 B CN103882005 B CN 103882005B
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streptomycete
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rifampicin
ntg
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张昺林
陈熙明
张威
张满效
陈拓
刘光琇
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Cold and Arid Regions Environmental and Engineering Research Institute of CAS
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Abstract

The invention discloses the mutagenesis screening method of streptomycete bacterial strain SPC6-50-1, it is, by nitrosoguanidine, streptomyces coelicolor and streptomycete Lzsg 6 are carried out mutation, obtain streptomycete mutant library, screen again through rifampicin and streptomycin, obtain re-recording system and bacterial strain that translation system is undergone mutation, and disclose streptomycete bacterial strain SPC6-50-1 and application thereof.The invention have the benefit that streptomycete SPC6-50-1 bacterial strain provided by the invention has the material of bacteriostatic activity, and the method for its NTG induction, rifampicin and streptomycin screening can activate some silent genes of streptomycete, obtain new metabolite, obtain antibiotics, simultaneously, above-mentioned induced screening method has simple to operate, cycle is short, the advantage that efficiency is high, may be not only suitable for streptomycete, it is also applied for slime bacteria etc. and has the microorganism of abundant secondary metabolite, have good value for applications.

Description

The mutagenesis screening method of streptomycete bacterial strain SPC6-50-1 and application
Technical field
The present invention relates to production of antibiotics field, be specifically related to the mutagenesis screening method of streptomycete bacterial strain SPC6-50-1, streptomycete bacterial strain SPC6-50-1 and application thereof.
Background technology
Since the nineteen twenty-eight mankind find antibiotic first---since penicillin, in these decades, antibiotic industry develops rapidly, and healthy for the mankind works it out huge contribution.But due to the irrational use antibiotic of people, especially China, the problem of antibiotic resistance breaks out out gradually.Solving this problem, except noting antibiotic reasonable employment, it is also very important for developing the new antibiotics that drug resistance pathogenic bacteria has inhibitory activity.
Streptomycete is that one is widely present in nature, particularly in soil, and protokaryon, gram positive bacteria, belong to actinomycetes door.Streptomycete has Morphological Differentiation and a cometabolism of complexity, and is nourishing and growing and the transition period of spore differentiation can produce multiple have bioactive secondary metabolite.The metabolite that this genus produces, except antibiotic, also includes the outer hydrolytic enzyme of antitumor agent, immunosuppressant, insect resistant agent and born of the same parents, such as amylase, chitinase cellulase, pectase, xylanase and protease etc..These secondary metabolites all have important application in the field such as medical treatment, agricultural, food.
So far more than 12000 found the forties in 20th century are planted in microbe-derived bioactive substance, and 55% is above being produced by streptomycete, it has been found that antibiotic in there are about 70% and produced by streptomycete.The antibiotic about 2/3rds applied clinically at present derives from streptomyces.
Sum up the antibiotic quantity obtained from streptomycete in the past few decades, since finding the nineties, it is found that the quantity of antibiotics sharply reduces, this illustrates that traditional method obtaining antibiotics by screening new streptomycete has encountered bottleneck (Berdy, 2005).Find a kind of new efficient acquisition antibiotics method necessary.
At present, several streptomycetes such as streptomyces coelicolor (Streptomycescoelicolor), Avid kyowamycin (Streptomycesavermitilis), streptomyces griseus (Streptomycesgrise μ s) have been completed genome sequencing.It was discovered by researchers that in the gene of streptomycete linear structure, the central core district gene of high conservative is main relevant to some important cell functions, and have in core space both sides substantial amounts of secondary metabolite gene cluster (Chenetal., 2002;Ohnishietal., 2008).Such as: at the genome analyzing streptomyces coelicolor, find there are 23 gene clusters participating in forming complicated secondary metabolite, account for the 5%(Bentleyetal. of genome total amount, 2002), it is the raw catabolite genes bunch of StreptomycescoelicolorA3 (2) parity as shown in table 1;Avid kyowamycin has 30 cometabolism gene clusters, accounts for the 6.6%(Ikedaetal. of full-length genome, 2003).Therefore, from this angle, whole streptomyces is exactly a huge potential secondary metabolite storehouse.Make streptomycete transcribe by some method, translate these genes, and the secondary metabolites filtering out active material is a kind of effective way (Zazopo μ losetal., 2003) obtaining antibiotics.
Table 1
In streptomycete, gene and the approach specific regulatory control protein gene Chang Liansuo of coding cometabolism particularly antibiotic biosynthesis pathway exist.Often comprising one in gene cluster to multiple approach specific regulatory control genes, they regulate and control one or more antibiotic biosynthesiss with common biological route of synthesis.As: actinorhodin and the gene expression of undecyl bacterium spirit red pigment that streptomyces coelicolor produces are regulated and controled (vanWezelandMcDowall, 2011) by its specific transcription activator protein ActII-ORF4 and RedD.In streptomycete, overwhelming majority secondary metabolites synthetic gene and specific regulatory control gene are all reticent, only when running into some special environment, corresponding special metabolic pathway controlling gene can be activated, produce new metabolite or the adjustment to its Physiology and biochemistry, streptomycete is helped to adapt to environment, pull through their difficulties (ChallisandHopwood, 2003).After the biological synthesis gene cluster finding certain potential compound, by the method for Combinatorial biosynthesis (CombinatorialBiosynthesis), as this gene cluster is carried out heterogenous expression;Or cut out with enzyme and to modify or to utilize the method for sudden change to change heredity metabolic process, it is possible to obtain new antibiotic (Medemaetal., 2011;Okamotoetal., 2003).Yu Zhibin et al. uses antibiotic that the inactive streptomycete B3054 deriving from ocean is screened the 1 strain chlorampenicol resistant and the streptomycin resistance strain that obtain anti-tumor activity.And through activated product analysis, separate from 1 strain streptomycin resistance strain fermented product and obtain 5 active compound for anti tumor, one of them is new natural product (Yu Zhibin, Zhu Tianjiao, Cui Chengbin etc., 2006).Therefore, obtaining antibiotics by change streptomycete heredity metabolic pathway is a kind of very effective way.
NTG is nitrosoguanidine, is a kind of very strong chemical mutagen.The sudden change that NTG brings out is mainly GC-AT conversion, additionally also has the disappearance of the excision of little scope, frameshift mutation and GC pair.The present invention first passes through superpower mutagenic agent NTG and streptomyces gene group is carried out mutation, and the induced mutation rate of NTG can reach tens percent, and is random, thus can obtain a more comprehensive streptomycete mutant library of ratio.Then recycling rifampicin screens with streptomycin.Rifampicin can be combined with the β subunit of the RNA polymerase relying on DNA, it is suppressed that the synthesis of bacteria RNA, it is prevented that this enzyme is connected with DNA, thus blocking rna transcription process, makes the synthesis of DNA and albumen stop.Screened by rifampicin, it is possible to obtain the bacterial strain that can grow on the flat board containing rifampicin because re-recording system is undergone mutation efficiently.These sudden changes change original re-recording system, and the sudden change of re-recording system may make the interior regulation and control disorder of antibacterial, some silent genes therefore can be made to be expressed, thus having an opportunity to obtain antibiotics.Streptomycin is a kind of aminoglycoside medicine, through active transport by bacterial cell membrane, with the specific receptor protein binding of bacterial ribosome 30S subunit, the formation of initiation complex between interference messenger ribonucleic acid and 30S subunit, it is suppressed that albumen synthesizes.Can obtaining, by the screening of streptomycin, the bacterial strain that translation system is undergone mutation, these bacterial strains there will be a degree of change for protein translation ability.
Summary of the invention
The purpose of the present invention is aiming at above-mentioned defect of the prior art, providing the mutagenesis screening method of streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1, streptomycete bacterial strain SPC6-50-1, this bacterial strain is that the method efficiently activating streptomycete silence antibiotic resistance gene metabolism gene cluster obtains.
To achieve these goals, technical scheme provided by the invention is: the mutagenesis screening method of streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1, it is, by nitrosoguanidine (NTG), streptomyces coelicolor (StreptomycescoelicolorA3 (2)) and streptomycete (Streptomycessp.) Lzsg6 are carried out mutation, obtain streptomycete mutant library, screen again through rifampicin and streptomycin, obtain re-recording system and bacterial strain that translation system is undergone mutation.
Further, the mutagenesis screening method of above-mentioned streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1, include following steps:
1) streptomycete is carried out mutation by chemical mutagen NTG: streptomyces coelicolor (StreptomycescoelicolorA3 (2)) and streptomycete (Streptomycessp.) Lzsg6 are prepared for bacteria suspension, with NTG mother solution, bacteria suspension is carried out mutation, obtain the NTG bacteria suspension processed;
2) select, in order to good fortune plansifter, the bacterial strain that re-recording system undergos mutation: the bacteria suspension that NTG step 1) obtained processed uniformly is applied on the Gause I culture medium flat plate containing 5 μ g/ml rifampicin, cultivate 7-15d for 30 DEG C, the single bacterium colony grown is connected in the test tube equipped with 5mL fluid medium YEME or TSBY respectively, then mutagenic bacteria suspension is prepared into again, carry out NTG mutation, the bacteria suspension processed uniformly is applied on the Gause I culture medium flat plate containing 10 μ g/ml rifampicin, repeatedly, carry out 15 μ g/ml, 20 μ g/ml, 25 μ g/ml, 30 μ g/ml, 35 μ g/ml, 40 μ g/ml, 45 μ g/ml, the rifampicin screening of 50 μ g/ml, obtain bacterial strain after rifampicin screens;
3) bacterial strain undergone mutation of translation system is screened with streptomycin: by step 2) bacterial strain carries out NTG mutation again after the rifampicin screening that obtains, method and step 2) identical, the bacteria suspension processed uniformly is applied to containing 5 μ g/mL streptomycins, on the Gause I culture medium flat plate of 50 μ g/mL rifampicin, cultivate 7-15d for 30 DEG C, the single bacterium colony grown is connected in the test tube equipped with 5mL fluid medium YEME or TSBY respectively, then mutagenic bacteria suspension is prepared into again, carry out NTG mutation, the bacteria suspension processed uniformly is applied to containing 10 μ g/mL streptomycins, on the Gause I culture medium flat plate of 50 μ g/mL rifampicin, repeatedly, Concentration of Rifampicin is 50 μ g/mL, streptomycin is 15 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/ml, 50 μ g/ml, 60 μ g/ml, 70 μ g/ml, 80 μ g/ml, 90 μ g/ml, 100 μ g/ml are incremented by successively, obtain streptomycin, bacterial strain after rifampicin screening.
Second purpose of the present invention there is provided streptomycete (Streptomyceslinzensis) the bacterial strain SPC6-50-1 that a strain obtains with above-mentioned mutagenesis screening method mutagenesis screening, described streptomycete (Streptomyceslinzensis) SPC6-50-1, it is preserved in (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18th, 2013, Institute of Microorganism, Academia Sinica), the deposit number of streptomycete SPC6-50-1 is CGMCCNo.8372.This bacterial strain is Gram-positive, can not form aerial mycelium, vegetative mycelium is yellow, cultivates one week for 37 DEG C, colony diameter about 7mm.
3rd purpose of the present invention there is provided above-mentioned streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1 application in preparing antibiotic.
4th purpose of the present invention there is provided the application in activating streptomycete silent gene of the mutagenesis screening method of above-mentioned streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1.
5th purpose of the present invention there is provided the application in preparing antibiotic of the mutagenesis screening method of above-mentioned streptomycete (Streptomyceslinzensis) bacterial strain SPC6-50-1.
Further, above-mentioned application, it is that the bacterial strain after streptomycin, rifampicin being screened is in the flat lining out of Gause I, collect Conidia preservation, it is connected in the triangular flask equipped with fluid medium YEME or TSBY, at 30 DEG C, the shaking table of 200r/min is cultivated 3-7d, 12000r/min is centrifuged 1min, takes supernatant.
Streptomyces coelicolor (StreptomycescoelicolorA3 (2)) and streptomycete (Streptomycessp.) Lzsg6 are carried out mutation by NTG by the method for the present invention, obtain a more comprehensive streptomycete mutant library of ratio, then pass through the bacterial strain that rifampicin and streptomycin filter out re-recording system and translation system is undergone mutation, the fermentation liquid of these bacterial strains is carried out bacteriostatic activity detection, find to change before its metabolite activity is relatively, this illustrates that the method can activate some silent genes of streptomycete, obtain new metabolite, thus obtaining antibiotics.
The invention have the benefit that streptomycete provided by the invention (Streptomyceslinzensis) bacterial strain SPC6-50-1 has the material of bacteriostatic activity, and the method for its NTG induction, rifampicin and streptomycin screening can activate some silent genes of streptomycete, obtain new metabolite, obtain antibiotics, simultaneously, above-mentioned induced screening method has simple to operate, cycle is short, the advantage that efficiency is high, may be not only suitable for streptomycete, it is also applied for slime bacteria etc. and has the microorganism of abundant secondary metabolite, have good value for applications.
Accompanying drawing explanation
Fig. 1 is the concrete technology flow chart of the mutagenesis screening method of streptomycete provided by the invention (Streptomyceslinzensis) bacterial strain SPC6-50-1.
Fig. 2 is the bacteriostatic activity comparing result schematic diagram of wild type streptomyces coelicolor (StreptomycescoelicolorA3 (2)) and mutant thereof.
Fig. 3 is the bacteriostatic activity comparing result schematic diagram of streptomycete (Streptomycessp.) Lzsg6 and mutant thereof.
Detailed description of the invention
Embodiment 1:
By chemical mutagen NTG, streptomycete is carried out mutation, as shown in Figure 1:
One, the processing method of NTG mutagenic species:
1, the preparation of culture medium and solution used:
TSBY (1L): Oxoid tryptone bean powder (TSB) 30g;Sucrose 340g;Oxoid yeast extract 5g;Distilled water is to 1000ml.
YEME (1L): Oxoid yeast extract 3g;Oxoid peptone Tryptone5g;Fructus Hordei Germinatus extract 3g;Glucose 10g;Sucrose 340g;MgCl2-6H2O (independent sterilizing);Distilled water is to 1000ml.
PH6.0 phosphate buffer: K2HPO42g/L, KH2PO48g/L, 0.1MPa sterilizing 20min.
The preparation of NTG mother solution: weigh 0.1gNTG and add cosolvent Methanamide or acetone 1mL, be subsequently adding 0.1mol/LpH6.0 phosphate buffer 9mL, be configured to the NTG mother solution of 10mg/mL.
2, prepared by strain:
Experimental strain is that streptomyces coelicolor StreptomycescoelicolorA3 (2) and Streptomycessp.Lzsg6(is isolatable from the husky raw phragmites communis Rhizosphere Soil of ecosystem in Linze, Gansu, China (N39 ° 22 ' 02.52 " E100 ° 07 ' 46.68 ")).
Bacterial strain is connected in the test tube equipped with 5mL fluid medium YEME or TSBY, at 30 DEG C, the shaking table of 200r/min is cultivated 3-7d, obvious cotton-shaped mycelium can be observed directly.
3, the preparation of mutagenic bacteria suspension:
Taking culture fluid to 2mLdoff pipe, 8000r/min is centrifuged 1min, washes twice with the 0.1mol/L phosphate buffer of pH6.0, is then resuspended in the buffer suspension of 2mLpH6.0.
Two, NTG mutagenic treatment screening:
Bacteria suspension adds NTG mother solution so that it is final concentration of 1mg/mL, process 60min.
It is disposed, by centrifugal for bacteria suspension 8000r/min 1min, washes twice with the 0.1mol/L phosphate buffer of pH6.0, be resuspended in the buffer suspension of 2mLpH6.0 afterwards.
Embodiment 2:
The bacterial strain that re-recording system is undergone mutation is screened by rifampicin:
The bacteria suspension processed by NTG is uniformly applied on the Gause I culture medium flat plate containing 5 μ g/ml rifampicin, cultivate 7-15d for 30 DEG C, the single bacterium colony grown is connected in the test tube equipped with 5mL fluid medium YEME or TSBY respectively, then mutagenic bacteria suspension is prepared into again, carry out NTG mutation, the bacteria suspension processed uniformly is applied on the Gause I culture medium flat plate containing 10 μ g/ml rifampicin, so repeatedly, carry out 15 μ g/ml, 20 μ g/ml, 25 μ g/ml, 30 μ g/ml, 35 μ g/ml, 40 μ g/ml, 45 μ g/ml, the rifampicin screening of 50 μ g/ml, obtain the bacterial strain that rifampicin is insensitive.
Embodiment 3:
By the bacterial strain that the screening translation system of streptomycin is undergone mutation:
Garbled for rifampicin bacterial strain is carried out NTG mutation again, method is ibid, the bacteria suspension processed uniformly is applied to containing 5 μ g/mL streptomycins, on the Gause I culture medium flat plate of 50 μ g/mL rifampicin, cultivate 7-15d for 30 DEG C, the single bacterium colony grown is connected in the test tube equipped with 5mL fluid medium YEME or TSBY respectively, then mutagenic bacteria suspension is prepared into again, carry out NTG mutation, the bacteria suspension processed uniformly is applied to containing 10 μ g/mL streptomycins, on the Gause I culture medium flat plate of 50 μ g/mL rifampicin, so repeatedly, Concentration of Rifampicin is 50 μ g/mL, streptomycin is 15 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/ml, 50 μ g/ml, 60 μ g/ml, 70 μ g/ml, 80 μ g/ml, 90 μ g/ml, 100 μ g/ml are incremented by successively, obtain streptomycin, the bacterial strain that rifampicin is all insensitive.
Embodiment 4:
Bacterial strain produces antibiotic detection:
One, the preparation of fermentation liquid:
By the bacterial strain that streptomycin, rifampicin is all insensitive that obtains at the flat lining out of the Gause I not having resistance, collect Conidia preservation, be connected in the triangular flask equipped with fluid medium YEME or TSBY, at 30 DEG C, the shaking table of 200r/min is cultivated the centrifugal 1min of 3-7d, 12000r/min, takes supernatant.
Two, Antibacterial Activity:
Supernatant Antibacterial Activity adopts Odontothrips loti.Experimental strain is escherichia coli (Escherichiacoli) and bacillus subtilis (Bacill μ ss μ btilis).The preparation beef extract-peptone agar plate containing both bacterium, adds fermented liquid supernatant obtained in the previous step in the cup of Oxford, cultivates 16h for 37 DEG C, measures inhibition zone size with ruler.With the fermented liquid supernatant of wild type StreptomycescoelicolorA3 (2) and Streptomycessp.Lzsg6 for comparison, result is as shown in Figures 2 and 3.In Fig. 2, with bacterial strain 2 fermented liquid supernatant to colibacillary antibacterial circle diameter for 100%, bacterial strain 1-9 is StreptomycescoelicolorA3 (2) 9 mutants obtained by the method.In Fig. 3, with the fermented liquid supernatant of wild type StreptomycescoelicolorA3 (2) to colibacillary antibacterial circle diameter for 100%, bacterial strain 1-6 is Streptomycessp.Lzsg6 6 mutants obtained by the method.
From the result of Fig. 2 and Fig. 3 it can be seen that after the processing of the method, wild type StreptomycescoelicolorA3 (2) bacteriostatic activity there occurs very big change, and the bacteriostatic activity of its mutant 2 is about 6 times of wild type.Wild type Streptomycessp.Lzsg6 itself is almost without bacteriostatic activity, and after the processing of the method, its mutant 2,4,5 has had obvious bacteriostatic activity, illustrates that mutant creates the material with bacteriostatic activity.This illustrates that the method can activate some silent genes of streptomycete, obtains new metabolite.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement.All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.

Claims (2)

1. the streptomycete that a strain obtains with mutagenesis screening method mutagenesis screening(Streptomyceslinzensis)Bacterial strain SPC6-50-1, it is characterised in that the preserving number of described streptomycete is CGMCCNo.8372.
2. the streptomycete described in claim 1(Streptomyceslinzensis)Bacterial strain SPC6-50-1 application in preparing antibiotic.
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