CN106191030A - A kind of high-throughput screening method of chlortetracycline superior strain - Google Patents
A kind of high-throughput screening method of chlortetracycline superior strain Download PDFInfo
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Abstract
The invention discloses the high-throughput screening method of a kind of chlortetracycline superior strain, belong to metabolic engineering technical field.The present invention is grown by streptomyces aureus on 5 kinds of plating mediums and is produced anti-amount and compares, and have selected optimal medium, and in order to set out, strain provides guarantee.The present invention passes through chlortetracycline and Sm3+Chromogenic reaction and chlortetracycline and hydrochloric acid heat altogether two kinds of prescreening methods of colorimetry compared with, select relatively simple and accurately chromogenic reaction as prescreening method.Demonstrate samaric nitrate chromogenic reaction as the repeatability of primary dcreening operation means and accuracy.
Description
Technical field
The present invention relates to a kind of method screening high yield chlortetracycline mutagenic strain, belong to micro-organisms technical field.
Background technology
Chlortetracycline (Chorotetracycline, i.e. CTC) belongs to a kind of broad ectrum antibiotic of Tetracyclines.Chlortetracycline energy
Enough the most antibacterial, growth promotion, efficiency of feed utilization high, in human body, residual quantity is low.Its production technology is the most ripe, and production cost is also
Relatively low, become the antimicrobial growth promoter that in current feed industry, consumption is maximum.The productivity of country facilities chlortetracycline is the most relatively low at present
(titer is at 18000 μ about g/mL), the productivity therefore improving chlortetracycline is necessary.Chlortetracycline is by streptomyces aureus
The secondary metabolite that (Streptomyces aureofaciens) fermentation obtains, its yield synthesis character is determined by polygenes,
Metabolism network is complicated.Affect the factor of Ferment of DM process also a lot, such as the quality of seed, the composition of culture medium, technique bar
Part and the Metabolism control etc. of sweat.Microbial Breeding technology develops into generation from mutation, reasoning, recombinant technique at present
Thank to the methods such as engineering, systems biology and heterologous organisms synthesis.But classic mutagenesis breeding still has many advantages.As need not know
Road biology genetic background, metabolic model, it is only necessary to obtain effective mutation library and directed screening.High flux screening is tied with routine mutagenesis
Close, it is adaptable to improve the industrial producing strain selection-breeding as target of the secondary metabolite yield, reduce leakage sieve phenomenon as far as possible.
The screening of existing chlortetracycline bacterial strain, is usually after using shake flask fermentation and detects by HPLC method, there is training
The defects such as foster cycle length, detection required time length, detection method are loaded down with trivial details.
Summary of the invention
In order to solve the problems referred to above, the invention provides the high-efficiency screening method of a kind of chlortetracycline superior strain.The present invention
Method combines atmospheric pressure at room plasma (ARTP), microplate reader detection and High Throughput Screening Assay, improves chlortetracycline Producing Strain
The screening efficiency of strain.
Described starting strain streptomyces aureus (Streptomyces aureofaciens) is by Shandong pharmacy (Inner Mongol)
The industrial producing strain that company limited provides.
The high-efficiency screening method of the chlortetracycline superior strain of the present invention, mainly comprises the following steps that
(1) streptomyces aureus bacteria suspension to be screened is coated in solid plate culture medium, selects after spore maturation
It is inoculated in and cultivates equipped with in 96 shallow bore hole plates of seed culture fluid；
(2) it is forwarded to parallel for the seed liquor obtained in 96 shallow bore hole plates equipped with fermentation training in 48 orifice plates of fermentation culture
Support；
(3), after fermentation ends, centrifuging and taking thalline, it is 0～20mg/L that Aspirate supernatant is diluted to chlortetracycline concentration, then takes
Diluent 100 μ L adds to and adds 40 μ L Tris-HCl buffer solution and 60 μ L Sm (NO) in advance3In 96 orifice plates of solution, shake
Swinging mixing, after 8min, colour developing is completely, and microplate reader measures the OD of each concentration405Value, according to y=0.0112x-0.009, R2=
0.99905 (wherein, x be chlortetracycline concentration concentration (μ g/mL), y be OD405Nm) it is calculated chlortetracycline concentration, and then according to dilute
Release multiple conversion and obtain the chlortetracycline concentration of fermentation liquid, i.e. obtain the bacterial strain that pan mycin is of a relatively high.
In one embodiment of the invention, the cultivation in the solid plate culture medium of described step (1) is at 34 DEG C
Cultivate 4d.
In one embodiment of the invention, the solid plate culture medium prescription (g/L) of described step (1): sucrose
20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
In one embodiment of the invention, the seed culture fluid (g/L) of described step (1): sucrose 30g, import ferment
Female powder 5g, MgSO40.5g, (NH4)2SO43g, NaCl 4g, KH2PO41g, CaCO30.2g。
In one embodiment of the invention, in 96 shallow bore hole plates of described step (1) cultivate be 750r/min, 30～
32 DEG C of shaking tables cultivate 23～25h.
In one embodiment of the invention, the fermentation culture (g/L) of described step (2): sucrose 30g,
MgSO40.5g, CaCO30.33g, citric acid 2.55g, (NH4)2SO43g, 1mL mixed liquor (0.3%CuSO4·5H2O, 0.5%
ZnSO4·7H2O, 0.25%MnSO4`4H2O)。
In one embodiment of the invention, the fermentation culture of described step (2) by seed liquor with 8% (v/v)
Inoculum concentration is forwarded in 48 orifice plates, in 750r/min, 30～31 DEG C of fermentation culture 96～110h.
In one embodiment of the invention, the centrifugal of described step (3) is to be centrifuged 15min at 4000r/min.
In one embodiment of the invention, described streptomyces aureus bacteria suspension to be screened, is by streptomyces aureus
Obtain after carrying out mutation.
In one embodiment of the invention, described mutation, can be physical mutagenesis or chemomorphosis, such as normal pressure
Room-temperature plasma (ARTP) mutation, ultraviolet mutagenesis, ethylmethane sulfonate (EMS) mutation, nitrosoguanidine (NTG) mutation, sulphuric acid
Diethylester (DES) mutation etc..
In one embodiment of the invention, in described step (1) 96 shallow bore hole plate, seed culture fluid addition is 180 μ
L/ hole.
In one embodiment of the invention, in described step (2) 48 orifice plate, the addition of fermentation culture is 1000 μ
L/ hole.
In one embodiment of the invention, the pH of described Tris-HCl buffer solution is 6.7, Sm (NO)3Solution
Concentration is 5 × 10-4mol/L。
Beneficial effects of the present invention:
The present invention establishes a kind of novel method combining chlortetracycline chromogenic reaction and high flux screening.The present invention grinds
Study carefully 5 kinds of chlortetracycline to grow and can detect the growth of plating medium of chlortetracycline and produce anti-amount situation, obtained
Optimal flat board is cultivated and formula, is effectively increased spore growth speed, is effectively increased follow-up screening efficiency.Meanwhile, the present invention
By chromogenic reaction reagent etc. is optimized, obtains the detection method that linear relationship is good, be effectively increased the efficiency of screening
And accuracy, it is therefore prevented that the problem that the best screening accuracy caused of extraction effect is not enough.
Detailed description of the invention
The mensuration of chlortetracycline: high performance liquid chromatography (HPLC)
Instrument: Agilent 1260 high performance liquid chromatograph (joins UV-vis detector, differential refraction detector and work
Stand), chromatographic condition:
Chromatographic column: ZORBAX LEclipse Lus Plus C18
Flowing phase: 28%N-N dimethylformamide+2%2mol/LH3PO4+ 70% deionized water
Flow velocity: 1mL/min
Column temperature: 35 DEG C
Sample size: 10 μ L
UV-detector wavelength: 365nm (detection chlortetracycline)
Prepared by sample: weigh fermentation liquid 1g in test tube, be settled to 50mL, ultrasonic 5min, 0.22 μ with 0.01mol/LHCl
After L inorganic system membrane filtration, carry out efficient liquid phase chromatographic analysis.
The calculating of fatality rate:
Wherein A represents the colony-forming units number on blank flat board, and B represents on mutagenic treatment rear plate
Colony-forming units number.
Culture medium:
Solid plate culture medium 1 (g/L): sucrose 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
Solid plate culture medium 2 (g/L): soluble starch 20g, import yeast powder 0.5g, KH2PO40.8g, agar
18g。
Solid plate culture medium 3 (g/L): glucose 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
Solid plate culture medium 4 (g/L): glycerol 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
Solid plate culture medium 5 (g/L): flour 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
Seed culture medium (g/L): sucrose 30g, import yeast powder 5g, MgSO40.5g, (NH4)2SO43g, NaCl 4g,
KH2PO41g, CaCO30.2g。
Orifice plate fermentation medium (g/L): sucrose 30g, MgSO40.5g, CaCO30.33g, citric acid 2.55g, (NH4)2SO43g, 1mL mixed liquor (0.3%CuSO4·5H2O, 0.5%ZnSO4·7H2O, 0.25%MnSO4`4H2O)。
Embodiment 1: optimal plating medium selects
The oblique plating medium of different formulations in preparation five, coats solid by the aseptic streptomyces aureus bacteria suspension after dilution
On body slant medium.Observe streptomyces aureus growing state on plating medium, and detect its anti-amount of product.Found that
The upper bacterial strain energy fast-growth of solid plate culture medium 1, spore growth is good, and plating medium color will not be to follow-up list
Clone selects instrument and chooses bacterium and interfere.Other culture medium, otherwise strain growth is relatively slow, otherwise plating medium color is for follow-up
Monoclonal is selected instrument and is chosen bacterium and interfere.
The mutation of embodiment 2:ARTP
Starting strain slant strains 34 DEG C cultivate 4-6d, scrape appropriate spore in band bead equipped with 2mL physiology salt
The test tube of water is broken up, takes 10 μ L on the most sterilized special slide glass, be put on the object stage of ARTP mutation instrument, adjust power
10-100w, irradiate 0 respectively, 10,20,30,40,50,60s, the slide glass tweezers after irradiating grip equipped with normal saline
In EP pipe, after earthquake device fully shakes, gradient dilution.Draw 100 μ L diluents to be spread evenly across on flat board.34 DEG C of constant temperature
After incubator cultivates 4d, count the colony-forming units number on every piece of flat board, then calculate each mutagenic treatment time
Fatality rate.Being 10-100W in incident power, under the conditions of helium gas flow is 1-10SLM, 0-25s irradiation time lures as optimal
Change processes the time, and fatality rate is the generation of about 80%-100%, beneficially positive mutating strain.
Embodiment 3: chlortetracycline chromogenic reaction
Chromogenic reaction according to chlortetracycline and samarium ion is as prescreening method.Chlortetracycline and samarium ion are under near-neutral sulfite deinking
Form light yellow coordination compound, at 405nm, have maximum light absorption.After considering fermentation ends, coloured former containing band in culture medium
Expect measurement result is produced certain impact.With producing fermentation liquor formulation, cultivate the most in the orifice plate and do not cultivate thalline.Result table
Bright, not cultivating thalline fermentation liquid also has certain OD value, illustrates that OD is worth mensuration to be implicitly present in shadow by the color of fermentation liquid itself
Ringing, but the fermentation liquid OD value cultivating thalline is more than the fermentation liquid not cultivating thalline, therefore fermentation liquid intrinsic colour is mould to screening gold
Element superior strain impact is little.Configuration concentration, at the chlortetracycline standard solution of 0.5～20mg/L, draws 40 μ L Tris-successively
HCl buffer solution, 60 μ L Sm (NO) 3 solution, the concussion mixing of 100 μ L each concentration standards solution, after 8min, colour developing is completely, enzyme
Mark instrument measures the OD of each concentration405Value.Determine OD405Regression equation with chlortetracycline concentration C (mg/L): y=0.0112x-
0.009, R2=0.99905, wherein x be sample concentration (μ g/mL), y be OD405Nm, standard concentration scope is 0～20mg/L.
Embodiment 4: the screening of superior strain
Bacteria suspension after ARTP mutagenic treatment is diluted to suitable concentration, and the bacteria suspension drawing 100 μ L is uniformly coated on
On flat board, in 34 DEG C of constant incubators, cultivate 4d, utilize QPix 420 instrument or similar bacterium colony selection equipment or manual picking
On flat board, mutation list bacterium colony is in 96 shallow bore hole plates (equipped with seed culture fluid 180 μ L), on 750r/min, the plate shaker of 31 DEG C
Cultivate 24h.With the inoculum concentration of 8% (v/v), by parallel for the seed liquor in 96 orifice plates 48 orifice plates that are forwarded to (equipped with fermentation culture
1000 μ L) in, 750r/min, 30 DEG C of fermentation culture 96h.Seed liquor 40% glycerol remained in 96 orifice plates is stored in 4 DEG C of ice
Case.
After fermentation ends, centrifuging and taking thalline, it is 0～20mg/L that Aspirate supernatant is diluted to chlortetracycline concentration, then takes dilute
Release liquid 100 μ L to add to and add 40 μ L Tris-HCl buffer solution (pH is 6.7) and 60 μ L Sm (NO) in advance3Solution (concentration
It is 5 × 10-4Mol/L), in 96 orifice plates, concussion mixing, after 8min, colour developing is completely, and microplate reader measures the OD of each concentration405Value, root
According to y=0.0112x-0.009, R2=0.99905 (wherein, x be chlortetracycline concentration concentration (μ g/mL), y be OD405Nm) calculate
To chlortetracycline concentration, and then obtain the chlortetracycline concentration of fermentation liquid according to extension rate conversion, i.e. obtain pan mycin relatively
High bacterial strain.
Embodiment 5: the Accuracy Verification of screening technique
The bacterial strain to be screened of embodiment 4 being used shake-flask culture and detects by HPLC method, result shows, embodiment
The result that 4 methods obtain, the result one_to_one corresponding obtained with shaking flask+HPLC method, the pan mycin that i.e. embodiment 4 obtains is relative
Higher bacterial strain is also the of a relatively high bacterial strain that shaking flask+HPLC method obtains.Illustrating, the method accuracy of embodiment 4 is good, can
Strong by property.
It addition, utilize relative standard deviation (RSD), investigate repeatability and the accuracy of prescreening method.According to microplate reader
The RSD of development process should control below 2.0%.Single bacterium colony starting strain high flux prescreening method is utilized to be repeated 6 times parallel examination
Testing, obtain OD405=0.07 ± 0.0022, RSD=0.144%, repeatability is good.The accuracy of verification method: use microplate reader
Detection method detectable concentration is 5,10, the standard solution of 15g/mL, each Concentration Testing twice, RSD is respectively 0.035%,
0.0322%, 0.057%, accuracy is good.
Additionally, inventor is investigated the extraction conditions impact on detection accuracy, it was found that use the side of the present invention
Method, it is possible to the most accurately detect chlortetracycline yield in the short period of time.Inventor attempted other adding proportions fermentation liquid,
Tris-HCl buffer solution, Sm (NO)3Solution, result shows inappropriate makes chlortetracycline concentration and OD than regular meeting405nmLinear
Relation is affected, and can affect testing result and ultimately result in the screened rejecting of part superior strain；The most once attempt using extending
Or substantially shorten developing time etc., found that can be to OD405nmSignificantly affect and cause final detection accuracy not enough.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (10)
1. the high-efficiency screening method of a chlortetracycline superior strain, it is characterised in that described method mainly comprises the following steps that
(1) streptomyces aureus bacteria suspension to be screened is coated in solid plate culture medium, after spore maturation, selects inoculation
Cultivate in equipped with 96 shallow bore hole plates of seed culture fluid；
(2) it is forwarded to parallel for the seed liquor obtained in 96 shallow bore hole plates equipped with fermentation culture in 48 orifice plates of fermentation culture；
(3), after fermentation ends, centrifuging and taking thalline, it is 0～20mg/L that Aspirate supernatant is diluted to chlortetracycline concentration, then takes dilution
Liquid 100 μ L adds to and adds 40 μ L Tris-HCl buffer solution and 60 μ L Sm (NO) in advance3In 96 orifice plates of solution, concussion is mixed
Even, after 8min, colour developing is completely, and microplate reader measures the OD of each concentration405Value, according to y=0.0112x-0.009, (wherein, x is that gold is mould
Element concentration (μ g/mL), y is OD405Nm) it is calculated chlortetracycline concentration, and then obtains fermentation liquid according to extension rate conversion
Chlortetracycline concentration, i.e. obtain the bacterial strain that pan mycin is of a relatively high.
Method the most according to claim 1, it is characterised in that the cultivation in the solid plate culture medium of described step (1)
It is to cultivate 4d at 34 DEG C.
Method the most according to claim 1, it is characterised in that the solid plate culture medium prescription (g/ of described step (1)
L): sucrose 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
Method the most according to claim 1, it is characterised in that the seed culture fluid (g/L) of described step (1): sucrose
30g, import yeast powder 5g, MgSO40.5g, (NH4)2SO43g, NaCl 4g, KH2PO41g, CaCO3 0.2g。
Method the most according to claim 1, it is characterised in that cultivating in 96 shallow bore hole plates of described step (1) is at 750r/
Min, 30～32 DEG C of shaking tables cultivate 23～25h.
Method the most according to claim 1, it is characterised in that the fermentation culture (g/L) of described step (2): sucrose
30g, MgSO40.5g, CaCO30.33g, citric acid 2.55g, (NH4)2SO43g, 1mL mixed liquor (0.3%CuSO4·5H2O、
0.5%ZnSO4·7H2O, 0.25%MnSO4`4H2O)。
Method the most according to claim 1, it is characterised in that the fermentation culture of described step (2) is with 8% by seed liquor
(v/v) inoculum concentration is forwarded in 48 orifice plates, in 750r/min, 30～31 DEG C of fermentation culture 96～110h.
Method the most according to claim 1, it is characterised in that described streptomyces aureus bacteria suspension to be screened, is by gold
Color streptomycete obtains after carrying out mutation.
Method the most according to claim 8, it is characterised in that described mutation is physical mutagenesis or chemomorphosis.
Method the most according to claim 9, it is characterised in that described mutation is that atmospheric pressure at room plasma (ARTP) lures
Change, ultraviolet mutagenesis, ethylmethane sulfonate (EMS) mutation, nitrosoguanidine (NTG) mutation or dithyl sulfate (DES) mutation.
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| CN112920962A (en) * | 2020-12-04 | 2021-06-08 | 湖南师范大学 | ARTP-NTG composite mutagenesis bacillus thuringiensis mutant strain and application thereof |
| CN113549579A (en) * | 2021-08-02 | 2021-10-26 | 金河生物科技股份有限公司 | Streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof |
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| CN113549579A (en) * | 2021-08-02 | 2021-10-26 | 金河生物科技股份有限公司 | Streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof |
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