CN109536399B - High-throughput screening method for virginiamycin high-producing strains - Google Patents
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Abstract
The invention relates to a high-throughput screening method of virginiamycin high-yield strains, and discloses a rapid virginiamycin detection method. Compared with the traditional screening method, the high-throughput screening method provided by the invention has the advantages of rapidness, high efficiency, simplicity, easiness and the like, can greatly shorten the screening period, improves the screening efficiency of the strains, greatly reduces the workload of breeding the high-yield Virginiamycin strains, and provides a powerful technical guarantee for breeding the high-yield strains.
Description
Technical Field
The invention relates to the field of microbial genetic breeding and biological fermentation, in particular to a high-throughput screening method of virginiamycin high-yield strains.
Background
Virginiamycin (virginiamycin) is a polypeptide antibiotic, contains a lactone ring in a molecular structure, and is separated from a streptomyces virginiae fermentation product. Virginiamycin has an inhibitory effect on gram-positive bacteria, and the action mechanism of virginiamycin is to inhibit the synthesis of protein in ribosome of bacteria. Virginiamycin is structurally composed of two factors, M (about 70%) and S (about 30%), which are different in structure and antibacterial range. The factor M is a macrocyclic lactone with the molecular formula C28H35N3O7The S factor is a cyclic polypeptide with the molecular formula of C43H49N7O10. The antibacterial activity of the antibacterial agent is enhanced by mixing S and M, and the antibacterial agent is not easy to generate drug resistance. The Smith Lklinr pharmaceutical factory in the united states developed the virginiamycin product Stafac (fast fertilizer), which consists of 70% M factor and 30% S factor. Because virginiamycin is not easy to be absorbed in animal intestinal tracts, has small residual quantity, low toxicity and better biodegradability, and has very stable structure (the action effect of virginiamycin is basically not changed after being stored at room temperature for 3 years), the activity of virginiamycin is still very stable after feed processing, so virginiamycin is known as one of the most promising antibiotics. However, the titer of the product produced by the virginiamycin fermentation is low at present, and the application and popularization of the virginiamycin are directly restricted. The performance of the streptomyces virginiae strain directly influences the yield of virginiamycin, so that the screening of the streptomyces virginiae high-yield strain is very important for popularization and application of the virginiamycin.
The currently reported method for screening Virginia production strains mainly comprises methods such as ultraviolet mutagenesis screening, guanidine nitrite mutagenesis screening and plate separation, however, the screening of mutant strains obtained by mutagenesis and mutant strains constructed by multiple genetic engineering has the problems of large strain quantity, long screening period, complex operation of the screening method and the like. The detection method of virginiamycin is mainly focused on HPLC detection, the detection period is long, single detection is only limited to one sample, the limitation on the number of the samples is large, and the method is not suitable for sampling detection at any time in the fermentation process. Therefore, there is a high-throughput screening method for developing virginiamycin-producing strains.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a high-throughput screening method of a virginiamycin high-producing strain.
The invention provides a high-throughput screening method of a virginiamycin high-producing strain, which comprises the following steps:
(1) carrying out fermentation culture on the virginiamycin production strain;
(2) extracting virginiamycin from the fermentation liquor to obtain a detection sample;
(3) adding a color developing agent into the detection sample for color development reaction, wherein the color developing agent comprises dimethylaminobenzaldehyde, isopropanol and strong acid;
(4) measuring the absorbance value at 595nm, and calculating the Virginia yield of the production strain according to the absorbance value.
Preferably, the strong acid is one or more of hydrochloric acid, sulfuric acid, perchloric acid.
Specifically, the color developing agent comprises the following components:
2 g/L-10 g/L of p-dimethylaminobenzaldehyde; 30-60% of isopropanol by volume; 1 to 10 percent of hydrochloric acid by volume percentage.
Preferably, the developer comprises the following components:
5 g/L-10 g/L of p-dimethylaminobenzaldehyde; 30-50% of isopropanol by volume; 1 to 5 percent of hydrochloric acid by volume percentage.
More preferably, the developer comprises the following components:
6 g/L-9 g/L of p-dimethylaminobenzaldehyde; 30-40% of isopropanol by volume; 1 to 3 percent of hydrochloric acid by volume percentage.
In the invention, the volume ratio of the detection sample to the color developing agent in the color development reaction is 1: 1-1: 5.
specifically, the color reaction is carried out for 5min to 30min at the temperature of 60 ℃ to 80 ℃; preferably, the color reaction is carried out at 70-80 ℃ for 10-20 min.
As a preferred embodiment of the present invention, the method of step (3) is as follows: and (3) adding the color reagent into the detection sample obtained in the step (2), shaking up, and carrying out water bath at 70-80 ℃ for 10-20 min.
In the invention, the virginiamycin in the fermentation liquor is extracted by adding the extracting solution into the fermentation liquor.
The extracting solution contains one or more of acetonitrile, acetone and methanol.
In a preferred embodiment of the present invention, the extraction liquid is acetonitrile.
Preferably, the volume ratio of the fermentation liquid to the extraction liquid is 1: 1-1: 5.
in a preferred embodiment of the present invention, the volume ratio of the fermentation liquid to the extraction liquid is 1: 1-1: 2.
the detection sample is prepared by adding acetonitrile into fermentation liquor to perform oscillation extraction for 20min to 100min, and separating supernatant and precipitate to obtain supernatant which is the detection sample.
Preferably, the time of the oscillation extraction is 30 min-60 min.
It should be understood by those skilled in the art that, in order to shorten the preparation time of the test sample, if conditions allow, ultrasonic treatment or stirring treatment can be performed during the extraction process, and these treatments are only used for accelerating the extraction of virginiamycin, and thus are all within the protection scope of the present invention.
In the high throughput screening method of the present invention, the culture medium for fermentation culture in step (1) comprises the following components:
5-30 g/L of soluble starch, 2-5 g/L of soybean meal, 1-5 g/L of yeast extract, 5-50 g/L of glucose, 1-5 g/L of linseed oil, 0.2-1 g/L of dipotassium hydrogen phosphate, 0.01-0.05g/L of ferrous sulfate, 0.2-2 g/L of ammonium sulfate and 1-5 g/L of calcium carbonate.
The pH value of the fermentation medium is 7.0-7.2.
Preferably, the fermentation culture is a culture in a multi-well plate.
More preferably, the multi-well plate is a deep-well culture plate.
The fermentation temperature is 30-32 ℃, and the rotating speed is 200-1000 rpm.
In a preferred embodiment of the present invention, the fermentation temperature is 32 ℃ and the rotation speed is 500 to 800 rpm.
The seeds cultured by fermentation are cultured by adopting a perforated plate, the temperature for seed culture is 28-32 ℃, and the rotating speed is 500-1000 rpm.
The seed culture medium comprises the following components: 10-15 g/L of soybean meal, 20-25 g/L of peanut meal, 30-40 g/L of glucose, 2-5 g/L of yeast extract, 15-20g/L of soluble starch and 2-3 g/L of calcium carbonate.
The pH value of the seed culture medium is 7.0-7.2.
The hypha growth amount (PMV) of the seed liquid obtained by seed culture is 20-40%, and the inoculation amount of fermentation culture is 5-20%.
Preferably, the PMV of the seed solution obtained by seed culture is 25-30%, and the inoculation amount of fermentation culture is 5-8%.
The virginiamycin production strain is a streptomyces microorganism, and is preferably streptomyces virginiae.
The virginiamycin production strain can be a strain which can accumulate virginiamycin and is obtained by mutation breeding, genetic engineering breeding or a combination method thereof.
The invention provides a high-throughput screening method of a virginiamycin high-yield strain, a quick and accurate product detection method is a premise and a basis for high-throughput screening of the strain, and aiming at the structure and the physicochemical properties of virginiamycin, an inventor screens a large amount of organic matters, inorganic matters and combinations thereof to obtain a color developing agent capable of generating a stable color reaction with the virginiamycin. The chromogenic product of the virginiamycin has a characteristic absorption peak at 595nm, the light absorption value has good linear correlation with the virginiamycin content in the fermentation liquor, and the chromogenic product has good positive correlation with the yield of the virginiamycin detected by HPLC. Further, by optimizing a culture method of a perforated plate and an extraction method of virginiamycin in fermentation liquor, the high-efficiency high-throughput screening method of the virginiamycin high-yield strain is finally obtained.
The invention has the beneficial effects that:
(1) the invention establishes a high-throughput screening method of the virginiamycin high-yield strain for the first time, combines multi-plate culture and a quick and simple detection method, can screen the virginiamycin production performance of a large number of strains through one-time operation, quickly eliminates negative mutant strains, and screens the high-yield strains.
(2) Compared with the traditional screening method, the high-throughput screening method has the advantages of rapidness, high efficiency, simplicity, feasibility and the like, can greatly shorten the screening period, improve the screening efficiency of the strains, greatly reduce the workload of breeding the high-yield Virginiamycin strains, and provide powerful technical support for breeding the high-yield strains.
Drawings
FIG. 1 is a standard curve for colorimetric detection of virginiamycin in example 2.
FIG. 2 is the correlation analysis of the results of the high throughput screening method in example 3 and the conventional screening method of shake flask fermentation-HPLC detection.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 mutagenesis treatment of Streptomyces virginiae
This example is exemplified by Streptomyces virginiae ATCC13161 (available from Shanghai Linyuan Biotech Co., Ltd.) and the mutagenesis treatment is carried out as follows.
1. Preparation of mature spores of streptomyces virginiae
(1) Preparation of single colony of streptomyces virginiae: under the aseptic condition, dissolving the freeze-dried powder of the streptomyces virginiae ATCC13161 by using aseptic normal saline, uniformly mixing by vortex shaking, uniformly coating on a plate culture medium after dilution, and standing and culturing for 8-10 days at the constant temperature of 26 ℃ for later use.
The composition of the plating medium was as follows: 30-35g/L of yeast extract, 8-10g/L of malt extract, 3-4g/L of glucose and 15-20g/L of agar powder.
(2) Slant culture of streptomyces virginiae: the full, robust and purple streptomyces virginiae spores are picked by an inoculating needle, inoculated on a slant culture medium, and statically cultured for 8-10 days at a constant temperature of 26 ℃ for later use.
The components of the slant culture medium are as follows: 30-35g/L of yeast extract, 8-10g/L of malt extract, 3-4g/L of glucose and 15-20g/L of agar powder.
2. Atmospheric Room Temperature Plasma (ARTP) mutagenesis treatment
Taking a mature inclined plane with good growth vigor in the step 1 of the embodiment, flushing spores with 0.9% sterile physiological saline, vortexing and shaking to break up the spores, absorbing 20 mu L of the spore to be added into a sample slide, placing the sample slide to be mutagenized on a mutagenesis objective table by using tweezers, rotating the sample slide to be mutagenized to a position under a plasma generator, adjusting the height of a positioning table to 5mm, controlling the nitrogen flow to be 5-12SLM, the air pressure to be 0.1-0.2MPa and the power to be 10-100w, respectively irradiating for 0, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60s, clamping the irradiated slide into an EP tube filled with the physiological saline by using the tweezers, fully shaking for 1 minute on the oscillator, and performing gradient dilution. Sucking 100 mu L of diluent, uniformly coating the diluent on a plate culture medium, culturing for 72h in a constant-temperature incubator at 26 ℃, counting colony forming units, making a lethality curve, selecting single colonies which are full, robust and purple in growth, uniformly transferring the single colonies to a slope, and storing.
Example 2 high throughput screening of Virginia mycin producing strains
The slant of the ARTP-mutagenized strain prepared in example 1 was subjected to high-throughput screening as follows.
1. Seed culture: preparing a seed culture medium according to the formula of the seed culture medium in proportion, carrying out constant-temperature water bath at the temperature of 80-90 ℃ for 20-30 minutes, cooling, and adjusting the pH value to 7.0-7.2. Seed medium was added to 96-well plates. Selecting a proper amount of mature spores of the ARTP mutagenic strain, inoculating the mature spores into a seed culture medium, and culturing at the constant temperature of 500rpm and 28 ℃ for 24 hours, wherein the PMV value of a seed solution is 25-30%.
The components of the seed culture medium are as follows: 15g/L of soybean meal, 20g/L of peanut meal, 30g/L of glucose, 4g/L of yeast extract, 15g/L of soluble starch and 3g/L of calcium carbonate.
2. Fermentation culture: preparing a fermentation culture medium according to the formula of the fermentation culture medium in proportion, carrying out constant-temperature water bath at the temperature of 80-90 ℃ for 20-30 minutes, cooling, and adjusting the pH value to 7.0-7.2. Fermentation medium was added to 48-well plates. Inoculating the seed liquid obtained in the step 1 into a fermentation culture medium in an inoculation amount of 6%, rotating at 650rpm, and culturing at a constant temperature of 32 ℃ for 72 hours.
The components of the fermentation medium were as follows: 20g/L of soluble starch, 4.5g/L of soybean meal, 2.5g/L of yeast extract, 10g/L of glucose, 5g/L of linseed oil, 0.4g/L of dipotassium phosphate, 0.03g/L of ferrous sulfate, 0.5g/L of ammonium sulfate and 3g/L of calcium carbonate.
3. Preparation of developer (100 mL): the developer is prepared in situ, 0.75g of p-dimethylaminobenzaldehyde is weighed, 30mL of isopropanol and 2mL of hydrochloric acid are added, and water is added to the mixture to be 100mL after the mixture is fully dissolved.
4. Colorimetric detection of virginiamycin:
(1) preparing a virginiamycin detection standard curve: weighing virginiamycin to prepare standard solutions with the concentrations of 100mg/L, 200mg/L, 400mg/L, 800mg/L, 1g/L and 2g/L respectively, adding a color developing agent with the same volume as the standard solutions into each standard solution respectively, shaking up, carrying out water bath at 75 ℃ for 15min, measuring the light absorption value at 595nm by using an enzyme labeling instrument, and making a standard curve according to the light absorption value as shown in figure 12The formula for the calculation of virginiamycin concentration is 0.9988, Y is 0.0025X +0.0033(X is absorbance, Y is virginiamycin concentration (mg/L)).
(2) Detection of a fermentation liquid sample: and after fermentation is finished, adding acetonitrile with the same volume into the fermentation liquor, oscillating and extracting for 30min, centrifuging and taking supernatant to obtain the detection sample. Adding an equal volume of color developing agent into a detection sample, shaking uniformly, carrying out water bath at 75 ℃ for 15min, measuring the light absorption value at 595nm by using an enzyme labeling instrument, and calculating the concentration of the virginiamycin in the fermentation liquid extract according to the formula that Y is 0.0025X +0.0033(X is absorbance, and Y is the concentration (mg/L) of the virginiamycin).
The obtained bacterial strain with high virginiamycin yield can be used for further re-screening.
Example 3 validation of high throughput screening method
In order to verify whether the high-throughput screening method can effectively screen the product production performance of the virginia producing strains, a part of mutant strains with different levels of virginiamycin yield obtained by screening in example 2 are selected and verified by a traditional strain screening method (shake flask experiment and HPLC detection).
1. Seed culture: preparing a seed culture medium according to a seed culture medium formula (the same as the seed culture medium in the example 2), carrying out constant-temperature water bath at 80-90 ℃ for 20-30 minutes, cooling, adjusting the pH value to 7.0-7.2, sterilizing at 115 ℃ for 20-30 minutes, and cooling for later use. 25mL of seed medium was added to a 250mL Erlenmeyer flask. Washing mature spores on a culture slant of the virginiamycin mutant strain by using 1-2mL of sterile physiological saline, inoculating 0.5-1mL of spore suspension into a seed culture medium, and culturing at constant temperature of 28 ℃ for 24 hours at the rotation speed of 200 and 220 rpm.
2. Preparing a fermentation culture medium according to a formula of the fermentation culture medium (the same as the fermentation culture medium in the example 2), carrying out constant-temperature water bath at 80-90 ℃ for 20-30 minutes, cooling, adjusting the pH value to 7.0-7.2, sterilizing at 115 ℃ for 20-30 minutes, and cooling for later use. Adding 30mL of fermentation medium into a 250mL triangular flask, transferring the seed liquid obtained in the step 1 into the fermentation medium at the inoculation amount of 6%, and culturing at constant temperature of 32 ℃ at the rotation speed of 200-220 rpm.
3. And (4) HPLC detection: and after the fermentation is finished, centrifuging to remove the supernatant, washing the thalli by ethyl acetate, centrifuging to remove the supernatant, adding acetonitrile with the same volume, performing vortex oscillation for 10 minutes to extract the virginiamycin, and detecting the fermentation titer unit of the virginiamycin by adopting HPLC.
The HPLC detection method is as follows:
(1) the instrument equipment comprises: shimadzu liquid chromatography system, the column is ODS-3C18 column (250.0mm 4.6 mm);
(2) detection wavelength: 230 nm;
(3) an elution system: binary high pressure gradient elution, mobile phase a (0.01M phosphoric acid); mobile phase B (acetonitrile); 0-0.2min, a mobile phase A94% and a mobile phase B6%; 0.2-35min, 15% of mobile phase A and 85% of mobile phase B; 35-45min, a mobile phase A94% and a mobile phase B6%; ending 45 min;
(4) flow rate: 1.0 mL/min.
The corresponding relation between the traditional screening method of the partial strains obtained by mutagenesis and the screening result of the high-throughput screening method is shown in figure 2, and the result shows that the yield of the strains obtained by the traditional screening method and the yield of the strains obtained by the high-throughput screening show good positive correlation, which indicates that the high-throughput screening method provided by the invention can be used for rapidly and efficiently screening the high-yield strains of the streptomyces virginiae.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (13)
1. A high-throughput screening method of virginiamycin high-producing strains is characterized by comprising the following steps:
(1) carrying out fermentation culture on the virginiamycin production strain;
(2) extracting virginiamycin from the fermentation liquor to obtain a detection sample;
(3) adding a color developing agent into the detection sample for color development reaction;
(4) measuring the light absorption value at 595nm, and calculating the Virginia yield of the production strain according to the light absorption value;
the color developing agent comprises the following components: p-dimethylaminobenzaldehyde, 7.5 g/L; isopropanol, the volume percentage content is 30%; hydrochloric acid, the volume percentage content is 2%.
2. The screening method according to claim 1, wherein the volume ratio of the detection sample to the color-developing agent in the color-developing reaction is 1: 1-1: 5.
3. the screening method according to claim 1 or 2, wherein the color reaction is carried out at 60 to 80 ℃ for 5 to 30 min.
4. The screening method according to claim 3, wherein the color reaction is carried out at 70 to 80 ℃ for 10 to 20 min.
5. The screening method according to claim 1, 2 or 4, wherein the extraction of virginiamycin from the fermentation broth is carried out by adding an extraction solution to the fermentation broth;
the extract is one or more of acetonitrile, acetone and methanol.
6. The screening method according to claim 5, wherein the volume ratio of the fermentation liquid to the extraction liquid is 1: 1-1: 5.
7. the screening method according to claim 1, 2 or 4, wherein the detection sample is prepared by adding acetonitrile into a fermentation liquid, performing shaking extraction for 20min to 100min, and separating a supernatant and a precipitate to obtain the supernatant as the detection sample.
8. The screening method according to claim 7, wherein the time for the shaking extraction is 30 to 60 min.
9. The screening method according to claim 1, 2, 4, 6 or 8, wherein the culture medium of the fermentation culture consists of: 5-30 g/L of soluble starch, 2-5 g/L of soybean meal, 1-5 g/L of yeast extract, 5-50 g/L of glucose, 1-5 g/L of linseed oil, 0.2-1 g/L of dipotassium hydrogen phosphate, 0.01-0.05g/L of ferrous sulfate, 0.2-2 g/L of ammonium sulfate and 1-5 g/L of calcium carbonate.
10. The screening method according to claim 9, wherein the fermentation culture is a culture in a multi-well plate.
11. The screening method according to claim 9, wherein the fermentation temperature is 30 to 32 ℃ and the rotation speed is 200 to 1000 rpm.
12. The screening method according to claim 1, 2, 4, 6, 8, 10 or 11, wherein the virginiamycin-producing strain is a microorganism of the genus streptomyces.
13. The screening method according to claim 12, wherein the microorganism belonging to the genus Streptomyces is Streptomyces virginiae (S.) (Streptomyces virginiae) 。
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