High-throughput screening method of high-yield lincomycin strain

Abstract

The invention relates to a high-throughput screening method of high-yield lincomycin strains, and discloses a rapid lincomycin detection method which comprises the following steps: adding a proper amount of oxalic acid into a detection sample under the condition of a phosphate buffer solution, and calculating the yield of lincomycin according to an absorbance value obtained by detection. By combining porous plate culture and shake flask fermentation re-screening of streptomyces lincomycin, high-throughput screening of high-yield lincomycin strains is realized. Compared with the traditional screening method, the high-throughput screening method provided by the invention has the advantages of rapidness, high efficiency, simplicity, feasibility and the like, the screening period can be shortened, the screening efficiency of the strain is improved, the workload of breeding the high-yield lincomycin strain is greatly reduced, and a powerful technical guarantee is provided for breeding the high-yield strain.

Description

Technical Field

[0001] The invention relates to the fields of microbial breeding and biological fermentation, and in particular to a high-throughput screening method for a high-yield lincomycin strain. Background Art

[0002] Streptomyces lincolnensis belongs to the genus Streptomyces. Its spores are oval, short rods with smooth surfaces. The secondary metabolite it produces is lincomycin. The molecular structure of lincomycin is C 18 H 34 N2O6S, molecular weight is 406.56. The chemical structure of lincomycin is mainly composed of two parts condensed and modified, one part is trans-N-methyl-4-n-propylproline (Propylproline, PPL) generated by L-tyrosine (L-Tyr) as a precursor, and the other part is methylthiolincosamide (Methylthiolincosamide, MTL) synthesized by 7-phosphate sedoheptulose (Sedoheptulose-7-phosphate, S7P) and 5-phosphate pentose (Ribose-5-phosphate, R5P) as precursors. Lincomycin has a very strong antibacterial effect on Gram-positive bacteria such as Staphylococcus epidermidis, Streptococcus pneumoniae and Streptococcus hemolyticus, and has no cross-resistance with penicillin, chloramphenicol, cephalosporins and tetracyclines, and has a wide range of selectivity in medical applications. In addition, lincomycin is cheap and has significant therapeutic effects on certain respiratory infections and other diseases. It currently still occupies a high market share. However, there is a large gap between China and foreign countries in terms of lincomycin A production. The performance of Streptomyces lincomycin strains directly affects the level of lincomycin production. Therefore, screening high-yield lincomycin strains is crucial for the promotion and application of lincomycin.

[0003] The method for screening Streptomyces lincomycin reported at present mainly includes methods such as ultraviolet mutagenesis screening, chemical mutagenesis, plate separation, etc., but no matter the mutant strain obtained by mutagenesis or the screening of the mutant strain constructed by multiple genetic engineering, there are problems such as large strain quantity, long screening cycle, and complicated operation of screening method. And the detection method of lincomycin is also mainly concentrated in HPLC detection, and the detection cycle is long, and single detection is only limited to one sample, and when the sample quantity is large, it is not suitable for sampling detection at any time of fermentation process. Therefore, it is necessary to develop a high-throughput screening method of high-yield lincomycin. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the object of the present invention is to provide a high-throughput screening method for high-yield lincomycin.

[0005] The present invention provides a high-throughput screening method for a high-yield lincomycin strain, comprising the following steps:

[0006] (1) fermenting Streptomyces lincomyces;

[0007] (2) extracting lincomycin from the fermentation broth to obtain a test sample;

[0008] (3) adding phosphate buffer and oxalic acid to the test sample;

[0009] (4) measuring the absorbance at 214 nm and calculating the lincomycin yield based on the absorbance;

[0010] Specifically, the present invention comprises the following components:

[0011] Phosphate buffer: 0.01-0.05 mol / L; Oxalic acid: 6-10 g / L.

[0012] In the present invention, the lincomycin in the fermentation broth is extracted by adding phosphate buffer into the fermentation broth.

[0013] Preferably, the volume ratio of the fermentation broth to the phosphate buffer is 1:1-1:1.5.

[0014] The preparation of the test sample of the present invention is to take lincomycin fermentation liquid and centrifuge it at 10000-12000rpm for 5-10min, separate the supernatant and the precipitate, and the obtained supernatant is the test sample.

[0015] In the high-throughput screening method of the present invention, the fermentation medium consists of the following components:

[0016] Glucose 100-120g / L, soybean powder 25-30g / L, corn steep liquor 2-5g / L, sodium chloride 5-10g / L, potassium dihydrogen phosphate 0.2-0.5g, ammonium sulfate 8-10g, sodium nitrate 8-10g / L, calcium carbonate 8-10g / L.

[0017] The pH of the fermentation medium is 6.9-7.0.

[0018] Preferably, the fermentation culture is cultured in a multi-well plate.

[0019] More preferably, the multi-well plate is a 24-deep-well culture plate.

[0020] The fermentation temperature is 30-32°C and the rotation speed is 220-250rpm.

[0021] As a preferred embodiment of the present invention, the fermentation temperature is 30°C and the rotation speed is 220-230rpm.

[0022] The seeds for fermentation culture of the present invention are cultured in a multi-well plate, the temperature of the seed culture is 30-32° C., and the rotation speed is 220-250 rpm.

[0023] The seed culture medium of the present invention comprises the following components: 10-15 g / L of glucose, 20-25 g / L of soybean powder, 30-35 g / L of corn starch, 2-5 g / L of ammonium sulfate and 5-10 g / L of calcium carbonate.

[0024] The pH of the seed culture medium is 6.9-7.0.

[0025] The inoculation amount of the fermentation culture is 5-20%.

[0026] Preferably, the inoculation amount of the fermentation culture is 5-10%.

[0027] The lincomycin production strain is a microorganism of the genus Streptomyces, namely Streptomyces lincomycin.

[0028] The lincomycin-producing strain may be a strain capable of accumulating lincomycin that is bred by mutation breeding, genetic engineering breeding or a combination thereof.

[0029] The invention provides a high-throughput screening method for high-yield lincomycin strains. A fast and accurate product detection method is the premise and basis for high-throughput screening of strains. With respect to the relevant properties of lincomycin, the inventors found that lincomycin produces an absorption peak at 214 nm in phosphate buffer, and the absorbance value has a good linear correlation with the lincomycin content in the fermentation broth, and also has a good positive correlation with the lincomycin yield detected by HPLC. Combined with a multi-well plate culture method and a method for extracting lincomycin from a fermentation broth, an efficient high-throughput screening method for high-yield lincomycin strains is finally obtained.

[0030] The beneficial effects of the present invention are:

[0031] (1) The present invention establishes a high-throughput screening method for high-yield lincomycin strains, which combines multi-well plate culture with a rapid and simple detection method, and can screen a large number of strains for lincomycin production performance in one operation, quickly eliminate negative mutant strains, and screen high-yield strains.

[0032] (2) Compared with traditional screening methods, the high-throughput screening method of the present invention has the advantages of being fast, efficient, simple and easy to operate, etc. It can greatly shorten the screening cycle, improve the screening efficiency of strains, and greatly reduce the workload of breeding high-yield lincomycin strains, providing a strong technical guarantee for the breeding of high-yield strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the standard curve of lincomycin detected by the ELISA method in Example 2.

[0034] Figure 2 This is a correlation analysis of the results of the high-throughput screening method in Example 3 and the traditional screening method of shake flask fermentation HPLC detection. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1 Mutagenesis of Streptomyces lincomyces

[0039] In this embodiment, Streptomyces lincolnensis (provided by Henan Tianfang Pharmaceutical Co., Ltd.) was used as an example to carry out mutagenesis treatment, and the specific method is as follows.

[0040] 1. Preparation of Streptomyces lincomyces by UV mutagenesis

[0041] First, add an appropriate amount of sterile water to the slant to prepare a spore suspension with a certain concentration, then drop the spore suspension into a culture dish, then irradiate with ultraviolet light on a magnetic stirrer, then dilute the ultraviolet-treated spore suspension with a spreading rod and separate it into a plate, culture it in a 30°C constant temperature incubator for 7 days, select a single colony and inoculate it on the slant, culture it in a 30°C constant temperature incubator for 7 days, and after it grows to maturity, carry out the subsequent screening process.

[0042] The components of the plate culture medium are as follows: soybean powder 5-10g / L; soluble starch 19-25g / L; potassium nitrate 1-5g / L; sodium chloride 0.5-1g / L; dipotassium hydrogen phosphate 0.5-1g / L; magnesium sulfate 0.5-1g / L; ferrous sulfate 0.01-0.05g; agar powder 1.2-1.5g / L.

[0043] The pH of the plate culture medium is 7.25-7.30.

[0044] Slant culture of Streptomyces lincomyces: Use a sterile bamboo stick to pick up a mature single colony on the plate in an ultra-clean bench, and streak it evenly and densely on the slant culture medium. Then, culture the slant at 30°C for about 7 days. During this period, observe the color of the slant culture medium and the changes in spore production.

[0045] The components of the slant culture medium are as follows: soybean powder 5-10g / L; soluble starch 19-25g / L; potassium nitrate 1-5g / L; sodium chloride 0.5-1g / L; dipotassium hydrogen phosphate 0.5-1g / L; magnesium sulfate 0.5-1g / L; ferrous sulfate 0.01-0.05g; agar powder 1.2-1.5g / L.

[0046] The pH of the slant culture medium is 7.25-7.30.

[0047] The ultraviolet mutagenized strain prepared in Example 1 was subjected to high-throughput screening, and the specific method is as follows.

[0048] 1. Seed culture: Prepare the seed culture medium in proportion according to the formula of the seed culture medium, sterilize it at 115-121℃ for 20-25min, and after cooling, use a pipette to dispense 1mL of shake flask seed culture medium into each well of a 24-deep-well plate. Use a sterilized white pipette tip to pick up a single colony that has been diluted and spread after mutagenesis, and culture it in a 24-deep-well plate constant temperature shaker at a temperature of 30℃ and a rotation speed of 220rpm for 2 days.

[0049] The components of the seed culture medium are as follows: glucose 10-15 g / L, soybean powder 20-25 g / L, corn starch 30-35 g / L, ammonium sulfate 2-5 g / L, and calcium carbonate 5-10 g / L.

[0050] The pH of the seed culture medium is 6.9-7.0.

[0051] 2. Fermentation culture: Use a pipette to dispense 1 mL of fermentation culture medium into each well of a 24-deep-well plate. Transfer the seeds cultured in the 24-deep-well plate to the fermentation medium at a 10% inoculation rate and culture in a 24-well plate constant temperature shaker at 30°C and 220 rpm for 7 days.

[0052] The pH of the seed culture medium is 6.9-7.0.

[0053] 3. Preparation of phosphate buffer: Accurately weigh 1.9068 g of borax and dissolve it in an appropriate amount of distilled water. Adjust the pH value to 6.0 with phosphoric acid and then make up to volume in a 100 mL volumetric flask.

[0054] 4. Preparation of oxalic acid solution: Accurately weigh 6g of borax and dissolve it in an appropriate amount of deionized water, then make up to volume in a 100mL volumetric flask.

[0055] 5. Detection of lincomycin by ELISA:

[0056] (1) Preparation of the standard curve for lincomycin detection: First, draw 10 mL of 5 mg / mL lincomycin fermentation broth, add 6% oxalic acid solution, centrifuge at 12000 rpm for 5 min, and take the supernatant. Use borax solution to gradient dilute the supernatant to obtain solutions with concentrations of 0.01, 0.005, 0.0025, 0.00125, 0.000625, and 0.000313 mol / L, respectively. Accurately draw 200 μL of samples of each concentration into a 96-well ELISA plate, add 200 μL of borax solution to the control wells, and use an ELISA reader to measure the absorbance value (OD) at 214 nm. The standard curve prepared based on the absorbance value is as follows: Figure 1 As shown in the figure, the results show that the absorbance value has a good linear relationship with lincomycin, y = 0.0007x + 0.0589, R 2 = 0.9976. (x is the absorbance value, y is the lincomycin concentration (μg / mL)).

[0057] (2) Detection of fermentation broth samples: After the fermentation is completed, add 6% oxalic acid solution, centrifuge at 12000rpm for 5min, and take the supernatant. Take 100μL of supernatant and 0.05mol / L borax solution respectively, place them in an ELISA plate, and use an ELISA reader to measure the absorbance value (OD) at 214nm. The concentration of lincomycin in the fermentation broth is calculated according to the formula y=0.0007x+0.0589 (x is the absorbance value, y is the lincomycin concentration (μg / mL)).

[0058] The obtained strain with high lincomycin production can be used for further screening.

[0059] Example 3 Validation of the high-throughput screening method

[0060] In order to verify whether the high-throughput screening method of the present invention can effectively screen the product production performance of Streptomyces lincomycin, some mutant strains with different levels of lincomycin production obtained by screening in Example 2 were selected and verified using traditional strain screening methods (shake flask experiment and HPLC detection).

[0061] 1. Seed culture: Prepare seed culture medium according to the seed culture medium formula, add 25 mL of seed culture medium to a 250 mL conical flask, and sterilize at 121°C for 20 minutes. Use a sterile iron shovel to dig out 0.5-1 cm2 of bacterial blocks on the slant culture medium and place them in the sterilized shake flask seed culture medium. Place in a shaker, set the temperature to 30°C, the speed to 220 r / min, and culture for 48 hours.

[0062] 2. Fermentation culture: Prepare the fermentation medium according to the fermentation medium formula, add 25 mL of fermentation medium into a 250 mL conical flask, and sterilize at 121°C for 20 min. After the grown seed medium is fully shaken, add 10% of the inoculation amount (2.5 mL) into the fermentation medium. Place in a shaker, set the temperature to 30°C, the speed to 220 r / min, and culture for 168 hours.

[0063] 3. HPLC detection: After the fermentation is completed, the supernatant is centrifuged and diluted with the mobile phase to mix the two evenly. Then, it is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is aspirated with a sterile syringe. Finally, the supernatant is filtered into the sample bottle with the help of a 0.22 μm organic filter.

[0064] The HPLC detection method is as follows:

[0065] (1) Instruments: Agilent liquid chromatography system, chromatographic column (Eclipse XDB-C18, 5 μm, 4.6 × 250 mm);

[0066] (2) Detection wavelength: 210nm;

[0067] (3) Elution system: mobile phase A (ultrapure water); mobile phase B (methanol); mobile phase D (V 乙酸铵 :V 甲醇 =2:3); mobile phase D 100%; 12 min.

[0068] (4) Flow rate: 0.6 mL / min.

[0069] The corresponding relationship between the screening results of the traditional mutagenesis method and the high-throughput screening method for some strains induced is shown in the figure. Figure 2 As shown, the results show that the yield of the strain obtained by the traditional screening method and the yield of the strain obtained by high-throughput screening present a good positive correlation, indicating that the high-throughput screening method provided by the present invention can be used to quickly and efficiently screen strains with high lincomycin production.

[0070] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A high-throughput screening method for high-yield lincomycin strains, It is characterized in that The following steps are involved: (1) fermenting Streptomyces lincomyces; (2) extracting lincomycin from the fermentation broth to obtain a test sample; (3) adding phosphate buffer and oxalic acid to the test sample; (4) measuring the absorbance at 214 nm and calculating the lincomycin yield based on the absorbance; Phosphate buffer: 0.01 mol / L; Oxalic acid: 6 g / L.

2. The screening method according to claim 1, It is characterized in that The volume ratio of the test sample to the phosphate buffer is 1:1-1:1.

5.

3. The screening method according to claim 1 or 2, It is characterized in that The test sample is prepared by centrifuging lincomycin fermentation broth at 10000-12000 rpm for 5-10 min, separating the supernatant and the precipitate, and the obtained supernatant is the test sample.

4. The screening method according to claims 1, 2 and 3, It is characterized in that The fermentation medium is composed of the following components: 100-120 g / L of glucose, 25-30 g / L of soybean powder, 2-5 g / L of corn steep liquor, 5-10 g / L of sodium chloride, 0.2-0.5 g of potassium dihydrogen phosphate, 8-10 g of ammonium sulfate, 8-10 g / L of sodium nitrate and 8-10 g / L of calcium carbonate.

5. The screening method according to claim 4, It is characterized in that The fermentation culture was carried out in a 24-deep-well plate.

6. The screening method according to claim 5, It is characterized in that The fermentation temperature is 30°C and the rotation speed is 200-220rpm.

7. The screening method according to claim 1, 2, 3, 5 or 6, It is characterized in that The lincomycin strain is a microorganism of the genus Streptomyces.

8. The screening method according to claim 7, It is characterized in that The microorganism of the genus Streptomyces is Streptomyces lincolnensis.

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