Lincomycin strain breeding method based on ARTP and ribosome engineering

Abstract

The invention relates to a lincomycin strain breeding method based on ARTP and ribosome engineering, which is named as lincomycin strain breeding method based on ARTP and ribosome engineering, and the lincomycin high-yield strain is successfully obtained by adopting an ARTP and ribosome engineering compounded mutagenesis method and then establishing a 48-hole deep-hole plate culture and screening method. A deep-well plate culture method of streptomyces lincolensis is established through correlation research of a shake flask and a 48-well plate; a streptomyces lincolensis deep-well plate screening method is established by combining a microbial turbidimetry taking staphylococcus aureus as sensitive bacteria. The final mutagenesis conditions are as follows: the ARTP treatment temperature is 20 DEG C, the input power is 120W, the distance between a plasma beam and a spore suspension is 2mm, the gas flow is 10L / min, and the treatment time is 80s; the treatment concentration of rifamycin is 5.0 mu g / mL, and the treatment concentration of streptomycin is 8.0 mu g / mL. According to the method, 18 high-yield strains are obtained and transferred into a shake flask to verify the yield of lincomycin A, the high-yield strain AR-5 is finally obtained, and the yield of lincomycin A reaches 2862 mu g / mL and is improved by 18.2% compared with that of a contrast (2423 mu g / mL).

Description

Technical Field

[0001] The invention provides a strain breeding and screening method, in particular to a lincomycin strain breeding method based on ARTP and ribosome engineering. Background Art

[0002] Plasma is a form different from solid, liquid and gas. It is composed of neutral particles and charged particles. It is divided into high-temperature plasma and low-temperature plasma according to the working temperature. The intensity varies with the working gas (commonly used inert gases, such as helium, argon, etc.), pressure, power and processing time. The results of quantitative analysis based on flow cytometry show that ARTP has higher DNA damage and mutation rates than conventional physical mutagenesis (ultraviolet light) or chemical mutagenesis. Based on the successful application of radio frequency atmospheric pressure glow discharge plasma (RF-APGD) in microbial breeding, an atmospheric pressure room temperature plasma (ARTP) mutagenesis system with helium as the working gas was established.

[0003] Through whole genome sequencing of several actinomycetes, it was found that each strain has the potential to produce dozens of important active substances or antibiotics, but under conventional culture conditions, only a small number of products can be expressed. The huge biosynthetic potential of Streptomyces has not yet been fully explored. At present, improving the production level of secondary metabolites can be achieved by changing laboratory culture conditions. 70% of antibiotics and their derivatives used in pharmaceuticals and agriculture come from members of the genus Actinomycetes. Activating silent or low-expressed genes, discovering new antibiotics, and improving high-yield strains are very important in applied microbiology. Therefore, scholars are very interested in how to activate these silent biosynthetic pathways and induce or enhance the expression of silent pathways.

[0004] Ribosome engineering is a new method to induce ribosome resistance mutations to activate the stringent response and increase the yield of actinomycetes. Mutations are introduced by producing resistance to antibiotics based on ribosome targeting (such as rifamycin, streptomycin, gentamicin, etc.). Although the mutation rate is low, mutation and screening can be achieved by simply inoculating spores or bacteria on resistance plates; in addition, there is no need to sequence the target strain, that is, directed mutagenesis of the strain can be completed without genomic information. Summary of the invention

[0005] Microplate Assay

[0006] Pour sterile water onto a Staphylococcus aureus plate or slant to prepare a sensitive bacterial suspension (OD 600 =1.5), inoculated into nutrient broth, inoculated at 5%, 490 μL / well dispensed into 48-well deep-well plates; after the sample was diluted to the linear range (10-100 mg / L), 50 μL of the sample was added to the nutrient broth containing bacteria, cultured at 37°C for 12 h, and the OD was detected by an enzyme-labeled instrument 600Calculate the sample concentration according to the standard curve.

[0007] Microplate culture method

[0008] The seed solution of Streptomyces lincomyces was inoculated into a 48-well plate, 1.2 mL per well, and cultured at 180 rpm and 30°C for 6-7 days.

[0009] Mutagenesis methods

[0010] Turn on the UV lamp in the ARTP and sterile room, irradiate for 30 minutes, ensure the environment is sterile, draw 10 μL of the prepared spore suspension and apply it to the metal slide, use helium as the working gas, control the temperature at 20°C, input power at 120W, the distance between the plasma beam and the spore suspension is 2mm, the gas flow rate is 10L / min, and irradiate for 80 seconds. Use tweezers to pick up the mutagenized metal slide and put it into a test tube containing 9.99mL sterile water, vortex to mix evenly, draw 100 μL and apply it on the solid plate, culture at 30°C for 6-7 days, and then transfer to 5-fold rifamycin and streptomycin resistance plates.

[0011] Initial screening results

[0012] A single colony from the above-mentioned resistance plate was picked into a 48-well deep-well plate containing 500 μL of Streptomyces lincomycin shake flask seed culture medium. After culturing at 30°C and 200 rpm for 2 days, 100 μL was pipetted into a 48-well fermentation deep-well plate containing 1100 μL of shake flask fermentation culture medium. After culturing at 30°C and 200 rpm for 7 days, the lincomycin production was detected by turbidimetry.

[0013] The results showed that the absorbance in 18 microplates was lower than that of the original strain (0.198), that is, the fermentation broth of the strains corresponding to these 18 microplates had a higher content of lincomycin. Considering that the color of the fermentation broth may affect the test results, the strains corresponding to the absorbance less than 0.15 were selected for rescreening (a total of 10 strains), and the seed liquid corresponding to these 10 strains was found, diluted and spread on a fresh plate, cultured at 30°C for 7 days, and then stored in a 4°C refrigerator for rescreening.

[0014] Rescreening results

[0015] The strains AS-7, AS-21, and AR-5 were obtained, and further transferred to shake flasks for rescreening and verification, and finally the high-yield lincomycin strain AR-5 was obtained. The shake flask fermentation results showed that the lincomycin production level of the mutant strain AR-5 (2862.0 mg / L) was increased by 18.2% compared with the parent strain (2423.5 mg / L), and the impurity component LinB content was reduced from 1.2% to 0.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Standard curve for microbial turbidimetry

[0017] Figure 2 The ARTP mortality curve

[0018] Figure 3 Determination of the Minimum Inhibitory Concentration of Rifamycin for Streptomyces lincomycin

[0019] Figure 4 Determination of the minimum inhibitory concentration of streptomycin for Streptomyces lincomycin

[0020] Figure 5 The results of the initial screening of mutant strains of Streptomyces lincomyces DETAILED DESCRIPTION

[0021] The feasibility of the microplate culture method for Streptomyces lincomyces was verified by selecting the sample loading volume and analyzing the parallelism between wells of the 48-well deep-well plate. The reliability of the microplate screening results for Streptomyces lincomyces was determined by studying the correlation between shake flasks and 48-well plates. A microplate screening method for Streptomyces lincomyces was established by combining microbial turbidimetry with Staphylococcus aureus as the sensitive bacteria.

[0022] The optimal sample volume for microplate culture of Streptomyces lincomycin was 1.2 mL. The results of parallelism analysis between wells showed that the RSD value of lincomycin production in each well was 3.85%. The correlation coefficients of bacterial concentration, pH, residual glucose concentration and lincomycin production of Streptomyces lincomycin in shake flasks and 48-deep-well plates were 0.995, 0.990, 0.990 and 0.989, respectively.

[0023] The ARTP mutagenesis conditions of Streptomyces lincomycin were as follows: temperature 20℃, input power 120W, distance between plasma beam and spore suspension 2mm, gas flow rate 10L / min, and treatment time 80s; the minimum inhibitory concentration (MIC) of streptomycin (Str) and rifamycin (Rif) against Streptomyces lincomycin were 1.6μg / mL and 1.0μg / mL, respectively.

[0024] Using ARTP and ribosome engineering as a combined mutagenesis method, a mutant library of Streptomyces lincomycin was obtained; through the established microplate screening method, strains AS-7, AS-21, and AR-5 were initially screened, and further transferred to shake flasks for rescreening and verification, and finally the lincomycin high-yielding strain AR-5 was obtained. The results of shake flask fermentation showed that the lincomycin production level of the mutant strain AR-5 (2862.0 mg / L) was increased by 18.2% compared with the parent strain (2423.5 mg / L), and the impurity component LinB content was reduced from 1.2% to 0.9%.

Claims

1. A method for breeding lincomycin strains based on ARTP and ribosome engineering, characterized in that: The steps include: (1) Place a 1 cm × 1 cm bacterial block in a grinder, add 2 mL of sterile water, grind thoroughly, and mix evenly with a vortex mixer. The spore suspension concentration is about 10 7 cfu / mL. (2) 10 μL of the prepared spore suspension was applied to a metal slide, and helium was used as the working gas. The temperature was controlled at 20°C, the input power was 120 W, the distance between the plasma beam and the spore suspension was 2 mm, the gas flow rate was 10 L / min, and the irradiation time was different to obtain the lethality curve. The optimal treatment time was 80 s. (3) The prepared spore suspension was diluted to 10 3 cfu / mL, pipette 100 μL to the resistance plate with different rifampicin and streptomycin concentrations, evenly spread it, and place it in a 30°C incubator for 6-7 days. The minimum inhibitory concentration (MIC) of rifampicin and streptomycin for Streptomyces lincomycin was 1.0 and 1.6 μg / mL, respectively. (4) After ARTP treatment for 80 seconds, the strain was treated with 10 times the MIC and the absorbance was detected by microbial turbidimetry. The strain with an absorbance lower than that of the parent strain was selected to complete the breeding of lincomycin high-yield strains.

2. The method for breeding lincomycin strains based on ARTP and ribosome engineering according to claim 1, characterized in that: Slant culture medium formula: Slant culture medium ratio (%): corn starch 2.0, soybean cake powder 0.5, KNO3 0.1, MgSO4 0.05, K2HPO4 0.05, NaCl 0.05, FeSO4 0.001, agar 2.0; pH 7.25-7.

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3. The method for breeding lincomycin strains based on ARTP and ribosome engineering according to claim 1, characterized in that: The proportion of shake flask fermentation medium (%): corn starch 8.0, soybean cake powder 2.0, corn steep liquor 0.15, glucose 6.0, NaCl 0.5, NaNO 3 0.8, K 2 HPO 4 0.01, (NH 4 ) 2 SO 4 0.6, CaCO 3 0.8, pH 7.6-7.

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4. The method for breeding lincomycin strains based on ARTP and ribosome engineering according to claim 1, characterized in that: In step (4), the 5 g / L lincomycin standard solution was accurately diluted to 20, 40, 60, 80, and 100 mg / L (3 parallels) using phosphate buffer for microbial turbidimetry. 5% of the prepared Staphylococcus aureus suspension (OD 600 =1.5), after mixing, dispense into 48 deep-well plates, 490 μL per well, add 10 μL of lincomycin standard of each concentration, incubate at 37°C, 200 rpm for 12 h, terminate when blank absorbance reaches 0.7 (blank without adding lincomycin standard), measure absorbance at 600 nm using an ELISA reader, and use the logarithm of LinA concentration as the horizontal axis, OD 600 Draw the standard curve with y as the vertical axis.

5. The method for breeding lincomycin strains based on ARTP and ribosome engineering according to claim 1, characterized in that: The culture was transferred from the shake flask to a 48-well square flat-bottom deep-well plate.

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