CN116814504A - Screening method for high-producing succinic acid strains - Google Patents

Abstract

The invention discloses a screening method of a high-yield succinic acid strain, which comprises the following steps: hybrid mutagenesis of strain BER308 using ARTP and microwaves; inoculating the strain BER308 after mutagenesis into succinic acid fermentation broth for anaerobic culture; inoculating the strain BER308 after anaerobic culture into LB culture medium with NaCl concentration above 0.6M for culture; selecting and mixing monoclone with good growth vigor; repeating steps (1) - (4), wherein after each mutagenesis, the concentration of NaCl in the NaCl-containing medium is higher than the concentration of NaCl in the NaCl-containing medium after the previous mutagenesis; until the NaCl concentration in the NaCl culture medium reaches 1-2M. The method can screen out the strain with the advantages of high succinic acid synthesis efficiency, high yield, good strain activity and the like, and the screened strain is suitable for industrial large-scale succinic acid fermentation synthesis.

Description

Screening method for high-producing succinic acid strains

Technical field

The invention relates to a method for screening strains with high succinic acid production.

Background technique

Succinic acid is a colorless crystal and an important organic acid with the chemical formula C4H6O4, also called succinic acid. Succinic acid is widely used in chemistry, medicine, food, cosmetics and other fields. The following are its main uses: Chemically, succinic acid can be used as a solvent, catalyst, intermediate, etc., and can be used to prepare a variety of organic substances. In medicine, succinic acid can be used to prepare drugs for the treatment of heart disease, liver disease, kidney disease, etc., and can also be used to treat anemia, fatigue syndrome and other diseases. In food, succinic acid can be used to make sour agents and flavoring agents, and can also be used as a nutritional enhancer for baked goods such as bread and biscuits. In cosmetics, succinic acid can be used to prepare skin care products, cosmetics, etc., with moisturizing, antioxidant and other effects. In the industrial field, succinic acid can be used to prepare resins, coatings, plastics, etc., and can also be used in industrial fields such as water treatment and paper processing.

The synthesis of succinic acid is mainly divided into two methods: chemical synthesis and biological synthesis. Chemical synthesis mainly produces acrylic acid through the oxidation reaction of styrene. Then, by reacting acrylic acid with ethylene glycol, dihydroxypropionic acid is produced. Finally, dihydroxypropionic acid can be converted to succinic acid through heating and dehydration reactions. This method is cumbersome to operate, has many by-products, and is difficult to separate and purify. Therefore, it is difficult to obtain high-quality and high-purity succinic acid.

During the biosynthesis of succinic acid, microorganisms generate succinic acid through the tricarboxylic acid cycle. In this process, microorganisms first convert glucose or other carbon sources into pyruvate and pyruvate carboxylic acid. Then, pyruvate reacts with pyruvate carboxylic acid to form succinic acid through carboxylation. Microorganisms that biosynthesize succinic acid include many types of bacteria and fungi, such as Escherichia coli, actinomycetes, yeast, etc. This biosynthetic method has the advantages of a wide range of raw materials, mild reaction conditions, and high product purity, so it is widely used in industrial production.

Contents of the invention

The object of the present invention is to provide a screening method for high-producing succinic acid strains.

The technical solution adopted by the present invention is: a screening method for high-producing succinic acid strains, which is characterized by including the following steps:

(1) Use ARTP and microwave to conduct mixed mutagenesis of strain BER308;

(2) Inoculate the mutated strain BER308 into the succinic acid fermentation broth for anaerobic culture;

(3) The strain BER308 after anaerobic culture is inoculated into LB medium with a NaCl concentration above 0.6M for culture;

(4) Select one or more monoclones with good growth and mix multiple monoclones;

(5) Repeat steps (1)-(4), where after each mutagenesis, the NaCl concentration in the NaCl-containing medium is higher than the NaCl concentration in the NaCl-containing medium after the previous mutagenesis, until the NaCl concentration in the NaCl-containing medium is The NaCl concentration reaches 1 to 2 M; the succinic acid fermentation broth used for inoculation after each mutagenesis in step (2) is the succinic acid fermentation broth of the previous mutagenesis anaerobic culture.

Preferably, the strain BER308 is the strain BER308 recorded in Chinese patent CN201710443617.4.

Preferably, ARTP and microwave mixed mutagenesis refers to first subjecting the strain to ARTP mutagenesis, culturing it in LB medium for a period of time after the mutagenesis is completed, and then using microwave mutagenesis, and cultivating it in the LB medium for a period of time after the mutagenesis is completed. time.

Preferably, the ARTP mutagenesis time of the strain is 30-60s, the power is 120W, the microwave mutagenesis time is 30-60s, the power is 100W, and the culture time in LB medium is 1-3 hours.

Preferably, the succinic acid fermentation liquid is the product of strain BER308 after fermenting succinic acid for 72 hours, and the supernatant fermentation liquid is obtained by removing the strain after centrifugation.

Preferably, the anaerobic culture conditions are to introduce sufficient CO ₂ and shake the culture at 37°C overnight.

Preferably, the LB medium containing NaCl is LB solid medium.

The method of the present invention can screen out strains with the advantages of fast succinic acid synthesis efficiency, high yield and good strain activity. The screened strains are suitable for large-scale industrial fermentation and synthesis of succinic acid.

Description of the drawings

Figure 1 Schematic diagram of the screening principle of high-producing succinic acid strains.

Figure 2 Comparison of the activity of strains in the late fermentation medium before and after screening.

Figure 3 Comparison of the production of strains in fermenting succinic acid before and after screening.

Detailed ways

The present invention will be further illustrated by examples below in conjunction with the accompanying drawings, but shall not be used as a limitation of the present invention. Specific materials used in embodiments of the invention and their sources are provided below. However, it should be understood that these are only exemplary and are not intended to limit the present invention. Materials that are the same or similar in type, model, quality, property or function to the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1

In order to improve the tolerance of BER308 to high concentrations of succinic acid and the utilization rate of glucose in the late fermentation period, we conducted mutagenesis and domestication of the E. coli strain BER308 recorded in Chinese patent CN201710443617.4. The domestication process is shown in Figure 1. BER308 was cultured in LB medium at 37°C with shaking at 220 rpm until the OD600 value reached 0.6. 10 ul was spread on an iron sheet and mutated at 120 W on an ARTP mutagenesis instrument for 40 s (about 80%-90% lethality). Put the iron piece into the EP tube, add 500ulBER308 succinic acid fermentation broth in the later stage of fermentation. The fermentation medium uses the fermentation medium described in CN201710443617.4, in which the carbon source is replaced with 10g/L glucose, and cultured to OD600 at 37°C and 220rpm with shaking. The value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 0.6M NaCl and incubate at 37°C overnight. Select single clones with better growth (the larger the clones, the better the growth, usually 1-10 single clones are selected) for fermentation verification.

At the same time, mix the single clones with better growth together and culture them in LB medium at 37°C and 220rpm with shaking until the OD600 value reaches 0.6. Take 10ul and spread it on the iron sheet. Mutagenize it at 120W on the ARTP mutagenesis instrument for 40s (about 80 seconds). %-90% fatality rate). Put the iron piece into the EP tube, add 500ul of the fermentation broth that was cultured anaerobically after the previous round of mutagenesis, and culture with shaking at 37°C and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 0.8M NaCl and incubate at 37°C overnight. Select single clones with better growth and conduct fermentation verification.

At the same time, mix the single clones with better growth together and culture them in LB medium at 37°C and 220rpm with shaking until the OD600 value reaches 0.6. Take 10ul and spread it on the iron sheet. Mutagenize it at 120W on the ARTP mutagenesis instrument for 40s (about 80 seconds). %-90% fatality rate). Put the iron piece into the EP tube, add 500ul of the fermentation broth that was cultured anaerobically after the previous round of mutagenesis, and culture with shaking at 37°C and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 1M NaCl and incubate at 37°C overnight. Select single clones with better growth and conduct fermentation verification.

At the same time, mix the single clones with better growth together and culture them in LB medium at 37°C and 220rpm with shaking until the OD600 value reaches 0.6. Take 10ul and spread it on the iron sheet. Mutagenize it at 120W on the ARTP mutagenesis instrument for 40s (about 80 seconds). %-90% fatality rate). Put the iron piece into the EP tube, add 500ul of the fermentation broth that was cultured anaerobically after the previous round of mutagenesis, and culture with shaking at 37°C and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 1.2M NaCl and incubate at 37°C overnight. Select single clones with better growth and conduct fermentation verification.

At the same time, mix the single clones with better growth together and culture them in LB medium at 37°C and 220rpm with shaking until the OD600 value reaches 0.6. Take 10ul and spread it on the iron sheet. Mutagenize it at 120W on the ARTP mutagenesis instrument for 40s (about 80 seconds). %-90% fatality rate). Put the iron piece into the EP tube, add 500ul of the fermentation broth that was cultured anaerobically after the previous round of mutagenesis, and culture with shaking at 37°C and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 1.4M NaCl and incubate at 37°C overnight. Select single clones with better growth and conduct fermentation verification.

At the same time, mix the single clones with better growth together and culture them in LB medium at 37°C and 220rpm with shaking until the OD600 value reaches 0.6. Take 10ul and spread it on the iron sheet. Mutagenize it at 120W on the ARTP mutagenesis instrument for 40s (about 80 seconds). %-90% fatality rate). Put the iron piece into the EP tube, add 500ul of the fermentation broth that was cultured anaerobically after the previous round of mutagenesis, and culture with shaking at 37°C and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, microwave in ice bath for 60 seconds (about 80%-90% lethality), and continue culturing until the OD600 value reaches 0.8. Spread on LB medium containing 1.6M NaCl and incubate at 37°C overnight. Select single clones with better growth and conduct fermentation verification.

The bacterial solution was divided into single colonies on LB solid medium plates, and cultured at 37°C for 15h-18h until larger single colonies grew. Select a single colony and inoculate it into a test tube containing 5 mL of LB medium. Cultivate it at 37°C and 200 rpm/min for about 8-12 hours as the first-level seed liquid (OD550 2-4) (the test tube needs to be placed tilted in the shaker). Transfer the primary seed liquid to a 1000mL Erlenmeyer flask containing 200mL LB medium at an inoculum volume of 1% (it is recommended to use a three-thorn Erlenmeyer flask), and culture at 37°C and 200rpm for 15-18h (OD550 4-8) to obtain secondary seeds. liquid. The secondary seed liquid was inoculated into the fermentation medium ((NH ₄ H ₂ PO ₄ 3.87g/L, KCl 0.3g/L, MgSO ₄ ·7H ₂ O 0.7g/L, 1/1000(v /v) trace elements (FeCl ₃ ·6H ₂ O 3.4g/L, CoCl ₂ ·2H ₂ O 1.3g/L, CuCl ₂ ·2H ₂ O 0.15g/L, ZnCl ₂ ·4H ₂ O 1g/L, MnCl ₂ ·4H ₂ O 1g/L), and the glucose concentration is controlled at 10g/L) for fermentation. The fermentation temperature is 37°C, pH 6.8, DO 20%. When the bacterial OD600 reaches 5, it will be transferred to anaerobic fermentation. Continue to pass Enter CO2, 37℃, 250rpm for anaerobic fermentation to the end. Glucose is sterilized separately and added in batches. The starting concentration of glucose in the aerobic stage is 30g/L. The glucose concentration and bacterial OD500 are detected every 4-6 hours. When glucose When the first concentration is 10g/L, continue to add 600g/L glucose to control the glucose concentration at around 10g/L. Continue to detect the glucose concentration and bacterial OD600 every 4-6 hours. It can end when the glucose is basically not consumed. Fermentation (about 100h). Through high performance liquid chromatography HPLC (High Performance Liquid Chromatography)

(UitiMate 3000HPLC system, Dionex, USA) was used to detect the concentration of succinic acid in the fermentation broth. The organic acid column model used for detection was Bio Rad Aminex HPX-87H. The mobile phase A used in the detection is 0.25g/L dilute sulfuric acid, which is a single mobile phase in a single pipeline, and the needle washing solution is 10% methanol. The results are shown in Figures 2 and 3. The results shown in Figures 2 and 3 are the 9 best growing single clones finally selected from the 1.6M NaCl medium. It can be seen that the strains after multiple induction and acclimation have better Good vitality and higher succinic acid production.

Claims (7)

1. The screening method of the high-yield succinic acid strain is characterized by comprising the following steps of:

(1) Hybrid mutagenesis of strain BER308 using ARTP and microwaves;

(2) Inoculating the strain BER308 after mutagenesis into succinic acid fermentation broth for anaerobic culture;

(3) Inoculating the strain BER308 after anaerobic culture into LB culture medium with NaCl concentration above 0.6M for culture;

(4) Selecting one or more of the monoclonal antibodies with good growth vigor, and mixing the monoclonal antibodies;

(5) Repeating the steps (1) - (4), wherein the NaCl concentration in the NaCl-containing medium after each mutagenesis is higher than that in the NaCl-containing medium after the previous mutagenesis until the NaCl concentration in the NaCl-containing medium reaches 1-2M; the succinic acid fermentation broth used in the inoculation after each mutagenesis in the step (2) is the succinic acid fermentation broth of the anaerobic culture of the previous mutagenesis.

2. The method for screening a high-yield succinic acid strain according to claim 1, wherein the strain BER308 is strain BER308 described in chinese patent CN 201710443617.4.

3. The method for screening a high-yield succinic acid strain according to claim 2, wherein the mixed mutagenesis of ARTP and microwave is to subject the strain to ARTP mutagenesis, culture it in LB medium for a while after the mutagenesis is completed, and culture it in LB medium for a while after the mutagenesis is completed by using microwave mutagenesis.

4. The method for screening a high-yield succinic acid strain according to claim 3, wherein the strain is subjected to ARTP mutagenesis for 30-60s at a power of 60-120W, microwave mutagenesis for 30-60s at a power of 50-100W, and cultured in LB medium for 1-3 hours.

5. The method for screening a high-yield succinic acid strain according to claim 2, wherein the succinic acid fermentation broth is a product of the strain BER308 after succinic acid fermentation for 72 hours, and the strain is removed after centrifugation, and the supernatant fermentation broth is obtained.

6. The method for screening a high-yield succinic acid strain according to claim 5, wherein the anaerobic culture is conducted under conditions in which CO is introduced in an amount sufficient for the culture ₂ Shake-culturing overnight at 37 ℃.

7. The method for screening a high-yield succinic acid strain according to claim 2, wherein the LB medium containing NaCl is an LB solid medium.