High-yield adipic acid strain as well as screening method and application thereof

Abstract

The invention discloses a high-yield adipic acid strain as well as a screening method and application thereof, and belongs to the technical field of bioengineering. According to the invention, engineering escherichia coli MG1655 into which an adipic acid synthesis route is introduced is taken as an original strain, and a strain capable of stably producing adipic acid at high yield is screened out by utilizing ARTP mutagenesis technology and is named as TB131. A mutant strain TB131 obtained through mutagenesis screening is placed in a shake flask containing a fermentation culture medium to be fermented to produce adipic acid, the yield of adipic acid is 3.41 g / L, and the yield is 0.43. The mutant strain TB131 is cultured in a 5L fermentation tank, the yield of adipic acid can reach 78g / L by supplementing glucose, and a solid foundation is laid for subsequent adipic acid research and large-scale industrial application of biologically producing adipic acid.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a high-yield adipic acid strain and a screening method and application thereof. Background Art

[0002] Adipic acid (adipate), also known as fatty acid, is an important organic dibasic acid and is widely used in chemical production, organic synthesis industry, medicine, lubricant manufacturing, etc.

[0003] At present, the main production method of adipic acid is chemical synthesis, but the product yield of this method is not high. In addition, in the chemical synthesis process of adipic acid, benzene is mainly used as the raw material. The raw materials and intermediate products are highly toxic, and a large amount of greenhouse gases such as N2O are produced in the process, which seriously pollutes the environment and is unsustainable.

[0004] In order to solve the above problems, people have focused their attention on the road of biosynthesis of adipic acid and have done a lot of basic work. The main methods for biosynthesis of adipic acid reported so far are biocatalysis and full biosynthesis. The main biocatalysis method mainly uses microorganisms to catalyze the synthesis of adipic acid precursor cis-cis-muconic acid, and then uses metal catalysts to catalyze the synthesis of adipic acid. The methods for full biosynthesis of adipic acid include: Escherichia coli uses glucose as a substrate to use acetyl CoA and succinyl CoA as substrates for full biosynthesis; Saccharomyces cerevisiae uses fatty acid oxidation to fully biosynthesize adipic acid. The full biosynthesis of adipic acid using glucose as a substrate has outstanding advantages such as simple process flow, low total input cost, and recyclability, so it is favored by researchers. However, in the process of producing adipic acid in the above reactions, the adipic acid yield cannot exceed 60g / L, which seriously limits the industrial application of biological adipic acid production. Summary of the invention

[0005] In view of this, in order to solve one of the above technical problems, the present invention provides a high-yield adipic acid strain and its screening method and application. The mutant strain of the present invention can continuously produce adipic acid, and the adipic acid yield is as high as 78g / L, and the production cost is low, which lays an application foundation for the precise application of biosynthetic adipic acid in industrialization.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0007] The invention provides a high-yield adipic acid strain TB131.

[0008] The invention uses the engineered Escherichia coli MG1655 introduced with adipic acid synthesis pathway as the starting strain, and obtains the mutant TB131 after mutagenesis treatment. Compared with the starting strain, the adipic acid yield of the mutant TB131 reaches 78g / L, and the genetic traits are stable within ten generations.

[0009] The invention provides a composition, which contains the strain TB131.

[0010] In one embodiment, the composition is a solid or liquid bacterial agent.

[0011] In one embodiment, the composition contains a bacterial concentration of ≥1×10 5 CFU / g or bacterial concentration ≥1×10 5 CFU / mL of strain TB131.

[0012] The present invention also provides a method for preparing adipic acid, which comprises inoculating the strain TB131 or the composition into a reaction system to perform fermentation preparation of adipic acid.

[0013] In one embodiment, the reaction system includes 8 g / L glucose, 5 g / L yeast powder, 20 g / L tryptone, 2.03 g / L magnesium chloride hexahydrate, 0.5 g / L sodium chloride, and 0.186 g / L potassium chloride.

[0014] The present invention also provides application of the strain TB131 or the composition in producing adipic acid.

[0015] In a specific embodiment, the present invention also provides a method for selecting mutant strains, wherein the method comprises performing ARTP mutagenesis, and selecting a mutant strain with high adipic acid production and genetic stability through primary screening, rescreening and verification.

[0016] The breeding method of the present invention comprises the following steps:

[0017] (1) Preparation of bacterial suspension: The starting strain was inoculated into the seed culture medium and cultured on a shaker until the logarithmic phase. The bacterial suspension was taken out and diluted with 0.9% NaCl solution to a bacterial suspension OD of 600 At 0.60.7; a suspension of mutagenic bacteria was obtained;

[0018] (2) ARTP mutagenesis;

[0019] (3) Primary screening: Select large, round, smooth-surfaced single colonies for activation and fermentation to produce adipic acid, and screen by comparing the adipic acid production with that of the starting strain.

[0020] (4) Rescreening: Select the mutant strain with the highest adipic acid production and the most stable propagation and identify the stability of the strain.

[0021] Preferably, the ARTP mutagenesis time is 75s.

[0022] The specific steps of the breeding method of the present invention are:

[0023] (1) ARTP mutagenesis treatment:

[0024] A loopful of the starting strain was picked from the solid culture medium and inoculated into a 250 mL conical flask containing 50 mL of seed solution. The suspension was cultured in a shaking incubator at 37 °C and 250 r / min until the logarithmic phase. The bacterial suspension in the conical flask was taken out and diluted with 0.9% NaCl solution to obtain a mutagenic bacterial suspension. The OD of the bacterial suspension was 0. 600 0.6 0.7. Pipette 20 μL of the bacterial suspension mixed with 5% (v / v) glycerol in a ratio of 1:1 and evenly spread it on the metal slide, place it in the ARTP mutagenizer, set the power to 120 W, the ventilation volume to 10 SLM, the treatment time to 75 s, put the treated metal gasket into an EP tube containing 980 μL sterile water, shake it thoroughly and dilute it tenfold, take 100 μL of the appropriate gradient dilution solution and spread it on the solid culture medium, and culture it in a 37°C incubator for 1 day.

[0025] (2) Screening of mutagenic strains

[0026] a. Preliminary screening: The induced colonies were preliminarily screened, and large, round, and smooth single colonies were selected and inoculated into the seed solution. The culture was continued at 37°C and 250 r / min in a shaking incubator until the late logarithmic phase. The seed solution cultured to the logarithmic phase was inoculated into 50 mL of liquid fermentation medium at an inoculation rate of 2%, and the culture was continued at 37°C and 250 r / min for 72 h to obtain the fermentation liquid. The fermentation liquid was diluted ten times by volume, and the adipic acid yield was detected by high performance liquid chromatography.

[0027] b. Rescreening: Activate and ferment the mutant strains whose adipic acid production changes significantly after the initial screening, collect fermentation broth samples of different strains, and rescreen; select the mutant strains with the highest adipic acid production and the most stable propagation.

[0028] (3) Determination of yield stability of superior mutant strains

[0029] The superior mutant strains were picked out from the solid plate and inoculated onto solid culture medium, cultured at 37°C for 1 day, which was recorded as the 1st generation. Under the same conditions, the solid culture medium plates were subcultured to the 10th generation. The yield of the superior mutant strains of the 1st to 10th generations was determined according to the primary screening and secondary screening methods of the mutagenic strains described in step (2).

[0030] The invention improves the mutation efficiency through ARTP mutagenesis, and the mutagenesis has strong purpose and high practicality. The optimized mutagenesis conditions of the invention are: the ARTP mutagenesis time is 75s. After ARTP mutagenesis, the mutagenesis lethality is ≥90%. Under this condition, it is easier to obtain a mutagenized strain with high adipic acid production.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention utilizes atmospheric pressure room temperature plasma mutagenesis technology (ARTP) to induce mutation in engineered Escherichia coli containing adipic acid synthesis pathway, and obtains a mutant strain TB131 with high adipic acid production. Compared with the starting strain, the adipic acid production produced by the strain is increased to 78g / L, and the mutant has stable genetic properties. The adipic acid produced can be used for the preparation of lubricants, chemical production or synthetic medicine. Therefore, the present invention lays an important theoretical and application foundation for the industrial production of adipic acid by biological method and its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a fatality rate graph;

[0034] Figure 2 This is a comparison chart of adipic acid production after initial screening of mutant strains;

[0035] Figure 3 It is a comparison chart of adipic acid production of the multiple screened mutant strains;

[0036] Figure 4 is the genetic stability graph of adipic acid production of mutant strain TB131 over ten passages;

[0037] Figure 5 This is a picture of the shake flask fermentation result of mutant strain TB131;

[0038] Figure 6 This is a graph showing the results of feeding the mutant strain TB131 in a 5L fermentation tank in batches; DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the embodiments: those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are all based on conventional conditions; the reagents and biological materials used, unless otherwise specified, can be obtained from commercial channels.

[0040] Starting strain: Engineered Escherichia coli MG1655 containing adipic acid synthesis pathway, which was constructed by our laboratory.

[0041] Seed culture medium: yeast powder 5g / L, trypsin 10g / L, sodium chloride 10g / L, glucose 8g / L.

[0042] Fermentation medium: glucose 8 g / L, yeast powder 5 g / L, tryptone 20 g / L, magnesium chloride hexahydrate 2.03 g / L, sodium chloride 0.5 g / L, potassium chloride 0.186 g / L.

[0043] The method for preparing adipic acid by fermentation is as follows: a single colony is picked and inoculated into a seed culture medium, and the seed solution cultured to the late logarithmic phase is obtained at 37°C and 250r / min. The seed solution cultured to the logarithmic phase is inoculated into 50mL of liquid fermentation medium at an inoculation rate of 2% (v / v), and the fermentation liquid is obtained by constant temperature culture at 37°C and 250r / min for 72h.

[0044] Determination method of adipic acid: HPLC detection conditions: Agilent liquid chromatograph, Aminex HPX-87H (300x7.8mm) column, column temperature: 35°C; mobile phase: 5mM sulfuric acid; flow rate: 0.6ml / min; injection volume: 20μL; detector: differential refractometer.

[0045] Example 1: Mutagenesis screening of strains

[0046] (1) ARTP mutagenesis

[0047] a. Preparation of mutagenic bacterial suspension: Pick a loop of the starting strain from the LB solid medium and inoculate it into a 250mL conical flask containing 50mL of seed medium. Cultivate it in a shaking incubator at 37°C and 250r / min until the logarithmic phase. Take out the bacterial suspension from the conical flask and dilute it with 0.9% NaCl solution to make the bacterial suspension OD 600 At 0.6 0.7, a mutagenic bacterial suspension was obtained.

[0048] b. ARTP mutagenesis: Mix the mutagenic bacterial suspension with 5% (v / v) glycerol at a ratio of 1:1, place in a vortex shaker under sterile conditions, take 20 μL of the bacterial suspension and evenly apply it on the metal slide and place it in the ARTP mutagenizer, set the power to 120W, the ventilation volume to 10SLM, and the treatment time to 15, 30, 45, 60, 75 or 90s. Place the treated metal gasket in an EP tube containing 980 μL of sterile water, shake it thoroughly, and dilute it tenfold. Take the dilution gradient of 10 4100 μL of the dilution was spread on the solid culture medium and cultured in a 37°C incubator for 1 day. The starting strain was used as a control and the optimal mutagenesis treatment conditions were determined based on the lethality curve. Lethality (%) = (number of colonies in the control group + number of colonies in the treatment group) / number of colonies in the control group × 100%.

[0049] Figure 1 is the fatality rate curve; Figure 1 It can be seen that when the ARTP mutagenesis time is 75s, the lethality rate is 91%; in order to ensure the survival of more strains in the future, the ARTP mutagenesis time of 75s is selected as the appropriate mutagenic dose in the subsequent mutagenesis process.

[0050] (2) Screening of mutagenic strains

[0051] a. Preliminary screening: Perform preliminary screening on the induced colonies, pick large, round, and smooth single colonies and inoculate them into seed culture medium, incubate at 37°C and 250r / min to obtain seed solution that has been cultured to the late logarithmic phase, inoculate the seed solution that has been cultured to the logarithmic phase into 50mL of liquid fermentation medium at an inoculation rate of 2% (v / v), and culture at 37°C and 250r / min for 72h to obtain fermentation solution. Collect the fermentation solution and centrifuge at 12000r / min for 5min, take the supernatant and perform high performance liquid chromatography (HPLC) detection at the same time;

[0052] After ARTP mutagenesis, a total of 265 mutant strains were obtained, of which 45 mutant strains with adipic acid production higher than that of the starting strain were preserved for subsequent verification. According to calculations, the adipic acid production of the starting strain was 2.37 g / L.

[0053] Figure 2 is the distribution diagram of adipic acid production after the initial screening of mutant strains; Figure 2 It can be seen that compared with the adipic acid production of the starting strain, the adipic acid production of 45 mutant strains changed significantly and can be used for subsequent rescreening.

[0054] b. Rescreening: Activate and ferment the 45 mutant strains that showed significant changes in adipic acid production after the initial screening, and collect fermentation broth samples of different strains. Collect the fermentation broth and centrifuge it at 12000r / min for 5min, and take the supernatant for high performance liquid chromatography (HPLC) detection;

[0055] The results are as follows Figure 3 As shown, the strain with the highest yield is TB131, with adipic acid content of 3.35 g / L in the fermentation broth and a yield of 0.42 g / g. The yield of adipic acid was calculated by comparing the peak area of ​​the HPLC test result of the sample with the peak area of ​​the HPLC test result of the 1 g / L adipic acid standard sample solution.

[0056] Figure 3is a graph of adipic acid production produced by the multiple screening mutant strains; Figure 3 It can be seen that the mutant strain TB131 has a adipic acid yield of 3.35 g / L, which is the highest adipic acid yield compared to most other mutant strains. It meets the requirements for industrial production of adipic acid, and the high-yield adipic acid mutant strain TB131 is selected as an excellent strain.

[0057] (3) Determination of genetic stability

[0058] The mutant strain TB131 screened in step (2) was picked out from the LB solid plate and inoculated into LB solid culture medium, cultured at 37°C for 1 day, which was recorded as the 1st generation. Under the same conditions, the LB solid culture medium plate was subcultured to the 10th generation, and then the 1st to 10th generation mutant strains TB131 were used to ferment and prepare adipic acid and measure the yield.

[0059] Figure 4 is a genetic stability diagram of adipic acid production of mutant strain TB131 within ten passages; Figure 4 It can be seen that the yield of mutant strain TB131 is relatively stable within ten generations. The highest adipic acid yield of mutant strain TB131 obtained is 3.44 g / L, and the average yield is 3.4 g / L.

[0060] Example 2: Fermentation production of adipic acid in shake flasks

[0061] Fermentation medium was selected for shake flask study on mutant strain TB131, with an initial glucose concentration of 8 g / L.

[0062] A single colony of the mutant strain TB131 screened in Example 1 was selected and inoculated into a seed culture medium, and the culture medium was cultured at 37°C and 250 r / min to obtain a seed solution cultured to the late logarithmic phase. 600 The seed liquid of 3-5% was added to 50mL / 250mL shake flask fermentation medium containing glucose at a 2% (v / v) inoculation rate. The fermentation conditions were temperature: 37°C, rotation speed: 250r / min, and fermentation for 72h. The fermentation broth was collected and centrifuged to obtain the supernatant. The adipic acid production in the fermentation broth was 3.41g / L and the yield was 0.43g / g. The results are as follows Figure 5 shown.

[0063] Example 3: 5L fed-batch tank production of adipic acid

[0064] The culture was grown to the logarithmic phase OD 600The seed liquid of 3-5 was inoculated at 5% (v / v) into a 5L fermentation tank containing 10g / L glucose and 2L fermentation medium. The fermentation conditions were as follows: temperature: 37°C; rotation speed: 400r / min for the first 24h, 300r / min for the next 48h; ventilation volume: 1vvm. After 8h of fermentation, 500g / L glucose was added at 2.2g / L / h. After 24h, the glucose flow rate was changed to 3.5g / L / h, and after 36h, it was changed to 4.5g / L / h. After 48h, 200mL of concentrated nitrogen source (containing 12g peptone and 24g yeast powder) was added. The fermentation broth was collected after 72h of fermentation. The results are as follows: Figure 6 As shown, the adipic acid content of the supernatant obtained by centrifugation was 78 g / L.

[0065] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A high-yield adipic acid strain TB131, characterized in that: The strain is obtained by mutagenesis of an engineered Escherichia coli containing an adipic acid production pathway.

2. A composition, characterized in that The composition contains the strain TB131 according to claim 1.

3. The composition according to claim 2, characterized in that The composition is a solid or liquid bacterial agent.

4. The combination according to claim 2 or 3, characterized in that The composition contains a bacterial concentration of ≥1×10 5 CFU / g or bacterial concentration ≥1×10 5 CFU / mL of strain TB131.

5. A method for preparing adipic acid, characterized in that: The method comprises inoculating the strain TB131 described in claim 1 or the composition described in any one of claims 2 to 4 into a reaction system to perform fermentation preparation of adipic acid.

6. The method according to claim 5, characterized in that The reaction system includes 8 g / L glucose, 5 g / L yeast powder, 20 g / L tryptone, 2.03 g / L magnesium chloride hexahydrate, 0.5 g / L sodium chloride, and 0.186 g / L potassium chloride.

7. A method for screening a high-yielding adipic acid strain, characterized in that: The engineered Escherichia coli containing adipic acid production pathway was used as the starting strain, and its bacterial suspension was subjected to ARTP mutagenesis to screen mutant strains with high adipic acid production.

8. The screening method according to claim 7, characterized in that The screening method comprises the following steps: (1) Preparation of bacterial suspension: The starting strain was inoculated into the seed culture medium and cultured on a shaking table until the logarithmic phase. The bacterial suspension was taken out and The bacterial suspension was diluted with 0.9% NaCl solution to make the bacterial suspension OD 600 At 0.60.7; a suspension of mutagenic bacteria was obtained; (2) ARTP mutagenesis; (3) Primary screening: Select large, round, and smooth single bacterial colonies for activation and fermentation to produce adipic acid, and screen by comparing the adipic acid production with that of the starting strain; (4) Rescreening: Select mutant strains with the highest adipic acid production and the most stable propagation, and identify the stability of the strains.

9. The screening method according to claim 7 or 8, characterized in that The ARTP mutagenesis time was 75 s.

10. Use of the strain TB131 according to claim 1 or the composition according to any one of claims 2 to 4 in producing adipic acid.

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