Method for improving beta-alanine production capacity of escherichia coli by utilizing adaptive evolution and escherichia coli engineering bacteria

Abstract

The invention discloses a method for improving the beta-alanine production capacity of escherichia coli by using adaptive evolution and escherichia coli engineering bacteria, which comprises the following steps: carrying out first round of test tube subculture on escherichia coli 630B serving as an original strain on the basis of an LB liquid culture medium containing 15g / L beta-alanine; the concentration of the beta-alanine is increased with the amplitude of 15-30 g / L for gradient culture, each beta-alanine is subjected to passage for 8-10 times, and escherichia coli engineering bacteria with high yield of the beta-alanine are obtained through screening; the engineering bacterium has higher beta-alanine production capacity, the yield of beta-alanine is obviously increased, and the engineering bacterium has important significance for subsequent screening of high-yield beta-alanine engineering bacteria and industrial application of production of beta-alanine.

Description

(I) TECHNICAL FIELD

[0001] The present application belongs to the field of microbial metabolic engineering, and particularly relates to a method for improving the ability of Escherichia coli to produce beta-alanine by adaptive evolution, and an engineered Escherichia coli strain with high beta-alanine production capacity obtained by the method. (II) BACKGROUND

[0002] Beta-alanine, also known as 3-amino propionic acid, is the only beta-type amino acid and non-protein amino acid in nature. Although beta-alanine does not participate in the synthesis of enzymes and proteins, it still has important physiological functions in the growth process of organisms. Plants and microorganisms can autonomously synthesize beta-alanine, while mammals need exogenous supplementation. Beta-alanine has a wide range of applications in medicine, cosmetics, food, and chemical products, and is an important precursor substance that can be used to synthesize pantothenic acid, calcium pantothenate, and carnosine, etc. At present, the global demand for D-calcium pantothenate is huge, resulting in a shortage of products, and the market demand for beta-alanine and its derivatives in other fields is also increasing.

[0003] At present, there are mainly three methods for producing beta-alanine at home and abroad: (1) chemical synthesis method, i.e. using nitrile substances such as propylene nitrile, propylene acid and beta-amino propionitrile to synthesize beta-alanine under the conditions of high pressure and high temperature, strong acid and strong base. However, the chemical synthesis method requires high energy consumption and high equipment requirements, and produces substances harmful to the environment and human body in the production process, and the by-products produced will bring great difficulties to subsequent separation and purification; (2) biological enzyme catalysis method, which uses aspartic acid as the substrate and aspartic acid decarboxylase from strains such as Bacillus subtilis and Corynebacterium glutamicum to express and catalyze the generation of beta-alanine in Escherichia coli. This method is more mild, safe and less polluting, and has attracted more attention from scholars; (3) microbial fermentation method, which uses synthetic biology, systems metabolic engineering, protein engineering, transcriptome and metabolome technologies to modify the metabolic process of the target product beta-alanine, so that more metabolic flow is directed to the direction of the target product. With the increasing severity of climate change and environmental problems, the use of cheap glucose as a carbon source to explore clean, environmentally friendly and low-energy production methods has attracted the attention of many scholars.

[0004] In the current research hotspot, Escherichia coli, Saccharomyces, Corynebacterium glutamicum, Bacillus subtilis and other strains are usually used as chassis strains to construct engineering strains. The metabolic genetic background and physiological characteristics of these chassis strains are relatively clear. Combined with mature gene editing technology and rational design of metabolic flux, the modification progress of strain metabolic pathway can be greatly accelerated, and the time to achieve the optimal production performance of the strain is shortened. Escherichia coli, as the most clear prokaryotic bacteria in current research, has the characteristics of rapid propagation, short fermentation period, high expression level of target gene, mature and perfect expression system and biological safety, and has been widely used in the industrial production of various biological products.

[0005] However, due to the inhibition of β-alanine on bacterial growth, large-scale production of β-alanine has the problems of low yield and the need for expensive culture medium additives (such as amino acids, inducers, antibiotics), etc., so it is challenging to directly ferment β-alanine. It is difficult to improve the synthesis of β-alanine by Escherichia coli strain using mutagenesis breeding, modifying key synthetic enzymes in the pathway and regulating metabolic nodes, and random mutagenesis strategy has the problem of individual genetic instability.

[0006] Adaptive evolution is an effective strain improvement technology, which can simulate natural selection to optimize the tolerance of microorganisms to stressors, accumulate beneficial mutations to respond to selection pressure by changing the phenotype and certain physiological characteristics of the strain in a short period of time, and thus obtain an evolved strain. Today, the most commonly used evolution method is to culture microorganisms under specific selection conditions to make the strain obtain phenotypes and physiological characteristics that are more suitable for the stress conditions.

[0007] However, the application of adaptive evolution has some condition restrictions in actual operation: (1) The effect of adaptive evolution is highly dependent on the type and intensity of the selection pressure. For β-alanine production, the tolerance of the strain to high-concentration β-alanine is a key selection pressure. However, not all strains can grow stably in a high-concentration β-alanine environment, and too high a selection pressure will lead to growth inhibition or even death of the strain, making it difficult to obtain an effective evolved strain. (2) Influence of strain genetic background: the genetic background and metabolic network of different starting strains differ, and the effect of adaptive evolution also differs. (3) Uncertainty of evolution direction: adaptive evolution is a random process, although the evolution direction can be guided by artificial selection pressure, but the phenotype of the finally obtained strain still has a certain uncertainty. Therefore, although adaptive evolution is an effective strain improvement technology, its application in β-alanine production still faces many challenges.

[0008] Therefore, it is urgent to screen an Escherichia coli strain with high β-alanine production capacity to meet the industrial demand for β-alanine production. (III)SUMMARY

[0009] The application aims to provide a method for improving the ability of Escherichia coli to produce beta-alanine by adaptive evolution, and an engineered Escherichia coli strain with high beta-alanine production screened by the method. The application uses Escherichia coli with an optimized beta-alanine metabolic pathway as a starting strain, and performs multiple rounds of test tube subculture pressure screening at a beta-alanine tolerance concentration gradient of 15-30 g / L to ensure that the strain can gradually adapt to a high-concentration beta-alanine environment, and to accumulate beneficial mutations by 8-10 times of subculture at the same beta-alanine concentration in each round of test tube subculture pressure screening, thereby improving the ability of the strain to biosynthesize beta-alanine. Ultimately, a strain with high product tolerance and substrate utilization rate is obtained, thereby laying a foundation for industrial production of beta-alanine.

[0010] The technical solution adopted by the application is as follows:

[0011] The application provides a method for improving the ability of Escherichia coli to produce beta-alanine by adaptive evolution. The method uses Escherichia coli 630B as a starting strain, and performs the first round of test tube subculture in LB liquid medium containing 15 g / L beta-alanine, and then performs multiple rounds of subculture by increasing the concentration of beta-alanine by 15-30 g / L, and each beta-alanine concentration is subcultured for 8-10 times, thereby screening an engineered Escherichia coli strain with high beta-alanine production. E. coli W3110Trc panD Trc ppc Delta pykA Delta cycA .

[0012] Preferably, the concentration of beta-alanine ranges from 15 g / L to 80 g / L.

[0013] Preferably, each beta-alanine concentration gradient is subcultured for 10 generations.

[0014] Preferably, the method for improving the ability of Escherichia coli to produce beta-alanine by adaptive evolution comprises the following steps: inoculating the starting strain Escherichia coli 630B into LB liquid medium containing 15 g / L beta-alanine, and continuously culturing for 10 generations to complete the first round of test tube subculture; inoculating it into LB liquid medium with a beta-alanine concentration gradient of 15-30 g / L for multiple rounds of subculture until the beta-alanine concentration in the medium increases to 80 g / L, and each beta-alanine concentration is subcultured for 10 times, thereby screening an Escherichia coli strain with high beta-alanine production; and the inoculation amount is 1‰ in volume concentration each time.

[0015] Preferably, the concentration of beta-alanine in the culture medium of each round of subculture can be 15 g / L, 30 g / L, 60 g / L, 80 g / L, respectively.

[0016] The application also provides the use of the high-yield beta-alanine E. coli engineering bacteria screened by the above method in the fermentation preparation of beta-alanine. The use is inoculating the high-yield beta-alanine E. coli engineering bacteria into a fermentation medium containing 48 mg / L IPTG and 50 mg / L kanamycin, culturing at 20-40 ℃ and 100-300 rpm (preferably 30 ℃ and 180 rpm) for 48 h to the end of fermentation, obtaining a fermentation liquor containing beta-alanine, and separating and purifying the fermentation liquor to obtain beta-alanine; the fermentation medium formula is as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 1 mL / L trace element solution, without adjusting pH value; wherein the components of the trace element solution are as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0017] Preferably, the high-yield beta-alanine E. coli engineering bacteria are E. coli engineering bacteria (E. coli) Escherichia coli ) JH9, with the preservation number of CCTCC NO: M 20252067.

[0018] Preferably, before fermentation, the high-yield beta-alanine E. coli engineering bacteria are inoculated into an LB liquid culture medium containing 50 mg / L kanamycin, cultured overnight at 37 ℃ and 180 rpm to prepare a seed liquor, and the seed liquor is inoculated into the fermentation medium at a volume concentration of 5%.

[0019] Preferably, the fermentation is carried out in a fermentation tank: the high-beta-alanine-producing E. coli engineering bacteria are inoculated on a LB plate containing 50 mg / L kanamycin, cultured at 37℃ overnight, a single colony is picked to an LB test tube containing 50 mg / L kanamycin, and cultured at 37℃, 150 rpm, overnight to prepare a seed solution; the seed solution is inoculated into LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, and cultured at 37℃, 150 rpm, overnight to obtain a secondary seed solution; the secondary seed solution is inoculated into a 5L fermentation tank containing 2L fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%, and fermented at 30℃, 300 rpm, 1.0 V / V·min, and pH 6.80; when the OD 600 of the fermentation tank reaches 10, 48 mg / L IPTG is added to induce; when the pH value is higher than 6.80 (the initial sugar in the fermentation tank is consumed), automatic feeding is started, and the feeding medium is added until the pH value is lower than 6.80; the culture is continued for 75 h to obtain a fermentation liquor containing beta-alanine; the fermentation medium formula is: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 1 mL / L trace element solution, and the pH value does not need to be adjusted; the trace element solution is composed of: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water; the feeding medium is composed of: glucose 600 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 48 mg / L IPTG, 50 mg / L kanamycin, 2 mL / L trace element solution, and the solvent is water; the pH value is adjusted to 6.8 by 14% ammonia water. The total amount of the feeding medium added is 1500 mL / 2L.

[0020] The application also provides a high-beta-alanine-producing E. coli engineering bacteria (JH9) Escherichia coli ) screened by the method, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20252067, and the preservation date is September 19, 2025.

[0021] Compared with the prior art, the beneficial effects of the present application mainly embody: the present application takes 630B as the starting strain, carries out adaptive evolution under aerobic conditions in the LB liquid medium rich in beta-alanine, and obtains an engineered E. coli strain with high beta-alanine yield through test tube passage pressure screening. The engineered E. coli strain with high beta-alanine yield has high product tolerance and substrate utilization rate, thereby promoting the beta-alanine biosynthesis ability of the strain. Experiments prove that the beta-alanine yield of strain JH9 after 48 h of shake flask fermentation is 5.42 g / L, which is increased by 15% than the yield of the initial strain 630B; after fed-batch fermentation in a 5 L fermenter, the beta-alanine yield of strain JH9 after 59.7 h is 105.54 g / L, which is increased by 22.51% than the yield of the initial strain 630B (86.15 g / L), indicating that the evolved strain JH9 has stronger beta-alanine production capacity, and the beta-alanine yield is obviously improved, which has important significance for subsequent screening of engineered bacteria with high beta-alanine yield and industrial application of beta-alanine production. (Four) DESCRIPTION OF DRAWINGS

[0022] Figure 1 Growth of the starting strain 630B of Example 2 in the LB liquid medium with different concentrations of beta-alanine or without beta-alanine.

[0023] Figure 2 Beta-alanine yield of strains JH1~JH10 of Example 3 after shake flask fermentation.

[0024] Figure 3 Beta-alanine yield of strains W1~W5 of Example 4 after shake flask fermentation.

[0025] Figure 4 Biomass OD of the starting strain 630B of Example 5 after fed-batch fermentation in a 5 L fermenter. 600 , residual sugar and beta-alanine concentration curve.

[0026] Figure 5 Biomass OD of strain JH9 of Example 5 after fed-batch fermentation in a 5 L fermenter. 600 , residual sugar and beta-alanine concentration curve. (Five) SPECIFIC EMBODIMENT

[0027] The present application will be further described below in conjunction with specific embodiments, but the protection scope of the present application is not limited to this:

[0028] In the embodiments of the present application, the methods used are conventional methods, and the reagents used can be obtained from commercial channels.

[0029] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, solvent is water, pH natural. The beta-alanine added to the LB medium was sterilized using a 0.22 pm filtration membrane.

[0030] LB solid medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L, solvent is water, pH natural.

[0031] Fermentation medium formula: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 1 mL / L trace element solution, without pH adjustment; the trace element solution consists of: 10 g / L CaCl2, 10 g / L FeSO4-7H2O, 1 g / L ZnSO4-7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2-7H2O, solvent is deionized water.

[0032] Strains E. coli W3110 from the Coli Genetic Stock Center of Yale University, deposited on August 5, 1975, with the accession number CGSC #4474, has been disclosed in the patents US 2009 / 0298135 A1, US 2010 / 0248311 A1.

[0033] Starting strain Escherichia coli 630B: E. coli W3110 Trc panD Trc ppc Delta pykA Delta cycA , constructed according to Li, B., Zhang, B., Wang, P., Cai, X., Chen, Y.Y., Yang, Y.F., Liu, Z.Q., Zheng, Y.G., 2022. Rerouting fluxes of the central carbon metabolism and relieving mechanism-based inactivation of L-aspartate-a-decarboxylase for fermentative production of beta-alanine in Escherichia coli. Biotechnology Advances 40, 108-123. Escherichia coliThe selected starting strain 630B of the present application is constructed by gene editing technology, and has a high β-alanine production capacity itself. Its metabolic pathway for producing β-alanine is optimized, and has specific gene knockout and expression regulation (Trc panD, Trc ppc, Δ pykA 、Δ cycA ): introduction of key aspartate-α-decarboxylase from Bacillus subtilis, increase of supply of precursor oxaloacetate, redistribution of carbon flux of pyruvate / phosphoenolpyruvate node, knockout of β-alanine efflux protein, and relief of mechanism based on inactivation of aspartate-α-decarboxylase, etc. In a specific metabolic background, compared with wild-type Escherichia coli (W3110), it is more suitable for synthesis of β-alanine, can make metabolic flow more effectively guided to the β-alanine synthesis pathway, and has higher product yield potential. E. coli

[0034] Example 1: β-alanine concentration determination

[0035] 1% 2,4-dinitrofluorobenzene configuration: 1 mL of 2,4-dinitrofluorobenzene was dissolved in 99 mL of acetonitrile.

[0036] 0.5 M NaHCO3 solution configuration: 21 g of NaHCO3 was dissolved in 500 mL of deionized water.

[0037] 0.2 M PB buffer: 8.74 g of Na2HPO4 12H2O and 2.43 g of Na2HPO4 2H2O were dissolved in 200 mL of deionized water, and were dissolved and stored for standby.

[0038] Sample treatment: the sample concentration was diluted to 0.1-1 g / L with ultrapure water.

[0039] Reaction conditions: 100 μL of sample, 100 μL of 0.5 M NaHCO3 solution and 100 μL of 1% 2,4-dinitrofluorobenzene were taken respectively, and were incubated at 60 ℃ for 60 min. Finally, 700 μL of 0.2 M PB buffer was added and mixed uniformly, and was filtered (polyvinylidene fluoride, organic membrane, 0.22 μm) for standby. The filtrate was detected by HPLC for β-alanine peak area, and the β-alanine concentration in the sample was calculated according to the standard curve of β-alanine standard peak area and concentration.

[0040] ​High performance liquid chromatography (HPLC) instrument model: Thermo Scientific Utimate 3000, chromatographic column model Welchrom C18 Column (4.6x250mm, 5μm). The HPLC detection wavelength is 360 nm, and gradient elution program is used to separate β-alanine, wherein the mobile phase A component is methanol: acetonitrile: ultrapure water = 45:45:10 (v:v:v); the mobile phase B component: 10 mM potassium dihydrogen phosphate, pH value is adjusted to 7.0 with KOH. The elution program is: 0-2.5 min 12% A, 88% B; 2.5-2.6 min A 12%→16%, B 88%→84%; 2.6-13 min A 16%→36%, B 84%→64%; 13-13.1 min A 36%→38% B 64%→62%; 13.1-28 min A 38%→100% B 62%→0; 28-28.1 min A 100%-10% B 0→90%; 28.1-32 min A 10%→12% B 90%→88%.

[0041] Example 2: Growth curve of β-alanine-producing strain 630B in LB medium with different concentrations of β-alanine or without β-alanine

[0042] The bacterial liquid of strain 630B in glycerol tube was streaked on LB solid medium plate and cultured at 37°C for 12 h, and a single colony visible to the naked eye was inoculated into 10 mL of LB liquid medium and activated at 37°C for 12 h. 1 mL of bacterial liquid was inoculated into a flask containing 50 mL of LB liquid medium containing 0, 15, 30, 60, 80 g / L of β-alanine with a final concentration, and cultured at 37°C, 180 rpm for 80 h. Every 4 h, sample was taken in a clean bench, and OD 600 was measured by spectrophotometer. The average growth amount (OD600nm) of strain during the culture process is shown in the following figure: 600 Figure 1 .

[0043] Example 3: Test tube passage pressure screening of strain 630B

[0044] 1. Strain passage

[0045] ​E. coli 630B as the starting strain for β-alanine production. The glycerol tube of strain 630B was inoculated into LB liquid medium test tube and incubated at 37°C overnight. The culture was inoculated into 10 mL LB liquid medium containing 15 g / L β-alanine at a volume concentration of 1‰ and incubated at 37°C. The culture was subcultured into fresh 10 mL LB liquid medium containing 15 g / L β-alanine at a volume concentration of 1‰ every 12 h and incubated at 37°C. The culture was subcultured for 10 generations to complete the first round of subculture. The culture was inoculated into LB liquid medium with the concentration of β-alanine increased to 30 g / L at a volume concentration of 1‰ and subcultured for 10 generations. The concentration of β-alanine was increased to 60 g / L and 80 g / L in the same manner. The culture in LB liquid medium containing 80 g / L β-alanine was spread on LB solid medium plate and incubated at 37°C for 1-2 days. Single colonies with different sizes were selected to obtain 10 evolved strains, which were designated as JH1-JH10, respectively.

[0046] 2. Shake flask fermentation

[0047] The 630B and the 10 evolved strains JH1-JH10 were subjected to shake flask fermentation according to the following procedure:

[0048] The single colonies were picked into LB liquid medium test tubes and incubated at 37°C and 180 rpm overnight to prepare seed liquid. 2.5 mL of the seed liquid was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and 0.5 g of sterile calcium carbonate and subjected to fermentation at 30°C and 180 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 1 min, and all the supernatant was discarded. 1 mL of distilled water was added to resuspend the bacterial cells and calcium carbonate, and the mixture was centrifuged at 12000 rpm for 1 min to discard the supernatant. 1 mL of 20% acetic acid aqueous solution was added to dissolve the calcium carbonate. The mixture was centrifuged at 12000 rpm for 1 min to discard the supernatant. 1 mL of distilled water was added to resuspend the bacterial cells. 100 μL of the resuspended bacterial cells was added to 1900 μL of distilled water to dilute 20-fold. Finally, the biomass OD 600 was determined by spectrophotometry, and the content of β-alanine was determined by the method of Example 1.

[0049] The fermentation results of strains JH1-JH10 are shown in Table 1. Figure 2 The yield of β-alanine of strain JH9 after shake flask fermentation for 48 h was 5.42 g / L, which was 15% higher than that of the starting strain 630B, indicating that the evolved strain JH9 had stronger β-alanine production capacity. Escherichia coliJH9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 20252067, on September 19, 2025.

[0050] Example 4: Wild-type Escherichia coli E. coli In vitro passage pressure screening of W3110

[0051] 1. Strains passage

[0052] wild-type Escherichia coli E. coli W3110 is the starting strain for β-alanine production. The strain is *Escherichia coli*. E. coli W3110 glycerol tubes were inoculated into LB liquid medium tubes and cultured overnight at 37°C. Then, at a concentration of 1‰ (v / v), the inoculum was transferred to 10 mL of LB liquid medium containing 10 g / L β-alanine and cultured at 37°C. During the culture process, every 12 hours, the inoculum was transferred to fresh 10 mL of LB liquid medium containing 10 g / L β-alanine for subculture. This process was repeated for 10 generations to complete the first round of culture. Next, the inoculum was transferred to LB liquid medium with a β-alanine concentration increased to 20 g / L at a concentration of 1‰ (v / v) for the next round of subculture. Each β-alanine concentration was subcultured 10 times, and this process was repeated until the β-alanine concentration in the LB liquid medium reached 30 and 40 g / L. The bacterial culture in LB liquid medium with a β-alanine concentration of 40 g / L was spread onto LB solid medium plates and incubated at 37°C for 1–2 days. Single colonies with significant differences in colony size were selected to obtain 5 individual evolutionary strains, which were designated as evolutionary strains W1–W5.

[0053] 2. Shake-flask fermentation

[0054] Pick E. coli W3110 and the above 5 evolved strains W1 to W5 were subjected to shake-flask fermentation according to the following steps:

[0055] The single colonies were picked into test tubes containing LB liquid medium, and cultured at 37°C, 180 rpm overnight to prepare seed liquid. 2.5 mL of the seed liquid was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and 0.5 g of sterile calcium carbonate, and the fermentation was carried out at 30°C, 180 rpm for 48 h. After the fermentation was completed, 1 mL of the fermentation liquid was centrifuged at 12000 rpm for 1 min, and all the supernatant was discarded. 1 mL of distilled water was added to resuspend the bacterial cells and the calcium carbonate, and the mixture was centrifuged at 12000 rpm for 1 min to discard the supernatant. Then, 1 mL of 20% acetic acid aqueous solution was added, and the mixture was allowed to stand at room temperature for 5 min to dissolve the calcium carbonate. The mixture was centrifuged at 12000 rpm for 1 min, and all the supernatant was discarded. 1 mL of distilled water was added to resuspend the bacterial cells, and 100 μL of the resuspended bacterial cells was added to 1900 μL of distilled water to dilute 20 times. Finally, the biomass OD 600 was determined by spectrophotometry.

[0056] The fermentation results of the strains W1-W5 are shown in Table 1. Figure 3 The β-alanine yield of the strain W4 after shake flask fermentation for 48 h was 1.64 g / L, which was significantly different from the evolution strains JH1-JH10 obtained in Example 3. This indicates that the adaptive evolution of the starting strain modified by gene editing can obtain a β-alanine production strain with good production performance.

[0057] Example 5: Batch fermentation of the adaptive evolution strain JH9 in a 5 L tank

[0058] This example provides the application of the high-yield β-alanine E. coli engineering strain, i.e., the evolution strain JH9, obtained in Example 3 in the preparation of β-alanine, and the specific process is as follows:

[0059] The strain JH9 in Example 3 was streaked from a glycerol tube to a LB solid medium plate containing 50 mg / L of kanamycin resistance, and cultured at 37°C overnight. A single colony was picked into a test tube containing LB liquid medium with 50 mg / L of kanamycin resistance, and cultured at 37°C, 180 rpm overnight to prepare seed liquid. The seed liquid was inoculated into 100 mL of LB liquid medium containing 50 mg / L of kanamycin resistance at a volume concentration of 5%, and cultured at 37°C, 180 rpm overnight as a secondary seed liquid. The secondary seed liquid was inoculated into a 5 L fermentation tank containing 2 L of fermentation medium with 50 mg / L of kanamycin at a volume concentration of 15%, and the fermentation was carried out at 30°C, 300 rpm, 1.0 V / V·min of aeration, and a pH value of 6.80. When the OD 600IPTG was added to a final concentration of 48 mg / L to induce expression. When the pH was higher than 6.80 (initial sugar consumption in the fermenter was completed), automatic feeding was started, and the feeding was stopped when the pH was lower than 6.80, and the residual sugar in the fermenter was maintained at a low level, and the sugar concentration was maintained at 0-2 g / L. The total amount of the feeding medium of the strain JH9 was 1500 mL, and the culture was performed for 74.5 h. The β-alanine yield in the fermentation broth was detected by the high performance liquid chromatography method of Example 1, the biomass OD 600 was detected by spectrophotometry, and the sugar concentration was detected by the DNS method, as shown in Figure 5 . Under the same conditions, the strain JH9 was replaced by the starting strain 630B, and the other operations were the same, and the results are shown in Figure 4 .

[0060] Figure 4 , Figure 5 It is shown that the β-alanine yield of the strain JH9 reached 105.54 g / L at 59.7 h, which was increased by 22.51% compared with the yield of the starting strain 630B (86.15 g / L), and the final biomass OD 600 was maintained at 70 (which was reduced by 36.36% compared with the starting strain 110). The fermentation results show that the β-alanine production strain obtained by adaptive evolution has good production performance, which lays a foundation for the industrial production of β-alanine.

[0061] In summary, the strain screened by the adaptive evolution method of test tube pressure passage can significantly improve the β-alanine yield, and has important significance for the subsequent screening of high-yield β-alanine and the industrial application of β-alanine production.

[0062] The above-described examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application.

Claims

1. A method for improving the ability of Escherichia coli to produce β-alanine using adaptive evolution, characterized by, The method is to take E. coli 630B as a starting strain, to take LB liquid medium containing 15 g / L β-alanine as a basis to carry out first round test tube subculture, to increase the concentration of β-alanine by 15-30 g / L in steps to carry out gradient subculture, to carry out subculture for 8-10 times for each concentration of β-alanine, and to screen to obtain E. coli engineering bacteria with high β-alanine yield; the E. coli 630B is: E. coli W3110Trc panD Trc ppc Δ pykA Δ cycA .

2. The method of claim 1, wherein, The concentration of beta-alanine used in the subculture ranges from 15 to 80 g / L.

3. The method of claim 1, wherein, Each beta-alanine concentration is subcultured for 10 times.

4. The method of claim 1, wherein, The method comprises the following steps: inoculating E. coli 630B into LB liquid medium containing 15 g / L beta-alanine, continuously culturing for 10 generations to complete the first round of test tube subculture; inoculating it into LB liquid medium with a gradient increase of beta-alanine concentration of 15-30 g / L for multiple rounds of subculture until the beta-alanine concentration in the medium increases to 80 g / L, and each beta-alanine concentration is subcultured for 10 times, and a high-yield beta-alanine E. coli is screened.

5. The method of claim 4, wherein, The concentrations of beta-alanine are 15 g / L, 30 g / L, 60 g / L, and 80 g / L, respectively.

6. The application of the high-yield beta-alanine E. coli engineering bacteria screened by the method of claim 1 in the fermentation preparation of beta-alanine.

7. Use according to claim 6, wherein The application is inoculating the high-yield beta-alanine E. coli engineering bacteria into fermentation medium containing 48 mg / L IPTG and 50 mg / L kanamycin, culturing at 20-40 ℃ and 100-300 rpm for 48 h until the fermentation is completed, obtaining a beta-alanine-containing fermentation broth, and separating and purifying the fermentation broth to obtain beta-alanine. The fermentation medium formula is as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 1 mL / L trace element solution, without pH adjustment; wherein the components of the trace element solution are as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

8. Use according to claim 7, wherein the compound is ###0002### Before fermentation, the high-yield beta-alanine E. coli engineering bacteria are inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 37 ℃ and 180 rpm overnight to prepare seed liquid, and the seed liquid is inoculated into the fermentation medium at a volume concentration of 5%.

9. The use according to claim 7, wherein the compound is ###0003### The fermentation is carried out in a fermenter: the high-yield beta-alanine E. coli engineering bacteria are inoculated into LB plates containing 50 mg / L kanamycin resistance, cultured at 37 ℃ overnight, a single colony is picked into LB test tubes containing 50 mg / L kanamycin resistance, cultured at 37 ℃ and 150 rpm overnight to prepare seed liquid; the seed liquid is inoculated into LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, cultured at 37 ℃ and 150 rpm overnight as secondary seed liquid; The secondary seed liquid was inoculated into the fermenter containing the fermentation medium with 50 mg / L kanamycin at a volume concentration of 15%, and the fermentation was carried out at 30°C, a rotation speed of 300 rpm, a ventilation amount of 1.0 V / V·min, and a pH value maintained at 6.

80. When the OD 600 of the fermentation liquid reached 10, 48 mg / L IPTG was added for induction; when the pH value was higher than 6.80, the automatic feeding was started, and the feeding medium was added until the pH value was lower than 6.80, and then the feeding was stopped. The culture was carried out for 75 h to obtain the fermentation liquid containing β-alanine. The fermentation medium formula was as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 1 mL / L trace element solution, without pH adjustment; the trace element solution was composed of 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent was deionized water; the feeding medium was composed of glucose 600 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB 12 , 48 mg / L IPTG, 50 mg / L kanamycin, 2 mL / L trace element solution, and the solvent was water, and the pH value was adjusted to 6.8 by 14% ammonia water.

10. An E. coli (Escherichia coli) strain screened by the method of claim 1. Escherichiacoli ) JH9, deposited with the China Center for Type Culture Collection on September 19, 2025, and assigned accession number CCTCC NO: M 20252067.

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