Application of transport protein to increase yield of beta-alanine and constructed probiotic escherichia coli

Abstract

The invention discloses application of transporter to increase yield of beta-alanine and constructed probiotic Escherichia coli, rhamnose transporter RhaT capable of increasing the yield of beta-alanine is obtained through screening, and the shake-flask fermentation yield of beta-alanine reaches 1.85 g / L through overexpression of rhamnose transporter rhaT gene, so that the yield of beta-alanine is increased, the yield of beta-alanine is increased, and the yield of beta-alanine is increased. And the content of beta-alanine in the fermentation tank is 73.11 g / L. Compared with a control strain, the shake-flask fermentation yield of beta-alanine is improved by 7.56%. In the future, related expression genes of serine and threonine of competitive branches of beta-alanine can be inhibited or knocked out by combining a protein structure optimization or directed evolution strategy, so that the yield of beta-alanine is further increased.

Description

(I) Technical Field

[0001] This invention relates to the application of a transport protein to increase β-alanine production and the construction of a probiotic Escherichia coli. (II) Background Technology

[0002] β-Alanine, also known as 3-aminopropionic acid, is the only β-type amino acid and non-protein amino acid found in nature. Although it does not participate in enzyme and protein synthesis, it still plays an important physiological role in the growth of organisms. In cellular metabolism, β-alanine can be used as a precursor to synthesize pantothenic acid (vitamin B5) and coenzyme A (CoA), and it is involved in various metabolic activities such as fatty acid metabolism and carbohydrate metabolism. Plants and microorganisms can synthesize β-alanine autonomously, while mammals require exogenous supplementation. Current research has found that β-alanine has important applications in various fields such as food, medicine, chemical industry, and animal feed.

[0003] In the pharmaceutical field, β-alanine is a key precursor in the production of pantothenic acid, calcium pantothenate, coenzyme A (CoA), and acyl carrier proteins (ACP), playing a crucial role in cell metabolism and the development of the central nervous system. β-alanine can also be used to synthesize drugs such as balsalazine and pamidronate disodium. In the food industry, β-alanine is used as a food additive, commonly in food flavorings to improve taste. Due to its antioxidant properties, it is also frequently used as a food preservative. Clinical studies have shown that β-alanine can also be used as a nutritional supplement for athletes; supplementation with β-alanine can significantly improve fatigue caused by prolonged high-intensity exercise and enhance endurance. In the chemical industry, β-alanine is not only used as a precursor in the production of pantothenic acid, calcium pantothenate, poly(3-hydroxypropionate), and poly(β-alanine), but it is also used as a precipitant in pharmaceuticals, an electroplating corrosion inhibitor, and an antidote for lead poisoning. In the feed industry, β-alanine is increasingly used in animal husbandry. Studies have found that adding β-alanine to feed can improve feed utilization in livestock and poultry, reduce feed intake, improve muscle antioxidant capacity, increase the content of myogenic active peptides, and effectively improve meat quality. Furthermore, β-alanine also demonstrates important roles in environmental and cosmetic fields.

[0004] Currently, there are three main methods for producing β-alanine both domestically and internationally: (1) Chemical synthesis, which uses nitrile substances such as acrylonitrile, acrylic acid, and β-aminopropionitrile to synthesize β-alanine under high pressure and high temperature, and strong acid and strong base conditions. However, chemical synthesis consumes a lot of energy, requires high-end equipment, and produces substances harmful to the environment and human body during the production process. At the same time, the by-products produced will bring great difficulties to the subsequent separation and purification. (2) Bioenzyme catalysis, which uses aspartic acid as a substrate and uses aspartic acid decarboxylase from strains such as Bacillus subtilis or Corynebacterium glutamicum to catalyze the production of β-alanine in Escherichia coli. This method is milder, safer, and less polluting, attracting more attention from scholars. However, bioenzyme catalysis is unstable, costly, and easily inhibited by the substrate. (3) Microbial fermentation to produce β-alanine. Microbial fermentation uses synthetic biology, systems metabolic engineering, protein engineering, transcriptomics, and metabolomics to modify the metabolic process of the target product β-alanine, so that more metabolic flux flows toward the target product. With climate change and increasingly severe environmental problems, the exploration of clean, environmentally friendly, and low-energy-consumption production methods using inexpensive glucose and other carbon sources has attracted the attention of many scholars.

[0005] The de novo synthesis pathway of β-alanine in probiotic E. coli includes glucose uptake, glycolysis, the TCA cycle, L-aspartate synthesis, and the β-alanine synthesis pathway. First, glucose undergoes phosphorylation in either a PTS or non-PTS system to synthesize glucose-6-phosphate. The carbon flux then enters the glycolysis pathway, where phosphoenolpyruvate, a precursor of β-alanine, is synthesized. Under the catalysis of phosphoenolpyruvate carboxylase, phosphoenolpyruvate is converted to oxaloacetate. Oxaloacetate is unstable and easily decomposes, converting to aspartate under the catalysis of aspartate transaminase, and subsequently to β-alanine under the catalysis of aspartate decarboxylase.

[0006] Regarding product efflux, although studies have reported the application of the transporter Ncgl0580 in enhancing β-alanine production, its effectiveness suggests the existence of other unexplored β-alanine transporters within microbial cells. These potential transporters may possess higher transport efficiency and broader substrate specificity, which would be crucial for further improving β-alanine production efficiency. (III) Summary of the Invention

[0007] The purpose of this invention is to provide an application of a transport protein to increase β-alanine production and a constructed probiotic Escherichia coli. By screening for β-alanine transport proteins with higher transport efficiency, the production of β-alanine can be further increased.

[0008] The technical solution adopted in this invention is:

[0009] In a first aspect, the present invention provides an application of a transporter protein in increasing β-alanine production, wherein the transporter protein is the rhamnose transporter RhaT.

[0010] Preferably, the nucleotide sequence of the transporter protein encoding gene rhaT is shown in SEQ ID NO.3.

[0011] Secondly, the present invention provides a probiotic Escherichia coli constructed from the aforementioned transporter protein, wherein the probiotic Escherichia coli uses strain E45 as the starting strain and is incorporated into plasmid pTrc99a-panD. BS -aspB CG -aspA-pyc CG The rhaT gene derived from E. coli Nissle 1917 was overexpressed; strain E45 is E. coli Nissle1917 / Trc-panD / Trc-aspC / Trc-sdhC / P fumA ::P flgC-ATG-fumA / Trc-ppc / ΔcycA.

[0012] The probiotic Escherichia coli described in this invention is constructed using the following method:

[0013] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The rhaT gene, expressed via codon-optimized plasmid pTrc99a-panD, was expressed. BS -aspB CG -aspA-pyc CG Using pTrc99a-line-F / pTrc99a-line-R as primers, the linearized plasmid pTrc99a-panD was obtained. BS -aspB CG -aspA-pyc CG -line; using the E. coli Nissle 1917 genome as a template, the rhaT gene fragment was obtained using rhaT-F / rhaT-R primers, and the linearized plasmid pTrc99a-panD was used. BS -aspB CG -aspA-pyc CG -line was ligated with the rhaT gene fragment to construct plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT and transformed into strain E45 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::PflgC-ATG-fumA In competent cells of Trc-ppc and ΔcycA, strain E54 (Trc-panD, Trc-aspC, Trc-sdhC, P) was constructed. fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT).

[0014] Thirdly, the present invention also relates to a method for preparing β-alanine by the aforementioned probiotic Escherichia coli microbial fermentation method, wherein the method comprises: inoculating the probiotic Escherichia coli into a fermentation medium containing 50 mg / L kanamycin, and culturing the cells at 30°C and 180 rpm until the OD reaches a certain level. 600 =0.5, at this point, add IPTG to a final concentration of 0.25mM to induce gene expression, continue culturing for 48h until fermentation ends, obtain fermentation broth containing β-alanine, separate and purify the fermentation broth to obtain β-alanine.

[0015] The fermentation medium formulation is as follows: glucose 18–22 g / L, (NH4)2SO4 14–18 g / L, yeast extract 2–3 g / L, KH2PO4 1–2 g / L, MgSO4 0.2–1 g / L, CaCO3 13–17 g / L, VB1 0.3–0.5 mg / L, VB 12 0.3–0.5 mg / L, trace element solution 0.5–1.5 mL / L, solvent is water, no pH adjustment required; further optimized to: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, CaCO3 15 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 A 1 mL / L trace element solution, with water as the solvent, requires no pH adjustment. The components of the trace element solution are as follows: 8–12 g / L CaCl2, 8–12 g / L FeSO4·7H2O, 0.8–1.2 g / L ZnSO4·7H2O, 0.1–0.3 g / L CuSO4, 0.01–0.04 g / L NiCl2·7H2O, with deionized water as the solvent; further optimized to: 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, with deionized water as the solvent.

[0016] Before fermentation, the Escherichia coli probiotics are inoculated into LB medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 180 rpm to prepare a seed culture. The seed culture is then inoculated into fermentation medium containing 50 mg / L kanamycin at a volume concentration of 5%.

[0017] Preferably, the fermentation is carried out in a fermenter: the *E. coli* probiotics are inoculated onto LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 37°C; single colonies are picked and transferred to LB test tubes containing 50 mg / L kanamycin resistance and incubated overnight at 37°C and 150-180 rpm to prepare a primary seed culture; the primary seed culture is inoculated at a volume concentration of 5% into LB medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 150-180 rpm to obtain a secondary seed culture. The secondary seed culture was inoculated at a volume concentration of 15% into a 5-L fermenter containing 2L of fermentation medium with 50mg / L kanamycin, and IPTG was added to a final concentration of 0.2mM. Fermentation was carried out at 30℃, 300-500rpm, and an aeration rate of 4V / V / min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), the automatic feeding was started. Feeding medium was added until the pH value was lower than 6.80, and then feeding was stopped. The culture was carried out for 70-81h to obtain a fermentation broth containing β-alanine.

[0018] Fermentation medium formulation in a 5-L fermenter: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 12 A 0.2 mg / L, 1 mL / L trace element solution, with deionized water as the solvent; the composition of the trace element solution is: 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, with deionized water as the solvent;

[0019] The fed culture medium composition is as follows: glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, VB1 0.5 mg / L, VB2 0.2 mg / L. 12 The solvent is water, and the pH is adjusted to 6.8 with 50% ammonia.

[0020] The feeding rate is 25 mL / h, and the total amount of feeding medium added is 1700 mL / 2 L.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] This invention screened and obtained the rhamnose transporter RhaT, which can increase β-alanine production. Overexpression of the rhamnose transporter rhaT gene resulted in a β-alanine yield of 1.85 g / L in shake-flask fermentation and a β-alanine content of 73.11 g / L in fermenter. Compared to the control strain, the β-alanine yield in shake-flask fermentation was increased by 7.56%. Future research could combine protein structure optimization or directed evolution strategies to suppress or knock out serine and threonine expression genes in the competitive pathway of β-alanine production, further increasing β-alanine yield. (iv) Description of the attached drawings

[0023] Figure 1 Biomass OD for strains E45, E52, E53, E54, E55, E56, E57, E58, E59, E60, E61 and E62 600 A bar chart showing the concentration of β-alanine.

[0024] Figure 2 Biomass OD of strain E54 in a 5-L feed fermenter 600 Concentration curves of residual sugar and β-alanine. (V) Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0026] Probiotic Escherichia coli Nissle 1917, i.e. wild strain E0, was purchased from Baosai Biotechnology, product number T0230.

[0027] In this embodiment of the invention, probiotic E. coli E45 (E0, Trc-panD, Trc-aspC, Trc-sdhC, P) is used. fumA ::P flgC-ATG-fumA The starting strain is Trc-ppc, ΔcycA.

[0028] All heterologous genes in this invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0029] plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG It was constructed with reference to patent application CN202411690856.6.

[0030] LB plate composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, water as solvent, natural pH value.

[0031] LB medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0032] 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, CaCO3 15 g / L, VB1 0.5 mg / L, VB2 0.2 mg / L. 12 A 1 mL / L trace element solution, with water as the solvent, does not require pH adjustment; 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, with deionized water as the solvent.

[0033] Table 1 Primers used in gene editing

[0034]

[0035]

[0036] SEQ ID NO.3(rhaT)

[0037]

[0038] Example 1: Construction of strain E52

[0039] β-Alanine synthesis is influenced by cellular metabolism and transport proteins. As β-alanine gradually accumulates and the strain's synthesis rate increases, the intracellular concentration of β-alanine remains higher than the extracellular concentration. Excessively high intracellular concentrations can inhibit cell growth. The inability to timely transport intracellular β-alanine to the extracellular space is another important factor limiting β-alanine production. Enhancing the efflux system of the target product can also increase product yield. Studies have reported that Ncgl0580 in *Corynebacterium glutamicum* is an efflux protein that promotes the transmembrane transport of β-alanine, and Ncgl0580 was initially identified for the efflux of L-serine. Therefore, this invention first introduces a codon-optimized Ncgl0580 gene from *Corynebacterium glutamicum*, resulting in strain E52.

[0040] 1. Construction of strains

[0041] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG Ncgl0580, derived from Corynebacterium glutamicum, has been expressed with codon-optimized expression. CG Genes (nucleotide sequences as shown in SEQ ID No. 1) were used to construct strain E52 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -Ncgl0580 CG ).

[0042] (1) Construction of pTrc99a-line plasmid: using plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG Using pTrc99a-line-F / pTrc99a-line-R as primers, the linearized plasmid fragment was amplified by PCR. After adding DpnI to the PCR product, the methylated vector was digested at 37°C. The product was then purified using a purification kit to obtain the linearized plasmid pTrc99a-panD. BS -aspB CG -aspA-pycCG -line.

[0043] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -Ncgl0580 CG Plasmid: Using a plasmid containing Ncgl0580 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, Ncgl0580... CG -F / Ncgl0580 CG -R is the primer that amplifies Ncgl0580 CG The fragment (nucleotide sequence as shown in SEQ ID NO.1) was purified and then cloned into the linearized vector pTrc99a-panD of step (1) in one step. BS -aspB CG -aspA-pyc CG Transformed into E. coli DH5α competent cells, the cells were plated onto LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12 h. Colony PCR was performed using pTrc99a-YZ-F / pTrc99a-YZ-R primers for verification, and sequencing confirmed the successful construction of a cell line containing pTrc99a-panD. BS -aspB CG -aspA-pyc CG -Ncgl0580 CG The strain containing the plasmid.

[0044] (3) Transformation plasmid: Extract the constructed pTrc99a-panD BS -aspB CG -aspA-pyc CG -Ncgl0580 CG The plasmid was transformed into competent cells of the plasmid-free E45 strain. The cells were plated onto LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12 h. Strains E52 (Trc-panD, Trc-aspC, Trc-sdhC, P) were obtained through screening. fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -Ncgl0580 CG ).

[0045] 2. Performance testing of strains in shake-flask fermentation

[0046] Single colonies of strain E52 were picked and cultured overnight at 37°C and 180 rpm in LB broth containing 50 mg / L kanamycin to obtain a seed culture. 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and 50 mg / L kanamycin, and cultured at 30°C and 180 rpm until the bacterial growth reached OD. 600 =0.5, add 0.25mM IPTG to a final concentration, and incubate at 30℃ and 180rpm for 48h with shaking. After centrifugation, the supernatant was analyzed using a spectrophotometer to measure the optical density (OD) at 600nm. 600 (i.e., biomass), and a separate sample of the supernatant was used to detect the β-alanine content, with strain E45 as a control. Figure 1 As shown, the biomass OD of strain E45 600 The OD value of strain E52 was 11.2, and the β-alanine content was 1.71 g / L; the biomass OD of strain E52 was... 600 The value was 12.03, and the β-alanine content was 1.71 g / L, indicating that the β-alanine production did not increase.

[0047] 3. Determination of β-alanine concentration:

[0048] Preparation of 1% 2,4-dinitrofluorobenzene: Dissolve 1 mL of 2,4-dinitrofluorobenzene in 99 mL of acetonitrile.

[0049] Preparation of 0.5M NaHCO3 solution: Dissolve 21g of NaHCO3 in 500mL of deionized water.

[0050] 0.2M PB buffer: Weigh 8.74g Na2HPO4·12H2O and 2.43g Na2HPO4·2H2O and dissolve them in 200mL of deionized water. Store the solution for later use.

[0051] Sample preparation: Dilute the sample with ultrapure water to between 0.1-1 g / L.

[0052] Reaction conditions: Take 100 μL of diluted sample, 100 μL of 0.5 M NaHCO3 solution and 100 μL of 1% 2,4-dinitrofluorobenzene respectively, incubate at 60℃ for 75 min, and finally add 700 μL of 0.2 M PB buffer and mix well. Filter through a membrane (polyvinylidene fluoride, organic membrane, 0.22 μm) for later use.

[0053] Detection conditions: HPLC model: Thermo Scientific Ultimate 3000, HPLC detection wavelength: 360 nm. β-alanine was separated using a gradient elution program. Mobile phase A consisted of methanol:acetonitrile:ultrapure water = 45:45:10 (v:v:v); mobile phase B consisted of 10 mM potassium dihydrogen phosphate, with pH adjusted to 7.0 using KOH. The elution program is as follows: 0-2.5 min: 10% A, 90% B; 2.5-2.6 min: A 10%→14%, B 90%→86%; 2.6-13 min: A 14%→34%, B 86%→66%; 13-13.1 min: A 34%→38%, B 66%→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%→14%, B 90%→86%.

[0054] Example 2: Construction of strain E53

[0055] Since Ncgl0580 belongs to the drug / metabolite (DMT) superfamily of transporters, it is a transporter that can encode a range of metabolites, including amino acids. Sequence alignment revealed that the cysteine / homoserine transporter (encoded by the eamA gene) in EcN is a homolog of Ncgl0580. Studies have reported that overexpression of the eamA gene can enhance L-cysteine ​​and L-homoserine production, and it also belongs to the EamA / RhaT transporter family within the DMT superfamily. Therefore, this embodiment selected overexpression of the eamA gene.

[0056] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The eamA gene (nucleotide sequence shown in SEQ ID No. 2) was overexpressed to construct strain E53 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -eamA).

[0057] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG-line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0058] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -eamA plasmid: Using the E0 genome of strain E0 as a template, the eamA fragment (nucleotide sequence as shown in SEQ ID No. 2) was amplified using eamA-F / eamA-R primers. The eamA fragment was then amplified with the linearized plasmid pTrc99a-panD from step (1) using the method in step (2) of Example 1. BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -eamA plasmid.

[0059] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -eamA was transformed into competent cells of E45 plasmid-free strain to construct strain E53 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -eamA).

[0060] (4) Using strain E45 as the control strain, strain E53 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E53 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E53 was [missing value]. 600 The value was 11.4, and the β-alanine content was 1.66 g / L. Compared with the control, overexpression of the eamA gene did not increase the yield of β-alanine.

[0061] The EamA / RhaT transporter family includes the following nine transporters: (1) Rhamnose: The H+ cotransporter (RhaT, encoded by the rhaT gene) is the only rhamnose transporter in E. coli; (2) Aromatic amino acid transporter (encoded by the yddG gene). Studies have reported that overexpression of the yddG gene not only promotes the efflux of L-tyrosine and L-tryptophan, but also promotes the efflux of L-phenylalanine and other amino acids, making it a broadly specific amino acid exporting protein; (3) L-threonine / L-homoserine transporter (encoded by the rhtA gene). Overexpression of this gene can promote the efflux of various amino acid analogs such as L-threonine and L-homoserine. Increasing the expression level of the rhtA gene can improve the cell's tolerance to β-alanine, indicating that the rhtA gene may be involved in β-alanine transport; (4) Cystine transporter (encoded by the yijE gene), which encodes an inner membrane protein mainly used for cystine transport. (5) Biotin transporter (encoded by the bioP gene), overexpression of which led to increased biotin in the wild-type strain. There were also four other unidentified members of the EamA / RhaT transporter family: (6) yedA, (7) yhbE, (8) yicL, and (9) ytfF. The following examples examined the effects of these nine transporters on β-alanine production.

[0062] Example 3: Construction of strain E54

[0063] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The rhaT gene (nucleotide sequence shown in SEQ ID No. 3) was overexpressed, and strain E54 (Trc-panD, Trc-aspC, Trc-sdhC, P) was constructed. fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT).

[0064] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0065] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -rhaT plasmid: Using the E0 genome of strain as a template, the rhaT fragment (nucleotide sequence as shown in SEQ ID No. 3) was amplified using rhaT-F / rhaT-R primers. The rhaT fragment was then amplified with the linearized plasmid pTrc99a-panD from step (1) using the method in step (2) of Example 1. BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT plasmid.

[0066] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT was transformed into competent cells of E45 plasmid-free strain to construct strain E54 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT).

[0067] (4) Using strain E45 as the control strain, strain E54 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E54 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E54 was [missing value]. 600 The value was 11.5, and the content of β-alanine was 1.85 g / L.

[0068] Example 4: Construction of strain E55

[0069] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CGThe yddG gene (nucleotide sequence shown in SEQ ID No. 4) was overexpressed to construct strain E55 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yddG).

[0070] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0071] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -yddG plasmid: Using the E0 genome of strain as a template, the yddG fragment was amplified using yddG-F / yddG-R primers. The yddG fragment was then amplified using the method in step (2) of Example 1, and the yddG fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -yddG plasmid.

[0072] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -yddG was transformed into competent cells of E45 plasmid-free strain to construct strain E55(Trc-panD,Trc-aspC,Trc-sdhC,P) fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG-yddG).

[0073] (4) Using strain E45 as the control strain, strain E55 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E55 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD value of strain E55 was [missing value]. 600 The value was 11.3, and the content of β-alanine was 1.59 g / L.

[0074] Example 5: Construction of strain E56

[0075] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The rhtA gene (nucleotide sequence shown in SEQ ID No. 5) was overexpressed to construct strain E56 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhtA).

[0076] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0077] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -rhtA plasmid: Using the E0 genome of strain as a template, the rhtA fragment was amplified using rhtA-F / rhtA-R primers. The rhtA fragment was then amplified using the method in step (2) of Example 1, and the rhtA fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panDBS -aspB CG -aspA-pyc CG -rhtA plasmid.

[0078] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhtA was transformed into competent cells of E45 plasmid-free strain to construct strain E56 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhtA).

[0079] (4) Using strain E45 as the control strain, strain E56 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E56 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E56 was [missing value]. 600 The value was 11.2, and the content of β-alanine was 1.64 g / L.

[0080] Example 6: Construction of strain E57

[0081] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The yiiE gene (nucleotide sequence shown in SEQ ID No. 6) was overexpressed to construct strain E57 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA /

[0082] pTrc99a-panD BS -aspB CG -aspA-pyc CG -yiiE).

[0083] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG-line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0084] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -yiiE plasmid: Using the E0 genome of strain as a template, the yiiE fragment was amplified using yiiE-F / yiiE-R primers. The yiiE fragment was then amplified using the method in step (2) of Example 1, and the yiiE fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -yiiE plasmid.

[0085] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -yiiE was transformed into competent cells of E45 plasmid-free strain to construct strain E57 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yiiE).

[0086] (4) Using strain E45 as the control strain, strain E57 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E57 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E57 was [missing value]. 600 The value was 10.7, and the content of β-alanine was 1.64 g / L.

[0087] Example 7: Construction of strain E58

[0088] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The bioP gene (nucleotide sequence shown in SEQ ID No. 7) was overexpressed to construct strain E58 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -bioP).

[0089] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0090] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -bioP plasmid: Using the E0 genome of strain as a template, the bioP fragment was amplified using bioP-F / bioP-R primers. The bioP fragment was then amplified using the method in step (2) of Example 1, and the bioP fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -bioP plasmid.

[0091] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -bioP was transformed into competent cells of E45 plasmid-free strain to construct strain E58 (Trc-panD,Trc-aspC,Trc-sdhC,P) fumA ::P flgC-ATG-fumA,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -bioP).

[0092] (4) Using strain E45 as the control strain, strain E58 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E58 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E58 was [missing value]. 600 The value was 11.8, and the content of β-alanine was 1.70 g / L.

[0093] Example 8: Construction of strain E59

[0094] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The yedA gene (nucleotide sequence shown in SEQ ID No. 8) was overexpressed to construct strain E59 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yedA).

[0095] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0096] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -yedA plasmid: Using the E0 genome of strain as a template, the yedA fragment was amplified using yedA-F / yedA-R primers. The yedA fragment was then amplified using the method in step (2) of Example 1, and the yedA fragment was combined with the linearized plasmid pTrc99a-panD from step (1).BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -yedA plasmid.

[0097] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -yedA was transformed into competent cells of E45 plasmid-free strain to construct strain E59 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yedA).

[0098] (4) Using strain E45 as the control strain, strain E59 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E59 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E59 was [missing value]. 600 The value was 12.3, and the content of β-alanine was 1.58 g / L.

[0099] Example 9: Construction of strain E60

[0100] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The yhbE gene (nucleotide sequence shown in SEQ ID No. 9) was overexpressed to construct strain E60 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yhbE).

[0101] (1) Construct pTrc99a-panD BS-aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0102] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -yhbE plasmid: Using the E0 genome of strain as a template, the yhbE fragment was amplified using yhbE-F / yhbE-R primers. The yhbE fragment was then amplified using the method in step (2) of Example 1, and the yhbE fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -yhbE plasmid.

[0103] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -yhbE was transformed into competent cells of E45 plasmid-free strain to construct strain E60 (Trc-panD,Trc-aspC,Trc-sdhC,P fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yhbE).

[0104] (4) Using strain E45 as the control strain, strain E60 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E60 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E60 was [missing value]. 600 The value was 10.7, and the content of β-alanine was 1.67 g / L.

[0105] Example 10: Construction of strain E61

[0106] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The yicL gene (nucleotide sequence shown in SEQ ID No. 10) was overexpressed to construct strain E61 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yicL).

[0107] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0108] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG -yicL plasmid: Using the E0 genome of strain as a template, the yicL fragment was amplified using yicL-F / yicL-R primers. The yicL fragment was then amplified using the method in step (2) of Example 1, and the yicL fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -yicL plasmid.

[0109] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -yicL was transformed into competent cells of E45 plasmid-free strain to construct strain E61(Trc-panD,Trc-aspC,Trc-sdhC,P)fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -yicL).

[0110] (4) Using strain E45 as the control strain, strain E61 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E61 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E61 was [missing value]. 600 The value was 10.5, and the content of β-alanine was 1.63 g / L.

[0111] Example 11: Construction of strain E62

[0112] In plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG The ytfF gene (nucleotide sequence shown in SEQ ID No. 11) was overexpressed to construct strain E62 (Trc-panD, Trc-aspC, Trc-sdhC, P). fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -ytfF).

[0113] (1) Construct pTrc99a-panD BS -aspB CG -aspA-pyc CG -line plasmid: The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 1. BS -aspB CG -aspA-pyc CG -line.

[0114] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA-pyc CG-ytfF plasmid: Using the E0 genome of strain as a template, the ytfF fragment was amplified using ytfF-F / ytfF-R primers. The ytfF fragment was then amplified using the method in step (2) of Example 1, and the ytfF fragment was combined with the linearized plasmid pTrc99a-panD from step (1). BS -aspB CG -aspA-pyc CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA-pyc CG -ytfF plasmid.

[0115] (3) Using the method in step (3) of Example 1, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -ytfF was transformed into competent cells of E45 plasmid-free strain to construct strain E62(Trc-panD,Trc-aspC,Trc-sdhC,P) fumA ::P flgC-ATG-fumA ,Trc-ppc,ΔcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG -ytfF).

[0116] (4) Using strain E45 as the control strain, strain E62 was subjected to shake-flask fermentation according to the method in Example 1, and the content of β-alanine and biomass OD were detected. 600 ,like Figure 1 As shown, the biomass OD of control strain E45 600 The OD value of strain E62 was 11.2, the β-alanine content was 1.71 g / L, and the biomass OD of strain E62 was [missing value]. 600 The value was 11.7, and the content of β-alanine was 1.57 g / L.

[0117] The above examples demonstrate that overexpression of the rhamnose:H+ cotransporter rhaT gene, i.e. strain E54, can increase β-alanine production. Overexpression of the other nine transporters mostly decreases β-alanine production. However, this does not mean that these genes are unrelated to β-alanine efflux, because some of these transporters are efflux proteins of serine and threonine. Overexpression of related genes will lead to enhanced efflux of these two amino acids. The synthesis pathways of serine and threonine are competitive branches of β-alanine and will compete with β-alanine for the same precursor.

[0118] Example 12: Fermentation of strain E54

[0119] Strain E54 was inoculated onto LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 37°C. Single colonies were picked and transferred to LB tubes containing 50 mg / L kanamycin resistance, and incubated overnight at 37°C and 150-180 rpm to prepare the primary seed culture. The primary seed culture was inoculated at a volume concentration of 5% into LB medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 180 rpm to obtain the secondary seed culture. The secondary seed culture was then inoculated at a volume concentration of 15% into a 5-L fermenter containing 2 L of fermentation medium containing 50 mg / L kanamycin. IPTG was added to a final concentration of 0.2 mM, and fermentation was carried out at 30°C, 500 rpm, and an aeration rate of 4 v / v / min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), automatic feeding was activated, and feeding medium was added until the pH was lower than 6.80, at which point feeding was stopped. The feeding rate was 25 mL / h, and the total amount of feeding medium added was 1700 mL / 2 L. Fermentation was carried out for a total of 75 h, and a fermentation broth containing β-alanine was obtained. The OD value was 75.02, and the β-alanine content was 73.11 g / L, as determined by the method in Example 1. The fermentation process is shown in [link to example]. Figure 2 As shown.

[0120] Fermentation medium formulation in a 5-L fermenter: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 12 A 0.2 mg / L, 1 mL / L trace element solution, with deionized water as the solvent; the composition of the trace element solution is: 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, with deionized water as the solvent;

[0121] The fed-batch culture medium composition is as follows: glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, VB1 0.5 mg / L, VB2 0.2 mg / L. 12 The solvent is water, and the pH is adjusted to 6.8 with 50% ammonia.

Claims

1. The application of a transporter protein in increasing β-alanine production, characterized in that, The transporter protein is the rhamnose transporter RhaT.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the transporter protein is shown in SEQ ID NO.

3.

3. A probiotic Escherichia coli constructed from the transporter protein of claim 1, characterized in that, The probiotic E. coli used strain E45 as the starting strain and was incorporated into plasmid pTrc99a-panD. BS -aspB CG -aspA-pyc CG The rhaT gene derived from E. coli Nissle 1917 was overexpressed; strain E45 is E. coli Nissle 1917 / Trc-panD / Trc-aspC / Trc-sdhC / P fumA ::P flgC-ATG-fumA / Trc-ppc / ΔcycA.

4. The probiotic Escherichia coli as described in claim 3, characterized in that, The probiotic E. coli was constructed as follows: in plasmid pTrc99a-panD... BS -aspB CG -aspA-pyc CG The rhaT gene, expressed via codon-optimized plasmid pTrc99a-panD, was expressed. BS -aspB CG -aspA-pyc CG Using this as a template, the linearized plasmid pTrc99a-panD was obtained. BS -aspB CG -aspA-pyc CG -line; Using the E. coli Nissle 1917 genome as a template, the rhaT gene fragment was obtained; the linearized plasmid pTrc99a-panD was then used to obtain the rhaT gene fragment. BS -aspB CG -aspA-pyc CG -line was ligated with the rhaT gene fragment to construct plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG -rhaT was transformed into E45 competent cells to construct probiotic Escherichia coli.

5. A method for preparing β-alanine by probiotic Escherichia coli microbial fermentation as described in claim 3, characterized in that, The method is as follows: The probiotic *E. coli* is inoculated into a fermentation medium containing 50 mg / L kanamycin, and the cells are cultured at 30°C and 180 rpm until the OD value reaches [value missing]. 600 =0.5, at this point, add IPTG to a final concentration of 0.25mM to induce gene expression, continue culturing for 48h until the end of fermentation, and obtain a fermentation broth containing β-alanine. Separate and purify the fermentation broth to obtain β-alanine; The fermentation medium formulation is as follows: glucose 18–22 g / L, (NH4)2SO4 14–18 g / L, yeast extract 2–3 g / L, KH2PO4 1–2 g / L, MgSO4 0.2–1 g / L, CaCO3 13–17 g / L, VB1 0.3–0.5 mg / L, VB 12 0.3–0.5 mg / L, trace element solution 0.5–1.5 mL / L, solvent is water; the components of the trace element solution are as follows: 8–12 g / L CaCl2, 8–12 g / L FeSO4·7H2O, 0.8–1.2 g / L ZnSO4·7H2O, 0.1–0.3 g / L CuSO4, 0.01–0.04 g / L NiCl2·7H2O, solvent is deionized water.

6. The method as described in claim 5, characterized in that, Before fermentation, the Escherichia coli probiotics are inoculated into LB medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 180 rpm to prepare a seed culture. The seed culture is then inoculated into fermentation medium containing 50 mg / L kanamycin at a volume concentration of 5%.

7. The method as described in claim 5, characterized in that, The fermentation is carried out in a fermenter: the *E. coli* probiotics are inoculated onto LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 37°C. Single colonies are picked and transferred to LB test tubes containing 50 mg / L kanamycin resistance and incubated overnight at 37°C and 150-180 rpm to prepare the primary seed culture. The primary seed culture is inoculated at a volume concentration of 5% into LB medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 150-180 rpm to obtain the secondary seed culture. The secondary seed culture was inoculated at a volume concentration of 15% into a 5-L fermenter containing 2L of fermentation medium with 50mg / L kanamycin, and IPTG was added to a final concentration of 0.2mM. Fermentation was carried out at 30℃, 300-500rpm, and an aeration rate of 4V / V / min. When the pH value was higher than 6.80, the automatic feeding was turned on. 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 70-81h to obtain a fermentation broth containing β-alanine. Fermentation medium formulation in a 5-L fermenter: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 12 A 0.2 mg / L, 1 mL / L trace element solution, with deionized water as the solvent; the composition of the trace element solution is: 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, with deionized water as the solvent; The fed culture medium composition is as follows: glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, VB1 0.5 mg / L, VB2 0.2 mg / L. 12 The solvent is water, and the pH is adjusted to 6.8 with 50% ammonia.

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