CN117778289A

CN117778289A - Recombinant genetically engineered bacterium for high yield of beta-alanine and application thereof

Info

Publication number: CN117778289A
Application number: CN202410005378.4A
Authority: CN
Inventors: 柳志强; 张义峰; 周海岩; 张昔; 龚子怡; 胡忠策; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2024-01-03
Filing date: 2024-01-03
Publication date: 2024-03-29

Abstract

The invention discloses a recombinant genetic engineering bacterium for high-yield beta-alanine and application thereof, wherein the recombinant genetic engineering bacterium takes escherichia coli B1 as an initial strain to carry out one or more of the following gene edits: knocking out pck gene, knocking out mdh gene and knocking out aspAGene, replacement poxB gene as EsaR gene, replacement ldhA gene as EsaI gene using artificial promoter Pbs1 to replace original promoter, replacement gltA gene promoter as P _esaS A promoter; in the fed-batch fermentation of the recombinant genetically engineered bacterium, the beta-alanine yield of the strain B8 reaches 111.86g/L at the level of the 5-L fermentation tank, and compared with the original strain B1, the beta-alanine yield of the recombinant genetically engineered bacterium is improved by 66.7%, and the sugar acid conversion rate reaches 37.8%.

Description

Recombinant genetically engineered bacterium for high yield of beta-alanine and application thereof

Field of the art

The invention relates to recombinant genetically engineered bacteria for high-yield beta-alanine and application thereof.

(II) background art

Beta-alanine, also known as 3-aminopropionic acid, is in nature the only beta-type amino acid, which is a protein amino acid, and although not involved in the synthesis of enzymes and proteins, has important physiological functions during the growth of organisms. In cellular metabolism, beta-alanine can be used as a precursor to synthesize pantothenic acid (vitamin B5) and coenzyme a (CoA), which are involved in various metabolic activities such as fatty acid metabolism, sugar metabolism, and the like. Plants and microorganisms can autonomously synthesize beta-alanine, while mammals require exogenous supplementation. According to the research at present, the beta-alanine has important application value in various fields such as food, medicine, chemical industry, feed and the like.

In the medical field, beta-alanine is a key precursor for the production of pantothenic acid, calcium pantothenate, coenzyme a (CoA), and Acyl Carrier Protein (ACP), and plays an important role in helping cell metabolism and development of the central nervous system. Meanwhile, the beta-alanine can also be used for synthesizing medicaments such as balsalazide and pamidronate disodium. In the food field, beta-alanine is used as a food additive, often in food flavors, to improve food flavor. Meanwhile, the compound is also commonly used as a food preservative due to the antioxidant property. According to the related clinical study, the beta-alanine can also be used as a nutritional supplement for athletes, and the human body can obviously improve fatigue generated by long-time high-strength exercise and improve the tolerance of the human body after the human body is supplemented with the beta-alanine. In the chemical industry, beta-alanine can be used as precursor to produce pantothenic acid and calcium pantothenate, poly 3-hydroxy propionate and poly beta-alanine, and can be used as precipitant in medicament, electroplating corrosion inhibitor and lead poisoning antidote. In addition, beta-alanine also plays an important role in the fields of environment, cosmetics and the like. In the field of feeds, beta-alanine is increasingly applied to animal feeding, and research shows that the addition of beta-alanine into the feeds can improve the utilization of the feeds by livestock and poultry and reduce the intake of the feeds; meanwhile, the antioxidation capability of the muscle can be improved, the content of the myogenic active peptide can be increased, and the meat quality can be effectively improved.

At present, the main production methods of beta-alanine at home and abroad are as follows: the chemical synthesis process is to synthesize beta-alanine with acrylonitrile, acrylic acid, beta-aminopropionitrile and other nitrile matter under the conditions of high pressure, high temperature, strong acid and strong alkali. However, the chemical synthesis method has high energy consumption and high equipment requirement, and generates substances harmful to the environment and human bodies in the production process, and the generated byproducts can cause great difficulty in subsequent separation and purification; the biological enzyme catalysis method uses aspartic acid as a substrate, and uses aspartic acid decarboxylase from strains such as bacillus subtilis or corynebacterium glutamicum to express and catalyze the generation of beta-alanine in escherichia coli. The method has milder conditions, safety and less pollution, and draws attention of more students; the microbial fermentation method is to modify the metabolic process of the target product beta-alanine by utilizing technologies such as synthetic biology, system metabolic engineering, protein engineering, transcriptome and metabolome and the like, so that more metabolic flows flow to the direction of the target product. With the increasing severity of climate change and environmental problems, the exploration of clean, environment-friendly and low-energy production methods by using inexpensive glucose and the like as carbon sources has attracted attention of a plurality of students.

With the development of biotechnology, the development of the system metabolic engineering technology makes human beings not limited by the traditional strain screening technologies such as mutagenesis, directed evolution and the like, and the production efficiency of target metabolites can be improved to the maximum extent and the production performance of the strain can be greatly improved by quantitatively analyzing the metabolic pathways and metabolic flux of the strain and carrying out purposeful metabolic pathway modification on the basis. Unlike traditional breeding techniques, it is an effective method for achieving improved genetic traits in organisms by rational design of cellular metabolic pathways and reconstruction of metabolic networks.

In the current research hot spot, engineering strains are generally constructed by taking strains such as escherichia coli, saccharomycetes, corynebacterium glutamicum, bacillus subtilis and the like as chassis strains, and the metabolic genetic background and the physiological characteristics of the chassis strains are clear. The combination of mature gene editing technology and reasonable design of metabolic flux can greatly accelerate the transformation progress of metabolic pathways of the strain, and shorten the time for achieving the optimal production performance of the strain. As prokaryotic bacteria with the most clear research at present, the escherichia coli has the characteristics of rapid propagation, short fermentation period, high target gene expression level, mature and complete expression system, biological safety and the like, and is widely applied to industrial production of various biological products at present.

De novo synthesis pathways for beta-alanine in E.coli include glucose uptake, glycolysis pathways, TCA cycle, L-aspartic acid synthesis and beta-alanine synthesis pathways. Firstly, glucose is phosphorylated under PTS or non PTS system to synthesize glucose-6-phosphate, carbon flow enters glycolysis path, beta-alanine precursor phosphoenolpyruvate is synthesized in glycolysis path, phosphoenolpyruvate carboxylase is catalyzed to produce oxaloacetate under phosphoenolpyruvate, wherein oxaloacetate is unstable and easy to decompose, aspartic acid is generated under aspartate aminotransferase catalysis, and beta-alanine is generated under aspartate decarboxylase catalysis. Among them, L-lysine, L-threonine, O-succinylhomoserine and homoserine belong to the amino acids of the aspartic acid family, and they share the precursor L-aspartic acid with beta-alanine, so attenuation of the competitive branch is an effective construction strategy. But there is still a need to explore the bottleneck that affects β -alanine production.

(III) summary of the invention

The invention aims to provide a recombinant genetic engineering bacterium for high-yield beta-alanine and application thereof in preparing beta-alanine by a microbial fermentation method, which lays a foundation for industrialized production of beta-alanine.

The technical scheme adopted by the invention is as follows:

the invention provides a recombinant genetically engineered bacterium for high-yield beta-alanine, which takes escherichia coli B1 as an initial strain to edit one or more of the following genes: the pck gene is knocked out, the mdh gene is knocked out, the aspA gene is knocked out, the poxB gene is replaced by the EsaR gene, the ldhA gene is replaced by the EsaI gene of which the original promoter is replaced by the artificial promoter Pbs1, and the gltA gene promoter is replaced by P _esaS A promoter; the starting strain B1: e.coli W3110TrcpanDTrcppcΔpykA ΔcycA/pTrc99a-panD _BS ^K104S -aspB _CG aspA, construction reference 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-aspalate- α -decarboxylase for fermentative production of β -alanine in Escherichia coli.

Further, the nucleotide sequence of the pck gene is shown as SEQ ID NO. 1; the nucleotide sequence of the mdh gene is shown as SEQ ID NO. 2; the nucleotide sequence of the aspA gene is shown as SEQ ID NO. 3; the nucleotide sequence of the poxB gene is shown as SEQ ID NO. 4; the nucleotide sequence of the EsaR gene is shown as SEQ ID NO. 5; the nucleotide sequence of the ldhA gene is shown in SEQ ID NO. 6; the nucleotide sequence of the Pbs1 promoter is shown in SEQ ID NO. 7; the nucleotide sequence of the EsaI gene is shown as SEQ ID NO. 8; the nucleotide sequence of the gltA gene is shown as SEQ ID NO. 9; the P is _esaS The nucleotide sequence of the promoter is shown as SEQ ID NO. 10.

Furthermore, the recombinant genetically engineered bacterium takes escherichia coli B1 as an initial strain, and the following gene editing is sequentially carried out: the pck gene is knocked out, the mdh gene is knocked out, the aspA gene is knocked out, the poxB gene is replaced by the EsaR gene, the ldhA gene is replaced by the EsaI gene of which the original promoter is replaced by the artificial promoter Pbs1, and the gltA gene promoter is replaced by P _esaS A promoter.

The recombinant genetically engineered bacterium disclosed by the invention is constructed according to the following method:

(1) Knocking out pck gene: the genome of E.coli W3110 was used as a template, and L-p was used as a template, respectivelyThe ck-F/L-pck-R and the R-pck-F/R-pck-R are used as primers to amplify upstream and downstream homology arms of pck genes, and the donor DNA is obtained through fusion; amplifying plasmids by taking pTarget plasmid as a template and pTarget-pck-F and pTarget-pck-R as primers and linearizing to obtain linearized pTarget-pck plasmid, connecting donorDNA and the linearized pTarget-pck plasmid and then converting into a starting strain B1 electrotransformation competent cell transferred into pCas9 plasmid, eliminating pTarget and pCas9 plasmids, and constructing a strain E.coli W3110Trc-ppcTrc-panDΔpykA ΔcycA Δpck/pTrc99a-panD _BS ^K104S -aspB _CG aspA, designated as strain B2.

(2) Knocking out mdh gene: amplifying the upstream and downstream homology arms of the mdh gene by taking the genome of E.coli W3110 as a template, respectively taking L-mdh-F/L-mdh-R and R-mdh-F/R-mdh-R as primers, and fusing to obtain the donor DNA; amplifying the plasmid by taking the pTarget plasmid as a template and taking the pTarget-mdh-F and the pTarget-mdh-R as primers and carrying out linearization treatment to obtain a linearization pTarget-mdh plasmid; ligation of the donarDNA and linearized pTarget-mdh plasmid and transformation into the pCas9 plasmid-transferred strain B2 electrotransformation competent cells, elimination of pTarget and pCas9 plasmids, construction of the strain E.coli W3110Trc-ppcTrc-panDΔpykA ΔcycApckΔmdh/pTrc99a-panD _BS ^K104S -aspB _CG aspA is designated as strain B3.

(3) Knocking out aspA gene: taking the genome of E.coli W3110 as a template, respectively taking L-aspA-F/L-aspA-R and R-aspA-F/R-aspA-R as primers, amplifying upstream and downstream homology arms of aspA genes, and fusing to obtain donor DNA; amplifying the plasmid by taking the pTarget plasmid as a template and taking the pTarget-aspA-F and the pTarget-aspA-R as primers and carrying out linearization treatment to obtain a linearization pTarget-aspA plasmid; ligation of the donarDNA and linearized pTarget-aspA plasmid and transformation into the pCas9 plasmid-transferred strain B3 electrotransformation competent cells, elimination of pTarget and pCas9 plasmids, construction of the strain E.coli W3110Trc-ppcTrc-panD ΔpykA ΔcycA Δpck Δmdh ΔaspA/pTrc99a-panD _BS ^K104S -aspB _CG Designated as strain B4.

(4) The replacement poxB gene is the EsaR gene: amplifying the upstream and downstream homology arms of the poxB gene by using the genome of E.coli W3110 as a template, L-poxB-F/L-poxB-R and R-poxB-F/R-poxB-R as primers, and esaR-F/esaR-R as primerssaR gene, fusing to obtain donor DNA; amplifying the plasmid by taking the pTarget plasmid as a template and taking the pTarget-poxB-F and the pTarget-poxB-R as primers and carrying out linearization treatment to obtain a linearization pTarget-poxB plasmid; the E.coli W3110Trc-ppcTrc-panD ΔpykA ΔcycA ΔpckΔmdh ΔaspA ΔpoxB:: esaR/pTrc99a-panD was constructed by ligating the donarDNA with the linearized pTarget-poxB plasmid and then transforming the strain B4 electrotransformation competent cells into the pCas9 plasmid, eliminating the pTarget and pCas9 plasmids _BS ^K104S -aspB _CG Designated as strain B5.

(5) Replacement of the ldhA Gene was the esaI gene whose promoter was pbs 1: the genome of E.coli W3110 is used as a template, L-ldhA-F/ldhA-FR-Pbs1 and Pbs1-esaI-F/esaI-R and R-ldhA-F/R-ldhA-R are respectively used as primers, an upstream and downstream homology arm of the ldhA gene and esaI gene containing a promoter Pbs1 are amplified, and the donor DNA is obtained through fusion; amplifying the plasmid by taking the pTarget plasmid as a template and taking the pTarget-ldhA-F and the pTarget-ldhA-R as primers and linearizing to obtain a linearized pTarget-ldhA plasmid; the dororDNA was ligated to the linearized pTarget-ldhA plasmid and then transformed into pCas9 plasmid-transferred strain B5 electrotransformation competent cells, pTarget and pCas9 plasmids were deleted and the strain E.coli W3110Trc-ppcTrc-panD ΔpykA ΔcycA Δpck Δmdh ΔaspA ΔpoxB::: esaR ΔldhA::: pbs1-EsaI/pTrc99a-panD was constructed _BS ^K104S -aspB _CG Designated as strain B6.

(6) Replacement of the gltA Gene promoter to P _esaS : amplifying upstream and downstream homology arms of the original promoter of the gltA gene by using the genome of E.coli W3110 as a template and using L-gltA-F/L-gltA-R and R-gltA-F/R-gltA-R as primers respectively; with P _esaS Plasmid of promoter gene is used as template, P is used as template _esaS -F/P _esaS Amplification of R as primer to obtain P _esaS Fragments; fusing a homology arm and a promoter fragment to obtain a donor DNA; amplifying the plasmid by taking the pTarget plasmid as a template and taking the pTarget-gltA-F and the pTarget-gltA-R as primers and carrying out linearization treatment to obtain a linearization pTarget-gltA plasmid; the dororDNA and linearized pTarget-gltA plasmids were ligated and transformed into strain B6 electrotransformation competent cells transformed into pCas9 plasmid, pTarget and pCas9 plasmids were deleted and strain E.coli W3110Trc-ppcTrc-panD ΔpykA ΔcycA Δpck Δmdh ΔaspA ΔpoxB:: esaR ΔldhA:: pbs1-EsaI ΔgltA was constructed::P _esaS -gltA/pTrc99a-panD _BS ^K104S -aspB _CG designated as strain B7.

(7) Substitution Strain B7 plasmid pTrc99a-panD _BS ^K104S -aspB _CG Substitution with the original Strain B1 plasmid pTrc99a-panD _BS ^K104S -aspB _CG Construction of the strain E.coli W3110Trc-ppcTrc-panDΔpykAΔcycApckΔmdhΔaspaΔpoxB:: esaRΔldhA::: pbs1-EsaI ΔgltA:: P _esaS -gltA/pTrc99a-panD _BS ^K104S -aspB _CG aspA, designated as strain B8.

The invention reforms the beta-alanine synthetic network of the escherichia coli, reduces the consumption of OAA by knocking out the encoding phosphoenolpyruvate carboxykinase gene pck and the malate dehydrogenase gene mdh, increases the carbon flow in the beta-alanine synthetic pathway, improves the concentration of oxaloacetic acid substrate pool by dynamically regulating the expression of gltA, and overcomes the defect of insufficient oxaloacetic acid substrate in the original strain, thereby increasing the yield of the beta-alanine.

The invention also relates to application of the recombinant genetically engineered bacterium in preparing beta-alanine by a microbial fermentation method, wherein the application is as follows: inoculating the recombinant genetically engineered bacterium into a fermentation medium containing 50mg/L kanamycin, and culturing the bacterial cells to grow to OD at 30 ℃ and 180rpm ₆₀₀ At this time, IPTG was added to a final concentration of 0.2mM to induce gene expression, and the culture was continued for 48 hours until the fermentation was completed, to obtain a fermentation broth containing β -alanine, and the fermentation broth was isolated and purified to obtain β -alanine.

The formula of the fermentation medium is as follows: glucose 18-22 g/L, (NH) ₄ ) ₂ SO ₄ 14-18 g/L, yeast extract 3-5 g/L, KH ₂ PO ₄ 0.5～1.5g/L，MgSO ₄ 0.2～1g/L，CaCO ₃ 13～17g/L，VB ₁ 0.3～05mg/L，VB ₁₂ 0.1-0.3 mg/L, trace element solution 0.5-1.5 mL/L, water as solvent, and no need of adjusting pH value; further preferred are: glucose 20g/L, (NH) ₄ ) ₂ SO ₄ 16g/L, yeast extract 4g/L, KH ₂ PO ₄ 1g/L，MgSO ₄ 0.5g/L，CaCO ₃ 15g/L，0.4mg/L VB ₁ ，0.2mg/L VB ₁₂ 1mL/L trace element solution, wherein the solvent is water, and the pH value is not required to be regulated; the trace element solution comprises the following components: 8-12 g/L CaCl ₂ ，8～12g/L FeSO ₄ ·7H ₂ O，0.8～1.2g/L ZnSO ₄ ·7H ₂ O，0.1～0.3g/L CuSO ₄ ，0.01～0.04g/L NiCl ₂ ·7H ₂ O, the solvent is deionized water; further preferred are: 10g/L CaCl ₂ ，10g/L FeSO ₄ ·7H ₂ O，1g/L ZnSO ₄ ·7H ₂ O，0.2g/L CuSO ₄ ，0.02g/L NiCl ₂ ·7H ₂ O, the solvent is deionized water.

Before fermentation, the recombinant genetically engineered bacteria are inoculated to LB culture medium containing 50mg/L kanamycin, seed liquid is prepared at 37 ℃ and 180rpm overnight, and the seed liquid is inoculated to fermentation culture medium containing 50mg/L kanamycin in an inoculum size of 5% of the volume concentration.

The fermentation is carried out in a fermenter: inoculating the recombinant genetically engineered bacteria to an LB plate containing 50mg/L kanamycin resistance, culturing overnight at 37 ℃, picking single colonies to an LB test tube containing 50mg/L kanamycin resistance, and culturing overnight at 150rpm at 37 ℃ to prepare seed liquid; inoculating the seed solution into LB medium containing 50mg/L kanamycin at a volume concentration of 5%, and culturing at 37 ℃ and 150rpm overnight to obtain a secondary seed solution; inoculating the secondary seed solution into a 5-L fermentation tank filled with 2L of fermentation medium containing 50mg/L kanamycin according to the volume concentration of 15%, adding 0.2mM IPTG with the final concentration, fermenting and culturing at 30 ℃ and 500rpm under the aeration condition of 0.5V/V/min, when the pH value is higher than 6.80 (the initial sugar consumption in the fermentation tank is finished), starting automatic feeding, and stopping feeding when the pH value is lower than 6.80, and culturing for 90-117 hours to obtain the fermentation liquid containing beta-alanine. The feed medium consists of: glucose 500g/L, (NH) ₄ ) ₂ SO ₄ 16g/L, yeast extract 2g/L, KH ₂ PO ₄ 14g/L，NaHCO ₃ 10 g/L，0.4mg/L VB ₁ ，0.2mg/L VB ₁₂ The solvent is water, and 12.5% of the solvent is usedAmmonia was used to adjust the pH to 6.8.

The feeding speed is 25mL/h, and the total addition amount of the feeding culture medium is 1800mL/2L.

The fermentation liquor separation and purification method comprises the following steps: firstly, removing thalli and other solid particles in fermentation liquor by centrifugation at 12000rpm and 4 ℃ for 10min, removing macromolecular proteins in the fermentation liquor by adopting a microfiltration method, removing inorganic salts in the fermentation liquor by adopting a deionization technology, adding activated carbon for decolorization, separating by using ion exchange resin to obtain beta-alanine, concentrating in vacuum, adding ethanol for cooling and crystallizing, and obtaining beta-alanine crystals.

Compared with the prior art, the invention has the beneficial effects that:

the invention knocks out pck gene and mdh gene, reduces carbon loss of gluconeogenesis pathway, and reduces carbon loss of oxaloacetate which is a key metabolic precursor substance. Knocking out byproduct genes poxB and ldhA, replacing in-situ by dynamic regulation elements, reducing loss of central metabolic carbon flow, introducing a dynamic regulation system, and replacing gltA gene promoter by P _esaS After that, the beta-alanine yield is obviously improved, and in fed-batch fermentation, the beta-alanine yield of the strain B8 on the 5-L fermentation tank level reaches 111.86g/L, and compared with the original strain B1, the sugar acid conversion rate is improved by 66.7 percent and reaches 37.8 percent.

(IV) description of the drawings

FIG. 1 shows the biomass OD of strains B1 and B2 ₆₀₀ And a bar graph of beta-alanine concentration.

FIG. 2 shows the biomass OD of strains B2 and B3 ₆₀₀ And a bar graph of beta-alanine concentration.

FIG. 3 shows the biomass OD of strains B3 and B4 ₆₀₀ And a bar graph of beta-alanine concentration.

FIG. 4 is a bar graph of biomass OD600 and beta-alanine concentration for strains B4, B8 and B9.

FIG. 5 shows the OD of biomass from the fed-batch fermentation of strain B8 in a 5-L fermenter ₆₀₀ Concentration profile of residual sugar and beta-alanine.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

strain e.coli W3110 was from the university of jerusalem CGSC collection (Coli Genetic Stock Center), 8 th month 5 th day of the collection date 1975, deposit number cgsc#4474, which is disclosed in patent US2009/0298135 A1,US2010/0248311 A1.

Coli B1:

E.coli W3110TrcpanDTrcppcΔpykAΔcycA/pTrc99a-panD _BS ^K104S -aspB _CG -aspA。

the construction of strain B1 was all referred 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- α -decarboxylase for fermentative production of β -alanine in Escherichia coli.

LB plate composition: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 20g/L of agar powder, water as a solvent and natural pH.

LB medium: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl, and the solvent is deionized water, and the pH value is natural.

The fermentation medium formulation is as follows: glucose 20g/L, (NH) ₄ ) ₂ SO ₄ 16g/L, yeast extract 4g/L, KH ₂ PO ₄ 1g/L，MgSO ₄ 0.5g/L，CaCO ₃ 15g/L，0.4mg/L VB ₁ ，0.2mg/L VB ₁₂ 1mL/L trace element solution, wherein the solvent is water, and the pH value is not required to be regulated; the trace element solution comprises the following components: 10g/L CaCl ₂ ，10g/L FeSO ₄ ·7H ₂ O，1g/L ZnSO ₄ ·7H ₂ O，0.2g/L CuSO ₄ ，0.02g/L NiCl ₂ ·7H ₂ O, the solvent is deionized water.

TABLE 1 Gene involved in Strain engineering and corresponding pathway

TABLE 2 primers used in Gene editing

Example 1, knockout of pck in Strain B1

1. Construction of Strain B2

The phosphoenolpyruvate carboxykinase gene pck (the nucleotide sequence is shown as SEQ ID No. 1) is knocked out by using CRISPR/Cas9 mediated gene editing technology, so that accumulation of oxaloacetate substrate pools is enhanced, and the specific operation is as follows:

(1) Construction of pTarget-pck plasmid: the plasmid pTarget is used as a template, pTarget-pck-F/pTarget-pck-R is used as a primer for amplification, a PCR product is added with DpnI and then subjected to heat preservation and digestion for 1h at 37 ℃, recovered and purified by a Clean up kit, then converted into E.coli DH5 alpha, coated on an LB solid plate containing 50mg/L spectinomycin, and subjected to inversion culture for 20h at 30 ℃, colony PCR is firstly subjected to primary verification, and then the correctness of pTarget-pck plasmid is verified by sequencing, so that the plasmid pTarget-pck is obtained. Then using pTD-line-F/pTD-line-R as a primer and using plasmid pTarget-pck as a template for amplification, adding DpnI into a PCR product, then carrying out heat preservation and digestion for 3h at 37 ℃, and recovering DNA fragments by using a clean up kit to obtain a linearized plasmid pTarget-pck.

(2) Construction of a Donor-containing plasmid pTD-pck: amplifying an upstream homology arm F1 by using a genome of E.coli W3110 as a template and L-pck-F/L-pck-R as a primer; the downstream homology arm R1 was amplified using R-pck-F/R-pck-R as primer. The PCR product was recovered using a Clean up purification kit. And (3) taking the upstream and downstream homology arms F1 and R1 as templates, taking the L-pck-F/R-pck-R as primers for fusion to obtain the upstream and downstream homology arms containing pck, and recovering DNA fragments through a Clean up kit to obtain fused fragments. And (2) constructing a plasmid pTDpck by using the fused fragment and the linearized plasmid pTarget-pck obtained in the step (1) through a one-step cloning method, coating the plasmid pTDpck into transformed E.coli DH5 alpha, culturing the plasmid on an LB solid plate containing 50mg/L spectinomycin in an inverted manner at 30 ℃ for 20 hours, screening and initially screening correct strains by using a primer T-pck-F/T-pck-R colony PCR, and finally verifying the correctness of the plasmid pTDpck through sequencing.

(3) Plasmid pCas9 was transformed into competent cell B1, plated onto LB solid plates containing 50mg/L kanamycin, cultured overnight at 30℃and single colonies were picked into LB tube medium containing 50mg/L kanamycin resistance, cultured overnight at 30 ℃. Inoculating into 100mL LB culture medium at 1% by volume, adding kanamycin resistance and 10mM L-arabinose, and culturing at 30 deg.C at 180rpm to OD ₆₀₀ Centrifugation at 4000rpm at 0.5,4 ℃. Washing with 4 deg.c cold ultrapure water for 2 times, washing with 10% cold glycerin for one time, and re-suspending with 10% glycerin for storage to obtain electrotransformation competent cells.

(4) Mixing 2. Mu.L of pTDpck plasmid constructed by the method of step (2) with 100. Mu.L of electrotransformation competent cells prepared by the method of step (3), transferring into a 2mm electric stump, ice-bathing for 45s, and using an electroporation device (MicroPluser) ^TM BIO-RAD) electric shock transformation, the selected voltage is 2500V, 700 mu L of LB culture medium precooled at 4 ℃ is added immediately after electric shock is finished, the mixture is immediately transferred into a new sterile 1.5mL EP tube after uniform mixing, shake culture is carried out for 3 hours at 30 ℃ and 150rpm, the mixture is coated on LB solid culture medium containing 50mg/L kanamycin and 50mg/L spectinomycin, inversion culture is carried out for 24 hours at 30 ℃, T-pck-F/T-pck-R is taken as a primer colony for PCR verification, sequencing verification is carried out, the construction correctness of the strain is carried out, and the pck knockout strain is successfully constructed.

(5) pTarget and pCas9 plasmid elimination: and (3) inoculating the positive single colony obtained in the step (4) into an LB liquid culture medium test tube containing 2mM IPTG and 50mg/L kanamycin, culturing overnight at 30 ℃, streaking a bacterial liquid to an LB solid culture medium containing 50mg/L kanamycin, reversely culturing at 30 ℃ for 20 hours until the single colony appears, picking the single colony to the LB solid culture medium containing 50mg/L spectinomycin, reversely culturing at 30 ℃ for 20 hours, and if no single colony appears, indicating that the pTarget plasmid has been eliminated in the strain. Selecting the strain without pTarget plasmid, inoculating into antibiotic-free LB liquid medium test tube, culturing at 42deg.C for 10 hr, dipping bacterial liquid, streaking onto LB solid medium containing 50mg/L kanamycin, culturing at 37deg.C for 10 hr in an inverted manner, if no single colony appearsThe successful elimination of the pCas9 plasmid, which finally gives a plasmid-free strain, is described for plasmid pTrc99a-panD _BS ^K104S -aspB _CG aspA transformation into plasmid-free, strain-transformed competent cells, resulting in strain B2: e.coli W3110TrcpanDTrcppcΔpykA ΔcycAΔpck/pTrc99a-panD _BS ^K104S -aspB _CG -aspA。

(6) The single colony of the strain B2 is picked into LB test tube culture medium containing 50mg/L kanamycin by taking the original strain B1 as a control strain, and is cultured at 37 ℃ and 180rpm overnight to obtain seed liquid. Inoculating 1mL of seed solution into 500mL of 50mL of fermentation medium containing 50mg/L kanamycin, culturing thallus at 30 ℃ and 180rpm to grow to OD ₆₀₀ =0.5, 0.25mM IPTG final concentration was added and shake culture was continued for 48h. After fermentation, 1mL of the fermentation broth was centrifuged at 12000rpm for 3min, the whole supernatant was discarded, 1mL of distilled water was added to resuspend the cells and calcium carbonate therein, the supernatant was discarded by centrifugation at 12000rpm for 3min, and 1mL of distilled water was added to resuspend the cells and calcium carbonate again, and the supernatant was discarded by centrifugation at 12000rpm for 3 min. Finally, 800. Mu.L of distilled water is added to resuspend the thalli and the calcium carbonate, and then 200. Mu.L of 20% acetic acid aqueous solution with volume concentration is added, and the mixture is left at room temperature for 5min to dissolve the calcium carbonate therein. Adding 50 μl of bacterial solution containing dissolved calcium carbonate into 1950 μl of distilled water, diluting 40 times, and measuring biomass OD with spectrophotometer ₆₀₀ . And then taking 1mL of fermentation liquor, centrifuging at 12000rpm for 3min, and taking supernatant as a sample for detecting the concentration of beta-alanine for later use. Biomass OD ₆₀₀ And beta-alanine content as shown in FIG. 1, biomass OD of control strain B1 ₆₀₀ =18.0, the content of β -alanine is 3.91g/L; biomass OD of strain B2 ₆₀₀ =20.5, the content of β -alanine was 3.95g/L, with a slight increase in β -alanine content.

(7) Determination of beta-alanine concentration:

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

0.5M NaHCO ₃ Solution preparation: 21g NaHCO is taken ₃ Dissolved in 500mL deionized water.

0.2M PB buffer: weigh 8.74g Na ₂ HPO ₄ ·12H ₂ O and 2.43g Na ₂ HPO ₄ ·2H ₂ O is dissolved in 200mL deionized water, and is dissolved and stored for standby.

Sample treatment: the sample concentration was diluted to between 0.1 and 1g/L with ultrapure water.

Reaction conditions: samples were taken at 100. Mu.L each, 0.5M NaHCO ₃ 100 mu L of the solution and 100 mu L of 1%2, 4-dinitrofluorobenzene are kept at 60 ℃ for 75min, and finally 700 mu L of 0.2M PB buffer solution is added for uniform mixing, and the mixture is subjected to film coating (polyvinylidene fluoride, organic film and 0.22 mu M) for standby.

Detection conditions: HPLC model: thermo Scientific Utimate the HPLC detection wavelength is 360nm. The β -alanine was isolated using a gradient elution procedure, wherein mobile phase a component, methanol: acetonitrile: ultrapure water=45:45:10 (v: v); mobile phase B component: 10mM potassium dihydrogen phosphate, the pH was adjusted to 7.0 with KOH. The elution procedure was 0-2.5min 10% A,90% B;2.5-2.6min A10% → 14%, B90% → 86%;2.6-13min A14% → 34%, B86% → 66%;13-13.1min A34% → 38% B66% → 62%;13.1-28min A38% → 100% B62% → 0;28-28.1min A100% →10% B0→90%;28.1-32min A10% → 14% B90% → 86%.

Example 2: knockout of mdh Gene in Strain B2

(1) The linearized plasmid pTarget-mdh was prepared by constructing the plasmid pTarget-mdh by the method of example 1.

(2) Construction of plasmid pTD-mdh: the E.coli W3110 genome is used as a template, and L-mdh-F/L-mdh-R and R-mdh-F/R-mdh-R are used as primers for amplification to obtain upstream and downstream homology arms. The plasmid pTD-mdh was constructed by transferring the upstream and downstream homology arms into the linearized plasmid pTarget-mdh of step (1) by the method of example 1 and verifying with the primers T-mdh-F/T-mdh-R.

(3) The pCas9 plasmid was introduced into strain B2 and competent cells were prepared by the method of example 1.

(4) Plasmid pTD-mdh was transferred into competent cells of step (3) using the procedure of example 1, and the mdh knockout strain was successfully constructed.

(5) Plasmid elimination: the procedure is as in example 1 to obtain the plasmid-free strain E.coli W3110 TrcppanDTrcppc.DELTA.pykA.DELTA.cycA.DELTA.mdh.

(6) Transformation of plasmids: extraction of plasmid pTrc99a-panD of starting Strain B1 _BS ^K104S aspAaspB _CG Transforming the plasmid into competent cells of the plasmid-free strain of step (5), constructing strain B3: e.coli W3110TrcpanDTrcppcΔpykA ΔcycAΔpckΔmdh/pTrc99a-panD _BS ^K104S -aspB _CG -aspA。

(7) The shake flask fermentation test was performed on the strains B2 and B3 in the manner of example 1, using the starting strain B2 as a control strain. The content of beta-alanine was detected according to example 1. Biomass OD ₆₀₀ And beta-alanine content as shown in FIG. 2, control strain B2 biomass OD ₆₀₀ 20.5, and the content of beta-alanine is 3.95g/L; strain B3 biomass OD ₆₀₀ The content of beta-alanine is 4.08g/L, and the yield of beta-alanine is improved by 3.3 percent.

Example 3: knockout of aspA Gene in Strain B3

(1) Construction of plasmid pTarget-aspA: the pTarget plasmid is used as a template, pTarget-aspA-F/pTarget-aspA-R is used as a primer for amplification, the PCR product is added with DpnI and then is incubated for 2 hours at 37 ℃, the methylation plasmid template is digested, and the methylation plasmid template is transformed into E.coli DH5 alpha competent cells after purification. Positive strains were screened using LB plates containing 50mg/L spectinomycin, and their correctness was verified by sequencing, and linearized plasmid pTarget-aspA was obtained by the method of example 1.

(2) Construction of plasmid pTD-aspA: the E.coli W3110 genome is used as a template, and L-aspA-F/L-aspA-R and R-aspA-F/R-aspA-R are used as primers to obtain upstream and downstream homology arms through amplification. Cloning the upstream and downstream homology arms to the linearized plasmid pTarget-aspA of step (1) by the method of example 1, and verifying by using the primer T-aspA-F/T-aspA-FR to obtain the plasmid pTD-aspA.

(3) Plasmid pCas9 was introduced into strain B3 and competent cells were prepared by the method of example 1.

(4) Plasmid pTD-aspA was transformed into competent cells of step (3) by the method of example 1 to construct strain E.coli W3110 TrcpanaDTrcppc.DELTA.pykA.DELTA.cycA.DELTA.pckΔmdh.aspA.

(5) Plasmid elimination: using example 1, the plasmids pTarget and pCas9 of step (4) were deleted to give plasmid-free strain E.coli W3110 TrcpanaDTrcppc.DELTA.pykA.DELTA.cycA.DELTA.pckDELTA.mdh.DELTA.aspA.

(6) Transformation of plasmids: the aspA gene on the original plasmid was removed and the plasmid pTrc99a-panD was used _BS ^K104S aspB _CG Transforming into competent cells of the plasmid-free strain in the step (5), and constructing a strain B4: e.coli W3110 TrcppanDTrcppcΔpykA ΔcycApckΔmdh ΔaspA/pTrc99a-panD _BS ^K104S aspB _CG 。

(7) The shake flask fermentation test was performed on the strains B3 and B4 in the manner of example 1, using the starting strain B3 as a control strain. The content of beta-alanine was detected according to example 1. Biomass OD ₆₀₀ And beta-alanine content as shown in FIG. 3, control strain B3 biomass OD ₆₀₀ 13.8, the content of beta-alanine is 4.08g/L; strain B4 biomass OD ₆₀₀ The content of beta-alanine is 9.8, the content of beta-alanine is 4.20g/L, and the yield of beta-alanine is improved by 3.4 percent.

Example 4: knockout of poxB Gene in Strain B4 genome and replacement with esaR at the site

(1) Construction of plasmid pTarget-poxB: the pTarget plasmid is used as a template, pTarget-poxB-F/pTarget-poxB-R is used as a primer for amplification, dpnI is added into a PCR product, the temperature is kept at 37 ℃ for 2 hours, the methylation plasmid template is digested, and the methylation plasmid template is purified and then is transformed into E.coli DH5 alpha competent cells. Positive strains were screened using LB plates containing 50mg/L spectinomycin, and after sequencing to verify their correctness, linearized plasmid pTarget-poxB was obtained by the method of example 1.

(2) Construction of plasmid pTD-poxB: the E.coli W3110 genome and the plasmid with esaR gene are used as templates, L-poxB-F/L-poxB-R and R-poxB-F/R-poxB-R are used as primers, upstream and downstream homology arms are obtained by amplification, and esaR-F/esaR-R is used as a primer, so that esaR gene is obtained by amplification. The plasmid pTD-poxB was obtained by cloning the ligated upstream and downstream homology arms and esaR gene into the linearized plasmid pTarget-poxB of step (1) by the method of example 1 and verifying it by using the primer T-poxB-F/T-poxB-R.

(3) Plasmid pCas9 was introduced into strain B4 and competent cells were prepared by the method of example 1.

(4) Plasmid pTD-poxB was transferred into competent cells of step (3) using the procedure of example 1 to construct strain E.coli W3110TrcpanDTrcppc ΔpykA ΔcycA Δpck Δmdh ΔaspA ΔpoxB:: esaR.

(5) Plasmid elimination: using example 1, the plasmids pTarget and pCas9 of the strains of step (4) were eliminated to give plasmid-free strain B5: e.coli W3110TrcpanDTrcppcΔpykA ΔcycApckΔmdh ΔaspA ΔpoxB:: esaR.

Example 5: the ldhA gene in the genome of strain B5 was knocked out and replaced in situ with the esaI gene whose promoter was Pbs 1.

(1) Construction of plasmid pTarget-ldhA: the pTarget plasmid is used as a template, pTarget-ldhA-F/pTarget-ldhA-R is used as a primer for amplification, dpnI is added into a PCR product, the temperature is kept at 37 ℃ for 2 hours, the methylation plasmid template is digested, and the methylation plasmid template is purified and then is transformed into E.coli DH5 alpha competent cells. Positive strains were selected using LB plate containing 50mg/L spectinomycin, and after verification of their correctness by sequencing, linearized plasmid pTarget-ldhA was obtained by the method of example 1.

(2) Construction of plasmid pTD-ldhA: the E.coli W3110 genome is used as a template, and L-ldhA-F/ldhA-FR-Pbs1 and R-ldhA-F/R-ldhA-R are used as primers, and an upstream homology arm and a downstream homology arm are obtained through amplification; the esaI gene is amplified by using a plasmid with the esaI gene as a template and Pbs1-esaI-F/esaI-R as a primer. The plasmid pTD-ldhA was obtained by cloning the linearized plasmid pTarget-ldhA of step (1) after ligation of the upstream and downstream homology arms and esaI gene by the method of example 1.

(3) Plasmid pCas9 was introduced into strain B5 and competent cells were prepared by the method of example 1.

(4) Plasmid pTD-ldhA was transformed into competent cells of step (3) by the method of example 1 to construct strain B6: e.coli W3110TrcpanDTrcppcΔpykA ΔcycApckΔmdh ΔaspA ΔpoxB:: esaRΔldhA:: pbs1-EsaI.

(5) Plasmid elimination: using example 1, the plasmids pTarget and pCas9 of step (4) were deleted to give plasmid-free strain B6:E.coli W3110TrcpanDTrcppc ΔpykA ΔcycA ΔpckΔmdh ΔaspA ΔpoxB:: esaR ΔldhA:: pbs1-EsaI.

Example 6: substitution of the gltA Gene promoter in the Strain B6 genome to P _esaS 。

(1) Construction of plasmid pTarget-gltA: the pTarget plasmid is used as a template, pTarget-gltA-F/pTarget-gltA-R is used as a primer for amplification, dpnI is added into a PCR product, the temperature is kept at 37 ℃ for 2 hours, the methylation plasmid template is digested, and the methylation plasmid template is purified and then is transformed into E.coli DH5 alpha competent cells. Positive strains were screened using LB plates containing 50mg/L spectinomycin, and after sequencing to verify their correctness, linearized plasmid pTarget-gltA was obtained by the method of example 1.

(2) Construction of plasmid pTD-gltA: and (3) using the E.coli W3110 genome as a template, and using L-gltA-F/L-gltA-R and R-gltA-F/R-gltA-R as primers to amplify to obtain upstream and downstream homology arms of the gltA original promoter. With P _esaS Plasmid of promoter is used as template, P is used as template _esaS -F/P _esaS R is a primer, and the promoter P is obtained by amplification _esaS The method of example 1 was used to combine the upstream and downstream homology arms with P _esaS The promoter was cloned after ligation to linearize the plasmid pTarget-gltA in step (1) to give the plasmid pTD-gltA.

(3) Plasmid pCas9 was introduced into strain B6 and competent cells were prepared by the method of example 1.

(4) Plasmid pTD-gltA was transformed into competent cells of step (3) using the procedure of example 1, to construct strain B7: e.coli W3110TrcpanDTrcppcΔpykAΔcycAΔpckΔmdhΔaspaΔpoxB:: esaRΔldhA::: pbs1-EsaI ΔgltA::: P _esaS -gltA。

(5) Plasmid elimination: using example 1, the plasmids pTarget and pCas9 of the strains of step (4) were eliminated to give plasmid-free strain B7: e.coli W3110TrcpanDTrcppcΔpykAΔcycAΔpckΔmdhΔaspaΔpoxB:: esaRΔldhA::: pbs1-EsaI ΔgltA::: P _esaS -gltA。

(6) Construction of Strain B8

Extraction of Strain B1 plasmid pTrc99a-panD _BS ^K104S -aspB _CG aspA and transfer into strain B7, creating strain B8: e.coli

W3110TrcpanDTrcppcΔpykAΔcycAΔpckΔmdhΔaspAΔpoxB::EsaRΔldhA::Pbs1-EsaIΔgltA::P _esaS -gltA/pTrc99a-panD _BS ^K104S -aspB _CG -aspA。

Example 7: overexpression of plasmid pTrc99a-panD in Strain B8 _BS ^K104S -aspB _CG -panD _BS ^K104S 。

(1) Construction of plasmid pTrc99a-panD _BS ^K104S -aspB _CG -panD _BS ^K104S : by pTrc99a-panD _BS ^K104S -aspB _CG The aspA plasmid is used as a template, pTrc99a-line-F/pTrc99a-line-R is used as a primer for amplification, dpnI is added into a PCR product, the temperature is kept at 37 ℃ for 2 hours, the methylation plasmid template is digested, and the recovery and purification are carried out to obtain a linearization vector pTrc99a-panD _BS ^K104S -aspB _CG 。

(2) Construction of plasmid pTrc99a-panD _BS ^K104S -aspB _CG -panD _BS ^K104S : by pTrc99a-panD _BS ^K104S -aspB _CG The aspA plasmid is used as a template, panD-F and panD-R are used as primers, and the panD is obtained by recovery and purification _BS ^K104S Fragment and linearized vector pTrc99a-panD using one-step cloning kit _BS ^K104S -aspB _CG With panD _BS ^K104S Ligation, transformation of ligation product into E.coli DH 5. Alpha. Competence, construction of vector pTrc99a-panD _BS ^K104S -aspB _CG -panD _BS ^K104S 。

(3) Construction of Strain B9

Extraction of plasmid pTrc99a-panD _BS ^K104S -aspB _CG -panD _BS ^K104S And transferring into a strain B7, and constructing to obtain a strain B9: e.coli W3110TrcpanDTrcppcΔpykAΔcycAΔpckΔmdhΔaspaΔpoxB:: esaRΔldhA::: pbs1-EsaI ΔgltA::: P _esaS -gltA/p Trc99a-panD _BS ^K104S -aspB _CG -panD _BS ^K104S 。

(4) The shake flask fermentation test was performed on the strains B8 and B9 as in example 1, using the strain B4 as a control. The content of beta-alanine was detected according to example 1. Biomass OD ₆₀₀ And beta-alanine content as shown in FIG. 4, control strain B4 biomass OD ₆₀₀ 9.8, and the content of beta-alanine is 4.20g/L; strain B8 biomass OD ₆₀₀ The content of beta-alanine is 13.8, the content of beta-alanine is 4.80g/L, and the yield of beta-alanine is improved by 14.3 percent; bacteria (fungus)Strain B9 biomass OD ₆₀₀ The content of beta-alanine was 7.4 and was 3.50g/L, the yield of beta-alanine was reduced by 17%.

Example 8 fed-batch fermentation in 5-L fermentors

The strain B8 of example 6 was streaked onto LB plates containing 50mg/L kanamycin resistance, cultured overnight at 37℃and single colonies were picked up to LB tubes containing 50mg/L kanamycin resistance, and cultured overnight at 37℃and 150rpm to prepare seed solutions. The seed solution was inoculated in an amount of 5% by volume into 100mL of LB medium containing 50mg/L kanamycin resistance, and cultured overnight at 37℃and 150rpm, to give a secondary seed solution. The secondary seed solution was inoculated at 15% by volume into a 5-L fermenter containing 50mg/L kanamycin 2L fermentation medium, and IPTG was added at a final concentration of 0.2 mM. Fermenting and culturing at 30deg.C and 500rpm under ventilation of 0.5V/V/min, when pH is higher than 6.80 (initial sugar consumption in fermentation tank is completed), automatic feeding is started, feeding medium containing 50mg/L kanamycin and final concentration of 0.2mM IPTG is fed at 25mL/h rate, feeding is stopped when pH is lower than 6.80, residual sugar in fermentation tank is maintained at lower level, and sugar concentration is maintained at 0-3g/L. The total addition amount of the feed medium of the strain B8 is 1800mL, the strain B8 is cultured for 117 hours, the beta-alanine yield in the fermentation broth is detected by adopting the high performance liquid chromatography method of the example 1, and the biomass OD is detected by adopting the spectrophotometry ₆₀₀ Detecting sugar concentration by adopting a DNS method; as shown in FIG. 5, the beta-alanine yield reached 111.86g/L at 91.5h, the final biomass OD ₆₀₀ Maintained at 74.4. The fermentation result shows that the beta-alanine producing strain subjected to metabolic engineering has good production performance, and lays a foundation for industrialized production of beta-alanine.

5-L fermentation tank culture medium formula: glucose 20g/L, (NH) ₄ ) ₂ SO ₄ 16g/L, yeast extract 2g/L, KH ₂ PO ₄ 2g/L，MgSO ₄ 0.5g/L，CaCO ₃ 15g/L，VB ₁ 0.4mg/L，VB ₁₂ 0.2mg/L,1mL/L trace element solution, deionized water as solvent; composition of trace element solution: 10g/L CaCl ₂ ，10g/L FeSO ₄ ·7H ₂ O，1g/L ZnSO ₄ ·7H ₂ O，0.2g/L CuSO ₄ ，0.02g/L NiCl ₂ ·7H ₂ O, the solvent is deionized water.

Feed medium: glucose 500g/L, (NH) ₄ ) ₂ SO ₄ 10g/L, yeast extract 2g/L, KH ₂ PO ₄ 14g/L，MgSO ₄ 8g/L，0.4mg/L VB ₁ ，0.2mg/L VB ₁₂ The pH was adjusted to 6.8 with 12.5% ammonia.

Claims

1. The recombinant genetically engineered bacterium for high-yield beta-alanine is characterized in that the recombinant genetically engineered bacterium takes escherichia coli B1 as an initial strain, and performs one or more of the following gene edits: the pck gene is knocked out, the mdh gene is knocked out, the aspA gene is knocked out, the poxB gene is replaced by the EsaR gene, the ldhA gene is replaced by the EsaI gene of which the original promoter is replaced by the artificial promoter Pbs1, and the gltA gene promoter is replaced by P _esaS A promoter; the starting strain B1: e.coli W3110TrcpanDTrcppcΔpykA ΔcycA/pTrc99a-panD _BS ^K104S -aspB _CG -aspA。

2. The recombinant genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the pck gene is shown in SEQ ID No. 1; the nucleotide sequence of the mdh gene is shown as SEQ ID NO. 2; the nucleotide sequence of the aspA gene is shown as SEQ ID NO. 3; the nucleotide sequence of the EsaR gene is shown as SEQ ID NO. 5; the nucleotide sequence of the Pbs1 promoter is shown in SEQ ID NO. 7; the nucleotide sequence of the EsaI gene is shown as SEQ ID NO. 8; the P is _esaS The nucleotide sequence of the promoter is shown as SEQ ID NO. 10.

3. The recombinant genetically engineered bacterium of claim 1, wherein the recombinant genetically engineered bacterium uses escherichia coli B1 as an initial strain, and the following genetic edits are sequentially performed: the pck gene is knocked out, the mdh gene is knocked out, the aspA gene is knocked out, the poxB gene is replaced by the EsaR gene, the ldhA gene is replaced by the EsaI gene of which the original promoter is replaced by the artificial promoter Pbs1, and the gltA gene promoter is replaced by P _esaS A promoter.

4. Use of the recombinant genetically engineered bacterium of claim 1 in the preparation of beta-alanine by microbial fermentation.

5. The application of claim 4, wherein the application is: inoculating the recombinant genetically engineered bacterium into a fermentation medium containing 50mg/L kanamycin, and culturing the bacterial cells to grow to OD at 30 ℃ and 180rpm ₆₀₀ And (2) adding IPTG to a final concentration of 0.2mM to induce gene expression, continuously culturing for 48 hours until fermentation is finished, obtaining fermentation liquor containing beta-alanine, and separating and purifying the fermentation liquor to obtain the beta-alanine.

6. The use according to claim 5, wherein the fermentation medium is formulated as follows: glucose 18-22 g/L, (NH) ₄ ) ₂ SO ₄ 14-18 g/L, yeast extract 3-5 g/L, KH ₂ PO ₄ 0.5～1.5g/L，MgSO ₄ 0.2～1g/L，CaCO ₃ 13～17g/L，VB ₁ 0.3～05mg/L，VB ₁₂ 0.1-0.3 mg/L, trace element solution 0.5-1.5 mL/L, water as solvent, and no need of adjusting pH value; the trace element solution comprises the following components: 8-12 g/L CaCl ₂ ，8～12g/L FeSO ₄ ·7H ₂ O，0.8～1.2g/L ZnSO ₄ ·7H ₂ O，0.1～0.3g/L CuSO ₄ ，0.01～0.04g/L NiCl ₂ ·7H ₂ O, the solvent is deionized water.

7. The use according to claim 5, wherein the recombinant genetically engineered bacterium is inoculated into LB medium containing 50mg/L kanamycin before fermentation, seed solution is prepared at 37 ℃ and 180rpm overnight, and the seed solution is inoculated into fermentation medium containing 50mg/L kanamycin in an inoculum size of 5% by volume.

8. The use according to claim 5, wherein the fermentation is carried out in a fermenter: inoculating the recombinant genetically engineered bacterium to a strain containing 50mg/L of a cardCulturing overnight at 37deg.C on natamycin resistant LB plate, picking single colony to LB test tube containing 50mg/L kanamycin resistance, culturing overnight at 37deg.C at 150rpm, and preparing seed solution; inoculating the seed solution into LB medium containing 50mg/L kanamycin at a volume concentration of 5%, and culturing at 37 ℃ and 150rpm overnight to obtain a secondary seed solution; inoculating the secondary seed liquid into a fermentation tank filled with a fermentation medium containing 50mg/L kanamycin according to the volume concentration of 15%, adding IPTG with the final concentration of 0.2mM, fermenting and culturing at 30 ℃ and 500rpm under the aeration of 0.5V/V/min, when the pH value is higher than 6.80, starting automatic feeding, stopping feeding when the pH value is lower than 6.80, and culturing for 90-117 hours to obtain a fermentation liquid containing beta-alanine; the feed medium consists of: glucose 500g/L, (NH) ₄ ) ₂ SO ₄ 16g/L, yeast extract 2g/L, KH ₂ PO ₄ 14g/L，NaHCO ₃ 10 g/L，0.4mg/L VB ₁ ，0.2mg/L VB ₁₂ The solvent was water and the pH was adjusted to 6.8 with 12.5% ammonia.

9. The use according to claim 8, wherein the feed rate is 25mL/h and the total feed medium addition is 1800mL/2L.