Genetically engineered bacterium of high-yield beta-alanine as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium of high-yield beta-alanine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli W3110 as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli W3110, the genetically engineered bacterium does not express proteins such as lactose operon repressor protein, pyruvate dehydrogenase, acetokinase and the like; protein such as aspartic acid-alpha-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase and the like is overexpressed; the phosphoenolpyruvate carboxykinase is replaced by phosphoenolpyruvate carboxykinase from Mannheim succinic acid producing bacteria. The genetically engineered bacterium BA-17 for high yield of beta-alanine, which is constructed by the invention, is fermented in a 5L fermentation tank for 60 hours, the yield of beta-alanine reaches 126.7 g / L, the conversion rate reaches 60.3%, and the production intensity reaches 2.11 g / L / h.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering and discloses a genetic engineering bacterium with high beta-alanine production, a construction method and an application thereof. Background Art

[0002] β-alanine, also known as 3-aminopropionic acid, is the only β-amino acid in nature. Although it does not participate in the synthesis of enzymes and proteins, it still has important physiological functions in the growth of organisms. In cell life activities, β-alanine can be used as a precursor to synthesize pantothenic acid (vitamin B5) and coenzyme A (CoA), which is also related to various metabolic activities such as fatty acid metabolism and sugar metabolism. Plants and microorganisms can synthesize β-alanine autonomously, while mammals need exogenous supplementation. According to current research, β-alanine has important application value in many fields such as food, medicine, chemical industry and feed.

[0003] Currently, Escherichia coli is widely used as the main industrial microorganism to produce β-alanine by fermentation. The recombinant Escherichia coli constructed by Chen Ling et al. (CN202310381712.1) contains simultaneously overexpressed Bacillus subtilis ( Bacillus subtilis ) and the gene encoding L-aspartate-α-decarboxylase ADC from the insect Tribolium castaneum ( Tribolium castaneum ) from L-aspartate-α-decarboxylase ADC, and also overexpressed panD Genes encoding maturation factors panZ (from Escherichia coli), the final strain produced 27.012 g / L of β-alanine at the shake flask level after 72 h, and the sugar-acid conversion rate was 0.45 g / g. Liu Zhiqiang et al. (CN202410005378.4) conducted a batch-fed fermentation, and the β-alanine production of strain B8 at the 5 L fermentation tank level reached 111.86 g / L after 91.5 h, which was 66.7% higher than that of the starting strain B1, and the OD 600 The above-mentioned engineered bacteria carry recombinant plasmids for overexpressing key genes, which undoubtedly makes the fermentation process and results unstable. In addition, antibiotics need to be added to ensure stable inheritance of plasmids, and inducers need to be added to activate gene expression, which increases production costs and damages cell growth, resulting in a longer fermentation cycle. In addition, the above-mentioned engineered bacteria also have shortcomings such as low conversion rate. Summary of the invention

[0004] In view of these defects, the present invention provides a genetically engineered bacterium with high yield of β-alanine and its construction method and application. The genetically engineered bacterium with high yield of β-alanine provided by the present invention has a β-alanine production of up to 126.7 g / L and a conversion rate of 60.3%, and no by-products are detected during the entire fermentation process, indicating that its metabolic pathway is highly specific and can efficiently convert glucose into β-alanine. At the same time, the strain does not carry a plasmid, avoiding the risks caused by plasmid loss or instability, ensuring the stability and reliability of the strain during long-term fermentation, and the strain is non-nutritional deficiency, and can stably grow and produce β-alanine without adding an inducer. Compared with the prior art, the genetically engineered bacteria of the present invention show significant advantages in terms of yield, conversion rate, metabolic specificity and strain stability, providing an efficient, stable and economically feasible solution for the industrial production of β-alanine.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a genetically engineered bacterium with high β-alanine production, wherein the genetically engineered bacterium is obtained by performing the following gene editing on the genome of Escherichia coli W3110 as a starting strain; the genetically engineered bacterium does not express lactose operon repressor protein, pyruvate dehydrogenase, acetate kinase, lactate dehydrogenase, DNA-binding transcription inhibitor, 4-aminobutyrate aminotransferase and amino acid uptake carrier protein; overexpresses aspartate-α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase, succinate dehydrogenase and β-alanine export carrier; and replaces its own phosphoenolpyruvate carboxykinase with phosphoenolpyruvate carboxykinase derived from Mannheim succinic acid producing bacteria.

[0006] The "non-expression" in the present invention means that the amount of the protease is significantly lower than the original level, for example, significantly reduced by at least 50%, 60%, 70%, 80%, 90%, 100%. Gene editing on the genome of the starting strain to make the protease non-expressed can be achieved by conventional means in the art, including but not limited to gene knockout, gene knock-in, point mutation, deletion mutation and their combination.

[0007] The present invention uses E. coli W3110 as the starting strain. In order to improve the fermentation production capacity of β-alanine, the genetic modification of the starting strain is mainly focused on optimizing and enhancing the precursor L-aspartate synthesis pathway, strengthening the synthesis and transportation system of β-alanine, cutting off the β-alanine intake pathway, and balancing cell growth and energy metabolism. Specifically, by knocking out the encoding gene of its own phosphoenolpyruvate carboxykinase, overexpressing the encoding gene of phosphoenolpyruvate carboxykinase from Mannheim succinic acid producing bacteria, and overexpressing the encoding gene of phosphoenolpyruvate carboxylase 、 Gene encoding pyruvate carboxylase 、 Aspartate aminotransferase encoding gene 、 The genes encoding PLP-dependent aspartate aminotransferase and glutamate dehydrogenase were replaced by the promoter of the gene encoding succinate dehydrogenase to increase the supply of oxaloacetate, fumarate and L-glutamate; the consumption of pyruvate and oxaloacetate was reduced by knocking out the genes encoding pyruvate dehydrogenase, acetate kinase, lactate dehydrogenase and DNA-binding transcription inhibitor; the L-aspartate synthesis pathway was strengthened by overexpressing the gene encoding pyridine nucleotide transhydrogenase to provide reducing power for the synthesis of L-aspartate, and the accumulation of L-aspartate, a precursor of β-alanine, was increased; and the accumulation of L-aspartate, a precursor of β-alanine, was increased by overexpressing The coding gene of aspartate-α-decarboxylase, a key enzyme in the synthesis of β-alanine, catalyzes the decarboxylation of L-aspartate to generate β-alanine; the coding gene of 4-aminobutyrate aminotransferase is knocked out to reduce the consumption of β-alanine; the coding gene of β-alanine export carrier is overexpressed to strengthen the β-alanine transport system and promote the transport of β-alanine to the extracellular space; the coding gene of amino acid uptake carrier protein is knocked out to destroy the β-alanine uptake pathway, reduce intracellular β-alanine, reduce the toxicity of β-alanine to cells, promote cell growth and β-alanine synthesis; the coding gene of lactose operon repressor protein is knocked out to make P trc Promoter overexpression gene constitutive expression. The present invention successfully constructed a high-yield β-alanine Escherichia coli genetically engineered bacteria through the above-mentioned gene editing. The experimental results show that the β-alanine production of the strain is significantly improved. After 60 h of fermentation in a 5 L fermenter, the β-alanine production reached 126.7 g / L, the conversion rate reached 60.3%, the production intensity reached 2.11 g / L / h, and no by-products were detected during the entire fermentation process.

[0008] The NCBI-Protein ID of the lactose operon repressor protein involved in the present invention is BAE76127; the NCBI-Protein ID of pyruvate dehydrogenase is BAA35585; the NCBI-Protein ID of acetate kinase is BAA16135; the NCBI-Protein ID of lactate dehydrogenase is BAA14990; the NCBI-Protein ID of DNA-binding transcription repressor is BAE78020; the NCBI-Protein ID of 4-aminobutyrate aminotransferase is BAA16525; the NCBI-Protein ID of amino acid uptake carrier protein is BAE78209; and the NCBI-Protein ID of 6-phosphoenolpyruvate carboxykinase is BAE77888.

[0009] The "overexpression" described in the present invention means that the amount of the protease or transport vector is significantly higher than the original level. Gene editing is performed on the genome of the starting strain, and the method of overexpressing the protease or transport vector can be achieved by conventional means in the art. It can be achieved by replacing the promoter of the corresponding gene with a strong promoter on the genome of the starting strain, or by inserting the corresponding gene into the genome, increasing the gene copy number, or a combination thereof. When selecting a promoter, a reasonable selection can be made based on the specific expression level requirements and the characteristics of the target gene.

[0010] Preferably, the overexpression of succinate dehydrogenase is carried out by replacing the promoter of its own gene with P trc promoter, the P trc The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.1.

[0011] Preferably, the aspartate-α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase 、 Overexpression of phosphoenolpyruvate kinase and β-alanine export vectors is achieved by integrating the coding genes of the corresponding enzymes themselves or heterologously into the genome of the starting strain.

[0012] P trc It is a strong promoter widely used in E. coli expression systems, consisting of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter. This hybrid promoter combines the strong promoter ability of the trp promoter and the regulatory characteristics of the lacUV5 promoter, and can efficiently promote gene expression in E. coli.

[0013] Preferably, the amino acid sequence of the aspartate-α-decarboxylase is shown in SEQ ID NO.2.

[0014] L-aspartate-α-decarboxylase is a key enzyme in the synthesis of β-alanine. Overexpression of L-aspartate-α-decarboxylase can significantly increase the yield of β-alanine. The encoding gene of the aspartate-α-decarboxylase can be derived from Bacillus subtilis 168. panD .

[0015] Preferably, the amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO.3.

[0016] Phosphoenolpyruvate carboxylase can catalyze the carboxylation of phosphoenolpyruvate to generate oxaloacetate. Overexpression of phosphoenolpyruvate carboxylase can increase the supply of oxaloacetate, thereby promoting the synthesis of β-alanine. The coding gene of the phosphoenolpyruvate carboxylase can be derived from the phosphoenolpyruvate carboxylase coding gene of Escherichia coli W3110. ppc .

[0017] Preferably, the amino acid sequence of the pyruvate carboxylase is shown in SEQ ID NO.4.

[0018] Pyruvate carboxylase can catalyze the carboxylation of pyruvate to generate oxaloacetate. Overexpression of pyruvate carboxylase can increase the supply of oxaloacetate, thereby promoting the synthesis of β-alanine. The coding gene of the pyruvate carboxylase can be derived from Bacillus subtilis 168. pycA .

[0019] Preferably, the amino acid sequence of the aspartate aminotransferase is shown in SEQ ID NO.5.

[0020] Aspartate aminotransferase can catalyze the synthesis of L-aspartate from fumarate, and overexpression thereof increases the accumulation of L-aspartate, a precursor of β-alanine. The encoding gene of the aspartate aminotransferase can be an aspartate aminotransferase encoding gene derived from Escherichia coli W3110. aspA .

[0021] Preferably, the amino acid sequence of the PLP-dependent aspartate aminotransferase is shown in SEQ ID NO.6.

[0022] Aspartate aminotransferase can catalyze the synthesis of L-aspartate from oxaloacetate, and overexpression thereof increases the accumulation of L-aspartate, a precursor of β-alanine. The coding gene of the PLP-dependent aspartate aminotransferase can be the coding gene of the aspartate aminotransferase derived from Escherichia coli W3110. aspC .

[0023] Preferably, the amino acid sequence of the pyrimidine nucleotide transhydrogenase is shown in SEQ ID NO.7.

[0024] Pyridine nucleotide transhydrogenase can adjust the ratio of NADH and NADPH according to the metabolic needs of the cell, ensure the balance of coenzymes in the cell, maintain normal metabolic activities, and provide reducing power for the synthesis of L-aspartic acid. The coding gene of the pyrimidine nucleotide transhydrogenase can be derived from the pyrimidine nucleotide transhydrogenase coding gene of Escherichia coli W3110. pntAB .

[0025] Preferably, the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.8.

[0026] Glutamate dehydrogenase catalyzes the synthesis of L-glutamate from α-ketoglutarate, and L-glutamate promotes the synthesis of aspartic acid. The coding gene of the glutamate dehydrogenase can be derived from the glutamate dehydrogenase coding gene of Bacillus subtilis 168. rocG .

[0027] Preferably, the amino acid sequence of the phosphoenolpyruvate carboxykinase is as shown in SEQ ID NO.9.

[0028] Phosphoenolpyruvate kinase can catalyze the conversion of phosphoenolpyruvate (PEP) and ADP into pyruvate and ATP, regulate the metabolic flow in the gluconeogenesis pathway, provide the carbon skeleton and energy for the synthesis of β-alanine, and thus participate in protein synthesis and the metabolic regulation of amino acids.

[0029] Preferably, the amino acid sequence of the β-alanine export vector is shown as SEQ ID NO.10.

[0030] The coding gene of the β-alanine export vector can be derived from the β-alanine export vector coding gene of Corynebacterium glutamicum ATCC 13032. Ncgl0580 .

[0031] More preferably, at least one of the genes encoding aspartate α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase and phosphoenolpyruvate kinase uses a promoter of P trc Promoter.

[0032] More preferably, the promoter used for overexpression of the coding gene of the β-alanine export vector is P lpp The promoter, whose nucleotide sequence is shown in SEQ ID NO.11.

[0033] P lpp The promoter is lpp The promoter of the gene, whose transcription level is lower than P trc Promoter. Using P lppThe promoter overexpresses the coding gene of the β-alanine export vector to avoid affecting the growth and metabolism of the bacteria due to its excessive expression level.

[0034] In a second aspect, the present invention also provides a method for constructing a genetically engineered bacterium with high β-alanine production, comprising the following steps: using Escherichia coli W3110 as a starting strain, knocking out the gene encoding the lactose operon repressor protein and the gene encoding the phosphoenolpyruvate carboxykinase in its genome 、 Gene encoding pyruvate dehydrogenase, gene encoding acetate kinase, gene encoding lactate dehydrogenase, gene encoding DNA binding transcription inhibitor, gene encoding 4-aminobutyrate aminotransferase 、 The coding gene of the amino acid uptake carrier protein is integrated; the coding gene of aspartate α-decarboxylase, the coding gene of phosphoenolpyruvate carboxylase, the coding gene of pyruvate carboxylase, the coding gene of aspartate aminotransferase, the coding gene of PLP-dependent aspartate aminotransferase, the coding gene of pyridine nucleotide transhydrogenase, the coding gene of glutamate dehydrogenase, the coding gene of phosphoenolpyruvate carboxykinase derived from Mannheim succinic acid bacteria, and the coding gene of β-alanine export carrier are replaced; the promoter of the coding gene of succinate dehydrogenase is replaced by P trc Promoter; there is no particular order in which the above gene editing can be performed.

[0035] When integrating the above genes, the gene insertion site can be reasonably selected based on the specific operational requirements and the characteristics of the target gene, taking into account factors such as PAM sequence, genomic environment, functional requirements and off-target risks.

[0036] Preferably, the insertion site of the gene encoding aspartate-α-decarboxylase is rph。

[0037] Preferably, the insertion site of the gene encoding phosphoenolpyruvate carboxylase is yjiT。

[0038] Preferably, the pyruvate carboxylase encoding gene insertion site is gltA。

[0039] Preferably, the insertion site of the gene encoding aspartate aminotransferase is mbhA。

[0040] Preferably, the insertion site of the gene encoding the PLP-dependent aspartate aminotransferase is ycgH。

[0041] Preferably, the insertion site of the pyridine nucleotide transhydrogenase encoding gene is gapc。

[0042] Preferably, the glutamate dehydrogenase encoding gene insertion site isilvE。

[0043] Preferably, the insertion site of the gene encoding phosphoenolpyruvate kinase is yeeP。

[0044] Preferably, the coding gene insertion site of the β-alanine export vector is yjiP .

[0045] Preferably, the gene editing technology for knockout, integration and replacement is CRISPR-Cas9 gene editing technology.

[0046] The present invention utilizes gene editing technology to overexpress and knock out the target gene of the starting strain, thereby constructing a genetically engineered bacterium with high beta-alanine production. In this process, the specific order of gene editing can be flexibly adjusted according to experimental requirements, and different editing orders can achieve the expected genetic engineering goals without affecting the performance of the genetically engineered bacterium and the yield of beta-alanine finally constructed.

[0047] Specifically, a method for constructing a genetically engineered bacterium for producing β-alanine is provided, comprising the following steps: (1) Using the Escherichia coli W3110 genome as a template, amplify the gene encoding the lactose operon repressor protein lacI The upstream homology arm and the downstream homology arm of lacI The upstream homology arm and the downstream homology arm are fused to obtain lacI The upstream homology arm and the downstream homology arm overlap fragment U lacI -D lacI ;Change U lacI -D lacI and containing lacI -gRNA vector was transformed into the starting strain Escherichia coli W3110 to obtain lacI Knockout strains E. coli W3110 ∆lacI ; (2) Using the wild-type Escherichia coli W3110 genome as a template, amplify the gene rph The upstream homology arm and downstream homology arm of the aspartate-α-decarboxylase encoding gene were obtained by PCR amplification. panD gene fragment; rph The upstream homology arm, downstream homology arm and panD Gene fragment fusion, get U rph -P trc - panD -D rph ; The resulting fusion fragment U rph - panD -D rph and containing rph -gRNA vector transformationE. coli W3110 ∆lacI strain, gene knocked out rph and in rph Aspartate α-decarboxylase gene panD Recombinant strains of (3) Using the recombinant strain obtained in the previous round as the basis for construction, the same method as step (2) was repeated to construct yjiT Phosphoenolpyruvate carboxylase gene ppc Recombinant strains 、 exist gltA Pyruvate carboxylase gene pycA Recombinant strains 、 exist mbhA Aspartate aminotransferase encoding gene aspA Recombinant strains 、 exist ycgH Site-integrated PLP-dependent aspartate aminotransferase encoding gene aspC The recombinant strains gapc Pyridine nucleotide transhydrogenase encoding gene pntAB The recombinant strains ilvE Glutamate dehydrogenase gene rocG The recombinant strains and yeeP Phosphoenolpyruvate kinase encoding gene pckA Recombinant strains of (4) Using CRISPR-Cas9 gene editing technology, the gene obtained in step (3) was yeeP Phosphoenolpyruvate kinase encoding gene pckA The genome of the recombinant strain sdhABCD The promoter of the gene was replaced by P trc Promoter, and obtain recombinant strain; (5) Knock out the phosphoenolpyruvate carboxykinase encoding gene based on the recombinant strain obtained in step (4) pck、 Pyruvate dehydrogenase encoding gene poxB , acetate kinase encoding gene ackA , lactate dehydrogenase encoding gene ldhA , DNA-binding transcriptional repressor encoding genes iclR , 4-aminobutyrate aminotransferase encoding gene gabT、 Amino acid uptake carrier protein encoding gene cycA , obtain a recombinant strain knocking out the above gene; (6) Based on the recombinant strain obtained in step (5), the same method as in step (2) was used to yjiP Site-integrated gene Ncgl0580, a genetically engineered bacterium with high β-alanine production was constructed.

[0048] Furthermore, in step (2), PCR amplification is performed to obtain the aspartate α-decarboxylase encoding gene panD The gene fragments and the gene fragments obtained by PCR amplification in step (3) are trc Promoter driven; the β-alanine export vector encoding gene obtained by PCR amplification in step (6) Ncgl0580 The gene fragment is composed of P lpp Promoter driven.

[0049] The above genes can be driven by the corresponding promoters by designing primers with corresponding promoter sequences.

[0050] Furthermore, the vector is a pGRB plasmid.

[0051] In a third aspect, the present invention also provides the use of the above-mentioned genetically engineered bacteria with high β-alanine production in the production of β-alanine by microbial fermentation.

[0052] In a fourth aspect, the present invention further provides a method for producing β-alanine by fermentation, using the genetically engineered bacteria mentioned above or the genetically engineered bacteria obtained according to the above construction method for fermentation.

[0053] The high-yield β-alanine genetically engineered bacteria provided by the present invention were used to ferment and produce β-alanine. After 60 h of fermentation in a 5 L fermenter, the β-alanine yield reached 112.2-126.7 g / L, and the conversion rate reached 48.2%-60.3%.

[0054] Preferably, the steps include: (1) inoculating the recombinant Escherichia coli on a seed culture medium to perform seed culture to obtain a seed culture; (2) inoculating the seed culture obtained in step (1) onto a fermentation medium at an inoculum rate of 10% to 20%, adding a glucose solution, and performing aerobic fermentation to obtain a fermentation broth containing β-alanine; The seed culture medium comprises: 15-30 g / L glucose, 2-5 g / L yeast powder, 1-5 g / L tryptone, 1.5-3 g / L KH2PO4, 5-15 mg / L FeSO4·7H2O, 10-12 mg / L MnSO4, and the rest is water; the pH value is 7.0-7.2.

[0055] The fermentation medium comprises: 10-20 g / L glucose, 2-6 g / L yeast powder, 1-5 g / L tryptone, 1-3 g / L KH2PO4, 0.5-2 g / L MgSO4·7H2O, 5-15 mg / L FeSO4·7H2O, 5-15 mg / L MnSO4, and the rest is water; the pH value is 6.8-7.2.

[0056] More preferably, in step (1), the recombinant Escherichia coli is inoculated into a fermentation tank containing a seed culture medium for seed culture, the pH of the fermentation liquid is adjusted to 6.8-7.2, the dissolved oxygen is maintained at 20%, and the ventilation volume is 2-4 m 3 / h, stirring speed 200-800 rpm, culturing at 30-35°C for 6-8 h to obtain seed culture.

[0057] More preferably, in step (2), the seed culture obtained in step (1) is inoculated into a fermentation tank containing a fermentation medium at an inoculation rate of 10%-20%, the fermentation temperature is 30-37°C, the ventilation volume is 2-4 m 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 20%-40%, glucose solution was added to maintain the residual sugar concentration at 0.1%-0.5%, the pH of the fermentation liquid was maintained at 6.8-7.2, and the fermentation period was 60 h.

[0058] More preferably, in step (1), the stirring speed is 200-800 rpm and the culture is carried out at 35° C. for 6 h.

[0059] More preferably, in step (2), the seed culture obtained in step (1) is inoculated with 20% of the seed culture into a fermentation tank filled with fermentation medium, the fermentation temperature is 37°C, the ventilation volume is 2-4 m 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 30%, 80% glucose solution was fed to maintain residual sugar concentration at 0.1%-0.5%, 25% ammonia water was fed to adjust the pH of the fermentation liquid to 6.8-7.2, and the fermentation period was 60 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 The diagram is a schematic diagram of the β-alanine biosynthesis pathway in Escherichia coli and the genetic engineering modification involved in the present invention. DETAILED DESCRIPTION

[0062] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] The Escherichia coli W3110, Bacillus subtilis 168, Corynebacterium glutamicum ATCC13032 and Mannheim succinic acid producing bacteria MBEL55E used in the examples of the present invention are all from the Metabolic Engineering Laboratory of Tianjin University of Science and Technology.

[0064] The recombination kit ClonExpress II One Step Cloning Kit was purchased from Nanjing Novogene Medical Technology Co., Ltd.

[0065] The detection method of amino acids and organic acids in the present invention is: The fermentation broth was centrifuged at 8000×g for 10 min, the supernatant was taken and diluted 20 times with deionized water, and then the fermentation broth was derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene, and the amino acid and organic acid contents were determined by high performance liquid chromatography.

[0066] The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information corresponding to the Protein ID number in NCBI provided in the above-mentioned Summary of the Invention section, or amplified using the microbial genome of the corresponding source as a template. The primers used in the embodiments of the present invention are shown in Table 1: Table 1 Primer sequences used in the examples of the present invention

[0067] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0068] Example 1 This embodiment provides a genetically engineered bacterium that produces high β-alanine, and the construction method thereof comprises the following steps: (1) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers lac-1 / lac-2 and lac-3 / lac-4 were used to amplify lacI The upstream homology arm and the downstream homology arm are then overlapped by PCR to obtain lacI Fusion fragment U of the upstream homology arm and the downstream homology armlacI -D lacI .

[0069] (2) Based on lacI Sequence design and synthesis of gRNA 20 bp forward sequence pGRB-lacI-S and reverse sequence pGRB-lacI-A, the two were annealed and connected to plasmid pGRB using the recombination kit ClonExpress II One Step Cloning Kit and transformed into E. coli The recombinant plasmid pGRB- lacI .

[0070] (3) Recombinant plasmid pGRB- lacI and fusion fragment U lacI -D lacI Electroporation into the pREDcas9 plasmid E. coli After resuscitation, W3110 competent cells were plated on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and cultured at 32°C overnight. The next day, colony PCR was performed using primers lac-1 / lac-4 to screen positive transformants. The transformants were activated, and arabinose was added at a final concentration of 0.2 mmol / L. The cells were shaken and cultured at 32°C overnight to make pGRB- lacI Loss; then shake culture at 42°C overnight to lose the pREDcas9 plasmid and obtain knockout lacI Recombinant strains E. coli W3110 ∆ lacI .

[0071] (4) Using the E. coli W3110 genome as a template, primers rph-1 / rph-2 and rph-3 / rph-4 were used to amplify rph The upstream and downstream homology arms of the gene were amplified by PCR using the genome of Bacillus subtilis 168 as a template and primers panD-1 / panD-2. trc Promoter driven panD Gene fragment P trc - panD PCR products were recovered and overlapped PCR was performed to obtain rph Upstream homology arm, downstream homology arm and P trc - panD Fusion fragment U rph -P trc - panD -D rph .

[0072] (5) Based on rph Sequence design and synthesis of gRNA 20 bp forward sequence PG-1 and reverse sequence PG-2, and obtain the recombinant plasmid pGRB1 using the same method as step (2).

[0073] (6) Recombinant plasmid pGRB1 and fusion fragment U rph -P trc - panD -D rph Electroporation into the pREDcas9 plasmid E. coli W3110 ∆lacI After recovery, the competent cells were spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin and cultured at 32°C overnight. The next day, colony PCR was performed using primers rph-1 / rph-4 to screen positive transformants. The pGRB1 plasmid and pREDcas9 plasmid were lost in the positive transformants using the same method as step (3) to obtain overexpression panD The recombinant strain BA-1.

[0074] (7) Using the E. coli W3110 genome as a template, primers yjiT-1 / yjiT-2 and yjiT-3 / yjiT-4 were used to amplify yjiT The upstream and downstream homology arms of P were amplified using primers ppc-1 / ppc-2. trc Promoter driven ppc Gene fragment P trc - ppc After the PCR products were recovered, overlapping PCR was performed to obtain yjiT Upstream homology arm, downstream homology arm and P trc - ppc Fusion fragment U yjiT -P trc - ppc -D yjiT .

[0075] (8) According to yjiT Sequence design and synthesis of gRNA 20 bp forward sequence PG-3 and reverse sequence PG-4, and obtain recombinant plasmid pGRB 2 using the same method as step (2).

[0076] (9) Recombinant plasmid pGRB2 and fusion fragment U yjiT -P trc - ppc -D yjiTElectrotransform into BA-1 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjiT-1 / yjiT-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 2 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression ppc The recombinant strain BA-2.

[0077] (10) Using the E. coli W3110 genome as a template, primers gltA-1 / gltA-2 and gltA-3 / gltA-4 were used to amplify gltA The upstream and downstream homology arms of P were amplified using the genome of Bacillus subtilis 168 as a template and primers pycA-1 / pycA-2. trc Promoter driven pycA Gene fragment P trc - pycA After the PCR products were recovered, overlapping PCR was performed to obtain gltA Upstream homology arm, downstream homology arm and P trc - pycA Fusion fragment U gltA -P trc - pycA -D gltA .

[0078] (11) According to gltA Sequence design and synthesis of gRNA 20 bp forward sequence PG-5 and reverse sequence PG-6, and obtain the recombinant plasmid pGRB 3 using the same method as step (2).

[0079] (12) The recombinant plasmid pGRB3 and the fusion fragment U gltA -P trc - pycA -D gltA Electrotransform into BA-2 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers gltA-1 / gltA-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 3 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression pycA The recombinant strain BA-3.

[0080] (13) Using the genome of Escherichia coli W3110 as a template, primers mbhA-1 / mbhA-2, mbhA-3 / mbhA-4, and aspA-1 / aspA-2 were used to amplify mbhA The upstream homology arm, downstream homology arm and P trc Promoter driven aspA Gene fragment P trc - aspA After the PCR products were recovered, overlapping PCR was performed to obtain mbhA Upstream homology arm, downstream homology arm and P trc - aspA Fusion fragment U mbhA -P trc - aspA- D mbhA .

[0081] (14) According to mbhA Sequence design and synthesis of gRNA 20 bp forward sequence PG-7 and reverse sequence PG-8, and obtain the recombinant plasmid pGRB 4 using the same method as step (2).

[0082] (15) The recombinant plasmid pGRB4 and the fusion fragment U mbhA -P trc - aspA- D mbhA Transform into competent cells of BA-3 containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers mbhA-1 / mbhA-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 4 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression aspA The recombinant strain BA-4.

[0083] (16) Using the E. coli W3110 genome as a template, primers ycgH-1 / ycgH-2, ycgH-3 / ycgH-4, and aspC-1 / aspC-2 were used to amplify ycgH The upstream homology arm, downstream homology arm and P trc Promoter driven aspC Gene fragment P trc - aspC After the PCR products were recovered, overlapping PCR was performed to obtain ycgH Upstream homology arm, downstream homology arm and P trc - aspC Fusion fragment U ycgH -P trc - aspC -DaspC .

[0084] (17) According to ycgH Sequence design and synthesis of gRNA 20 bp forward sequence PG-9 and reverse sequence PG-10, and obtain the recombinant plasmid pGRB 5 using the same method as step (2).

[0085] (18) The recombinant plasmid pGRB5 and the fusion fragment U ycgH -P trc - aspC -D aspC Electrotransform into BA-4 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers ycgH-1 / ycgH-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 5 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression aspC The recombinant strain BA-5.

[0086] (19) Using the E. coli W3110 genome as a template, primers gapc-1 / gapc-2 and gapc-3 / gapc-4 were used to amplify gapc The upstream and downstream homology arms of the gene were amplified using primers pntAB-1 / pntAB-2. trc Promoter driven pntAB Gene fragment P trc - pntAB After the PCR products were recovered, overlapping PCR was performed to obtain pntAB Upstream homology arm, downstream homology arm and P trc - pntAB Fusion fragment U gapc -P trc - pntAB- D gapc .

[0087] (20) According to gapc Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-11 and reverse sequence PG-12, and the recombinant plasmid pGRB 6 was obtained using the same method as step (2).

[0088] (21) The recombinant plasmid pGRB6 and the fusion fragment U gapc -P trc - pntAB- D gapcTransform into competent cells of BA-5 containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers gapc-1 / gapc-4 to screen positive transformants. Use the same method as step (3) to make the pGRB 6 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression pntAB The recombinant strain BA-6.

[0089] (22) Using the E. coli W3110 genome as a template, primers ilvE-1 / ilvE-2 and ilvE-3 / ilvE-4 were used to amplify ilvE The upstream and downstream homology arms of the gene were amplified using the genome of Bacillus subtilis 168 as a template and primers rocG-1 / rocG-2. trc Promoter driven rocG Gene fragment P trc - rocG After the PCR products were recovered, overlapping PCR was performed to obtain ilvE Gene upstream homology arm, downstream homology arm and P trc - rocG Fusion fragment U ilvE -P trc - rocG- D ilvE .

[0090] (23) According to ilvE Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-13 and reverse sequence PG-14, and the recombinant plasmid pGRB 7 was obtained using the same method as step (2).

[0091] (24) The recombinant plasmid pGRB7 and the fusion fragment U ilvE -P trc - rocG- D ilvE Transform into competent cells of BA-6 containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers ilvE-1 / ilvE-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 7 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression rocG The recombinant strain BA-7.

[0092] (25) Using the E. coli W3110 genome as a template, primers yeeP-1 / yeeP-2 and yeeP-3 / yeeP-4 were used to amplify yeeP The upstream and downstream homology arms of the gene were amplified using the genome of Mannheim succinic acid producing bacteria MBEL55E as a template and primers pckA-1 / pckA-2. trc Promoter driven pckA Gene fragment P trc - pckA After the PCR products were recovered, overlapping PCR was performed to obtain yeeP Gene upstream homology arm, downstream homology arm and P trc - pckA Fusion fragment U yeeP -P trc - pckA- D yeeP .

[0093] (26) According to yeeP Gene sequence design and synthesis of gRNA 20 bp forward sequence PG-15 and reverse sequence PG-16, and the recombinant plasmid pGRB 8 was obtained using the same method as step (2).

[0094] (27) The recombinant plasmid pGRB8 and the fusion fragment U yeeP -P trc - pckA- D yeeP Transform into competent cells of BA-7 containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yeeP-1 / yeeP-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB 8 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression pckA The recombinant strain BA-8.

[0095] (28) Using the E. coli W3110 genome as a template, primers sdhABCD-1 / sdhABCD-2 and sdhABCD-3 / sdhABCD-4 were used to amplify the gene that does not contain its own promoter sequence. sdhABCD The upstream homology arm of the gene and the trc The downstream homology arm of the promoter was recovered and the PCR product was obtained by overlapping PCR. sdhABCD The fusion fragment U of the upstream homology arm and the downstream homology arm of the gene sdhABCD -P trc - D sdhABCD .

[0096] (29) According to sdhABCD The promoter gene sequence was designed and the gRNA 20 bp forward sequence PG-17 and reverse sequence PG-18 were synthesized, and the recombinant plasmid pGRB 9 was obtained using the same method as step (2).

[0097] (30) The recombinant plasmid pGRB9 and the fusion fragment P trc - sdhABCD Transform into competent cells of BA-8 containing pREDcas9 plasmid, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin after recovery, and culture at 32°C overnight. sdhABCD -1 / sdhABCD -4. Perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB9 plasmid and pREDcas9 plasmid in the positive transformants lose and obtain overexpression sdhABCD The recombinant strain BA-9.

[0098] (31) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers pck-1 / pck-2 and pck-3 / pck-4 were used to amplify pck The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. pck Fusion fragment U of the upstream homology arm and the downstream homology arm pck -D pck .

[0099] (32) According to pck Sequence design and synthesis of gRNA 20 bp forward sequence PG-19 and reverse sequence PG-20, and the recombinant plasmid pGRB- pck .

[0100] (33) The recombinant plasmid pGRB- pck and fusion fragment U pck -D pck Electrotransform into BA-9 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification with primers pck-1 / pck-4 to screen positive transformants. Use the same method as step (3) to make pGRB- pck Plasmid and pREDcas9 plasmid lost, knockout obtained pck The recombinant strain BA-10.

[0101] (34) Using wild-type Escherichia coliE. coli W3110 genome was used as template and primers poxB-1 / poxB-2 and poxB-3 / poxB-4 were used to amplify poxB The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. poxB Fusion fragment U of the upstream homology arm and the downstream homology arm poxB -D poxB .

[0102] (35) According to poxB Sequence design and synthesis of gRNA 20 bp forward sequence PG-21 and reverse sequence PG-22, and the recombinant plasmid pGRB- poxB .

[0103] (36) The recombinant plasmid pGRB- poxB and fusion fragment U poxB -D poxB Electrotransform into BA-9 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification with primers poxB-1 / poxB-4 to screen positive transformants. Use the same method as step (3) to make pGRB- poxB Plasmid and pREDcas9 plasmid lost, knockout obtained poxB The recombinant strain BA-11 was constructed.

[0104] (37) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers ackA-1 / ackA-2 and ackA-3 / ackA-4 were used to amplify ackA The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. ackA Fusion fragment U of the upstream homology arm and the downstream homology arm ackA -D ackA .

[0105] (38) According to ackA Sequence design and synthesis of gRNA 20 bp forward sequence PG-23 and reverse sequence PG-24, and the recombinant plasmid pGRB- ackA .

[0106] (39) The recombinant plasmid pGRB- ackA and fusion fragment U ackA -D ackAElectrotransform into BA-11 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers ackA-1 / ackA-4 to screen positive transformants. Use the same method as step (3) to make pGRB- ackA Plasmid and pREDcas9 plasmid lost, knockout obtained ackA The recombinant strain BA-12 was constructed.

[0107] (40) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers ldhA-1 / ldhA-2 and ldhA-3 / ldhA-4 were used to amplify ldhA The upstream homology arm and the downstream homology arm are then overlapped by PCR to obtain l dhA Fusion fragment U of the upstream homology arm and the downstream homology arm ldhA -D ldhA .

[0108] (41) According to ldhA Sequence design and synthesis of gRNA 20 bp forward sequence PG-25 and reverse sequence PG-26, and the recombinant plasmid pGRB- ldhA .

[0109] (42) The recombinant plasmid pGRB- ldhA and fusion fragment U ldhA -D ldhA Electrotransform into BA-12 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers ldhA-1 / ldhA-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make pGRB- ldhA Plasmid and pREDcas9 plasmid lost, knockout obtained ldhA The recombinant strain BA-13 was constructed.

[0110] (43) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers iclR-1 / iclR-2 and iclR-3 / iclR-4 were used to amplify iclR The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. iclR Fusion fragment U of the upstream homology arm and the downstream homology arm iclR -D iclR .

[0111] (44) According to iclR Sequence design and synthesis of gRNA 20 bp forward sequence PG-27 and reverse sequence PG-28, and the recombinant plasmid pGRB- iclR .

[0112] (45) The recombinant plasmid pGRB- iclR and fusion fragment U iclR -D iclR Electrotransform into BA-13 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers iclR-1 / iclR-4 to screen positive transformants. Use the same method as step (3) to make pGRB- iclR Plasmid and pREDcas9 plasmid lost, knockout obtained iclR The recombinant strain BA-14 was constructed.

[0113] (46) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers gabT-1 / gabT-2 and gabT-3 / gabT-4 were used to amplify gabT The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. gabT Fusion fragment U of the upstream homology arm and the downstream homology arm gabT -D gabT .

[0114] (47) According to gabT Sequence design and synthesis of gRNA 20 bp forward sequence PG-29 and reverse sequence PG-30, and the recombinant plasmid pGRB- gabT .

[0115] (48) The recombinant plasmid pGRB- gabT and fusion fragment U gabT -D gabT Electrotransform into BA-14 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification with primers gabT-1 / gabT-4 to screen positive transformants. Use the same method as step (3) to make pGRB- gabT Plasmid and pREDcas9 plasmid lost, knockout obtained gabT The recombinant strain BA-15.

[0116] (49) Using wild-type Escherichia coli E. coli W3110 genome was used as template and primers cycA-1 / cycA-2 and cycA-3 / cycA-4 were used to amplify cycA The upstream homology arm and the downstream homology arm are then obtained by overlapping PCR. cycA Fusion fragment U of the upstream homology arm and the downstream homology arm cycA -D cycA .

[0117] (50) According to cycA Sequence design and synthesis of gRNA 20 bp forward sequence PG-31 and reverse sequence PG-32, and the recombinant plasmid pGRB- cycA .

[0118] (51) The recombinant plasmid pGRB- cycA and fusion fragment U cycA -D cycA Electrotransform into BA-15 competent cells containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, perform colony PCR identification using primers cycA-1 / cycA-4 to screen positive transformants. Use the same method as step (3) to make pGRB- cycA Plasmid and pREDcas9 plasmid lost, knockout obtained gabT The recombinant strain BA-16 was constructed.

[0119] (52) Using the E. coli W3110 genome as a template, primers yjip-1 / yjip-2 and yjip-3 / yjip-4 were used to amplify yjip The upstream homology arm and downstream homology arm of glutamicum ATCC 13032 genome were used as template and primers Ncgl0580-1 / Ncgl0580-2 were used to amplify the P lpp Promoter driven Ncgl0580 Gene fragment P lpp - Ncgl0580 After the PCR products were recovered, overlapping PCR was performed to obtain yjip Upstream homology arm, downstream homology arm and P lpp - Ncgl0580 Fusion fragment U yjip -P lpp - Ncgl0580 -D yjip .

[0120] (53) According to yjipSequence design and synthesis of gRNA 20 bp forward sequence PG-33 and reverse sequence PG-34, and obtain recombinant plasmid pGRB17 using the same method as step (2).

[0121] (54) The recombinant plasmid pGRB17 and the fusion fragment U yjip -P lpp - Ncgl0580 -D yjip Transform into competent cells of BA-16 containing pREDcas9 plasmid, and after recovery, spread on LB solid medium containing 100 μg / mL spectinomycin and ampicillin, and culture at 32°C overnight. The next day, use primers yjip-1 / yjip-4 to perform colony PCR identification and screen positive transformants. Use the same method as step (3) to make the pGRB17 plasmid and pREDcas9 plasmid in the positive transformants lose, and obtain overexpression Ncgl0580 The recombinant strain BA-17 is a genetically engineered bacterium with high β-alanine production.

[0122] The schematic diagram of the genetic modification of the high-yield β-alanine genetic engineering bacteria is as follows Figure 1 shown.

[0123] Example 2 Shake flask fermentation (1) Seed cultivation The β-alanine-producing recombinant strains BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA-7, BA-8, BA-9, BA-10, BA-11, BA-12, BA-13, BA-14, BA-15, BA-16 and BA-17 obtained in Example 1 were inoculated onto the LB solid culture medium slant, with BA-1 as the control, and cultured at 37 ° C for 12 h. Then, single colonies were picked and inoculated into 30 mL seed culture medium, and cultured at 37 ° C, 220 rpm shaking for 8 h. The components of the seed culture medium used were 20 g / L glucose, 5 g / L yeast extract, 2 g / L tryptone, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, and 10 mg / L MnSO4. Add 2.0% phenol red to the seed culture medium as a pH indicator, adjust the pH to 7.0-7.2, and sterilize with high-pressure steam at 115°C for 15 min before use.

[0124] (2) Fermentation culture The fermentation medium was inoculated with 10% inoculum, and the culture was shaken at 35℃ and 220 rpm for 48 h. During the fermentation process, when the glucose in the medium was exhausted, 60% glucose was supplemented 2-3 times to maintain the residual sugar concentration of 0.1%-0.5%, and 1 mL was supplemented each time. The pH was adjusted to 6.9-7.1 with ammonia water. After the fermentation, the β-alanine content in the fermentation broth was detected. The β-alanine content in the fermentation broth of each recombinant strain is shown in Table 2. The fermentation medium used was composed of 20 g / L glucose, 3 g / L yeast extract, 1 g / L tryptone, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, and 10 mg / L MnSO4. 2.0% phenol red was added to the fermentation medium as a pH indicator, and the pH was adjusted to 6.8-7.2. It was sterilized by high-pressure steam at 115℃ for 15 min before use.

[0125] Table 2 β-alanine content in the fermentation broth of the recombinant strain

[0126] From the results in Table 2, it can be seen that the β-alanine content in the fermentation broth of the recombinant strains BA-1 to BA-17 showed an overall upward trend, indicating that the ability of the strains to produce β-alanine gradually increased. Among them, the recombinant strain BA-17 had the strongest ability to produce β-alanine, and its shake flask fermentation production of β-alanine could reach 54.3 mg / L, which was about 9 times that of BA-1.

[0127] Example 3 This embodiment provides a method for producing β-alanine by fermentation using a 5L fermenter.

[0128] (1) Seed cultivation The activated recombinant strain BA-17 (provided in Example 1) was inoculated into a 5 L fermenter containing 3 L of seed culture medium at an inoculation rate of 10%, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, the dissolved oxygen was maintained at 20%, and the ventilation rate was 2-4 m 3 / h, stirring speed 200-700 rpm, culturing at 32℃ for 7 h to obtain seed culture. The seed culture medium used is composed of 15g / L glucose, 3g / L yeast powder, 5g / L tryptone, 1.5g / L KH2PO4, 5mg / L FeSO4·7H2O, 12mg / LMnSO4, pH is adjusted to 7.0-7.2, and high pressure steam sterilization at 115℃ for 15 min before use.

[0129] (2) Fermentation in a fermentation tank The seed culture of step (1) was inoculated with 10% of the inoculum into a 5 L fermentation tank containing 3 L of fermentation medium for fermentation at a temperature of 30 °C and a ventilation volume of 2-4 m 3 / h, stirring speed 300-600 rpm, dissolved oxygen maintained at 20%, 80% glucose solution was added, residual sugar concentration was maintained at 0.1%-0.5%, 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, and the fermentation cycle was 60 h. The fermentation medium used was composed of 10 g / L glucose, 2 g / L yeast powder, 5 g / L tryptone, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4, pH was adjusted to 6.8-7.2, and high pressure steam sterilization at 115℃ for 15 min was used.

[0130] After fermentation, the contents of β-alanine and other amino acids and organic acids in the fermentation broth were detected. The β-alanine content in the fermentation broth of the recombinant strain BA-17 reached 112.2 g / L, the conversion rate reached 48.2%, and no other amino acids and organic acids were detected.

[0131] Example 4 This embodiment provides a method for producing β-alanine by fermentation using a 5L fermenter.

[0132] (1) Seed cultivation The activated recombinant strain BA-17 (provided in Example 1) was inoculated into a 5 L fermenter containing 3 L of seed culture medium at an inoculation rate of 10%, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, the dissolved oxygen was maintained at 20%, and the ventilation rate was 2-4 m 3 / h, stirring speed 200-600 rpm, culturing at 30℃ for 8 h to obtain seed culture. The seed culture medium used is composed of 30g / L glucose, 5g / L yeast powder, 1g / L tryptone, 3g / L KH2PO4, 15mg / L FeSO4·7H2O, 12mg / LMnSO4, pH is adjusted to 7.0-7.2, and high pressure steam sterilization at 115℃ for 15 min before use.

[0133] (2) Fermentation in a fermentation tank The seed culture of step (1) was inoculated with 15% of the inoculum into a 5 L fermentation tank containing 3 L of fermentation medium for fermentation at a temperature of 35 °C and a ventilation volume of 2-4 m 3 / h, stirring speed 300-800 rpm, dissolved oxygen maintained at 40%, 80% glucose solution was added, residual sugar concentration was maintained at 0.1%-0.5%, 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, and the fermentation cycle was 60 h. The fermentation medium used was composed of 20 g / L glucose, 6 g / L yeast powder, 1 g / L tryptone, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 15 mg / L FeSO4·7H2O, 15 mg / L MnSO4, pH was adjusted to 6.8-7.2, and high pressure steam sterilization at 115℃ for 15 min was used.

[0134] After fermentation, the content of β-alanine and other amino acids and organic acids in the fermentation broth was detected. The β-alanine content in the fermentation broth of the recombinant strain BA-17 reached 119.9 g / L, the conversion rate reached 55%, and no other amino acids and organic acids were detected.

[0135] Example 5 This embodiment provides a method for producing β-alanine by fermentation using a 5L fermenter.

[0136] (1) Seed cultivation The activated recombinant strain BA-17 (provided in Example 1) was inoculated into a 5 L fermenter containing 3 L of seed culture medium at an inoculation rate of 10%, and 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, the dissolved oxygen was maintained at 20%, and the ventilation rate was 2-4 m 3 / h, stirring speed 200-800rpm, culturing at 35℃ for 6 hours to obtain seed culture. The seed culture medium used is composed of 25 g / L glucose, 3 g / L yeast powder, 4 g / L tryptone, 2 g / L KH2PO4, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4, pH is adjusted to 7.0-7.2, and high pressure steam sterilization at 115℃ for 15 minutes before use.

[0137] (2) Fermentation in a fermentation tank The seed culture of step (1) was inoculated with 20% of the inoculum into a 5 L fermentation tank containing 3 L of fermentation medium for fermentation at a temperature of 37 °C and a ventilation volume of 2-4 m 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 30%, 80% glucose solution was added, residual sugar concentration was maintained at 0.1%-0.5%, 25% ammonia water was added to adjust the pH of the fermentation liquid to 6.8-7.2, and the fermentation cycle was 60 h. The fermentation medium used was composed of 15 g / L glucose, 3.5 g / L yeast powder, 4 g / L tryptone, 2 g / L KH2PO4, 1.5 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4, pH was adjusted to 6.8-7.2, and high pressure steam sterilization at 115℃ for 15 min was used.

[0138] After fermentation, the content of β-alanine and other amino acids and organic acids in the fermentation broth was detected. The β-alanine yield in the fermentation broth of the recombinant strain BA-17 reached 126.7 g / L, and the conversion reached 60.3%. No other amino acids and organic acids were detected.

[0139] In summary, the present invention is based on E. coli W3110 is the starting strain. By replacing the promoter, the β-alanine synthesis pathway is dynamically regulated to balance cell growth and β-alanine production. The β-alanine synthesis flux is enhanced, the NADPH level is optimized, and the β-alanine transport system is modified by means of system metabolic engineering. Finally, by blocking the β-alanine intake pathway, a non-nutrition-deficient, inducer-free, plasmid-free high-yield β-alanine genetically engineered bacterium is constructed. At the same time, the fermentation process of β-alanine fermentation using this strain is simple, easy to control, and has low production cost, which is conducive to the promotion and application of industrial production.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for high production of β-alanine, characterized in that: The genetically engineered bacteria is obtained by performing gene editing on the genome of Escherichia coli W3110 with the Escherichia coli W3110 as the starting strain; the genetically engineered bacteria does not express lactose operon repressor protein, pyruvate dehydrogenase, acetate kinase, lactate dehydrogenase, DNA binding transcription inhibitor, 4-aminobutyrate aminotransferase and amino acid uptake carrier protein; overexpresses aspartate-α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase, succinate dehydrogenase and β-alanine export carrier; and replaces its own phosphoenolpyruvate carboxykinase with phosphoenolpyruvate carboxykinase derived from Mannheim succinic acid producing bacteria.

2. The genetically engineered bacterium according to claim 1, characterized in that: The overexpression of succinate dehydrogenase is carried out by replacing the promoter of its own gene with P trc promoter, the P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.1; and / or The aspartate-α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase 、 Overexpression of phosphoenolpyruvate kinase and β-alanine export vectors is achieved by integrating the coding genes of the corresponding enzymes themselves or heterologously into the genome of the starting strain.

3. The genetically engineered bacterium according to claim 1, characterized in that The amino acid sequence of the aspartate-α-decarboxylase is shown in SEQ ID NO.2; and / or The amino acid sequence of the phosphoenolpyruvate carboxylase is shown in SEQ ID NO.3; and / or The amino acid sequence of the pyruvate carboxylase is shown in SEQ ID NO.4; and / or The amino acid sequence of the aspartate aminotransferase is shown in SEQ ID NO.5; and / or The amino acid sequence of the PLP-dependent aspartate aminotransferase is shown in SEQ ID NO.6; and / or The amino acid sequence of the pyrimidine nucleotide transhydrogenase is shown in SEQ ID NO.7; and / or The amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.8; and / or The amino acid sequence of the phosphoenolpyruvate carboxykinase is shown in SEQ ID NO.9; and / or The amino acid sequence of the β-alanine export vector is shown in SEQ ID NO.

10.

4. The genetically engineered bacteria according to claim 2, characterized in that: At least one of the genes encoding aspartate α-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, PLP-dependent aspartate aminotransferase, pyridine nucleotide transhydrogenase, glutamate dehydrogenase and phosphoenolpyruvate kinase uses a promoter of P trc promoter; and / or The promoter used by the gene encoding the β-alanine export vector is P lpp The promoter, whose nucleotide sequence is shown in SEQ ID NO.

11.

5. A method for constructing a genetically engineered bacterium with high β-alanine production, characterized in that: The following steps are involved: Escherichia coli W3110 was used as the starting strain, and the gene encoding the lactose operon repressor protein and the gene encoding phosphoenolpyruvate carboxykinase were knocked out from its genome. 、 Gene encoding pyruvate dehydrogenase, gene encoding acetate kinase, gene encoding lactate dehydrogenase, gene encoding DNA binding transcription inhibitor, gene encoding 4-aminobutyrate aminotransferase 、 The coding gene of the amino acid uptake carrier protein was integrated with the coding gene of aspartate-α-decarboxylase, the coding gene of phosphoenolpyruvate carboxylase, the coding gene of pyruvate carboxylase, the coding gene of aspartate aminotransferase, the coding gene of PLP-dependent aspartate aminotransferase, the coding gene of pyridine nucleotide transhydrogenase, the coding gene of glutamate dehydrogenase, the coding gene of phosphoenolpyruvate carboxykinase derived from Mannheim succinic acid producing bacteria and the coding gene of β-alanine export carrier, and the promoter of the coding gene of succinate dehydrogenase was replaced with P trc Promoter; there is no particular order in which the above gene editing can be performed.

6. The method for constructing a genetically engineered bacterium with high β-alanine production according to claim 5, characterized in that: The insertion site of the aspartate α-decarboxylase coding gene is r ; and / or The insertion site of the phosphoenolpyruvate carboxylase encoding gene is Yj ; and / or The insertion site of the pyruvate carboxylase coding gene is gltA ; and / or The insertion site of the aspartate aminotransferase coding gene is mbA ; and / or The insertion site of the gene encoding the PLP-dependent aspartate aminotransferase is HkDJ ; and / or The insertion site of the pyridine nucleotide transhydrogenase encoding gene is gapc ; and / or The insertion site of the glutamate dehydrogenase coding gene is ilvE ; and / or The insertion site of the coding gene of phosphoenolpyruvate kinase is yeeP ; and / or The coding gene insertion site of the β-alanine export vector is Yj .

7. Use of the genetically engineered bacterium with high β-alanine production according to any one of claims 1 to 4 in the production of β-alanine by microbial fermentation.

8. A method for producing β-alanine by fermentation, characterized in that: The fermentation is carried out using the genetically engineered bacteria according to any one of claims 1 to 4 or the genetically engineered bacteria obtained by the construction method according to claim 5 or 6.

9. The method according to claim 8, characterized in that The following steps are involved: (1) inoculating the recombinant Escherichia coli on a seed culture medium to perform seed culture to obtain a seed culture; (2) inoculating the seed culture obtained in step (1) onto a fermentation medium at an inoculum rate of 10% to 20%, adding a glucose solution, and performing aerobic fermentation to obtain a fermentation broth containing β-alanine; The seed culture medium comprises: 15-30 g / L glucose, 2-5 g / L yeast powder, 1-5 g / L tryptone, 1.5-3 g / L KH2PO4, 5-15 mg / L FeSO4·7H2O, 10-12 mg / L MnSO4, and the rest is water; the pH value is 7.0-7.

2. The fermentation medium comprises: 10-20 g / L glucose, 2-6 g / L yeast powder, 1-5 g / L tryptone, 1-3 g / L KH2PO4, 0.5-2 g / L MgSO4·7H2O, 5-15 mg / L FeSO4·7H2O, 5-15 mg / L MnSO4, and the rest is water; the pH value is 6.8-7.

2.

10. The method according to claim 9, characterized in that In step (1), the recombinant Escherichia coli is inoculated into a fermentation tank containing a seed culture medium for seed culture, the pH of the fermentation liquid is adjusted to 6.8-7.2, the dissolved oxygen is maintained at 20%, and the ventilation volume is 2-4 m 3 / h, stirring speed 200-800 rpm, culturing at 30-35°C for 6-8 h to obtain a seed culture; and / or In step (2), the seed culture obtained in step (1) is inoculated into a fermentation tank filled with fermentation medium at an inoculation rate of 10%-20%, the fermentation temperature is 30-37°C, and the ventilation volume is 2-4 m 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 20%-40%, glucose solution was added to maintain the residual sugar concentration at 0.1%-0.5%, the pH of the fermentation liquid was maintained at 6.8-7.2, and the fermentation period was 60h.

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