Escherichia coli probiotics with high yield of beta-alanine and application of escherichia coli probiotics

Abstract

The invention discloses Escherichia coli probiotics with high yield of beta-alanine and application of the Escherichia coli probiotics. The recombinant genetically engineered bacterium takes wild type Escherichia coli probiotics as a starting strain, and edits one or more of the following genes: a Trc promoter enhanced panD gene, an aspC gene, an sdhC gene and a ppc gene; a panD gene from bacillus subtilis, an aspB gene and an aspA gene from corynebacterium glutamicum and a pyc gene from corynebacterium glutamicum are over-expressed on a plasmid pTrc99a, and a cycA gene is knocked out. Compared with the existing reported Escherichia coli probiotic beta-alanine production strain, the OD600 of the recombinant genetic engineering strain is 4.4 times of that of the existing reported strain, the yield of beta-alanine is 6 times of that of the existing reported strain, and the recombinant genetic engineering strain has huge industrial application potential.

Description

(I) Technical field

[0001] The invention relates to an Escherichia coli probiotic with high beta-alanine production and application thereof. (II) Background technology

[0002] β-alanine, also known as 3-aminopropionic acid, is the only β-type amino acid and non-protein 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 metabolism, β-alanine can be used as a precursor to synthesize pantothenic acid (vitamin B5) and coenzyme A (CoA), which is related to a variety of metabolic activities such as fatty acid metabolism and sugar metabolism. Plants and microorganisms can synthesize β-alanine autonomously, while mammals need exogenous supplements. According to current research, β-alanine has important application value in many fields such as food, medicine, chemical industry and feed.

[0003] In the pharmaceutical field, β-alanine is a key precursor for the production of pantothenic acid, calcium pantothenate, coenzyme A (CoA), and acyl carrier protein (ACP). It plays an important role in helping cell metabolism and the development of the central nervous system. At the same time, β-alanine can also be used to synthesize drugs such as balsalazide and pamidronate disodium. In the food field, β-alanine can be used as a food additive and is often used in food flavors to improve food flavor. At the same time, due to its antioxidant properties, it is also often used as a food preservative. According to relevant clinical studies, β-alanine can also be used as a nutritional supplement for athletes. After supplementing β-alanine, the human body can significantly improve fatigue caused by long-term high-intensity exercise and improve the body's tolerance. In the chemical industry, β-alanine can not only be used as a precursor to produce chemical products such as pantothenic acid and calcium pantothenate, as well as poly 3-hydroxypropionate and poly β-alanine, but can also be used as a precipitant in medicines, an electroplating corrosion inhibitor, and a lead poisoning antidote. In the field of feed, β-alanine is increasingly used in animal husbandry. Studies have found that adding β-alanine to feed can improve livestock and poultry's utilization of feed and reduce feed intake; it can also improve muscle antioxidant capacity, increase the content of myogenic active peptides, and effectively improve meat quality. In addition, β-alanine also plays an important role in the fields of environment and cosmetics.

[0004] At present, there are three main methods for producing β-alanine at home and abroad: (1) Chemical synthesis, that is, using nitrile substances such as acrylonitrile, acrylic acid and β-aminopropionitrile to synthesize β-alanine under high pressure and high temperature under strong acid and strong base conditions. However, the chemical synthesis method consumes a lot of energy, has high requirements for equipment, and produces substances that are harmful to the environment and human body during the production process. At the same time, the by-products produced will bring great difficulties to the subsequent separation and purification. (2) Bioenzyme catalysis, using aspartic acid as a substrate, using aspartate decarboxylase from strains such as Bacillus subtilis or Corynebacterium glutamicum to express in Escherichia coli to catalyze the production of β-alanine. This method has milder conditions, is safer and less polluting, and has attracted the attention of more scholars. However, the bioenzyme catalysis method is unstable, costly, and easily inhibited by substrates. (3) Microbial fermentation method produces β-alanine. The microbial fermentation method uses synthetic biology, system metabolic engineering, protein engineering, transcriptome and metabolome technologies to transform the metabolic process of the target product β-alanine, so that more metabolic flow flows to the direction of the target product. As climate change and environmental problems become increasingly severe, the use of cheap glucose and other carbon sources to explore clean, environmentally friendly and low-energy production methods has attracted the attention of many scholars.

[0005] With the development of biotechnology, the emergence of system metabolic engineering technology has made humans no longer limited to traditional strain screening technologies such as mutagenesis and directed evolution. Through quantitative analysis of strain metabolic pathways and metabolic fluxes, targeted metabolic pathway transformation can be carried out on this basis to maximize the production efficiency of target metabolites and significantly improve the production performance of strains. Different from traditional breeding technology, it is an effective method to improve the genetic traits of organisms by rationally designing cell metabolic pathways and reconstructing metabolic networks.

[0006] In the current research hotspots, strains such as Escherichia coli, yeast, Corynebacterium glutamicum, and Bacillus subtilis are usually used as chassis bacteria to construct engineered strains. The metabolic genetic background and physiological characteristics of these chassis strains are relatively clear. Combining mature gene editing technology and the rational design of metabolic flux can greatly accelerate the transformation of strain metabolic pathways and shorten the time to achieve the optimal production performance of strains. As the most clearly studied prokaryotic bacteria, Escherichia coli has the characteristics of rapid reproduction, short fermentation cycle, high expression level of target genes, and mature and perfect expression system. It has been widely used in the industrial production of various biological products. Escherichia coli probiotics Escherichia coli Nissle 1917, referred to as EcN. As a subspecies of Escherichia coli, it is a Gram-negative bacterium. In addition to the advantages of Escherichia coli itself, EcN also has biosafety, but it has rarely been studied for the production of industrial metabolites. Therefore, EcN has good application prospects in the fields of metabolic engineering and synthetic biology.

[0007] The de novo synthesis pathway of β-alanine in Escherichia coli probiotics includes glucose uptake, glycolysis pathway, TCA cycle, L-aspartate synthesis and its β-alanine synthesis pathway. First, glucose completes the phosphorylation of glucose under the PTS or non-PTS system to synthesize glucose-6-phosphate, and the carbon flow enters the glycolysis pathway. Phosphoenolpyruvate, the precursor of β-alanine, is synthesized in the glycolysis pathway. Under the catalysis of phosphoenolpyruvate carboxylase, phosphoenolpyruvate generates oxaloacetate. Oxaloacetate is unstable and easy to decompose. It generates aspartate under the catalysis of aspartate aminotransferase, and then generates β-alanine under the catalysis of aspartate decarboxylase. Among them, L-lysine, L-threonine, O-succinylhomoserine and homoserine belong to the aspartate family of amino acids. They and β-alanine have a common precursor L-aspartate, so weakening the competition branch is an effective construction strategy. However, it is still necessary to further explore the bottlenecks that affect the production of β-alanine in Escherichia coli probiotics. E. coli probiotics contain two highly stable cryptic plasmids, namely pMUT1 (NCBI No. MW240712, 3.2kb) and pMUT2 (NCBI No. CP023342, 5.6kb). Due to the presence of these two plasmids, its potential as an engineered bacterium is limited. Not only is the expression of heterologous genes restricted, but the effect of protein expression is also poor. Due to plasmid incompatibility, it is also difficult for exogenous plasmids to be transferred into it and successfully expressed. How to study and transform cryptic plasmids is the key to improving the efficiency of exogenous plasmid transformation. (III) Summary of the invention

[0008] The invention aims to provide an Escherichia coli probiotic with high beta-alanine production and application in preparing beta-alanine by microbial fermentation, thereby laying a foundation for industrial production of beta-alanine.

[0009] The technical solution adopted by the present invention is:

[0010] The invention provides an Escherichia coli probiotic with high beta-alanine production. The Escherichia coli probiotic takes a wild-type Escherichia coli probiotic as a starting strain and performs one or more of the following gene editing: a Trc promoter is used to strengthen a panD gene, an aspC gene, an sdhC gene and a ppc gene; a panD gene derived from Bacillus subtilis, an aspB gene and an aspA gene derived from Corynebacterium glutamicum, and a pyc gene derived from Corynebacterium glutamicum are overexpressed on a plasmid pTrc99a; and a cycA gene is knocked out.

[0011] Furthermore, the nucleotide sequence of the panD gene is shown in SEQ ID NO.3; the nucleotide sequence of the aspC gene is shown in SEQ ID NO.7; the nucleotide sequence of the aspB gene is shown in SEQ ID NO.8; the nucleotide sequence of the aspA gene is shown in SEQ ID NO.9; the nucleotide sequence of the sdhC gene is shown in SEQ ID NO.10; the nucleotide sequence of the ppc gene is shown in SEQ ID NO.11; the nucleotide sequence of the pyc gene is shown in SEQ ID NO.12; the nucleotide sequence of the cycA gene is shown in SEQ ID NO.13; and the nucleotide sequence of the Trc promoter is shown in SEQID NO.14.

[0012] Furthermore, the wild-type Escherichia coli probiotic is E. coli Nissle 1917.

[0013] Furthermore, the Escherichia coli probiotics are based on wild-type Escherichia coli probiotics as the starting strain, and the following gene editing is performed in sequence: Trc promoter strengthens the panD gene, overexpresses the panD gene from Bacillus subtilis on the plasmid pTrc99a, Trc promoter strengthens the aspC gene, overexpresses the aspB gene from Corynebacterium glutamicum on the plasmid pTrc99a, overexpresses the aspA gene on the plasmid pTrc99a, Trc promoter strengthens the sdhC gene, Trc promoter strengthens the ppc gene, introduces exogenous PC catalytic reaction, and knocks out the cycA gene.

[0014] The E. coli probiotics of the present invention are constructed as follows:

[0015] (1) Trc promoter strengthens panD gene: Using the genome of E. coli Nissle 1917 as a template, Trc promoter was designed as homology arm on primers, and panD-FF / panD-FR and panD-RF / panD-RR were used as primers to amplify the upstream and downstream homology arms of the original promoter of panD gene, and fused to obtain donor DNA; using pTarget plasmid as a template, pTarget-panD-F and pTarget-panD-R were used as primers to amplify the plasmid and linearize it to obtain linearized pTarget-panD plasmid, and donor DNA and linearized pTarget-panD plasmid were connected and transformed into competent cells of wild-type Escherichia coli probiotics E. coli Nissle1917 (denoted as starting strain E0) into which pCas9 plasmid had been introduced, pTarget and pCas9 plasmids were eliminated, and strain E1 (Trc-panD) was constructed.

[0016] (2) Overexpression of the codon-optimized panD gene from Bacillus subtilis on plasmid pTrc99a: Using plasmid pTrc99a as a template and pTrc99a-line-F / pTrc99a-line-R as primers, linearized plasmid pTrc99a-line was obtained; BS The pTrc99a plasmid was used as a template and panD BS -F / panD BS -R is the primer to obtain the fragment panD BS , linearized plasmid pTrc99a-line and panD BS Gene fragment ligation to construct plasmid pTrc99a-panD BS The cells were transformed into competent cells of strain E1 (Trc-panD) to construct strain E4 (Trc-panD / pTrc99a-panD BS ).

[0017] (3) In the plasmid pTrc99a-panD BS Overexpression of the codon-optimized aspB gene from Corynebacterium glutamicum: plasmid pTrc99a-panD BS As a template, pTrc99a-D BS -line-F / pTrc99a-D BS -line-R is the primer to obtain the linearized plasmid pTrc99a-panD BS -line; containing codon-optimized heterologous aspB CG pTrc99a plasmid, with aspB CG -F / aspB CG -R is the primer to obtain fragment aspB CG , linearized plasmid pTrc99a-panD BS -line and aspB CG Gene fragment ligation to construct plasmid pTrc99a-panD BS -aspB CG and transformed into strain E8 (Trc-panDTrc-aspC / pTrc99a-panD BS ) competent cells, construct strain E10 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG ).

[0018] (4) In plasmid pTrc99a-panD BS -aspB CG Overexpression of aspA gene: plasmid pTrc99a-panD BS -aspB CG As a template, pTrc99a-D BS B CG -line-F / pTrc99a-D BS B CG -line-R is the primer to obtain the linearized plasmid pTrc99a-panD BS -aspB CG -line; using the wild-type E. coli probiotic E. coli Nissle 1917 genome as a template, using aspA-F / aspA-R as primers to obtain the fragment aspA, and linearizing the plasmid pTrc99a-panD BS -aspB CG -line and aspA gene fragment to construct plasmid pTrc99a-panD BS -aspB CG -aspA and transformed into strain E10 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG ) competent cells, construct strain E11 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG -aspA).

[0019] (5) Trc promoter enhancement of sdhC gene: Using the genome of E. coli Nissle 1917 as a template, the Trc promoter was designed as a homology arm on the primers, and sdhC-FF / sdhC-FR and sdhC-RF / sdhC-RR were used as primers to amplify the upstream and downstream homology arms of the original promoter of the sdhC gene, respectively, and fused to obtain donor DNA; using the pTarget plasmid as a template, pTarget-sdhC-F and pTarget-sdhC-R were used as primers to amplify the plasmid and linearize it to obtain a linearized pTarget-sdhC plasmid. The donor DNA and the linearized pTarget-sdhC plasmid were connected and transformed into the strain E11 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG-aspA) competent cells, pTarget and pCas9 plasmids were eliminated, and strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD BS -aspB CG -aspA).

[0020] (6) Trc promoter to strengthen ppc gene: Using the genome of E. coli Nissle 1917 as a template, the Trc promoter was designed as a homology arm on the primers, and ppc-FF / ppc-FR and ppc-RF / ppc-RR were used as primers to amplify the upstream and downstream homology arms of the original promoter of the ppc gene, and fused to obtain donor DNA; using pTarget plasmid as a template, pTarget-ppc-F and pTarget-ppc-R were used as primers to amplify the plasmid and linearize it to obtain a linearized pTarget-ppc plasmid. The donor DNA and the linearized pTarget-ppc plasmid were connected and transformed into the strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD BS -aspB CG -aspA) competent cells, pTarget and pCas9 plasmids were eliminated, and strain E13 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA).

[0021] (7) In plasmid pTrc99a-panD BS -aspB CG -aspA overexpression of the codon-optimized pyc gene from Corynebacterium glutamicum: plasmid pTrc99a-panD BS -aspB CG -aspA as template, using pTrc99a-D BS B CG A-line-F / pTrc99a-D BS B CG A-line-R is the primer to obtain the linearized plasmid pTrc99a-panD BS -aspB CG -aspA-line; containing codon-optimized heterologous pyc CG The pTrc99a plasmid was used as a template and pyc CG -F / pycCG -R is the primer to obtain the fragment pyc CG , linearized plasmid pTrc99a-panD BS -aspB CG -aspA-line and pyc CG Gene fragment ligation to construct plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG and transformed into strain E13 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA) competent cells, construct strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0022] (8) Knockout of cycA gene: Using the genome of E. coli Nissle 1917 as a template, cycA-FF / cycA-FR and cycA-RF / cycA-RR as primers were used to amplify the upstream and downstream homologous arms of the cycA gene, and fused them to obtain donor DNA; using the pTarget plasmid as a template, pTarget-cycA-F and pTarget-cycA-R were used to amplify the plasmid and linearize it to obtain a linearized pTarget-cycA plasmid. The donor DNA and the linearized pTarget-cycA plasmid were connected and transformed into the strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ) competent cells, pTarget and pCas9 plasmids were eliminated, and strain E15 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc△cycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0023] The present invention transforms the β-alanine synthesis network of EcN. The key enzyme of β-alanine biosynthesis is aspartate-α-decarboxylase (ADC, encoded by panD gene). Since the catalytic activity of endogenous ADC in EcN is not high, it can only meet the needs of normal growth and limit the accumulation of β-alanine, so it is not suitable for constructing β-alanine production strains. At the same time, in EcN, ADC catalyzes aspartate to be converted into β-alanine, and aspartate is a precursor substance for the synthesis of threonine, lysine and homoserine. Therefore, in the process of constructing β-alanine production strains, the panD gene from Bacillus subtilis with excellent performance is screened out, which can provide sufficient metabolic pulling force for the synthesis of β-alanine, thereby enhancing the competition for the precursor substance aspartate. L-aspartic acid, as a direct precursor for the synthesis of β-alanine, can be synthesized in EcN through two routes, the oxaloacetic acid transamination route, i.e., the TCA reduction branch pathway, and the fumaric acid amination route, i.e., the TCA oxidation branch pathway. In the oxaloacetate transamination route, the intermediate oxaloacetate of the TCA cycle uses L-glutamate as an amino donor and can generate L-aspartate under the catalysis of aspartate transaminase (encoded by the aspC gene). In the fumarate amination route, aspartate ammonia lyase (encoded by the aspA gene) catalyzes the reversible conversion of the intermediate fumarate of the TCA cycle with aspartate. This route is one of the effective means to extract carbon flux from the TCA cycle. The transamination reaction catalyzed by the aspC gene is affected by the substrate, product and cofactor in the reaction system, and the enzyme activity is not high. By overexpressing the aspB gene from Corynebacterium glutamicum on the plasmid, oxaloacetate can be efficiently catalyzed into aspartate. At the same time, overexpressing the aspA gene on the plasmid can strengthen the amination route of fumarate, reduce the carbon flux residing in the TCA cycle, and prevent the accumulation of intermediates in the TCA cycle. By strengthening succinate dehydrogenase (encoded by the sdhC gene), carbon flux can be extracted from the TCA cycle to enrich the aspartate substrate pool. By strengthening phosphoenolpyruvate carboxylase (encoded by the ppc gene), phosphoenolpyruvate PEP is directly converted into oxaloacetate, thereby reducing the carbon flux entering the oxidative branch of the TCA cycle. During the glucose uptake process, PTS is used as a phosphate donor, and part of PEP is consumed in PTS to form pyruvate, referred to as PYR. However, using ATP instead of PEP as a phosphate donor will increase the metabolic burden in the cell. At the same time, the accumulation of pyruvate is one of the main reasons for carbon loss during β-alanine synthesis.Therefore, in order to reduce carbon loss during β-alanine synthesis and use pyruvate for the synthesis of β-alanine, the effective distribution of carbon flux at the PEP and PYR nodes is the key to promoting the synthesis of the precursor oxaloacetate. By introducing exogenous PC catalytic reactions, the carbon flux at the PEP / PYR nodes is redistributed, which can not only directly enrich the carbon flux for β-Ala synthesis and reduce carbon loss, but also avoid the metabolic burden and increase in organic acids caused by replacing PTS with non-PTS. At the same time, the β-alanine transporter (encoded by the cycA gene) is knocked out to further increase the production of β-alanine.

[0024] The present invention also relates to the use of the Escherichia coli probiotics in the preparation of β-alanine by microbial fermentation, wherein the use is as follows: the Escherichia coli probiotics are inoculated into a fermentation medium containing 50 mg / L kanamycin, and the bacteria are cultured at 30°C and 180 rpm to grow to an OD of 600 =0.5, at this time, IPTG was added to a final concentration of 0.25 mM to induce gene expression, and the culture was continued for 48 h until the fermentation was completed to obtain a fermentation broth containing β-alanine, and the fermentation broth was separated and purified to obtain β-alanine.

[0025] The fermentation medium is formulated as follows: glucose 18-22 g / L, (NH4)2SO4 14-18 g / L, yeast extract 2-3 g / L, KH2PO4 1-2 g / L, MgSO4 0.2-1 g / L, CaCO3 13-17 g / L, VB1 0.3-0.5 mg / L, VB 12 0.3~0.5mg / L, trace element solution 0.5~1.5mL / L, solvent is water, no need to adjust pH; further optimized as: glucose 20g / L, (NH4)2SO4 16g / L, yeast extract 2g / L, KH2PO4 2g / L, MgSO4 0.5g / L, CaCO3 15g / L, 0.5mg / L VB1, 0.2mg / LVB 12 , 1mL / L trace element solution, the solvent is water, and there is no need to adjust the pH value; the components of the trace element solution are as follows: 8-12g / L CaCl2, 8-12g / L FeSO4·7H2O, 0.8-1.2g / L ZnSO4·7H2O, 0.1-0.3g / L CuSO4, 0.01-0.04g / L NiCl2·7H2O, and the solvent is deionized water; further optimized: 10g / L CaCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.2g / LCuSO4, 0.02g / L NiCl2·7H2O, and the solvent is deionized water.

[0026] Before fermentation, the E. coli probiotics were inoculated into LB medium containing 50 mg / L kanamycin at 37° C. and 180 rpm overnight to prepare seed solution, which was inoculated into a fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%.

[0027] The fermentation is carried out in a fermenter: the E. coli probiotics are inoculated onto an LB plate containing 50 mg / L kanamycin resistance, cultured overnight at 37° C., a single colony is picked into an LB test tube containing 50 mg / L kanamycin resistance, cultured overnight at 37° C. and 150-180 rpm to prepare a primary seed solution; the primary seed solution is inoculated into an LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, cultured overnight at 37° C. and 150-180 rpm to serve as a secondary seed solution; and the secondary seed solution is inoculated into an LB medium containing 50 mg / L kanamycin at a volume concentration of 5%. The first-grade seed liquid was inoculated into a 5-L fermentation tank filled with 2L of fermentation medium containing 50mg / L kanamycin at a volume concentration of 15%, and IPTG with a final concentration of 0.2mM was added. The fermentation was carried out at 30°C, 300-500rpm, and aeration volume of 4V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermentation tank was consumed), automatic feeding was started, and feeding medium was added until the pH was lower than 6.80, and feeding was stopped. The culture was carried out for 70-81h to obtain a fermentation broth containing β-alanine.

[0028] The fermentation medium formula in the 5-L fermenter was as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 12 0.2mg / L, 1mL / L trace element solution, the solvent is deionized water; the composition of the trace element solution: 10g / L CaCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.2g / LCuSO4, 0.02g / L NiCl2·7H2O, the solvent is deionized water;

[0029] The feed medium composition: glucose 500g / L, (NH4)2SO4 10g / L, yeast extract 2g / L, KH2PO4 14g / L, MgSO4 8g / L, 0.5mg / L VB1, 0.2mg / L VB 12 The solvent is water, and the pH is adjusted to 6.8 with 14% ammonia water.

[0030] The feeding rate was 25 mL / h, and the total amount of feed medium added was 1700 mL / 2L.

[0031] The fermentation broth separation and purification method comprises the following steps: firstly, centrifuging at 12000 rpm and 4° C. for 10 min to remove the bacteria and other solid particles in the fermentation broth, then using a microfiltration method to remove the macromolecular protein therein, then using a deionization technique to remove the inorganic salt therein, adding activated carbon for decolorization, separating β-alanine with an ion exchange resin, vacuum concentrating, adding ethanol for cooling and crystallization, and obtaining β-alanine crystals.

[0032] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0033] The invention selects a panD gene from Bacillus subtilis with excellent performance, enhances competition for the precursor substance aspartic acid, provides sufficient metabolic pulling force for the synthesis of beta-alanine, overexpresses the aspB gene from Corynebacterium glutamicum on a plasmid, can efficiently catalyze the conversion of oxaloacetate into aspartic acid, and overexpresses the aspA gene on the plasmid to strengthen the amination route of fumaric acid, reduce the carbon flux residing in the TCA cycle, prevent the accumulation of intermediates in the TCA cycle, strengthen the sdhC gene, extract the carbon flux from the TCA cycle to enrich the aspartic acid substrate pool, strengthen the ppc gene, and promote Phosphoenolpyruvate PEP is directly converted into oxaloacetate, thereby reducing the carbon flux entering the oxidation branch of the TCA cycle, while increasing the carbon flow of the key metabolic precursor oxaloacetate. The introduction of exogenous PC catalytic reaction directly enriches the carbon flux of β-Ala synthesis, reduces the loss of central metabolic carbon flow, and avoids the metabolic burden and increase of organic acids caused by replacing PTS with non-PTS. The cycA gene was knocked out, further increasing the production of β-alanine. In batch fed fermentation, the β-alanine production of strain E15 reached 70.4g / L at the 5-L fermenter level, and the sugar-acid conversion rate reached 37%. Compared with the existing reported β-alanine-producing strains of E. coli probiotics, the OD of this E. coli probiotic is 2.3477 g / L, which is 0.33% higher than that of the E. coli probiotics. 600 The yield of β-alanine is 6 times that of the reported strains, which shows great potential for industrial application. (IV) Description of the drawings

[0034] Figure 1 is the biomass OD of strains E0, E1, E2, E3, E4, E5, E6 and E7 600 and bar graphs of β-alanine concentrations.

[0035] Figure 2 is the biomass OD of strains E4, E8, E9, E10 and E11 600 and bar graphs of β-alanine concentrations.

[0036] Figure 3 is the biomass OD of strains E11 and E12 600and bar graphs of β-alanine concentrations.

[0037] Figure 4 is the biomass OD of strains E12 and E13 600 and bar graphs of β-alanine concentrations.

[0038] Figure 5 is the biomass OD of strains E13 and E14 600 and bar graphs of β-alanine concentrations.

[0039] Figure 6 is the biomass OD of strains E14 and E15 600 and bar graphs of β-alanine concentrations.

[0040] Figure 7 is the biomass OD of strain E15 in a 5-L fermenter fed-batch fermentation. 600 , concentration curves of residual sugar and β-alanine. (V) Specific implementation methods

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0042] The wild-type Escherichia coli probiotic strain Escherichia coli Nissle 1917, i.e. the wild strain E0, comes from Hangzhou BioSci Biotech Collection Center, with a collection date of August 5, 2020 and a collection number of BSBCC T0023.

[0043] LB plate composition: 10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 15g / L agar powder, solvent is water, pH value is natural.

[0044] LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0045] The fermentation medium formula is as follows: glucose 20g / L, (NH4)2SO4 16g / L, yeast extract 2g / L, KH2PO42g / L, MgSO4 0.5g / L, CaCO3 15g / L, 0.5mg / L VB1, 0.2mg / L VB 12 , 1mL / L trace element solution, the solvent is water, and there is no need to adjust the pH value; the components of the trace element solution are as follows: 10g / L CaCl2, 10g / L FeSO4·7H2O, 1g / LZnSO4·7H2O, 0.2g / L CuSO4, 0.02g / L NiCl2·7H2O, and the solvent is deionized water.

[0046] Table 1 Genes involved in strain transformation and their corresponding pathways

[0047]

[0048] Table 2 Primers used in gene editing

[0049]

[0050]

[0051]

[0052] Example 1: The panD gene in strain E0 was enhanced by Trc promoter to construct strain E1 (pTarget-Trc panD).

[0053] Using CRISPR / Cas9-mediated gene editing technology, the original promoter of the aspartate-α-decarboxylase panD gene (nucleotide sequence shown in SEQ ID No. 1) on the genome of the wild strain E0 was replaced with the Trc promoter (nucleotide sequence shown in SEQ ID No. 14) derived from pTrc99a to enhance β-alanine accumulation. The specific operation is as follows:

[0054] (1) Construction of pTarget-panD-line plasmid: Using plasmid pTarget as template and pTarget-panD-F / pTarget-panD-R as primers, N20 mutant product was amplified. After adding DpnI to the PCR product, it was digested at 37°C for 1 hour. The methylated plasmid template was digested and purified by purification kit, then transformed into E. coli DH5α competent cells, plated on LB solid plate containing 50 mg / L spectinomycin, and cultured at 37°C for 12 hours. Colony PCR was used for preliminary verification, and then sequencing was used to verify the correctness of pTarget-panD-PAM plasmid to obtain plasmid pTarget-panD-PAM. Then, pTarget-line-F / pTarget-line-R were used as primers and plasmid pTarget-panD-PAM was used as template to amplify plasmid linearized fragment. After adding DpnI to the PCR product, it was digested at 37°C for 3 hours. The DNA fragment was recovered by purification kit to obtain linearized plasmid pTarget-panD-line.

[0055] (2) Construction of the plasmid pTarget-panD containing Donor: Using the genome of E. coli Nissle 1917 as a template, panD-FF / panD-FR as primers were used to amplify the upstream homology arm F1 (the F1 primer contained the Trc promoter sequence); panD-RF / panD-RR as primers were used to amplify the downstream homology arm R1 (the R1 primer contained the Trc promoter sequence). The PCR product was recovered using a purification kit, which was the upstream and downstream fragments of Trc-panD. The upstream and downstream homology arms F1 and R1 were used as templates, and panD-FF / panD-RR was used as primers to fuse the upstream and downstream homology arm DNA fragments containing Trc-panD. The DNA fragments were recovered using a purification kit to obtain the fused fragments. The fused fragment and the plasmid pTarget-panD-line linearized in step (1) were then used to construct the plasmid pTarget-Trc panD through a one-step cloning method, transformed into E. coli DH5α competent cells, spread on LB solid plates containing 50 mg / L spectinomycin, and cultured inverted at 37°C for 12 h. The correct strain was preliminarily screened using primers pTarget-YZ-F / pTarget-YZ-R colony PCR, and finally the correctness of the plasmid pTarget-Trc panD was verified by sequencing.

[0056] (3) The plasmid pCas9 was transformed into competent cells E0, spread on LB solid plates containing 50 mg / L kanamycin, cultured at 30°C overnight, and single colonies were picked into LB test tube culture medium containing 50 mg / L kanamycin resistance, and cultured at 30°C overnight. Then, the cells were inoculated into 100 mL of LB culture medium at a volume concentration of 1%, and kanamycin resistance at a final concentration of 50 mg / L and 10 mM L-arabinose were added, and cultured at 180 rpm and 30°C until the OD 600 = 0.5, centrifuged at 4°C and 4000 rpm. Wash once with a 4°C cold 0.1M calcium chloride aqueous solution, then wash once with a 0.1M calcium chloride solution containing 15% glycerol, and finally resuspend with 15% glycerol calcium chloride by volume concentration, aliquot and store to obtain transfection competent cells containing plasmid pCas9 for later use.

[0057] (4) Take 2 μL of the pTarget-Trc panD plasmid constructed by the method in step (2) and mix it with 100 μL of the competent cells prepared by the method in step (3), put it in an ice bath for 30 minutes, and use a constant temperature water bath (HH-4) for heat shock transformation. The selected temperature is 42°C. After the heat shock is completed, immediately add 600 μL of LB medium pre-cooled at 4°C, mix well, and immediately transfer to 30°C, 150 rpm shaking culture for 3 hours, spread it on LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin, and invert and culture at 30°C for 20 hours. Use panD-YZ-F / panD-YZ-R as primers for colony PCR verification, and sequence to verify the correctness of the strain construction.

[0058] (5) Elimination of pTarget and pCas9 plasmids: Pick the positive single colony in step (4) and inoculate it into a LB liquid culture medium test tube containing 2mM IPTG and 50mg / L kanamycin, culture it overnight at 30℃, dip the bacterial liquid and streak it onto LB solid culture medium containing 50mg / L kanamycin, invert and culture it at 30℃ for 20h until a single colony appears, pick a single colony onto LB solid culture medium containing 50mg / L spectinomycin, invert and culture it at 30℃ for 20h, if no single colony appears, it means that the pTarget plasmid has been eliminated in this strain. Pick the strain that eliminated the pTarget plasmid and inoculate it into a non-antibiotic LB liquid culture medium test tube, culture it at 42℃ for 10h, dip the bacterial liquid and streak it onto LB solid culture medium containing 50mg / L kanamycin, invert and culture it at 37℃ for 10h, if no single colony appears, it means that the pCas9 plasmid has been successfully eliminated, and make it into a plasmid-free competent state for use, and finally obtain the plasmid-free strain E1 (pTarget-Trc panD).

[0059] (6) Using the wild strain E0 as the control bacteria, a single colony of strain E1 was picked into an antibiotic-free LB test tube medium and cultured overnight at 37°C and 180rpm to obtain seed liquid. 1mL of seed liquid was inoculated into a 500mL shake flask containing 50mL of fermentation medium without antibiotics, and cultured at 30°C and 180rpm for 48h. After the fermentation was completed, 1mL of fermentation liquid was centrifuged at 12000rpm for 3min, all the supernatant was discarded, 1mL of distilled water was added to resuspend the bacteria and the calcium carbonate therein, centrifuged at 12000rpm for 3min, and the supernatant was discarded. 1mL of distilled water was added again to resuspend the bacteria and calcium carbonate, and centrifuged at 12000rpm for 3min to discard the supernatant. Finally, 800μL of distilled water was added to resuspend the bacteria and calcium carbonate, and then 200μL of 20% acetic acid aqueous solution was added, and the solution was left at room temperature for 5min to dissolve the calcium carbonate therein. Take 100 μL of the bacterial solution dissolved in calcium carbonate and add it to 1900 μL of distilled water, dilute it 20 times, and finally measure the biomass OD using a spectrophotometer. 600. Take 1 mL of fermentation broth and centrifuge at 12000 rpm for 3 min. Take the supernatant as a sample for detecting β-alanine concentration and set aside. 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E0 600 =12.02, the content of β-alanine was 0 g / L; the biomass OD of strain E1 600 =11.95, the content of β-alanine is 0.01g / L, and the content of β-alanine is not significantly increased.

[0060] (7) Determination of β-alanine concentration:

[0061] 1% 2,4-Dinitrofluorobenzene preparation: Dissolve 1 mL of 2,4-Dinitrofluorobenzene in 99 mL of acetonitrile.

[0062] Preparation of 0.5M NaHCO3 solution: Dissolve 21g NaHCO3 in 500mL deionized water.

[0063] 0.2M PB buffer: weigh 8.74g Na2HPO4·12H2O and 2.43g Na2HPO4·2H2O and dissolve them in 200mL deionized water. Dissolve and store for later use.

[0064] Sample treatment: Dilute the sample with ultrapure water to between 0.1-1 g / L.

[0065] Reaction conditions: Take 100 μL of sample, 100 μL of 0.5 M NaHCO3 solution and 100 μL of 1% 2,4-dinitrofluorobenzene respectively, keep warm at 60°C for 75 min, finally add 700 μL of 0.2 M PB buffer and mix evenly, pass through a membrane (polyvinylidene fluoride, organic membrane, 0.22 μm) for later use.

[0066] Detection conditions: HPLC model: Thermo Scientific Utimate 3000, HPLC detection wavelength is 360nm. β-alanine was separated by gradient elution, wherein mobile phase A component, methanol: acetonitrile: ultrapure water = 45:45:10 (v:v:v); mobile phase B component: 10mM potassium dihydrogen phosphate, pH adjusted to 7.0 with KOH. The elution program was 0-2.5 min 10% A, 90% B; 2.5-2.6 min A 10%→14%, B 90%→86%; 2.6-13 min A 14%→34%, B 86%→66%; 13-13.1 min A34%→38% B 66%→62%; 13.1-28 min A 38%→100% B 62%→0; 28-28.1 min A 100%→10% B 0→90%; 28.1-32 min A 10%→14% B 90%→86%.

[0067] Example 2: Overexpression of endogenous panD in strain E0 on pTrc99a plasmid ECN gene (nucleotide sequence as shown in SEQ ID No. 1), construct strain E2 (Trc-panD / pTrc99a-panD ECN ).

[0068] (1) Construction of pTrc99a-line plasmid: plasmid pTrc99a was used as a template and pTrc99a-line-F / pTrc99a-line-R were used as primers to amplify the linearized fragment of the plasmid. DpnI was added to the PCR product and the methylated vector was digested at 37°C. The product was recovered and purified using a purification kit to obtain the linearized plasmid pTrc99a-line.

[0069] (2) Construction of pTrc99a-panD containing Donor ECN Plasmid: wild-type strain E0 was used as template and panD ECN -F / panD ECN -R is the primer that amplifies panD ECN The fragment was purified and cloned into the linearized vector pTrc99a-line in step (1), transformed into E. coli DH5α competent cells, plated on LB plates containing 50 mg / L kanamycin, and cultured at 37°C for 12 h. Colony PCR was performed using pTrc99a-YZ-F / pTrc99a-YZ-R as primers, and sequencing was performed to verify the correctness of the strain construction. pTrc99a-panD was successfully constructed. ECN Plasmid.

[0070] (3) Transformation plasmid: Extract the constructed pTrc99a-panD ECN The plasmid was transformed into the competent cells of the E1 plasmid-free strain, plated on LB plates containing 50 mg / L kanamycin, and cultured at 37°C for 12 h to construct strain E2 (Trc-panD / pTrc99a-panD ECN ).

[0071] (4) Using strain E1 as the control bacteria, strain E1 was subjected to shake flask fermentation test according to the method of Example 1. A single colony of strain E2 was picked into LB test tube medium containing 50 mg / L kanamycin, and cultured overnight at 37°C and 180 rpm to obtain seed liquid. 1 mL of seed liquid was inoculated into 500 mL shake flasks containing 50 mL fermentation medium and 50 mg / L kanamycin, and the bacteria were cultured at 30°C and 180 rpm to grow to OD 600 =0.5, add IPTG at a final concentration of 0.25 mM, and culture at 30°C and 180 rpm for 48 h. Detect the β-alanine content according to the method in Example 1. Biomass OD 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E1 600 was 11.95, the content of β-alanine was 0.01 g / L; the biomass OD of strain E2 600 It is 11.84, the content of β-alanine is 0.09g / L, and the content of β-alanine is slightly increased.

[0072] Example 3: Overexpression of panD gene from Bacillus tequilensis (nucleotide sequence as shown in SEQ ID No. 2) on pTrc99a plasmid to construct strain E3 (Trc-panD / pTrc99a-panD BT ).

[0073] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method of step (1) of Example 2.

[0074] (2) Construction of pTrc99a-panD containing Donor BT Plasmid: containing codon-optimized heterologous panD BT The pTrc99a plasmid was used as a template and panD BT -F / panD BT -R is the primer that amplifies panD BT The panD fragment was prepared by the method of step (2) of Example 2. BTThe fragment was cloned and verified with the linearized plasmid pTrc99a-line in step (1) to construct pTrc99a-panD BT Plasmid.

[0075] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BT Transformed into E1 plasmid-free strain competent cells to construct strain E3 (Trc-panD / pTrc99a-panD BT ).

[0076] (4) Using strain E2 as the control strain, strains E2 and E3 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E2 600 was 11.84, the content of β-alanine was 0.09 g / L; the biomass OD of strain E3 600 It is 11.37, and the content of β-alanine is 0.23g / L.

[0077] Example 4: Overexpression of panD gene from Bacillus subtilis (nucleotide sequence as shown in SEQ ID No. 3) on pTrc99a plasmid to construct strain E4 (Trc-panD / pTrc99a-panD BS ).

[0078] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method of step (1) of Example 2.

[0079] (2) Construction of pTrc99a-panD containing Donor BS Plasmid: containing codon-optimized heterologous panD BS The pTrc99a plasmid was used as a template and panD BS -F / panD BS -R is the primer that amplifies panD BS The panD fragment was prepared by the method of step (2) of Example 2. BS The fragment was cloned and verified with the linearized plasmid pTrc99a-line in step (1) to construct pTrc99a-panD BS Plasmid.

[0080] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BSTransformed into E1 plasmid-free competent cells to construct strain E4 (Trc-panD / pTrc99a-panD BS ).

[0081] (4) Using strain E2 as the control strain, strains E2 and E4 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E2 600 was 11.84, the content of β-alanine was 0.09 g / L; the biomass OD of strain E4 600 It is 11.63, and the content of β-alanine is 0.4g / L.

[0082] Example 5: Overexpression of panD gene from Caulobacter crescentus (nucleotide sequence shown in SEQ ID No. 4) on pTrc99a plasmid to construct strain E5 (Trc-panD / pTrc99a-panD CC ).

[0083] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method of step (1) of Example 2.

[0084] (2) Construction of pTrc99a-panD containing Donor CC Plasmid: containing codon-optimized heterologous panD CC The pTrc99a plasmid was used as a template and panD CC -F / panD CC -R is the primer that amplifies panD CC The panD fragment was prepared by the method of step (2) of Example 2. CC The fragment was cloned and verified with the linearized plasmid pTrc99a-line in step (1) to construct pTrc99a-panD CC Plasmid.

[0085] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD CC Transformed into E1 plasmid-free competent cells to construct strain E5 (Trc-panD / pTrc99a-panD CC ).

[0086] (4) Using strain E2 as the control strain, strains E2 and E5 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E2 600 was 11.84, the content of β-alanine was 0.09 g / L; the biomass OD of strain E5 600 It is 10.33, and the content of β-alanine is 0.19g / L.

[0087] Example 6: Overexpression of panD gene from Mycobacterium tuberculosis H37RV (nucleotide sequence as shown in SEQ ID No. 5) on pTrc99a plasmid to construct strain E6 (Trc-panD / pTrc99a-panD MB ).

[0088] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method of step (1) of Example 2.

[0089] (2) Construction of pTrc99a-panD containing Donor MB Plasmid: containing codon-optimized heterologous panD MB The pTrc99a plasmid was used as a template and panD MB -F / panD MB -R is the primer that amplifies panD MB The panD fragment was prepared by the method of step (2) of Example 2. MB The fragment was cloned and verified with the linearized plasmid pTrc99a-line in step (1) to construct pTrc99a-panD MB Plasmid.

[0090] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD MB Transformed into E1 plasmid-free competent cells to construct strain E6 (Trc-panD / pTrc99a-panD MB ).

[0091] (4) Using strain E2 as the control strain, strains E2 and E6 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E2 600 was 11.84, the content of β-alanine was 0.09 g / L; the biomass OD of strain E6 600 It is 11.73, and the content of β-alanine is 0.24g / L.

[0092] Example 7: Overexpression of panD gene from Helicobacter pylori Puno135 (nucleotide sequence as shown in SEQ ID No. 6) on pTrc99a plasmid to construct strain E7 (Trc-panD / pTrc99a-panD HP ).

[0093] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method of step (1) of Example 2.

[0094] (2) Construction of pTrc99a-panD containing Donor HP Plasmid: containing codon-optimized heterologous panD HP The pTrc99a plasmid was used as a template and panD HP -F / panD HP -R is the primer that amplifies panD HP The panD fragment was prepared by the method of step (2) of Example 2. HP The fragment was cloned and verified with the linearized plasmid pTrc99a-line in step (1) to construct pTrc99a-panD HP Plasmid.

[0095] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD HP Transformed into E1 plasmid-free competent cells to construct strain E7 (Trc-panD / pTrc99a-panD HP ).

[0096] (4) Using strain E2 as the control strain, strains E2 and E7 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 1 As shown, the biomass OD of the control strain E2 600 was 11.84, the content of β-alanine was 0.09 g / L; the biomass OD of strain E7 600 It is 10.92, and the content of β-alanine is 0.17g / L.

[0097] Example 8: Trc promoter enhanced aspartate aminotransferase aspC gene in strain E4 (nucleotide sequence as shown in SEQ ID No. 7), and constructed strain E8 (Trc-panDTrc-aspC / pTrc99a-panD BS ).

[0098] (1) Construction of pTarget-aspC-line plasmid: Using plasmid pTarget as a template and pTarget-aspC-F / pTarget-aspC-R as primers, the N20 mutant product was amplified, and the method of step (1) of Example 1 was used for purification and transformation, and then the correctness of the pTarget-aspC-PAM plasmid was verified by sequencing to obtain plasmid pTarget-aspC-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain a linearized plasmid pTarget-aspC-line.

[0099] (2) Construction of plasmid pTarget-aspC containing Donor: Using the genome of E. coli Nissle 1917 as a template, aspC-FF / aspC-FR as primers were used to amplify the upstream homology arm F1 (the F1 primer contained the Trc promoter sequence); aspC-RF / aspC-RR as primers were used to amplify the downstream homology arm R1 (the R1 primer contained the Trc promoter sequence). The upstream and downstream homology arms were cloned into the linearized plasmid pTarget-aspC-line in step (1) using the method of step (1) of Example 1, and transformed and verified to construct the pTarget-aspC plasmid.

[0100] (3) Using the method of step (3) of Example 1, the plasmid pCas9 was transformed into a plasmid without pTrc99a-panD BS Competent cells E4 and prepare competent cells.

[0101] (4) Using the method of step (4) of Example 1, the plasmid pTarget-aspC was transformed into the competent cells of step (3) and verified, and the Trc-aspC strain was successfully constructed.

[0102] (5) Elimination of pTarget and pCas9 plasmids: The method of step (5) of Example 1 was used to obtain a plasmid-free strain.

[0103] (6) Transformation of pTrc99a-panD BS Plasmid: Extract plasmid pTrc99a-panD from strain E4 BS The plasmid was transformed into competent cells of a plasmid-free strain to construct strain E8 (Trc-panDTrc-aspC / pTrc99a-panD BS ).

[0104] (7) Using strain E4 as the control strain, strains E4 and E8 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 2As shown, the biomass OD of the control strain E4 600 was 11.63, the content of β-alanine was 0.4 g / L; the biomass OD of strain E8 600 It is 11.1, the content of β-alanine is 0.4g / L, and the content of β-alanine has not increased.

[0105] Example 9: pTrc99a-panD BS Overexpression of endogenous aspC in strain E0 ECN gene (nucleotide sequence as shown in SEQ ID No.7), and construct strain E9 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspC ECN ).

[0106] (1) Construction of pTrc99a-D BS -line plasmid: plasmid pTrc99a-panD BS As a template, pTrc99a-D BS -line-F / pTrc99a-D BS -line-R is a primer for amplifying the linearized fragment of the plasmid, and the method of step (1) of Example 2 is used to obtain the linearized plasmid pTrc99a-panD BS -line.

[0107] (2) Construction of pTrc99a-panD containing Donor BS -aspC ECN Plasmid: wild-type strain E0 was used as template and aspC ECN -F / aspC ECN -R is the primer that amplifies aspC ECN fragment, using the method of step (2) of Example 2, aspC ECN Fragment and step (1) linearization plasmid pTrc99a-panD BS -line was cloned and verified in one step to construct pTrc99a-panD BS -aspC ECN Plasmid.

[0108] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BS -aspC ECN Transformed into E8 plasmid-free competent cells to construct strain E9 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspC ECN ).

[0109] (4) Using strain E8 as the control strain, strains E8 and E9 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 2 As shown, the biomass OD of the control strain E8 600 was 11.1, the content of β-alanine was 0.4 g / L; the biomass OD of strain E9 600 It is 10.6, the content of β-alanine is 0.35g / L, and the content of β-alanine has decreased slightly.

[0110] Example 10: pTrc99a-panD BS The plasmid was used to overexpress the aspB gene from Corynebacterium glutamicum (nucleotide sequence shown in SEQ ID No. 8), and strain E10 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG ).

[0111] (1) Construction of pTrc99a-D BS -line plasmid: Use the method of step (1) of Example 9 to obtain the linearized plasmid pTrc99a-D BS -line.

[0112] (2) Construction of pTrc99a-panD containing Donor BS -aspB CG Plasmid: containing codon-optimized heterologous aspB CG The pTrc99a plasmid was used as a template and aspB CG -F / aspB CG -R is the primer that amplifies aspB CG fragment, using the method of step (2) of Example 2, aspB CG Fragment and step (1) linearization plasmid pTrc99a-panD BS -line was cloned and verified in one step to construct pTrc99a-panD BS -aspB CG Plasmid.

[0113] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BS -aspB CG Transformed into E9 plasmid-free competent cells to construct strain E10 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspBCG ).

[0114] (4) Using strain E9 as the control strain, strains E9 and E10 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 2 As shown, the biomass OD of the control strain E9 600 was 10.6, the content of β-alanine was 0.35 g / L; the biomass OD of strain E10 600 It is 10.3, and the content of β-alanine is 0.7g / L.

[0115] Example 11: pTrc99a-panD BS -aspB CG The endogenous aspA gene in strain E0 was overexpressed on the plasmid (nucleotide sequence is shown in SEQ ID No. 9), and strain E11 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG -aspA).

[0116] (1) Construction of pTrc99a-panD BS -aspB CG -line plasmid: plasmid pTrc99a-panD BS -aspB CG As a template, pTrc99a-D BS B CG -line-F / pTrc99a-D BS B CG -line-R is a primer for amplifying the linearized fragment of the plasmid, and the method of step (1) of Example 2 is used to obtain the linearized plasmid pTrc99a-D BS B CG -line.

[0117] (2) Construction of pTrc99a-panD containing Donor BS -aspB CG -aspA plasmid: Using the wild-type strain E0 as a template and aspA-F / aspA-R as primers, the aspA fragment was amplified. The aspA fragment was amplified with the linearized plasmid pTrc99a-D in step (1) using the method of step (2) of Example 2. BS B CG -line was cloned and verified in one step to construct pTrc99a-panD BS -aspB CG -aspA plasmid.

[0118] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BS -aspB CG -aspA was transformed into E10 plasmid-free strain competent cells to construct strain E11 (Trc-panDTrc-aspC / pTrc99a-panD BS -aspB CG -aspA).

[0119] (4) Using strain E10 as the control strain, strains E10 and E11 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 2 As shown, the biomass OD of the control strain E10 600 was 10.3, the content of β-alanine was 0.7 g / L; the biomass OD of strain E11 600 It is 11.62, and the content of β-alanine is 1.04g / L.

[0120] Example 12: Trc promoter enhanced succinate dehydrogenase sdhC gene (nucleotide sequence as shown in SEQ ID No. 10) in strain E11, and constructed strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD BS -aspB CG -aspA).

[0121] (1) Construction of pTarget-sdhC-line plasmid: Using plasmid pTarget as template and pTarget-sdhC-F / pTarget-sdhC-R as primers, the N20 mutant product was amplified, and the method of step (1) of Example 1 was used for purification and transformation, and then the correctness of the pTarget-sdhC-PAM plasmid was verified by sequencing to obtain plasmid pTarget-sdhC-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain a linearized plasmid pTarget-sdhC-line.

[0122] (2) Construction of plasmid pTarget-sdhC containing Donor: Using the genome of E. coli Nissle 1917 as a template, sdhC-FF / sdhC-FR as primers were used to amplify the upstream homology arm F1 (the F1 primer contained the Trc promoter sequence); sdhC-RF / sdhC-RR as primers were used to amplify the downstream homology arm R1 (the R1 primer contained the Trc promoter sequence). The upstream and downstream homology arms were cloned into the linearized plasmid pTarget-sdhC-line of step (1) using the method of step (1) of Example 1, and transformation and verification were performed to construct the pTarget-sdhC plasmid.

[0123] (3) Using the method of step (3) of Example 1, the plasmid pCas9 was transformed into a plasmid without pTrc99a-panD BS -aspB CG -aspA competent cells E11 and prepare competent cells.

[0124] (4) Using the method of step (4) of Example 1, the plasmid pTarget-sdhC was transformed into the competent cells of step (3) and verified, and the Trc-sdhC strain was successfully constructed.

[0125] (5) Elimination of pTarget and pCas9 plasmids: The method of step (5) of Example 1 was used to obtain a plasmid-free strain.

[0126] (6) Transformation of pTrc99a-panD BS -aspB CG -aspA plasmid: Extract plasmid pTrc99a-panD from strain E11 BS -aspB CG -aspA, and transform the plasmid into competent cells of a plasmid-free strain to construct strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD BS -aspB CG -aspA).

[0127] (7) Using strain E11 as the control strain, strains E11 and E12 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 3 As shown, the biomass OD of the control strain E11 600 was 11.62, the content of β-alanine was 1.04 g / L; the biomass OD of strain E12 600 It is 12.51, and the content of β-alanine is 1.36g / L.

[0128] Example 13: Trc promoter enhanced phosphoenolpyruvate carboxylase ppc gene in strain E12 (nucleotide sequence as shown in SEQ ID No. 11), and constructed strain E13 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA).

[0129] (1) Construction of pTarget-ppc-line plasmid: Using plasmid pTarget as template and pTarget-ppc-F / pTarget-ppc-R as primers, the N20 mutant product was amplified, and the method of step (1) of Example 1 was used for purification and transformation, and then the correctness of the pTarget-ppc-PAM plasmid was verified by sequencing to obtain plasmid pTarget-ppc-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain a linearized plasmid pTarget-ppc-line.

[0130] (2) Construction of plasmid pTarget-ppc containing Donor: Using the genome of E. coli Nissle 1917 as a template, ppc-FF / ppc-FR as primers were used to amplify the upstream homology arm F1 (the F1 primer contained the Trc promoter sequence); ppc-RF / ppc-RR as primers were used to amplify the downstream homology arm R1 (the R1 primer contained the Trc promoter sequence). The upstream and downstream homology arms were cloned into the linearized plasmid pTarget-ppc-line in step (1) using the method of step (1) of Example 1, and transformation and verification were performed to construct the pTarget-ppc plasmid.

[0131] (3) Using the method of step (3) of Example 1, the plasmid pCas9 was transformed into a plasmid without pTrc99a-panD BS -aspB CG -aspA competent cells E12 and prepare competent cells.

[0132] (4) Using the method of step (4) of Example 1, the plasmid pTarget-ppc was transformed into the competent cells of step (3) and verified, and the Trc-ppc strain was successfully constructed.

[0133] (5) Elimination of pTarget and pCas9 plasmids: The method of step (5) of Example 1 was used to obtain a plasmid-free strain.

[0134] (6) Transformation of pTrc99a-panD BS -aspB CG -aspA plasmid: Extract plasmid pTrc99a-panD from strain E11BS -aspB CG -aspA, and transform the plasmid into competent cells of a plasmid-free strain to construct strain E13 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA).

[0135] (7) Using strain E12 as the control strain, strains E12 and E13 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 4 As shown, the biomass OD of the control strain E12 600 was 12.51, and the content of β-alanine was 1.36 g / L; the biomass OD of strain E13 600 It is 11.66, and the content of β-alanine is 1.57g / L.

[0136] Example 14: pTrc99a-panD BS -aspB CG The pyc gene from Corynebacterium glutamicum (nucleotide sequence as shown in SEQ ID No. 12) was overexpressed on the -aspA plasmid to construct strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0137] (1) Construction of pTrc99a-panD BS -aspB CG -aspA-line plasmid: plasmid pTrc99a-panD BS -aspB CG -aspA as template, using pTrc99a-D BS B CG A-line-F / pTrc99a-D BS B CG A-line-R is a primer for amplifying the linearized fragment of the plasmid. The method of step (1) of Example 2 is used to obtain the linearized plasmid pTrc99a-panD BS -aspB CG -aspA-line.

[0138] (2) Construction of pTrc99a-panD containing DonorBS -aspB CG -aspA-pyc CG Plasmid: containing codon-optimized heterologous pyc CG The pTrc99a plasmid was used as a template and pyc CG -F / pyc CG -R is the primer to amplify pyc CG fragment, using the method of step (2) of Example 2, pyc CG Fragment and step (1) linearization plasmid pTrc99a-panD BS -aspB CG -aspA-line was cloned and verified in one step to construct pTrc99a-panD BS -aspB CG -aspA-pyc CG Plasmid.

[0139] (3) Using the method of step (3) of Example 2, plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG Transformed into E13 plasmid-free competent cells to construct strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0140] (4) Using strain E13 as the control strain, strains E13 and E14 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 5 As shown, the biomass OD of the control strain E13 600 was 11.66, the content of β-alanine was 1.57 g / L; the biomass OD of strain E14 600 It is 10.67, and the content of β-alanine is 1.87g / L.

[0141] Example 15: Knockout of the transporter cycA gene in strain E14 (nucleotide sequence as shown in SEQ ID No. 13), construct strain E15 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc△cycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0142] (1) Construction of pTarget-cycA-line plasmid: Using plasmid pTarget as template and pTarget-cycA-F / pTarget-cycA-R as primers, the N20 mutant product was amplified, and the method of step (1) of Example 1 was used for purification and transformation, and then the correctness of the pTarget-cycA-PAM plasmid was verified by sequencing to obtain plasmid pTarget-cycA-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain a linearized plasmid pTarget-cycA-line.

[0143] (2) Construction of plasmid pTarget-cycA containing Donor: Using the genome of E. coli Nissle 1917 as a template, cycA-FF / cycA-FR as primers to amplify the upstream homology arm; cycA-RF / cycA-RR as primers to amplify the downstream homology arm. The upstream and downstream homology arms were cloned into the linearized plasmid pTarget-cycA-line in step (1) using the method of step (1) of Example 1, and transformed and verified to construct the pTarget-cycA plasmid.

[0144] (3) Using the method of step (3) of Example 1, the plasmid pCas9 was transformed into a plasmid without pTrc99a-panD BS -aspB CG -aspA-pyc CG Competent cells were prepared in E14 medium.

[0145] (4) Using the method of step (4) of Example 1, the plasmid pTarget-cycA was transformed into the competent cells of step (3) and verified, and the cycA knockout strain was successfully constructed.

[0146] (5) Elimination of pTarget and pCas9 plasmids: The method of step (5) of Example 1 was used to obtain a plasmid-free strain.

[0147] (6) Transformation of pTrc99a-panD BS -aspB CG -aspA-pyc CG Plasmid: Extract plasmid pTrc99a-panD from strain E14 BS -aspB CG -aspA-pyc CG The plasmid was transformed into competent cells of the plasmid-free strain to construct strain E15 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc△cycA / pTrc99a-panD BS -aspBCG -aspA-pyc CG ).

[0148] (7) Using strain E14 as the control strain, strains E14 and E15 were subjected to shake flask fermentation test according to the method of Example 2. The content of β-alanine was detected according to the method of Example 1. Biomass OD 600 and β-alanine content as Figure 6 As shown, the biomass OD of control strain E14 600 was 10.67, the content of β-alanine was 1.87 g / L; the biomass OD of strain E15 600 It is 11.1, and the content of β-alanine is 2.17g / L.

[0149] Example 16: Fed-batch fermentation in a 5-L fermenter

[0150] The strain E15 in Example 15 was streaked onto an LB plate containing 50 mg / L kanamycin resistance, cultured overnight at 37°C, and a single colony was picked into an LB test tube containing 50 mg / L kanamycin resistance, and cultured overnight at 37°C, 150 rpm to prepare a primary seed solution. The primary seed solution was inoculated into 100 mL of LB medium containing 50 mg / L kanamycin resistance at a volume concentration of 5%, and cultured overnight at 37°C, 150 rpm as a secondary seed solution. The secondary seed solution was inoculated into a 5-L fermentation tank containing 2 L fermentation medium at a volume concentration of 15%, and a final concentration of 0.2 mM IPTG was added. Fermentation was carried out at 30°C, 300 rpm, and aeration of 4 V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), the automatic feeding was turned on, and the feed medium was added at a rate of 25 mL / h until the pH was lower than 6.80, and the feeding was stopped. The residual sugar in the fermenter was maintained at a low level, and the sugar concentration was maintained below 1 g / L. The total amount of feed medium added to strain E15 was 1700 mL, and the culture was carried out for 81 hours. The β-alanine yield in the fermentation broth was detected by the method in Example 1, and the biomass OD was detected by spectrophotometry. 600 , the DNS method was used to detect sugar concentration; Figure 7 As shown in the figure, the β-alanine yield reached 70.4 g / L at 81 h, and the final biomass OD 600 Maintained at 74.3. The fermentation results showed that the metabolically engineered E. coli probiotics had good performance in producing β-alanine, laying a foundation for the industrial production of β-alanine.

[0151] The fermentation medium formula in the 5-L fermenter was as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 120.2mg / L, 1mL / L trace element solution, the solvent is deionized water; the composition of the trace element solution: 10g / L CaCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.2g / LCuSO4, 0.02g / L NiCl2·7H2O, the solvent is deionized water.

[0152] Feed medium: glucose 500g / L, (NH4)2SO4 10g / L, yeast extract 2g / L, KH2PO4 14g / L, MgSO4 8g / L, 0.5mg / L VB1, 0.2mg / L VB 12 The solvent was deionized water, and the pH was adjusted to 6.8 with 14% ammonia water.

Claims

1. A probiotic Escherichia coli with high β-alanine production, characterized in that: The Escherichia coli probiotics are based on wild-type Escherichia coli probiotics as the starting strain, and one or more of the following gene editing is performed: Trc promoter is used to strengthen panD gene, aspC gene, sdhC gene, and ppc gene, and panD gene from Bacillus subtilis, aspB gene from Corynebacterium glutamicum, aspA gene, and pyc gene from Corynebacterium glutamicum are overexpressed on plasmid pTrc99a, and cycA gene is knocked out.

2. The Escherichia coli probiotic according to claim 1, characterized in that The nucleotide sequence of the panD gene is shown in SEQ ID NO.3; the nucleotide sequence of the aspC gene is shown in SEQ ID NO.7; the nucleotide sequence of the aspB gene is shown in SEQ ID NO.8; the nucleotide sequence of the aspA gene is shown in SEQ ID NO.9; the nucleotide sequence of the sdhC gene is shown in SEQ ID NO.10; the nucleotide sequence of the ppc gene is shown in SEQ ID NO.11; the nucleotide sequence of the pyc gene is shown in SEQ ID NO.12; the nucleotide sequence of the cycA gene is shown in SEQ ID NO.13; and the nucleotide sequence of the Trc promoter is shown in SEQ ID NO.

14.

3. The Escherichia coli probiotic according to claim 1, characterized in that The wild-type Escherichia coli probiotic is E. coli Nissle1917.

4. The Escherichia coli probiotic according to claim 1, characterized in that The recombinant genetically engineered bacteria uses wild-type Escherichia coli probiotics as a starting strain, and performs the following gene editing in sequence: Trc promoter strengthens the panD gene, overexpresses the panD gene from Bacillus subtilis on the plasmid pTrc99a, Trc promoter strengthens the aspC gene, overexpresses the aspB gene from Corynebacterium glutamicum on the plasmid pTrc99a, overexpresses the aspA gene on the plasmid pTrc99a, Trc promoter strengthens the sdhC gene, Trc promoter strengthens the ppc gene, introduces exogenous PC catalytic reaction, and knocks out the cycA gene.

5. Use of the Escherichia coli probiotic according to claim 1 in preparing β-alanine by microbial fermentation.

6. The use according to claim 5, characterized in that The application is: inoculating the E. coli probiotics into a fermentation medium containing 50 mg / L kanamycin, and culturing the bacteria at 30°C and 180 rpm to grow to OD 600 =0.5, add IPTG to a final concentration of 0.25 mM to induce gene expression, continue culturing for 48 h until the fermentation is completed, obtain a fermentation broth containing β-alanine, separate and purify the fermentation broth to obtain β-alanine; The fermentation medium is formulated as follows: glucose 18-22 g / L, (NH4)2SO4 14-18 g / L, yeast extract 2-3 g / L, KH2PO4 1-2 g / L, MgSO4 0.2-1 g / L, CaCO3 13-17 g / L, VB1 0.3-0.5 mg / L, VB 12 0.3~0.5mg / L, trace element solution 0.5~1.5mL / L, the solvent is water, and there is no need to adjust the pH value; the components of the trace element solution are as follows: 8~12g / L CaCl2, 8~12g / L FeSO4·7H2O, 0.8~1.2g / L ZnSO4·7H2O, 0.1~0.3g / LCuSO4, 0.01~0.04g / L NiCl2·7H2O, and the solvent is deionized water.

7. The use according to claim 6, characterized in that Before fermentation, the E. coli probiotics were inoculated into LB medium containing 50 mg / L kanamycin at 37° C. and 180 rpm overnight to prepare seed solution, which was inoculated into a fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%.

8. The use according to claim 6, characterized in that The fermentation is carried out in a fermenter: the E. coli probiotics are inoculated onto an LB plate containing 50 mg / L kanamycin resistance, cultured overnight at 37° C., a single colony is picked into an LB test tube containing 50 mg / L kanamycin resistance, cultured overnight at 37° C. and 150-180 rpm to prepare a primary seed solution; the primary seed solution is inoculated into an LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, cultured overnight at 37° C. and 150-180 rpm to serve as a secondary seed solution; The secondary seed liquid is inoculated into a fermentation tank containing a fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%, and IPTG with a final concentration of 0.2 mM is added. The fermentation is carried out at 30° C., 300-500 rpm, and a ventilation volume of 4 V / V·min. When the pH value is higher than 6.80, automatic feeding is started, and feeding medium is added until the pH is lower than 6.80, and feeding is stopped. The culture is carried out for 70-81 hours to obtain a fermentation liquid containing β-alanine; Formula of fermentation medium in fermenter: glucose 20g / L, (NH4)2SO4 16g / L, yeast extract 2g / L, KH2PO42g / L, MgSO4 0.5g / L, VB1 0.5mg / L, VB 12 0.2mg / L, 1mL / L trace element solution, the solvent is deionized water; the composition of the trace element solution: 10g / L CaCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.2g / L CuSO4, 0.02g / L NiCl2·7H2O, the solvent is deionized water; The feed medium composition: glucose 500g / L, (NH4)2SO4 10g / L, yeast extract 2g / L, KH2PO4 14g / L, MgSO4 8g / L, 0.5mg / L VB1, 0.2mg / L VB 12 The solvent is water, and the pH is adjusted to 6.8 with 14% ammonia water.

9. The use according to claim 8, characterized in that The feeding rate was 25 mL / h, and the total amount of feed medium added was 1700 mL / 2L.

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