Recombinant genetically engineered bacterium for producing beta-alanine by dynamically regulating and controlling competitive pathway and application of recombinant genetically engineered bacterium

Abstract

The invention discloses a recombinant genetically engineered bacterium for producing beta-alanine by dynamically regulating and controlling a competitive pathway and application of the recombinant genetically engineered bacterium, heterologous key genes pycCG and NCgl0580 are over-expressed on plasmids, accumulation of a beta-alanine precursor oxaloacetic acid pool is increased, and efflux of beta-alanine is enhanced; a heterologous gene gapN is introduced to adjust a glycolytic pathway, so that the efficiency of the glycolytic pathway is enhanced, and the carbon flow in a beta-alanine synthesis pathway is increased; the expression activity of the gene gltBD is up-regulated so as to enhance the supply of an amino donor when oxaloacetic acid is converted into aspartic acid; an EsaI / EsaR group sensing system is introduced to dynamically regulate and control an aspartic acid competition branch; after the thrA gene promoter is replaced by PesaS, the situation that excessive metabolic flow enters an aspartic acid competition branch is effectively weakened, the carbon flow is pulled to the synthesis direction of beta-alanine to the maximum extent, and the yield of beta-alanine is obviously increased.

Description

(1) Technical field

[0001] The present invention relates to a recombinant genetic engineering bacterium for producing beta-alanine by dynamically regulating a competitive pathway and its application. (2) Background technology

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

[0003] In the pharmaceutical field, β-alanine is a key precursor for the production of substances such as pantothenic acid, calcium pantothenate, coenzyme A (CoA), and acyl carrier protein (ACP). It plays a vital role in cellular metabolism and the development of the central nervous system. β-alanine can also be used to synthesize drugs such as balsalazide and pamidronate disodium. In the food industry, β-alanine can be used as a food additive and is often included in food flavorings to improve flavor. Due to its antioxidant properties, it is also commonly used as a food preservative. Clinical studies have shown that β-alanine can also be used as a nutritional supplement for athletes. Supplementation with β-alanine can significantly reduce fatigue caused by prolonged, high-intensity exercise and improve human endurance. In the chemical industry, β-alanine can serve as a precursor for the production of chemical products such as pantothenic acid and calcium pantothenate, as well as poly(3-hydroxypropionate) and poly(β-alanine). It can also be used as a precipitant in pharmaceuticals, as an electroplating corrosion inhibitor, and as an antidote for lead poisoning. β-alanine also plays an important role in environmental and cosmetic applications. In the feed field, 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; at the same time, it can improve the antioxidant capacity of muscles, increase the content of muscle-derived active peptides, and effectively improve meat quality.

[0004] At present, there are three main methods for producing β-alanine at home and abroad: (1) Chemical synthesis, which uses nitrile substances such as acrylonitrile, acrylic acid and β-aminopropionitrile to synthesize β-alanine under high pressure and high temperature under strong acid and strong base conditions. However, the chemical synthesis method consumes a lot of energy and has high requirements for equipment. In addition, it 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) Bio-enzyme catalysis, using aspartic acid as the 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 has less pollution, and has attracted more attention from scholars; (3) Microbial fermentation method produces β-alanine. Microbial fermentation method uses synthetic biology, system metabolic engineering, protein engineering, transcriptomics and metabolomics technologies to transform the metabolic process of the target product β-alanine, so that more metabolic flow is directed towards 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 systems metabolic engineering has freed humanity from the constraints of traditional strain screening techniques such as mutagenesis and directed evolution. By quantitatively analyzing metabolic pathways and fluxes within strains, and then conducting targeted metabolic engineering based on this, we can maximize the production efficiency of target metabolites and significantly enhance strain productivity. Unlike traditional breeding techniques, it is an effective approach to improve the genetic traits of organisms by rationally designing cellular metabolic pathways and reconstructing metabolic networks.

[0006] In current research hotspots, engineered strains are typically constructed using strains such as Escherichia coli, yeast, Corynebacterium glutamicum, and Bacillus subtilis as the base bacteria. The metabolic genetic background and physiological characteristics of these base strains are already relatively clear. Combining mature gene editing techniques with the rational design of metabolic flux can greatly accelerate the transformation of strain metabolic pathways and shorten the time to achieve optimal production performance. As the most clearly studied prokaryotic bacterium, Escherichia coli boasts rapid reproduction, short fermentation cycles, high levels of target gene expression, a mature and comprehensive expression system, and biosafety. It is currently widely used in the industrial production of various bioproducts.

[0007] The de novo β-alanine synthesis pathway in Escherichia coli involves glucose uptake, glycolysis, the TCA cycle, L-aspartate synthesis, and finally the β-alanine biosynthesis pathway. First, glucose is phosphorylated under either the PTS or non-PTS system to produce glucose-6-phosphate. This carbon flux then enters the glycolysis pathway, where the β-alanine precursor, phosphoenolpyruvate, is synthesized. Phosphoenolpyruvate carboxylase catalyzes the conversion of phosphoenolpyruvate to oxaloacetate. Oxaloacetate is unstable and easily decomposes, which is then converted to aspartate by aspartate aminotransferase. Subsequently, aspartate decarboxylase catalyzes the conversion to β-alanine. L-lysine, L-threonine, O-succinylhomoserine, and homoserine belong to the aspartate family of amino acids and share the same precursor, L-aspartate, with β-alanine. Therefore, reducing the competing branch is an effective strategy. However, further investigation is needed to identify bottlenecks that affect β-alanine production. (3) Summary of the invention

[0008] The present invention aims to provide a recombinant genetically engineered bacterium capable of producing high β-alanine by dynamically regulating a competitive pathway and its application in preparing β-alanine by a microbial fermentation method, thereby laying a foundation for the industrial production of β-alanine.

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

[0010] The present invention provides a recombinant genetically engineered bacterium for high-yield β-alanine by dynamically regulating a competitive pathway. The recombinant genetically engineered bacterium uses Escherichia coli 630B1 as a starting strain and performs one or more of the following gene editing: overexpressing pyc on the plasmid pTrc99a; CG The yeeP gene and the NCgl0580 gene were replaced with the gapN gene, the gltBD promoter was replaced with the Trc promoter, the yjiV gene was replaced with the EsaR gene, the yjiT gene was replaced with the EsaI gene using the artificial promoter Pbs to replace the original promoter, and the thrA gene promoter was replaced with the PesaS promoter; the starting strain 630B1: E. coli W3110 TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG-aspA, constructed according to Li, B., Zhang, B., Wang, P., Cai,

[0011] Further, the pyc CG The nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the NCgl0580 gene is shown in SEQ ID NO.2; the nucleotide sequence of the yeeP gene is shown in SEQ ID NO.3; the nucleotide sequence of the gapN gene is shown in SEQ ID NO.4; the nucleotide sequence of the gltBD gene is shown in SEQ ID NO.5; the nucleotide sequence of the yjiV gene is shown in SEQ ID NO.6; the nucleotide sequence of the EsaR gene is shown in SEQ ID NO.7; the nucleotide sequence of the yjiT gene is shown in SEQ ID NO.8; the nucleotide sequence of the Pbs promoter is shown in SEQ ID NO.9; the nucleotide sequence of the EsaI gene is shown in SEQ ID NO.10; the nucleotide sequence of the thrA gene is shown in SEQ ID NO.11; the P esaS The nucleotide sequence of the promoter is shown in SEQ ID NO.12; the nucleotide sequence of the Trc promoter is shown in SEQ ID NO.13.

[0012] Furthermore, the recombinant genetically engineered bacteria were subjected to the following gene editing in sequence using Escherichia coli 630B1 as the starting strain: overexpression of pyc CG The yeeP gene and the NCgl0580 gene were replaced by the gapN gene, the gltBD promoter was replaced by the Trc promoter, the yjiV gene was replaced by the EsaR gene, the yjiT gene was replaced by the EsaI gene using the artificial promoter Pbs to replace the original promoter, and the thrA gene promoter was replaced by P esaS promoter.

[0013] The recombinant genetically engineered bacteria of the present invention are constructed as follows:

[0014] (1) Overexpression of codon-optimized pyc from Corynebacterium glutamicum ATCC 13032 on plasmid pTrc99a CG : Plasmid pTrc99a-panD BS K104S -aspB CG -aspA was used as a template and the linearized plasmid pTrc99a-panD was obtained using primers pTrc99a-line-F / pTrc99a-line-R. BS K104S -aspB CG -aspA; linearized plasmid pTrc99a-panD BS K104S -aspB CG -aspA and the codon-optimized pyc shown in SEQ ID NO.1 CG Gene fragment ligation to construct plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG The plasmid was transformed into competent cells of strain 630B to construct strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG , recorded as strain 630B2; strain 630B: E. coli W3110TrcpanDTrcppcΔpykAΔcycA.

[0015] (2) Overexpression of codon-optimized NCgl0580 from Corynebacterium glutamicum ATCC 13032 on plasmid pTrc99a: BS K104S -aspB CG -aspA-pyc CG As a template, the linearized plasmid pTrc99a-panD was obtained using primers pTrc99apyc-line-F / pTrc99apyc-line-R. BS K104S -aspB CG -aspA-pyc CG ; Linearize the plasmid pTrc99a-panD BS K104S-aspB CG -aspA-pyc CG The codon-optimized NCgl0580 gene fragment shown in SEQ ID NO.2 was ligated to construct plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580 and transformed into strain 630B competent cells to construct strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B3.

[0016] (3) Replace the yeeP gene with the gapN gene: Using the genome of E. coli W3110 as a template, the upstream and downstream homologous arms of the yeeP gene were amplified using L-yeeP-F / L-yeeP-R and R-yeeP-F / R-yeeP-R primers, respectively, and the gapN gene was amplified using gapN-F / gapN-R primers, and the donor DNA was obtained by fusion; using the pTarget plasmid as a template, the plasmid was amplified using pTarget-yeeP-F and pTarget-yeeP-R primers and linearized to obtain a linearized pTarget-yeeP plasmid; the donor DNA and the linearized pTarget-yeeP plasmid were ligated and transformed into the starting strain 630B electroporation competent cells, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapN / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B4.

[0017] (4) Replacement of gltBD promoter with Trc promoter: Using pTarget plasmid as template and pTarget-gltBD-F / pTarget-gltBD-R as primers, linearized plasmid pTarget-gltBD was obtained by amplification. Using E. coli W3110 genome as template and L-gltBD-F / L-gltBD-R and R-gltBD-F / R-gltBD-R as primers, upstream and downstream homology arms of the original promoter were amplified. Using CRISPR / Cas9 gene editing technology, upstream and downstream homology arms and linearized plasmid pTarget-gltBD were transferred into competent cells of strain 630B4, pTarget and pCas9 plasmids were eliminated, and CRISPR / Cas9 gene editing technology was used to replace the gltBD promoter on the genome of strain 630B4 with Trc promoter to construct strain E. coli W3110. W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B5.

[0018] (5) Replace the yjiV gene with the EsaR gene: Using the genome of E. coli W3110 as a template, the upstream and downstream homology arms of the yjiV gene were amplified using L-yjiV-F / L-yjiV-R and R-yjiV-F / R-yjiV-R primers, respectively, and the EsaR gene was amplified using esaR-F / esaR-R primers, and the donor DNA was obtained by fusion; using the pTarget plasmid as a template, the plasmid was amplified using pTarget-yjiV-F and pTarget-yjiV-R primers and linearized to obtain the linearized pTarget-yjiV plasmid; the donor DNA and the linearized pTarget-yjiV plasmid were connected and transformed into the starting strain E. coli Competent cells were electroporated with W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD, and the pTarget and pCas9 plasmids were eliminated to construct the E. coli strain W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaR, which was designated as strain 630B6.

[0019] (6) Replace the yjiT gene with the esaI gene with the pbs promoter: Using the genome of E. coli W3110 as a template, L-yjiT-F / yjiT-FR-Pbs and R-yjiT-F / R-yjiT-R were used as primers to amplify the upstream and downstream homologous arms of the yjiT gene, and using the plasmid containing the pbs promoter gene as a template, the primers Pbs-esaI-F / esaI-R were used to amplify the esaI gene containing the promoter pbs, and the donor DNA was obtained by fusion; using the pTarget plasmid as a template, the plasmid was amplified with pTarget-yjiT-F and pTarget-yjiT-R as primers and linearized to obtain a linearized pTarget-yjiT plasmid; the donor DNA and the linearized pTarget-yjiT plasmid were connected and transformed into the starting strain 630B6 electroporation competent cells, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli. W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaI, denoted as strain 630B7.

[0020] (7) Replace the thrA gene promoter with P esaS :The genome of E. coli W3110 was used as a template, and the upstream and downstream homology arms of the original promoter of thrA gene were amplified with primers L-thrA-F / L-thrA-R and R-thrA-F / R-thrA-R, respectively. esaS The plasmid containing the promoter gene was used as a template, and P esaS -F / P esaS -R is the primer amplification to obtain P esaS The homology arm was fused with the promoter fragment to obtain donor DNA; the pTarget plasmid was used as a template, and the plasmid was amplified with pTarget-thrA-F and pTarget-thrA-R as primers and linearized to obtain the linearized pTarget-thrA plasmid; the donor DNA and the linearized pTarget-thrA plasmid were ligated and transformed into the electroporation competent cells of strain 630B7, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -th rA, designated as strain 630B8;

[0021] (8) Extraction of plasmid pTrc99a-panD from strain 630B3 BSK104S -aspB CG -aspA-pyc CG -NCgl0580 and transformed into strain 630B8 to construct strain 630B9: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -th rA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580.

[0022] The present invention transforms the β-alanine synthesis network of Escherichia coli by overexpressing the codon-optimized pyc gene from Corynebacterium glutamicum ATCC 13032 on the plasmid pTrc99a. CG The researchers optimized the NCgl0580 gene, which promoted the conversion of pyruvate to oxaloacetate and increased the accumulation of the precursor oxaloacetate. They also overexpressed the codon-optimized NCgl0580 gene from Corynebacterium glutamicum ATCC 13032 on the plasmid pTrc99a to enhance the expression of the β-alanine efflux protein and increase the accumulation of β-alanine. They introduced the heterologous gene gapN to adjust the glycolysis pathway, enhancing its efficiency and increasing the carbon flux in the β-alanine synthesis pathway. They also upregulated the expression activity of the gene gltBD to enhance the supply of amino donors when oxaloacetate is converted to aspartate. They also dynamically downregulated the expression of thrA by introducing a quorum sensing system to effectively reduce the excessive metabolic flux entering the aspartate competition branch, maximizing the carbon flux toward β-alanine synthesis, thereby increasing β-alanine production.

[0023] The present invention also relates to the use of the recombinant genetically engineered bacteria in the preparation of β-alanine by microbial fermentation. The use comprises: inoculating the recombinant genetically engineered bacteria into a fermentation medium containing 48 mg / L IPTG and 50 mg / L kanamycin, culturing at 20-40° C. and 100-300 rpm (preferably 30° C. and 180 rpm) for 48 hours until the fermentation is completed, obtaining a fermentation broth containing β-alanine, and separating and purifying the fermentation broth to obtain β-alanine.

[0024] The fermentation medium has the following formula: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, and 1 mL / L trace element solution, without the need to adjust the pH value; the trace element solution comprises the following components: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0025] Before fermentation, the recombinant genetically engineered bacteria were inoculated into LB medium containing 50 mg / L kanamycin at 37° C. and 180 rpm overnight to prepare seed liquid, which was then inoculated into a fermentation medium containing 50 mg / L kanamycin at an inoculum concentration of 5% by volume.

[0026] The fermentation is carried out in a fermenter: the recombinant genetically engineered bacteria are inoculated onto an LB plate containing 50 mg / L kanamycin resistance, cultured at 37° C. overnight, a single colony is picked and placed in an LB test tube containing 50 mg / L kanamycin resistance, cultured at 37° C. and 150 rpm overnight to prepare a seed solution; the seed solution is inoculated into an LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, cultured at 37° C. and 150 rpm overnight to serve as a secondary seed solution; the secondary seed solution is inoculated into a 5-L fermenter containing 2 L of a fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%, and fermented and cultured at 30° C., a rotation speed of 300 rpm, a ventilation volume of 1.0 V / V / min, and a pH value maintained at 6.80, and the OD value is maintained at 6.80. 600 When the pH reaches 10, IPTG is added to a final concentration of 48 mg / L for induction. When the pH value is higher than 6.80 (the initial sugar in the fermenter is consumed), automatic feeding is started, and feeding medium is added until the pH is lower than 6.80. Feeding is stopped and cultured for 60-64 hours to obtain a fermentation broth containing β-alanine. The fermentation medium formula includes: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, and 1 mL / L trace element solution, without pH adjustment; the trace element solution is composed of: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0027] Feed medium composition: 600 g / L glucose, 16 g / L (NH₄)₂SO₄, 2 g / L yeast extract, 14 g / L KH₂PO₄, 8 g / L MgSO₄, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, 48 mg / L IPTG, 50 mg / L kanamycin, 2 mL / L trace element solution. The solvent was water, and the pH was adjusted to 6.8 with 14% aqueous ammonia. The total feed medium volume was 1400 mL / 2 L.

[0028] The fermentation broth separation and purification method comprises the following steps: first, centrifuging at 12,000 rpm and 4° C. for 10 minutes to remove bacterial cells and other solid particles in the fermentation broth; then, using microfiltration to remove macromolecular proteins; and finally, using deionization technology to remove inorganic salts. Activated carbon is added for decolorization, and ion exchange resin is used for separation to obtain β-alanine. The broth is vacuum concentrated, and ethanol is added for cooling and crystallization to obtain β-alanine crystals.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] The present invention utilizes the strategy of "push, pull and block" to strengthen the synthesis pathway of β-alanine, and utilizes genetic engineering and metabolic engineering technology to transform the β-alanine biosynthesis pathway of Escherichia coli. CG , increasing the accumulation of the β-alanine precursor oxaloacetate pool and strengthening the efflux of β-alanine. The heterologous gene gapN was introduced to adjust the glycolysis pathway, enhancing the efficiency of the glycolysis pathway and increasing the carbon flow in the β-alanine synthesis pathway; the expression activity of the gene gltBD was upregulated to enhance the supply of amino donors when oxaloacetate was converted to aspartate; and the EsaI / EsaR quorum sensing system in the corn bacterial wilt fungus Pantoea stewartia strain was introduced to dynamically regulate the aspartate competition branch. After replacing the thrA gene promoter with P esaS Afterwards, the excessive metabolic flow entering the aspartate competition branch was effectively weakened, and the carbon flow was pulled to the synthesis direction of β-alanine to the maximum extent. The β-alanine production was significantly improved. In the batch fed fermentation, the β-alanine production of strain 630B9 reached 122.06 g / L at the 5-L fermenter level, which was 81.91% higher than that of the starting strain 630B1, and the sugar-acid conversion rate reached 45.77%. (IV) Description of the accompanying drawings

[0031] Figure 1 is the biomass OD of strains 630B1 and 630B2 600 and β-alanine concentrations.

[0032] Figure 2is the biomass OD of strains 630B2 and 630B3 600 and β-alanine concentrations.

[0033] Figure 3 is the biomass OD of strains 630B3 and 630B4 600 and β-alanine concentrations.

[0034] Figure 4 is the biomass OD of strains 630B4 and 630B5 600 and β-alanine concentrations.

[0035] Figure 5 is the biomass OD of strains 630B5 and 630B9 600 and β-alanine concentrations.

[0036] Figure 6 is the biomass OD of strain 630B9 in a 5-L fermenter fed-batch fermentation. 600 , concentration curves of residual sugar and β-alanine. (V) Specific implementation methods

[0037] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0038] The strain E. coli W3110 was obtained from the Coli Genetic Stock Center of Yale University, with a deposit date of August 5, 1975, and a deposit number of CGSC#4474, and has been disclosed in patents US2009 / 0298135 A1 and US2010 / 0248311 A1.

[0039] Escherichia coli 630B:

[0040] E. coli W3110TrcpanDTrcppcΔpykAΔcycA.

[0041] Escherichia coli 630B1:

[0042] E.coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA.

[0043] Plasmid pTrc99a-panD BS K104S -aspB CG-aspA, strains 630B and 630B1 were constructed according to Li, B., Zhang, B., Wang, P., Cai, ofβ-alanine in Escherichia coli.

[0044] All heterologous genes in the present invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0045] LB plate composition: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 20g / L, solvent is water, pH natural.

[0046] LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0047] Fermentation medium: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, 1 mL / L trace element solution, no pH adjustment required; the trace element solution composition is as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0048] Table 1 Genes involved in strain transformation and their corresponding pathways

[0049]

[0050]

[0051] Table 2 Primers used in the gene editing process

[0052]

[0053]

[0054] Example 1: β-Alanine Concentration Determination

[0055] Preparation of 1% 2,4-dinitrofluorobenzene: Dissolve 1 mL of 2,4-dinitrofluorobenzene in 99 mL of acetonitrile.

[0056] Preparation of 0.5M NaHCO3 solution: Dissolve 21g NaHCO3 in 500mL deionized water.

[0057] 0.2 M PB buffer: Weigh 8.74 g Na2HPO4·12H2O and 2.43 g Na2HPO4·2H2O and dissolve them in 200 mL deionized water. Dissolve and store for later use.

[0058] Sample treatment: Dilute the sample concentration to between 0.1-1 g / L with ultrapure water.

[0059] Reaction conditions: 100 μL of sample, 100 μL of 0.5 M NaHCO₃ solution, and 100 μL of 1% 2,4-dinitrofluorobenzene were each incubated at 60°C for 60 min. Finally, 700 μL of 0.2 M PB buffer was added, mixed thoroughly, and passed through a polyvinylidene fluoride (PVDF) membrane (0.22 μm). The permeate was analyzed by HPLC for peak area at 19 min. β-alanine concentration was calculated using a calibration curve obtained previously using the same conditions, comparing β-alanine standard concentration to peak area.

[0060] HPLC model: Thermo Scientific Ultimate 3000, HPLC detection wavelength: 360 nm, gradient elution program for separation of β-alanine, wherein the mobile phase A component is methanol: acetonitrile: ultrapure water = 45:45:10 (v:v:v); mobile phase B component: 10 mM potassium dihydrogen phosphate, pH adjusted to 7.0 with KOH.

[0061] The elution program is: 0-2.5min 12% A, 88% B; 2.5-2.6min A 12%→16%, B 88%→84%; 2.6-13min A 16%→36%, B 84%→64%; 13-13.1min A36%→38% B 64%→62%; 13.1-28min A 38%→100% B62%→0; 28-28.1min A 100%-10% B 0→90%; 28.1-32min A 10%→12% B 90%→88%.

[0062] Example 2: Overexpression of pTrc99a-panD in strain 630B BS K104S -aspB CG -aspA-pycCG Construction of strain and its shake flask fermentation

[0063] (1) Construction of pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG Plasmid: plasmid pTrc99a-panD BS K104S -aspB CG -aspA was used as a template and pTrc99a-line-F / pTrc99a-line-R as primers for PCR amplification. The PCR product was digested with Dpn I and then digested at 37°C for 2 h to remove the methylated vector. The DNA fragment was then recovered using a Clean up kit and set aside. The genome of Corynebacterium glutamicum ATCC13032 was used as a template and primers pyc-F and pyc-R were used to amplify pyc CG Fragment (nucleotide sequence as shown in SEQ ID NO.1), and then use Clean up kit to recover DNA fragments for future use. (One step cloning kit, VazymeBiotech, Nanjing, China) was used to linearize pTrc99a-panD BS K104S -aspB CG -aspA plasmid, fragment pyc CG The ligation products were transformed into E. coli DH5α competent cells; finally, clones were selected and verified by sequencing to obtain pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG plasmid.

[0064] (2) Prepare 630B competent cells, pick a single colony and place it in LB tube culture medium, culture at 30℃ overnight. Then inoculate it into 100mL LB medium at a volume concentration of 1% and culture at 180rpm and 30℃ until OD 600 = 0.5, centrifuge at 4000 rpm at 4°C, wash twice with ultrapure water at 4°C, wash once with cold 10% glycerol aqueous solution, and finally resuspend in 10% glycerol aqueous solution, aliquot and store to obtain electrocompetent cells for later use.

[0065] (3) construct pTrc99a-panD in step (1) BS K104S -aspBCG -aspA-pyc CG The plasmid was transformed into competent cells of strain 630B in step (2) to obtain the final constructed strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG , recorded as strain 630B2.

[0066] (4) Using the starting strain 630B1 as the control, a single colony of strain 630B2 was picked into LB test tube culture medium containing 50 mg / L kanamycin and cultured overnight at 37°C and 180 rpm to obtain seed liquid. 2.5 mL of seed liquid was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium containing 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 48 mg / L, and continue shaking culture for 48 hours. After the fermentation is completed, take 1 mL of fermentation liquid and centrifuge at 12000 rpm for 3 minutes, discard all the supernatant, add 1 mL of distilled water to resuspend the bacteria and the calcium carbonate therein, centrifuge at 12000 rpm for 3 minutes and discard the supernatant, add 1 mL of distilled water to resuspend the bacteria and calcium carbonate again, centrifuge at 12000 rpm for 3 minutes and discard the supernatant. Finally, add 800 μL of distilled water, resuspend the bacteria and calcium carbonate, and then add 200 μL of 20% acetic acid aqueous solution by volume, and let it stand at room temperature for 5 minutes to dissolve the calcium carbonate therein. Take 50 μL of the bacterial solution with dissolved calcium carbonate and add it to 1950 μL of distilled water, dilute 40 times, and finally measure the biomass OD with a spectrophotometer. 600 Then take 1 mL of fermentation broth and centrifuge at 12000 rpm for 3 min. The supernatant is used as a sample for detecting β-alanine concentration. 600 and β-alanine content as Figure 1 As shown in the figure, overexpression of pyc from Corynebacterium glutamicum on the plasmid enhanced the expression of key enzymes in the OAA biosynthesis pathway, which was beneficial to the synthesis of β-alanine. As a result, the production of β-alanine increased from 3.75g / L to 4.72g / L.

[0067] Example 3: Overexpression of pTrc99a-panD in strain 630B BS K104S -aspB CG -aspA-pyc CG -Construction of NCgl0580 strain and its shake flask fermentation

[0068] (1) Construction of pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580 plasmid: plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG As a template, PCR amplification was performed with pTrc99apyc-line-F / pTrc99apyc-line-R as primers, wherein the PCR product was digested with Dpn I and then digested at 37°C for 2h to remove the methylated vector, and then the DNA fragment was recovered with a Clean up kit and set aside. Then, the genome of Corynebacterium glutamicum ATCC13032 was used as a template, and primers NCgl0580-F and NCgl0580-R were used to PCR amplify the NCgl0580 fragment (nucleotide sequence shown in SEQ ID NO.2), and then the DNA fragment was recovered with a Clean up kit and set aside. The pTrc99a-panD was linearized according to the instructions of the One step cloning kit (Vazyme Biotech, Nanjing, China). BS K104S -aspB CG -aspA-pyc CG The plasmid and fragment NCgl0580 were connected together, and the ligation product was transformed into E. coli DH5α competent cells; finally, clones were selected and verified by sequencing to obtain pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -pyc CG -NCgl0580 plasmid.

[0069] (2) Preparation of 630B competent cells, same as in Example 2.

[0070] (3) construct pTrc99a-panD in step (1) BS K104S -aspB CG -aspA-pyc CG The plasmid was transformed into competent cells of strain 630B in step (2) to obtain the final constructed strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BSK104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B3.

[0071] (4) Using 630B2 as the control strain, strains 630B2 and 630B3 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was determined according to Example 1. Biomass OD 600 and β-alanine content as Figure 2 As shown, the results showed that the β-alanine content of strain 630B3 reached 5.03 g / L, which showed that the transporter encoded by NCgl0580 indeed had the function of a β-alanine exporter.

[0072] Example 4: Knockout of the yeeP gene in the genome of strain 630B4 and replacement with gapN at the original site

[0073] (1) Construction of plasmid pTarget-yeeP: Using the pTarget plasmid as a template and pTarget-yeeP-F / pTarget-yeeP-R as primers, amplification was performed. DpnI was added to the PCR product and incubated at 37°C for 2 h to digest the methylated plasmid template. After purification, the methylated plasmid template was transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin. After sequencing, their correctness was verified to obtain plasmid pTarget-yeeP. Then, pTD-line-F / pTD-line-R were used as primers and plasmid pTarget-yeeP was used as a template for amplification. The DNA fragment was recovered using a Clean Up kit to obtain the linearized plasmid pTarget-yeeP.

[0074] (2) Construction of plasmid pTD-yeeP: Using the E. coli W3110 genome as a template, L-yeeP-F / L-yeeP-R and R-yeeP-F / R-yeeP-R as primers, the upstream and downstream homology arms F1 and R1 were amplified; and using a plasmid carrying the gapN gene (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, gapN-F / gapN-R as primers, the gapN gene fragment (nucleotide sequence shown in SEQ ID NO.4) was amplified. The PCR product was recovered using a Clean up purification kit. The upstream and downstream homology arms F1, R1 and the gapN gene fragment were used as templates, and L-yeeP-F / R-yeeP-R were used as primers to fuse the upstream and downstream homology arms containing gapN. The DNA fragment was recovered using a Clean up kit to obtain the fused fragment. The fused fragment and the plasmid pTarget-yeeP linearized in step (1) were then used to construct the plasmid pTD-yeeP through a one-step cloning method, transformed into E. coli DH5α, spread on an LB solid plate containing 50 mg / L spectinomycin, and cultured inverted at 37°C for 12 hours. The correct strain was preliminarily screened using primers T-pTarget-F / T-pTarget-R colony PCR, and finally the correctness of the plasmid pTD-yeeP was verified by sequencing.

[0075] (3) The plasmid pCas9 was transformed into competent cells 630B, spread onto LB solid plates containing 50 mg / L kanamycin, and cultured at 30°C overnight. A single colony was picked and placed in an 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. The cells were cultured at 180 rpm and 30°C until the OD 600 = 0.5, centrifuge at 4000 rpm at 4°C, wash twice with ultrapure water at 4°C, wash once with cold 10% glycerol aqueous solution, and finally resuspend in 10% glycerol aqueous solution, aliquot and store to obtain electrocompetent cells for later use.

[0076] (4) Take 2 μL of the pTD-yeeP plasmid constructed in step (2) and mix it with 100 μL of the electroporation competent cells prepared in step (3), transfer it into a 2 mm electroporation cup, and place it in an ice bath for 45 seconds. TM, BIO-RAD) electroporation transformation with a voltage of 2500 V. 700 μL of 4°C pre-cooled LB medium was immediately added after electroporation, mixed evenly, and immediately transferred to a new sterile 1.5 mL EP tube. The tube was shaken and cultured at 30°C and 150 rpm for 3 h, and then spread on LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin. The tube was inverted and cultured at 30°C for 24 h. Colony PCR was performed using T-yeeP-F / T-yeeP-R as primers, and the correctness of the strain construction was verified by sequencing. The yeeP-replacing gapN strain was successfully constructed.

[0077] (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 48 mg / L IPTG and 50 mg / L kanamycin, culture it at 30℃ overnight, dip the bacterial liquid and streak it onto LB solid culture medium containing 50 mg / L kanamycin, invert and culture it at 30℃ for 20h until a single colony appears, pick a single colony and transfer it to LB solid culture medium containing 50 mg / 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 has 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 50 mg / 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 finally a plasmid-free strain is obtained.

[0078] (6) Transformation plasmid: plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580 was transformed into the plasmid-free competent cells prepared in step (5) to construct strain 630B4: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapN / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580

[0079] (7) Using strain 630B3 as the control strain, strains 630B3 and 630B4 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was determined according to Example 1. Biomass OD 600 and β-alanine content as Figure 3As shown, the β-alanine yield of 630B4 was 5.21 g / L, which was 3.58% higher than that of 630B3. The results indicate that the introduction of the heterologous gene gapN to adjust the glycolysis pathway has a certain degree of enhancement effect on the anabolism of β-alanine.

[0080] Example 5: Replacement of the gltBD gene promoter with P in the genome of strain 630B4 Trc

[0081] (1) Construction of the pTarget-gltBD plasmid: Using the plasmid pTarget as a template and primers pTarget-gltBD-F / pTarget-gltBD-R as amplification primers, Dpn I was added to the PCR product and incubated at 37°C for 1 hour to eliminate the methylated plasmid template. The product was transformed into E. coli DH5α competent cells, plated onto LB solid culture medium containing 50 mg / L spectinomycin, and cultured at 37°C for 12 hours until single colonies appeared. Colony PCR was performed for preliminary verification, and finally sequencing was used to verify the correctness of the constructed plasmid. Linearization was performed as in Example 4 to obtain the linearized plasmid pTarget-gltBD.

[0082] (2) Construction of plasmid pTD-gltBD: Using the E. coli W3110 genome as a template and L-gltBD-F / L-gltBD-R and R-gltBD-F / R-gltBD-R as primers, PCR amplification was performed to obtain upstream and downstream homology arms, which were then cloned into the linearized plasmid pTarget-gltBD. The remaining construction steps were the same as step (2) of Example 4 to obtain plasmid pTD-gltBD.

[0083] (3) The pCas9 plasmid was introduced into the strain 630B4 in Example 4, and competent cells of 630B4 were prepared in the same manner as in Example 4.

[0084] (4) Using the method of Example 4, the plasmid pTD-gltBD was transferred into the competent cells of step (3) to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD.

[0085] (5) Plasmid elimination: The plasmids pTarget and pCas9 of the strain in step (4) were eliminated using the method of Example 4 to obtain a plasmid-free strain.

[0086] (6) Transformation plasmid: plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG-NCgl0580 was transformed into the plasmid-free competent cells prepared in step (5) to construct strain 630B5: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580.

[0087] (7) Using strain 630B4 as a control, strains 630B4 and 630B5 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was determined according to Example 1. Biomass OD 600 and β-alanine content as Figure 4 As shown, the β-alanine yield of 630B4 was 5.50 g / L, a 5.57% increase compared to 630B3. These results suggest that upregulating the expression activity of the gltBD gene to enhance the supply of amino donors for the conversion of oxaloacetate to aspartate is a regulatory strategy that favors β-alanine accumulation.

[0088] Example 6: Deletion of the yjiV gene in the genome of strain 630B5 and replacement with esaR at the original site

[0089] (1) Construction of plasmid pTarget-yjiV: Using the pTarget plasmid as a template and pTarget-yjiV-F / pTarget-yjiV-R as primers, amplification was performed. DpnI was added to the PCR product and incubated at 37°C for 2 h to digest the methylated plasmid template. After purification, the product was transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin. After sequencing, the linearized plasmid pTarget-yjiV was obtained using the method of Example 4.

[0090] (2) Construction of plasmid pTD-yjiV: Using the E. coli W3110 genome as a template and L-yjiV-F / L-yjiV-R and R-yjiV-F / R-yjiV-R as primers, upstream and downstream homology arms were amplified; and using a plasmid carrying the esaR gene (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template and esaR-F / esaR-R as primers, the esaR gene (nucleotide sequence shown in SEQ ID NO. 7) was amplified. The upstream and downstream homology arms and the esaR gene were ligated using the method of Example 4 and then cloned into the linearized plasmid pTarget-yjiV in step (1) to obtain plasmid pTD-yjiV.

[0091] (3) Using the method of Example 4, the plasmid pCas9 was introduced into the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD and competent cells were prepared.

[0092] (4) Using the method of Example 4, the plasmid pTD-yjiV was transferred into the competent cells of step (3) to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaR.

[0093] (5) Plasmid elimination: Using Example 4, the strain pTarget and pCas9 plasmids of step (4) were eliminated to obtain the plasmid-free strain 630B6: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaR.

[0094] Example 7: Knockout of the yjiT gene in the genome of strain 630B6 and replacement of the original site with the esaI gene with the Pbs promoter

[0095] (1) Construction of plasmid pTarget-yjiT: Using the pTarget plasmid as a template and primers pTarget-yjiT-F / pTarget-yjiT-R as amplification primers, DpnI was added to the PCR product and incubated at 37°C for 2 h to digest the methylated plasmid template. After purification, the product was transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin. After sequencing, the linearized plasmid pTarget-yjiT was obtained using the method of Example 4.

[0096] (2) Construction of plasmid pTD-yjiT: Using the E. coli W3110 genome as a template and L-yjiT-F / L-yjiT-R and R-yjiT-F / R-yjiT-R as primers, upstream and downstream homology arms were amplified; and using a plasmid carrying the esaI gene (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template and Pbs-esaI-F / esaI-R as primers, the esaI gene (nucleotide sequence shown in SEQ ID NO. 10) was amplified. The upstream and downstream homology arms and the esaI gene were ligated using the method of Example 4 and then cloned into the linearized plasmid pTarget-yjiT in step (1) to obtain plasmid pTD-yjiT.

[0097] (3) Using the method of Example 4, the plasmid pCas9 was introduced into the strain 630B6 and competent cells were prepared.

[0098] (4) Using the method of Example 4, the plasmid pTD-yjiV was transferred into the competent cells of step (3) to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaI.

[0099] (5) Plasmid elimination: Using Example 4, the strain pTarget and pCas9 plasmids of step (4) were eliminated to obtain the plasmid-free strain 630B7: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaI.

[0100] Example 8: Replacement of the thrA gene promoter with P in the genome of strain 630B7 esaS .

[0101] (1) Construction of plasmid pTarget-thrA: Using the pTarget plasmid as a template and pTarget-thrA-F / pTarget-thrA-R as primers, amplification was performed. DpnI was added to the PCR product and incubated at 37°C for 2 h to digest the methylated plasmid template. After purification, the product was transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin. After verification by sequencing, the linearized plasmid pTarget-thrA was obtained using the method of Example 4.

[0102] (2) Construction of plasmid pTD-thrA: Using the E. coli W3110 genome as a template, L-thrA-F / L-thrA-R and R-thrA-F / R-thrA-R as primers, the upstream and downstream homology arms of the thrA original promoter were amplified. esaS The promoter plasmid (synthesized by Beijing Qingke Biotechnology Co., Ltd.) was used as a template, and P esaS -F / P esaS -R is a primer, which amplifies the promoter P esaS (nucleotide sequence as SEQ ID NO.12), the upstream and downstream homology arms and P were separated by the method of Example 4. esaS The promoter was ligated and cloned into the linearized plasmid pTarget-thrA in step (1) to obtain plasmid pTD-thrA.

[0103] (3) Using the method of Example 4, the plasmid pCas9 was introduced into the strain 630B7 and competent cells were prepared.

[0104] (4) Using the method of Example 4, the plasmid pTD-thrA was transferred into the competent cells of step (3) to construct strain 630B8.

[0105] : E.coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -th rA.

[0106] (5) Plasmid elimination: Using Example 4, eliminate the pTarget and pCas9 plasmids of the strain in step (4) to obtain a plasmid-free strain: E. coli

[0107] W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -th rA.

[0108] (6) Construction of strain 630B9

[0109] Extract plasmid pTrc99a-panD from strain 630B3 BS K104S -aspB CG -aspA-pyc CG -NCgl0580 and transformed into strain 630B8 to construct strain 630B9: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -th rA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580.

[0110] (7) Using strain 630B5 as a control, strains 630B5 and 630B9 were subjected to shake flask fermentation tests according to the method of Example 2. The content of β-alanine was determined according to Example 1. Biomass OD 600 and β-alanine content as Figure 5As shown, the results showed that strain 630B9 exhibited normal cell growth and β-alanine production reached 5.68 g / L. The results showed that using the quorum sensing system to dynamically regulate the expression of the thrA gene can further increase β-alanine production.

[0111] Example 9: Fed-batch fermentation in a 5-L fermenter

[0112] Streak the strain 630B9 in Example 8 onto an LB plate containing 50 mg / L kanamycin resistance, culture at 37°C overnight, pick a single colony to an LB test tube containing 50 mg / L kanamycin resistance, culture at 37°C, 150 rpm overnight to prepare a seed solution. Inoculate the seed solution into 100 mL of LB medium containing 50 mg / L kanamycin resistance at a volume concentration of 5%, culture at 37°C, 150 rpm overnight to serve as a secondary seed solution. Inoculate the secondary seed solution into a 5-L fermentation tank containing 2 L of fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%, and ferment at 30°C, 300 rpm, 1.0 V / V / min of ventilation, and maintain the pH at 6.80 until the OD 600 When the pH value reaches 10, IPTG is added at a final concentration of 48 mg / L for induction. When the pH value is higher than 6.80 (the initial sugar in the fermenter is consumed), automatic feeding is started, and feeding medium is added until the pH is lower than 6.80, and feeding is stopped. The residual sugar in the fermenter is maintained at a low level, and the sugar concentration is maintained at 0-2 g / L. The total amount of feeding medium added is 1400 mL, and the culture is carried out for 64 hours. The β-alanine production in the fermentation broth is detected by high-performance liquid chromatography according to Example 1, and the biomass OD is detected by spectrophotometry. 600 , the DNS method was used to detect sugar concentration; Figure 6 As shown in the figure, the β-alanine production reached 122.06 g / L at 60 h, and the final biomass OD 600 The fermentation results showed that the metabolically engineered β-alanine-producing strain 630B9 had good production performance, laying a foundation for the industrial production of β-alanine.

[0113] The medium formula of the 5-L fermentation tank is as follows: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, and 1 mL / L trace element solution; the composition of the trace element solution is 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0114] Feed medium formula: glucose 600 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB12, 48 mg / L IPTG, 50 mg / L kanamycin, 2 mL / L trace element solution, the solvent is water, and the pH is adjusted to 6.8 with 14% ammonia water.

Claims

1. A recombinant genetically engineered bacterium for producing β-alanine by dynamically regulating a competitive pathway, characterized in that: The recombinant genetically engineered bacteria were based on Escherichia coli 630B1 and were subjected to one or more of the following gene editing: overexpression of pyc on plasmid pTrc99a. CG The yeeP gene and the NCgl0580 gene were replaced with the gapN gene, the gltBD promoter was replaced with the Trc promoter, the yjiV gene was replaced with the EsaR gene, the yjiT gene was replaced with the EsaI gene using the artificial promoter Pbs to replace the original promoter, and the thrA gene promoter was replaced with the PesaS promoter; the starting strain 630B1: E. coli W3110 TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA.

2. The recombinant genetically engineered bacterium according to claim 1, wherein The pyc CG The nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the NCgl0580 gene is shown in SEQ ID NO.

2.

3. The recombinant genetically engineered bacterium according to claim 1, wherein The nucleotide sequence of the gapN gene is shown in SEQ ID NO.4; the nucleotide sequence of the gltBD gene is shown in SEQ ID NO.5; the nucleotide sequence of the EsaR gene is shown in SEQ ID NO.7; and the nucleotide sequence of the EsaI gene is shown in SEQ ID NO.

10.

4. The recombinant genetically engineered bacterium according to claim 1, wherein The nucleotide sequence of the Pbs promoter is shown in SEQ ID NO.9; esaS The nucleotide sequence of the promoter is shown in SEQ ID NO.12; the nucleotide sequence of the Trc promoter is shown in SEQ ID NO.

13.

5. The recombinant genetically engineered bacterium according to claim 1, wherein The nucleotide sequence of the yeeP gene is shown in SEQ ID NO.3; the nucleotide sequence of the yjiV gene is shown in SEQ ID NO.6; the nucleotide sequence of the yjiT gene is shown in SEQ ID NO.8; and the nucleotide sequence of the thrA gene is shown in SEQ ID NO.

11.

6. The recombinant genetically engineered bacterium according to claim 1, wherein The recombinant genetic engineering bacteria used Escherichia coli 630B1 as the starting strain, and the following gene editing was performed in sequence: pyc was overexpressed on the plasmid pTrc99a. CG The yeeP gene and the NCgl0580 gene were replaced by the gapN gene, the gltBD promoter was replaced by the Trc promoter, the yjiV gene was replaced by the EsaR gene, the yjiT gene was replaced by the EsaI gene using the artificial promoter Pbs to replace the original promoter, and the thrA gene promoter was replaced by P esaS promoter.

7. The recombinant genetically engineered bacterium according to claim 6, wherein The recombinant genetically engineered bacteria were constructed as follows: (1) Overexpression of codon-optimized pyc from Corynebacterium glutamicum ATCC 13032 on plasmid pTrc99a CG : Plasmid pTrc99a-panD BS K104S -aspB CG -aspA was used as a template to obtain the linearized plasmid pTrc99a-panD BS K104S -aspB CG -aspA; linearized plasmid pTrc99a-panD BS K104S -aspB CG -aspA and the codon-optimized pyc shown in SEQ ID NO.1 CG Gene fragment ligation to construct plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG The plasmid was transformed into competent cells of strain 630B to construct strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG , recorded as strain 630B2; strain 630B: E. coli W3110 Trcpan DTrcppc ΔpykA ΔcycA; (2) Overexpression of codon-optimized NCgl0580 from Corynebacterium glutamicum ATCC 13032 on plasmid pTrc99a: BS K104S -aspB CG -aspA-pyc CG The linearized plasmid pTrc99a-panD was obtained as a template BS K104S -aspB CG -aspA-pyc CG ; Linearize the plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG The codon-optimized NCgl0580 gene fragment shown in SEQ ID NO.2 was ligated to construct plasmid pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580 and transformed into strain 630B competent cells to construct strain E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B3; (3) Replace the yeeP gene with the gapN gene: Use the genome of E. coli W3110 as a template to amplify the upstream and downstream homology arms of the yeeP gene and fuse them with the gapN gene to obtain donor DNA; The donor DNA and linearized pTarget-yeeP plasmid were ligated and transformed into the starting strain 630B electroporation competent cells, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapN / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B4; (4) Replacement of gltBD promoter with Trc promoter: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the original promoter were amplified; the upstream and downstream homology arms and the linearized plasmid pTarget-gltBD were transferred into the competent cells of strain 630B4 using CRISPR / Cas9 gene editing technology, the pTarget and pCas9 plasmids were eliminated, and the gltBD promoter on the genome of strain 630B4 was replaced with the Trc promoter using CRISPR / Cas9 gene editing technology to construct the strain E. coli W3110 Trc-ppc Trc-panD ΔpykA ΔcycA ΔyeeP:: gapNTrc-gltBD / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580, designated as strain 630B5; (5) Replace the yjiV gene with the EsaR gene: Using the genome of E. coli W3110 as a template, amplify the upstream and downstream homology arms of the yjiV gene and fuse them with the EsaR gene to obtain donor DNA; The donor DNA and linearized pTarget-yjiV plasmid were ligated and transformed into the starting strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBD electroporation competent cells, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaR, which was designated as strain 630B6. (6) Replace the yjiT gene with the esaI gene with the promoter pbs: Using the genome of E. coli W3110 as a template, amplify the upstream and downstream homology arms of the yjiT gene and fuse them with the esaI gene containing the promoter pbs to obtain donor DNA; The donor DNA and linearized pTarget-yjiT plasmid were ligated and transformed into electrocompetent cells of the starting strain 630B6, and the pTarget and pCas9 plasmids were eliminated to construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaI, which was designated as strain 630B7. (7) Replace the thrA gene promoter with P esaS :Using the genome of E.coli W3110 as a template, the upstream and downstream homology arms of the original promoter of thrA gene were amplified and the promoter P esaS Fragments are fused to obtain donor DNA; The donor DNA and linearized pTarget-thrA plasmid were ligated and transformed into electroporation competent cells of strain 630B7 to eliminate the pTarget and pCas9 plasmids and construct the strain E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -thrA, designated as strain 630B8; (8) Extraction of plasmid pTrc99a-panD from strain 630B3 BS K104S -aspB CG -aspA-pyc CG -NCgl0580 and transformed into strain 630B8 to construct strain 630B9: E. coli W3110Trc-ppcTrc-panDΔpykAΔcycAΔyeeP::gapNTrc-gltBDΔyjiV::EsaRΔyjiT::Pbs-EsaIΔthrA::P esaS -thrA / pTrc99a-panD BS K104S -aspB CG -aspA-pyc CG -NCgl0580.

8. Use of the recombinant genetically engineered bacteria according to claim 1 in the production of β-alanine by microbial fermentation.

9. The use according to claim 8, characterized in that The application comprises: inoculating the recombinant genetically engineered bacteria into a fermentation medium containing 48 mg / L IPTG and 50 mg / L kanamycin, culturing at 20-40°C and 100-300 rpm for 48 hours until the fermentation is complete, obtaining a fermentation broth containing β-alanine, and separating and purifying the fermentation broth to obtain β-alanine; The fermentation medium has the following formula: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, and 1 mL / L trace element solution, without the need to adjust the pH value; the trace element solution comprises the following components: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

10. The use according to claim 8, characterized in that The fermentation is carried out in a fermentor: the recombinant genetically engineered bacteria are inoculated onto an LB plate containing 50 mg / L kanamycin resistance, cultured at 37°C overnight, a single colony is picked and transferred to an LB medium containing 50 mg / L kanamycin resistance, cultured at 37°C and 150 rpm overnight to prepare a seed solution; the seed solution is inoculated into an LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, cultured at 37°C and 150 rpm overnight to serve as a secondary seed solution; The secondary seed solution was inoculated into a 5-L fermentation tank containing 2 L of fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%. The fermentation was carried out at 30°C, a speed of 300 rpm, a ventilation volume of 1.0 V / V / min, and a pH value of 6.

80. 600 When the pH reaches 10, IPTG is added at a final concentration of 48 mg / L for induction; 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, feeding is stopped, and the culture is continued for 60-64 hours to obtain a fermentation broth containing β-alanine; The fermentation medium formula is as follows: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.5 g / L anhydrous betaine, 5 mg / L VB1, 2 mg / L VB12, 1 mL / L trace element solution, no pH adjustment required; the trace element solution composition is: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water; The feed medium composition was as follows: glucose 600 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, anhydrous betaine 0.5 g / L, 5 mg / L VB1, 2 mg / L VB12, 48 mg / L IPTG, 50 mg / L kanamycin, 2 mL / L trace element solution, the solvent was water, and the pH was adjusted to 6.8 with 14% ammonia water.

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