Recombinant genetically engineered bacteria for producing beta-alanine and application thereof

Abstract

The application discloses three kinds of recombinant genetically engineered bacteria for producing beta-alanine and application thereof, and belongs to the field of biotechnology.The recombinant genetically engineered bacteria A2 is constructed by knocking out a pck gene from an escherichia coli A1 as a starting strain; the recombinant genetically engineered bacteria B2 is constructed by knocking out the pck gene from an escherichia coli B1 as the starting strain; and the recombinant genetically engineered bacteria C2 is constructed by knocking out the pck gene from an escherichia coli C1 as the starting strain, wherein the escherichia coli C1 is an escherichia coli containing a Trc promoter, ppc, panD and glK genes, and knocking out pykA, aspA and pykF genes, and introducing a pTrc99a plasmid containing a panD gene of bacillus subtilis. Compared with the strain without knocking out the pck gene, the beta-alanine production capacity is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of recombinant genetically engineered bacteria for producing β-alanine, specifically to three types of recombinant genetically engineered bacteria for producing β-alanine and their applications. Background Technology

[0002] β-Alanine, also known as 3-aminopropionic acid, is the only naturally occurring β-type non-protein amino acid. It is a crucial precursor for the synthesis of D-pantothenic acid and coenzyme A in organisms, playing a vital physiological role in maintaining cell growth. As an important 3-carbon platform compound, β-alanine is widely used in food, feed additives, fine chemicals, and pharmaceuticals, and is one of the 12 most promising 3-carbon chemical products. In the pharmaceutical field, β-alanine is mainly used to synthesize pantothenic acid and calcium pantothenate. It is a precursor to acyl carrier proteins (ACP) and coenzyme A (CoA), playing important physiological roles in cell regeneration, assisting in the normal development of the central nervous system, and participating in the metabolism of proteins, fats, and sugars in the body. Furthermore, β-alanine can also serve as a raw material for drugs such as vitamin B5, balsalazine, and carnosine. In the food industry, β-alanine can be used not only as a food additive to improve food flavor but also as a nutritional supplement for athletes to improve physical function. In the fine chemical industry, β-alanine can be used to prepare precipitants in pharmaceuticals, electroplating corrosion inhibitors, and antidotes for lead poisoning. In addition, it can also be used directly to produce poly-β-alanine, which is widely used in cosmetics, water purification and construction.

[0003] Currently, there are three main methods for producing β-alanine both domestically and internationally: chemical synthesis, which utilizes nitrile substances such as acrylonitrile, acrylic acid, and β-aminopropionitrile to synthesize β-alanine under high pressure and high temperature, and strong acid and strong base conditions. However, chemical synthesis consumes a lot of energy, requires sophisticated equipment, and generates substances harmful to the environment and human health during the production process. Furthermore, the byproducts produced can significantly hinder subsequent separation and purification. The second method is bio-enzymatic catalysis, which uses aspartic acid as a substrate and utilizes aspartic acid decarboxylase derived from strains such as Bacillus subtilis or Corynebacterium glutamicum to catalyze the production of β-alanine in Escherichia coli. This method offers milder conditions, is safer, and produces less pollution, attracting increasing attention from researchers. The third method is microbial fermentation, which utilizes synthetic biology, systems metabolic engineering, protein engineering, and transcriptomics and metabolomics techniques to modify the metabolic process of the target product, β-alanine, directing more metabolic flux towards the desired product. With the increasing severity of climate change and environmental problems, exploring clean, environmentally friendly, and low-energy-consumption production methods using inexpensive carbon sources such as glucose has attracted considerable attention from researchers.

[0004] With a deeper understanding of microbial metabolic processes and the continuous development of metabolic engineering technologies (systems metabolic engineering, synthetic biology, promoter engineering, protein engineering, transcriptomics, and metabolomics), the production of various high-value-added products, including β-alanine, using inexpensive glucose and other carbon sources has broad prospects for application. However, due to the physiological characteristics of wild-type *E. coli*, the β-alanine synthesized in *E. coli* cells can only meet their own growth needs and will not accumulate in large quantities. Therefore, it is necessary to rationally modify the β-alanine synthesis pathway to construct a stable and efficient cell factory. High-yielding β-alanine strains can be obtained through strategies such as genetic engineering and metabolic engineering. However, in the process of constructing genetically engineered bacteria, the relationship between different metabolic pathways must be considered. While increasing the metabolic throughput of the target product, normal cell growth must not be affected. Therefore, rational metabolic engineering modification from multiple perspectives is required.

[0005] The de novo synthesis pathway of β-alanine in *E. coli* includes glucose uptake, glycolysis, the TCA cycle, L-aspartate synthesis, and the β-alanine synthesis pathway. First, glucose undergoes phosphorylation in either a PTS or non-PTS system to synthesize glucose-6-phosphate. The carbon flux then enters the glycolysis pathway, where the precursor of β-alanine, phosphoenolpyruvate, is synthesized. Phosphoenolpyruvate carboxylase catalyzes the production of oxaloacetate from phosphoenolpyruvate. Oxaloacetate is unstable and easily decomposes, generating aspartate under the catalysis of aspartate transaminase. Subsequently, aspartate decarboxylase catalyzes the formation of β-alanine. L-lysine, L-threonine, O-succinylhomoserine, and homoserine belong to the aspartate group of amino acids, sharing the same precursor L-aspartate as β-alanine. Therefore, weakening competing pathways is an effective synthesis strategy. However, further investigation is needed to identify the bottlenecks affecting β-alanine production. Summary of the Invention

[0006] The purpose of this invention is to construct three recombinant genetically engineered bacteria for producing β-alanine using metabolic engineering and gene editing technologies, and to demonstrate the application of these bacteria in the microbial fermentation production of β-alanine. This overcomes the drawbacks of high energy consumption and high pollution associated with chemical methods for producing β-alanine, as well as the high cost associated with enzymatic methods. This invention lays the foundation for the industrial production of β-alanine.

[0007] The technical solution adopted in this invention is as follows:

[0008] Escherichia coli A1: The panD gene was replaced with the panD gene of Bacillus subtilis containing the Trc promoter, the ppc, glk, and gltBD genes containing the Trc promoter, the ptsG, galR, cycA, and iclR genes were knocked out, the lysC and thrA genes with the start codon ATG replaced with GTG were replaced, and the pTrc99a plasmid containing the panD gene of Bacillus subtilis and the aspB gene of Corynebacterium glutamicum was introduced.

[0009] Escherichia coli B1: Escherichia coli containing the panD and ppc genes with the Trc promoter, the pykA and cycA genes knocked out, and the pTrc99a plasmid containing the panD gene of Bacillus subtilis and the aspA and aspB genes of Corynebacterium glutamicum, with the panD gene at position 104 mutated to serine.

[0010] Escherichia coli C1: This is an Escherichia coli containing the ppc, panD, and glK genes with the Trc promoter, the pykA, aspA, and pykF genes knocked out, and the pTrc99a plasmid containing the panD gene of Bacillus subtilis introduced.

[0011] Escherichia coli A1: E. coli W3110TrcpanD (bs) ::panDTrcppcΔptsGTrcglkΔgalRΔcycAΔiclRlysC*thrA*Trcglt BD / pTrc99a-panD (bs) -aspB (cg) Construction reference Wang, P., Zhou, HY, Li, B., Ding, WQ, Liu, ZQ, Zheng, YG, 2021. Multiplex modification of Escherichiacoli for enhanced β-alanine biosynthesis through metabolic engineering.

[0012] Escherichia coli B1: E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD (bs) K104S aspAaspB (cg)Constructed with reference to Li, B., Zhang, B., Wang, P., Cai,

[0013] Recombinant genetically engineered bacteria C2 is derived from Escherichia coli C1: E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk / pTrc99a-panD (bs) The strain was constructed by knocking out the pck gene.

[0014] This invention provides three recombinant genetically engineered bacteria:

[0015] Recombinant genetically engineered bacteria A2, B2, and C2 were constructed by knocking out the pck gene from Escherichia coli A1, B1, and C1.

[0016] The nucleotide sequence of the pck gene is shown in SEQ ID NO.1; the nucleotide sequence of the ppc gene is shown in SEQ ID NO.2; the nucleotide sequence of the panD gene is shown in SEQ ID NO.3; the nucleotide sequence of the pykA gene is shown in SEQ ID NO.4; the nucleotide sequence of the aspA gene is shown in SEQ ID NO.5; the nucleotide sequence of the pykF gene is shown in SEQ ID NO.6; the nucleotide sequence of the glk gene is shown in SEQ ID NO.7; the nucleotide sequence of the panD gene is shown in SEQ ID NO.7; the nucleotide sequence of the pck 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 aspA gene is shown in SEQ ID NO.1; the nucleotide sequence of the ppc gene is shown in SEQ ID NO.2; the nucleotide sequence of the panD gene is shown in SEQ ID NO.3; the (bs) The gene nucleotide sequence is shown in SEQ ID NO.8, and the Trc promoter nucleotide sequence is shown in SEQ ID NO.9.

[0017] The recombinant genetically engineered bacteria of this invention are constructed according to the following method:

[0018] (1) Construction of strains A2 / B2: Using the genome of E. coli W3110 as a template, donor DNA was amplified using L-pck-F / L-pck-R and R-pck-F / R-pck-R primers, respectively; using pTarget plasmid as a template, the plasmid was amplified using pTarget-pck-F and pTarget-pck-R primers and linearized to obtain linearized pTarget-pck plasmid; donor DNA and linearized pTarget-pck plasmid were ligated and transformed into electroporation competent cells of the starting strain A1 / B1, and pTarget and pCas9 plasmids were eliminated to construct strains A2 and B2.

[0019] (2) Construction of strain C2:

[0020] Using E. coli W3110 as the starting strain, the ppc gene in the genome was overexpressed using CRISPR / Cas9 gene editing technology to obtain the engineered strain E. coli W3110 Trcppc;

[0021] The panD gene promoter in the genome of engineered E. coli W3110 Trcppc was replaced with the trc promoter using CRISPR / Cas9 gene editing technology, resulting in engineered E. coli W3110 Trcppc TrcpanD;

[0022] The pykA gene was knocked out in the genome of engineered E. coli W3110 Trcppc TrcpanD using CRISPR / Cas9 gene editing technology, resulting in engineered E. coli W3110 Trcppc TrcpanDΔpykA.

[0023] Using E. coli W3110 TrcppcTrcpanDΔpykA as the chassis strain, the panD gene from Bacillus subtilis was overexpressed on the pTrc99a plasmid to obtain the engineered strain E. coli W3110 TrcppcTrcpanDΔpykA / pTrc99a-panD (bs) ;

[0024] The engineered E. coli W3110 Trcppc TrcpanDΔpykA / pTrc99a-panD strain was knocked out using CRISPR / Cas9 gene editing technology. (bs) The aspA gene in the genome was used to obtain the engineered E. coli W3110 TrcppcTrcpanDΔpykAΔaspA / pTrc99a-panD (bs) ;

[0025] The engineered E. coli W3110 strain was knocked out using CRISPR / Cas9 gene editing technology. The gene sequence was: Trcppc TrcpanDΔpykAΔaspA / pTrc99a-panD. (bs) The pykF gene in the genome was used to obtain the engineered strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykF / pTrc99a-panD (bs) ;

[0026] E.coliW3110 TrcppcTrcpanDΔpykAΔaspAΔpykF / pTrc99a-panD (bs) The glk gene promoter was replaced with the trc promoter in the genome, resulting in the engineered strain C1: E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk / pTrc99a-panD (bs) ;

[0027] Using the genome of E. coli W3110 as a template, donor DNA was amplified using L-pck-F / L-pck-R and R-pck-F / R-pck-R primers, respectively. Using pTarget plasmid as a template, the plasmid was amplified using pTarget-pck-F and pTarget-pck-R primers and linearized to obtain linearized pTarget-pck plasmid. The donor DNA and linearized pTarget-pck plasmid were ligated and transformed into the starting strain A1 / B1 electroporation competent cells to eliminate pTarget and pCas9 plasmids and construct strain C2.

[0028] This invention modifies the β-alanine synthesis network of Escherichia coli by knocking out the gene pck encoding phosphoenolpyruvate carboxykinase, reducing OAA consumption, increasing carbon flux in the β-alanine synthesis pathway, and increasing the oxaloacetate substrate pool concentration. This overcomes the deficiency of insufficient oxaloacetate substrate in the original strain, thereby increasing the yield of β-alanine.

[0029] This invention also relates to the application of the recombinant genetically engineered bacteria in the microbial fermentation preparation of β-alanine. The application involves: inoculating the recombinant genetically engineered bacteria into a kanamycin fermentation medium, continuing the culture until fermentation is complete, obtaining a fermentation broth containing β-alanine, and then separating and purifying the fermentation broth to obtain β-alanine. More preferably, the recombinant genetically engineered bacteria are inoculated into a fermentation medium containing 50 mg / L kanamycin and cultured at 30°C and 180 rpm until the bacterial cells reach OD. 600=0.5, at this point, add IPTG to a final concentration of 0.2mM to induce gene expression, continue culturing for 48h until fermentation ends, and obtain fermentation broth containing β-alanine. Separate and purify the fermentation broth to obtain β-alanine.

[0030] The fermentation medium formulation is as follows: glucose 18-22 g / L, (NH4)2SO4 14-18 g / L, yeast extract 3-5 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.2-1 g / L, CaCO3 13-17 g / L, VB1 0.3-0.5 mg / L, VB 12 0.1–0.3 mg / L, trace element solution 0.5–1.5 mL / L, solvent is water, no pH adjustment required; further preferred: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 4 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, CaCO3 15 g / L, 0.4 mg / L VB1, 0.2 mg / L VB 12 A 1 mL / L trace element solution, with water as the solvent, requires no pH adjustment.

[0031] The trace element solution comprises the following components: 8–12 g / L CaCl2, 8–12 g / L FeSO4·7H2O, 0.8–1.2 g / L ZnSO4·7H2O, 0.1–0.3 g / L CuSO4, and 0.01–0.04 g / L NiCl2·7H2O, with deionized water as the solvent; more preferably: 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, with deionized water as the solvent.

[0032] Before fermentation, the recombinant genetically engineered bacteria are inoculated into LB medium and incubated overnight at 37°C and 180 rpm to prepare a seed culture. The seed culture is then inoculated into the fermentation medium at a volume concentration of 5%.

[0033] The fermentation is carried out in a fermenter: the recombinant genetically engineered bacteria are inoculated onto LB agar plates containing 50 mg / L kanamycin resistance and cultured overnight at 37°C. Single colonies are picked and cultured overnight at 37°C and 150 rpm in LB test tubes containing 50 mg / L kanamycin resistance to prepare seed culture. The seed culture is inoculated into LB medium at a volume concentration of 5% and cultured overnight at 37°C and 150 rpm to obtain secondary seed culture. The secondary seed culture is inoculated into a 5-L fermenter containing 2 L of fermentation medium at a volume concentration of 15%, and IPTG is added to a final concentration of 0.2 mM. Fermentation is carried out at 30°C, 500 rpm, and an aeration rate of 0.5 V / V·min. When the pH value is higher than 6.80 (the initial sugar in the fermenter is consumed), automatic feeding is started. Feeding medium is added until the pH value is lower than 6.80 and then feeding is stopped. After culturing for 90 h, a fermentation broth containing β-alanine is obtained. The fed culture medium consisted of: glucose 500 g / L, (NH4)2SO4 16 g / L, yeast extract 4 g / L, KH2PO4 14 g / L, NaHCO3 10 g / L, VB1 0.4 mg / L, and VB2 0.2 mg / L. 12 The solvent is water, and the pH is adjusted to 6.8 with 50% ammonia.

[0034] The feeding rate is 25 mL / h, and the total amount of feeding medium added is 800 mL / 2 L.

[0035] The fermentation broth separation and purification method is as follows: First, centrifuge at 12000 rpm and 4℃ for 10 min to remove the bacterial cells and other solid particles in the fermentation broth. Then, use microfiltration to remove the macromolecular proteins. Next, use deionization technology to remove the inorganic salts. Add activated carbon for decolorization. Separate β-alanine with ion exchange resin. Concentrate under vacuum. Add ethanol and cool to crystallize to obtain β-alanine crystals.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] Traditional chemical methods for synthesizing β-alanine require toxic substances such as nitriles, high temperature and pressure conditions, and strong acids and bases, resulting in demanding equipment, high energy consumption, and high pollution. Bioenzymatic synthesis of β-alanine suffers from substrate inhibition, low yield, and high separation and purification costs. Furthermore, the substrates aspartic acid or fumaric acid are expensive, further increasing production costs. Microbial fermentation for β-alanine production offers low-cost glucose as a raw material, mild conditions, and environmental friendliness. Pck encodes phosphoenolpyruvate carboxykinase, a key enzyme in the gluconeogenesis pathway. Knocking out Pck blocks gluconeogenesis while activating the glyoxylate cycle. This reduces carbon loss in the gluconeogenesis pathway and, conversely, activates the glyoxylate cycle. Validation in three E. coli strains with different genetic backgrounds showed that Pck knockout significantly increased β-alanine production: 41.2% in strain A2, 17.4% in strain B2, and 45.9% in strain C2. Attached Figure Description

[0038] Figure 1 Biomass OD of strains A1 and A2 600 A bar chart showing the concentration of β-alanine.

[0039] Figure 2 Biomass OD of strains B1 and B2 600 A bar chart showing the concentration of β-alanine.

[0040] Figure 3 Biomass OD of strains C1 and C2 600 A bar chart showing the concentration of β-alanine.

[0041] Figure 4 Biomass OD of strain C1 in a 5-L fermenter 600 Concentration curves of residual sugar and β-alanine.

[0042] Figure 5 Biomass OD of strain C2 in a 5-L fermenter 600 Concentration curves of residual sugar and β-alanine. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0044] In the examples, the final concentration of kanamycin in both liquid and solid culture media was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of IPTG was 0.2 mM.

[0045] The strain E. coli W3110 was deposited at the Coli Genetic Stock Center (CGSC) of Yale University on August 5, 1975, with accession number CGSC#4474, and has been disclosed in patents US 2009 / 0298135 A1 and US 2010 / 0248311 A1.

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

[0047]

[0048] Table 2 Primers used in gene editing

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Example 1: In strain A1: E. coli W3110TrcpanD (bs) ::panDTrcppcΔptsGTrcglkΔgalRΔcycAΔiclRlysC*thrA*TrcgltBD / pTrc99a-panD (bs) -aspB (cg) pck knockout

[0055] 1. Construction of strain A2

[0056] Using CRISPR / Cas9-mediated gene editing technology, the phosphoenolpyruvate carboxykinase gene pck (nucleotide sequence shown in SEQ ID No. 1) was knocked out to enhance the accumulation of oxaloacetate substrate pools. The specific operation is as follows:

[0057] (1) Construction of pTarget-pck plasmid: Using plasmid pTarget as a template and pTarget-pck-F / pTarget-pck-R as primers, the PCR product was amplified. After adding DpnI, the product was digested at 37℃ for 1 h. The DNA fragments were recovered and purified using a Cleanup kit and then transformed into E. coli DH5α. The product was plated on LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 30℃ for 20 h. Colony PCR was used for preliminary verification, and then sequencing was used to verify the correctness of the pTarget-pck plasmid, thus obtaining the pTarget-pck plasmid. Then, using pTD-line-F / pTD-line-R as primers and pTarget-pck as a template, the product was amplified. After adding DpnI, the product was digested at 37℃ for 3 h. The DNA fragments were recovered using a Cleanup kit to obtain the linearized pTarget-pck plasmid.

[0058] (2) Construction of plasmid pTD-pck containing Donor: Using the genome of E. coli W3110 as a template, the upstream homologous arm F1 was amplified using L-pck-F / L-pck-R primers; the downstream homologous arm R1 was amplified using R-pck-F / R-pck-R primers. The PCR products were recovered using a Clean Up purification kit. Using the upstream and downstream homologous arms F1 and R1 as templates, the upstream and downstream homologous arms containing pck were fused using L-pck-F / R-pck-R primers. The DNA fragments were recovered using a Clean Up kit to obtain the fused fragment. The fused fragment and the linearized plasmid pTarget-pck from step (1) were then used to construct plasmid pTDpck using a one-step cloning method. The plasmid was transformed into E. coli DH5α, plated onto LB solid plates containing 50 mg / L spectinomycin, and incubated upside down at 30°C for 20 h. Colony PCR was used to preliminarily screen for correct strains, and finally, the correctness of plasmid pTDpck was verified by sequencing.

[0059] (3) Transform plasmid pCas9 into competent cells A1, spread them onto LB agar plates containing 50 mg / L kanamycin, and incubate overnight at 30°C. Pick single colonies and transfer them to LB test tubes containing 50 mg / L kanamycin resistance, and incubate overnight at 30°C. Then, inoculate 1% (v / v) into 100 mL of LB medium, add kanamycin resistance at a final concentration of 50 mg / L and 10 mM L-arabinose, and incubate at 180 rpm and 30°C until OD500. 600 =0.5, centrifuged at 4℃ and 4000rpm. Washed twice with cold ultrapure water at 4℃, then washed once with cold 10% glycerol, and finally resuspended in 10% glycerol, aliquoted and stored to obtain electrocompetent cells for later use.

[0060] (4) Take 2 μL of the pTDpck plasmid constructed in step (2) and mix it with 100 μL of the electrocompetent cells prepared in step (3). Transfer the mixture into a 2 mm electroporation cuvette, incubate on ice for 45 s, and then electroporate using a MicroPluser. TM BIO-RAD electroporation was performed at a voltage of 2500V. Immediately after electroporation, 700μL of pre-cooled LB medium at 4℃ was added and mixed thoroughly. The mixture was then immediately transferred to a new sterile 1.5mL EP tube and cultured at 30℃ and 150rpm for 3 hours. The culture was then spread on LB solid medium containing 50mg / L kanamycin and 50mg / L spectinomycin and incubated upside down at 30℃ for 24 hours. Colony PCR was performed using T-pck-F / T-pck-R primers to verify the colony structure, and sequencing was used to verify the correctness of the strain construction. The pck knockout strain was successfully constructed.

[0061] (5) Elimination of pTarget and pCas9 plasmids: Pick a positive single colony from step (4) and inoculate it into a test tube containing 2 mM IPTG and 50 mg / L kanamycin. Incubate overnight at 30°C. Streak the bacterial solution onto LB solid medium containing 50 mg / L kanamycin. Incubate upside down at 30°C for 20 h until a single colony appears. Pick a single colony onto LB solid medium containing 50 mg / L spectinomycin. Incubate upside down at 30°C for 20 h. If no single colony appears, it means that the pTarget plasmid has been eliminated from this strain. Pick the strain with the pTarget plasmid eliminated and inoculate it into an antibiotic-free LB liquid medium test tube. Incubate at 42°C for 10 h. Streak the bacterial solution onto LB solid medium containing 50 mg / L kanamycin. Incubate upside down at 37°C for 10 h. If no single colony appears, it means that the pCas9 plasmid has been successfully eliminated. Finally, a plasmid-free strain is obtained. Plast the plasmid pTrc99a-panD. (bs) -aspB (cg) Transformed into plasmid-free competent cells, strain A2 was obtained: E. coli W3110 (TrcpanD) (bs) ::panDTrcppcΔptsGTrcglkΔgalRΔcycAΔiclRlysC*thrA*TrcgltBDΔpck) / pTrc99a-panD (bs) -aspB (cg) .

[0062] (6) Using the starting strain A1 as the control, single colonies of strain A2 were picked and cultured overnight at 37°C and 180 rpm in LB test tubes to obtain seed culture. 1 mL of seed culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and 0.25 mM IPTG was added. The flask was then cultured at 30°C and 180 rpm for 48 h with shaking. After fermentation, 1 mL of fermentation broth was centrifuged at 12000 rpm for 3 min, and the supernatant was discarded. 1 mL of distilled water was added to resuspend the bacterial cells and calcium carbonate, and the mixture was centrifuged at 12000 rpm for 3 min, and the supernatant was discarded. Another 1 mL of distilled water was added to resuspend the bacterial cells and calcium carbonate, and the mixture was centrifuged at 12000 rpm for 3 min, and the supernatant was discarded. Finally, 800 μL of distilled water was added to resuspend the bacterial cells and calcium carbonate, and then 200 μL of 20% acetic acid aqueous solution was added. The mixture was left at room temperature for 5 min to dissolve the calcium carbonate. Add 50 μL of bacterial culture containing dissolved calcium carbonate to 1950 μL of distilled water, dilute 40 times, and finally measure the biomass OD using a spectrophotometer. 600 Take another 1 mL of fermentation broth and centrifuge at 12000 rpm for 3 min, then set aside.

[0063] (7) Determination of β-alanine concentration:

[0064] Preparation of 1% 2,4-dinitrofluorobenzene: Dissolve 1 mL of 2,4-dinitrofluorobenzene in 99 mL of acetonitrile.

[0065] Preparation of 0.5M NaHCO3 solution: Dissolve 21g of NaHCO3 in 500mL of deionized water.

[0066] 0.2M PB buffer: Weigh 8.74g Na2HPO4·12H2O and 2.43g Na2HPO4·2H2O and dissolve them in 200mL of deionized water. Store the solution for later use.

[0067] Sample preparation: Dilute the sample concentration with ultrapure water to between 0.1-1 g / L.

[0068] Reaction conditions: Take 100 μL of sample, 100 μL of 0.5 M NaHCO3 solution and 100 μL of 1% 2,4-dinitrofluorobenzene, respectively, incubate at 60℃ for 75 min, and finally add 700 μL of 0.2 M PB buffer and mix well. Filter through a membrane (polyvinylidene fluoride, organic membrane, 0.22 μm) for later use.

[0069] Detection conditions: HPLC model: Thermo Scientific Ultimate 3000, HPLC detection wavelength: 360 nm. β-alanine was separated using a gradient elution program. Mobile phase A consisted of methanol:acetonitrile:ultrapure water = 45:45:10 (v:v:v); mobile phase B consisted of 10 mM potassium dihydrogen phosphate, with pH adjusted to 7.0 using KOH. The elution program is as follows: 0-2.5 min: 10% A, 90% B; 2.5-2.6 min: A 10%→14%, B 90%→86%; 2.6-13 min: A 14%→34%, B 86%→66%; 13-13.1 min: A 34%→38%, B 66%→62%; 13.1-28 min: A 38%→100%, B 62%→0; 28-28.1 min: A 100%→10%, B 0→90%; 28.1-32 min: A 10%→14%, B 90%→86%.

[0070] Using strain A1 as a control, strains A1 and A2 were subjected to shake-flask fermentation tests according to the method in step (6). The content of β-alanine was determined according to Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 1 As shown, the biomass OD of control strain A1 600 =13.8, β-alanine content is 2.74 g / L; biomass OD of strain A2 600 =15.9, the content of β-alanine is 3.87 g / L. The content of β-alanine increased by 41.2%.

[0071] Example 2: In strain B1: E. coli W3110TrcpanDTrcppcΔpykAΔcycA / pTrc99a-panD (bs) K104S aspAaspB (cg) pck knockout

[0072] (1) The plasmid pTarget-pck was constructed using the method in Example 1, and the linearized plasmid pTarget-pck was prepared.

[0073] (2) Construction of plasmid pTD-pck: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were amplified using L-pck-F / L-pck-R and R-pck-F / R-pck-R primers. The upstream and downstream homologous arms were then transferred into the linearized plasmid pTarget-pck from step (1) using the method described in Example 1 to construct plasmid pTD-pck.

[0074] (3) Using the method in Example 1, the pCas9 plasmid was introduced into strain B1 and competent cells were prepared.

[0075] (4) Using the method in Example 1, the plasmid pTD-pck was transferred into the competent cells in step (3) to successfully construct the pck knockout strain.

[0076] (5) Plasmid elimination: The method is the same as in Example 1, and a plasmid-free strain E. coli W3110 (TrcpanDTrcppcΔpykAΔcycAΔpck) is obtained.

[0077] (6) Transformation plasmid: Extract plasmid pTrc99a-panD (bs) K104S aspAaspB (cg) The plasmid was transformed into competent cells of a plasmid-free strain to construct strain B2: E. coli W3110(TrcpanDTrcppcΔpykAΔcycAΔpck) / pTrc99a-panD (bs) K104S aspAaspB (cg) .

[0078] (7) Using the starting strain B1 as the control, strains B1 and B2 were subjected to shake-flask fermentation tests according to the method in Example 1. The content of β-alanine was determined according to Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 2 As shown, the biomass OD of control strain B1 600 The OD value of strain B2 was 18.0, and the β-alanine content was 3.45 g / L; the biomass OD of strain B2 was... 600 The concentration was 20.5, the β-alanine content was 4.05 g / L, and the yield of β-alanine increased by 17.4%.

[0079] Example 3: In strain C1: E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykF Trcglk / pTrc99a-panD (bs) pck knockout

[0080] 1. Construct strain E. coli W3110Trcppc

[0081] (1) Construction of plasmid pTarget-ppc: Using pTarget plasmid as a template and pTarget-ppc-F / pTarget-ppc-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-ppc was obtained using the method in Example 1.

[0082] (2) Construction of plasmid pTD-ppc: Using the E. coli W3110 genome as a template, and L-ppc-F / L-ppc-R and R-ppc-F / R-ppc-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-ppc of step (1) using the method in Example 1 to obtain plasmid pTD-ppc.

[0083] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110 and competent cells were prepared.

[0084] (4) Using the method in Example 1, the plasmid pTD-ppc was transferred into the competent cells of step (3) to construct the strain E. coli W3110Trc-ppc.

[0085] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of the strain in step (4) were eliminated to obtain the plasmid-free strain E.coli W3110Trc-ppc.

[0086] 2. Construct strain E. coli W3110TrcppcTrcpanD

[0087] (1) Construction of plasmid pTarget-panD: Using pTarget plasmid as a template and pTarget-panD-F / pTarget-panD-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-panD was obtained using the method in Example 1.

[0088] (2) Construction of plasmid pTD-panD: Using the E. coli W3110 genome as a template, and L-panD-F / L-panD-R and R-panD-F / R-panD-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-panD of step (1) using the method in Example 1 to obtain plasmid pTD-panD.

[0089] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110Trcppc and competent cells were prepared.

[0090] (4) Using the method in Example 1, the plasmid pTD-panD was transferred into the competent cells of step (3) to construct the strain E. coli W3110TrcppcTrcpanD.

[0091] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of strain (4) were eliminated to obtain the plasmid-free strain E.coli W3110TrcppcTrcpanD.

[0092] 3. Construct strain E. coli W3110TrcppcTrcpanDΔpykA

[0093] (1) Construction of plasmid pTarget-pykA: Using pTarget plasmid as a template, and pTarget-pykA-F / pTarget-pykA-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-pykA was obtained using the method in Example 1.

[0094] (2) Construction of plasmid pTD-pykA: Using the E. coli W3110 genome as a template, and L-pykA-F / L-pykA-R and R-pykA-F / R-pykA-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-pykA of step (1) using the method in Example 1 to obtain plasmid pTD-pykA.

[0095] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110TrcppcTrcpanD and competent cells were prepared.

[0096] (4) Using the method in Example 1, the plasmid pTD-pykA was transferred into the competent cells of step (3) to construct the strain E. coli W3110TrcppcTrcpanDΔpykA.

[0097] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of strain (4) were eliminated to obtain the plasmid-free strain E.coli W3110TrcppcTrcpanDΔpykA.

[0098] 4. Construct strain E. coli W3110TrcppcTrcpanDΔpykAΔaspA

[0099] (1) Construction of plasmid pTarget-aspA: Using pTarget plasmid as a template and pTarget-aspA-F / pTarget-aspA-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-aspA was obtained using the method in Example 1.

[0100] (2) Construction of plasmid pTD-aspA: Using the E. coli W3110 genome as a template, and L-aspA-F / L-aspA-R and R-aspA-F / R-aspA-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-aspA of step (1) using the method in Example 1 to obtain plasmid pTD-aspA.

[0101] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110TrcppcTrcpanDΔpykA and competent cells were prepared.

[0102] (4) Using the method in Example 1, the plasmid pTD-pykA was transferred into the competent cells of step (3) to construct the strain E. coli W3110TrcppcTrcpanDΔpykAΔaspA.

[0103] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of the strain in step (4) were eliminated to obtain the plasmid-free strain E. coli W3110TrcppcTrcpanDΔpykAΔaspA.

[0104] 5. Construct strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykF

[0105] (1) Construction of plasmid pTarget-pykF: Using pTarget plasmid as a template, and pTarget-pykF-F / pTarget-pykF-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-pykF was obtained using the method in Example 1.

[0106] (2) Construction of plasmid pTD-pykF: Using the E. coli W3110 genome as a template, and L-pykF-F / L-pykF-R and R-pykF-F / R-pykF-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-pykF of step (1) using the method in Example 1 to obtain plasmid pTD-pykF.

[0107] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110 and competent cells were prepared.

[0108] (4) Using the method in Example 1, the plasmid pTD-pykF was transferred into the competent cells of step (3) to construct the strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykF.

[0109] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of the strain in step (4) were eliminated to obtain the plasmid-free strain E.coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykF.

[0110] 6. Construct strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk

[0111] (1) Construction of plasmid pTarget-glk: Using pTarget plasmid as a template and pTarget-glk-F / pTarget-glk-R as primers for amplification, DpnI was added to the PCR product and incubated at 37℃ for 2 h. The methylated plasmid template was digested, purified, and transformed into E. coli DH5α competent cells. Positive strains were screened using LB plates containing 50 mg / L spectinomycin, and their correctness was verified by sequencing. The linearized plasmid pTarget-glk was obtained using the method in Example 1.

[0112] (2) Construction of plasmid pTD-glk: Using the E. coli W3110 genome as a template, and L-glk-F / L-glk-R and R-glk-F / R-glk-R as primers, upstream and downstream homologous arms were amplified. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-glk of step (1) using the method in Example 1 to obtain plasmid pTD-glk.

[0113] (3) Using the method in Example 1, plasmid pCas9 was introduced into strain E. coli W3110 and competent cells were prepared.

[0114] (4) Using the method in Example 1, the plasmid pTD-glk was transferred into the competent cells of step (3) to construct the strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk.

[0115] (5) Plasmid elimination: Using Example 1, the pTarget and pCas9 plasmids of strain (4) were eliminated to obtain the plasmid-free strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk.

[0116] 7. Plasmid pTrc99a-panD (bs) Construction

[0117] Using the panD gene from Bacillus subtilis as a template, and using panD... (bs) -F and panD (bs) -R is used as a primer for amplification. The pTrc99a plasmid is linearized using pTrc99a-line-F and pTrc99a-line-R primers. The fragment is then ligated using the method described in Example 1 to obtain the plasmid pTrc99a-panD. (bs) The ligation product was transformed into E. coli DH5α competent cells to construct the vector pTrc99a-panD. (bs) .

[0118] 8. Construct strain E. coli W3110TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglkΔpck / pTrc99a-panD (bs)

[0119] (1) The plasmid pTarget-pck was constructed using the method in Example 1, and the linearized plasmid pTarget-pck was prepared.

[0120] (2) Construction of plasmid pTD-pck: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were amplified using L-pck-F / L-pck-R and R-pck-F / R-pck-R primers. The upstream and downstream homologous arms were then transferred into the linearized plasmid pTarget-pck from step (1) using the method described in Example 1 to construct plasmid pTD-pck.

[0121] (3) Using the method in Example 1, the pCas9 plasmid was introduced into strain E.coli W3110 (TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglk) and competent cells were prepared.

[0122] (4) Using the method in Example 1, the plasmid pTD-pck was transferred into the competent cells in step (3) to successfully construct the pck knockout strain.

[0123] (5) Plasmid elimination: The method is the same as in Example 1, and plasmid-free strain E. coli W3110 (TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglkΔpck) is obtained.

[0124] (6) Extraction of plasmid pTrc99a-panD (bs) The plasmid was transformed into plasmid-free strain competent cells to construct strain C2: E. coli W3110(TrcppcTrcpanDΔpykAΔaspAΔpykFTrcglkΔpck / pTrc99a-panD) (bs) ).

[0125] (7) Using the starting strain C1 as the control, strain C2 was subjected to shake-flask fermentation according to the method in Example 1. The β-alanine content was determined according to Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 3 As shown, the biomass OD of control strain C1 600 The OD value of strain C2 was 20.1, and the β-alanine content was 4.01 g / L; 600 The concentration was 19.0, the β-alanine content was 5.85 g / L, and the yield of β-alanine increased by 45.9%.

[0126] Example 4: Fed-feed fermentation in a 5-L fermenter

[0127] The strain from Example 3 was streaked onto LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 37°C. Single colonies were picked and transferred to LB tubes containing 50 mg / L kanamycin resistance, and incubated overnight at 37°C and 150 rpm to prepare the seed culture. The seed culture was inoculated into 100 mL of LB medium at a concentration of 5% (v / v) and incubated overnight at 37°C and 150 rpm to obtain the secondary seed culture. The secondary seed culture was inoculated into a 5-L fermenter containing 2 L of fermentation medium at a concentration of 15% (v / v), and IPTG was added to a final concentration of 0.2 mM. Fermentation was carried out at 30°C, 500 rpm, and an aeration rate of 0.5 V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), automatic feeding was activated, and feeding medium was added at a rate of 25 mL / h until the pH was lower than 6.80. Feeding was then stopped, maintaining a low residual sugar level in the fermenter, with a sugar concentration of 3 g / L. The total amount of feed culture medium added was 800 mL, and the culture was carried out for 117 h. The yield of β-alanine in the fermentation broth was determined by high performance liquid chromatography (HPLC) as described in Example 1, and the biomass OD was determined by spectrophotometry. 600 Sugar concentration was detected using the DNS method; Figure 5 As shown, the β-alanine yield was 40.2 g / L after 62 h, and the final biomass OD 600 The concentration was maintained at 80.4. Fermentation results showed that the metabolically engineered β-alanine-producing bacteria had good production performance, and no accumulation of other amino acids was detected in the fermentation broth, laying the foundation for the industrial production of β-alanine.

[0128] 5-L fermenter culture medium formula: glucose 20g / L, (NH4)2SO4 16g / L, yeast extract 4g / L, KH2PO4 1g / L, MgSO4 0.5g / L, CaCO3 15g / L, VB1 0.4mg / L, VB 12 0.2 mg / L, 1 mL / L trace element solution; composition of trace element solution: 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, solvent is deionized water.

[0129] Feeding medium: glucose 500 g / L, (NH4)2SO4 16 g / L, yeast extract 4 g / L, KH2PO4 14 g / L, NaHCO3 10 g / L, VB1 0.4 mg / L, VB2 0.2 mg / L 12 Adjust the pH to 6.8 using 50% ammonia solution.

[0130] Biomass OD of strain C1 in a 5-L feed fermenter 600The concentration curves of residual sugar and β-alanine are shown in the figure. Figure 4 As shown.

[0131] Biomass OD of strain C2 in a 5-L feed fermenter 600 The concentration curves of residual sugar and β-alanine are shown in the figure. Figure 5 As shown.

Claims

1. A recombinant genetically engineered bacterium for producing β-alanine, characterized in that, The recombinant genetically engineered bacteria mentioned are recombinant genetically engineered bacteria A2, recombinant genetically engineered bacteria B2, or recombinant genetically engineered bacteria C2; Recombinant genetically engineered bacteria A2 is based on Escherichia coli A1, with the gene knocked out... pck The gene was constructed after gene generation, and the genotype of the *E. coli* A1 is: E. coli W3110Trc panD (bs) ::panD Trc PPC Δ ptsG Trc glk Δ galR Δ cycA Δ iclRlysC*thrA* Trc gltBD / pTrc99a -panD (bs) -aspB (cg) ; Recombinant genetically engineered bacteria B2 is based on Escherichia coli B1, with the knockout strain... pck The gene was constructed after gene generation, and the genotype of the *E. coli* B1 is: E. coli W3110Trc panD Trc PPC Δ pykA Δ cycA / pTrc99a- panD (bs) K104S - aspA (cg) -aspB (cg) ; Recombinant genetically engineered bacteria C2 is based on Escherichia coli C1, with the knockout of... pck The gene was constructed later, and the E. coli C1 contained the Trc promoter. PPC , panD , glK Gene knockout pykA , aspA , pykF Genes, and the introduction of Bacillus subtilis-containing genes. panD E. coli containing the pTrc99a plasmid of the gene; The pck The nucleotide sequence of the gene is shown in SEQ ID NO. 1; Knockout pck Genes specifically include: Knockout on the genome using CRISPR / Cas9 gene editing technology pck Gene.

2. The application of the recombinant genetically engineered bacteria for producing β-alanine as described in claim 1 in the preparation of β-alanine by microbial fermentation.

3. The application as described in claim 2, characterized in that, Specifically, it includes: The recombinant genetically engineered bacteria were inoculated into kanamycin fermentation medium and cultured until fermentation was completed to obtain a fermentation broth containing β-alanine. The fermentation broth was then separated and purified to obtain β-alanine.

4. The application as described in claim 3, characterized in that, The fermentation medium formulation is as follows: glucose 18-22 g / L, (NH4)2SO4 14-18 g / L, yeast extract 3-5 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.2-1 g / L, CaCO3 13-17 g / L, VB1 0.3-0.5 mg / L, VB 12 0.1~0.3 mg / L, trace element solution 0.5~1.5 mL / L, solvent is water, no pH adjustment required.

5. The application as described in claim 4, characterized in that, The components of the trace element solution are as follows: 8~12 g / L CaCl2, 8~12 g / L FeSO4·7H2O, 0.8~1.2 g / L ZnSO4·7H2O, 0.1~0.3 g / L CuSO4, 0.01~0.04 g / L NiCl2·7H2O, and the solvent is deionized water.

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