A plasmid-free genetically engineered bacterium with high yield of L-2-amino butyric acid and its application

Abstract

The present invention relates to the field of genetic engineering technology, and discloses a plasmid-free genetically engineered bacterium with high yield of L-2-amino butyric acid and its application. The plasmid-free genetically engineered bacterium for producing L-2-amino butyric acid provided by the present invention is obtained by modifying Escherichia coli THR as a chassis cell through metabolic engineering and gene editing technology. This genetically engineered bacterium strain can use glucose as a raw material and obtain L-2-amino butyric acid through fermentation culture. Without adding exogenous enzyme activity by plasmids, the batch-fed fermentation yield in a 5L fermenter reaches 13.81 g / L. It has low fermentation cost, high product concentration, no by-products affecting, and the product is easy to purify, and is suitable for industrial applications.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid and its application. Background Art

[0002] L-2-aminobutyric acid (L-ABA) is an unnatural chiral α-amino acid, and as a key chiral intermediate, it can be used to synthesize a variety of chiral drugs, such as the tuberculosis treatment drug ethambutol, and the anti-epileptic drugs levetiracetam and brivaracetam.

[0003] The molecular formula of L-2-aminobutyric acid is C4H9NO2, the relative molecular weight is 103.12, the melting point is 270 - 280 °C, 22.7 g can be dissolved in every 100 g of water at 25 °C, slightly soluble in ethanol, ether, etc., and the isoelectric point is 6.05 (25 °C). It is an odorless, slightly sweet white flaky crystal. The structural formula of L-2-aminobutyric acid is shown as follows:

[0004]

[0005] Currently, L-2-aminobutyric acid is mainly synthesized industrially by chemical methods, but it faces problems such as harsh reaction conditions and environmental unfriendliness.

[0006] To avoid the problems caused by the chemical synthesis of L-2-aminobutyric acid, therefore, in recent years, many biosynthetic pathways of L-2-aminobutyric acid have been developed, mainly through enzymatic catalysis methods such as dehydrogenases or transaminases. Using keto acids and glutamic acid as substrates, L-2-aminobutyric acid is generated under the action of transaminases, but the yield of this method is low; using L-threonine as the raw material, a one-pot method with a three-enzyme system containing amino acid dehydrogenase is used to prepare L-2-aminobutyric acid. Although a coenzyme recycling system is added, an appropriate amount of coenzymes such as nicotinamide adenine dinucleotide, NAD + or NADH are still required. Due to the high price of coenzymes, it limits its application at the industrial level, and there are by-products affecting the purification of the product; the amino acid oxidase method is to use D-amino acid oxidase to resolve the racemic substrate under the action of a metal catalyst to prepare L-2-aminobutyric acid. This method has a high cost and is not suitable for large-scale industrial applications. In addition, there are also aminoacylase methods, etc., but there are problems such as too high production costs and enzyme activity being inhibited by substrates, so the industrial application effect is not good.

[0007] Therefore, the research team intends to develop a new biosynthetic method for the synthesis of L-2-aminobutyric acid to avoid problems such as harsh chemical synthesis reaction conditions, and at the same time avoid the problems of high cost and low yield of existing biosynthetic methods. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid and its application. The object of the present invention is to transform Escherichia coli through metabolic engineering and gene editing techniques to obtain a recombinant Escherichia coli strain with high yield of L-2-aminobutyric acid. Specifically, the object of the present invention is to provide a plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid, its construction method, and the application of this genetically engineered bacterium in the microbial fermentation for preparing L-2-aminobutyric acid, using Escherichia coli THR constructed by this research team as the chassis cell.

[0009] The specific technical solution of the present invention is as follows:

[0010] On the one hand, the present invention provides a plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid, which is constructed by the following method: (1) Using Escherichia coli THR as the chassis bacterium, replace the ptsG gene in the genome of the strain Escherichia coli THR with the ilvA* gene and the leuDH gene to obtain the recombinant strain Escherichia coli THRΔptsG::ilvA*-leuDH, denoted as ABA-1;

[0011] (2) Inoculate the strain Escherichia coli THRΔptsG::ilvA*-leuDH into a 2-ketobutyric acid liquid medium for subculture, and screen the strains that can grow normally in a liquid medium with a 2-ketobutyric acid concentration of not less than 50 g / L to obtain the strain Escherichia coli THRΔptsG::ilvA*-leuDHMut, denoted as ABA-2;

[0012] (3) Replace the adjacent ltaE gene and poxB gene in the genome of the strain Escherichia coli THRΔptsG::ilvA*-leuDHMut with the leuDH gene to obtain the recombinant strain Escherichia coli THRΔptsG::ilvA*-leuDHMutΔltaEΔpoxB::leuDH, denoted as ABA-3;

[0013] (4) Knock out the focA gene and pflB gene in the genome of the strain Escherichia coli THRΔptsG::ilvA*-leuDHMutΔltaEΔpoxB::leuDH to obtain the recombinant strain Escherichia coli THRΔptsG::ilvA*-leuDHMutΔltaEΔpoxB::leuDHΔfocAΔpflB, denoted as ABA-4;

[0014] (5) Replace the adjacent pta gene and ackA gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB with the ilvA* gene and leuDH gene to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH, denoted as ABA-5;

[0015] (6) Replace the mgsA gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH with the ilvA* gene and leuDH gene to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH△mgsA::ilvA*-leuDH, denoted as ABA-6, which is the plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid.

[0016] Escherichia coli THR is described in Fermentative production ofthe unnatural amino acid L-2-aminobutyric acid based on metabolic engineering (Microb Cell Fact. 2019, 18(1):43). The plasmid-free recombinant Escherichia coli for producing L-2-aminobutyric acid provided by the present invention uses Escherichia coli THR as the chassis cell and is constructed by the above steps (1)-(6). Using this plasmid-free recombinant Escherichia coli for producing L-2-aminobutyric acid has the advantage of high yield. Specifically:

[0017] In step (1), the relevant genes on the L-2-aminobutyric acid synthesis pathway of Escherichia coli are edited. The ptsG gene in the glucose phosphotransferase system (PTS) is replaced with the ilvA* gene that relieves the feedback inhibition of L-isoleucine and the leucine dehydrogenase leuDH gene derived from T. intermedius by using the CRISPR-Cas9 gene editing technology, so that Escherichia coli can ferment and produce L-2-aminobutyric acid without plasmid on the one hand, and on the other hand, the deletion of the ptsG gene can reduce the consumption of phosphoenolpyruvate.

[0018] In step (2), using laboratory adaptive evolution technology, the strains were respectively transferred to LB media containing different concentrations of 2-ketobutyric acid for subculture. Wait until the strains can grow normally at a 2-ketobutyric acid concentration of 50 g / L to obtain adaptive strains with a high 2-ketobutyric acid concentration.

[0019] In step (3), the genes ltaE and poxB related to by-products were replaced with the leucine dehydrogenase leuDH gene from T. intermedius using CRISPR-Cas9 gene editing technology to reduce the degradation of threonine and the accumulation of acetic acid, so that more metabolism flows towards the production of L-2-aminobutyric acid.

[0020] In step (4), the genes focA and pflB related to the formic acid pathway were deleted using CRISPR-Cas9 gene editing technology to reduce the accumulation of by-products, extend the fermentation cycle, and improve the fermentation production of L-2-aminobutyric acid.

[0021] In step (5), the genes pta and ackA related to the acetic acid pathway were replaced with the ilvA* gene that relieves L-isoleucine feedback inhibition and the leucine dehydrogenase leuDH gene from T. intermedius using CRISPR-Cas9 gene editing technology to reduce the accumulation of acetic acid, extend the continuous fermentation time, the ilvA* gene improves the catabolism of threonine, increases the production of α-ketobutyric acid, and produces L-2-aminobutyric acid through the leuDH gene.

[0022] In step (6), the gene mgsA encoding methylglyoxal synthase was replaced with the ilvA* gene that relieves L-isoleucine feedback inhibition and the leucine dehydrogenase leuDH gene from T. intermedius using CRISPR-Cas9 gene editing technology, and finally a recombinant Escherichia coli strain ABA-6 with high-yield fermentation production of L-2-aminobutyric acid was constructed.

[0023] The plasmid-free recombinant Escherichia coli for high-yield production of L-2-aminobutyric acid uses Escherichia coli THR as the chassis cell, edits the relevant genes in the L-2-aminobutyric acid synthesis pathway of Escherichia coli, replaces the ptsG gene in the glucose phosphotransferase system (PTS) with the ilvA* gene that relieves the feedback inhibition of L-isoleucine and the leucine dehydrogenase leuDH gene from T. intermedius. On one hand, Escherichia coli can ferment and produce L-2-aminobutyric acid without a plasmid. On the other hand, the deletion of the ptsG gene can reduce the consumption of phosphoenolpyruvate. Laboratory adaptive evolution is carried out with the tolerance to high concentrations of 2-ketobutyric acid as the screening to obtain a strain highly tolerant to the toxic metabolite 2-ketobutyric acid, so that its growth is no longer inhibited by 2-ketobutyric acid. The genes ltaE and poxB related to by-products are replaced with the leucine dehydrogenase leuDH gene from T. intermedius to reduce the degradation of threonine and the accumulation of acetic acid, so that more metabolism flows towards the production of L-2-aminobutyric acid. The related genes focA and pflB in the formic acid pathway are deleted to reduce the accumulation of by-products, extend the fermentation cycle, and improve the fermentation production of L-2-aminobutyric acid. The related genes pta and ackA in the acetic acid pathway are replaced with the ilvA* gene that relieves the feedback inhibition of L-isoleucine and the leucine dehydrogenase leuDH gene from T. intermedius to reduce the accumulation of acetic acid and extend the continuous fermentation time. The ilvA* gene improves the catabolism of threonine and increases the production of 2-ketobutyric acid, and L-2-aminobutyric acid is produced through the leuDH gene. The gene mgsA encoding methylglyoxal synthase is replaced with the ilvA* gene that relieves the feedback inhibition of L-isoleucine and the leucine dehydrogenase leuDH gene from T. intermedius. Finally, a recombinant Escherichia coli strain with higher yield of L-2-aminobutyric acid fermentation production is constructed. This recombinant Escherichia coli strain can use glucose as a raw material to obtain L-2-aminobutyric acid through fermentation culture. Without adding exogenous enzyme activity with a plasmid, the batch fed-batch fermentation yield in a 5 L fermenter reaches 13.81 g / L.

[0024] Specifically, as a preference for the above technical solution of the present invention, the ilvA* gene sequence is as shown in SEQ ID No.1; the leuDH gene is a gene encoding leucine dehydrogenase, and its gene sequence is as shown in SEQ ID No.2; the ptsG gene sequence is as shown in SEQ ID No.3; the ltaE gene sequence is as shown in SEQ ID No.4, the poxB gene sequence is as shown in SEQ ID No.5; the focA gene sequence is as shown in SEQ ID No.6, the pflB gene sequence is as shown in SEQ ID No.7; the pta gene sequence is as shown in SEQ ID No.8; the ackA gene sequence is as shown in SEQ ID No.9; the mgsA gene sequence is as shown in SEQ ID No.10.

[0025] On the other hand, the present invention provides a method for constructing the above plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid, comprising the following steps:

[0026] (1) Using E. coli THR as the chassis bacterium, applying the CRISPR-Cas9 gene editing technology, replacing the ptsG gene in the genome of the strain E. coli THR with the ilvA* gene that relieves L-isoleucine feedback inhibition and the leucine dehydrogenase leuDH gene derived from T. intermedius to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDH, denoted as ABA-1;

[0027] (2) Using the laboratory adaptive evolution technology, transferring the E. coli THR△ptsG::ilvA*-leuDH strain to LB media containing different 2-ketobutyric acid concentrations for subculture respectively. Wait until the strain can grow normally at a 2-ketobutyric acid concentration of 50 g / L to obtain the strain E. coli THR△ptsG::ilvA*-leuDHMut that tolerates high concentrations of 2-ketobutyric acid, denoted as ABA-2;

[0028] (3) Applying the CRISPR-Cas9 gene editing technology, replacing the ltaE gene and the poxB gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut with the leucine dehydrogenase leuDH gene derived from T. intermedius to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH, denoted as ABA-3;

[0029] (4) Using CRISPR-Cas9 gene editing technology, the focA gene and the pflB gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH were knocked out to obtain the recombinant strain E. coliTHR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB, which was recorded as ABA-4;

[0030] (5) Using CRISPR-Cas9 gene editing technology, the pta gene and ackA gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB were replaced with the ilvA* gene that relieves L-isoleucine feedback inhibition and the leuDH gene of leucine dehydrogenase from T. intermedius to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH, denoted as ABA-5;

[0031] (6) Using CRISPR-Cas9 gene editing technology, the mgsA gene in the genome of the strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH was replaced with the ilvA* gene that relieves the feedback inhibition of L-isoleucine and the leuDH gene of leucine dehydrogenase from T. intermedius to obtain the recombinant strain E. coli THR△ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA::ilvA*-leuDH△mgsA::ilvA*-leuDH, denoted as ABA-6, which is the high-yield genetically engineered bacterium of L-2-aminobutyric acid.

[0032] At the same time, the present invention also provides the application of the above-mentioned plasmid-free genetic engineering bacteria in the preparation of L-2-aminobutyric acid by microbial fermentation and a method thereof.

[0033] Specifically, the application method includes the following steps: inoculating the above-mentioned plasmid-free recombinant Escherichia coli into a fermentation medium containing IPTG, fermenting and culturing for more than 48 hours at 30-35°C and 100-200rpm, and after the fermentation is completed, taking the supernatant of the fermentation liquid for separation and purification to obtain L-2-aminobutyric acid.

[0034] Preferably, the fermentation medium is composed as follows: IPTG 0.1 mM, glucose 50 ± 10 g / L, yeast extract 6 ± 1 g / L, magnesium sulfate heptahydrate 2 ± 0.3 g / L, potassium dihydrogen phosphate 4 ± 0.5 g / L, ammonium sulfate 14 ± 1 g / L, betaine hydrochloride 1 ± 0.2 g / L, citric acid 4 ± 1 g / L, L-methionine 0.149 ± 0.2 g / L, L-lysine 0.164 ± 0.2 g / L, metal salt ion solution 5 ± 1 mL / L, calcium carbonate 30 ± 5 g / L, the solvent is deionized water, and the pH value is natural; the metal salt ion solution is composed of: FeSO4·7H2O 10 ± 1.5 g / L, CaCl2 1.35 ± 0.6 g / L, ZnSO4·7H2O 2.25 ± 0.8 g / L, MnSO4·4H2O 0.5 ± 0.1 g / L, CuSO4·5H2O 1 ± 0.5 g / L, (NH4)6Mo7O 24 ·4H2O 0.106 ± 0.05 g / L, Na2B4O7·10H2O 0.23 ± 0.08 g / L, 35% HCl 10 ± 1 mL / L, and the solvent is water.

[0035] Preferably, before the fermentation of the plasmid-free genetically engineered bacterium, it is first inoculated into LB medium and cultured overnight on a shaker at a temperature of 37 °C and a rotation speed of 200 rpm, and then inoculated into the fermentation medium at an inoculation amount of 3-5% by volume for culture.

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

[0037] The plasmid-free genetically engineered bacterium for producing L-2-aminobutyric acid provided by the present invention can use glucose as a raw material, and without adding exogenous enzyme activity by plasmid, the fermentation yield in a 5 L fermenter reaches 13.81 g / L. The fermentation cost is low, the product concentration is high, there is no by-product influence, and the product is easy to purify, which is very suitable for industrial application. Description of the Drawings

[0038] Figure 1 For Example 1 of the present invention, OD 600 and the fermentation result diagram of the L-2-aminobutyric acid content;

[0039] Figure 2 For Example 3 of the present invention, OD 600 and the fermentation result diagram of the L-2-aminobutyric acid content;

[0040] Figure 3 For Example 4 of the present invention, OD 600 and the fermentation result diagram of the L-2-aminobutyric acid content;

[0041] Figure 4 For Example 5 of the present invention, OD600 and the fermentation result graph of L-2-amino butyric acid content;

[0042] Figure 5 This is the OD of Example 6 of the present invention 600 and the fermentation result graph of L-2-amino butyric acid content. Detailed implementation mode

[0043] The present invention will be further described below in conjunction with the embodiments.

[0044] In the following embodiments, the final concentration of kanamycin in the culture medium is 0.05 mg / L, and the final concentration of spectinomycin in the culture medium is 0.05 mg / L.

[0045] The strain E. coli W3110 is from the Coli Genetic Stock Center of Yale University, with a preservation date of August 5, 1975 and a preservation number of CGSC#4474, and has been disclosed in patents US2009 / 0298135 A1 and US2010 / 0248311A1.

[0046] The parental strain E. coli THR is from the laboratory collection and is a derivative of E. coli W3110. Its genotype is described as follows (E. coliW3110 / △lacl / thrA C1034T / lysC C1055T / Tac-thrABC / △lysA / △metA / △tdh / △ilcR / Trc-ppc).

[0047] In the following embodiments, the ilvA* gene sequence is as shown in SEQ ID No. 1; the leuDH gene is the gene encoding leucine dehydrogenase, and its gene sequence is as shown in SEQ ID No. 2; the ptsG gene sequence is as shown in SEQ ID No. 3; the ltaE gene sequence is as shown in SEQ ID No. 4, and the poxB gene sequence is as shown in SEQ ID No. 5; the focA gene sequence is as shown in SEQ ID No. 6, and the pflB gene sequence is as shown in SEQ ID No. 7; the pta gene sequence is as shown in SEQ ID No. 8; the ackA gene sequence is as shown in SEQ ID No. 9; the mgsA gene sequence is as shown in SEQ ID No. 10.

[0048] The primer sequence information used in the following embodiments is shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052]

[0053] Among them, X deletion-sgRNA-F / R is the mutation primer of the pTatget plasmid, where X is the 20 bp sequence before the PAM site (NGG) contained in the target gene carrying the genome; X D-Arm1-F / R are the upstream and downstream primers of the upstream homologous arm of the target gene; X D-Arm2-F / R are the upstream and downstream primers of the downstream homologous arm of the target gene; X deletion-VF / VR are the verification primers for editing the target gene.

[0054] The detection methods for L-2-aminobutyric acid and L-threonine in the following examples are as follows:

[0055] Take 1 mL of the fermentation broth in an EP tube, centrifuge at 12000 rpm for 2 min, take the supernatant and dilute it to an appropriate multiple with sterilized ddH2O, perform a derivatization reaction at 60 °C for 60 min, add phosphate buffer to make the volume up to 1 mL, and detect by high performance liquid chromatography (HPLC) after filtering through a 0.22 μm organic filter membrane.

[0056] Detection method: The chromatographic column is a C18 column (150×4.6 mm), the column temperature is 33 °C, the flow rate is 1 mL / min, gradient elution is used, the injection volume is 10 μL, the chromatographic retention time is 30 min, and the detection wavelength is 360 nm. The elution program is shown in Table 2.

[0057] Table 2

[0058] Serial number Time (min) Mobile phase A Mobile phase B 1 0.00 16 84 2 0.18 16 84 3 2.40 30 70 4 4.20 34 66 5 7.20 43 57 6 13.30 55 45 7 15.00 55 45 8 20.40 98 2 9 21.30 16 84 10 30.00 16 84

[0059] Among them, the preparation method of the mobile phase: Mobile phase A: 50% acetonitrile; Mobile phase B: Weigh 4.1 g of anhydrous sodium acetate and dissolve it in 800 mL of ddH2O, adjust the pH to 6.4 with acetic acid, and then make the volume up to 1 L.

[0060] Construction and fermentation of strain ABA-1 with the ilvA* and leuDH genes replacing the ptsG gene in Example 1

[0061] The threonine deaminase gene ilvA is from Escherichia coli W3110 (NCBI accession number: AP009048); the leucine dehydrogenase gene leuDH is from Thermoactinomyces intermedius.

[0062] Using the high-threonine-producing strain E. coli THR (i.e., E. coli W3110 / thrA* / lysC* / Tac-thrABC / △lysA / △metA / △tdh / ilcR / Trc-ppc) as the starting strain, the ptsG gene (SEQ ID No. 3) was replaced with the ilvA* (SEQ ID No. 1) and leuDH genes (SEQ ID No. 2) on the genome using the CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al. 2015 Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental Microbiology. 81:2506-2514), enabling Escherichia coli to ferment and produce L-2-aminobutyric acid without plasmids:

[0063] Construction of the recombinant vector pTarget-ptsG::ilvA*-leuDH

[0064] Using the pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed with ptsG deletion-sgRNA-F / ptsG deletion-sgRNA-R as primers. The obtained PCR product was digested with Dpn I at 37°C for 1 h, and then transformed into competent E. coli DH5α cells. Screening was carried out on a spectinomycin (SD) plate, and the correct pTarget-ptsG::leuDH plasmid was obtained by sequencing verification for subsequent ligation of Donor DNA.

[0065] 2. Construction of the recombinant vector pTD-ptsG::ilvA*-leuDH

[0066] Using the E. coli W3110 genome as a template, the upstream homologous arm of the donor DNA amplified with ptsG D-Arm1-F and ptsG D-Arm1-R primers was denoted as Arm1, the downstream homologous arm of the donor DNA amplified with ptsG D-Arm2-F and ptsG D-Arm2-R primers was denoted as Arm2, and the donor DNA ilvA*-leuDH gene amplified with In ptsG-F-ilvA*-leuDH and In ptsG-R-ilvA*-leuDH primers was denoted as In ilvA*-leuDH. The DNA fragments were recovered using a Clean up kit to obtain the homologous arms Arm1, Arm2 and the donor DNA In ilvA*-leuDH. The pTarget-ptsG::ilvA*-leuDH plasmid was PCR amplified with ptarget-F and ptarget-R primers. The resulting PCR product was digested with Dpn I at 37 °C for 1 - 2 h, and the DNA fragment was recovered using a Clean up kit. According to the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China), the pTarget-ptsG::ilvA*-leuDH plasmid fragment, homologous arms Arm1, Arm2 and the donor DNA In leuDH were ligated together and introduced into competent E. coli DH5α cells. Positive clones were screened by colony PCR and verified by sequencing to obtain the pTD-ptsG::ilvA*-leuDH plasmid.

[0067] PCR system: 0.5 μL of genomic template, 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP, 2 μL each of forward and reverse primers (10 μM), 1 μL of PhantaMax DNA polymerase, and deionized water was added to make up to 50 μL. PCR program: Denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 1.5 min, for a total of 30 cycles; finally, extension at 72 °C for 10 min. After electrophoresis identification, this fragment was cleaned-up for later use.

[0068] Ligation process: The One Step Cloning Kit (purchased from Vazyme) was used for ligation. In a sterilized PCR tube, 2× Mix 5 μL of the linearized vector after digestion, X μL of the linear amplified fragment, and Y μL (the optimal amount of cloning vector to use = [0.02 × the number of base pairs of the cloning vector] ng (0.03 pmol), and the optimal amount of insert fragment to use = [0.04 × the number of base pairs of the insert fragment] ng (0.06 pmol)). Make up to 10 μL with sterilized ddH2O and react at 50 °C for 20 minutes. Transform the ligation product into DH5α E. coli competent cells by the calcium chloride method, screen using spectinomycin resistance, pick positive transformants for sequencing verification, and finally obtain the pTD-ptsG::leuDH plasmid.

[0069] 3. Preparation of electrocompetent cells

[0070] Introduce the pCas plasmid (Addgene Plasmid #62225) into the 19031 electrocompetent cells. Pick a positive clone and transfer it to an LB test tube containing 0.05 mg / L kanamycin, and culture overnight at 30 °C; then inoculate it into a 250 mL flask containing 50 mL of LB medium at an inoculation volume concentration of 1%, and add 500 μL of L-arabinose with a concentration of 1 mol / L. Culture at 150 rpm and 30 °C until the OD600 reaches 0.4 - 0.6; centrifuge at 4000 rpm and 4 °C for 10 min to collect the cells and prepare electrocompetent cells. The detailed procedure is described in (Molecular Cloning: A Laboratory Manual, 3rd Edition, 99 - 102).

[0071] 4. Electroporation editing and verification

[0072] Mix 300 - 600 ng of the pTD-ptsG::ilvA*-leuDH plasmid with 100 - 150 μL of electrocompetent cells, incubate on ice for 3 - 4 min, transfer to a pre-cooled 2 mm electroporation cuvette, incubate on ice for about 45 s, and perform electroporation using an electroporator (MicroPluser TM, BIO-RAD). Immediately add 1 mL of pre-cooled LLB medium after electroporation and immediately aspirate it, transfer to a 2 mL EP tube, resuscitate at 30 °C for 3 - 4 h, then take 400 μL and spread it on an LB plate containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin, and culture inverted at 30 °C for 14 - 18 h. Use ptsG deletion-VF and ptsG deletion-VR as verification primers for colony PCR verification. If a fragment with a size of about 2100 kb can be successfully cloned and verified by sequencing, it proves that the single colony is a positive colony of E. coli THR△ptsG::ilvA*-leuDH, that is, the editing is successful, and a new strain ABA-1 is obtained.

[0073] 5. Plasmid curing

[0074] Inoculate the positive clone into a 10 mL LLB test tube containing 1 mM IPTG and 0.05 mg / L kanamycin, and culture it at 30 °C with 180 rpm for 12 - 18 h. Streak it on an LLB solid medium containing 0.05 mg / L kanamycin and culture it at 30 °C overnight. Use a sterilized toothpick to pick the numbered small single colonies and streak them on the corresponding area of the LLB solid medium containing 0.05 mg / L spectinomycin, and culture it at 30 °C overnight. The single colonies that cannot grow in the corresponding area of the LLB solid medium containing 0.05 mg / L spectinomycin are the clones in which pTarget-ptsG::ilvA*-leuDH has been successfully eliminated.

[0075] Inoculate the clone in which pTarget-ptsG::ilvA*-leuDH has been successfully eliminated into a 10 mL antibiotic-free LLB test tube, and culture it at 42 °C with 180 rpm for 12 - 18 h. Streak it on an antibiotic-free LLB solid medium and culture it at 37 °C overnight. Use a sterilized toothpick to pick the numbered small single colonies and streak them on the corresponding area of the LLB solid medium containing 0.05 mg / L kanamycin, and culture it at 30 °C overnight. The single colonies that cannot grow in the corresponding area of the LLB solid medium containing 0.05 mg / L kanamycin are the clones in which pCas has been successfully eliminated, and the plasmid-free strain E. coli THR△ptsG::ilvA*-leuDH (denoted as ABA-1) is obtained.

[0076] LLB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, the solvent is deionized water, and the pH value is natural.

[0077] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, the solvent is deionized water, and the pH value is natural.

[0078] 6. Fermentation of the strain E. coli THR△ptsG::ilvA*-leuDH in a 5 L fermenter

[0079] Streak the constructed strain ABA-1 on a plate of antibiotic-free LB solid medium and culture it at 37 °C overnight. The next day, pick single colonies from the plate into 10 mL of liquid LB medium and culture it at 37 °C with 150 rpm overnight. Then transfer it to a 500 mL shake flask containing 100 mL of LB medium at an inoculation amount of 2% and culture it at 37 °C for about 8 hours to obtain a seed solution. Subsequently, transfer it to a 5 L fermenter (the liquid volume of the medium in the fermenter is 3 L) at an inoculation amount of 3% - 5% for fermentation.

[0080] Fermentation conditions: The fermentation adopted a low-sugar feeding method. The fermentation temperature was 35°C. The pH was automatically adjusted to 6.8 with ammonia water at a volume ratio of 50%. The dissolved oxygen was controlled at 10%-30%. The initial aeration rate was 4 ppm, 400 rpm. The dissolved oxygen concentration was adjusted by the rotation speed. When the initial glucose was exhausted and the pH and dissolved oxygen concentration began to rise, feeding started, and the glucose concentration in the tank was always maintained at about 5 g / L. When the OD of the strain growth reached the maximum value, IPTG with a final concentration of 0.1 mM was added. During the fermentation process, samples were taken every 3 h to measure the glucose concentration in the fermentation broth. At the same time, the content of L-2-aminobutyric acid in the fermentation broth was measured by HPLC, and the OD was detected by a spectrophotometer. 600 To determine the growth of the strain, the results are shown in Figure 1 .

[0081] Fermentation medium: IPTG 0.1 mM, glucose 50±10 g / L, yeast powder 6±1 g / L, magnesium sulfate heptahydrate 2±0.3 g / L, potassium dihydrogen phosphate 4±0.5 g / L, ammonium sulfate 14±1 g / L, betaine hydrochloride 1±0.2 g / L, citric acid 4±1 g / L, L-methionine 0.149±0.2 g / L, L-lysine 0.164±0.2 g / L, metal salt ion solution 5±1 mL / L, calcium carbonate 30±5 g / L, the solvent was deionized water, and the pH value was natural; the composition of the metal salt ion solution was: FeSO4·7H2O 10±1.5 g / L, CaCl2 1.35±0.6 g / L, ZnSO4·7H2O 2.25±0.8 g / L, MnSO4·4H2O 0.5±0.1 g / L, CuSO4·5H2O 1±0.5 g / L, (NH4)6Mo7O 24 ·4H2O 0.106±0.05 g / L, Na2B4O7·10H2O 0.23±0.08 g / L, 35% HCl 10±1 mL / L, and the solvent was water.

[0082] Feeding medium: glucose 500±10 g / L, yeast powder 12±2 g / L, potassium dihydrogen phosphate 12±2 g / L, ammonium sulfate 30±2.5 g / L, L-methionine 0.2±0.05 g / L, L-lysine 0.24±0.5 g / L, L-isoleucine 0.15±0.05 g / L.

[0083] It can be seen from Figure 1 that by using gene editing means to exogenously add the ilvA* and leuDH genes to replace the ptsG gene, the growth of the ABA-1 strain was not significantly inhibited, but it enabled Escherichia coli to ferment and produce L-2-aminobutyric acid without a plasmid, increasing the titer of L-2-aminobutyric acid from 0 g / L to 1.19 g / L. This indicates that the genomic replacement of the ilvA* and leuDH genes endows Escherichia coli with the ability to synthesize L-2-aminobutyric acid.

[0084] Example 2 Construction of Escherichia coli Strains with Adaptive Evolution to Tolerate High Concentrations of 2-Ketobutyric Acid

[0085] Configuration of Adaptive Evolution Medium

[0086] Prepare 20 mL of LB medium in a 250 mL shake flask, add 2-ketobutyric acid, and set the concentration gradients of 2-ketobutyric acid to 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, and 50 g / L. Adjust the pH of all media to 6.8 ± 0.1 before sterilization.

[0087] Inoculation

[0088] Transfer the E. coli THR△ptsG::ilvA*-leuDH strain to LB media containing different concentrations of 2-ketobutyric acid for subculture. The culture conditions are 37 °C, 180 rpm, and 16 - 18 h. Obtain the strain that can grow normally at a 2-ketobutyric acid concentration of 50 g / L, which is the strain E. coli THR△ptsG::ilvA*-leuDHMutt that tolerates high concentrations of 2-ketobutyric acid, denoted as ABA-2.

[0089] Example 3 Construction and Fermentation of Strain ABA-3 with the leuDH Gene Replacing the ltaE Gene and the poxB Gene 1. Construction of the recombinant vector pTarget-ltaEpoxB::leuDH Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, perform PCR amplification with ltaE-poxB deletion-sgRNA-F / ltaE-poxB deletion-sgRNA-R as primers. The obtained PCR product is digested with Dpn I at 37 °C for 1 h, and then transformed into competent E. coli DH5α cells. Screen on a spectinomycin plate and verify by sequencing to obtain the correct pTarget-ltaE poxB::leuDH plasmid for subsequent ligation with Donor DNA.

[0090] 2. Construction of the recombinant vector pTD-ltaEpoxB::leuDH Using the E. coli W3110 genome as a template, with ltaE-poxB D-Arm1-F, ltaE-poxB D-Arm1-R, ltaE-poxB D-Arm2-F, ltaE-poxB D-Arm2-R, InptsG-F-leuDH, and In ptsG-R-leuDH as primers. The construction steps are the same as in Example 1(2) to obtain the pTD-ltaEpoxB::leuDH plasmid.

[0091] 3. Preparation of electrocompetent cells

[0092] The pCas plasmid (Addgene Plasmid #62225) was introduced into the electrocompetent cells of strain ABA-2 obtained by adaptive evolution in Example 2. The method for preparing electrocompetent cells of strain ABA-2 was the same as that in Example 1(3).

[0093] 4. Electroporation editing and verification

[0094] Positive colonies of strain ABA-3 were constructed, and the construction method was the same as that in Example 1(4).

[0095] 5. The plasmid curing method was the same as that in Example 1(5) to obtain plasmid-free strain ABA-3.

[0096] 6. Fermentation of strain ABA-3 in a 5L fermenter

[0097] The constructed strain ABA-3 production strain was streaked from the glycerol tube onto an LB plate, and a single colony was picked and inoculated into 10 mL of LB medium. Using the strain ABA-1 constructed in Example 1 as a control, the implementation method was the same as that in Example 1(6), and the fermentation results are shown in Figure 2 .

[0098] From Figure 2 It can be seen that by using gene editing means to replace the ltaE gene (SEQ ID No.4) and the poxB gene (SEQ ID No.5) with the leuDH gene (SEQ ID No.2), the growth of strain ABA-3 was improved, the OD increased to 36.16, and at the same time, the yield of L-2-aminobutyric acid could be increased, making the titer of L-2-aminobutyric acid increase from 1.25 g / L to 4.35 g / L. This indicates that increasing the leuDH copy is beneficial to the synthesis of L-2-aminobutyric acid in Escherichia coli.

[0099] Construction and fermentation of strain ABA-4 with the focA gene and pflB gene deleted in Example 4

[0100] 1. Construction of recombinant vector pTarget-focApflB

[0101] Using pTarget F plasmid (Addgene Plasmid #62226) as a template and focA-pflB deletion-sgRNA-F / focA-pflB deletion-sgRNA-R as primers, PCR amplification was performed, and the obtained PCR product was digested with Dpn I at 37°C for 1 h, then transformed into E. coli DH5α competent cells, screened on spectinomycin plates, and sequenced to verify the correct pTarget-focApflB plasmid for subsequent ligation of Donor DNA.

[0102] 2. Construction of the recombinant vector pTD-focApflB Using the E. coli W3110 genome as a template, focA-pflB D-Arm1-F, focA-pflB D-Arm1-R, focA-pflB D-Arm2-F, focA-pflB D-Arm2-R, InfocA-pflB-F and InfocA-pflB-R as primers. The construction steps were the same as in Example 1 (2) to obtain the pTD-focApflB plasmid.

[0103] 3. Preparation of competent cells by electroporation The pCas plasmid (Addgene Plasmid #62225) was introduced into the competent cells of the strain ABA-3 obtained in Example 2. The method for preparing the competent cells of the strain ABA-3 was the same as that in Example 1 (3).

[0104] 4. Electroporation transformation, editing and verification The positive colonies of strain ABA-4 were obtained by the same construction method as in Example 1 (4).

[0105] 5. The plasmid elimination method is the same as in Example 1 (5) to obtain the plasmid-free strain ABA-4.

[0106] 6. Strain ABA-45L fermentation tank fermentation The constructed strain ABA-4 production strain was streaked from the glycerol tube onto the LB plate, and a single colony was picked and inoculated into 10 mL of LB medium. The strain ABA-3 constructed in Example 2 was used as a control. The implementation method was the same as Example 1 (6). The fermentation results are shown in FIG. Figure 3 .

[0107] Depend on Figure 3 It can be seen that deleting the focA gene (SEQ ID No. 6) and the pflB gene (SEQ ID No. 7) by gene editing has no obvious inhibitory effect on the growth of the ABA-4 strain, but can increase the production of L-2-aminobutyric acid, and the titer of L-2-aminobutyric acid increases from 4.35 g / L to 4.95 g / L, which indicates that the knockout of the by-product pathway is beneficial to the synthesis of L-2-aminobutyric acid in Escherichia coli.

[0108] Construction and Fermentation of Strain ABA-5 with Replacement of pta Gene and ackA Gene by ilvA* Gene and leuDH Gene in Example 5

[0109] 1. Construction of Recombinant Vector pTarget-pta ackA::ilvA*-leuDH

[0110] Using the pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed with pta-ackA deletion-sgRNA-F / pta-ackA deletion-sgRNA-R as primers. The obtained PCR product was digested with Dpn I at 37°C for 1 h, and then transformed into competent E. coli DH5α cells. Screening was carried out on a spectinomycin plate, and the correct pTarget-pta ackA::ilvA*-leuDH plasmid was obtained by sequencing verification for subsequent ligation of Donor DNA.

[0111] 2. Construction of Recombinant Vector pTD-pta ackA::ilvA*-leuDH

[0112] Using the E. coli W3110 genome as a template, pta-ackA D-Arm1-F, pta-ackA D-Arm1-R, pta-ackA D-Arm2-F, pta-ackA D-Arm2-R, Trc-F, and Trc ilvA*-leuDH-R were used as primers. The construction steps were the same as in Example 1(2) to obtain the pTD-pta ackA::ilvA*-leuDH plasmid.

[0113] 3. Preparation of Electrocompetent Cells

[0114] The pCas plasmid (Addgene Plasmid#62225) was introduced into the competent cells of strain ABA-4 obtained in Example 3. The method for preparing competent cells of strain ABA-4 was the same as in Example 1(3).

[0115] 4. Electroporation Editing and Verification

[0116] Positive colonies of strain ABA-5 were constructed, and the construction method was the same as in Example 1(4).

[0117] 5. Plasmid Elimination

[0118] The implementation method was the same as in Example 1(5) to obtain plasmid-free strain ABA-5.

[0119] 6. Fermentation of Strain ABA-5 in a 5 L Fermenter

[0120] The constructed strain ABA-4 production strain was streaked from a glycerol tube onto an LB plate, and a single colony was picked and inoculated into 10 mL of LB medium. Using the strain ABA-4 constructed in Example 3 as a control, the implementation method was the same as that in Example 1(6). The fermentation results are shown in Figure 4 .

[0121] It can be seen from Figure 4 that by using gene editing means to replace the pta gene (SEQ ID No.8) and ackA gene (SEQ ID No.9) with the ilvA* gene (SEQ ID No.1) and leuDH gene (SEQ ID No.2), there is no obvious inhibitory effect on the growth of the ABA-5 strain. However, it can increase the yield of L-2-aminobutyric acid, making the titer of L-2-aminobutyric acid increase from 4.95 g / L to 11.59 g / L. This shows that increasing the copy number of the ilvA* gene and leuDH gene and knocking out the by-product pathway are beneficial to the synthesis of L-2-aminobutyric acid in Escherichia coli.

[0122] Example 6 Construction and fermentation of strain ABA-6 with the ilvA* gene and leuDH gene replacing the mgsA gene 1. Construction of the recombinant vector pTarget-mgsA::ilvA*-leuDH Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, PCR amplification was carried out with mgsA deletion-sgRNA-F / mgsA deletion-sgRNA-R as primers. The obtained PCR product was digested with Dpn I at 37 °C for 1 h, and then transformed into E. coli DH5α competent cells. Screening was carried out on a spectinomycin plate, and sequencing verification was performed to obtain the correct pTarget-mgsA::ilvA*-leuDH plasmid for subsequent ligation of Donor DNA.

[0123] 2. Construction of the recombinant vector pTD-mgsA::ilvA*-leuDH Using the E. coli W3110 genome as a template, mgsAD-Arm1-F, mgsA D-Arm1-R, mgsA D-Arm2-F, mgsA D-Arm2-R, Trc-F and Trc ilvA*-leuDH-R as primers. The construction steps were the same as those in Example 1(2) to obtain the pTD-mgsA::ilvA*-leuDH plasmid.

[0124] 3. Preparation of electrotransformation competent cells

[0125] The pCas plasmid (Addgene Plasmid #62225) was introduced into the competent cells of the strain ABA-5 obtained in Example 4. The method for preparing the competent cells of the strain ABA-5 was the same as that in Example 1(3).

[0126] 4. Electrotransformation Editing and Verification

[0127] The positive colony of strain ABA-6 was constructed, and the construction method was the same as that in Example 1(4).

[0128] 5. Plasmid Elimination

[0129] The implementation method was the same as that in Example 1(5), and the plasmid-free strain ABA-6 was obtained.

[0130] 6. Fermentation of Strain ABA-6 in a 5L Fermenter

[0131] The constructed strain ABA-6 production strain was streaked from the glycerol tube onto an LB plate, and a single colony was picked and inoculated into 10 mL of LB medium. Using the strain ABA-4 constructed in Example 3 as a control, the implementation method was the same as that in Example 1(6), and the results are shown in Figure 5 .

[0132] As can be seen from Figure 5 , by using gene editing means to replace the mgsA gene (SEQ ID No.10) with the ilvA* gene (SEQ ID No.1) and the leuDH gene (SEQ ID No.2), the growth of strain ABA-6 was not affected, and the yield of L-2-aminobutyric acid increased, from 11.59 g / L to 13.81 g / L. This indicates that increasing the copy number of the ilvA* gene and the leuDH gene and knocking out the by-product pathway are beneficial to the synthesis of L-2-aminobutyric acid in Escherichia coli.

[0133] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art unless otherwise specified; the methods used in the present invention are all conventional methods in the art unless otherwise specified.

[0134] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid, characterized in that: Constructed by the following method: (1) Using E. coli THR as the chassis bacterium, replace the E.coli gene in the THR genome with the ptsG gene, the ilvA* gene and the leuDH gene to obtain the recombinant strain E. coli THR△ ptsG :: ilvA*-leuDH ; (2) Inoculate the strain E. coli THR△ ptsG :: ilvA*-leuDH into a 2-ketobutyric acid liquid medium for subculture, and screen for strains that can grow normally in a liquid medium with a 2-ketobutyric acid concentration of not less than 50 g / L to obtain the strain E. coli THR△ ptsG :: ilvA*-leuDHMut ; (3) The strain E. coli THR△ ptsG :: ilvA*-leuDHMut Adjacent in the genome ltaE Gene and poxB Gene leuDH Gene replacement to obtain recombinant strains E. coli THR△ ptsG :: ilvA*-leuDHMut △ ltaE △ poxB :: leuDH ; (4) The strain E. coli THR△ ptsG :: ilvA*-leuDHMut △ ltaE △ poxB :: leuDH in the genome and focA gene and pflB gene are knocked out to obtain the recombinant strain E. coli THR△ ptsG :: ilvA*-leuDHMut △ ltaE △ poxB :: leuDH △ focA △ pflB ; (5) Replace the adjacent E. coli THR△ ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△ pflB genes in the pta genome and ackA genes with ilvA* genes and leuDH genes to obtain the recombinant strain E. coli THR △ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ackA:: ilvA*-leuDH ; (6) Replace the E. coli THR △ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB △pta △ackA::ilvA*-leuDH gene in the mgsA genome with the ilvA* gene and the leuDH gene to obtain the recombinant strain E. coli THR △ptsG::ilvA*-leuDHMut△ltaE△poxB::leuDH△focA△pflB△pta△ ackA::ilvA*-leuDH△mgsA::ilvA*-leuDH , namely the plasmid-free genetically engineered bacterium with high-yield L-2-aminobutyric acid; Among them, the ilvA* gene sequence is as shown in SEQ ID No.

1.

2. The plasmid-free genetically engineered bacterium for highly producing L-2-amino butyric acid according to claim 1, wherein: The said leuDH gene sequence is as shown in SEQ ID No.

2.

3. The plasmid-free genetically engineered bacterium for highly producing L-2-amino butyric acid according to claim 1, characterized in that: The said ptsG gene sequence is as shown in SEQ ID No.

3.

4. The plasmid-free genetically engineered bacterium for highly producing L-2-aminobutyric acid according to claim 1, characterized in that: The said ltaE gene sequence is as shown in SEQ ID No. 4, and the said poxB gene sequence is as shown in SEQ ID No.

5.

5. The plasmid-free genetically engineered bacterium for highly producing L-2-aminobutyric acid according to claim 1, characterized in that: The focA gene sequence is as shown in SEQ ID No. 6, and the pflB gene sequence is as shown in SEQ ID No.

7.

6. The plasmid-free genetically engineered bacterium for highly producing L-2-amino butyric acid according to claim 1, wherein: The said pta gene sequence is as shown in SEQ ID No.8, and the said ackA gene sequence is as shown in SEQ ID No.

9.

7. The plasmid-free genetically engineered bacterium for highly producing L-2-amino butyric acid according to claim 1, characterized in that: The said mgsA gene sequence is as shown in SEQ ID No.

10.

8. Use of the plasmid-free genetically engineered bacterium according to any one of claims 1-7 in the preparation of L-2-aminobutyric acid by microbial fermentation.

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