CN114456995B - Genetically engineered bacterium for high yield of L-2-aminobutyric acid, construction method and application - Google Patents
Genetically engineered bacterium for high yield of L-2-aminobutyric acid, construction method and application Download PDFInfo
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- CN114456995B CN114456995B CN202210091140.9A CN202210091140A CN114456995B CN 114456995 B CN114456995 B CN 114456995B CN 202210091140 A CN202210091140 A CN 202210091140A CN 114456995 B CN114456995 B CN 114456995B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01009—Leucine dehydrogenase (1.4.1.9)
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Abstract
The invention relates to a genetic engineering bacterium for high yield of L-2-aminobutyric acid, a construction method thereof and application thereof in microbial fermentation preparation of L-2-aminobutyric acid. According to the invention, related competitive genes of glucose PTS (PTS) of escherichia coli are knocked out, so that the consumption of phosphoenolpyruvic acid is reduced, the expression condition of main path synthesis pathway genes is improved by over-expressed genes ilvA and leuDH, and finally, the L-2-aminobutyric acid engineering bacteria with higher yield are obtained, exogenous enzyme is not required to be introduced into plasmids to enhance the key enzyme activity, and the fermentation yield of a 5L fermentation tank can reach 15.11g/L when an inducer IPTG is added; the repressor coding gene lacI is knocked out to form constitutive expression, and after the culture medium is optimized, fed-batch fermentation is carried out in a 5L fermentation tank, and the yield reaches 10.86g/L when inducer IPTG is not needed to be added.
Description
Technical Field
The invention relates to a genetically engineered bacterium for high yield of L-2-aminobutyric acid, a construction method and application thereof.
Background
2-aminobutyric acid (2-aminobutyric acid) is a four-carbon aliphatic amino acid, and two optical isomers are D-2-aminobutyric acid and L-2-aminobutyric acid respectively, wherein L-2-aminobutyric acid (L-2-aminobutyric acid) is naturally distributed and exists in animal and plant tissues, and is called L-ABA for short. The molecular formula of L-ABA is C4H9NO2, the relative molecular weight is 103.12, the melting point is 270-280 oC, 22.7 g can be dissolved in water of every 100 g at 25 oC, the isoelectric point is 6.05, the L-ABA is slightly dissolved in organic solvents such as ethanol, diethyl ether and the like, and the L-ABA is odorless slightly sweet white flaky crystal and is a non-protein chiral alpha-amino acid.
As important chemical raw materials and medical precursors, L-ABA is widely applied to the chemical industry and the medical industry and is used for synthesizing chemicals and medicines. L-ABA can generate (S) -2-aminobutanamide (S) -2-aminobutyramid) through amidation, is a key intermediate for preparing antiepileptic drugs of Brivaracetam (Brivaracetam) and Levetiracetam (LEV), and can also be used for preparing antituberculosis drugs of Ethambutol (Ethambutol) through reduction of terminal carboxyl groups to synthesize (S) -2-aminobutanol). Levetiracetam belongs to a pyrrolidone derivative, is a piracetam analogue, is a novel antiepileptic drug, has stronger pharmacokinetic properties and tolerance due to a unique antiepileptic mechanism, and has high-efficiency and safe clinical effect, thus becoming one of the novel antiepileptic drugs with the most application prospect at present. Ethambutol has antibacterial and antituberculosis effects, and is mainly used for human and bovine type tubercle bacillus to inhibit ribonucleic acid synthesis, and has good inhibitory effect on other mycobacteria. However, when ethambutol is used alone, the drug resistance of bacteria is rapidly generated, so the ethambutol should be used together with other antitubercular drugs, and is mainly applied to the treatment of pulmonary tuberculosis and tubercular meningitis clinically.
With the increasing demand of medicines such as levetiracetam, buvaracetam, ethambutol and the like in the international market, the demand of L-ABA which is a key intermediate for synthesizing the medicines is also increasing, and the production of L-ABA is receiving more and more attention. Chemical methods and biological methods are main ways for synthesizing L-ABA, the chemical methods comprise a desulfurization reaction method, a 2-KB reduction method, an ammonolysis method of alpha-halogenated acid, an ammonolysis method and the like, and the biological methods comprise a microbial fermentation method, an enzyme catalysis method and the like.
In recent years, the biological method for synthesizing L-ABA has been widely focused, and compared with the chemical method, the biological method has obvious advantages, such as strong stereoselectivity, high catalytic efficiency, wide raw material sources, low price, mild reaction conditions, environmental protection and the like, and has very wide application prospects.
Disclosure of Invention
The invention aims to provide a genetically engineered bacterium with high L-2-aminobutyric acid yield and a construction method thereof through a metabolic engineering technology, and application of the genetically engineered bacterium in microbial fermentation preparation of L-2-aminobutyric acid.
The technical scheme adopted by the invention is as follows:
the genetically engineered bacterium for producing the L-2-aminobutyric acid at high yield is constructed and obtained by the following method:
(1) To be used forE.coli 19031 is Chaetomium, and ilvIH gene in genome of strain 19031 is derived fromT. intermediusLeuDH was replaced with the leucine dehydrogenase of 19031ilvIH, designated 19031-1;
(2) The ptsG gene in the LeuDH genome was treated with ilvA which released feedback inhibition by L-isoleucine * Alternatively, a recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA was obtained * Designated 19031-2;
(3) Strain 19031ilvIH:: leuDH/ptsG:: ilvA was used * The rhtA gene in the genome is derived fromT. intermediusThe leucine dehydrogenase leuDH of (E) was replaced to give recombinant strain 19031ilvIH:: leuDH/ptsG::: ilvA * rhtA: leuDH, designated 19031-3;
(4) Strain 19031ilvIH:: leuDH/ptsG:: ilvA was used * The lacI gene in the leuDH genome is knocked out to obtain recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA * And rhtA is leuDH/[ delta ] lacI, which is 19031-4, namely the genetically engineered bacterium for high-yield L-2-aminobutyric acid.
The chassis fungusE.coli 19031 can be constructed by the following method:
(1) To be used forEscherichia.coliW3110 as starting strain, strainEscherichia.coliThe thrA gene in the W3110 genome was replaced with thrA mutant gene (C1034T, S345F) to give a recombinant strainEscherichia.coliW3110/thrA * Denoted THR1;
(2) The lysC gene in the genome of the strain THR1 was subjected to mutation with lysC gene (C1055T, T342I) to give a recombinant strainEscherichia.coli W3110/ thrA * /lysC * Denoted THR2;
(3) The strain THR2 genomeThe thrABC gene promoter is replaced by Tac promoter to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC is designated THR3;
(4) Knocking out lysA gene in THR3 genome of strain to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA, designated THR4;
(5) Knocking out metA gene in THR4 genome of strain to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.meta, designated THR5;
(6) Knocking out tdh gene in strain DPA5 genome to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh, designated THR6;
(7) Knocking out ilcR gene in strain DPA6 genome to obtain recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR, designated THR7;
(8) Replacing the ppc gene promoter in the THR7 genome of the strain with Trc promoter to obtain a recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc, designated THR8, i.e.E.coli 19031。
The invention is characterized in that: (1) Editing related genes of L-2-aminobutyric acid synthesis pathway of escherichia coli, and adopting CRISPR-Cas9 gene editing technology to synthesize related genes ilvIH of L-2-aminobutyric acid on genome to be derived fromT. intermediusThe leucine dehydrogenase leuDH gene is replaced, so that the escherichia coli can ferment and produce the L-2-aminobutyric acid without plasmids; (2) ilvA for releasing feedback inhibition of L-isoleucine for ptsG gene related to Phosphatase Transfer System (PTS) of glucose using CRISPR-Cas9 gene editing technique * Replacement, deletion of ptsG gene reduces consumption of phosphoenolpyruvate, increases accumulation of pyruvate, and makes more reaction progress to TCA cycle, ilvA * The gene improves the catabolic capacity of threonine,increased production of alpha-ketobutyric acid; (3) Use of CRISPR-Cas9 Gene editing technology to derive L-threonine transport-related genes rhtA in the genomeT. intermediusThe leucine dehydrogenase leuDH is replaced, so that the synthesis capability of the escherichia coli on the L-2-aminobutyric acid is further enhanced, meanwhile, the outward transport of L-threonine is reduced, and the accumulation of L-threonine is increased; (4) The CRISPR-Cas9 gene editing technology is used for knocking out the gene lacI, and finally, a genetically engineered bacterium 19031-4 for producing the L-2-aminobutyric acid in a higher yield is constructed. (5) The No. 19031-4 strain is subjected to 5L fermentation tank expansion culture, and the yield is further improved to 10.86g/L.
The invention also relates to a method for the genetically engineered bacterium, which comprises the following steps:
(1) To be used forEscherichia.coliW3110 as starting strain, strainEscherichia.coliThe thrA gene in the W3110 genome was replaced with thrA mutant gene (C1034T, S345F) to give a recombinant strainEscherichia.coliW3110/thrA * Denoted THR1;
(2) The lysC gene in the genome of the strain THR1 was subjected to mutation with lysC gene (C1055T, T342I) to give a recombinant strainEscherichia.coli W3110/ thrA * /lysC * Denoted THR2;
(3) Replacing thrABC gene promoter in THR2 genome of strain with Tac promoter to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC is designated THR3;
(4) Knocking out lysA gene in THR3 genome of strain to obtain recombinant strainEscherichia.coliW3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA, designated THR4;
(5) Knocking out metA gene in THR4 genome of strain to obtain recombinant strainEscherichia.coliW3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.meta, designated THR5;
(6) Knocking out tdh gene in strain DPA5 genome to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh, is designated asTHR6；
(7) Knocking out ilcR gene in strain DPA6 genome to obtain recombinant strainEscherichia.coliW3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR, designated THR7;
(8) Replacing the ppc gene promoter in the THR7 genome of the strain with Trc promoter to obtain a recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc, designated THR8, i.eE.coli 19031；
(9) To be used forE.coli 19031 is Chaetomium, and ilvIH gene in genome of strain 19031 is derived from CRISPR-Cas9 gene editing technologyT. intermediusThe leucine dehydrogenase leuDH gene is replaced to obtain a recombinant strain 19031ilvIH, namely leuDH, which is recorded as 19031-1;
(10) By using CRISPR-Cas9 gene editing technology, the ptsG gene in the strain 19031ilvIH:: leuDH genome is treated with ilvA which releases feedback inhibition by L-isoleucine * Alternatively, a recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA was obtained * Designated 19031-2;
(11) Strain 19031 ilvIH::: leuDH/ptsG::: ilvA was obtained by using CRISPR-Cas9 gene editing technique * The rhtA gene in the genome is derived fromT. intermediusThe leucine dehydrogenase leuDH of (E) was replaced to give recombinant strain 19031ilvIH:: leuDH/ptsG::: ilvA * rhtA: leuDH, designated 19031-3;
(12) Strain 19031 ilvIH::: leuDH/ptsG::: ilvA was obtained by using CRISPR-Cas9 gene editing technique * The lacI gene in the leuDH genome is knocked out to obtain recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA * And rhtA is leuDH/[ delta ] lacI, which is 19031-4, namely the genetically engineered bacterium for high-yield L-2-aminobutyric acid.
Preferably, the nucleotide sequence of the Tac promoter is shown as SEQ ID NO.1, and the nucleotide sequence of the Trc promoter is shown as SEQ ID NO. 2.
Preferably, the thrA mutant gene sequence is shown as SEQ ID No.5, the lysC mutant gene sequence is shown as SEQ ID No.4, and the leuDH geneThe sequence is shown as SEQ ID No.7, the ilvA * The gene sequence is shown as SEQ ID No. 8.
The invention also relates to application of the genetically engineered bacterium in microbial fermentation preparation of L-2-aminobutyric acid.
Specifically, the application is as follows: inoculating the genetically engineered strain into a fermentation culture medium, and fermenting and culturing at 30-37 ℃ and 150-300 rpm until OD 600 When the fermentation liquid is 10-30, adding IPTG with the final concentration of 0.1mM, continuously culturing for 48 hours, and taking the supernatant of the fermentation liquid after the fermentation is finished, and separating and purifying to obtain the L-2-aminobutyric acid.
The fermentation medium comprises the following components: 30-50 g/L of glucose, 5-8 g/L of yeast powder, 1-3 g/L of magnesium sulfate, 3-4 g/L of dipotassium hydrogen phosphate, 10-20 g/L of ammonium sulfate, 0.5-1.5 g/L of betaine hydrochloride, 0.05-0.5 g/L of L-methionine, 0.05-0.5 g/L of L-lysine, 1-5 mL/L of metal salt ion solution, 20-30 g/L of calcium carbonate, water as a solvent and natural pH value; the metal salt ion solution comprises the following components: feSO 4 ·7H 2 O 10g/L，CaCl 2 1.35g/L，ZnSO 4 ·7H 2 O 2.25g/L，MnSO 4 ·4H 2 O 0.5g/L，CuSO 4 ·5H 2 O 1g/L，（NH 4 ）6Mo 7 O 24 ·4H 2 O 0.106g/L，Na 2 B 4 O 7 ·10H 2 O0.23 g/L,35% HCl 10mL/L, and water as solvent.
Preferably, the fermentation medium is composed of: 50g/L of glucose, 6g/L of yeast powder, 2g/L of magnesium sulfate, 4g/L of dipotassium hydrogen phosphate, 14g/L of ammonium sulfate, 1g/L of betaine hydrochloride, 0.149 g/L of L-methionine, 0.164 g/L of L-lysine, 5mL of metal salt ion solution, caCO 3 30g/L, and the solvent is water.
Typically, the genetically engineered strain is inoculated into LB medium before fermentation, cultured overnight on a shaker at 37℃and 200rpm, and then inoculated into fermentation medium at an inoculum size of 5% by volume for fermentation culture.
The beneficial effects of the invention are mainly as follows: the invention edits the genes related to the synthesis path of the L-2-aminobutyric acid of the escherichia coli to obtain the genesThe gene ilvIH related to the synthesis of L-2-aminobutyric acid on genome is derived fromT. intermediusThe leucine dehydrogenase leuDH gene of (2) was replaced to allow L-2-aminobutyric acid to be produced by fermentation of E.coli without the need for a plasmid, and the ptsG gene, which is a gene related to the Phosphatase Transfer System (PTS) of glucose, was used as ilvA for releasing feedback inhibition by L-isoleucine * Replacement, deletion of ptsG gene reduces consumption of phosphoenolpyruvate, increases accumulation of pyruvate, and makes more reaction progress to TCA cycle, ilvA * The gene improves the catabolism capability of threonine, increases the production of alpha-ketobutyrate, and uses the L-threonine transport related gene rhtA in the genome to be derived fromT. intermediusThe leucine dehydrogenase leuDH is replaced, the synthesis capacity of escherichia coli on L-2-aminobutyric acid is further enhanced, meanwhile, the outward transport of L-threonine is reduced, the accumulation of L-threonine is increased, finally, the repressor protein coding gene lacI is knocked out, and finally, the L-2-aminobutyric acid engineering bacteria with higher yield is obtained, and under the condition that a plasmid primer is not needed, exogenous enzyme is used for enhancing the key enzyme activity, and no inducer is added, the fed-batch fermentation yield of a 5L fermentation tank reaches 10.86g/L.
Drawings
FIG. 1 shows the fermentation result OD 600 and the L-2-aminobutyric acid content in the supernatant of the fermentation broth of example 2;
FIG. 2 shows the fermentation result OD 600 and the L-2-aminobutyric acid content in the supernatant of the fermentation broth in example 3;
FIG. 3 shows the fermentation result OD 600 and the L-2-aminobutyric acid content in the supernatant of the fermentation broth of example 4;
FIG. 4 shows the results of fermentation in example 5, OD 600 and L-2-aminobutyric acid content in the supernatant of the fermentation broth.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
in the examples, the final concentration of kanamycin in the medium was 0.05mg/L, and the final concentration of spectinomycin in the medium was 0.05mg/L.
The strain Escherichia coli W3110 is from the university of Yersinia CGSC collection (Coli Genetic StockCenter), the date of deposit 1975, month 8 and day 5, and the deposit number CGSC#4474, which is disclosed in patent US 2009/0298135 A1,US2010/0248311A 1.
The parent strain E.coli 19031 is from laboratory preservation, is an E.coli W3110 derivative, and has the genotype of the following descriptionE.coliW3110/thrA * /lysC * /Tac-thrABC/△lysA/△metA/△tdh/ilcR/Trc-ppc）
The primer sequence information used in examples 2 to 5 is shown in Table 1:
table 1: primer sequences
pT-X-F/R is a mutation primer of pTatget plasmid, wherein X is a 20bp sequence before PAM locus (NGG) contained in target gene carrying genome; x P1/P2 is an upstream primer and a downstream primer of an upstream homology arm of a target gene; pTD-X P3/P4 is a downstream primer of a downstream homology arm of a target gene; x VF/VR is a verification primer for editing target genes.
HPLC method for measuring L-2-aminobutyric acid content in fermentation liquor:
sample treatment: taking 1mL fermentation liquor, centrifuging at 12000rpm for 1min at room temperature, diluting supernatant by a proper multiple, and keeping D-pantothenic acid content between 0.1g/L and 2.0 g/L;
chromatographic conditions: the chromatographic column is C18 column (150×4.6. 4.6 mm), column temperature 33 o C, the flow rate is 1 mL/min, gradient elution is adopted, the sample injection amount is 10 mu L, and the detection wavelength is 360 nm.
Table 2: gradient elution procedure
Mobile phase: mobile phase a:50% acetonitrile, mobile phase B: anhydrous sodium acetate, 4.1 g, was weighed into 800 mL deionized water, pH adjusted to 6.4 with acetic acid, and then to 1L with deionized water.
Data acquisition time: 30min.
Example 1: construction of Chaetomium 19031
The chassis fungusE.coli 19031 is constructed by the following method:
(1) To be used forEscherichia.coliW3110 is taken as a starting strain, and CRISPR-Cas9 gene editing technology is applied to the starting strainEscherichia.coliThe thrA gene in the W3110 genome was replaced with thrA mutant gene (C1034T, S345F) (SEQ ID No. 5) to give a recombinant strainEscherichia.coli W3110/thrA * Denoted THR1;
(2) The lysC mutant gene (C1055T, T342I) (SEQ ID No. 4) is used for lysC gene in the genome of the strain THR1 by using CRISPR-Cas9 gene editing technology to obtain a recombinant strainEscherichia.coli W3110/ thrA * /lysC * Denoted THR2;
(3) The CRISPR-Cas9 gene editing technology is used to replace thrABC gene promoter in the THR2 genome of the strain with Tac promoter (SEQ ID No. 1) to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC is designated THR3;
(4) Knocking out lysA gene in THR3 genome of strain by using CRISPR-Cas9 gene editing technology to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA, designated THR4;
(5) The CRISPR-Cas9 gene editing technology is used for knocking out the metA gene in the THR4 genome of the strain to obtain a recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.meta, designated THR5;
(6) The tdh gene in the DPA5 genome of the strain is knocked out by using CRISPR-Cas9 gene editing technology to obtain a recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh, designated THR6;
(7) Knocking out ilcR genes in a strain DPA6 genome by using a CRISPR-Cas9 gene editing technology to obtain a recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR, designated THR7;
(8) The CRISPR-Cas9 gene editing technology is used to replace the ppc gene promoter in the THR7 genome of the strain with Trc promoter (SEQ ID No. 2) to obtain recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc, as THR8, as 19031.
Example 2: construction and fermentation of strain 19031-1 with leuDH gene replacing ilvIH gene
By genetically engineering bacteria 19031 (i.eE.coli W3110 thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc) as the starting strain, CRISPR-Cas9 mediated Gene Editing technique (Yujiang et al 2015Multigene Editing in theEscherichia coliGenome via theCRISPR-Cas9 System. Applied Environmental microbiology.81:2506-2514) substitution of the ilvIH gene (SEQ ID No. 6) with the leuDH gene (SEQ ID No. 7) on the genome, allows the fermentative production of L-2-aminobutyric acid by E.coli without plasmids:
(1) Construction of pTarget-ilvIH: : leuDH plasmid: the pTarget F Plasmid (Addgene Plasmid # 62226) was used as a template, pT-ilvIH: : leuDH F/pT-ilvIH: : the leuDH R is used as a primer for PCR amplification, the obtained PCR product is subjected to Dpn I heat preservation and digestion for 30min at 37 ℃, then is transformed into E.coli DH5 alphaization transformation competence, spectinomycin (SD) plate screening is carried out, and sequencing verification is carried out to obtain the correct pTarget-ilvIH: : the leuDH plasmid was used for subsequent ligation of Donor DNA.
(2) Construction of pTD-ilvIH: : leuDH plasmid: to be used forE.coliW3110 genome is template, ilvIH: : leuDH P1 with ilvIH: : the upstream homology arm for the primer amplification of leuDH P2 to give the donor DNA was designated L, ilvIH: : leuDH P3 and ilvIH: : the downstream homology arm of the primer amplified douDH P4 to give the douor DNA was designated as R, ilvIH: : leuDH P5 and ilvIH: : the primer amplification of the leuDH P6 to obtain a donor DNA leuDH gene is marked as D, and the DNA fragment is recovered by using a Clean up kit to obtain a homology arm A, a homology arm C and a donor DNA B; pTarget-ilvIH: : the leuDH plasmid is amplified by PCR with pT+D CFcom and pT+D CRcom as primers, the obtained PCR product is digested by Dpn I at 37 ℃ for 1-2h under heat preservation, and DN is recovered by Clean up kitFragment A; pTarget-ilvIH was performed according to the instructions of (One step clone kit, vazyme Biotech, nanjing, china): : the leuDH plasmid, the homology arm A+C and the donor DNA B are connected together and introducedE.coliIn DH5 alphation transformation competence, colony PCR is used for screening positive clones, and pTD-ilvIH is obtained through sequencing verification: : the leuDH plasmid.
(3) The pCas Plasmid (Addgene Plasmid # 62225) was introduced into 19031 transformation competence, positive clones were picked and transferred into LB tubes containing 0.05mg/L kanamycin, and cultured overnight at 30 ℃; inoculating the strain with 1% of the volume concentration into a 250mL shaking flask containing 50mL of LB culture medium, adding 500 μl of 1 mol/L-arabinose, and culturing at 150rpm and 30 ℃ to OD 600-0.4-0.6; cells were harvested by centrifugation at 4000rpm at 4℃for 10min to prepare electrotransformation competence, as described in detail in (Molecular Cloning: A Laboratory Manual,3ed edition, 99-102).
(4) 200-500 ng of pTD-ilvIH was taken: : mixing the leuDH plasmid with 100-200 mu L of electrotransformation competent cells, transferring into a precooled 2mm electric shock cup, carrying out electric shock transformation by using an electroporation apparatus (MicroPluser TM, BIO-RAD) for about 1min, immediately adding precooled 1mL of LB medium after electric shock is finished, immediately sucking out, transferring into a 2mLEP tube, resuscitating at 30 ℃ for 3-4 h, coating an LB plate containing 0.05mg/L kanamycin and 0.05mg/L spectinomycin, inversely culturing at 37 ℃ for 14-18 h, and carrying out ilvIH: : leuDH VF and ilvIH: : the leuDH VR is used as a verification primer for colony PCR verification, and if a fragment of about 1500 can be successfully cloned, and sequencing verification is successful, the single colony is proved to be 19031/ilvIH: : positive colonies of leuDH, i.e.successful editing, gave a new strain 19031-1.
(5) Plasmid elimination: positive single colonies were picked up and inoculated into LB tubes containing 1mM IPTG and 0.05mg/L kanamycin, incubated overnight at 30℃and the next day bacterial solution streaked on LB plates containing 0.05mg/L kanamycin, incubated at 30℃for 24h, and single colonies were picked up and streaked on LB plates containing 0.05mg/L spectinomycin, but not pTarget-ilvIH on single colonies of LB plates containing 0.05mg/L spectinomycin: : the leuDH plasmid was successfully eliminated and pTarget-ilvIH was picked up: : single colonies with successful elimination of the leuDH plasmid were incubated in LB tubes at 37℃overnight, the next day broth streaked on LB plates and incubated at 37℃for 12h, single colonies were picked up streaked on LB plates containing 0.05mg/L kanamycin, and pCas plasmids could not be successfully eliminated in single colonies on LB plates containing 0.05mg/L kanamycin, resulting in plasmid-free strain 19031/ilvIH: : leuDH (abbreviated as 19031-1).
LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, deionized water as solvent, and natural pH.
TPM fermentation medium: 50g/L of glucose, 6g/L of yeast powder, 2g/L of magnesium sulfate, 4g/L of dipotassium hydrogen phosphate, 14g/L of ammonium sulfate, 1g/L of betaine hydrochloride, 0.149 g/L of L-methionine, 0.164 g/L of L-lysine, 5mL of metal salt ion solution, 30g/L of CaCO3, deionized water as a solvent and natural pH value; metal salt ion solution FeSO 4 ·7H 2 O 10g/L，CaCl 2 1.35g/L，ZnSO 4 ·7H 2 O 2.25g/L，MnSO 4 ·4H 2 O 0.5g/L，CuSO 4 ·5H 2 O 1g/L，（NH 4 ）6Mo 7 O 24 ·4H 2 O 0.106g/L，Na 2 B 4 O 7 ·10H 2 O0.23 g/L,35% HCl 10mL, and deionized water as solvent.
(6) The constructed 19031-1 strain is streaked from an glycerol pipe to an LB plate, and a single colony is picked and inoculated into 10mL of LB culture medium to obtain a wild strainE.coliW3110 (DE 3) as a control, cultured at 37℃and 200rpm was used as seed solution; after 8-12 h, inoculating 1mL of seed solution into a 250mL shaking flask filled with 30mL of TPM fermentation medium, then culturing and fermenting at 35 ℃ and 200rpm until the strain grows to OD 600 = 0.8-1.0, adding IPTG with the final concentration of 0.1mM, and continuing culturing for 48h; after fermentation, 1mL of fermentation liquor is taken to determine OD 600, 1mL of fermentation liquor is taken, the fermentation liquor is centrifuged at 12000rpm at room temperature for 2min, the fermentation supernatant is diluted 5-10 times, the content of L-2-aminobutyric acid is detected by HPLC according to the method, the growth condition of the strain is determined by detecting OD 600 by a spectrophotometer, and the result is shown in figure 1.
As can be seen from FIG. 1, the leucine dehydrogenase gene leuDH is exogenously added by using a gene editing means to replace ilvIH gene, and the growth of 19031-1 strain has no obvious inhibition effect, but can make Escherichia coli ferment to produce L-2-aminobutyric acid under the condition of no plasmid, so that the titer of L-2-aminobutyric acid is increased from 0g/L to 3.35g/L, which indicates that the genome replacement of the leuDH gene can make Escherichia coli have the synthesis capability of L-2-aminobutyric acid.
Example 3: ilvA * Construction and fermentation of Gene replacement ptsG Gene Strain 19031-2
(1) Construction of pTarget-ptsG: : ilvA * Plasmid: pTarget F Plasmid (Addgene Plasmid # 62226) was used as a template, pTarget-ptsG: : ilvA * F/ pTarget-ptsG：：ilvA * PCR amplification with R as primer, digestion with Dpn I at 37 deg.c for 30min, and conversion toE.coliIn DH5 alphation transduction competence, spectinomycin (SD) plates were screened and sequencing verified to obtain the correct pTarget-ptsG: : ilvA * Plasmid for subsequent ligation of the Donor DNA.
(2) Construction of pTD-ptsG: : ilvA * Plasmid: to be used forE.coliW3110 genome is template, ptsG: : ilvA * P1、 ptsG：：ilvA * P2、 ptsG：：ilvA * P3、ptsG：：ilvA * P4、 ptsG：：ilvA * P5 and ptsG: : ilvA * P6 is a primer. The construction procedure was the same as in example 2 (2), to give pTD-ptsG: : ilvA * A plasmid.
(3) The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain 19031-1 obtained in example 2, and the competence of strain 19031-1 was prepared in the same manner as in example 2 (3).
(4) The strain 19031-2 positive colony was constructed in the same manner as in example 2 (4).
(5) Plasmid elimination: the procedure was followed in example 2 (5) to obtain plasmid-free strain 19031-2.
(6) Shaking and fermenting: the strain 19031-2 was streaked from the glycerol tube to the LB plate, and a single colony was picked and inoculated into 10mL of LB medium, and the method was carried out as in example 2 (6) using the strain 19031-1 constructed in example 4 as a control, and the results are shown in FIG. 2.
As can be seen from FIG. 2, ilvA was obtained by gene editing means * Replacement of ptsG Gene with Gene (SEQ ID No. 8)SEQ ID No. 9), 19031-2 strain had no remarkable inhibitory effect on growth, but could increase the yield of L-2-aminobutyric acid, so that the L-2-aminobutyric acid titer was increased from g/L to 6.35g/L, indicating that the gene ilvA * Is beneficial to the synthesis of the L-2-aminobutyric acid of the escherichia coli.
Example 4: construction and fermentation of bacterial strain 19031-3 with leuDH gene replacing rhtA gene
(1) Construction of pTarget-rhtA: : leuDH plasmid: pTarget F Plasmid (Addgene Plasmid # 62226) was used as a template, pTarget-rhtA: : leuDH F/pTarget-rhtA: : the leuDH R is used as a primer for PCR amplification, and the obtained PCR product is digested for 30min at 37 ℃ by Dpn I and then is converted intoE.coliIn DH5 alphation transduction competence, spectinomycin (SD) plate screening, sequencing verification to obtain the correct pTarget-rhtA: : the leuDH plasmid was used for subsequent ligation of Donor DNA.
(2) Construction of pTD-rhtA: : leuDH plasmid: to be used forE.coliW3110 genome is template, rhtA: : leuDH P1, rhtA: : leuDH P2, rhtA: : leuDH P3, rhtA: : leuDH P4, rhtA: : leuDH P5 and rhtA: : leuDH P6 is the primer. The construction procedure was the same as in example 3 (2) to give pTD-rhtA: : the leuDH plasmid.
(3) The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain 19031-2 obtained in example 2, and the competence of strain 19031-2 was prepared in the same manner as in example 2 (3).
(4) The positive colony of the strain 19031-3 was constructed in the same manner as in example 2 (4).
(5) Plasmid elimination: the procedure was followed in example 2 (5) to obtain plasmid-free strain 19031-3.
(6) Shaking and fermenting: the constructed strain 19031-3 producing strain was streaked from the glycerol tube to the LB plate, and a single colony was picked up and inoculated into 10mL of LB medium, and the method was carried out in the same manner as in example 2 (6) with the strain 19031-2 constructed in example 3 as a control, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the substitution of the rhtA gene with the leucine dehydrogenase gene leuDH gene by means of gene editing means did not significantly inhibit the growth of the 19031-3 strain, but increased the yield of L-2-aminobutyric acid, resulting in an increase in the titer of L-2-aminobutyric acid from g/L to 15.11g/L, which indicated that overexpression of the gene leuDH was beneficial to E.coli L-2-aminobutyric acid synthesis.
Example 5: construction of an inducer-free L-2-aminobutyric acid production strain, knockout of the Gene lacI on the genome, construction of Strain 19031-4 and fermentation
(1) Construction of pTarget-DeltalacI plasmid: PCR amplification is carried out by taking pTarget F Plasmid (Addgene Plasmid # 62226) as template, taking pTarget-delta lacI F/pTarget-delta lacI R as primer, and digestion for 1-2h at 37 ℃ by Dpn I, and then converting intoE.coliIn DH5 alphation transgenic competent cells, spectinomycin plates were screened and sequencing verified to obtain the correct pTarget-DeltalacI plasmid for subsequent ligation of Donor DNA.
(2) Construction of pTD-DeltalacI plasmid: to be used forE.coliThe W3110 genome was used as a template, and DeltalacI P1, deltalacI P2, deltalacI P3 and DeltalacI P4 were used as primers, and the procedure of example 2 (2) was followed to obtain DeltalacI plasmid.
(3) The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain 19031-3 obtained in example 11, and the competence of strain 19031-3 was prepared in the same manner as in example 2 (3).
(4) The positive colony of the strain 19031-4 was constructed in the same manner as in example 2 (4).
(5) Plasmid elimination: the procedure was carried out in the same manner as in example 2 (5), to obtain strain 19031-4.
(6) The constructed strain 19031-4 producing strain was streaked from the glycerol tube to the LB plate, a single colony was picked up and inoculated into 10mL of LB medium, and shake flask test and detection were performed in accordance with the method of example 2 (6) using the strain 19031-3 constructed in example 4 as a control, but strain 19031-4 was not added with the inducer IPTG. The OD 600 of the fermentation results and the L-2-aminobutyric acid content in the supernatant of the fermentation broth are shown in FIG. 4.
As can be seen from FIG. 4, the lacI gene (SEQ ID No. 10) was knocked out on the genome of the strain 19031-3 by gene editing means, and the strain 19031-4 was constructed, and the fermentation result showed that the growth of the strain was slightly improved after the lacI gene was knocked out, and the yield of L-2-aminobutyric acid of 19031-4 was 10.86g/L, which indicates that the knocking out of the lacI gene had a certain effect on the production of the strain, but no inducer was added during the fermentation.
Sequence listing
<110> Zhejiang university of industry
<120> genetically engineered bacterium for high yield of L-2-aminobutyric acid, construction method and application
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> Unknown (Unknown)
<400> 1
ttgacaatta atcatcggct cgtataatg 29
<210> 2
<211> 73
<212> DNA
<213> Unknown (Unknown)
<400> 2
tgacaattaa tcatccggct cgtataatgt gtggaattgt gagcggataa caatttcaca 60
caggaaacag acc 73
<210> 3
<211> 888
<212> DNA
<213> Unknown (Unknown)
<400> 3
atgcctggtt cattacgtaa aatgccggtc tggttaccaa tagtcatatt gctcgttgcc 60
atggcgtcta ttcagggtgg agcctcgtta gctaagtcac tttttcctct ggtgggcgca 120
ccgggtgtca ctgcgctgcg tctggcatta ggaacgctga tcctcatcgc gttctttaag 180
ccatggcgac tgcgctttgc caaagagcaa cggttaccgc tgttgtttta cggcgtttcg 240
ctgggtggga tgaattatct tttttatctt tctattcaga cagtaccgct gggtattgcg 300
gtggcgctgg agttcaccgg accactggcg gtggcgctgt tctcttctcg tcgcccggta 360
gatttcgtct gggttgtgct ggcggttctt ggtctgtggt tcctgctacc gctggggcaa 420
gacgtttccc atgtcgattt aaccggctgt gcgctggcac tgggggccgg ggcttgttgg 480
gctatttaca ttttaagtgg gcaacgcgca ggagcggaac atggccctgc gacggtggca 540
attggttcgt tgattgcagc gttaattttc gtgccaattg gagcgcttca ggctggtgaa 600
gcactctggc actggtcggt tattccattg ggtctggctg tcgctattct ctcgaccgct 660
ctgccttatt cgctggaaat gattgccctc acccgtttgc caacacggac atttggtacg 720
ctgatgagca tggaaccggc gctggctgcc gtttccggga tgattttcct cggagaaaca 780
ctgacaccca tacagctact ggcgctcggc gctatcatcg ccgcttcaat ggggtctacg 840
ctgacagtac gcaaagagag caaaataaaa gaattagaca ttaattaa 888
<210> 4
<211> 1350
<212> DNA
<213> Unknown (Unknown)
<400> 4
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca cggcgagctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaatccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 5
<211> 2463
<212> DNA
<213> Unknown (Unknown)
<400> 5
atgcgagtgt tgaagttcgg cggtacatca gtggcaaatg cagaacgttt tctgcgtgtt 60
gccgatattc tggaaagcaa tgccaggcag gggcaggtgg ccaccgtcct ctctgccccc 120
gccaaaatca ccaaccacct ggtggcgatg attgaaaaaa ccattagcgg ccaggatgct 180
ttacccaata tcagcgatgc cgaacgtatt tttgccgaac ttttgacggg actcgccgcc 240
gcccagccgg ggttcccgct ggcgcaattg aaaactttcg tcgatcagga atttgcccaa 300
ataaaacatg tcctgcatgg cattagtttg ttggggcagt gcccggatag catcaacgct 360
gcgctgattt gccgtggcga gaaaatgtcg atcgccatta tggccggcgt attagaagcg 420
cgcggtcaca acgttactgt tatcgatccg gtcgaaaaac tgctggcagt ggggcattac 480
ctcgaatcta ccgtcgatat tgctgagtcc acccgccgta ttgcggcaag ccgcattccg 540
gctgatcaca tggtgctgat ggcaggtttc accgccggta atgaaaaagg cgaactggtg 600
gtgcttggac gcaacggttc cgactactct gctgcggtgc tggctgcctg tttacgcgcc 660
gattgttgcg agatttggac ggacgttgac ggggtctata cctgcgaccc gcgtcaggtg 720
cccgatgcga ggttgttgaa gtcgatgtcc taccaggaag cgatggagct ttcctacttc 780
ggcgctaaag ttcttcaccc ccgcaccatt acccccatcg cccagttcca gatcccttgc 840
ctgattaaaa ataccggaaa tcctcaagca ccaggtacgc tcattggtgc cagccgtgat 900
gaagacgaat taccggtcaa gggcatttcc aatctgaata acatggcaat gttcagcgtt 960
tctggtccgg ggatgaaagg gatggtcggc atggcggcgc gcgtctttgc agcgatgtca 1020
cgcgcccgta ttttcgtggt gctgattacg caatcatctt ccgaatacag catcagtttc 1080
tgcgttccac aaagcgactg tgtgcgagct gaacgggcaa tgcaggaaga gttctacctg 1140
gaactgaaag aaggcttact ggagccgctg gcagtgacgg aacggctggc cattatctcg 1200
gtggtaggtg atggtatgcg caccttgcgt gggatctcgg cgaaattctt tgccgcactg 1260
gcccgcgcca atatcaacat tgtcgccatt gctcagggat cttctgaacg ctcaatctct 1320
gtcgtggtaa ataacgatga tgcgaccact ggcgtgcgcg ttactcatca gatgctgttc 1380
aataccgatc aggttatcga agtgtttgtg attggcgtcg gtggcgttgg cggtgcgctg 1440
ctggagcaac tgaagcgtca gcaaagctgg ctgaagaata aacatatcga cttacgtgtc 1500
tgcggtgttg ccaactcgaa ggctctgctc accaatgtac atggccttaa tctggaaaac 1560
tggcaggaag aactggcgca agccaaagag ccgtttaatc tcgggcgctt aattcgcctc 1620
gtgaaagaat atcatctgct gaacccggtc attgttgact gcacttccag ccaggcagtg 1680
gcggatcaat atgccgactt cctgcgcgaa ggtttccacg ttgtcacgcc gaacaaaaag 1740
gccaacacct cgtcgatgga ttactaccat cagttgcgtt atgcggcgga aaaatcgcgg 1800
cgtaaattcc tctatgacac caacgttggg gctggattac cggttattga gaacctgcaa 1860
aatctgctca atgcaggtga tgaattgatg aagttctccg gcattctttc tggttcgctt 1920
tcttatatct tcggcaagtt agacgaaggc atgagtttct ccgaggcgac cacgctggcg 1980
cgggaaatgg gttataccga accggacccg cgagatgatc tttctggtat ggatgtggcg 2040
cgtaaactat tgattctcgc tcgtgaaacg ggacgtgaac tggagctggc ggatattgaa 2100
attgaacctg tgctgcccgc agagtttaac gccgagggtg atgttgccgc ttttatggcg 2160
aatctgtcac aactcgacga tctctttgcc gcgcgcgtgg cgaaggcccg tgatgaagga 2220
aaagttttgc gctatgttgg caatattgat gaagatggcg tctgccgcgt gaagattgcc 2280
gaagtggatg gtaatgatcc gctgttcaaa gtgaaaaatg gcgaaaacgc cctggccttc 2340
tatagccact attatcagcc gctgccgttg gtactgcgcg gatatggtgc gggcaatgac 2400
gttacagctg ccggtgtctt tgctgatctg ctacgtaccc tctcatggaa gttaggagtc 2460
tga 2463
<210> 6
<211> 2219
<212> DNA
<213> Unknown (Unknown)
<400> 6
atggagatgt tgtctggagc cgagatggtc gtccgatcgc ttatcgatca gggcgttaaa 60
caagtattcg gttatcccgg aggcgcagtc cttgatattt atgatgcatt gcataccgtg 120
ggtggtattg atcatgtatt agttcgtcat gagcaggcgg cggtgcatat ggccgatggc 180
ctggcgcgcg cgaccgggga agtcggcgtc gtgctggtaa cgtcgggtcc aggggcgacc 240
aatgcgatta ctggcatcgc caccgcttat atggattcca ttccattagt tgtcctttcc 300
gggcaggtag cgacctcgtt gataggttac gatgcctttc aggagtgcga catggtgggg 360
atttcgcgac cggtggttaa acacagtttt ctggttaagc aaacggaaga cattccgcag 420
gtgctgaaaa aggctttctg gctggcggca agtggtcgcc caggaccagt agtcgttgat 480
ttaccgaaag atattcttaa tccggcgaac aaattaccct atgtctggcc ggagtcggtc 540
agtatgcgtt cttacaatcc cactactacc ggacataaag ggcaaattaa gcgtgctctg 600
caaacgctgg tagcggcaaa aaaaccggtt gtctacgtag gcggtggggc aatcacggcg 660
ggctgccatc agcagttgaa agaaacggtg gaggcgttga atctgcccgt tgtttgctca 720
ttgatggggc tgggggcgtt tccggcaacg catcgtcagg cactgggcat gctgggaatg 780
cacggtacct acgaagccaa tatgacgatg cataacgcgg atgtgatttt cgccgtcggg 840
gtacgatttg atgaccgaac gacgaacaat ctggcaaagt actgcccaaa tgccactgtt 900
ctgcatatcg atattgatcc tacttccatt tctaaaaccg tgactgcgga tatcccgatt 960
gtgggggatg ctcgccaggt cctcgaacaa atgcttgaac tcttgtcgca agaatccgcc 1020
catcaaccac tggatgagat ccgcgactgg tggcagcaaa ttgaacagtg gcgcgctcgt 1080
cagtgcctga aatatgacac tcacagtgaa aagattaaac cgcaggcggt gatcgagact 1140
ctttggcggt tgacgaaggg agacgcttac gtgacgtccg atgtcgggca gcaccagatg 1200
tttgctgcac tttattatcc attcgacaaa ccgcgtcgct ggatcaattc cggtggcctc 1260
ggcacgatgg gttttggttt acctgcggca ctgggcgtca aaatggcgtt gccagaagaa 1320
accgtggttt gcgtcactgg cgacggcagt attcagatga acatccagga actgtctacc 1380
gcgttgcaat acgagttgcc cgtactggtg gtgaatctca ataaccgcta tctggggatg 1440
gtgaagcagt ggcaggacat gatctattcc ggccgtcatt cacaatctta tatgcaatcg 1500
ctacccgatt tcgtccgtct ggcggaagcc tatgggcatg tcgggatcca gatttctcat 1560
ccgcatgagc tggaaagcaa acttagcgag gcgctggaac aggtgcgcaa taatcgcctg 1620
gtgtttgttg atgttaccgt cgatggcagc gagcacgtct acccgatgca gattcgcggg 1680
ggcggaatgg atgaaatgtg gttaagcaaa acggagagaa cctgattatg cgccggatat 1740
tatcagtctt actcgaaaat gaatcaggcg cgttatcccg cgtgattggc cttttttccc 1800
agcgtggcta caacattgaa agcctgaccg ttgcgccaac cgacgatccg acattatcgc 1860
gtatgaccat ccagaccgtg ggcgatgaaa aagtacttga gcagatcgaa aagcaattac 1920
acaaactggt cgatgtcttg cgcgtgagtg agttggggca gggcgcgcat gttgagcggg 1980
aaatcatgct ggtgaaaatt caggccagcg gttacgggcg tgacgaagtg aaacgtaata 2040
cggaaatatt ccgtgggcaa attatcgatg tcacaccctc gctttatacc gttcaattag 2100
caggcaccag cggtaagctt gatgcatttt tagcatcgat tcgcgatgtg gcgaaaattg 2160
tggaggttgc tcgctctggt gtggtcggac tttcgcgcgg cgataaaata atgcgttga 2219
<210> 7
<211> 1104
<212> DNA
<213> unknown (T. Inter media)
<400> 7
atgggtaaaa tcttcgacta catggaaaaa tacgactacg aacaactggt gatgtgccag 60
gacaaagaat ccggtctgaa ggctatcatc tgcatccacg tgactaccct gggtccggct 120
ctgggtggta tgcgtatgtg gacgtacgct tccgaagaag aggcgattga agatgctctg 180
cgcctgggtc gtggcatgac ctataaaaac gcggctgcag gtctgaacct gggtggcggc 240
aaaaccgtaa ttattggtga cccgcgtaaa gacaaaaacg aggcgatgtt tcgtgctctg 300
ggtcgcttca tccagggtct gaacggtcgt tacattaccg ccgaggatgt tggtaccact 360
gtcgaagaca tggacatcat ccacgaagaa acccgttacg taaccggcgt ttccccagcc 420
tttggctctt ctggcaaccc gagcccggtg actgcctacg gcgtttatcg tggtatgaaa 480
gcagcagcga aagaagcatt cggcgatgac tctctggaag gtaaagtcgt tgctgttcaa 540
ggtgtgggcc acgtcgcgta tgaactgtgc aaacatctgc acaacggcgg tgcgaaactg 600
atcgtaacgg acatcaacaa agaaaacgcg gatcgtgcgg tgcaagaatt tggtgcggag 660
ttcgtgcacc cggataaaat ctatgatgtt gaatgcgaca tcttcgcccc gtgcgcactg 720
ggtgctatca tcaacgacga aactatcgaa cgcctgaaat gtaaagtagt cgctggctcc 780
gcgaacaacc agctgaaaga agaacgtcat ggcaaaatgc tggaggaaaa aggtattgta 840
tatgcccctg attacgttat caacgctggt ggcgtgatca acgtagcgga cgagctgctg 900
ggctataacc gcgaacgcgc gatgaagaag gttgaaggca tctacgacaa aattctgaaa 960
gttttcgaaa tcgcaaaacg cgatggtatc ccgagctacc tggctgccga ccgcatggcc 1020
gaagaacgta tcgaaatgat gcgtaagacc cgcagcacct ttctgcagga ccaacgtaac 1080
ctgatcaact tcaacaacaa ataa 1104
<210> 8
<211> 1545
<212> DNA
<213> Unknown (Unknown)
<400> 8
atggctgact cgcaacccct gtccggtgct ccggaaggtg ccgaatattt aagagcagtg 60
ctgcgcgcgc cggtttacga ggcggcgcag gttacgccgc tacaaaaaat ggaaaaactg 120
tcgtcgcgtc ttgataacgt cattctggtg aagcgcgaag atcgccagcc agtgcacagc 180
tttaagctgc gcggcgcata cgccatgatg gcgggcctga cggaagaaca gaaagcgcac 240
ggcgtgatca ctgcttctgc gggtaaccac gcgcagggcg tcgcgttttc ttctgcgcgg 300
ttaggcgtga aggccctgat cgttatgcca accgccaccg ccgacatcaa agtcgacgcg 360
gtgcgcggct tcggcggcga agtgctgctc cacggcgcga actttgatga agcgaaagcc 420
aaagcgatcg aactgtcaca gcagcagggg ttcacctggg tgccgccgtt cgaccatccg 480
atggtgattg ccgggcaagg cacgctggcg ctggaactgc tccagcagga cgcccatctc 540
gaccgcgtat ttgtgccagt cggcggcggc ggtctggctg ctggcgtggc ggtgctgatc 600
aaacaactga tgccgcaaat caaagtgatc gccgtagaag cggaagactc cgcctgcctg 660
aaagcagcgc tggatgcggg tcatccggtt gatctgccgc gcgtagggct atttgctgaa 720
ggcgtagcgg taaaacgcat cggtgacgaa accttccgtt tatgccagga gtatctcgac 780
gacatcatca ccgtcgatag cgatgcgatc tgtgcggcga tgaaggattt attcgaagat 840
gtgcgcgcgg tggcggaacc ctctggcgcg ctggcgctgg cgggaatgaa aaaatatatc 900
gccctgcaca acattcgcgg cgaacggctg gcgcatattc tttccggtgc caacgtgaac 960
ttccacggcc tgcgctacgt ctcagaacgc tgcgaactgg gcgaacagcg tgaagcgttg 1020
ttggcggtga ccattccgga agaaaaaggc agcgccctca aattctgcca actgcttggc 1080
gggttctcgg tcaccgagtt caactaccgt tttgccgatg ccaaaaacgc ctgcatcttt 1140
gtcggtgtgc gcctgagccg cggcctcgaa gagcgcaaag aaattttgca gatgctcaac 1200
gacggcggct acagcgtggt tgatctctcc gacgacgaaa tggcgaagct acacgtgcgc 1260
tatatggtcg gcggacgtcc atcgcatccg ttgcaggaac gcctctacag cttcgaattc 1320
ccggaatcac cgggcgcgct gctgcgcttc ctcaacacgc tgggtacgta ctggaacatt 1380
tctttgttcc actatcgcag ccatggcacc gactacgggc gcgtactggc ggcgttcgaa 1440
cttggcgacc atgaaccgga tttcgaaacc cggctgaatg agctgggcta cgattgccac 1500
gacgaaacca ataacccggc gttcaggttc tttttggcgg gttag 1545
<210> 9
<211> 1434
<212> DNA
<213> Unknown (Unknown)
<400> 9
atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcgctgat gctgccggta 60
tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120
cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180
ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240
gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300
ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360
gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420
cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480
gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540
accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600
atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660
cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720
atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780
ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840
ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900
gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960
ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020
ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080
ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140
ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200
atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260
tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320
aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380
actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434
<210> 10
<211> 1083
<212> DNA
<213> Unknown (Unknown)
<400> 10
gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 60
tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 120
gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 180
tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 240
gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 300
cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 360
gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 420
actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 480
ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 540
caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 600
tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 660
agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 720
gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 780
gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 840
tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 900
gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 960
gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 1020
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 1080
tga 1083
Claims (5)
1. The genetically engineered bacterium for producing the L-2-aminobutyric acid at high yield is constructed and obtained by the following method:
(1) To be used forEscherichia.coliW3110 as starting strain, strainEscherichia.coliThe thrA gene in the W3110 genome was replaced with thrA mutant gene (C1034T, S345F) to give a recombinant strainEscherichia.coliW3110/thrA * The thrA mutant gene is marked as THR1, and the sequence of the thrA mutant gene is shown as SEQ ID No. 5;
(2) The lysC gene in the genome of the strain THR1 was subjected to mutation with lysC gene (C1055T, T342I) to give a recombinant strainEscherichia.coli W3110/ thrA * /lysC * The lysC mutant gene is marked as THR2, and the sequence of the lysC mutant gene is shown as SEQ ID No. 4;
(3) Replacing thrABC gene promoter in THR2 genome of strain with Tac promoter to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * The Tac-thrABC is marked as THR3, and the nucleotide sequence of the Tac promoter is shown as SEQ ID NO. 1;
(4) Knocking out lysA gene in THR3 genome of strain to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA, designated THR4;
(5) Knocking out metA gene in THR4 genome of strain to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.meta, designated THR5;
(6) Knocking out tdh gene in strain DPA5 genome to obtain recombinant strainEscherichia.coli W3110/ thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh, designated THR6;
(7) Knocking out ilcR gene in strain DPA6 genome to obtain recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR, designated THR7;
(8) Replacing the ppc gene promoter in the THR7 genome of the strain with Trc promoter to obtain a recombinant strainEscherichia.coli W3110/thrA * /lysC * Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc, designated THR8, i.eE.coli19031, the nucleotide sequence of the Trc promoter is shown in SEQ ID NO. 2;
(9) To be used forE.coli19031 is Chaetomium, and ilvIH gene in genome of strain 19031 is derived from CRISPR-Cas9 gene editing technologyT. intermediusThe leucine dehydrogenase leuDH gene of the strain is replaced to obtain a recombinant strain 19031ilvIH, wherein the recombinant strain is marked as 19031-1, and the leuDH gene sequence is shown as SEQ ID No. 7;
(10) By using CRISPR-Cas9 gene editing technology, the ptsG gene in the strain 19031ilvIH:: leuDH genome is treated with ilvA which releases feedback inhibition by L-isoleucine * Alternatively, a recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA was obtained * Designated 19031-2, the ilvA * The gene sequence is shown as SEQ ID No. 8;
(11) Applying CRISPR-Cas9 gene editing technology to strain19031ilvIH::leuDH /ptsG:: ilvA * The rhtA gene in the genome is derived fromT. intermediusThe leucine dehydrogenase leuDH of (E) was replaced to give recombinant strain 19031ilvIH:: leuDH/ptsG::: ilvA * rhtA: leuDH, designated 19031-3;
(12) Strain 19031 ilvIH::: leuDH/ptsG::: ilvA was obtained by using CRISPR-Cas9 gene editing technique * The lacI gene in the leuDH genome is knocked out to obtain recombinant strain 19031ilvIH:: leuDH/ptsG:: ilvA * And rhtA is leuDH/[ delta ] lacI, which is 19031-4, namely the genetically engineered bacterium for high-yield L-2-aminobutyric acid.
2. The use of the genetically engineered bacterium of claim 1 in microbial fermentation to prepare L-2-aminobutyric acid.
3. The application according to claim 2, characterized in that it is: inoculating the genetically engineered strain into a fermentation culture medium, and fermenting and culturing at 30-37 ℃ and 150-300 rpm until OD 600 When the fermentation liquid is 10-30, adding IPTG with the final concentration of 0.1mM, continuously culturing for 48 hours, and taking the supernatant of the fermentation liquid after the fermentation is finished, and separating and purifying to obtain the L-2-aminobutyric acid.
4. Use according to claim 3, characterized in that the fermentation medium consists of: 30-50 g/L of glucose, 5-8 g/L of yeast powder, 1-3 g/L of magnesium sulfate, 3-4 g/L of dipotassium hydrogen phosphate, 10-20 g/L of ammonium sulfate, 0.5-1.5 g/L of betaine hydrochloride, 0.05-0.5 g/L of L-methionine, 0.05-0.5 g/L of L-lysine, 1-5 mL/L of metal salt ion solution, 20-30 g/L of calcium carbonate, water as a solvent and natural pH value; the metal salt ion solution comprises the following components: feSO 4 ·7H 2 O 10g/L，CaCl 2 1.35g/L，ZnSO 4 ·7H 2 O 2.25g/L，MnSO 4 ·4H 2 O 0.5g/L，CuSO 4 ·5H 2 O 1g/L，（NH 4 ）6Mo 7 O 24 ·4H 2 O 0.106g/L，Na 2 B 4 O 7 ·10H 2 O0.23 g/L,35% HCl 10mL/L, and water as solvent.
5. The use according to claim 4, characterized in that the fermentation medium consists of: 50g/L of glucose, 6g/L of yeast powder, 2g/L of magnesium sulfate, 4g/L of dipotassium hydrogen phosphate, 14g/L of ammonium sulfate, 1g/L of betaine hydrochloride, 0.149 g/L of L-methionine, 0.164 g/L of L-lysine, 5mL of metal salt ion solution, caCO 3 30g/L, and the solvent is water.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215291A (en) * | 2012-01-18 | 2013-07-24 | 中国科学院上海生命科学研究院 | Vector, engineering strain and method for producing L(+)-2-aminobutyric acid |
| CN106148259A (en) * | 2015-04-28 | 2016-11-23 | 中国科学院微生物研究所 | Produce recombinant bacterium of C4H9NO2 and preparation method and application |
| CN106497895A (en) * | 2016-09-30 | 2017-03-15 | 浙江工业大学 | Leucine dehydrogenase mutant, encoding gene, carrier, engineering bacteria and its application |
| CN109679978A (en) * | 2018-12-12 | 2019-04-26 | 浙江工业大学 | A kind of recombinant co-expression system and its application being used to prepare C4H9NO2 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215291A (en) * | 2012-01-18 | 2013-07-24 | 中国科学院上海生命科学研究院 | Vector, engineering strain and method for producing L(+)-2-aminobutyric acid |
| CN106148259A (en) * | 2015-04-28 | 2016-11-23 | 中国科学院微生物研究所 | Produce recombinant bacterium of C4H9NO2 and preparation method and application |
| CN106497895A (en) * | 2016-09-30 | 2017-03-15 | 浙江工业大学 | Leucine dehydrogenase mutant, encoding gene, carrier, engineering bacteria and its application |
| CN109679978A (en) * | 2018-12-12 | 2019-04-26 | 浙江工业大学 | A kind of recombinant co-expression system and its application being used to prepare C4H9NO2 |
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