CN116103215A

CN116103215A - Plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid and application thereof

Info

Publication number: CN116103215A
Application number: CN202310210617.5A
Authority: CN
Inventors: 柳志强; 李雪峰; 周俊平; 徐建妙; 张博; 黄良刚; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-05-12

Abstract

The invention relates to the technical field of genetic engineering, and discloses plasmid-free genetic engineering bacteria for high-yield L-2-aminobutyric acid and application thereof. The plasmid-free genetically engineered bacterium for producing L-2-aminobutyric acid is obtained by taking Escherichia coli THR as a chassis cell and modifying the genetically engineered bacterium by metabolic engineering and genetic editing technology, the genetically engineered bacterium strain can be used for obtaining the L-2-aminobutyric acid by fermentation culture by taking glucose as a raw material, under the condition of no plasmid increasing exogenous enzyme activity, the fed-batch fermentation yield of a 5L fermentation tank reaches 13.81g/L, the fermentation cost is low, the product concentration is high, no byproduct influence exists, the product is easy to purify, and the genetically engineered bacterium strain is suitable for industrial application.

Description

Plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to plasmid-free genetic engineering bacteria for high-yield L-2-aminobutyric acid and application thereof.

Background

L-2-aminobutyric acid (L-2-Aminobutyric acid, L-ABA for short) is an unnatural chiral alpha-amino acid, and can be used as a key chiral intermediate for synthesizing various chiral medicines, such as ethambutol which is a tuberculosis treatment medicine, and levetiracetam and brivaracetam which are antiepileptics medicines.

Molecular formula C of L-2-aminobutyric acid ₄ H ₉ NO ₂ The relative molecular weight is 103.12, the melting point is 270-280 ℃, 22.7g of the white flaky crystal can be dissolved in 100g of water at 25 ℃, the white flaky crystal is slightly dissolved in ethanol, diethyl ether and the like, the isoelectric point is 6.05 (25 ℃), and the white flaky crystal is odorless and slightly sweet. The structural formula of the L-2-aminobutyric acid is shown as the following formula:

at present, the L-2-aminobutyric acid is mainly synthesized industrially by a chemical method, but the method has the defects of harsh reaction conditions, unfriendly environment and the like.

In order to avoid the problem of the chemical synthesis of L-2-aminobutyric acid, various biosynthesis pathways for L-2-aminobutyric acid have been developed in recent years, and the synthesis is mainly carried out by an enzyme-catalyzed method such as dehydrogenase or transaminase. The keto acid and the glutamic acid are used as substrates, and L-2-aminobutyric acid is generated under the action of transaminase, but the yield of the method is low; l-threonine is used as raw material, and three-enzyme system one-pot method containing amino acid dehydrogenase is used to prepare L-2-aminobutyric acid, and although coenzyme circulation system is added, proper amount of coenzyme such as nicotinamide adenine dinucleotide and NAD is needed ⁺ Or NADH, etc., which limits the application thereof in industrial level due to the high price of the coenzyme and has byproducts to influence the purification of the product; the amino acid oxidase method is to split a racemized substrate by using D-amino acid oxidase under the action of a metal catalyst to prepare L-2-aminobutyric acid, and has high cost and is not suitable for large-scale industrial application. In addition, the aminoacylase method and the like have problems of excessively high production cost and inhibition of enzyme activity by a substrate, and therefore, the industrial application effect is not good.

Therefore, the research team aims to develop a novel biosynthesis method for synthesizing L-2-aminobutyric acid so as to avoid the problems of harsh chemical synthesis reaction conditions and the like, and simultaneously avoid the problems of high cost and low yield of the existing biosynthesis method.

Disclosure of Invention

In order to solve the technical problems, the invention provides a plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid and application thereof. The invention aims to reform escherichia coli by metabolic engineering and gene editing technology to obtain recombinant escherichia coli strain with high yield of L-2-aminobutyric acid, and particularly aims to provide plasmid-free genetically engineered bacterium with high yield of L-2-aminobutyric acid by taking Escherichia coli THR constructed by the research team as a chassis cell and a construction method thereof, and application of the genetically engineered bacterium in microbial fermentation preparation of L-2-aminobutyric acid.

The specific technical scheme of the invention is as follows:

on one hand, the invention provides a plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid, which is constructed by the following method: (1) The E.coli THR is taken as chassis fungus, ptsG genes in the genome of the strain E.coli THR are replaced by ilvA genes and leuDH genes, and a recombinant strain E.coli THR delta ptsG is obtained, wherein ilvA-leuDH is marked as ABA-1;

(2) Inoculating ilvA-leuDH to 2-ketobutyrate liquid medium for subculture, screening strains capable of growing normally in the liquid medium with the concentration of 2-ketobutyrate not lower than 50g/L, and obtaining strain E.coli THR delta ptsG:: ilvA-leuDHMut, which is marked as ABA-2;

(3) The adjacent ltaE gene and poxB gene in the genome of the strain E.coli THR delta ptsG:: ilvA-leuDHMut are replaced by leuDH genes to obtain a recombinant strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH, which is marked as ABA-3;

(4) Knocking out the focA gene and pflB gene in the bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH genome to obtain a recombinant bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB, and marking as ABA-4;

(5) The adjacent pta genes and ackA genes in the bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB genome are replaced by ilvA-genes and leuDH genes to obtain a recombinant bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB delta ptaackA::: ilvA-leuDH, and the recombinant bacterial strain E.coli THR delta ptsG:: leuDH is marked as ABA-5;

(6) The mgsA gene in the genome of the strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB delta pta delta ackA:: ilvA-leuDH is replaced by ilvA-gene and leuDH gene, and the recombinant strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH delta focA delta pflA-leuDH is marked as ABA-6, namely the plasmid for high-yield L-2-aminobutyric acid is obtained.

Coli Escherichia coli THR is described in Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering (Microb Cell face.2019, 18 (1): 43). The plasmid-free recombinant escherichia coli for producing the L-2-aminobutyric acid provided by the invention takes Escherichia coli THR as a chassis cell, is constructed by the steps (1) - (6), and has the advantage of high yield when the plasmid-free recombinant escherichia coli is used for producing the L-2-aminobutyric acid. Specifically:

editing related genes on a path of synthesizing L-2-aminobutyric acid by using escherichia coli, replacing ptsG genes in a glucose Phosphatase Transfer System (PTS) by ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.inter media by using a CRISPR-Cas9 gene editing technology, so that the escherichia coli can produce the L-2-aminobutyric acid without plasmid fermentation on one hand, and on the other hand, the consumption of phosphoenolpyruvic acid can be reduced by deleting the ptsG genes.

And (2) respectively transferring the strains into LB culture media containing different 2-ketobutyrate concentrations by utilizing a laboratory self-adaptive evolution technology, and subculturing until the strains can normally grow under the 2-ketobutyrate concentration of 50g/L, so as to obtain the adaptive strains with high 2-ketobutyrate concentration.

And (3) replacing genes ltaE and poxB related to byproducts by using a CRISPR-Cas9 gene editing technology with leucine dehydrogenase leuDH genes derived from T.inter media, so that threonine degradation and acetic acid accumulation are reduced, and more metabolism flows to produce L-2-aminobutyric acid.

And (4) deleting related genes focA and pflB of the formate pathway by using a CRISPR-Cas9 gene editing technology, reducing byproduct accumulation, prolonging fermentation period and improving fermentation production of L-2-aminobutyric acid.

And (5) replacing related genes pta and ackA of an acetic acid pathway by ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.inter-medium by using a CRISPR-Cas9 gene editing technology, reducing acetic acid accumulation, prolonging continuous fermentation time, improving catabolism of threonine by the ilvA genes, increasing generation of alpha-ketobutyrate, and producing L-2-aminobutyric acid by the leuDH genes.

And (6) replacing the gene mgsA encoding the methylglyoxal synthase by ilvA gene for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH gene from T.inter media by using CRISPR-Cas9 gene editing technology, and finally constructing a high-yield L-2-aminobutyric acid fermentation production recombinant escherichia coli strain ABA-6.

According to the plasmid-free recombinant escherichia coli with high yield of L-2-aminobutyric acid, escherichia coli THR is taken as a chassis cell, related genes on a path of synthesizing the L-2-aminobutyric acid by the escherichia coli are edited, ptsG genes in a glucose Phosphatase Transfer System (PTS) are replaced by ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.inter media, so that the escherichia coli can produce the L-2-aminobutyric acid without plasmid fermentation on one hand, and consumption of phosphoenolpyruvic acid can be reduced due to deletion of the ptsG genes on the other hand; performing laboratory self-adaptive evolution by taking high-concentration-tolerant 2-ketobutyrate as a screening to obtain a high-toxicity-tolerant metabolite 2-ketobutyrate strain, so that the growth of the strain is not inhibited by the 2-ketobutyrate; the genes ltaE and poxB related to byproducts are replaced by leucine dehydrogenase leuDH gene derived from T.intermedium, so that threonine degradation and acetic acid accumulation are reduced, and more metabolism flows to produce L-2-aminobutyric acid; related genes focA and pflB of the formic acid pathway are deleted, so that byproduct accumulation is reduced, the fermentation period is prolonged, and the fermentation production of L-2-aminobutyric acid is improved; the related genes pta and ackA of the acetic acid pathway are replaced by ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.inter media, so that acetic acid accumulation is reduced, the continuous fermentation time is prolonged, the ilvA genes improve threonine catabolism, the generation of 2-ketobutyrate is increased, and L-2-aminobutyric acid is produced through the leuDH genes; the gene mgsA for coding the methylglyoxal synthase is replaced by ilvA gene for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH gene from T.inter medium, and a recombinant escherichia coli strain with higher yield of L-2-aminobutyric acid is finally constructed, and can be obtained by fermenting and culturing glucose as a raw material, wherein the fed-batch fermentation yield of a 5L fermentation tank reaches 13.81g/L under the condition of no plasmid increasing exogenous enzyme activity.

Specifically, as a preferable mode of the technical scheme, the ilvA gene sequence is shown as SEQ ID No. 1; the leuDH gene is a leucine dehydrogenase encoding gene, and the gene sequence of the leuDH gene is shown as SEQ ID No. 2; the ptsG gene sequence is shown in SEQ ID No. 3; the ltaE gene sequence is shown as SEQ ID No.4, and the poxB gene sequence is shown as SEQ ID No. 5; the sequence of the focA gene is shown as SEQ ID No.6, and the sequence of the pflB gene is shown as SEQ ID No. 7; the pta gene sequence is shown as SEQ ID No. 8; the ackA gene sequence is shown as SEQ ID No. 9; the mgsA gene sequence is shown as SEQ ID No. 10.

On the other hand, the invention provides a construction method of the plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid, which comprises the following steps:

(1) Using E.coli THR as chassis bacteria, applying CRISPR-Cas9 gene editing technology, replacing ptsG genes in a bacterial strain E.coli THR genome with ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.inter media to obtain recombinant bacterial strain E.coli THR delta ptsG, wherein ilvA-leuDH is marked as ABA-1;

(2) E.coli THR delta ptsG:: ilvA-leuDHMut is transferred to LB culture media containing different 2-ketobutyrate concentrations respectively for subculture, and the strain can normally grow under the 2-ketobutyrate concentration of 50g/L to obtain a strain E.coli THR delta ptsG:: ilvA-leuDHMut which tolerates high concentration of 2-ketobutyrate, and is marked as ABA-2;

(3) The CRISPR-Cas9 gene editing technology is applied, the ltaE gene and the poxB gene in the genome of the strain E.coli THR delta ptsG:: ilvA x-leuDHMut are replaced by leucine dehydrogenase leuDH gene from T.intermedia, and the recombinant strain E.coli THR delta ptsG::: ilvA x-leuDHMut delta ltaE delta poxB::: leuDH is marked as ABA-3;

(4) Knocking out the focA gene and pflB gene in the bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH genome to obtain a recombinant bacterial strain E.coli THR delta ptsG:::: ilvA-leuDHMut delta ltaE delta poxB::: leuDH delta focA delta pflB, which is marked as ABA-4;

(5) By using CRISPR-Cas9 gene editing technology, replacing pta genes and ackA genes in a bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta pflA delta pacdA: vA-leuDH is obtained by using ilvA genes for relieving feedback inhibition of L-isoleucine and leucine dehydrogenase leuDH genes from T.intemedia, and obtaining a recombinant bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH delta pflB delta pflA: ABA-leuDH;

(6) By using CRISPR-Cas9 gene editing technology, the mgsA gene in the genome of the strain E.coli THR delta ptsG: ilvA-leuDHMut delta ltaE delta poxB: leuDH delta paca: ilvA-leuDH is replaced by the ilvA gene for relieving feedback inhibition of L-isoleucine and the leucine dehydrogenase leuDH gene from T.intemedia, and the recombinant strain E.coli THR delta ptsG: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta paca: ilvA-leuDH delta mgA: ilvA-leuDH is obtained, namely the strain is the strain with high yield of the amino acid-producing strain E.coli 2.

Meanwhile, the invention also provides application of the plasmid-free genetically engineered bacterium in preparing L-2-aminobutyric acid by microbial fermentation and a method thereof.

Specifically, the application method comprises the following steps: inoculating the plasmid-free recombinant escherichia coli into a fermentation culture medium containing IPTG, fermenting and culturing for more than 48 hours at the temperature of 30-35 ℃ and the rpm of 100-200 rpm, and separating and purifying a fermentation liquor supernatant after fermentation to obtain the L-2-aminobutyric acid.

Preferably, the fermentation medium is composed as follows: IPTG 0.1mM, 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, deionized water as solvent, and pH value is natural; the metal salt ion solution comprises the following components: feSO ₄ ·7H ₂ O10±1.5g/L，CaCl ₂ 1.35±0.6g/L，ZnSO ₄ ·7H ₂ O 2.25±0.8g/L，MnSO ₄ ·4H ₂ O 0.5±0.1g/L，CuSO ₄ ·5H ₂ O 1±0.5g/L，(NH ₄ )6Mo ₇ O ₂₄ ·4H ₂ O 0.106±0.05g/L，Na ₂ B ₄ O ₇ ·10H ₂ O0.23+/-0.08 g/L,35% HCl 10+/-1 mL/L, and water as solvent.

Preferably, before the plasmid-free genetically engineered bacteria are fermented, the plasmid-free genetically engineered bacteria are inoculated into LB culture medium, cultured overnight on a shaking table at 37 ℃ and a rotating speed of 200rpm, and then inoculated into fermentation culture medium for culture at an inoculum size of 3-5% by volume.

Compared with the prior art, the invention has the following technical effects:

the plasmid-free genetically engineered bacterium for producing L-2-aminobutyric acid provided by the invention can take glucose as a raw material, and under the condition that exogenous enzyme activity is not increased by plasmid, the fermentation yield of a 5L fermentation tank reaches 13.81g/L. The fermentation cost is low, the concentration of the product is high, no influence of byproducts is caused, and the product is easy to purify and is very suitable for industrial application.

Drawings

FIG. 1 is an OD of example 1 of the present invention ₆₀₀ A fermentation result diagram of the L-2-aminobutyric acid content;

FIG. 2 is an OD of example 3 of the present invention ₆₀₀ A fermentation result diagram of the L-2-aminobutyric acid content;

FIG. 3 is an OD of example 4 of the present invention ₆₀₀ A fermentation result diagram of the L-2-aminobutyric acid content;

FIG. 4 is an OD of example 5 of the present invention ₆₀₀ A fermentation result diagram of the L-2-aminobutyric acid content;

FIG. 5 is an OD of example 6 of the present invention ₆₀₀ And (3) a graph of fermentation results of L-2-aminobutyric acid content.

Detailed Description

The invention is further described below with reference to examples.

In the following 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.

Strain e.coli W3110 was from the university of jerusalem CGSC collection (Coli Genetic Stock Center), 8 th month 5 th day of the collection date 1975, deposit number cgsc#4474, which is disclosed in patent US 2009/0298135 A1,US2010/0248311 A1.

The parent strain E.coli THR was from laboratory preservation as an E.coli W3110 derivative, the genotype of which is described below (E.coli W3110/thrA/lysC/Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc).

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

The primer sequence information used in the following examples is shown in Table 1.

TABLE 1

/>

Wherein X release-sgRNA-F/R is a mutation primer of pTatget plasmid, and X is a 20bp sequence before a PAM locus (NGG) contained in a target gene carrying genome; x D-Arm1-F/R is an upstream and downstream primer of an upstream homology Arm of a target gene; x D-Arm2-F/R is a downstream homologous Arm upstream and downstream primer of the target gene; x release-VF/VR is a verification primer for editing target genes.

The detection method of L-2-aminobutyric acid and L-threonine in the following examples is:

taking 1mL of fermentation broth, centrifuging at 12000rpm for 2min in an EP tube, taking supernatant, and sterilizing with ddH ₂ O is diluted to proper multiple, derivatization is carried out for 60min at 60 ℃, phosphate buffer solution is added to fix the volume to 1mL, and after the membrane is covered by a 0.22 mu m organic filter membrane, the membrane is detected by High Performance Liquid Chromatography (HPLC).

The detection method comprises the following steps: the chromatographic column is C18 column (150×4.6mm), the column temperature is 33 ℃, the flow rate is 1mL/min, gradient elution is adopted, the sample injection amount is 10 μL, the chromatographic retention time is 30min, and the detection wavelength is 360nm. The elution procedure is shown in Table 2.

TABLE 2

Sequence 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

Wherein the flow matching method comprises the following steps: mobile phase A, 50% acetonitrile; mobile phase B4.1 g anhydrous sodium acetate was weighed into 800ML ddH ₂ In O, the pH was adjusted to 6.4 with acetic acid and then the volume was set to 1L.

Example 1 construction of strain ABA-1 with the ptsG gene replaced by ilvA and leuDH gene the fermented threonine deaminase gene ilvA was from e.coli W3110 (NCBI accession number: AP 009048); leucine dehydrogenase gene leuDH is derived from Thermoactinomyces intermedius T.

The threonine-producing strain E.coli THR (i.e., E.coli W3110/thrA/lysC/Tac-thrABC/. DELTA.lysA/. DELTA.metA/. DELTA.tdh/ilcR/Trc-ppc) was used as starting strain, and the gene editing technique mediated by CRISPR-Cas9 (Yu Jiang et al 2015-Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental microbiology 81:2506-2514) was used to replace the ptsG gene (SEQ ID No. 3) with ilvA (SEQ ID No. 1) and leuDH gene (SEQ ID No. 2) on the genome, allowing E.coli to ferment L-2-aminobutyric acid without the need of plasmids:

construction of the recombinant vector pTarget-ptsG:: ilvA x-leuDH

The pTarget F Plasmid (Addgene Plasmid # 62226) is used as a template, ptsG degradation-sgRNA-F/ptsG degradation-sgRNA-R is used as a primer for PCR amplification, the obtained PCR product is subjected to Dpn I heat preservation and digestion for 1h at 37 ℃, then is transformed into E.coli DH5 alpha conversion competence, spectinomycin (SD) plate screening is carried out, and sequencing verification is carried out to obtain the correct pTarget-ptsG:: -leuDH Plasmid for subsequent connection with the Donor DNA.

2. Construction of the recombinant vector pTD-ptsG:: ilvA x-leuDH

Taking E.coli W3110 genome as a template, taking ptsG D-Arm1-F and ptsG D-Arm1-R as primers for amplification to obtain an upstream homology Arm of the donor DNA, taking ptsG D-Arm2-F and ptsG D-Arm2-R as primers for amplification to obtain a downstream homology Arm of the donor DNA, taking Arm2 as the primer, taking In ptsG-F-ilvA-leuDH and In ptsG-R-ilvA-leuDH as primers for amplification to obtain the donor DNA ilvA-leuDH genes, taking In ilvA-leuDH as the InilvA-leuDH, and taking DNA fragments by a Clean up kit for recovery to obtain homology arms Arm1, arm2 and donor DNA In ilvA; the ilvA-leuDH plasmid is amplified by taking pTarget-F and pTarget-R as primers, the obtained PCR product is digested for 1-2h by Dpn I at 37 ℃ in a heat preservation way, and DNA fragments are recovered by a clean up kit; pTarget-ptsG::: ilvA-leuDH plasmid fragment, homology arms Arm1, arm2 and donor DNA In leuDH were ligated together according to the instructions of (One step clone kit, vazyme Biotech, nanjing, china), introduced into E.coli DH 5. Alpha. Transformation competence, colonies were PCR screened for positive clones, and the pTD-ptsG::: ilvA-leuDH plasmid was obtained by sequencing verification.

PCR system: genome template 0.5. Mu.L, 2X Phanta Max Buffer. Mu.L, dNTP 1. Mu.L, forward and reverse primer 2. Mu.L (10. Mu.M) each, phanta Max DNA polymerase 1. Mu.L, deionized water to 50. Mu.L. PCR procedure: denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 57℃for 30s, extension at 72℃for 1.5min for 30 cycles; finally, the fragment is extended for 10min at 72 ℃, and the fragment is clear-up for standby after electrophoresis identification.

The connection process comprises the following steps: ligation was performed using One Step Cloning Kit (available from Vazyme) and added to the sterilized PCR tube

Mix 5 μl, linear vector X μl after cleavage and linear amplified fragment Y μl (optimum cloning vector usage= [0.02×cloning vector base pair number)]ng (0.03 pmol), optimum amount of insert = [0.04 x base pair number of insert]ng(0.06pmol))，With sterilized ddH ₂ O was added to 10. Mu.L, and the mixture was reacted at 50℃for 20 minutes. The connection product is transformed into DH5 alpha escherichia coli competence by a calcium chloride method, spectinomycin resistance screening is utilized, positive transformant sequencing is selected for verification, and pTD-ptsG is finally obtained.

3. Electrotransformation competent preparation

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 mu L of L-arabinose with the concentration of 1mol/L, 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. Electric shock conversion editing and verification

Mixing ilvA-leuDH plasmid with 100-150 mu L of electrotransformation competent cells, ice-bathing for 3-4min, transferring into a pre-chilled 2mm electric shock cup, carrying out electric shock transformation for about 45s in ice-bathing, immediately adding pre-chilled 1mL LLB culture medium after electric shock is finished and immediately sucking out, transferring into a 2mL EP tube, resuscitating for 3-4 h at 30 ℃, taking 400 mu L of LB plate coated with spectinomycin containing 0.05mg/L kanamycin and 0.05mg/L, inversely culturing for 14-18 h at 30 ℃, carrying out colony PCR verification by taking ptsG delete-VF and ptsG delete-VR as verification primers, and if a fragment with the size of about 2100kb can be successfully cloned, and sequencing verification is successful, proving that the single colony is E.coli THR delta ptsG positive for the ilvA-leuDH is successfully edited, namely a new colony of 1 is obtained.

5. Plasmid elimination

Positive clones were inoculated into 10mL LLB tubes containing 1mM IPTG and 0.05mg/L kanamycin, and incubated at 30℃for 12-18h at 180 rpm. Streaking was performed on a solid medium containing 0.05mg/L kanamycin LLB, and incubated overnight at 30 ℃. The numbered small single colonies were picked with sterilized toothpicks, streaked onto corresponding areas on LLB solid medium containing 0.05mg/L spectinomycin, and incubated overnight at 30 ℃. Single colonies which failed to grow in the region corresponding to LLB solid medium containing 0.05mg/L spectinomycin were pTarget-ptsG:: ilvA. Times. -leuDH was successfully deleted.

Clones successfully deleted by pTarget-ptsG:: ilvA x-leuDH were inoculated into 10mL non-anti-LLB tubes and incubated at 42℃for 12-18h at 180 rpm. Streaking on LLB non-antibiotic solid medium, and culturing overnight at 37 ℃. The numbered small single colonies were picked with sterilized toothpicks, streaked onto corresponding areas on LLB solid medium containing 0.05mg/L kanamycin, and incubated overnight at 30 ℃. A single colony incapable of growing in the corresponding region of LLB solid medium containing 0.05mg/L kanamycin was a clone successfully eliminated by pCas to obtain a plasmid-free strain E.coli THR delta ptsG:: ilvA x-leuDH (recorded as ABA-1).

LLB medium: 10g/L peptone, 5g/L yeast extract, 5g/L NaCl, deionized water as solvent, and natural pH.

LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, deionized water as solvent, and natural pH.

6. The strain E.coli THR delta ptsG:: ilvA-leuDH 5L fermenter fermentation

The constructed strain ABA-1 is streaked on a flat plate without an anti-LB solid medium, cultured overnight at 37 ℃, single colony is picked from the flat plate on the next day in 10mL of liquid LB medium, cultured overnight at 37 ℃ and at 150rpm, and then transferred into a 500mL shake flask of 100mL of LB medium according to the inoculation amount of 2%, and cultured at 37 ℃ for about 8 hours, so as to obtain seed liquid. Then transferring the mixture into a 5L fermentation tank (the culture medium liquid loading amount of the fermentation tank is 3L) according to the inoculation amount of 3-5 percent for fermentation.

Fermentation conditions: the fermentation adopts a low-sugar feeding method, the fermentation temperature is 35 ℃, the pH value is automatically adjusted to 6.8 by using ammonia water with the volume ratio of 50 percent, and the dissolved oxygen is controlled to be 10-30 percent. The initial aeration was 4ppm at 400rpm, the dissolved oxygen concentration was adjusted by the rotational speed, and when the initial glucose was consumed and the pH and dissolved oxygen concentration began to rise, feed was started, always maintaining the glucose concentration in the tank at about 5g/L. When the strain growth OD reached a maximum, IPTG was added at a final concentration of 0.1 mM. During fermentation, sampling is carried out every 3h to determine the concentration of glucose in the fermentation liquid, and HPLC is used for determining the content of L-2-aminobutyric acid in the fermentation liquidOptical photometer for detecting OD ₆₀₀ The growth of the strain was determined and the results are shown in FIG. 1.

Fermentation medium: IPTG 0.1mM, 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, deionized water as solvent, and pH value is natural; the metal salt ion solution comprises the following components: feSO ₄ ·7H ₂ O 10±1.5g/L，CaCl ₂ 1.35±0.6g/L，ZnSO ₄ ·7H ₂ O 2.25±0.8g/L，MnSO ₄ ·4H ₂ O 0.5±0.1g/L，CuSO ₄ ·5H ₂ O 1±0.5g/L，(NH ₄ )6Mo ₇ O ₂₄ ·4H ₂ O 0.106±0.05g/L，Na ₂ B ₄ O ₇ ·10H ₂ O0.23+/-0.08 g/L,35% HCl 10+/-1 mL/L, and water as solvent.

Feed 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, and L-isoleucine 0.15+ -0.05 g/L.

As can be seen from FIG. 1, the exogenous addition of ilvA and leuDH genes to replace ptsG genes by gene editing means did not significantly inhibit the growth of the ABA-1 strain, but could make E.coli ferment to produce L-2-aminobutyric acid without plasmids, so that the L-2-aminobutyric acid titer increased from 0g/L to 1.19g/L, which indicates that genome replacement of ilvA and leuDH genes made E.coli have the L-2-aminobutyric acid synthesis capability.

EXAMPLE 2 construction of E.coli adaptive evolution of strains resistant to high concentrations of 2-ketobutyrate

Adaptive evolution medium configuration

20mL of LB medium was placed in a 250mL flask, 2-ketobutyric acid was added thereto, and the concentration gradient of 2-ketobutyric acid was set to 2g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L. All media were pH adjusted to 6.8.+ -. 0.1 before sterilization.

Inoculation of

E.coli THR delta ptsG:. IlvA-leuDH strains were transferred to LB medium containing different concentrations of 2-ketobutyrate for subculture at 37℃at 180rpm for 16-18h. The strain which can grow normally under the concentration of 50 g/L2-ketobutyrate is obtained, namely the strain E.coli THR delta ptsG which tolerates high concentration 2-ketobutyrate, wherein ilvA-leuDHMutt is marked as ABA-2.

Example 3 construction and fermentation of Strain ABA-3 with LeuDH Gene replacing the ltaE Gene and poxB Gene 1, construction of recombinant vector pTarget-ltaE poxB by using pTarget F Plasmid (Addge Plasmid # 62226) as template and ltaE-poxB degradation-sgRNA-F/ltaE-poxB degradation-sgRNA-R as primer for PCR amplification, digestion of the obtained PCR product with Dpn I at 37℃for 1h, transformation into E.coli DH 5. Alpha. Transformation competent, spectinomycin plate screening, sequencing to verify that the correct pTarget-ltaE poxB: leuDH Plasmid was obtained for subsequent ligation with Donor DNA.

2. The construction of the recombinant vector pTD-ltaE poxB was carried out using the E.coli W3110 genome as template and the ltaE-poxB D-Arm1-F, ltaE-poxB D-Arm1-R, ltaE-poxB D-Arm2-F, ltaE-poxB D-Arm2-R, inptsG-F-leuDH and InptsG-R-leuDH as primers. The construction procedure was as in example 1 (2) to give pTD-ltaE poxB:: leuDH plasmid.

3. Electrotransformation competent preparation

The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain ABA-2 obtained by adaptive evolution of example 2, and the preparation of competence of strain ABA-2 was the same as in example 1 (3).

4. Electric shock conversion editing and verification

The strain ABA-3 positive colony was constructed in the same manner as in example 1 (4).

5. Plasmid elimination was performed in the same manner as in example 1 (5), to obtain a plasmid-free strain ABA-3.

6. Strain ABA-3 5L fermentation tank fermentation

The constructed strain ABA-3 production strain is streaked from a glycerol tube to an LB plate, a single colony is selected and inoculated into 10mL of LB culture medium, the method is carried out by taking the strain ABA-1 constructed in example 1 as a control, and the fermentation result is shown in figure 2.

As can be seen from FIG. 2, the gene editing means was used to replace the ltaE gene (SEQ ID No. 4) and poxB gene (SEQ ID No. 5) with the leuDH gene (SEQ ID No. 2), the growth of the strain ABA-3 was promoted, the OD was increased to 36.16, and the yield of L-2-aminobutyric acid was increased, so that the titer of L-2-aminobutyric acid was increased from 1.25g/L to 4.35g/L, which indicated that increasing the copy of leuDH was beneficial to the synthesis of E.coli L-2-aminobutyric acid.

EXAMPLE 4 construction and fermentation of Strain ABA-4 deleted for the focA Gene and pflB Gene

1. Construction of recombinant vector pTarget-focA pflB

The pTarget F Plasmid (Addgene Plasmid # 62226) is used as a template, focA-pflB degradation-sgRNA-F/focA-pflB degradation-sgRNA-R is used as a primer for PCR amplification, the obtained PCR product is subjected to Dpn I heat preservation and digestion for 1h at 37 ℃, then is transformed into E.coli DH5 alphaization transformation competence, spectinomycin plates are screened, and sequencing verification is carried out to obtain the correct pTarget-focA pflB Plasmid for subsequent connection with the Donor DNA.

2. The recombinant vector pTD-focA pflB was constructed using E.coli W3110 genome as 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 In focA-pflB-R as primers. The construction procedure was the same as in example 1 (2), to obtain pTD-focA pflB plasmid.

3. Electrotransformation competent preparation pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competent strain ABA-3 obtained in example 2, and the competent preparation of strain ABA-3 was carried out in the same manner as in example 1 (3).

4. Electric shock transformation editing and verification construction are carried out to obtain bacterial strain ABA-4 positive colonies, and the construction method is the same as that of the example 1 (4).

5. Plasmid elimination was performed in the same manner as in example 1 (5), to obtain a plasmid-free strain ABA-4.

6. Fermentation of strain ABA-4 in 5L fermenter the constructed strain ABA-4 producing strain was streaked from glycerol tubes to LB plates, single colonies were picked up and inoculated into 10mL of LB medium, the method was carried out with the strain ABA-3 constructed in example 2 as a control, and the fermentation results are shown in FIG. 3.

As can be seen from FIG. 3, deletion of the focA gene (SEQ ID No. 6) and pflB gene (SEQ ID No. 7) by gene editing means showed no obvious inhibition of growth of the ABA-4 strain, but increased the yield of L-2-aminobutyric acid, resulting in an increase in the L-2-aminobutyric acid titer from 4.35g/L to 4.95g/L, indicating that knockout of the by-product pathway is favorable for E.coli L-2-aminobutyric acid synthesis.

EXAMPLE 5 construction and fermentation of strain ABA-5 in which pta Gene and ackA Gene were replaced with ilvA Gene and leuDH Gene

1. Construction of the recombinant vector pTarget-ptaackA:: ilvA-leuDH

The pTarget F Plasmid (Addgene Plasmid # 62226) is used as a template, pta-ackA degradation-sgRNA-F/pta-ackA degradation-sgRNA-R is used as a primer for PCR amplification, the obtained PCR product is subjected to Dpn I heat preservation and digestion for 1h at 37 ℃, and then is converted into E.coli DH5 alpha conversion competence, spectinomycin is screened by a plate, and sequencing verification is carried out to obtain the correct pTarget-pta ackA:: ilvA-leuDH Plasmid for subsequent connection with Donor DNA.

2. The recombinant vector pTD-ptaackA is constructed by taking E.coli W3110 genome as a template, and 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 as primers. The construction procedure was as in example 1 (2) to give pTD-pta ackA:: ilvA x-leuDH plasmid.

3. Electrotransformation competent preparation

The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain ABA-4 obtained in example 3, and the competence of strain ABA-4 was prepared in the same manner as in example 1 (3).

4. Electric shock conversion editing and verification

The strain ABA-5 positive colony is constructed and the construction method is the same as that of the example 1 (4).

5. Plasmid elimination

The procedure was followed in the same manner as in example 1 (5) to obtain a plasmid-free strain ABA-5.

6. Fermentation of strain ABA-5L fermentation tank

The constructed strain ABA-4 producing strain was streaked from glycerol tubes to LB plates, single colonies were picked and inoculated into 10mL of LB medium, the method of the comparative example was carried out with the strain ABA-4 constructed in example 3 as a control, and the fermentation results are shown in FIG. 4.

As can be seen from FIG. 4, the use of gene editing means to replace pta gene (SEQ ID No. 8) and ackA gene (SEQ ID No. 9) with ilvA (SEQ ID No. 1) and leuDH gene (SEQ ID No. 2) did not significantly inhibit the growth of the strain ABA-5, but could increase the yield of L-2-aminobutyric acid, resulting in an increase in the titer of L-2-aminobutyric acid from 4.95g/L to 11.59g/L, indicating that increasing the copy number of ilvA and leuDH genes and knockout of the by-product pathway was beneficial for E.coli L-2-aminobutyric acid synthesis.

Example 6 construction of strain ABA-6 with replacement of mgsA Gene by ilvA Gene and leuDH Gene and fermentation 1, construction of recombinant vector pTarget-mgsA:: construction of ilvA-leuDH with pTarget F Plasmid (Addgene Plasmid # 62226) as template and mgsA degradation-sgRNA-F/mgsA degradation-sgRNA-R as primer were PCR amplified, the obtained PCR product was digested with Dpn I at 37℃for 1h, and then transformed into E.coli DH 5. Alpha. Transformation competence, spectinomycin plate screening, sequencing verification to obtain the correct pTarget-mgsA:: ilvA-leuDH Plasmid for subsequent ligation to Donor DNA.

2. Construction of recombinant vector pTD-mgsA. IlvA. Mu. DH was carried out using E.coli W3110 genome as template, mgsA D-Arm1-F, mgsA D-Arm1-R, mgsA D-Arm2-F, mgsA D-Arm2-R, trc-F and Trc ilvA. Mu. DH-R as primers. The construction procedure was the same as in example 1 (2) to give pTD-mgsA:: ilvA x-leuDH plasmid.

3. Electrotransformation competent preparation

The pCas Plasmid (Addgene Plasmid # 62225) was introduced into the competence of strain ABA-5 obtained in example 4, and the competence of strain ABA-5 was prepared in the same manner as in example 1 (3).

4. Electric shock conversion editing and verification

The strain ABA-6 positive colony is constructed and the construction method is the same as that of the example 1 (4).

5. Plasmid elimination

The procedure was followed in the same manner as in example 1 (5) to obtain a plasmid-free strain ABA-6.

6. Strain ABA-6 5L fermentation tank fermentation

The constructed strain ABA-6 producing strain was streaked from glycerol tubes to LB plates, single colonies were picked and inoculated into 10mL of LB medium, and the method was carried out in the same manner as in example 1 (6) using the strain ABA-4 constructed in example 3 as a control, and the results are shown in FIG. 5.

As can be seen from FIG. 5, the mgsA gene (SEQ ID No. 10) was replaced with ilvA gene (SEQ ID No. 1) and leuDH gene (SEQ ID No. 2) by gene editing means, the growth of the strain ABA-6 was not affected, and the yield of L-2-aminobutyric acid was increased from 11.59g/L to 13.81g/L, which suggests that increasing the copy number of ilvA gene and leuDH gene and knockout of the by-product pathway was favorable for E.coli L-2-aminobutyric acid synthesis.

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

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid is characterized in that: the method is constructed by the following steps:

(1) The E.coli THR is taken as chassis fungus, ptsG genes in the genome of the strain E.coli THR are replaced by ilvA genes and leuDH genes, and a recombinant strain E.coli THR delta ptsG is obtained;

(2) Inoculating ilvA-leuDH to 2-ketobutyrate liquid culture medium for subculturing, screening strains capable of growing normally in the liquid culture medium with the concentration of 2-ketobutyrate not lower than 50g/L, and obtaining strain E.coli THR delta ptsG;

(3) Replacing adjacent ltaE genes and poxB genes in an ilvA-leuDHMut genome by leuDH genes to obtain a recombinant strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH;

(4) Knocking out the focA gene and pflB gene in the bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH genome to obtain a recombinant bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB;

(5) The adjacent pta genes and ackA genes in the bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB genome are replaced by ilvA-genes and leuDH genes, so as to obtain a recombinant bacterial strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta focA delta pflB delta ptaackA::: ilvA-leuDH;

(6) The mgsA gene in the genome of the strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB:: leuDH delta flocA delta pflB delta ackA:: ilvA-leuDH is replaced by ilvA-gene and leuDH gene, and the recombinant strain E.coli THR delta ptsG:: ilvA-leuDHMut delta ltaE delta poxB::: leuDH delta focA delta pflA-leuDH delta mgsA: ilvA-leuDH is obtained, namely the genetically-free plasmid for producing L-2-aminobutyric acid.

2. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the ilvA gene sequence is shown as SEQ ID No. 1.

3. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the leuDH gene is a leucine dehydrogenase encoding gene.

4. The plasmid-free genetically engineered bacterium for high production of L-2-aminobutyric acid according to claim 1 or 3, wherein: the leuDH gene sequence is shown as SEQ ID No. 2.

5. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the ptsG gene sequence is shown in SEQ ID No. 3.

6. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the ltaE gene sequence is shown as SEQ ID No.4, and the poxB gene sequence is shown as SEQ ID No. 5.

7. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the sequence of the focA gene is shown as SEQ ID No.6, and the sequence of the pflB gene is shown as SEQ ID No. 7.

8. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the pta gene sequence is shown as SEQ ID No.8, and the ackA gene sequence is shown as SEQ ID No. 9.

9. The plasmid-free genetically engineered bacterium for high yield of L-2-aminobutyric acid according to claim 1, wherein the genetically engineered bacterium comprises: the mgsA gene sequence is shown as SEQ ID No. 10.

10. Use of the plasmid-free genetically engineered bacterium of any one of claims 1-9 in the preparation of L-2-aminobutyric acid by microbial fermentation.