CN115404224A - L-threonine producing genetic engineering bacterium and construction method and application thereof - Google Patents
L-threonine producing genetic engineering bacterium and construction method and application thereof Download PDFInfo
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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
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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Abstract
The invention provides an L-threonine producing genetic engineering bacterium and a construction method and application thereof. The engineering bacteria introduce mutation into the genome of the bacteria with threonine production capacity by using genetic engineering means, so that the encoded C4-dicarboxylate transport protein comprises an A12G or F14L mutation site, or both mutation sites. The shake flask fermentation experiment shows that the Escherichia coli containing the DcuA protein variant can obviously improve the threonine production capacity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a gene engineering bacterium for producing L-threonine and a construction method and application thereof.
Background
L-threonine is one of 8 amino acids essential for human and animal growth, and is widely used in feed, food additives, preparation of auxiliary materials for medicines, and the like. Currently, L-threonine is mainly produced by fermentation of microorganisms, and various bacteria are available for L-threonine production, such as mutant strains induced by wild-type strains of Escherichia coli, corynebacterium, serratia, and the like, as production strains. Specific examples include amino acid analogue-resistant mutants or various auxotrophs such as methionine, lysine, isoleucine and the like (Japanese patent application laid-open No. 224684/83; korean patent application laid-open No. 8022/87). However, in the conventional mutation breeding, the strain grows slowly and generates more byproducts due to random mutation, so that a high-yield strain is not easy to obtain.
With the increasing demand of global threonine, the construction and modification of high-yield threonine strains are particularly important. In the Chinese patent CN03811059.8 applied by Korea CJ corporation in 2003, the expression of thrABC, a key gene for threonine synthesis, was enhanced by deleting 39bp from-56 to-18 of the threonine operon sequence using E.coli, and the threonine productivity was improved by 22%. Kwang Ho Lee (Kwang Ho Lee et al, systems metabolism engineering for L-threonine production, mol Syst biol.2007; 3. In the Chinese patent ZL201611250306.8 applied by the plum blossom group in 2020, MHZ-0215-2 strain is obtained by strengthening pntAB gene and introducing pyc gene heterologously, and the strain has threonine yield of 12.4g/L, transformation rate of about 16.2% and no plasmid burden. In the Chinese patent application CN112481179A, the yield of the biotin-enhanced strain MHZ-0217-4 constructed by starting from MHZ-0215-2 strain is 20.2g/L, the conversion rate is 23.8 percent, and no plasmid burden is caused.
DcuA is a C4-dicarboxylate transporter, and Escherichia coli dcuA gene is constitutively expressed under both aerobic and anaerobic conditions. DcuA is the major transporter for uptake of L-aspartic acid by E.coli under aerobic conditions, and it takes up L-aspartic acid as a nitrogen source under aerobic conditions. DcuA mediates the export of succinate while absorbing fumarate, malate or aspartate. Furthermore, the DcuA transporter is active under anaerobic conditions and may play a role in C4-dicarboxylate transport during the conversion of cells from microaerophilic to anaerobic conditions. DcuA is a transmembrane protein with 10 transmembrane helices with an 80 residue central cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm. The topology of the DcuA protein has been studied in the art. In which transmembrane helix 2 contains a highly conserved and repetitive region GGIGL, but its function is unknown. No study report is available on the influence of amino acid changes in the conserved region on the yield of threonine in Escherichia coli. In the threonine synthesis pathway of Escherichia coli, aspartic acid is a direct precursor for threonine synthesis; while malic acid produces oxaloacetate, a precursor for the synthesis of aspartic acid. Considering that DcuA protein can absorb aspartic acid and malic acid, dcuA protein variants may have an effect on the synthesis of E.coli threonine. Similarly, based on the threonine synthesis pathway of E.coli, it was deduced that DcuA protein variants also had an effect on the production of downstream amino acids of threonine, such as isoleucine and glycine.
Disclosure of Invention
The invention aims to provide a gene engineering bacterium for producing L-threonine and a construction method and application thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a DcuA protein mutant, which is any one of the following (1) to (3):
(1) the 12 th amino acid of the DcuA protein is mutated from A to G;
(2) the 14 th amino acid of the DcuA protein is mutated from F to L;
(3) the 12 th amino acid of the DcuA protein is mutated from A to G, and the 14 th amino acid is mutated from F to L.
Compared with wild DcuA protein, the enzyme activity of the DcuA protein mutant is enhanced.
In the present invention, the reference sequence number of the DcuA protein on NCBI is NP-418561.1.
In a second aspect, the invention provides nucleic acid molecules encoding said mutant DcuA protein.
In a third aspect, the invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.
In a fourth aspect, the invention provides any one of the following uses of the nucleic acid molecule or a biological material containing the nucleic acid molecule:
(1) The method is used for amino acid fermentation production;
(2) Used for improving the fermentation yield of amino acid;
(3) Used for constructing gene engineering bacteria for producing amino acid.
In the present invention, the amino acid is at least one selected from the group consisting of L-threonine, isoleucine, glycine and the like.
In a fifth aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces L-threonine by introducing a mutation into the genome of a bacterium that has threonine-producing ability by genetic engineering means such that the encoded C4-dicarboxylate transporter contains the A12G or F14L mutation site, or both of them.
Preferably, the bacterium is an Escherichia (Escherichia) species, more preferably Escherichia coli (Escherichia coli), such as strain MHZ-0217-4 (see CN 112481179A), strain MHZ-0215-2 (see ZL 201611250306.8).
Strain MHZ-0219-1 (dcuA) A12G ) The construction method comprises the following steps:
A. pTargetF-N20 (dcuA) plasmid and Donor DNA construction
A1: amplifying a pTF linear plasmid with N20 by using a pTARgetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (A12G) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying up-dcuA-down full-length fragment, also called Donor DNA-1, by using dcuA-UF/dcuA-DR primer pair with the (1) and (2) as templates;
wherein dcu A Is a C4-dicarboxylate transporter gene;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-1 into MHZ-0217-4 (pCas) competent cells, coating the competent cells on an LB plate containing spectinomycin and kanamycin, and performing static culture at 30 ℃ until a single colony is visible;
C. reconstitution validation
C1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G) -F1/dcuA-R; c2: using a primer pair dcuA-F/dcuA-R to amplify a target fragment, and sequencing an amplification product to verify the integrity of a sequence;
D. construction of related plasmid losses
D1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin; d2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: selecting a single colony to be spotted on an LB plate containing kanamycin and an LB plate without resistance, if the single colony can not grow on the LB plate containing kanamycin and can not grow on the LB plate without resistance, showing that pCas plasmid is lost, obtaining the L-threonine producing genetic engineering bacteria which are marked as MHZ-0219-1 (dcuA) A12G )。
Strain MHZ-0219-2 (dcuA) F14L ) The construction method comprises the following steps:
A. construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-2
A1: amplifying a pTF linear plasmid with N20 by using a pTARgetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (F14L) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying up-dcu by using the primer pair dcuA-UF/dcuA-DR and taking the (1) and the (2) as templates A -down full length fragment, also known as Donor DNA-2;
wherein, dcu A Is a C4-dicarboxylate transporter gene;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-2 into MHZ-0217-4 (pCas) competent cells, coating the competent cells on an LB plate containing spectinomycin and kanamycin, and performing static culture at 30 ℃ until a single colony is visible;
C. reconstitution validation
C1: performing colony PCR verification on the single colony by using a primer pair dcuA (F14L) -F1/dcuA-R; c2: using a primer to amplify the dcuA-F/dcuA-R target fragment, and sequencing an amplification product to verify the integrity of the sequence;
D. construction of related plasmid losses
D1: selecting single colony with correct sequencing verification, inoculating into 5mL LB test tube containing kanamycin and 0.5mM IPTG, and culturing at 30 deg.C overnightAfter cultivation, streaking on an LB plate containing kanamycin; d2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: selecting a single colony to be spotted on an LB plate containing kanamycin and an LB plate without resistance, if the single colony can not grow on the LB plate containing kanamycin and can not grow on the LB plate without resistance, showing that pCas plasmid is lost, obtaining the L-threonine producing genetic engineering bacteria which are marked as MHZ-0219-2 (dcuA) F14L )。
Strain MHZ-0219-3 (dcuA) A12G,F14L ) The construction method comprises the following steps:
A. construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-3
A1: amplifying a pTF linear plasmid with N20 by using a pTARgetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G, F14L) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (A12G, F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying up-dcuA-down full-length fragment, also called Donor DNA-3, by using dcuA-UF/dcuA-DR primer pair with the (1) and (2) as templates;
wherein, dcu A Is a C4-dicarboxylate transporter gene;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ at 200r/min to OD 650 Is 0.4 post-production electrotransferA competent cell; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-3 into MHZ-0217-4 (pCas) competent cells, coating on an LB plate containing spectinomycin and kanamycin, and standing and culturing at 30 ℃ until a single colony is visible;
C. recombination verification
C1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G, F14L) -F1/dcuA-R; c2: using a primer to amplify the dcuA-F/dcuA-R target fragment, and sequencing an amplification product to verify the integrity of the sequence;
D. construction of related plasmid losses
D1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin; d2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: picking single colony spot on an LB plate containing kanamycin and an LB plate without resistance, if the colony can not grow on the LB plate containing kanamycin, the colony grows on the LB plate without resistance, which shows that pCas plasmid is lost, and obtaining the L-threonine producing genetically engineered bacterium which is marked as MHZ-0219-3 (dcuA) A12G,F14L )。
The sequences of the primers used in the above method are shown in Table 1.
In a sixth aspect, the invention provides the genetically engineered bacterium producing L-threonine constructed according to the above method.
In a seventh aspect, the invention provides an application of the genetic engineering bacterium for producing L-threonine in L-threonine fermentation production or L-threonine fermentation yield improvement.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention takes MHZ-0217-4 as an original strain to carry out genome transformationAnd (3) transforming, introducing point mutation into the dcuA gene so as to generate different DcuA protein variants. Carrying out shake flask fermentation on threonine Escherichia coli producing bacteria containing DcuA variants, wherein the fermentation result shows that the Escherichia coli producing bacteria contain dcuA A12G The yield of the modified strain MHZ-0219-1 threonine is 23.36g/L, the shake flask conversion rate is 27.48%, the yield is increased by 15.47% compared with that of the original strain, and the sugar acid conversion rate is increased by 15.46%. Containing dcuA F14L The yield of the modified strain MHZ-0219-2 threonine is 23.85g/L, the shake flask conversion rate is 28.05%, the yield is improved by 17.89% compared with that of the original strain, and the sugar acid conversion rate is improved by 17.86%. Meanwhile, the yield of the threonine of the MHZ-0219-3 strain containing A12G and F14L point mutations is 26.12G/L, the shake flask conversion rate is 30.73%, the yield is increased by 29.12% compared with that of the original strain, and the sugar acid conversion rate is increased by 29.12%. The above results indicate that E.coli containing DcuA protein variants can significantly improve threonine productivity.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning Laboratory Manual, sambrook, et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or following the conditions recommended by the manufacturer's instructions.
In the following examples, MHZ-0217-4 and MHZ-0215-2 were used as starting strains, and the genomes thereof were modified to introduce point mutations into the dcuA gene to generate different DcuA protein variants (enzyme activity enhancement). The DcuA variant-containing threonine Escherichia coli-producing strain was subjected to shake flask fermentation, and the effect on threonine-producing ability was observed.
For Genome Editing of Escherichia coli, reference is mainly made to CRISPR-Cas9 gene Editing technology (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al.
In the following examples, kanamycin (Kanamycin) was present in the medium at a final concentration of 50. Mu.g/mL, and Spectinomycin (Spectinomycin) was present in the medium at a final concentration of 50. Mu.g/mL.
The reagents used in the following examples are all commercially available. The parent strain of the threonine producing strain with high conversion rate provided by the invention is MHZ-0217-4, and belongs to W3110 (Escherichia).
The sequences of the primers involved in the present invention are shown in Table 1.
TABLE 1
The protein coded by the gene related to the invention is as follows:
dcuA: a C4-dicarboxylate transporter;
a bioA: ademetionine-8-amino-7-oxononanoic acid aminotransferase;
and (3) bioB: a biotin synthase;
and (3) bioF: 8-amino-7-oxononanoic acid synthase;
and (3) bioC: malonyl carrier protein methyltransferase;
and (3) bioD: a desthiobiotin synthase;
birA: DNA-binding transcription repressor/biotin- [ acetyl-CoA-carboxylase ] ligase;
pyc: a pyruvate carboxylase.
Example 1 construction of a plasmid containing dcuA A12G Gene strain MHZ-0219-1
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-1
Step1: amplifying a pTF linear plasmid with N20 by using a pTF-sgRNA-F/pTF-sgRNA-R primer pair and using a seamless assembly ClonExpress kit to assemble the linear plasmid at 37 ℃ by using a pTargetF plasmid as a template (derived from Multigene Editing in the Escherichia coli Genome the CRISPR-Cas9 System, jiang Y, chen B, et al, apple. Environ Microbiol, 2015), and then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA) and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (A12G) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying an up-dcuA-down full-length fragment, which is also called Donor DNA-1, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: the pCas plasmid (derived from Multigene differentiation in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) was electroporated into MHZ-0217-4 competent cells (the transformation method and the competent preparation method are referred to as molecular clone III), and positive MHZ-0217-4 (pCas) was obtained; step2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ at 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see "molecular clone III" for a competent preparation method). Step3: the pTargetF-N20 (dcuA) plasmid and the Donor DNA-1 constructed in (1) were simultaneously electroporated into MHZ-0217-4 (pCas) competent cells (electrotransformation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plate containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.
(3) Recombination verification
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of the relevant plasmid loss
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; step2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: picking a single colony to point on an LB plate containing kanamycin and an LB plate without resistance, if the colony can not grow on the LB plate containing kanamycin and can not grow on the LB plate without resistance, indicating that pCas plasmid is lost, and obtaining MHZ-0219-1 (dcuA) A12G ) And (3) strain.
Example 2 construction of a plasmid containing dcuA F14L Gene strain MHZ-0219-2
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-2
Step1: amplifying a pTF linear plasmid with N20 by using a pTF-sgRNA-F/pTF-sgRNA-R primer pair and using a seamless assembly ClonExpress kit to assemble the linear plasmid at 37 ℃ by using a pTargetF plasmid as a template (derived from Multigene Editing in the Escherichia coli Genome the CRISPR-Cas9 System, jiang Y, chen B, et al, apple. Environ Microbiol, 2015), and then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA) and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (F14L) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying an up-down full-length fragment up-dcuA-down, also called Donor DNA-2, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and the (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: the pCas plasmid (derived from Multigene differentiation in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) was electroporated into MHZ-0217-4 competent cells (the transformation method and the competent preparation method are referred to as molecular clone III), and positive MHZ-0217-4 (pCas) was obtained; step2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: mixing pTargetF-N20 (dcuA) substanceThe DNA of Donor constructed in (1) and the plasmid were simultaneously electroporated into MHZ-0217-4 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plate containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colony was visible.
(3) Reconstitution validation
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (F14L) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that the pTargetF-N20 (dcuA) plasmid is lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: picking a single colony to point on an LB plate containing kanamycin and an LB plate without resistance, if the colony can not grow on the LB plate containing kanamycin and can not grow on the LB plate without resistance, indicating that pCas plasmid is lost, and obtaining MHZ-0219-2 (dcuA) F14L ) And (3) strain.
Example 3 construction of a plasmid containing dcuA A12G,F14L Gene strain MHZ-0219-3
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-3
Step1: amplifying a pTF linear plasmid with N20 by using a pTF-sgRNA-F/pTF-sgRNA-R primer pair and using a seamless assembly ClonExpress kit to assemble the linear plasmid at 37 ℃ by using a pTargetF plasmid as a template (derived from Multigene Editing in the Escherichia coli Genome the CRISPR-Cas9 System, jiang Y, chen B, et al, apple. Environ Microbiol, 2015), and then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA) and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G, F14L) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (A12G, F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying the up-dcuA-down full-length fragment, which is also called Donor DNA-3, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and the (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: electrically transferring pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0217-4 competent cells (the transformation method and the competence preparation method refer to molecular clone III), and obtaining positive MHZ-0217-4 (pCas); step2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTargetF-N20 (dcuA) plasmid and the Donor DNA constructed in (1) were simultaneously electroporated into MHZ-0217-4 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.
(3) Reconstitution validation
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G, F14L) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; step2: single colonies were picked and spotted on LB plates containing kanamycin and spectinomycin and LB plates containing kanamycin only, and cultured overnight at 30 ℃ if they could not grow on LB plates containing kanamycin and spectinomycinGrowth on LB plates of kanamycin showed that the pTargetF-N20 (dcuA) plasmid had been lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colony spots were picked on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, yielding MHZ-0219-3 (dcuA) A12G，F14L ) And (3) strain.
The threonine-producing genetically modified strains obtained in examples 1 to 3 are shown in Table 2.
TABLE 2 genetically engineered bacteria constructed according to the present invention
Example 4 shaking flask fermentation experiment of genetically engineered bacteria producing L-threonine
Step1, taking 4 strains of MHZ-0217-4, MHZ-0219-1, MHZ-0219-2 and MHZ-0219-3 from a frozen tube, marking and activating on an LB plate, and culturing at 37 ℃ for 18-24 hours; step2, the cells were scraped off the plate in a loop, inoculated into a 500mL shaking flask containing 50mL of seed medium (Table 3), incubated at 37 ℃ and 90rpm for about 5 hours to OD 650 Controlling the content within 2; step3, transferring 2mL of seed solution into a 500mL shaking flask containing 20mL of fermentation medium (Table 4), performing fermentation culture at 37 ℃ by a reciprocating shaking table and 100rpm until residual sugar is exhausted, and measuring OD (optical density) of a sample after fermentation is finished 650 And the content of L-threonine is measured by using HPLC, and the residual sugar content is measured by using a biosensor method. To ensure the reliability of the experiment, the shaking flasks were subjected to 3 replicates and the average results for acid production and conversion are shown in table 5.
TABLE 3 seed culture Medium (g/L)
| Composition (I) | Concentration of |
| Glucose | 25 |
| Corn steep liquor | 25 |
| Soybean meal hydrolysate | 7.7 |
| Yeast extract | 2.5 |
| KH 2 PO 4 | 1.4 |
| Magnesium sulfate heptahydrate | 0.5 |
| FeSO 4 、MnSO 4 | 20mg/L |
| pH | 7.0 |
TABLE 4 fermentation Medium (g/L)
| Composition (A) | Concentration of |
| Glucose | 85 |
| Corn steep liquor | 6 |
| Soybean meal hydrolysate | 7.7 |
| Magnesium sulfate heptahydrate | 0.5 |
| KH 2 PO 4 | 1.0 |
| Aspartic acid | 10 |
| FeSO 4 、MnSO 4 | 30mg/L |
| Biotin | 50μg |
| Thiamine | 500μg |
| pH | 7.2 |
TABLE 5 comparison of the productivity of the threonine producing genetically engineered bacteria
Note: * P value <0.01, indicating a significant difference from the control.
As can be seen from Table 5, the novel Escherichia coli strains according to the present invention all had higher L-threonine yields than the control strains, which contained dcuA A12G The yield of the modified strain MHZ-0219-1 threonine is 23.36g/L, the shake flask conversion rate is 27.48%, the yield is increased by 15.47% compared with that of the original strain, and the sugar-acid conversion rate is increased by 15.46%. Containing dcuA F14L The yield of the modified strain MHZ-0219-2 threonine is 23.85g/L, the shake flask conversion rate is 28.05%, the yield is improved by 17.89% compared with that of the original strain, and the sugar acid conversion rate is improved by 17.86%. The yield of the dCuA strain containing two point mutations and MHZ-0219-3 threonine is 26.12g/L, the shake flask conversion rate is 30.73%, the yield is increased by 29.12% compared with that of the original strain, and the saccharic acid conversion rate is increased by 29.12%. From the shake flask results, it can be concluded that E.coli containing DcuA protein variants can significantly improve threonine productivity. In addition, the content of isoleucine in the fermentation liquor of the downstream product of threonine is also obviously improved, wherein the content of isoleucine in the modified strain MHZ-0219-3 is improved by 50.78% compared with that in the control strain. Therefore, the introduction of the DcuA mutant into an isoleucine-producing strain can obviously improve the yield of isoleucine and the sugar-acid conversion rate.
Example 5 construction of a microorganism containing dcuA with MHZ-0215-2 as Chassis bacterium A12G Gene strain MHZ-0220-1
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-1
Step1: amplifying a pTF linear plasmid with N20 by using pTARGEtF plasmid as a template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015), using pTF-sgRNA-F/pTF-sgRNA-R primer pair to assemble the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA), and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (A12G) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying up-dcuA-down full-length fragment, also called Donor DNA-1, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: electrically transferring pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and the competence preparation method refer to molecular clone III), and obtaining positive MHZ-0215-2 (pCas); step2: a single MHZ-0215-2 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ at 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTargetF-N20 (dcuA) plasmid and the Donor DNA-1 constructed in (1) were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.
(3) Reconstitution validation
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that the pTargetF-N20 (dcuA) plasmid is lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were picked on both kanamycin-containing LB plates and non-resistant LB plates, if they failed to grow on kanamycin-containing LB plates, they were left withoutGrowth on an anti-LB plate, indicating loss of pCas plasmid, resulting in MHZ-0220-1 (dcuA) A12G ) And (3) strain.
Example 6 construction of a microorganism containing dcuA with MHZ-0215-2 as Chassis bacterium F14L Gene strain MHZ-0220-2
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-2
Step1: amplifying a pTF linear plasmid with N20 by using a pTF-sgRNA-F/pTF-sgRNA-R primer pair and using a seamless assembly ClonExpress kit to assemble the linear plasmid at 37 ℃ by using a pTargetF plasmid as a template (derived from Multigene Editing in the Escherichia coli Genome the CRISPR-Cas9 System, jiang Y, chen B, et al, apple. Environ Microbiol, 2015), and then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA) and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (F14L) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying an up-down full-length fragment up-dcuA-down, also called Donor DNA-2, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and the (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: the pCas plasmid (derived from Multigene differentiation in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) was electroporated into MHZ-0215-2 competent cells (the transformation method and the competent preparation method are referred to as molecular clone III), and positive MHZ-0215-2 (pCas) was obtained; step2: a single MHZ-0215-2 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ at 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see "molecular clone III" for a competent preparation method). Step3: the pTargetF-N20 (dcuA) plasmid and the Donor DNA constructed in (1) were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.
(3) Recombination verification
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (F14L) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin; step2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colony spots were picked on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, yielding MHZ-0220-2 (dcuA) F14L ) And (3) strain.
Example 7 construction of a microorganism containing dcuA with MHZ-0215-2 as Chassis bacterium A12G,F14L Gene strain MHZ-0220-3
(1) Construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-3
Step1: amplifying a pTF linear plasmid with N20 by using pTARGEtF plasmid as a template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015), using pTF-sgRNA-F/pTF-sgRNA-R primer pair to assemble the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain pTargetF-N20 (dcuA), and carrying out PCR identification and sequencing verification; step2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G, F14L) -UR primer pair by using a W3110 genome as a template; step3: amplifying a downstream homology arm (2) by using a dcuA (A12G, F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; step4: and (3) amplifying the up-dcuA-down full-length fragment, which is also called Donor DNA-3, by using the dcuA-UF/dcuA-DR primer pair by using the (1) and the (2) as templates.
(2) Competent cell preparation and electrotransformation
Step1: electrically transferring pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, jiang Y, chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and the competence preparation method refer to molecular clone III), and obtaining positive MHZ-0215-2 (pCas); step2: a single MHZ-0215-2 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTargetF-N20 (dcuA) plasmid and the Donor DNA constructed in (1) were simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5kV, 200. Omega., 25. Mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until single colonies were visible.
(3) Reconstitution validation
Step1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G, F14L) -F1/dcuA-R; step2: and (3) amplifying the target fragment by using a primer pair dcuA-F/dcuA-R, and sequencing an amplification product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that the pTargetF-N20 (dcuA) plasmid is lost; step3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight;step4: picking single colony to point on LB plate containing kanamycin and LB plate without resistance, if they can not grow on LB plate containing kanamycin, they can grow on LB plate without resistance, indicating that pCas plasmid is lost, and obtaining MHZ-0220-3 (dcuA) A12G，F14L ) And (3) strain.
The threonine-producing genetically modified strains obtained in examples 5 to 7 are shown in Table 6.
TABLE 6 genetically engineered bacteria constructed according to the present invention
| Strain numbering | Genotype of a plant |
| MHZ-0215-2 | W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc) |
| MHZ-0220-1 | W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc，dcuA A12G ) |
| MHZ-0220-2 | W3110(thrA*(S345P),tdh::thrA*BC,Ptac-pntAB,IS4::P1-pyc,dcuA F14L ) |
| MHZ-0220-3 | W3110(thrA*(S345P),tdh::thrA*BC，Ptac-pntAB，IS4::P1-pyc，dcuA A12G，F14L ) |
EXAMPLE 8 Shake flask fermentation experiment of genetically engineered bacteria producing L-threonine
Step1, extracting MHZ-0215-2, MHZ-0220-1, MHZ-0220-2 and MHZ-0220-3 from a frozen tubeStreaking and activating 4 strains of bacteria on an LB (Langmuir-Blodgett) plate, and culturing at 37 ℃ for 18-24h; step2, the cells were scraped from the plate and inoculated into a 500mL shaking flask containing 50mL of seed medium (Table 3), and the mixture was cultured at 37 ℃ and 90rpm for about 5 hours to OD 650 Controlling the content within 2; step3, transferring 2mL of the seed solution into a 500mL shaking flask containing 20mL of a fermentation medium (Table 4), performing fermentation culture by a reciprocating shaker at 37 ℃ and 100rpm until residual sugar is exhausted, and determining OD (optical density) of a sample after fermentation 650 And the content of L-threonine is measured by using HPLC, and the residual sugar content is measured by using a biosensor method. To ensure the reliability of the experiment, 3 replicates of the flasks were run and the average acid production and conversion results are shown in Table 7.
TABLE 7 comparison of productivity of threonine-producing genetically engineered bacteria
Note: * P value <0.01, indicating a significant difference from the control.
As can be seen from Table 7, the recombinant E.coli L-threonine produced higher than the control strain by introducing the same DcuA protein variant after replacement of the underpan cells. It contains dcuA A12G The yield of the modified strain MHZ-0220-1 threonine is 16.37g/L, the shake flask conversion rate is 19.26%, the yield is improved by 18.62% compared with that of the original strain, and the sugar acid conversion rate is improved by 18.67%. Containing dcuA F14L The yield of the modified strain MHZ-0220-2 threonine is 16.91g/L, the shake flask conversion rate is 19.89%, the yield is increased by 22.54% compared with that of the original strain, and the sugar acid conversion rate is increased by 22.55%. The yield of the dCuA strain containing two point mutations and MHZ-0220-3 threonine is 18.82g/L, the shake flask conversion rate is 22.14%, the yield is improved by 36.38% compared with that of the original strain, and the sugar acid conversion rate is improved by 36.41%. It was again demonstrated from the shake flask results that E.coli containing DcuA protein variants could significantly improve threonine productivity. Similarly, the content of isoleucine in the fermentation liquor of the downstream product of threonine is obviously increased, wherein the content of isoleucine in the modified strain MHZ-0220-3 is increased by 75.56% compared with that in the control strain. From this, it can be deduced that the DcuA mutant is introduced into the mutantThe leucine producing bacteria can also obviously improve the yield of isoleucine and the sugar-acid conversion rate.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
- A DcuA protein mutant, which is characterized in that it is any one of the following (1) to (3):(1) the 12 th amino acid of the DcuA protein is mutated from A to G;(2) the 14 th amino acid of the DcuA protein is mutated from F to L;(3) the 12 th amino acid of the DcuA protein is mutated from A to G, and the 14 th amino acid is mutated from F to L;among them, the reference sequence number of the DcuA protein on NCBI is NP-418561.1.
- 2. A nucleic acid molecule encoding a mutant DcuA protein of claim 1.
- 3. Biological material comprising a nucleic acid molecule according to claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.
- 4. Use of the nucleic acid molecule of claim 2 or the biomaterial of claim 3 for any one of:(1) The method is used for amino acid fermentation production;(2) Used for improving the fermentation yield of amino acid;(3) Used for constructing gene engineering bacteria for producing amino acid;wherein the amino acid is selected from at least one of L-threonine, isoleucine and glycine.
- 5. A method for constructing L-threonine-producing genetic engineering bacteria is characterized in that a genetic engineering means is utilized to introduce mutation into a bacterial genome with threonine production capacity, so that the encoded C4-dicarboxylate transport protein contains A12G or F14L mutation sites, or contains the two mutation sites at the same time;preferably, the bacterium is an Escherichia (Escherichia) species, more preferably Escherichia coli (Escherichia coli).
- 6. The method of claim 5, comprising the steps of:A. pTargetF-N20 (dcuA) plasmid and Donor DNA constructionA1: amplifying a pTF linear plasmid with N20 by using a pTARgetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (A12G) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying an up-dcuA-down full-length fragment, which is also called Donor DNA-1, by using the dcuA-UF/dcuA-DR primer pair by taking the (1) and the (2) as templates;wherein dcuA is a C4-dicarboxylate transporter gene;B. competent cell preparation and electrotransformationB1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-1 into MHZ-0217-4 (pCas) competent cells, coating the competent cells on an LB plate containing spectinomycin and kanamycin, and performing static culture at 30 ℃ until a single colony is visible;C. reconstitution validationC1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G) -F1/dcuA-R; c2: using a primer pair dcuA-F/dcuA-R to amplify a target fragment, and sequencing an amplification product to verify the integrity of a sequence;D. construction of the relevant plasmid lossD1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; d2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: selecting a single colony to be spotted on an LB plate containing kanamycin and an LB plate without resistance, if the single colony can not grow on the LB plate containing kanamycin and can not grow on the LB plate without resistance, showing that pCas plasmid is lost, obtaining the L-threonine producing genetic engineering bacteria which are marked as MHZ-0219-1 (dcuA) A12G )；The sequences of the primers used in the above method are shown in Table 1.
- 7. The method of claim 5, comprising the steps of:A. construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-2A1: amplifying a pTF linear plasmid with N20 by using a pTargetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly ClonExpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (F14L) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying up-dcuA-down full-length fragment, also called Donor DNA-2, by using dcuA-UF/dcuA-DR primer pair with the (1) and (2) as templates;wherein dcuA is a C4-dicarboxylate transport protein gene;B. competent cell preparation and electrotransformationB1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-2 into MHZ-0217-4 (pCas) competent cells, coating the competent cells on an LB plate containing spectinomycin and kanamycin, and performing static culture at 30 ℃ until a single colony is visible;C. reconstitution validationC1: performing colony PCR verification on the single colony by using a primer pair dcuA (F14L) -F1/dcuA-R; c2: using a primer pair dcuA-F/dcuA-R to amplify a target fragment, and sequencing an amplification product to verify the integrity of a sequence;D. construction of the relevant plasmid lossD1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and a final concentration of 0.5mM IPTG, culturing overnight at 30 ℃, and streaking on an LB flat plate containing kanamycin; d2: picking a single colony to be spotted on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing the colony overnight at the temperature of 30 ℃, wherein if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, the colony grows on the LB plate containing kanamycin and the spectinomycin, which indicates that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: selecting a single colony point to be spotted on an LB plate containing kanamycin and an LB plate without resistance, if the single colony point can not grow on the LB plate containing kanamycin, the single colony point grows on the LB plate without resistance, which shows that pCas plasmid is lost, and the gene engineering bacteria producing L-threonine is obtained and is marked as MHZ-0219-2 (dcuA) F14L )；The sequences of the primers used in the above method are shown in Table 1.
- 8. The method according to claim 5, characterized in that it comprises the following steps:A. construction of pTargetF-N20 (dcuA) plasmid and Donor DNA-3A1: amplifying a pTF linear plasmid with N20 by using a pTARgetF plasmid as a template and a pTF-sgRNA-F/pTF-sgRNA-R primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20 (dcuA) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (1) by using a dcuA-UF/dcuA (A12G, F14L) -UR primer pair by using a W3110 genome as a template; a3: amplifying a downstream homology arm (2) by using a dcuA (A12G, F14L) -DF/dcuA-DR primer pair by using a W3110 genome as a template; a4: amplifying up-dcuA-down full-length fragment, also called Donor DNA-3, by using dcuA-UF/dcuA-DR primer pair with the (1) and (2) as templates;wherein dcuA is a C4-dicarboxylate transport protein gene;B. competent cell preparation and electrotransformationB1: electrically transferring the pCas plasmid into MHZ-0217-4 competent cells to obtain a positive transformant MHZ-0217-4 (pCas); b2: a single MHZ-0217-4 (pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ at 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20 (dcuA) plasmid and Donor DNA-3 into MHZ-0217-4 (pCas) competent cells, coating the competent cells on an LB plate containing spectinomycin and kanamycin, and performing static culture at 30 ℃ until a single colony is visible;C. reconstitution validationC1: performing colony PCR verification on the single colony by using a primer pair dcuA (A12G, F14L) -F1/dcuA-R;c2: using a primer to amplify the dcuA-F/dcuA-R target fragment, and sequencing an amplification product to verify the integrity of the sequence;D. construction of related plasmid lossesD1: single colonies with correct sequencing were picked and inoculated into 5mL of L containing kanamycin and 0.5mM IPTG to a final concentrationIn the test tube B, after overnight culture at 30 ℃, streaked on an LB plate containing kanamycin; d2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that the pTargetF-N20 (dcuA) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20 (dcuA) plasmid, inoculating into an anti-LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: picking single colony spot on an LB plate containing kanamycin and an LB plate without resistance, if the colony can not grow on the LB plate containing kanamycin, the colony grows on the LB plate without resistance, which shows that pCas plasmid is lost, and obtaining the L-threonine producing genetically engineered bacterium which is marked as MHZ-0219-3 (dcuA) A12G,F14L )；The sequences of the primers used in the above method are shown in Table 1.
- 9. The L-threonine-producing genetically engineered bacterium constructed according to any one of claims 5 to 8.
- 10. Use of the genetically engineered bacterium producing L-threonine of claim 9 for the fermentative production of L-threonine or for increasing the fermentative production of L-threonine.
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