CN115029289A - Genetic engineering bacterium for high yield of L-threonine and construction method and application thereof - Google Patents
Genetic engineering bacterium for high yield of L-threonine and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a genetic engineering bacterium for high yield of L-threonine and a construction method and application thereof. The present invention also provides a method for increasing the fermentation yield of threonine, which comprises attenuating the isocitrate dehydrogenase gene in a fermentative strain, i.e., a bacterium having threonine-producing ability. The invention reduces the synthesis of isocitrate dehydrogenase of the strain and reduces CO from isocitrate to alpha-ketoglutarate and succinyl coenzyme A by weakening the isocitrate dehydrogenase gene in the fermentation strain 2 Thereby reducing the loss of carbon source and improving the threonine producing capacity and the sugar acid conversion rate of the strain.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a genetic engineering bacterium for high yield of 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. 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 threonine in the world, the construction and modification of high-yield threonine strains are particularly important. In chinese patent CN03811059.8 applied by korean CJ corporation in 2003, the expression of threonine synthesis key gene thrABC was enhanced by deleting 39bp sequence from-56 to-18 of threonine operon sequence using escherichia coli, and 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; 3:149) and the like utilize a system metabolic engineering strategy, the feedback inhibition of products is relieved by mutating the genes thrA and lysC for encoding aspartate kinase I and III, byproducts glycine and isoleucine are removed by knocking out tdh and weakening ilvA, more precursors and the like are provided for threonine synthesis by inactivating competitive pathway genes metA and lysA, and the finally obtained TH28C (pBRThrABCR3) strain can produce 82.4g/L of acid after being fermented for 50h, and the conversion rate of the acid is 39.3%. In Chinese patent ZL201611250306.8 applied by the plum blossom group in 2016, MHZ-0215-2 strain is obtained by strengthening pntAB gene and heterogeneously introducing pyc gene, and the strain has threonine yield of 12.4g/L, conversion rate of about 16.2% and no plasmid load. The icd gene codes for isocitrate dehydrogenase IDH, which is mostly present as a homodimer with one active center per subunit, two binding sites. IDH is the rate-limiting enzyme of the bacterial TCA cycle, and plays an important role in the regulation of energy and substance metabolism, and its oppositeCatalyzing the oxidative decarboxylation of isocitric acid to generate alpha-ketoglutaric acid and CO 2 。
Disclosure of Invention
The invention aims to provide a genetic engineering bacterium for high yield of L-threonine and a construction method and application thereof.
To achieve the objects of the present invention, in a first aspect, the present invention provides a method for increasing the fermentation yield of L-threonine, the method comprising weakening an isocitrate dehydrogenase gene or a gene encoding an isocitrate dehydrogenase that has 80% or more homology with the amino acid sequence of the isocitrate dehydrogenase and expresses the same functional protein in a fermentative strain; the fermentation strain is a bacterium having threonine producing ability.
Further, the bacterium is an Escherichia (Escherichia) or Corynebacterium (Corynebacterium) strain, preferably Escherichia coli (Escherichia coli) or Corynebacterium glutamicum (Corynebacterium glutamicum), more preferably strain MHZ-0215-2 with the preservation number of CGMCC No. 13403. See ZL 201611250306.8; more preferably Corynebacterium glutamicum SMCT 033.
The strain SMCT033 is constructed according to a cereal bar classical method (C.glutamicum Handbook, Charpter 23), Corynebacterium glutamicum ATCC13032 is used as an initial strain, feedback inhibition of lysC genes is relieved and expression of the lysC genes is enhanced, feedback inhibition of hom and thrB is relieved and expression of the thrC genes is enhanced, and pck and pyk genes are inactivated. Wherein, the reference sequence numbers of lysC, hom, thrB, thrC, pck and pyk on NCBI are 1021294, 1019166, 1019167, 1020172, 1020806 and 1020040 respectively.
In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium producing L-threonine in high yield, which comprises weakening or inactivating the isocitrate dehydrogenase gene in Escherichia coli or Corynebacterium glutamicum, and using the resulting gene-weakened strain in the fermentative production of threonine.
Wherein, the reference sequence number of the escherichia coli isocitrate dehydrogenase Gene (icd) on the NCBI is U00096.3NC _000913.3(1195123..1196373), Gene ID: 945702.
gene ID of Corynebacterium glutamicum isocitrate dehydrogenase Gene at NCBI: 1018663.
the attenuation or inactivation may be at least one selected from the group consisting of attenuation of the initiation codon of the isocitrate dehydrogenase gene, attenuation of a promoter, attenuation or inactivation of an RBS sequence, and the like.
Preferably, the initiation codon attenuation is the replacement of the initiation codon ATG of the isocitrate dehydrogenase gene with TTG.
Preferably, the promoter attenuation is the replacement of the promoter of the isocitrate dehydrogenase gene with a weak promoter.
Preferably, the RBS sequence attenuation is the replacement of the RBS sequence of the isocitrate dehydrogenase gene with an RBS sequence which is regulated by threonine.
In the above method, the Escherichia coli is strain MHZ-0215-2.
The corynebacterium glutamicum is a strain SMCT 033.
In one embodiment of the present invention, a method for constructing a genetically engineered bacterium with high L-threonine productivity comprises the following steps:
A. construction of pTargetF-N20(icd) plasmid and Donor DNA-1
A1: amplifying a pTF linear plasmid with N20 by using a pTF-sgRNA-F1/pTF-sgRNA-R1 primer pair by using a pTargetF plasmid as a template, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargetF-N20(icd) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (i) of the icd by using a W3110 genome as a template and an icd-UF/icd-UR primer pair; a3: using a W3110 genome as a template, and selecting an icd-DF/icd-DR primer pair to amplify a downstream homology arm II of icd; a4: using the first and the second as templates, and using an icd-UF/icd-DR primer pair to amplify an up-icd-down fragment, which is also called Donor DNA-1;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0215-2 competent cells to obtain a positive transformant MHZ-0215-2 (pCas); b2: a single MHZ-0215-2(pCas) colony was picked up and cultured in 5mL of LB tube containing kanamycin and arabinose at a final concentration of 10mM (LB tube: tube containing LB liquid medium) at 30 ℃ at 200r/min to OD 650 After 0.4, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20(icd) plasmid and Donor DNA-1 into MHZ-0215-2(pCas) competent cells, coating on LB plate containing spectinomycin and kanamycin, and standing at 30 ℃ for culturing until a single colony is visible;
C. recombination verification
C1: carrying out colony PCR verification on the single colony by using a primer pair icd-F/icd-R; c2: amplifying the target fragment by using a primer pair icd-F/icd-R, and sequencing the amplified 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 single colony spot on LB plate containing kanamycin and spectinomycin and LB plate containing only kanamycin, culturing overnight at 30 ℃, if the colony can not grow on LB plate containing kanamycin and spectinomycin, and growing on LB plate containing kanamycin, indicating that pTargetF-N20(icd) plasmid is lost; d3: picking positive colonies lost by pTargetF-N20(icd) plasmid, inoculating into non-anti LB test tube (non-anti LB test tube: test tube filled with LB liquid culture medium without antibiotic), culturing at 42 deg.C for 8h, streaking on LB plate, and culturing at 37 deg.C overnight; d4: and selecting a single colony to spot on an LB plate containing kanamycin and an LB plate without resistance, if the single colony cannot grow on the LB plate containing kanamycin, growing on the LB plate without resistance, indicating that pCas plasmid is lost, and obtaining the genetically engineered bacterium with high threonine yield, which is named MHZ-0221-1.
In another embodiment of the present invention, a method for constructing a genetically engineered bacterium that produces L-threonine at a high yield comprises the steps of:
A. construction of pTargetF-N20(icd) plasmid and Donor DNA-2
A1: amplifying a pTF linear plasmid with N20 by using a pTargitF plasmid as a template and a pTF-sgRNA-F2/pTF-sgRNA-R2 primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargitF-N20 (icd) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm I containing a zwf promoter by using a Pzwf-UF/Pzwf-UR primer pair by using a W3110 genome as a template; a3: amplifying a zwf promoter by using a Pzwf-F/Pzwf-R primer pair by using a W3110 genome as a template; a4: amplifying a downstream homology arm (c) containing a zwf promoter by using a Pzwf-DF/Pzwf-DR primer pair by using a W3110 genome as a template; a5: amplifying up-Pzwf-down fragments, which are also called Donor DNA-2, by using Pzwf-UF/Pzwf-DR primer pairs by taking the first, the second and the third as templates;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0215-2 competent cells; b2: 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, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20(icd) plasmid and Donor DNA-2 into MHZ-0215-2(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. recombination verification
C1: performing colony PCR verification on the single colony by using a primer pair Pzwf-AF/Pzwf-AR; c2: amplifying a target fragment by using a primer pair Pzwf-AF/Pzwf-AR, 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(icd) plasmid is lost; d3: selecting positive colonies lost by pTargetF-N20(icd) 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: and selecting a single colony to spot on an LB plate containing kanamycin and an LB plate without resistance, if the single colony cannot grow on the LB plate containing kanamycin, growing on the LB plate without resistance, indicating that pCas plasmid is lost, and obtaining the genetically engineered bacterium with high threonine yield, namely the MHZ-0221-2 strain.
In still another embodiment of the present invention, a method for constructing a genetically engineered bacterium that produces L-threonine at a high yield comprises the steps of:
A. construction of pTargetF-N20(icd) plasmid and Donor DNA-3
A1: amplifying a pTF linear plasmid with N20 by using a pTargitF plasmid as a template and a pTF-sgRNA-F3/pTF-sgRNA-R3 primer pair, assembling the linear plasmid at 37 ℃ by using a seamless assembly Clonexpress kit, then transforming Trans1-T1 competent cells to obtain a pTargitF-N20 (icd) plasmid, and carrying out PCR identification and sequencing verification; a2: amplifying an upstream homology arm (i) containing RBSthrR by using a W3110 genome as a template and an RBSthrR-UF/RBSthrR-UR primer pair; a3: using W3110 genome as template, and using RBSthrR-F/RBSthrR-R primer pair to amplify RBSthrR; a4: using W3110 genome as template, selecting RBSthrR-DF/RBSthrR-DR primer pair to amplify downstream homology arm (③) containing RBSthrR; a5: amplifying up-RBSthrR-down fragment, also called Donor DNA-3, by using RBSthrR-UF/RBSthrR-DR primer pair with the first, second and third as template;
B. competent cell preparation and electrotransformation
B1: electrically transferring the pCas plasmid into MHZ-0215-2 competent cells; b2: 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, preparing electrotransferase competent cells; b3: simultaneously transferring pTargetF-N20(icd) plasmid and Donor DNA-3 into MHZ-0215-2(pCas) competent cells, coating on 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 RBSthrR-AF/RBSthrR-AR; c2: amplifying the target fragment of the RBSthrR-AF/RBSthrR-AR by using a primer, and sequencing the amplified 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 single colony spot on LB plate containing kanamycin and spectinomycin and LB plate containing only kanamycin, culturing overnight at 30 ℃, if the colony can not grow on LB plate containing kanamycin and spectinomycin, and growing on LB plate containing kanamycin, indicating that pTargetF-N20(icd) plasmid is lost; d3: picking positive colonies lost by pTargetF-N20(icd) plasmid, inoculating the positive colonies in a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; d4: and selecting a single colony to spot on an LB plate containing kanamycin and an LB plate without resistance, if the single colony cannot grow on the LB plate containing kanamycin, growing on the LB plate without resistance, indicating that pCas plasmid is lost, and obtaining the genetically engineered bacterium with high threonine yield, namely MHZ-0221-3 strain.
The sequences of the primers used in the above method are shown in Table 1.
In a third aspect, the present invention provides a genetically engineered bacterium that produces L-threonine with high yield, constructed according to the above method.
In a fourth aspect, the invention provides a method for improving the fermentation yield of L-threonine, or an application of the genetically engineered bacterium with high L-threonine yield, which is constructed according to the method, in the fermentation production of L-threonine.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention reduces the synthesis of isocitrate dehydrogenase of the strain and reduces CO from isocitrate to alpha-ketoglutarate and succinyl coenzyme A by weakening the isocitrate dehydrogenase gene in the fermentation strain 2 Thereby reducing the loss of carbon source, and improving the L-threonine producing capacity and the sugar-acid conversion rate of the strain. Wherein, the average acid yield of the bacterial strain MHZ-0221-1 shake flask after the initiation codon is weakened is 16.03g/L, which is increased by 41.23 percent compared with that of the starting bacterial strain; the average acid production of the bacterial strain MHZ-0221-2 after the weakening of the promoter in a shake flask is 12.47g/L, which is improved by 9.86 percent compared with that of the starting bacterial strain; the strain MHZ-0221-3 after RBS replacement produces acid on average in a shake flask by 13.24g/L, which is 16.65 percent higher than that of the original strain. The average acid production of the corynebacterium glutamicum icd inactivated strain SMCT034 is 22.4g/L, and the threonine yield is increased by 11.44% compared with that of the original strain, which shows that the inactivation of icd can improve the threonine production capacity of corynebacterium glutamicum.
Detailed Description
The present invention provides a method for constructing a threonine (L-threonine) producing strain with an improved sugar acid conversion rate and a method for producing threonine (L-threonine). The following technical scheme is adopted:
the invention takes recombinant Escherichia coli MHZ-0215-2 or Corynebacterium glutamicum SMCT033 as an original strain, and the MHZ-0215-2 preservation number is CGMCC No.13403, see ZL 201611250306.8. The strain MHZ-0215-2 belongs to W3110 (Escherichia genus), and SMCT033 belongs to ATCC13032 (Corynebacterium genus). According to the metabolic pathway of L-threonine in escherichia coli and corynebacterium glutamicum and the genetic background of starting strains MHZ-0215-2 and SMCT033, related transformation is carried out on genomes of the L-threonine and the corynebacterium glutamicum so as to weaken isocitrate dehydrogenase synthesis of the strains. The method specifically comprises the following steps: attenuation of the start codon or replacement of the original promoter with a weak promoter or replacement of the original RBS sequence with a threonine-regulated RBS sequence and inactivation of the icd sequence. For example, the expression of isocitrate dehydrogenase (icd gene-encoded) is attenuated by replacing the original initiation codon ATG with a weak initiation codon TTG and using the promoter P zwf Replacement of the original promoter P icd And replacing the original RBSicd, icd gene sequence deletion by RBSthrR (consisting of an RBS sequence of thrL, a thrL sequence and an RBS sequence of thrA). Reduction of CO in isocitrate to alpha-ketoglutarate and succinyl-CoA 2 Thereby reducing carbon source loss, to improve the ability of the strain to produce threonine.
The Genome Editing method of Escherichia coli mainly refers to the CRISPR-Cas9 gene Editing technology (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. The gene editing method of corynebacterium glutamicum mainly refers to the classical method of corynebacterium glutamicum (c.glutamicum Handbook, Charpter 23).
In the following examples, the final concentration of Kanamycin (Kanamycin) in the medium was 50. mu.g/mL, and the final concentration of spectinomycin (spectinomycin) in the medium was 50. mu.g/mL.
The reagents used in the following examples are all commercially available. pTargetF plasmid and pCas plasmid are provided by the plum blossom group species research laboratory. The construction method of pTargetF plasmid and pCas plasmid can refer to CRISPR-Cas9 gene Editing technology reported by Jiang Y et al (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. The sequences of the primers used in the present invention are shown in Table 1.
TABLE 1
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.
Example 1 preparation of a Strain attenuated for the icd Gene MHZ-0221-1(icd initiation codon point mutation A1T)
1. Construction of pTargetF-N20(icd) plasmid and Donor DNA-1
Step 1: using pTargetF plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015), selecting pTF-sgRNA-F1/pTF-sgRNA-R1 primer pair, amplifying pTF linear plasmid with N20, assembling the linear plasmid at 37 ℃ by using seamless assembly ClonExpress kit, then transforming Trans1-T1 competent cells to obtain pTargetF-N20(icd), and carrying out PCR identification and sequencing verification; step 2: using a W3110 genome as a template, and selecting an icd-UF/icd-UR primer pair to amplify an upstream homology arm (i) of icd; step 3: using a W3110 genome as a template, and selecting an icd-DF/icd-DR primer pair to amplify a downstream homology arm II of icd; step 4: using the first and the second as templates, and selecting an icd-UF/icd-DR primer pair to amplify an up-icd-down fragment, which is also called Donor DNA-1.
2. Competent cell preparation and electrotransformation
Step 1: the 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) was electroporated into MHZ-0215-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); step 2: 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). Step 3: pTargetF-N20(icd) plasmid and Donor DNA-1 were simultaneously electroporated into MHZ-0215-2(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 a single colony was visible.
3. Reconstitution validation
Step 1: carrying out colony PCR verification on the single colony by using a primer pair icd-F/icd-R; step 2: and (3) amplifying the target fragment by using the primer pair icd-F/icd-R, and sequencing the amplified product to verify the integrity of the sequence.
4. Construction of related plasmid losses
Step 1: 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; step 2: picking single colony spot on LB plate containing kanamycin and spectinomycin and LB plate containing only kanamycin, culturing overnight at 30 ℃, if the colony can not grow on LB plate containing kanamycin and spectinomycin, and growing on LB plate containing kanamycin, indicating that pTargetF-N20(icd) plasmid is lost; step 3: picking positive colonies lost by pTargetF-N20(icd) plasmid, inoculating the positive colonies in a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step 4: single colonies were picked and spotted 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, resulting in MHZ-0221-1 strain.
Example 2 preparation of icd Gene-attenuated Strain MHZ-0221-2 (promoter attenuated)
1. Construction of pTargetF-N20(icd) plasmid and Donor DNA-2
Step 1: pTargetF plasmid is taken as a template (from Multigene edition in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015), pTF-sgRNA-F2/pTF-sgRNA-R2 primer pair is selected to amplify pTF linear plasmid with N20, the linear plasmid is assembled at 37 ℃ by using a seamless assembly Clonexpress kit, then Trans1-T1 competent cells are transformed to obtain pTargetF-N20(icd), and PCR identification and sequencing verification are carried out; step 2: using a W3110 genome as a template, and selecting a Pzwf-UF/Pzwf-UR primer pair to amplify an upstream homology arm (I) containing a zwf promoter; step 3: using a W3110 genome as a template, and selecting a Pzwf-F/Pzwf-R primer pair to amplify a zwf promoter II; wherein, the reference sequence number of the zwf Gene on NCBI is U00096.3NC _000913.3(1934839..1936314), Gene ID: 946370, respectively; step 4: using a W3110 genome as a template, and selecting a Pzwf-DF/Pzwf-DR primer pair to amplify a downstream homology arm (c) containing a zwf promoter; step 5: using the first, the second and the third as templates, selecting Pzwf-UF/Pzwf-DR primer pair, and amplifying up-Pzwf-down fragment, also called Donor DNA-2.
2. Competent cell preparation and electrotransformation
Step 1: the 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) was electroporated into MHZ-0215-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); 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). Step 3: pTargetF-N20(icd) plasmid and Donor DNA-2 were simultaneously electroporated into MHZ-0215-2(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 colonies were visible.
3. Recombination verification
Step 1: performing colony PCR verification on the single colony by using a primer pair Pzwf-AF/Pzwf-AR; step 2: and (3) amplifying the target fragment by using a primer pair Pzwf-AF/Pzwf-AR, and sequencing the amplified product to verify the integrity of the sequence.
4. Construction of related plasmid losses
Step 1: 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; step 2: 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(icd) plasmid is lost; step 3: picking positive colonies lost by pTargetF-N20(icd) plasmid, inoculating the positive colonies in a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step 4: single colonies were picked and spotted 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, resulting in MHZ-0221-2 strain.
Example 3 preparation of an icd Gene-attenuated Strain MHZ-0221-3(RBS Regulation)
1. Construction of pTargetF-N20(icd) plasmid and Donor DNA-3
Step 1: using pTargetF plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015), selecting pTF-sgRNA-F3/pTF-sgRNA-R3 primer pair, amplifying pTF linear plasmid with N20, assembling the linear plasmid at 37 ℃ by using seamless assembly ClonExpress kit, then transforming Trans1-T1 competent cells to obtain pTargetF-N20(icd), and carrying out PCR identification and sequencing verification; step 2: using W3110 genome as template, selecting RBSthrR-UF/RBSthrR-UR primer pair, and amplifying to obtain upstream homology arm (I) containing RBSthrR; step 3: using W3110 genome as template, selecting RBSthrR-F/RBSthrR-R primer pair to amplify RBSthrR; step 4: using W3110 genome as template, selecting RBSthrR-DF/RBSthrR-DR primer pair to amplify downstream homology arm (③) containing RBSthrR; step 5: using primer pair RBSthrR-UF/RBSthrR-DR as template to amplify up-RBSthrR-down fragment, also called Donor DNA-3.
2. Competent cell preparation and electrotransformation
Step 1: the 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) was electroporated into MHZ-0215-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); step 2: 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). Step 3: pTargetF-N20(icd) plasmid and Donor DNA-3 were simultaneously electroporated into MHZ-0215-2(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 colonies were visible.
3. Recombination verification
Step 1: performing colony PCR verification on the single colony by using a primer pair RBSthrR-AF/RBSthrR-AR; step 2: and (3) amplifying the target fragment by using a primer pair RBSthrR-AF/RBSthrR-AR, and sequencing an amplification product to verify the integrity of the sequence.
4. Construction of related plasmid losses
Step 1: selecting single colony with correct sequencing verification, inoculating to 5mL of kanamycin-containing and final concentrationIn an LB test tube with the concentration of 0.5mM IPTG, after overnight culture at 30 ℃, streaking on an LB flat plate containing kanamycin; step 2: picking single colony spot on LB plate containing kanamycin and spectinomycin and LB plate containing only kanamycin, culturing overnight at 30 ℃, if the colony can not grow on LB plate containing kanamycin and spectinomycin, and growing on LB plate containing kanamycin, indicating that pTargetF-N20(icd) plasmid is lost; step 3: picking positive colonies lost by pTargetF-N20(icd) plasmid, inoculating the positive colonies in a non-anti LB test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step 4: single colonies were picked and spotted 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, resulting in MHZ-0221-3 strain. Example 4 recombinant plasmid pK18mobsacB-P sod -lysC V1M-T311I Construction of (2) and substitution in ATCC13032
(1)pK18mobsacB-P sod -lysC V1M-T311I Construction of plasmids
Taking ATCC13032 genome as a template, carrying out PCR amplification by using a primer pair P21/P22 to obtain an upstream homology arm up, and carrying out PCR amplification by using a primer pair P23/P24 to obtain a promoter fragment P sod PCR amplification with primer pair P25/P26 to obtain lysC V1M-T311I And performing PCR amplification by using a primer pair P27/P28 to obtain a downstream homology arm dn. The primer pair P21/P24 is used as up and P sod Performing fusion PCR to obtain fragment up-P sod . The primer pair P21/P28 is used as up-P sod 、lysC V1M-T311I And dn as template for fusion PCR to obtain full-length segment up-P sod -lysC V1M-T311I -dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two enzyme digestion products are connected by T4 DNA ligase, and the Trans1T1 competent cell is transformed, thus obtaining the recombinant plasmid pK18mobsacB-P sod -lysC V1M-T311I 。
(2) Replacement of lysC in ATCC13032
ATCC13032 competent cells were prepared according to the classical method of cereal rods (c. glutamicum Handbook, Charpter 23). Recombinant plasmid pK18mobsacB-P sod -lysC V1M-T311I Transformation of the sensation by electroporationThe cells were kept in the normal state, and transformants were selected on a selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the temperature of 30 ℃ under the condition of shaking at 220rpm of a rotary shaking table. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) -2 Continuously diluting to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10 percent of sucrose, and the brain heart infusion culture medium is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target sequence is amplified through PCR, and the obtained mutant strain is named as SMCT018 through nucleotide sequencing analysis. Example 5 recombinant plasmid pK18mobsacB-P cspB -hom G378E Construction of (3) and replacement in SMCT018
(1)pK18mobsacB-P cspB -hom G378E Construction of plasmids
Taking ATCC13032 genome as a template, carrying out PCR amplification by using a primer pair P29/P30 to obtain an upstream homology arm up, and carrying out PCR amplification by using a primer pair P31/P32 to obtain a promoter fragment P cspB PCR amplification with primer pair P33/P34 to obtain hom G378E And performing PCR amplification by using a primer pair P35/P36 to obtain a downstream homology arm dn. The primer pair P29/P32 is used as up and P scpB Performing fusion PCR to obtain fragment up-P cspB . The primer pair P29/P36 is used as up-P cspB 、hom G378E And dn as template for fusion PCR to obtain full-length segment up-P cspB -hom G378E -dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two enzyme digestion products are connected by T4 DNA ligase, and the Trans1T1 competent cells are transformed to obtain the recombinant plasmid pK18mobsacB-P cspB -hom G378E 。
(2) Hom substitutions at SMCT018
SMCT018 competent cells were prepared according to the cereal bar classical method (c. glutamicum Handbook, Charpter 23). Recombinant plasmid pK18mobsacB-P cspB -hom G378E Transforming the competent cells by electroporationCells, and transformants were selected on a selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and performing shaking culture on a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. Cultures were serially diluted in gradient (10) -2 Continuously diluting to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target mutant strain is obtained by PCR amplification of a target sequence and nucleotide sequencing analysis and is named as SMCT 019.
Example 6 recombinant plasmid pK18mobsacB-P sod -thrC V1M Construction of (2) and substitution in SMCT019
(1)pK18mobsacB-P sod -thrC V1M Construction of plasmids
Taking ATCC13032 genome as a template, carrying out PCR amplification by using a primer pair P37/P38 to obtain an upstream homology arm up, and carrying out PCR amplification by using a primer pair P39/P40 to obtain a promoter fragment P sod -thrC V1M The dn was obtained by PCR amplification with primer pair P41/P42. The primer pair P37/P42 is used as up and P sod -thrC V1M And dn as template to perform fusion PCR to obtain fragment up-P sod -thrC V1M -dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two enzyme digestion products are connected by T4 DNA ligase to transform Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-P is obtained sod -thrC V1M 。
(2) thrC replacement at SMCT019
SMCT019 competent cells were prepared according to the cereal bar classical method (c. glutamicum Handbook, Charpter 23). Recombinant plasmid pK18mobsacB-P sod -thrC V1M The competent cells were transformed by electroporation, and transformants were selected on a selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. To-be-screenedThe obtained transformant is cultured in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and is subjected to shaking culture by a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) -2 Continuously diluting to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target mutant strain is obtained by PCR amplification of a target sequence and nucleotide sequencing analysis and is named as SMCT 021.
Example 7 construction of recombinant plasmid pK18mobsacB- Δ pck and inactivation of pck in SMCT021
(1) Construction of pK18mobsacB- Δ pck plasmid
Taking an ATCC13032 genome as a template, carrying out PCR amplification by using a P5/P6 primer pair to obtain an upstream fragment up, carrying out PCR amplification by using a P7/P8 primer pair to obtain a downstream fragment dn, and carrying out fusion PCR by using a P5/P8 primer pair to obtain a fragment up-dn by using up and dn as templates. The up-dn fragment was double digested with BamHI, HindIII and pK18mobsacB with the same enzymes. The two digestion products are connected by T4 DNA ligase, and Trans1T1 competent cells are transformed, so that the recombinant plasmid pK18 mobsacB-delta pck is obtained.
(2) Knock-out of pck in SMCT021
SMCT021 competent cells were prepared according to the classical method of cereal bars (c. glutamicum Handbook, Charpter 23). The competent cells were transformed with the recombinant plasmid pK18 mobsacB-. DELTA.pck by electroporation, and transformants in which the gene of interest was inserted into the chromosome due to homology were selected on a selection medium containing 15mg/L kanamycin. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 30 ℃ and performing shaking culture on a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. Cultures were serially diluted in gradient (10) -2 Diluting continuously to 10 -4 ) Coating the diluted solution on common solid brain heart infusion culture medium containing 10% sucrose, and standing at 33 deg.CAnd (5) cultivating for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target sequence is amplified by PCR, and the obtained mutant strain is named as SMCT023 by nucleotide sequencing analysis.
Example 8 construction of recombinant plasmid pK18 mobsacB-Delta pyk and inactivation pyk in SMCT023
(1) Construction of pK18 mobsacB-Delta pyk plasmid
Taking an ATCC13032 genome as a template, carrying out PCR amplification by using a P13/P14 primer pair to obtain an upstream fragment up, carrying out PCR amplification by using a P15/P16 primer pair to obtain a downstream fragment dn, and carrying out fusion PCR by using a P13/P16 primer pair to obtain a fragment up-dn by using up and dn as templates. The up-dn fragment was digested simultaneously with BamHI and HindIII, and pK18mobsacB was digested simultaneously with the same enzymes. The two enzyme products are connected by T4 DNA ligase, and Trans1T1 competent cells are transformed, thus obtaining the recombinant plasmid pK18 mobsacB-delta pyk.
(2) Knock-out pyk in SMCT023
SMCT023 competent cells were prepared according to the classical method of cereal rods (c. glutamicum Handbook, Charpter 23). The recombinant plasmid pK18 mobsacB-. DELTA. pyk transformed the competent cells by electroporation, and transformants were selected on selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the temperature of 30 ℃ under the condition of shaking at 220rpm of a rotary shaking table. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) -2 Diluting continuously to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10 percent of sucrose, and the brain heart infusion culture medium is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target mutant strain is obtained by PCR amplification of a target sequence and nucleotide sequencing analysis and is named as SMCT031
EXAMPLE 9 construction of plasmid pEKEx2-P sod -lysC V1M-T311I ,P cspB -hom G378E ,P sod -thrC V1M And is overexpressed in SMCT031
(1)pEKEx2-P sod -lysC V1M-T311I ,P cspB -hom G378E thrB,P sod -thrC V1M Construction of plasmids
Performing PCR amplification by using the SMCT021 genome as a template and a primer pair P17/P18 to obtain P sod -lysC V1M-T311I Carrying out PCR amplification on the fragment by using a primer pair P19/P20 to obtain a fragment P cspB -hom G378E thrB, fragment P was obtained by PCR amplification with primer pair P43/P44 sod -thrC V1M . Taking P17/P44 as a template and P sod -lysC V1M-T311I 、P cspB -hom G378E thrB、P sod -thrC V1M Performing fusion PCR to obtain P as template sod -lysC V1M-T311I -P cspB -hom G378E thrB-P sod -thrC V1M And (3) fragment. The fragment was digested with BamHI and pEKEx2 was digested with the same enzyme. The two digestion products are connected by T4 DNA ligase, and Trans1T1 competent cells are transformed to obtain a plasmid pEKEx2-P sod -lysC V1M-T311I ,P cspB -hom G378E thrB,P sod -P sod -thrC V1M， Named pMCT 1.
(2) Expression of the above plasmid in SMCT031
SMCT031 competent cells were prepared according to the cereal bar classical method (c. glutamicum Handbook, Charpter 23). Plasmid pEKEx2-P sod -lysC V1M-T311I ,P cspB -hom G378E thrB,P sod -thrC V1M The competent cells were transformed by electroporation and transformants were selected on selection medium containing 50mg/L kanamycin. The obtained mutant strain was named SMCT 033.
EXAMPLE 10 construction of icd deleted Corynebacterium glutamicum
(1) Construction of pK18 mobsacB-Delta icd plasmid
Taking Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 genome as a template, carrying out PCR amplification by using a Dicd-UF/Dicd-UR primer pair to obtain an upstream fragment up, carrying out PCR amplification by using a Dicd-DF/Dicd-DR primer pair to obtain a downstream fragment dn, and carrying out fusion PCR by using a Dicd-UF/Dicd-DR primer pair and up and dn as templates to obtain the fragment up-dn. The up-dn fragment was double digested with BamHI, HindIII and pK18mobsacB with the same enzymes. The two digestion products are connected by T4 DNA ligase, and Trans1T1 competent cells are transformed, so that the recombinant plasmid pK18 mobsacB-delta icd is obtained.
(2) Knock-out of icd in SMCT033
SMCT033 competent cells were prepared according to the cereal bar classical method (c. glutamicum Handbook, Charpter 23). The competent cells were transformed with the recombinant plasmid pK18mobsacB- Δ icd by electroporation, and transformants were selected on selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the temperature of 30 ℃ under the condition of shaking at 220rpm of a rotary shaking table. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) -2 Continuously diluting to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Strains grown on sucrose medium do not carry inserted vector sequences in their genome. The target mutant strain is obtained by PCR amplification of a target sequence and nucleotide sequencing analysis and is named as SMCT 034.
The threonine producing genetically modified strains constructed in examples 1 to 4 are shown in Table 2.
TABLE 2 genetically engineered bacteria constructed according to the present invention
Example 11 verification of shaking flask fermentation of genetically engineered bacteria producing L-threonine by Escherichia coli
Step1, taking 4 strains of MHZ-0215-2, MHZ-0221-1, MHZ-0221-2 and MHZ-0221-3 from a frozen tube, streaking and activating on an LB plate, and culturing for 24 hours at 37 ℃; step2, the cells were scraped from the plate and inoculated into a shaking flask containing 50mL of seed medium (Table 3) and cultured at 37 ℃ and 180rpm for about 5 hours to OD 650 Is controlled at 2The content of the compound is less than the content of the compound; step3, transferring 1mL of seed solution into a shake flask containing 50mL of fermentation medium (shown in Table 4), performing fermentation culture at 115rpm with a reciprocating shaker at 37 ℃ until residual sugar is exhausted, and measuring OD of a sample after fermentation is finished 650 And the content of L-threonine was measured by HPLC, and the amount of residual sugar was measured by biosensing. 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)
TABLE 4 fermentation Medium (g/L)
| Composition (I) | Concentration of |
| Glucose | 70 |
| Corn steep liquor | 10 |
| Soybean meal hydrolysate | 10 |
| Magnesium sulfate heptahydrate | 1.5 |
| KH 2 PO 4 | 1.0 |
| Aspartic acid | 10 |
| FeSO 4 | 30mg/L |
| MnSO 4 | 30mg/L |
| pH | 7.0 |
TABLE 5 comparison of productivity of threonine-producing genetically engineered bacteria
As can be seen from Table 5, the L-threonine yield of the novel Escherichia coli provided by the invention is higher than that of a control strain, wherein the average acid yield of the strain MHZ-0221-1 shake flask after the initiation codon is weakened is 16.03g/L, which is increased by 41.23% compared with that of the starting strain; the average acid production of the bacterial strain MHZ-0221-2 after the weakening of the promoter in a shake flask is 12.47g/L, which is improved by 9.86 percent compared with that of the starting bacterial strain; the average acid yield of the strain MHZ-0221-3 after RBS replacement in a shake flask is 13.24g/L, which is improved by 16.65 percent compared with that of the original strain. According to the results of the shake flask, the threonine production capacity of the modified strain is obviously superior to that of the starting strain MHZ-0215-2, so that the threonine production capacity can be obviously improved due to the weakened expression of the isocitrate dehydrogenase icd.
EXAMPLE 12L-threonine production by fermentation of C.glutamicum
1. Culture medium
Seed activation medium: BHI 3.7%, agar 2%, pH 7.
Seed culture medium: 5/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 16g/L of ammonium sulfate, 8g/L of urea, 10.4g/L of potassium dihydrogen phosphate, 21.4g/L of dipotassium hydrogen phosphate, 5mg/L of biotin and 3g/L of magnesium sulfate. Glucose 50g/L, pH 7.2.
Fermentation medium: 50mL/L of corn steep liquor, 50g/L of glucose, 4g/L of ammonium sulfate, 30g/L of MOPS, 10g/L of monopotassium phosphate, 20g/L of urea, 10mg/L of biotin, 6g/L of magnesium sulfate, 1g/L of ferrous sulfate, 40mg/L of VB1 & HCl, 50mg/L of calcium pantothenate, 40mg/L of nicotinamide, 1g/L of manganese sulfate, 20mg/L of zinc sulfate, 20mg/L of copper sulfate and pH 7.2.
2. Engineering bacteria shake flask fermentation production of L-threonine
(1) Seed culture: selecting the SMCT021, SMCT023, SMCT031, SMCT033 and SMCT034 slant seeds 1, circularly inoculating the slant seeds into a 500mL triangular flask filled with 20mL of seed culture medium, and carrying out shaking culture at 30 ℃ and 220r/min for 16 h.
(2) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 20mL of the fermentation medium, and shaking-cultured at 33 ℃ and 220r/min for 24 hours.
(3) 1mL of the fermentation broth was centrifuged (12000rpm, 2min), the supernatant was collected, and L-threonine was detected by HPLC in the fermentation broths of the engineered and control bacteria, the concentrations of which are shown in Table 6.
TABLE 6 comparison of the threonine-producing ability of Corynebacterium glutamicum
As can be seen from Table 6, the average acid production of the icd inactivated strain was 22.4g/L, and the threonine production thereof was increased by 11.44% compared to the original strain, indicating that inactivation of icd can improve the threonine producing ability of Corynebacterium glutamicum. As shown by the performance verification results of the attenuation or inactivation of the icd modified bacteria by the escherichia coli and the corynebacterium glutamicum, the attenuation or inactivation of the icd can improve the threonine production capacity of the strain in both escherichia coli and the corynebacterium glutamicum.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gtaccggacg caggcagatg ta 22
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tcgcagcgtg gggaagccaa c 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cggaagataa ccatatcggt c 21
<210> 21
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
agtacgcaaa cgcatatgca acggttttag agctagaaat agcaa 45
<210> 22
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cgttgcatat gcgtttgcgt actagtatta tacctaggac tgagct 46
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tacgcaaacg catatgcaac gg 22
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
agggtagggc catcatttaa g 21
<210> 25
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gtaattttta tctgtctgtg cgctattaga gcaatttttt gttaatgatt tg 52
<210> 26
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caaatcatta acaaaaaatt gctctaatag cgcacagaca gataaaaatt ac 52
<210> 27
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ccggaacaac tactttactt tccatggttg ttacctcgtt acctttggtc 50
<210> 28
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaccaaaggt aacgaggtaa caaccatgga aagtaaagta gttgttccgg 50
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aatcactttc tcggcgtcgg c 21
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
taaccacgcg ggttcgtc 18
<210> 31
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cgataccaca atgttccg 18
<210> 32
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cggggatccg cgcgcatcct cgaagacct 29
<210> 33
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gcgctttttg tgaagagact gagtctcctt ggttgatggg 40
<210> 34
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cccatcaacc aaggagactc agtctcttca caaaaagcgc 40
<210> 35
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gccaagcttt ggagaatccg ctgatgatg 29
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tccacgtggg cggcgtctgc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ttggaaatcc tctttaagag 20
<210> 38
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cgcggatcca taacctttgg ggtaacgtg 29
<210> 39
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gcagttctta agcgtgaact tacttctcca gattttgtgt c 41
<210> 40
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gacacaaaat ctggagaagt aagttcacgc ttaagaactg c 41
<210> 41
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cccaagctta ctcctccaac gttgcagaac 30
<210> 42
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cggggatccg gccgagtatt tgcactctac 30
<210> 43
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tttttgaaag atttaagcga aagcctagta cgtcattccc 40
<210> 44
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gggaatgacg tactaggctt tcgcttaaat ctttcaaaaa 40
<210> 45
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tgcctgcagc ccacaaaacg tgagcgtga 29
<210> 46
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ggatcctagc tgccaattat tccggg 26
<210> 47
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tagacttaac ctcgggctac ttagcgtccg gtgcctgcat 40
<210> 48
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
atgcaggcac cggacgctaa gtagcccgag gttaagtcta 40
<210> 49
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gcccggaata attggcagct actaaggttg gttaacttca a 41
<210> 50
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cgcggatcca gcggcagcgt gaacatcag 29
<210> 51
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
cccggaataa ttggcagcta ctttctgcac ctttcgatct 40
<210> 52
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
agatcgaaag gtgcacaaag tagctgccaa ttattccggg 40
<210> 53
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tatttctgta cgaccagggc catgggtaaa aaatcctttc gta 43
<210> 54
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tacgaaagga ttttttaccc atggccctgg tcgtacagaa ata 43
<210> 55
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tcggaacgag ggcaggtgaa gatgatgtcg gtggtgccgt ctt 43
<210> 56
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
aagacggcac caccgacatc atcttcacct gccctcgttc cga 43
<210> 57
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cgcggatccg gtgcctccag cggagaaca 29
<210> 58
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cgggatccgc agtgagcgtg gcgtttccg 29
<210> 59
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tagacttaac ctcgggctac gattctccaa aaataatggc 40
<210> 60
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gcgattattt ttggagaatc gtagcccgag gttaagtcta 40
<210> 61
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggggcagatg ctgaggtcat atgtatatct ccttctgcag 40
<210> 62
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
ctgcagaagg agatatacat atgacctcag catctgcccc 40
<210> 63
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ctagccaatt cagccaaaac ctccacgcga tcttccacat cca 43
<210> 64
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tggatgtgga agatcgcgtg gaggttttgg ctgaattggc tag 43
<210> 65
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cgggatccga gactgcggaa tgttgttg 28
<210> 66
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cgggatcctc acagaaggat cagggtagcc c 31
<210> 67
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
cccggaataa ttggcagcta ggatataacc ctatcccaag 40
<210> 68
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
cttgggatag ggttatatcc tagctgccaa ttattccggg 40
<210> 69
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tcacgcgtcg aaatgtagtc catgggtaaa aaatcctttc gta 43
<210> 70
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tacgaaagga ttttttaccc atggactaca tttcgacgcg tga 43
<210> 71
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cgggatcctc aaggccaaac atctgtgct 29
<210> 72
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ttgaagttaa ccaaccttag tagctgccaa ttattccggg c 41
<210> 73
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cgggatcctt acttcacgga agtgtttg 28
Claims (9)
1. A method for increasing the fermentation yield of L-threonine, comprising weakening or inactivating an isocitrate dehydrogenase gene or a coding gene having homology of 80% or more with the amino acid sequence of isocitrate dehydrogenase and expressing the same functional protein in a fermentative strain; the fermentation strain is a bacterium having threonine producing ability.
2. The method of claim 1, wherein the bacteria are Escherichia (Escherichia) species or Corynebacterium (Corynebacterium) species.
3. The method according to claim 2, wherein the bacterium is Escherichia coli (Escherichia coli) or Corynebacterium glutamicum (Corynebacterium glutamicum).
4. A method for constructing genetic engineering bacteria for high yield of L-threonine is characterized in that the method comprises weakening or inactivating isocitrate dehydrogenase genes in escherichia coli or corynebacterium glutamicum, and the obtained gene weakening or inactivating strains are used for fermentation production of L-threonine;
gene ID of the E.coli isocitrate dehydrogenase Gene at NCBI: 945702, respectively;
GeneID of Corynebacterium glutamicum isocitrate dehydrogenase gene at NCBI: 1018663.
5. the method according to claim 4, wherein the attenuation or inactivation is selected from at least one of the group consisting of attenuation of the start codon of the isocitrate dehydrogenase gene, attenuation of a promoter, attenuation or inactivation of an RBS sequence.
6. The method according to claim 5, wherein the initiation codon attenuation is replacement of the initiation codon ATG of isocitrate dehydrogenase gene with TTG;
promoter attenuation is the replacement of the promoter of the isocitrate dehydrogenase gene with a weak promoter;
RBS sequence attenuation is the replacement of the RBS sequence of the isocitrate dehydrogenase gene with an RBS sequence which can be regulated by threonine.
7. The method of claim 6, wherein the escherichia coli is strain MHZ-0215-2; and/or
The corynebacterium glutamicum is a strain SMCT 033; the strain SMCT033 is constructed according to a cereal bar classical method, corynebacterium glutamicum ATCC13032 is used as an initial strain, and feedback inhibition of lysC genes is relieved and expression of the lysC genes is strengthened, feedback inhibition of hom and thrB is relieved and expression of the thrC genes is strengthened, expression of the pck genes and pyk genes are inactivated; wherein, the reference sequence numbers of lysC, hom, thrB, thrC, pck and pyk on NCBI are 1021294, 1019166, 1019167, 1020172, 1020806 and 1020040 respectively.
8. The genetically engineered bacterium with high L-threonine yield, which is constructed by the method of any one of claims 4 to 7.
9. The method of any one of claims 1 to 3, or the use of the genetically engineered bacterium with high L-threonine productivity of claim 8 in the fermentation production of L-threonine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104862329A (en) * | 2015-04-23 | 2015-08-26 | 上海工业生物技术研发中心 | L-threonine genetic engineering production bacteria |
Non-Patent Citations (3)
| Title |
|---|
| AMNA MHAMDI ET AL.: "Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves", 《PLANT, CELL AND ENVIRONMENT》, pages 3 * |
| NG SHYH-CHANG ET AL.: "Influence of threonine metabolism on S-adenosylmethionine and histone methylation", 《SCIENCE》, pages 222 - 226 * |
| 解晓鹏等: "L-苏氨酸产生菌的选育", 《安徽农业科学》, pages 10417 - 10419 * |
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