CN111019878B - Recombinant Escherichia coli with improved L-threonine yield, construction method and application thereof - Google Patents
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Abstract
The invention discloses recombinant escherichia coli with improved L-threonine yield and a construction method and application thereof. The invention uses Escherichia coli CICC20905 as a starting strain, adopts error-prone PCR and CRISPR-Cas9 gene editing technology to knock out a key gene dapA of an L-threonine synthesis competition pathway and a threonine dehydrogenase coding gene tdh, and then utilizes a gene random mutation kit to randomly mutate a promoter sequence P of a L-threonine synthesis related gene thrAthrA5 recombinant bacteria ECT1, ECR2 and ECT 3-1-ECT 3-3 are constructed, wherein the ECT3-2 enables the fermentation tank yield of the L-threonine to be remarkably improved and reach 120g/L, and a foundation is laid for further metabolic engineering transformation of escherichia coli to produce the L-threonine.
Description
Technical Field
The invention relates to recombinant escherichia coli with improved L-threonine yield and a construction method and application thereof, belonging to the technical field of genetic engineering.
Background
L-threonine is one of eight major essential amino acids, an essential amino acid for humans and animals, which cannot be synthesized by itself. L-threonine has the functions of balancing amino acid composition, regulating metabolism balance in vivo, improving the absorption and utilization rate of cereal protein by organisms, promoting growth and development of the organisms and the like, and is widely applied to the feed, medicine and food industries. At present, the production of L-threonine mainly comprises a chemical synthesis method, a protein hydrolysis method and a microbial fermentation method, wherein the microbial fermentation method has low production cost, high production intensity and small environmental pollution, thereby becoming the most widely applied method for industrial production of L-threonine at present.
Escherichia coli (Escherichia coli) has been used for the industrial fermentative production of various amino acids. Therefore, the construction of recombinant Escherichia coli by metabolic engineering is an effective way to produce L-threonine. At present, the overexpression or attenuation of key enzyme genes in an amino acid synthesis path and a competition path mediated by expression plasmids is a main means for carrying out genetic modification on Escherichia coli. However, the use of expression plasmid mediated gene overexpression must introduce antibiotic resistance gene into Escherichia coli cells and add certain antibiotic during growth, which raises the doubt of antibiotic use. Therefore, the method for genetically modifying the escherichia coli is safe and efficient, and has important significance for the field of amino acid production.
In recent years, with the development of synthetic biology, artificially synthesized functional elements have shown great application potential in the field of metabolic engineering. The metabolic flux of a cell is mainly controlled by the transcription level, and a promoter is an important regulatory element of the transcription level, so the promoter is listed as one of important functional elements of synthetic biology.
The error-prone PCR increases the mutation rate of amplification by adjusting the concentration of magnesium ions in a PCR reaction system, adding manganese ions, changing the concentration of 4 dNTPs, adding DNA polymerase with poor fidelity and the like, thereby introducing mutation sites. Error-prone PCR is a simple and effective in vitro random mutagenesis technique, and is an effective method for obtaining sequence diversity. The mutation introduced by error-prone PCR is random mutation, can occur at any position of a promoter region, and provides guarantee for constructing a larger library.
At present, the yield of L-threonine in Escherichia coli for producing L-threonine is not high enough, and the requirement of industrial production cannot be met.
Disclosure of Invention
In order to solve the technical problems, the invention provides a recombinant Escherichia coli with improved L-threonine yield, which is prepared by knocking out a key gene dapA of an L-threonine synthesis competition pathway and a threonine dehydrogenase coding gene tdh by using an Escherichia coli CICC20905 as an initial strain, and then randomly mutating a promoter sequence P of a L-threonine synthesis related gene thrA by using a gene random mutation kitthrAAnd screening out the optimal combination to obtain the recombinant Escherichia coli with high L-threonine yield.
The first object of the present invention is to provide a recombinant E.coli having an improved L-threonine production, which has a dihydrodipicolinate synthetase encoding gene dapA and a threonine dehydrogenase encoding gene tdh deleted from an E.coli host strain.
Further, the recombinant Escherichia coli also has homoserine dehydrogenase thrA promoter sequence PthrAThe mutation is one of the sequences shown as SEQ ID NO. 6-8.
Further, the escherichia coli host bacterium is escherichia coli CICC 20905.
Further, the nucleotide sequence of the dapA gene is shown in ECK2474, and the nucleotide sequence of the tdh gene is shown in ECK 3606.
Further, the nucleotide sequence of the thrA gene is shown as ECK 0002.
The second purpose of the invention is to provide the construction method of the recombinant Escherichia coli, which comprises the following steps:
(1) construction of an integration fragment of the sequence mutant promoter: synthesizing thrA upstream and downstream homologous arm fragments containing PmthrA gene and fusing to obtain fragment mthrA- (1-3);
(2) constructing a recombinant plasmid: respectively connecting the 3 recombinant fragments, namely, the mthrA- (1-3), with a linearized vector containing the sgRNA to obtain recombinant plasmids containing the mthrA-1, the mthrA-2 and the mthrA-3;
(3) constructing high-yield L-threonine recombinant escherichia coli: the method comprises the steps of transforming a plasmid containing cas9 protein into escherichia coli CICC20905, transforming the recombinant plasmid pTDAP into escherichia coli to obtain recombinant escherichia coli ECT-1, transforming the recombinant plasmid pTTDH into escherichia coli ECT-1 to obtain recombinant escherichia coli ECT-2, transforming the recombinant plasmid pT-thrA (1-3) into escherichia coli ECT-2 respectively, and removing foreign plasmids to obtain recombinant escherichia coli ECT3- (1-3).
Further, in step (2), the linearized vector comprises lpTdap or lptdd or pT-thrA.
Further, the plasmid containing cas9 protein includes pCas9 plasmid.
The third purpose of the invention is to provide the application of the recombinant Escherichia coli in the production of L-threonine.
Further, the application is that the recombinant Escherichia coli is cultured in a fermentation medium for 50-70 h to obtain the L-threonine, wherein the fermentation medium (g/L): 25-35 parts of cane sugar, 1.5-2.0 parts of monopotassium phosphate, 1.3-1.8 parts of betaine, 0.3-0.7 part of potassium chloride, 3-7 parts of ammonium sulfate, 0.8-1.2 parts of magnesium sulfate heptahydrate, 2-4 parts of corn steep liquor dry powder, and FeSO4·7H2O0.08～0.12，MnSO4·H2O0.08～0.12。
The invention has the beneficial effects that:
(1) according to the invention, 5 recombinant bacteria ECT1, ECR2 and ECT 3-1-ECT 3-3 are constructed by modifying genes related to an L-threonine biosynthesis pathway, wherein the yield of the L-threonine is obviously improved by ECT3-2, and the yield of a fermentation tank reaches 120g/L and is 2 times of the yield of an original strain.
(2) The invention provides a method for improving L-threonine biosynthesis pathway in microorganism to increase L-threonine yield, and provides theoretical basis for constructing high-yield L-threonine strains.
Drawings
FIG. 1 shows the results of colony PCR gel validation of recombinant DAP-fragment E.coli.
FIG. 2 shows the results of colony PCR gel verification of recombinant TDH fragment E.coli.
FIG. 3 shows the results of colony PCR gel validation of recombinant plasmids.
FIG. 4 shows relative fluorescence intensity when the mutant promoter expresses green fluorescent protein.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The Escherichia coli CICC20905 disclosed by the invention is high-yield L-threonine Escherichia coli and is disclosed in a patent CN 201811465330.2.
Culturing and fermenting recombinant escherichia coli seeds:
plate medium (g/L): sucrose 1, beef extract 10, peptone 10, sodium chloride 5, agar 20, pH adjusted to 7.0.
Seed medium (g/L): 20 parts of cane sugar, 4 parts of yeast extract, 5 parts of peptone, 3 parts of ammonium sulfate, 1 part of magnesium sulfate heptahydrate and 2 parts of potassium dihydrogen phosphate.
(II) fermentation method under fermentation tank condition:
the seed medium (50L in 13L) formulation is shown in Table 1.
TABLE 1 seed culture Medium formulation
| Ingredients | Concentration (g/L) |
| Sucrose (fen xiao) | 30 |
| |
5 |
| |
2 |
| |
1 |
| Corn steep liquor |
3 |
| FeSO4·7H2O | 0.1 |
| MnSO4·H2O | 0.1 |
Remarking: sterilizing at 121 deg.C for 13min, and adjusting pH to 7.0 after sterilization.
2 fermentation
The medium formulation is shown in table 2.
TABLE 2 fermentation Medium formulation
| Ingredients | Concentration (g/L) |
| Sucrose (Sterilization alone) | 30 |
| Potassium dihydrogen phosphate | 1.7 |
| Betaine | 1.5 |
| Potassium chloride | 0.5 |
| |
5 |
| |
1 |
| Corn steep liquor |
3 |
| FeSO4·7H2O | 0.1 |
| MnSO4·H2O | 0.1 |
(III) method for measuring L-threonine:
1) sample treatment: 1mL of the fermentation broth was taken, centrifuged to remove the cells and the supernatant was taken. The supernatant was diluted appropriately with 5% trichloroacetic acid at 12000rpm, centrifuged for 10min, and then filtered through a filter with a pore size of 0.22. mu.m.
2) The analysis method comprises the following steps: OPA boric acid pre-column derivatization, with 9.478min elution peak being threonine
3) Chromatographic conditions are as follows:
(1) a chromatographic column: column C18 (250X 4.6) mm
(2) Column temperature: 40 deg.C
(3) Mobile phase A: weighing 3.01g of anhydrous sodium acetate in a beaker, adding deionized water to dissolve the anhydrous sodium acetate and fixing the volume to 1L, then adding 200 mu L of triethylamine, and adjusting the pH to 7.20 +/-0.05 by using 5% acetic acid; after suction filtration, 5mL of tetrahydrofuran was added and mixed for further use. Mobile phase B: weighing 3.01g of anhydrous sodium acetate in a beaker; adding deionized water to dissolve and fixing the volume to 200 mL; adjusting pH to 7.20 + -0.05 with 5% acetic acid; after suction filtration, 400mL of acetonitrile and 400mL of methanol were added to the solution, and the mixture was mixed for use.
(4) Flow rate: 1.0 ml/min;
(5) an ultraviolet detector: 338 nm;
(6) column temperature: 40 ℃;
in the following examples, conventional molecular biological experiments are used, not much described.
The primer sequences were designed with reference to table 3.
TABLE 3 primer sequence Listing
Example 1: construction of recombinant fragments
Designing primers pT-DAP-1F, pT-DAP-1R, pT-DAP-2F and pT-DAP-2R according to sequence information of escherichia coli, and respectively amplifying homologous arm gene sequences at two sides of a dapA gene from the genome of escherichia coli CICC20905 by using the primers, so as to obtain fragments DAP1 and DAP2 (the sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2); primers pT-TDH-1F, pT-TDH-1R, pT-TDH-2F and pT-TDH-2R are designed according to sequence information of escherichia coli, and the primers are used for amplifying homologous arm gene sequences on two sides of a TDH gene from an escherichia coli CICC20905 genome respectively by 600bp to obtain fragments TDH1 and TDH2 (the sequences are shown as SEQ ID NO.3 and SEQ ID NO. 4).
Example 2: construction of recombinant plasmid
Based on the sequence information of the vector pTarget, a primer pT-dap-F, pT-dap-R is designed for PCR to obtain a linearized vector lpTdap containing sgRNA. The fragments DAP1, DAP2 and lpTdap were ligated to construct recombinant plasmid pTDAP. Based on the sequence information of the vector pTarget, a primer pT-tdh-F, pT-tdh-R is designed for PCR to obtain a linearized vector lpTtdh containing sgRNA. The fragments TDH1, TDH2 and lpTtdh were ligated to construct a recombinant plasmid pTTDH.
Example 3: construction of recombinant DAP fragment E.coli
Coli CICC20905 was transformed with pCas9 plasmid containing cas9 protein. Screening successfully transformed recombinant Escherichia coli ECC9 by using a Kana resistance plate, then transforming the recombinant plasmid pTDAP into Escherichia coli ECC9, screening to confirm that fragments DAP1 and DAP2 are successful, adding 0.05mM IPTG to induce for 12h at 30 ℃, removing the recombinant plasmid pTDAP, selecting transformants by using primers pT-DAP-1F and pT-DAP-2R for colony PCR, generating a band of about 1200bp (see figure 1), and obtaining the recombinant Escherichia coli ECT-1 with the dapA gene knocked out after correct sequencing.
Example 4: construction of recombinant TDH fragment E.coli
Transforming the recombinant plasmid pTTDH into Escherichia coli ECT-1, screening to confirm that the fragments TDH1 and TDH2 succeed, adding 0.05mM IPTG to induce at 30 ℃ for 12h, removing the recombinant plasmid pTTDH, selecting a transformant by selecting primers pT-TDH-1F and pT-TDH-2R to perform colony PCR, generating a band of about 1200bp (see figure 2), and obtaining the recombinant Escherichia coli ECT-2 with TDH gene knocked out after the sequencing is correct.
Example 5: construction of promoter libraries
The upstream promoter sequence P of the thrA gene is amplified by taking an escherichia coli CICC 23604 genome as a template and adopting primers thrA. FOR, thrA. REV and a Quick Mutation gene random kitthrA(the sequence is shown as SEQ ID NO. 5), and the PCR conditions are as follows: pre-denaturation at 94 deg.C for 3min, then denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 0.5min for 30 cycles in total, cutting gel, and recovering fragments with correct size to obtain PthrAMixed promoter fragments of different sequences PmthrA.
Example 6: obtaining vectors containing different promoter sequences
Vector pET-20b (purchased from EMD Biosciences (Novagen)) containing eGFP was linearized with primers zttha.for, zttha.rev, PCR conditions: pre-denaturation at 98 deg.C for 3min, then denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 5s, and extension at 72 deg.C for 2min for 34 cycles in total, cutting gel, and recovering the correct size fragment to obtain linearized vector fragment ZTTHRA. The vector fragment and the mixed promoter fragment obtained in example 5 were ligated to each other, and E.coli JM109 was transformed to extract a mixed plasmid.
Example 7: transformation of Mixed plasmids
The mixed plasmid obtained in example 6 was transformed into E.coli ECT-2 competent cells. The method comprises the following specific steps:
(1) the plasmid constructed in the example 6 is used for electrically transforming competent cells of Escherichia coli ECT-2, the addition amount is 100-200ng, and the electric transformation conditions are as follows: the voltage is 25kV, the electric shock time is 5ms, the ampicillin-resistant LB plate with the final concentration of 10 mug/mL is recovered for 2h at 37 ℃, and the anaerobic culture is carried out for 12h at 37 ℃. The Escherichia coli which is transformed successfully is positive in ampicillin resistance.
(2) The single colony growing on the plate was selected, colony PCR was verified using primers thrayz.for, thrayz.rev, and the amplified fragment after the substitution was 1000bp in length (see fig. 3). And sequencing is performed.
Example 8: detection of fluorescence intensity of recombinant strain
10 strains with correct sequencing are selected, 96 shallow-well plate culture is carried out at 37 ℃ for 20h, the fluorescence intensity of each recombinant strain fermentation liquid is detected at 523nm by a microplate reader (see figure 4), and RNA is extracted to detect the relative transcription intensity.
Example 9: construction of homologous recombination fragments
In integration of PthrASelecting a strain with stronger fluorescence intensity from strains of the mixed promoter fragments PmthA with different sequences, selecting 3 different mutant sequences (the sequences are respectively shown as SEQ ID NO. 6-8) after sequencing, respectively re-synthesizing with the fragment of 800bp at the upstream of the thrA locus, and designing a primer thrA-1-F, thrA-1-R for amplification; designing thrA-2-F and thrA-2-R according to the DNA sequence information of Escherichia coli CICC 23604 genome, amplifying the gene sequence of the downstream homology arm of thrA from the Escherichia coli CICC 23604 genome, and fusing the obtained 2 fragments by a fusion PCR technology to obtain a recombinant fragment mthrA- (1-3).
Example 10: construction of homologous recombination plasmids
A primer zhthhA-F, zhthrA-R is designed according to the sequence information of the vector pTarget, PCR is carried out by using the primer to obtain a linearized vector pT-thrA containing sgRNA, and the linearized vector pT-thrA is connected with a recombinant fragment mthrA- (1-3) to construct a recombinant plasmid pT-thrA (1-3).
Example 11: recombination of PmthrAOf the promoter Escherichia coliConstruction of
Respectively transforming Escherichia coli ECT-2 with the recombinant plasmid pT-thrA (1-3) to obtain recombinant Escherichia coli with original promoter of thrA gene replaced, selecting transformant with primer thrA-1-F and thrA-1-R to perform colony PCR, sequencing after 1000bp band appears to construct successfully recombinant Escherichia coli, adding 0.01M IPTG for induction, culturing at 30 deg.C for 24 hr to remove pT-thrA (1-3) plasmid, culturing at 37 deg.C for 24 hr to remove pCas9 plasmid, and naming as ECT3- (1-3).
The genotypes of the strains constructed in examples 3, 4 and 11 are shown in Table 4.
TABLE 4 genotype of the strains
Example 12: using recombinant PmthrAPromoter strain fermentation production of L-threonine
(1) Seed liquid preparation
The starting strain, Corynebacterium glutamicum CICC20905, the recombinant bacterium ECT1 constructed in example 3, the recombinant bacterium ECT1 constructed in example 4, and the recombinant bacterium ECT3- (1-3) constructed in example 11 were inoculated into a seed medium, respectively.
Controlling the seed culture process:
a)37℃，8～10h；
b) air volume: 0.4m 3/h;
c) dissolving oxygen: the early stage is 20-40%, and the rotation speed is increased by 20-50 rpm each time;
d) rotating speed: 200-700 rpm;
e) and (3) tank pressure: 0.05 to 0.08 MPa;
f) pH: controlling the pH value to be 7.0 by ammonia water;
g) inoculation amount: 10%, 13L inoculation 1.2L;
h) the residual sugar is controlled at 10g/L, the sugar supplement is started when the residual sugar is about 15g/L, and the sugar supplement is started generally for 7 h;
i) the seed loading of a 50L tank is 13L, the dissolved oxygen is 100 percent, and the correction conditions are as follows: 600rpm, 0.05MPa, 0.4m3/h。
And (4) finishing standard: culturing for about 8-10 h.
(2) Fermentation culture
Transferring the seed liquid obtained in the step (1) into a fermentation medium, and taking a fermentation liquid to determine the content of the L-threonine (see table 4).
After sterilization, the pH was adjusted to 7.0. And (3) sterilization: 121 ℃ for 3 min. And (3) process control:
initial volume of fermentation 20L, culture conditions:
a)37℃；
b) air volume: 0.75m 3/h;
c) dissolving oxygen: controlling DO in the fermentation process to be 40% -60%;
d) rotating speed: 500 rpm;
e) and (3) tank pressure: 0.05 to 0.08 MPa;
f) pH: the pH of the solution was controlled to 6.9 with ammonia.
g) Inoculation amount: 20%, 20L inoculation 4L
h) Residual sugar (total sugar): and (3) starting sugar supplement when the total sugar content is lower than 5g/L, and controlling the sugar supplement by determining the total sugar content: the target is 5-8 g/L.
Culturing for 60h, taking the fermentation liquor to determine the content of L-threonine, and the result is shown in Table 5.
TABLE 5 fermentative production of L-threonine by the strains
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university in south of the Yangtze river, Wuxi Zhongliang engineering science and technology Co., Ltd
<120> recombinant escherichia coli with improved L-threonine yield, construction method and application thereof
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ggtaaggcgc tggacattcg tccaccagcc cagccgctgg cactggtttc tggcgcgcgt 300
acccagttca cgggcgatac cgcttcattg ctggtggaaa atggtcgtgg caatactctg 360
tggccgcagg tggttagcgt gctgcaggcg aaaaactaca ccatcaccca acgtgatgat 420
gctggtcaga cactgaccac cgattgggta caatggaacc gtctggacga agacgagcag 480
tatcgtggtc gttatcaaat ctctgttaag ccgcagggtt atcagcaggc ggttacggtt 540
aaactgctga acctggaaca ggcgggcaaa ccggttgcag acgcggcttc catgcagc 598
<210> 3
<211> 602
<212> DNA
<213> (Artificial sequence)
<400> 3
aatatgatgc cctggtgatg gtagacgact cccacgcggt cggttttgtc ggtgaaaatg 60
gtcgtggttc ccatgaatac tgcgatgtga tgggccgggt cgatattatc accggtacgc 120
ttggtaaagc gctgggcggg gcttctggtg gttataccgc ggcgcgcaaa gaagtggttg 180
agtggctgcg ccagcgttct cgtccgtacc tgttctccaa ctcgctggca ccggccattg 240
ttgccgcgtc catcaaagta ctggagatgg tcgaagcggg cagcgaactg cgtgaccgtc 300
tgtgggcgaa cgcgcgtcag ttccgtgagc aaatgtcggc ggcgggcttt accctggcgg 360
gagccgatca cgccattatt ccggtcatgc ttggtgatgc ggtagtggcg cagaaatttg 420
cccgtgagct gcaaaaagag ggcatttacg ttaccggttt cttctatccg gtcgttccga 480
aaggtcaggc gcgtattcgt acccagatgt ctgcggcgca tacccctgag caaattacgc 540
gtgcagtaga agcatttacg cgtattggta aacaactggg cgttatcgcc tgaggatgtg 600
ag 602
<210> 4
<211> 638
<212> DNA
<213> (Artificial sequence)
<400> 4
cgaacaaggg ctggtattcc agccctttta tctgaggata atctgttaaa tatgtaaaat 60
cctgtcagtg taataaagag ttcgtaattg tgctgatctc ttatatagct gctctcatta 120
tctctctacc ctgaagtgac tctctcacct gtaaaaataa tatctcacag gcttaatagt 180
ttcttaatac aaagcctgta aaacgtcagg ataacttcag aggtcgtcgg taatttatga 240
tgaacagcac caataaactt agtgttatta ttccgttata taatgcgggc gatgatttcc 300
gcacttgtat ggaatcttta attacgcaaa cctggactgc tctggaaatc attattatta 360
acgatggttc aacggataat tctgttgaaa tagcaaagta ttacgcagaa aactatccgc 420
acgttcgttt gttgcatcag gcgaatgctg gcgcatcggt ggcgcgtaat cgtgggattg 480
aagtggcaac gggcaaatat gtcgcttttg tcgatgctga cgatgaagtc tatcccacca 540
tgtacgaaac gctgatgacc atggcgttag aggacgacct cgacgtggcg cagtgcaacg 600
ctgactggtg ttttcgtgaa acgggagaaa cctggcaa 638
<210> 5
<211> 81
<212> DNA
<213> (Artificial sequence)
<400> 5
cgcgtacagg aaacacagaa aaaagcccgc acctgacagt gcgggctttt tttttcgacc 60
aaaggtaacg aggtaacaac c 81
<210> 6
<211> 81
<212> DNA
<213> (Artificial sequence)
<400> 6
cgcgtacagg aaacacagaa aaaagcccgc acctgacaat ctgggctttt tttttcgacc 60
aaaggtaacg aggtaacaac c 81
<210> 7
<211> 81
<212> DNA
<213> (Artificial sequence)
<400> 7
cgcgtacagg aaacacagaa aaaggtccgc acctgacagt gcgggctttt tttttcgacc 60
aactttaacg aggtaacaac c 81
<210> 8
<211> 81
<212> DNA
<213> (Artificial sequence)
<400> 8
cgcgtacagg aaacacactt aaaagcccgc acctgacagt gcggaccttt tttttcgacc 60
aaaggtaacg aggtaacaac c 81
<210> 9
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 9
atgcggggct gtgggcggt 19
<210> 10
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 10
aacagagtaa gccatcaaat ctccctaggg ccatcctctg tgcaaa 46
<210> 11
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 11
tagggagatt tgatggctta ctctgtt 27
<210> 12
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 12
gctgcatgga agccgcgtct gcaac 25
<210> 13
<211> 17
<212> DNA
<213> (Artificial sequence)
<400> 13
aatatgatgc cctggtg 17
<210> 14
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 14
ctcacatcct caggcgataa cg 22
<210> 15
<211> 37
<212> DNA
<213> (Artificial sequence)
<400> 15
cgcctgagga tgtgagcgaa caagggctgg tattcca 37
<210> 16
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 16
ttgccaggtt tctcccgttt c 21
<210> 17
<211> 45
<212> DNA
<213> (Artificial sequence)
<400> 17
cggttgcaga cgcggcttcc atgcagcttt gcaacagtgc cgttg 45
<210> 18
<211> 36
<212> DNA
<213> (Artificial sequence)
<400> 18
taaccgccca cagccccgca ttagtcggtg gtgata 36
<210> 19
<211> 50
<212> DNA
<213> (Artificial sequence)
<400> 19
tgaaacggga gaaacctggc aatttgcaac agtgccgttg atcgtgctat 50
<210> 20
<211> 41
<212> DNA
<213> (Artificial sequence)
<400> 20
tcaccagggc atcatattta gtcggtggtg ataaacttat c 41
<210> 21
<211> 15
<212> DNA
<213> (Artificial sequence)
<400> 21
cgcgtacagg aaaca 15
<210> 22
<211> 17
<212> DNA
<213> (Artificial sequence)
<400> 22
ggttgttacc tcgttac 17
<210> 23
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 23
tggcatggat gaactataca aatagacaag cttgcggccg cactcg 46
<210> 24
<211> 47
<212> DNA
<213> (Artificial sequence)
<400> 24
gtgtttcctg tacgcgcgac ggagctcgaa ttcggatccg aattaat 47
<210> 25
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 25
tacgactcac tatagggaga ccaca 25
<210> 26
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 26
cgagtgcggc cgcaagcttg t 21
<210> 27
<211> 16
<212> DNA
<213> (Artificial sequence)
<400> 27
cattactacg ccacgc 16
<210> 28
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 28
gaacttcaac actcgcatgg ttgttacctc gttacctt 38
<210> 29
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 29
atgcgagtgt tgaagttcgg cggt 24
<210> 30
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 30
ggggtgaaga actttagcgc 20
<210> 31
<211> 47
<212> DNA
<213> (Artificial sequence)
<400> 31
gcgctaaagt tcttcacccc tttgcaacag tgccgttgat cgtgcta 47
<210> 32
<211> 35
<212> DNA
<213> (Artificial sequence)
<400> 32
gcgtggcgta gtaatgtagt cggtggtgat aaact 35
Claims (8)
1. The recombinant Escherichia coli with improved L-threonine yield is characterized in that a dihydrodipicolinate synthetase encoding gene is knocked out from an Escherichia coli host bacteriumdapAAnd threonine dehydrogenase-encoding genetdh；
The recombinant Escherichia coli also dehydrogenase homoserinethrAPromoter sequence PthrAThe mutation is a sequence shown as SEQ ID NO. 7;
the escherichia coli host bacterium is escherichia coli CICC 20905.
2. The recombinant Escherichia coli of claim 1, wherein said recombinant Escherichia coli isdapAThe nucleotide sequence of the gene is shown in ECK2474,tdhthe nucleotide sequence of the gene is shown in ECK 3606.
3. The recombinant Escherichia coli of claim 1, wherein said recombinant Escherichia coli isthrAThe nucleotide sequence of the gene is shown in ECK 0002.
4. A method for constructing recombinant Escherichia coli according to any one of claims 1 to 3, comprising the steps of:
(1) construction of an integration fragment of the sequence mutant promoter: synthesizing thrA upstream and downstream homologous arm fragments containing PmthrA gene and fusing to obtain fragment mthrA- (1-3);
(2) constructing a recombinant plasmid: respectively connecting the 3 recombinant fragments, namely, the mthrA- (1-3), with a linearized vector containing the sgRNA to obtain recombinant plasmids containing the mthrA-1, the mthrA-2 and the mthrA-3;
(3) constructing high-yield L-threonine recombinant escherichia coli: the method comprises the steps of transforming a plasmid containing cas9 protein into escherichia coli CICC20905, transforming the recombinant plasmid pTDAP into escherichia coli to obtain recombinant escherichia coli ECT-1, transforming the recombinant plasmid pTTDH into escherichia coli ECT-1 to obtain recombinant escherichia coli ECT-2, transforming the recombinant plasmid pT-thrA (1-3) into escherichia coli ECT-2 respectively, and removing foreign plasmids to obtain recombinant escherichia coli ECT3- (1-3).
5. The method of claim 4, wherein in step (2), the linearized vector comprises lpTdap or lptdd or pT-thrA.
6. The method according to claim 4, wherein the plasmid containing cas9 protein comprises pCas9 plasmid.
7. Use of the recombinant E.coli of any one of claims 1 to 3 for the production of L-threonine.
8. The use of claim 7, wherein said use comprises culturing said recombinant E.coli in a fermentation medium for 50-70 hours to obtain said L-threonine, wherein said fermentation medium: 25-35 g/L of sucrose, 1.5-2.0 g/L of monopotassium phosphate, 1.3-1.8 g/L of betaine, 0.3-0.7 g/L of potassium chloride, 3-7 g/L of ammonium sulfate, 0.8-1.2 g/L of magnesium sulfate heptahydrate, 2-4 g/L of corn steep liquor dry powder, FeSO4•7H2O 0.08~0.12 g/L，MnSO4•H2O 0.08~0.12g/L。
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| CN111471638B (en) * | 2020-05-22 | 2021-11-23 | 江南大学 | Construction and application of corynebacterium glutamicum mutant strain capable of producing L-homoserine |
| WO2022094847A1 (en) * | 2020-11-05 | 2022-05-12 | 中国科学院深圳先进技术研究院 | Cell lysis solution of engineering bacterium and use thereof in tumor therapy |
| CN114317389B (en) * | 2021-12-22 | 2023-06-13 | 南京工业大学 | Method for producing L-threonine by co-culture fermentation of recombinant escherichia coli |
| CN116536310A (en) * | 2022-01-26 | 2023-08-04 | 廊坊梅花生物技术开发有限公司 | Promoter, threonine-producing recombinant microorganism and application thereof |
| CN115011620B (en) * | 2022-05-19 | 2023-11-07 | 江南大学 | Escherichia coli recombinant nucleic acid, recombinant Escherichia coli and culture method, and method for biosynthesizing L-threonine |
| CN115960801B (en) * | 2022-09-28 | 2024-07-09 | 浙江大学杭州国际科创中心 | Genetically engineered bacterium for high-yield L-threonine and application thereof |
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