CN111154706B - Recombinant escherichia coli with improved L-tryptophan yield as well as construction method and application thereof - Google Patents
Recombinant escherichia coli with improved L-tryptophan yield as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses recombinant escherichia coli with improved L-tryptophan yield, and a construction method and application thereof. The invention takes Escherichia coli CICC10303 as an initial strain, adopts error-prone PCR and CRISPR-Cas9 gene editing technology, knocks out a key gene trpR to eliminate feedback repression regulation and control on tryptophan synthesis and transportation key enzyme, knocks out a predhenyl dehydrogenase encoding gene pheA to eliminate competition of a phenylalanine synthesis path, and randomly mutates a promoter sequence P of a L-tryptophan synthesis related gene aroK by using a gene random mutation kitaroK5 recombinant bacteria ECTR1, ECTR2 and ECTR 3-1-ECTR 3-3 are constructed, wherein the ECTR3-2 enables the fermentation tank yield of the L-tryptophan to be obviously improved and reach 40g/L, and a foundation is laid for further metabolic engineering transformation of escherichia coli to produce the L-tryptophan.
Description
Technical Field
The invention relates to recombinant escherichia coli with improved L-tryptophan yield and a construction method and application thereof, belonging to the technical field of genetic engineering.
Background
L-tryptophan is one of eight essential amino acids for human body as a very important aromatic amino acid. In organisms, L-tryptophan can synthesize important bioactive substances such as 5-hydroxytryptamine, nicotinic acid, pigment, alkaloid, coenzyme, indoleacetic acid and the like, plays an important role in the growth and development of human and animals, and is widely applied to the aspects of food, medicine, feed and the like. The production of L-tryptophan was originally mainly based on chemical synthesis and protein hydrolysis, but these methods have the disadvantages of limited material source, long period, complex process, etc., and thus are gradually eliminated. Due to the characteristics of low cost, wide raw material sources, small environmental pollution and the like, the method for producing the L-tryptophan by the microbial method is widely applied.
At present, the microorganism method for producing L-tryptophan has the defects of low yield, complex operation and the like. In 2001, Wangjian and the like use diethyl sulfate (DES) as a mutagen, select a tyrosine and phenylalanine double-auxotroph strain starting strain, breed an L-tryptophan production strain after multiple mutagenesis treatment, and continuously ferment for 64 hours in batches, wherein the accumulation amount of the L-tryptophan can reach 7.28 g/L. In 2007, Chenjunfeng and the like utilize a separation method of multiple mutagenesis, a corynebacterium glutamicum strain is used as an initial strain to obtain a phenylalanine and tyrosine double-auxotroph L-tryptophan structural analogue resistant mutant strain, and after the strain is fermented in a shake flask for 96 hours, the accumulation amount of L-tryptophan reaches 10.82 g/L.
Escherichia coli (Escherichia coli) has been used for the industrial fermentative production of various amino acids. Therefore, the construction of recombinant Escherichia coli by using metabolic engineering means is an effective way for producing L-tryptophan. 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.
The yield of the L-tryptophan of the prior Escherichia coli for producing the L-tryptophan 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-tryptophan yield, wherein escherichia coli CICC10303 is used as an original strain, an L-tryptophan feedback repression inhibition coding gene trpR and a pre-benzene dehydrogenase coding gene pheA are knocked out, and then a gene random mutation kit is used for randomly mutating L-tryptophan to synthesize a related genePromoter sequence P of aroKaroKAnd screening out the optimal combination to obtain the recombinant Escherichia coli ECTR3-2 with high L-tryptophan yield.
The first purpose of the invention is to provide a recombinant Escherichia coli with improved L-tryptophan yield, wherein an L-tryptophan feedback repression coding gene trpR and a pre-benzene dehydrogenase coding gene pheA are knocked out from an Escherichia coli host bacterium.
Furthermore, the recombinant escherichia coli also has a shikimic acid kinase coding gene aroK promoter sequence ParoKThe mutation is a sequence shown as SEQ ID NO. 7.
Further, the escherichia coli host bacterium is escherichia coli CICC 10303.
Furthermore, the nucleotide sequence of the trpR gene is shown as ECK4385, and the nucleotide sequence of the pheA gene is shown as ECK 2596.
Further, the nucleotide sequence of the aroK gene is shown as ECK 3377.
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 aroK upstream and downstream homologous arm segments containing PmaroK gene, and fusing to obtain a segment maroK- (1-3);
(2) constructing a recombinant plasmid: respectively connecting 3 recombinant fragments maroK- (1-3) with a linear vector containing sgRNA to obtain recombinant plasmids containing maroK-1, maroK-2 and maroK-3;
(3) constructing high-yield L-tryptophan recombinant escherichia coli: transforming the plasmid containing cas9 protein into escherichia coli CICC10303 to obtain recombinant escherichia coli ECC 9; the recombinant plasmid pTTRP is transformed into Escherichia coli ECC9 to obtain recombinant Escherichia coli ECTR1, the recombinant plasmid pTPHEA is transformed into Escherichia coli ECTR-1 to obtain recombinant Escherichia coli ECTR2, the recombinant plasmid pT-aroK (1-3) is respectively transformed into Escherichia coli ECTR-2, and exogenous plasmids are removed to obtain recombinant Escherichia coli ECTR3- (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-tryptophan.
Further, the application is that the recombinant escherichia coli is cultured in a fermentation medium for 40-50 h to obtain the L-tryptophan, and the fermentation medium (g/L): 50-70 parts of glucose, 14-18 parts of magnesium sulfate heptahydrate, 22-26 parts of ammonium sulfate, 8-12 parts of yeast extract powder, 14-18 parts of trisodium citrate dihydrate and 5-6 parts of dipotassium phosphate.
The invention has the beneficial effects that:
(1) according to the invention, 5 recombinant bacteria ECTR1, ECTR2 and ECTR 3-1-ECTR 3-3 are constructed by modifying genes related to the biosynthetic pathway of L-tryptophan, wherein the yield of L-tryptophan is obviously improved by ECTR3-2, and the yield of a fermentation tank reaches 40g/L, which is 1.54 times of the yield of an original strain.
(2) The invention provides a method for improving L-tryptophan biosynthesis pathway in microorganism to improve L-tryptophan yield, and provides theoretical basis for constructing high-yield strain of L-tryptophan.
Drawings
FIG. 1 is a recombinant plasmid pTTRP.
FIG. 2 shows the recombinant plasmid pTPHEA.
FIG. 3 shows the recombinant plasmid pET20 b-PpheA.
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 CICC10303 disclosed by the invention is high-yield L-tryptophan escherichia coli and is disclosed in a patent CN 201811465696. X.
Recombinant escherichia coli seed culture and fermentation medium:
plate medium (g/L): tryptone 10, yeast powder 5, sodium chloride 5 and agar powder 15, and the pH is adjusted to 7.0.
Seed medium (g/L): 30 parts of glucose, 15.2 parts of yeast powder, 24 parts of dipotassium hydrogen phosphate, 5 parts of ammonium sulfate, 9.6 parts of monopotassium phosphate and 1 part of magnesium sulfate heptahydrate.
Fermentation medium (g/L): 60 parts of glucose, 16 parts of magnesium sulfate heptahydrate, 24 parts of ammonium sulfate, 10 parts of yeast extract powder, 16 parts of trisodium citrate dihydrate and 5.6 parts of dipotassium phosphate.
The culture conditions are as follows: picking single colony from the plate cultured at 37 ℃ for 24h to a seed culture medium, culturing at 37 ℃ and 220rpm for 10h, inoculating 10% of inoculum size to a fermentation culture medium, and culturing at 35 ℃ and 220rpm for 42 h.
(III) the method for measuring L-tryptophan comprises the following steps:
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, centrifuged at 12000rpm/min 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, wherein the peak eluted at 13.768min is tryptophan
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 1.
TABLE 1 primer sequence Listing
Example 1: construction of recombinant fragments
Designing primers pT-TRP-1F, pT-TRP-1R, pT-TRP-2F and pT-TRP-2R according to sequence information of escherichia coli, and respectively amplifying homologous arm gene sequences on both sides of a trpR gene from an escherichia coli CICC10303 genome by using the primers, so as to obtain fragments TRP1 and TRP2 (the sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2); primers pT-PHEA-1F, pT-PHEA-1R, pT-PHEA-2F and pT-PHEA-2R are designed according to sequence information of escherichia coli, and the homologous arm gene sequences at two sides of the pheA gene are amplified respectively by 600bp from the escherichia coli CICC10303 genome by using the primers to obtain fragments PHEA1 and PHEA2 (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-TRP-F, pT-TRP-R is designed for PCR to obtain a linearized vector lpTtrp containing sgRNA. The fragments TRP1, TRP2 and lpTtrp were ligated to construct a recombinant plasmid pTTRP (see fig. 1). Based on the sequence information of the vector pTarget, a primer pT-pheA-F, pT-pheA-R is designed for PCR to obtain a linearized vector lpTpheA containing sgRNA. The fragments PHEA1, PHEA2 and lppTpheA were ligated to construct a recombinant plasmid pTPHEA (see FIG. 2).
Example 3: construction of recombinant TRP fragment E.coli
Coli cic 10303 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 pTTRP into Escherichia coli ECC9, screening to confirm that the fragments TRP1 and TRP2 are successful, adding 0.05mM IPTG to induce for 12h at 30 ℃, removing the recombinant plasmid pTTRP, selecting transformants by using primers pT-TRP-1F and pT-TRP-2R to carry out colony PCR, generating a band of about 1200bp, and obtaining the recombinant Escherichia coli ECTR-1 with the trpR gene knocked out after correct sequencing.
Example 4: construction of recombinant PHEA fragment E.coli
Transforming the recombinant plasmid pTPHEA into Escherichia coli ECTR-1, screening to confirm that the fragments PHEA1 and PHEA2 succeed, adding 0.05mM IPTG to induce for 12h at 30 ℃, removing the recombinant plasmid pTPHEA, selecting a transformant by selecting a primer pT-PHEA-1F and pT-PHEA-2R to perform colony PCR, generating a band of about 1200bp, and obtaining the recombinant Escherichia coli ECTR-2 with the pheA gene knocked out after correct sequencing.
Example 5: construction of promoter libraries
The upstream promoter sequence P of aroK gene is amplified by taking Escherichia coli CICC10303 genome as a template and adopting primers aroK.FOR, aroK.REV and QuickMutation gene random mutation kitaroK(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 ParoKMixed promoter fragments of different sequences PmaroK.
Example 6: obtaining vectors containing different promoter sequences
Linearizing a vector pET-20b containing eGFP by using primers ZTaroK. FOR, ZTaroK. REV, and carrying out PCR conditions: pre-denaturation at 98 ℃ for 3min, then denaturation at 98 ℃ for 10s and annealing at 55 ℃ for 5s and 72 ℃ for 2min, totally 34 cycles, cutting gel and recovering fragments with correct sizes to obtain a linearized vector fragment ZTaroK. 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 (see FIG. 3).
Example 7: transformation of Mixed plasmids
The mixed plasmid obtained in example 6 was transformed into E.coli ECTR-2 competent cells. The method comprises the following specific steps:
(1) the plasmid constructed in the example 6 is used for electrically transforming the competent cells of the Escherichia coli ECTR-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) Selecting a single colony growing on the plate, carrying out colony PCR verification by using primers AROKYZ. FOR and AROKYZ.REV, and obtaining a fragment with the length of 1000bp after the substitution and the amplification. 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 ParoKSelecting strains with stronger fluorescence intensity from the strains of the mixed promoter fragments PmyoK with different sequences, selecting 3 different mutant sequences after sequencing, respectively resynthesizing with the fragment of 800bp at the upstream of the aroK locus, and designing a primer aroK-1-F, aroK-1-R for amplification; according to the sequence information of genome DNA of Escherichia coli CICC 23604, aroK-2-F and aroK-2-R are designed, the gene sequence of aroK downstream homology arm is amplified from Escherichia coli CICC 23604 genome, and the obtained 2 segments are fused by fusion PCR technology to obtain a recombinant segment maroK- (1-3) (the sequences are respectively shown as SEQ ID NO. 6-8).
Example 10: construction of homologous recombination plasmids
A primer zharoK-F, zharoK-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-aroK containing sgRNA, and the linearized vector pT-aroK is connected with a recombinant fragment maroK- (1-3) to construct a recombinant plasmid.
Example 11: recombination of PmaroKConstruction of promoter E.coli
Respectively transforming the recombinant plasmid pT-aroK (1-3) into Escherichia coli ECTR-2 to obtain recombinant Escherichia coli with original aroK gene promoter replaced, selecting transformants by using a primer aroK-1-F and aroK-1-R to perform colony PCR, sequencing after 1000bp bands appear to successfully construct the recombinant Escherichia coli, adding 0.01M IPTG for induction, culturing at 30 ℃ for 24h to remove the pT-aroK (1-3) plasmid, and culturing at 37 ℃ for 24h to remove the pCas9 plasmid, wherein the named ECTR3- (1-3).
The genotypes of the strains constructed in examples 3, 4 and 11 are shown in Table 2.
TABLE 2 genotype of the strains
Example 12: using recombinant PmaroKPromoter strain fermentation production of L-tryptophan
(1) Seed liquid preparation
The original strain, Corynebacterium glutamicum CICC10303, and the recombinant bacterium ECTR3- (1-3) constructed in example 11 were inoculated into seed culture medium, respectively.
Culturing recombinant Escherichia coli CICC 10303-TRYP in plate culture medium at 37 deg.C for 24h, picking single colony to seed culture medium, and culturing at 37 deg.C and 220rpm for 10 h.
(2) Fermentation culture
Inoculating 10% of the inoculum size into fermentation medium, and culturing at 35 deg.C and 220rpm for 42 h. And measuring the L-tryptophan in the supernatant of the fermentation liquor by using HPLC high performance liquid chromatography. The results are shown in Table 3.
TABLE 3 fermentative production of L-tryptophan 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
Recombinant escherichia coli with improved L-tryptophan yield and construction method and application thereof
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<213> (Artificial sequence)
<400> 5
acagccgtaa aagcggtaat gtttttacgc tgaacgtgtt tcatctattt gacgcgcgca 60
ggtatttagc atacaaggag taccgatttg agagttggtg ctcttcgctg cctgcgttcc 120
atgatgatga tttatcattc aggcggcatt ttgctgtctt ttttacgcta atcttacccg 180
gtgatttatc gccagagcgg tggtagcaag gcagcgcgct tgcagcgacc agatatgcag 240
agggatgggt gatttattca gttgccaaac ccgctggagt attgagataa ttttcagtct 300
gactctcgca atatcttatg aggtttcagt tcatgtcctg cggcgctctc tgagcgaagc 360
gggtttatca ttaacgaata gtcttagtag taccgaaaaa 400
<210> 6
<211> 400
<212> DNA
<213> (Artificial sequence)
<400> 6
acagccgtaa aagcggtaat gtttttacgc tgaacgtgtt tcatctattt gacgcgcgca 60
ggtatttagc atacaaggag taccgatttg agagttggtg ctcttcgctg cctgcgttcc 120
atgatgatga tttatcattc aggcggcatt ttgctgtctt ttttacgcta atcttacccg 180
gtgatttatc gccagagcgg tggtagcaag gcagcgcgct tgcagcgacc agatatgcag 240
agggatgggt gatttattca gttgccaaac ccgctggagt attgagatac ttttcagtct 300
gactctcgca atatcttatg aggtttcagt tcatgtaatc cggcgctctc tgagtccagc 360
gggtttatca ttaacgaata gtcttagtag taccgaaaaa 400
<210> 7
<211> 400
<212> DNA
<213> (Artificial sequence)
<400> 7
acagccgtaa aagcggtaat gtttttacgc tgaacgtgtt tcatctattt gacgcgcgca 60
ggtatttagc atacaaggag taccgatttg agagttggtg ctcttcgctg cctgcgttcc 120
atgatgatga tttatcattc aggcggcatt ttgctgtctt ttttacgcta atcttacccg 180
gtgatttatc gccagagcgg tggtagcaag gcagcgcgct tgcagcgacc agatatgcag 240
agggatgggt gatttattca gttgccaaac ccgctggacc tatgagataa ttttcagtct 300
gactctcgca atatcttatg aggaagcagt tcatgtcctg cggcgctctc tgagcgaagc 360
gggtttatca ttaacgaata gtcttagtag taccgaaaaa 400
<210> 8
<211> 401
<212> DNA
<213> (Artificial sequence)
<400> 8
acagccgtaa aagcggtaat gtttttacgc tgaacgtgtt tcatctattt gacgcgcgca 60
ggtatttagc atacaaggag taccgatttg agagttggtg ctcttcgctg cctgcgttcc 120
atgatgatga tttatcattc aggcggcatt ttgctgtctt ttttacgcta atcttacccg 180
gtgatttatc gccagagcgg tggtagcaag gcagcgcgct tgcagcgacc agatatgcag 240
agggatgggt gatttattca gttgccaaac ccgctggaag gttcgagata attttcagtc 300
tgactctcgc aatatcttat gaggtttcag ttcatgggtc gcggcgctct ctgagcgaag 360
cgggtttatc attaacgaat agtcttagta gtaccgaaaa a 401
<210> 9
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 9
ttcaggcaac gatcgccggg 20
<210> 10
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 10
actcgctaaa gagtacgata 20
<210> 11
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 11
tatcgtactc tttagcgagt tttgtaggcc tgataagacg tggcgcatca ggca 54
<210> 12
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 12
catttcacga gatcttcggc 20
<210> 13
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 13
tgaattcacc aagacgggaa 20
<210> 14
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 14
aggccccccg atttgggag 19
<210> 15
<211> 40
<212> DNA
<213> (Artificial sequence)
<400> 15
cctcccaaat cggggggcct aaaggtgccg gatgatgtga 40
<210> 16
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 16
ggcgcatgat ggtccggt 18
<210> 17
<211> 49
<212> DNA
<213> (Artificial sequence)
<400> 17
cgccgaagat ctcgtgaaat gtttgcaaca gtgccgttga tcgtgctat 49
<210> 18
<211> 45
<212> DNA
<213> (Artificial sequence)
<400> 18
cccggcgatc gttgcctgaa tagtcggtgg tgataaactt atcat 45
<210> 19
<211> 42
<212> DNA
<213> (Artificial sequence)
<400> 19
caccggacca tcatgcgcct ttgcaacagt gccgttgatc gt 42
<210> 20
<211> 41
<212> DNA
<213> (Artificial sequence)
<400> 20
ttcccgtctt ggtgaattca tagtcggtgg tgataaactt a 41
<210> 21
<211> 14
<212> DNA
<213> (Artificial sequence)
<400> 21
acagccgtaa aagc 14
<210> 22
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 22
tttttcggta ctactaaga 19
<210> 23
<211> 69
<212> DNA
<213> (Artificial sequence)
<400> 23
ttaccgcttt tacggctgtc gacggagctc gaattcggat ccgaattaat tccgatatcc 60
atggccatc 69
<210> 24
<211> 52
<212> DNA
<213> (Artificial sequence)
<400> 24
atagtcttag tagtaccgaa aaaatgagta aaggagaaga acttttcact gg 52
<210> 25
<211> 15
<212> DNA
<213> (Artificial sequence)
<400> 25
tcgagtgcgg ccgca 15
<210> 26
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 26
cgaattcgag ctccgtcg 18
<210> 27
<211> 15
<212> DNA
<213> (Artificial sequence)
<400> 27
aattccatat caggt 15
<210> 28
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 28
tttttcggta ctactaag 18
<210> 29
<211> 47
<212> DNA
<213> (Artificial sequence)
<400> 29
tcttagtagt accgaaaaaa tggcagagaa acgcaatatc tttctgg 47
<210> 30
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 30
ctgctcgccg tcagggagg 19
<210> 31
<211> 40
<212> DNA
<213> (Artificial sequence)
<400> 31
cctccctgac ggcgagcagt ttgcaacagt gccgttgatc 40
<210> 32
<211> 40
<212> DNA
<213> (Artificial sequence)
<400> 32
acctgatatg gaatttagtc ggtggtgata aacttatcat 40
Claims (6)
1. The recombinant Escherichia coli with improved L-tryptophan yield is characterized in that an L-tryptophan feedback repression inhibition coding gene is knocked out in an Escherichia coli host bacteriumtrpRAnd genes encoding a prephenyl dehydrogenasepheA；
The recombinant escherichia coli also encodes shikimate kinase genesaroKPromoter sequence ParoKThe mutation is a sequence shown as SEQ ID NO. 7;
the escherichia coli host bacterium is escherichia coli CICC 10303.
2. A method for constructing recombinant Escherichia coli according to claim 1, comprising the steps of:
(1) construction of an integration fragment of the sequence mutant promoter: synthesizing aroK upstream and downstream homologous arm segments containing PmaroK gene, and fusing to obtain a segment maroK- (1-3);
(2) constructing a recombinant plasmid: respectively connecting 3 recombinant fragments maroK- (1-3) with a linear vector containing sgRNA to obtain recombinant plasmids containing maroK-1, maroK-2 and maroK-3;
(3) constructing high-yield L-tryptophan recombinant escherichia coli: transforming the plasmid containing cas9 protein into escherichia coli CICC10303 to obtain recombinant escherichia coli ECC 9; transforming Escherichia coli ECC9 with the recombinant plasmid pTTRP to obtain recombinant Escherichia coli ECTR1, transforming Escherichia coli ECTR-1 with the recombinant plasmid pTPHEA to obtain recombinant Escherichia coli ECTR2, respectively transforming Escherichia coli ECTR-2 with the recombinant plasmid pT-aroK (1-3), and removing foreign plasmid to obtain recombinant Escherichia coli ECTR3- (1-3);
the recombinant escherichia coli encodes shikimate kinase coding genearoKPromoter sequence ParoKThe mutation is a sequence shown as SEQ ID NO. 7.
3. The method of claim 2, wherein in step (2), the linearized vector comprises lpTdap or lptdd or pT-thrA.
4. The method according to claim 2, wherein the plasmid containing cas9 protein comprises pCas9 plasmid.
5. Use of the recombinant E.coli of claim 1 for the production of L-tryptophan.
6. The use of claim 5, wherein the recombinant Escherichia coli is cultured in a fermentation medium for 40-50 h to obtain the L-tryptophan, and the fermentation medium: 50-70 g/L of glucose, 14-18 g/L of magnesium sulfate heptahydrate, 22-26 g/L of ammonium sulfate, 8-12 g/L of yeast extract powder, 14-18 g/L of trisodium citrate dihydrate and 5-6 g/L of dipotassium phosphate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140431A (en) * | 2010-12-21 | 2011-08-03 | 大成生化科技(松原)有限公司 | L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same |
| CN109486737A (en) * | 2018-12-03 | 2019-03-19 | 江南大学 | A kind of recombination bacillus coli and its construction method of high yield L-Trp |
-
2020
- 2020-01-13 CN CN202010033337.8A patent/CN111154706B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140431A (en) * | 2010-12-21 | 2011-08-03 | 大成生化科技(松原)有限公司 | L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same |
| CN109486737A (en) * | 2018-12-03 | 2019-03-19 | 江南大学 | A kind of recombination bacillus coli and its construction method of high yield L-Trp |
Non-Patent Citations (4)
| Title |
|---|
| Development of L-tryptophan production strains by defined genetic modification in Escherichia coli;Zhi-Jun Zhao;《J Ind Microbiol Biotechnol》;20110504;第1921-1929页 * |
| Effect of gene knockouts of l-tryptophan uptake system on the production of l-tryptophan in Escherichia coli;Zhijun Zhao;《Process Biochemistry》;20111122;第340-344页 * |
| Zhi-Jun Zhao.Development of L-tryptophan production strains by defined genetic modification in Escherichia coli.《J Ind Microbiol Biotechnol》.2011,第1921-1929页. * |
| Zhijun Zhao.Effect of gene knockouts of l-tryptophan uptake system on the production of l-tryptophan in Escherichia coli.《Process Biochemistry》.2011,第340-344页. * |
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