CN108531439A - Escherichia coli genetic engineering bacterium and construction method and application thereof - Google Patents

Abstract

The invention discloses an Escherichia coli genetically engineered bacterium, which is introduced with a recombinant plasmid carrying the fimH gene. The invention also discloses a construction method of the genetically engineered bacteria. The invention further discloses the application of genetically engineered bacteria in the fermentation production of L-threonine. In the present invention, by constructing the Escherichia coli genetic engineering bacteria overexpressing the fimH gene, the biofilm production of Escherichia coli is increased in the process of fermenting and producing L-threonine, and the adhesion is increased, the number of bacteria groups is increased, and the L-threonine is improved. The yield and sugar conversion rate are high, and the fermentation period is shortened.

Description

A kind of Escherichia coli genetically engineered bacteria and its construction method and application

technical field

The invention relates to the fermentation of L-threonine, in particular to a genetic engineering bacterium of Escherichia coli and its construction method and application.

Background technique

L-threonine is an amino acid isolated and identified from fibrin hydrolyzate by W.C. Rose in 1935. It has been proved to be the last essential amino acid discovered. The human body cannot synthesize it by itself and must be ingested from food. As a food additive, L-threonine is widely used in the feed industry, health food and pharmaceutical industries. Due to the wide range of functions of L-threonine, the global market demand for threonine has grown rapidly in recent years, and the future threonine market will remain will increase rapidly, and the demand for threonine will also continue to increase.

The preparation methods of L-threonine mainly include protein hydrolysis, chemical synthesis, and microbial fermentation. Microbial fermentation has the advantages of saving resources, low cost, and less environmental pollution, and has been widely used in the production of L-threonine. With the rapid development of genetic engineering technology in modern society, the demand for industrial microbes and industrial technology applications is increasing, especially the construction of industrial microbe carrier systems, which provides an excellent source for the screening of excellent L-threonine production strains and the improvement of acid production levels. Reliable technical support makes the production of L-threonine by microbial direct fermentation a cheap industrial production method. At present, microorganisms capable of producing threonine include Escherichia, Corynebacterium, and Serratia. Threonine produced by Escherichia coli is now widely used in microbial fermentation experiments. Its advantages are fast propagation of strains, short fermentation cycle, and low cost. However, compared with the demand of the amino acid industry, although the output has increased steadily, the speed is still relatively slow. .

Contents of the invention

Purpose of the invention: In order to solve the problems of low yield and long fermentation cycle when producing L-threonine by fermentation of the existing E. The construction method of the present invention further provides the application of the Escherichia coli genetic engineering bacteria in the fermentation production of L-threonine.

Technical solution: a genetic engineering bacterium of Escherichia coli described in the present invention, which introduces a recombinant plasmid carrying the fimH gene. The present invention starts from the medical field. Most bacterial infections are closely related to the formation of biofilms. In medicine, reducing the formation of biofilms is used to reduce bacterial infections; the present invention firstly proposes to increase the amount of biofilms of Escherichia coli to increase the stickiness of Escherichia coli. Adhesive, can adhere to the non-biological surface, improving the yield of L-threonine and the fermentation cycle in the process of fermenting and producing L-threonine.

Wherein, the original Escherichia coli is CCTCC NO:M 2015233; the recombinant plasmid is the PET-28a plasmid carrying the fimH gene.

The present invention further provides the construction method of described escherichia coli genetic engineering bacterium, comprises the following steps:

(1) extracting the genome of the original Escherichia coli;

(2) using the genome extracted in step (1) as a template, and using PCR technology to amplify the fimH gene;

(3) inserting the fimH gene obtained in step (2) between the NcoI/XbaI restriction sites of the PET-28a plasmid to obtain a recombinant plasmid carrying the fimH gene;

(4) transforming the recombinant plasmid obtained in step (3) into competent cells of E.coli DH5α, and extracting the recombinant plasmid from E.coliDH5α;

(5) transforming the recombinant plasmid extracted in step (4) into the competent cells of the original Escherichia coli to obtain the Escherichia coli genetically engineered bacteria.

Wherein, the original Escherichia coli described in step (1) was donated from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and the Escherichia coli has applied for patent 201510199036.1 and has been preserved. The preservation number is CCTCC NO: M 2015233, and the strain number is CIBTS1688.

Wherein, the primers used in PCR described in step (2) are as follows:

fimH-F: GGATAACAATTCCCCTCTAGAATGAAACGAGTTATTACCCTGTTTGC

fimH-R: GATGATGGCTGCTGCCCATGGTTATTGATAAACAAAAGTCACGCCA.

The present invention further provides the application of the Escherichia coli genetically engineered bacterium in fermentative production of L-threonine.

Wherein, the fermentation conditions are as follows: the fermentation temperature is 25-37° C., and the fermentation time is 30-32 hours.

Beneficial effects: the present invention increases the yield of biofilm in the process of fermenting and producing L-threonine in Escherichia coli by constructing the Escherichia coli genetic engineering bacteria that overexpresses the fimH gene, increases the adhesion, increases the number of bacterial groups, and improves the L-threonine production process. The production of amino acid and the conversion rate of sugar shorten the fermentation cycle.

Description of drawings

Fig. 1 is the electropherogram of the original Escherichia coli genome;

Fig. 2 is the electropherogram of fimH gene;

Fig. 3 is the electrophoresis figure of recombinant plasmid;

Fig. 4 is the schematic diagram of recombinant plasmid;

Fig. 5 is the biofilm electron micrograph of bacterial strain CIBTS1688;

Fig. 6 is the biofilm electron micrograph of bacterial strain CIBTS1688fimH*;

Fig. 7 is the fermentation result of strain CIBTS1688 and strain CIBTS1688fimH*.

Detailed ways

Example 1

1. Extraction of the original Escherichia coli CIBTS1688 genome

Use takara company to extract bacterial genome kit (takara minibest bacteria genomicDNA extractionkit ver.3.0)

(1) Glycerol bacteria activation: 10 μL of the original strain CIBTS1688+5mL LB liquid medium, cultured overnight;

Among them, the formula of LB liquid medium is as follows: sodium chloride 10g/L, tryptone 10g/L, yeast powder 5g/L, sterilized at 121°C for 20min;

(2) Use a 1.5mL centrifuge tube to collect the bacterial solution (1.2mL bacterial solution per 1.5mL centrifuge tube), centrifuge at 12000rpm for 2min, and discard the supernatant;

(3) Add 180 μL of buffer GL, 20 μL of Proteinase K (20 mg/mL) and 10 μL of RNase A (10 mg/mL) to the cells obtained in step (2), shake and mix well, and warm in a water bath at 56 °C for 10 min. At this time, the solution is transparent and clear;

(4) Add 200 μL of buffer GB and 200 μL of absolute ethanol to the system obtained in step (3), and fully mix by pipetting;

(5) Install the Spin Column on the Collection Tube, move the solution obtained in step (4) to the SpinColumn, centrifuge at 12000rpm for 2min, and discard the filtrate;

(6) Add 500 μL of Buffer WA to the Spin Column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

(7) Add 700 μL of Buffer WB to the Spin Column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

(8) Add Buffer WB along the wall of the Spin Column to help completely flush the salt attached to the wall of the tube;

(9) repeat operation step (8);

(10) Place the Spin Column on the Collection Tube and centrifuge at 12000rpm for 2min;

(11) Place the Spin Column on a new 1.5mL centrifuge tube, add 50-200 μL of sterilized water or Elution Buffer to the center of the Spin Column membrane, and let it stand at room temperature for 5 minutes; among them, heat the sterilized water to 65°C The use is beneficial to improve the elution efficiency;

(12) Then centrifuge at 12000rpm for 2min to elute the DNA. If more yield is required, re-add the supernatant to the center of the Spin Column membrane or add 50-200 μL of sterilized water, let stand at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes to elute the DNA. The original E. coli DNA The electropherogram is shown in Figure 1.

2. Using PCR technology to amplify the target gene fimH

(1) Using the DNA obtained in step 1 as a template, use PCR technology to amplify the target gene fimH, and the reaction system is shown in Table 1;

fimH-F: GGATAACAATTCCCCTCTAGAATGAAACGAGTTATTACCCTGTTTGC

fimH-R: GATGATGGCTGCTGCCCATGGTTATTGATAAACAAAAGTCACGCCA

Table 1 PCR reaction system

The above PCR product (fimH gene fragment) was electrophoresed using 0.8% (0.8g/100mL) agarose gel, and the results are shown in FIG. 2 .

(2) Gel recovery and purification of fragment fimH

Use takara kit (TaKaRa MiniBEST Agarose Gel DNA Extraction KitVer.4.0)

2.1 Use TBE buffer to make agarose gel, and then perform agarose gel electrophoresis on the target fragment fimH;

2.2 Cut out the agarose gel containing the target fragment fimH under ultraviolet light, and absorb the surface liquid with a paper towel;

2.3 Cut up the rubber block and weigh the rubber block. When calculating the volume, calculate as 1 mg = 1 μL;

2.4 Add the dissolving solution Buffer GM to the rubber block. The amount of Buffer GM should not exceed the rubber block. After mixing evenly, the room temperature is 15-25°C to dissolve the rubber block. At this time, shake and mix intermittently to fully dissolve the rubber block;

2.5 Place the Spin Colum in the kit on the Collection Tube;

2.6 Transfer the solution obtained in step 2.4 to the Spin Column, centrifuge at 1200000rpm for 1min, and discard the filtrate;

2.7 Add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

2.8 Repeat step 2.7;

2.9 Place the Spin Column on the Collection Tube, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

2.10 Place the Spin Column on a new 1.5mL centrifuge tube, add 30μL sterilized water to the center of the Spin Column membrane, and let stand at room temperature for 1min;

2.11 Centrifuge at 12,000 rpm at room temperature for 1 min to elute fimH fragments;

2.12 Perform agarose gel electrophoresis verification on the purified fimH target fragment, and calculate the concentration; the nucleotide sequence of the fimH gene is shown in SEQ ID NO:1.

3. Extract plasmid PET-28a

Using takara kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0)

(1) Select a single colony of E.coli DH5α from the plate medium (containing PET-28a, from Kelei Biotechnology Co., Ltd., its nucleotide sequence is shown in SEQ ID NO: 2), wherein E.coli DH5α was purchased from Shanghai Sangon Bioengineering (Shanghai) Co., Ltd., NO.B528413, the preservation method of PET-28a refers to the method in "Molecular Cloning Experiment Guide" (translated by Huang Peitang, China, Science Press, 2002, third edition) ; Then inoculate into 5 mL of LB liquid medium containing antibiotics (the resistance is 50 μg/mL of kanamycin), and culture overnight at 37°C for 12-16 hours;

(2) Take 1-4 mL of the overnight culture, centrifuge at 12000 rpm for 2 min, and discard the supernatant;

(3) Fully suspend the bacterial pellet with 250 μL of Solution Ⅰ (containing RNase A);

(4) Add 250 μL of Solution II and mix gently up and down for 5-6 times to fully lyse the bacteria and form a transparent solution;

(5) Add 350 μL of Solution III pre-cooled at 4°C, gently invert and mix 5 to 6 times until a compact agglutination is formed, and then stand at room temperature for 2 minutes;

(6) centrifuge at 12000rpm at room temperature for 10min, and take the supernatant;

(7) Place the Spin Column in the kit on the Collection Tube;

(8) Transfer the supernatant obtained in step (6) to the Spin Column, centrifuge at 12000rpm for 1min, and discard the filtrate;

(9) Add 500 μL of Buffer WA to the Spin Column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

(10) Add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

(11) repeat operation step (10);

(12) Place the Spin Column on the Collection Tube again, centrifuge at 12000rpm for 1min, and remove the residual washing solution;

(13) Place the Spin Column on a new 1.5mL centrifuge tube, add 30-50μL of sterilized water at 65°C to the center of the Spin Column membrane, and let stand at room temperature for 1min;

(14) centrifuge at 12000rpm for 1min to elute DNA;

(15) Perform agarose gel electrophoresis verification on the extracted plasmid PET-28a, and calculate the concentration.

4. Using restriction endonucleases NcoI and XbaI to double-digest plasmid PET28a

Table 2 Enzyme hydrolysis system

5. Connect the target gene fimH and the double-digested plasmid PET28a to obtain the recombinant plasmid PET28a+fimH

Using Novizan One-Step Cloning Kit The following reaction system was prepared in an ice-water bath, see Table 3.

Table 3 enzyme-linked system

6. Transform the recombinant plasmid PET28a+fimH into E.coli DH5α

(1) Take out a tube of 200 μL E.coli DH5α competent cell suspension (Shanghai Sangon Bioengineering (Shanghai) Co., Ltd. NO.B528413)) from the -80°C refrigerator, thaw it at room temperature, and place it on ice immediately after thawing. superior;

(2) Add the recombinant plasmid PET-28a+fimH solution obtained in Step 5 to the E.coli DH5α competent cell suspension, shake gently, and place on ice for 30 minutes;

(3) Heat shock in a water bath at 42°C for 90 seconds, and immediately place on ice to cool for 5 minutes;

(4) After adding 1 mL of LB liquid medium and mixing, shake culture at 37°C for 40 minutes to restore the normal growth state of the bacteria;

(5) Shake the above bacterial solution, take 100 μL and spread it on the LB resistance plate, invert the culture dish, and incubate in a constant temperature incubator at 37°C for 12 hours;

The formula of the LB resistance plate is as follows: sodium chloride 10g/L, tryptone 10g/L, yeast powder 5g/L, agar powder 20g/L, sterilize at 121°C for 20min, add kanamycin to the final concentration after cooling 50μg/mL.

(6) Pick a single colony from the resistance plate, after preservation, extract the plasmid PET-28a+fimH according to step 3, perform agarose gel electrophoresis and sequencing verification on the extracted plasmid PET-28a+fimH, and the result is correct , see Figure 3, the map of plasmid PET-28a+fimH is shown in Figure 4, and its nucleotide sequence is shown in SEQ ID NO:3.

7. Transform the recombinant plasmid PET-28a+fimH into the original Escherichia coli

Transform the verified recombinant plasmid PET-28a+fimH into the competent cells of the original strain CIBTS1688 according to step 6, pick a single colony and extract the plasmid according to step 3 for sequencing verification, and obtain the constructed recombinant Escherichia coli CIBTS1688fimH*.

Among them, the competent cells of CIBTS1688 are prepared according to the following steps:

(1) Transfer 500 μL of the fresh overnight CIBTS1688 cell culture solution obtained from the activation of glycerol in step 1 into 50 mL of LB liquid medium, shake and shake at 37°C for 3 hours (shaking table 200r/min), measure OD ₆₀₀ =0.8-1.0 or so ;

(2) Under sterile conditions, transfer 1.5 mL of bacterial culture solution to an ice-precooled centrifuge tube, and place on ice for 10 min;

(3) Centrifuge at 4000r/min for 10min at 4°C;

(4) Discard the supernatant and invert the centrifuge tube for 1 min to drain the remaining culture medium;

(5) Add 150 μL of ice-precooled 0.1M CaCl ₂ aqueous solution to resuspend the cells, combine the two tubes, and ice-bath for 10 minutes;

(6) Centrifuge at 4000r/min for 10min at 4°C;

(7) Discard the supernatant, and invert the centrifuge tube for 1 min, so that the remaining traces of the culture solution are exhausted;

(8) First add 800 μL of ice-precooled 0.1M CaCl ₂ aqueous solution to resuspend the cells, then add 25 μL of pre-cooled 30% v/v glycerol, and store in -80°C for later use.

Example 2

(1) Take 100 μL of glycerol bacteria CIBTS1688 and CIBTS1688fimH* and add them into sterilized 5 mL LB liquid medium for overnight culture and activation;

(2) Then dilute at 1:100, and shake slowly at 37°C for 5 hours to make it reach the logarithmic phase _OD600 0.8-1.0;

(3) Take 2 mL of the bacterial solution to measure the absorbance value at _OD600 , and dilute the bacterial solution with sterilized distilled water so that the _OD600 of the diluted bacterial solution is 0.01;

(4) Take 200 μL of bacterial solution and add it to a 96-well plate, and LB liquid medium as a control, and incubate at 37°C for 24 hours;

(5) Pour out the bacterial solution from the 96-well plate, buffer with pure water for 3 times, and pat dry;

(6) Add 200 μL of 1% crystal violet solution into a 96-well plate, stain for 10 minutes, rinse with tap water, and dry in the air;

(7) Take 200 μL of 33% glacial acetic acid and add it to a 96-well plate to dissolve, shake gently, measure the biofilm production rate at OD ₆₀₀ , and take the average value: CIBTS1688 is cultured in a 96-well plate for 24 hours. The OD value of the well plate cultured for 24 hours was 2.6-2.7, and the electron micrographs of the biofilm are shown in Figure 5 and Figure 6 respectively.

Example 3

(1) Inoculate 10 μL of CIBTS1688 and modified CIBTS1688fimH* glycerol bacteria into 5 mL of LB liquid medium, and culture overnight at 37°C and 200 rpm;

(2) Inoculate the bacterial solution obtained in step (1) into 50 mL of LB liquid medium at a volume ratio of 1:10, cultivate at 37° C. and 200 rpm for 2 hours, and the concentration of the bacterial solution reaches about OD ₆₀₀ =1.0;

(3) Inoculate the bacterial liquid in the fermentation medium at a ratio of 5% by volume, ferment at 37° C., 200 to 250 rpm, and the reaction ends after the glucose is exhausted, and measure L-threonine in the fermentation liquid with a high-performance liquid chromatograph Acid content, the results are shown in Table 5, Figure 7.

Among them, the formula of the fermentation medium is as follows: glucose monohydrate 33g/L, sodium chloride 0.8g/L, ammonium sulfate 22g/L, dipotassium hydrogen phosphate trihydrate 2.65g/L, magnesium sulfate heptahydrate 0.8g/L, no Copper sulfate water 0.02g/L, iron sulfate heptahydrate 0.02g/L, thiamine hydrochloride 0.002g/L, yeast powder 1.0g/L, calcium carbonate 30g/L, pH7.2.

Wherein, the HPLC detection method is as follows:

Chromatographic conditions: Sepax AA special column, 4.6*150mm, detection wavelength 254nm, column temperature: 36°C, injection volume 5μL.

Derivative agent configuration: triethylamine acetonitrile solution: take 1.4mL triethylamine, add 2mL acetonitrile and mix well;

Phenyl isothiocyanate in acetonitrile solution: Take 25 μL of phenyl isothiocyanate and add 2 mL of acetonitrile and mix well.

Mobile phase A: Weigh 15.2g of sodium acetate, add 1850mL of water, adjust the pH value to 6.5 with glacial acetic acid after dissolving.

Mobile phase B: 80% acetonitrile (v/v);

Flow rate: 0.8mL/min, data collection time 50min.

Table 4 is the HPLC gradient elution program

Table 5 is the L-threonine output of genetically engineered bacteria

Note: The number of bacterial flora is measured after the fermentation is stable. The threonine production of CIBTS1688 is stable and reaches the highest value after 36 hours of fermentation, and the sugar consumption is over. The threonine production of CIBTS1688fimH* is stable and reaches the highest value after 32 hours of fermentation, and the sugar consumption is over.

Example 4

The method is the same as in Example 3, except that the glucose concentration is 100g/L, and the results are shown in Table 6.

Table 6 is the L-threonine output of genetically engineered bacteria when the glucose concentration is 100g/L

sequence listing

<110> Nanjing University of Technology

<120> A kind of Escherichia coli genetically engineered bacteria and its construction method and application

<160> 3

<170> SIPOSequenceListing 1.0

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<213> FimH

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ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 3480

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 3540

cacgaaccccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 3600

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 3660

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 3720

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3780

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3840

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3900

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact 3960

gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 4020

ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 4080

tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 4140

gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 4200

ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 4260

tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt 4320

attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 4380

ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 4440

cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 4500

gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc 4560

accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 4620

gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 4680

tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 4740

aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 4800

gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac 4860

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata 4920

ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 4980

aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 5040

cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 5100

ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 5160

cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 5220

ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 5280

gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 5340

cgccgctaca gggcgcgtcc cattcgcca 5369

<210> 3

<211> 6227

<212>DNA

<213> PET-28a+fimH

<400> 3

atccggatat agttccctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata 240

tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcttatt 300

gataaacaaa agtcacgcca ataatcgatt gcacattccc tgcagtcacc tgccctccgg 360

tacgtgcata atttgccgtt aatcccagac tcaccgccga agtccctact gctcctaacg 420

ataccgtgtt attcgctgga ataatcgtac cgttgcgcgt caactgtacg ccgacgccct 480

gtgcaggtga aaacgacgcg gtattggtga aaatcgagtt gcccgcatct gcggttgtgc 540

cggagaggta ataccccagg ttttggcttt tcgcacaata aacggtaaga ggaattggca 600

ctgaaccagg gtagtccggc agagtaacgg tgacatcacg agcagaaaca tcgcagccgc 660

cagtaggcac caccacatca ttattggcgt aaatattcca cacaaactgg aaatcatcgc 720

tgttatagtt gttggtctgt cgcaaaataa gcacggcaat taatgagcca gctttaatcg 780

ccaccccgcc cgcactgctc acaggcgtca aataaagcgc caccggccac ggcttatccg 840

ttctcgaatt ataaacaacg cgcggcgttt cgctggtggt aggaaatgga tagctactgc 900

cactatattt tacggtcccg gaaaaattag ataacacgcc gccataagcc gagcctcgtt 960

gcagtgtgac atagtctgta atggtttccg gataatcgtt atggcaaaag atttgcgtcg 1020

aaagatccac gaccaggttt tgccccacat tcacgacggg cgcaaggttt acataaacat 1080

tggcgctgcc accgccaata gggatagcgg taccattggc ggttttacag gcgaatgacc 1140

aggcatttac cgaccagccc atcagcagta cagcaaacag ggtaataact cgtttcatgg 1200

ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag 1260

atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt 1320

gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc 1380

atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc 1440

gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta 1500

ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat 1560

cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag 1620

ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctctta 1680

tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa 1740

agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc 1800

gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc 1860

gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc 1920

gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca 1980

acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga 2040

agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa 2100

cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt 2160

gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg 2220

tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga 2280

aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat 2340

cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat 2400

taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga 2460

agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg 2520

gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca 2580

gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc 2640

ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 2700

aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat 2760

ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2820

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2880

cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg 2940

gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc 3000

ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg 3060

tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg 3120

gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt 3180

ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa 3240

cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc 3300

taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt 3360

ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc 3420

gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca 3480

ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa 3540

ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca 3600

acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg 3660

agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 3720

agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 3780

agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg 3840

atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 3900

ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 3960

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 4020

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 4080

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4140

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4200

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4260

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4320

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4380

tccaagctgg gctgtgtgca cgaaccccccc gttcagcccg accgctgcgc cttatccggt 4440

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4500

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4560

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4620

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4680

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4740

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggatttg 4800

gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca 4860

tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt 4920

atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt 4980

gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa 5040

tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac 5100

catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg 5160

gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc 5220

gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag 5280

cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc 5340

gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca 5400

taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa 5460

ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc 5520

agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt 5580

acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt 5640

tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat 5700

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5760

aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 5820

tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 5880

tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 5940

acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 6000

aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 6060

cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 6120

cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 6180

cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca 6227

Claims (8)

1. an escherichia coli genetically engineered bacterium, is characterized in that, original escherichia coli has introduced the recombinant plasmid that carries fimH gene. 2. A kind of escherichia coli genetic engineering bacterium according to claim 1, is characterized in that, described original escherichia coli is CCTCC NO: M 2015233. 3. a kind of escherichia coli genetic engineering bacterium according to claim 1, is characterized in that, described recombinant plasmid is the PET-28a plasmid that carries fimH gene. 4. the construction method of Escherichia coli genetic engineering bacterium described in any one of claim 1-3, is characterized in that, comprises the following steps: (1) extracting the genome of the original Escherichia coli; (2) using the genome extracted in step (1) as a template, and using PCR technology to amplify the fimH gene; (3) inserting the fimH gene obtained in step (2) between the NcoI/XbaI restriction sites of the PET-28a plasmid to obtain a recombinant plasmid carrying the fimH gene; (4) transforming the recombinant plasmid obtained in step (3) into competent cells of E.coli DH5α, and extracting the recombinant plasmid from E.coli DH5α; (5) transforming the recombinant plasmid extracted in step (4) into the competent cells of the original Escherichia coli to obtain the Escherichia coli genetically engineered bacteria. 5. The construction method according to claim 4, characterized in that the original Escherichia coli described in step (1) is CCTCCNO:M 2015233. 6. construction method according to claim 4, is characterized in that, the primer that PCR described in step (2) uses is as follows: fimH-F: GGATAACAATTCCCCTCTAGAATGAAACGAGTTATTACCCTGTTTGC fimH-R: GATGATGGCTGCTGCCCATGGTTATTGATAAACAAAAGTCACGCCA. 7. the application of the Escherichia coli genetically engineered bacterium described in any one of claims 1-3 in the fermentative production of L-threonine. 8. The application according to claim 7, characterized in that the fermentation conditions are as follows: the fermentation temperature is 25-37° C., and the fermentation time is 30-32 hours.