CN114958705A

CN114958705A - Method for improving immobilized fermentation of escherichia coli by applying flagellin motA to quorum sensing dynamic regulation and control system

Info

Publication number: CN114958705A
Application number: CN202210712627.4A
Authority: CN
Inventors: 陈勇; 侯安奇; 应汉杰; 陈天鹏; 孙文俊; 余斌; 柳东; 牛欢青
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2022-08-30

Abstract

The invention discloses a method for improving immobilized fermentation of escherichia coli by applying flagellin motA to a quorum sensing dynamic regulation and control system. The Escherichia coli genetic engineering bacteria are pUC19-PJ23119-MCS plasmid containing esaI/R system introduced into Escherichia coli W1688, and the plasmid is combined with motion gene motA. The invention combines the motion gene motA related to colibacillus membrane formation with a quorum sensing system esaI/R for the first time, controls the formation, decomposition and diffusion of the biological membrane through signal transmission among quorum sensing signal molecules, establishes a specific regulation and control mode, accurately regulates and controls the formation process of the biological membrane in the fermentation process, strengthens the formation of the biological membrane in the early and middle stages of the fermentation, weakens the decomposition and diffusion in the later stage of the fermentation, and improves the immobilization efficiency and the catalytic efficiency of the biological membrane.

Description

Method for improving immobilized fermentation of escherichia coli by applying flagellin motA to quorum sensing dynamic regulation and control system

Technical Field

The invention relates to the field of biology, in particular to a method for improving immobilized fermentation of escherichia coli by applying flagellin motA to a quorum sensing dynamic regulation and control system.

Background

Biofilms (bifilms) are a form of microbial community in nature. The microorganisms gather together and secrete organic substances, such as polysaccharides, to cover the surface thereof, forming a film-like substance. The microorganisms can better adapt to the external environment by forming the colony structure, and resist factors which are not beneficial to survival. In the medical field, the drug resistance of the bacteria is improved because the pathogenic bacteria often form a biological membrane, and the research on the biological membrane lasts for a long time. In the field of industrial fermentation applications, the mechanism of biofilm formation is relatively poorly studied. In particular to the molecular research aiming at the fermentation characteristics of the immobilized cells.

Flagella play an important role in the survival and virulence of many pathogenic bacteria. Bacteria propel themselves using rotating flagella, the ability of which is critical to the survival and pathogenicity of the bacteria, consisting of a long outer filament acting as a propeller, a flexible connection structure, a hook, and a motor embedded in the cell envelope. Unlike other biomacromolecule systems that produce mechanical motion (e.g., myosin in muscle), bacterial flagellar motion does not directly use the energy of ATP hydrolysis. Flagellar rotation is powered by the energy of the electrochemical gradient across the cytoplasm. The bacterial flagella movement serves as a membrane-embedded ion-powered rotary engine consisting of a rotor surrounded by a ring of stator protein complexes (MotAB) powering its rotation. The motor is bidirectional: the chemoattractant signal can cause a conformational change in the rotor, known as "switching," which can result in a change in the direction of rotation of the motor. Each motA motB complex is thought to contain two proton conducting channels, spanning the cytoplasmic membrane.

In the early stages of biofilm development, escherichia coli cells are sessile due to adhesion genes attached to the surface, thus inhibiting flagella synthesis. Several small molecules such as cyclic diguanylic acid (c-di-GMP) are responsible for the transition from a planktonic state to a sessile state. The concentration of c-di-GMP is lower in the locomotor state and increases as the biofilm becomes mature. The motA motB compound plays an important role in the movement of escherichia coli and the irreversible attachment of thalli and the surface.

Quorum Sensing (QS) is a bacterial cell-to-cell communication system, where microorganisms sense changes in the density of bacterial populations through secretion of diffusible small molecule signals, thereby causing coordinated expression of a set of specific genes at the transcriptional level. This communication allows the bacteria to express different physiological behaviors including regulation of bioluminescence, biofilm formation, toxin production, spore formation, and the like.

With the intensive research on quorum sensing action mechanism, the biological characteristic can be used for developing a cell density-dependent self-induced dynamic regulation tool. In E.coli, lacking genes responsible for the synthesis of AHLs with functions similar to LuxI, it is not capable of producing AHL signal molecules by itself, but they encode LuxR homologous proteins and therefore can sense and respond to AHLs produced by other bacteria.

The AHL-mediated quorum sensing systems currently most studied and most widely used are mainly the luxI/luxR system from Vibrio fischeri and the esaI/esaR system from bacterial wilt disease of maize. Among gram-negative bacteria, the QS system, consisting of the luxI/luxR bi-component, is the most widely studied. Whereas the esaI/esaR system is homologous to the luxI/luxR system and originates from the maize pathogen Pantoea stewartii subspecies. Unlike most LuxR homologues, esaR can act as both a transcriptional activator and repressor, independent of whether binding to AHL signaling molecule occurs. In addition, Pesar is a native promoter that is repressed by esaR, and Pesas is a native promoter that is activated by esaR. At low cell densities, the cells produce less AHL signaling molecule and esaR binds to its target DNA sequence (esa box) turning on P _esaS Expression of downstream genes, turning off P _esaR Expression of a gene downstream of the promoter. At high cell densities, the cells produced more AHL signaling molecules and esaR released esa box binding to AHL, at which time promoter P _esaR Is opened and P _esaS And closing.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a genetically engineered Escherichia coli strain aiming at the defects of the prior art.

The technical problem to be solved by the invention is to provide a construction method of the escherichia coli genetic engineering bacteria.

The technical problem to be solved by the invention is to provide the application of the escherichia coli genetic engineering bacteria.

The invention idea is as follows: the esaI/esaR system is homologous with the luxI/luxR system and has a quorum sensing system with a bidirectional regulation function, and the motor gene motA is proved to promote the formation of an escherichia coli biofilm; therefore, the invention selects to utilize the esaI/esaR system and the motA to realize the aim of dynamic regulation.

In order to solve the first technical problem, the invention discloses an escherichia coli genetic engineering bacterium containing quorum sensing dynamic regulation and control biofilm formation, and pUC19-PJ23119-MCS plasmids containing esaI/R systems are introduced into escherichia coli W1688 and combined with a motor gene motA influencing escherichia coli film formation, so that the escherichia coli genetic engineering bacterium is applied to increase of escherichia coli film formation amount, and fermentation efficiency is further increased in immobilized fermentation.

Wherein, the gene contained in the esaI/R system is a transcription regulation factor with the nucleotide sequence respectively shown as SEQ ID NO.1 and SEQ ID NO. 2: esaI, esaR; the nucleotide sequences of the promoter are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4: p _esaS 、P _esaR-P (ii) a The nucleotide sequences of the reporter genes are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6: red fluorescent protein mCherry, green fluorescent protein EGFP.

Wherein the nucleotide sequence of the motion gene motA is shown as SEQ ID NO. 7.

In order to solve the second technical problem, the invention discloses a construction method of the escherichia coli genetic engineering bacterium, which comprises the following steps:

(1) amplifying to obtain esaI and esaR genes by taking pseudomonas aeruginosa genome DNA as a template; the gene P is synthesized by general biosynthesis _esaS 、P _esaR-P The PUC19 plasmid is used as a template to obtain P through amplification _esaS 、P _esaR-P A gene; amplifying by taking a plasmid pET28a-mCherry as a template to obtain an mCheerry gene; amplifying by taking the plasmid pET-28a-EGFP as a template to obtain an EGFP gene;

(2) taking the fragment obtained in the step (1) as a template, and carrying out overlap PCR amplification to obtain an esaI + esaR fragment and P _esaS + motA + mCherry fragment, P _esaR-P + an EGFP fragment;

(3) cloning the gene fragment obtained in the step (2) between Hind III and Sac I restriction sites of plasmid pUC19-PJ23119-MCS to obtain recombinant plasmid pUC19-PJ23119-MCS-esaI/R, wherein the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 8;

(4) transforming the recombinant plasmid obtained in the step (3) into an escherichia coli T1 competence, and extracting, purifying and recovering the recombinant plasmid from escherichia coli T1;

(5) and (3) transforming the recombinant plasmid extracted in the step (4) into the competence of Escherichia coli W1688 to obtain the Escherichia coli genetic engineering bacteria.

In the step (1), primers for amplifying esaI and esaR genes are esaI-F, esaI-R, esaR-F, esaR-R with nucleotide sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 respectively.

In step (1), P is amplified _esaS 、P _esaR-P The primers of the gene are P with nucleotide sequences shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 respectively _esaS -F、P _esaS -R、P _esaR-P -F、P _esaR-P -R。

In the step (1), primers for amplifying mCherry and EGFP genes are mChery-F, mCherry-R, EGFP-F, EGFP-R with nucleotide sequences shown as SEQ ID No.19, SEQ ID No.20, SEQ ID No.21 and SEQ ID No.22 respectively.

In order to solve the third technical problem, the invention discloses application of the large Escherichia coli genetic engineering bacteria in L-threonine production.

The application comprises the steps of replacing threonine synthetase thrC with a nucleotide sequence shown as SEQ ID No.23 to the position of an EGFP gene in the large escherichia coli genetic engineering bacteria, inoculating the bacteria into an LB culture medium for culture to obtain a bacterial liquid, diluting the bacterial liquid until OD is 0.05-0.15, and then inoculating the bacterial liquid into a fermentation culture medium containing an immobilized carrier for immobilized fermentation to produce xylanase.

Wherein the fermentation medium comprises 30g/L of glucose, 0.8g/L of sodium chloride, 20-22g/L of ammonium sulfate, 2g/L of anhydrous potassium dihydrogen phosphate, 0.8g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate pentahydrate, 0.02g/L of ferric sulfate heptahydrate, 10.002g/L of vitamin B, 1g/L of yeast powder and 15-30g/L of calcium carbonate.

Wherein the immobilized carrier is cotton fiber.

Wherein the dosage of the immobilized carrier is 3-7 g/100mL of fermentation medium.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention combines the motion gene motA related to colibacillus membrane formation with a quorum sensing system esaI/R for the first time, controls the formation, decomposition and diffusion of the biological membrane through signal transmission among quorum sensing signal molecules, establishes a specific regulation and control mode, accurately regulates and controls the formation process of the biological membrane in the fermentation process, strengthens the formation of the biological membrane in the early and middle stages of the fermentation, weakens the decomposition and diffusion in the later stage of the fermentation, and improves the immobilization efficiency and the catalytic efficiency of the biological membrane.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows motA, esaI, esaR, P in example 1 _esaS 、P _esaR-P Agarose gel electrophoresis of genes mCherry and EGFP.

FIG. 2 depicts the esaI + esaR fragment, P, after OVERLAP in example 1 _esaS + motA + mCherry fragment and P _esaR-P Agarose gel electrophoresis of the P + EGFP fragment.

FIG. 3 is a schematic diagram of the construction of a recombinant expression plasmid in example 1.

FIG. 4 is a PCR scheme of plasmid-transformed colonies in example 1.

FIG. 5 is a graph showing the results of fluorescence microscopy in example 2.

FIG. 6 is a graph showing the results of fluorescence intensity detection by the microplate reader in example 3.

FIG. 7 is a graph showing the results of the crystal violet staining experiment in example 4.

FIG. 8 is a graph showing the results of L-threonine production in example 5.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

In the following embodiment, the Escherichia coli strain selected for use in the present invention is W1688, and Pseudomonas aeruginosa strain selected for use in the present invention is ATCC 15692.

Example 1: construction of the target Strain

First, amplification of target Gene

(1) Using the original genome of escherichia coli as a template, and using a common PCR (polymerase chain reaction) to amplify by using motA-F, motA-R (sequences are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10) as a primer to obtain motA; using pseudomonas aeruginosa genome DNA as a template, and using common PCR to amplify by using esaI-F, esaI-R, esaR-F, esaR-R (sequences are respectively shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14) as a primer to obtain esaI and esaR; the gene P is synthesized by general biosynthesis _esaS 、P _esaR-P The pUC19 plasmid was used as a template, and P was used as a template in the conventional PCR _esaS -F、P _esaS -R、P _esaR-P -F、P _esaR-P -R (the sequences are respectively shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18) is used as a primer for amplification to obtain P _esaS 、P _esaR-P A gene; using plasmid pET28a-mCherry as a template, and using a common PCR to amplify by using mCherry-F, mCherry-R (sequences are respectively shown as SEQ ID NO.19 and SEQ ID NO. 20) as a primer to obtain a reporter gene mCheerry; the reporter gene EGFP is obtained by using plasmid pET-28a-EGFP as a template and EGFP-F, EGFP-R (sequences are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22) as a primer through common PCR (polymerase chain reaction).

The PCR reaction system is shown in Table 1 below, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 10 s; annealing at 60 ℃ for 30 s; extending for 2min at 68 ℃; the three steps of denaturation, annealing and extension are circulated for 35 times; further extension was carried out at 68 ℃ for 10 min. Wherein the annealing temperature depends on the Tm value of the primer, and the extension time at 68 ℃ depends on the length of the amplified fragment (1 kb/min). After the PCR is finished, the products are subjected to agarose gel electrophoresis, as shown in figure 1, and the target genes motA, esaI, esaR and P are obtained by cutting and recovering the gel _esaS 、P _esaR-P 、mCherry、EGFP。

TABLE 1 PCR reaction System

(2) Using the esaI gene and the esaR gene obtained in the step (1) as templates, using esaI-F as an upper primer and esaR-R as a lower primer, and amplifying by using an overlap PCR technology to obtain an esaI + esaR fragment; with P obtained in step (1) _esaS The gene, motA gene and mCherry gene are used as templates and P is used _esaS F is an upper primer, mchery-R is a lower primer, and P is obtained by applying overlap PCR technology to amplify _esaS + motA + mCherry fragment; with P obtained in step (1) _esaR-P Gene and EGFP gene as template, using P _esaR-P F is an upper primer, EGFP-R is a lower primer, and P is obtained by amplification by using overlap PCR technology _esaR-P + an EGFP fragment;

the PCR reaction system is shown in Table 3 below, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 10 s; annealing at 60 ℃ for 30 s; extending for 2min at 68 ℃; three steps of denaturation, annealing and extension are cycled for 30 times; further extension was carried out at 68 ℃ for 10 min. Wherein the annealing temperature depends on the Tm value of the primer, and the extension time at 68 ℃ depends on the length of the amplified fragment (1 kb/min). After the PCR was completed, the product was subjected to agarose gel electrophoresis, see FIG. 2, and the three fragment modules esaI + esaR fragment and P were recovered by cutting the gel _esaS + motA + mCherry fragment and P _esaR-P + EGFP fragment.

TABLE 2 PCR reaction System

Second, construction of recombinant plasmid

(1) Plasmid extracting body

Coli DH 5. alpha. glycerol strain (containing plasmid pUC19-PJ23119-MCS) was inoculated to liquid LB (ampicillin resistant, 100. mu.g/ml) and cultured at 37 ℃ for 12 hours. The cells cultured in the previous step were collected in a 1.5mL centrifuge tube, centrifuged at 10000rpm for 2min, and the supernatant was removed. Plasmid pUC19-PJ23119-MCS was extracted according to the AxyPrep plasmid extraction kit of Corning Life sciences Ltd.

(2) Linearization of vectors

To introduce a target geneFragment (esaI + esaR, P) _esaS + mCherry and P _esaR-P + EGFP) was ligated to plasmid pUC19-PJ23119-MCS, which was digested in a double digestion reaction system (20. mu.L) of 0.1% BSA 2. mu.L, 1. mu.M buffer 2. mu.L, 0.5. mu.L each of HindIII and SacI enzymes (from TAKARA), plasmid pUC19-PJ23119-MCS 10. mu.L, sterile water 5. mu.L. The enzyme digestion reaction is carried out for 2h at 37 ℃. And (4) carrying out gel recovery after enzyme digestion for subsequent experiments.

Wherein, the buffer: 10 × Loading buffer: 0.9% SDS, 50% glycocol, 0.05% Bromophenol Blue;

the Hind iii enzyme: restriction enzyme

The SacI enzyme: restriction enzyme

Definition of enzyme activity: the reaction was carried out at 37 ℃ for 1 hour in 50. mu.L of the reaction solution, and the amount of the enzyme that completely decomposed 1. mu.g of lambda.DNA was defined as 1 activity unit (U).

(3) Construction of plasmid pUC19-PJ23119-MCS-esaI/R

The three fragments (esaI + esaR, P) _esaS + mCherry and P _esaR-P + EGFP) was ligated to the purified linear vector following the procedures described in the instructions for the multicegment one-step cloning of the enzyme by NEB to obtain the recombinant plasmid pUC19-PJ23119-MCS-esaI/R (SEQ ID NO. 8). The one-step cloning reaction system was as shown in Table 3, and was immediately ice-cooled for 5 minutes after being water-bathed at 50 ℃ for 15 minutes and stored at-20 ℃. A schematic diagram of the construction of the recombinant expression plasmid is shown in FIG. 3.

TABLE 3 Multi-fragment one-step cloning reaction System

Wherein the plasmid pUC19-PJ23119-MCS after double digestion, the purified gene fragment esaI + esaR and the purified geneFragment P _esaS + motA + mCherry, purified gene fragment P _esaR-P + EGFP concentration 100 ng/. mu.l.

(4) Transformation and selection of recombinant plasmids

T1 (purchased from all-gold) competent cells were thawed to liquid state on ice, and competent cells and recombinant plasmid pUC19-PJ23119-MCS-esaI/R at a ratio of 10:1v/v were added to a pre-cooled centrifuge tube. Standing on ice for 30min, and performing heat shock in 42 deg.C water bath for 90 s. The ice box was cooled for 3 minutes, added with 1mL LB at 37 degrees C, 200rpm culture for 1 hours, centrifugal removal of most of the supernatant heavy suspension bacteria liquid and ampicillin resistance plate. After overnight incubation in an incubator at 37 ℃, single spots on the plates were picked, 100. mu.g/ml ampicillin resistance in the shake tube was added, and then the plasmid pUC19-PJ23119-MCS-esaI/R was extracted for sequencing validation.

Third, construction of recombinant strains

After the recombinant plasmid is successfully verified, a system strain containing esaI/R is constructed and named as Escherichia coli W1688-P. The successfully verified recombinant plasmid is transferred into the escherichia coli W1688 competence by the same transformation method, and a single colony is picked for colony PCR verification. The results of colony PCR experiments are shown in FIG. 4, and it can be seen that the transformation was successful.

Example 2: performing fluorescence microscope observation on the original bacterium W1688 and the modified bacterium W1688-P

(1) Glycerol bacterium WT (original bacterium W1688) and modified bacterium W1688-P (the strain containing esaI/R system constructed in example 1) were each added in an amount of 100. mu.L to a sterilized LB liquid medium of 5mL overnight for culture (ampicillin resistance, 100. mu.g/mL), and activated;

(2) according to the volume ratio of 1: 10, transferring the activated bacterial liquid obtained in the step (1) to 100mL of LB liquid medium (ampicillin resistance, 100. mu.g/mL; 3mL of 1M L-arabinose was added when appropriate) and continuing the culture at 37 ℃ and 220rpm for 13 hours;

(3) and (3) directly observing the cultured bacterial liquid in the step (2) under a fluorescence microscope after sampling, wherein the fluorescence microscope result is shown in figure 5, which shows that the original transformed bacterium has no luminescence, and the transformed bacterium has luminescence, and shows that the esaI/R system of the transformed bacterium W1688-P is constructed without errors.

Example 3: fluorescence intensity detection experiment of enzyme labeling instrument for transformed bacterium W1688-P

(1) Since the original bacterium W1688 did not emit light in the above fluorescence microscope observation, only the modified bacterium was detected in the fluorescence intensity detection experiment. The transformed strain W1688-P (the strain containing the esaI/R system constructed in example 1) was added to a sterilized 5mL LB liquid medium in an amount of 100. mu.L for overnight culture (ampicillin resistance, 100. mu.g/mL), and activated;

(2) according to the volume ratio of 1: 10, transferring the activated bacterial liquid obtained in the step (1) to 100mL of LB liquid medium (ampicillin resistance, 100 mu g/mL), continuously culturing at 37 ℃ and 220rpm, and taking samples every 4 hours for 48 hours;

(3) the well-taken sample is diluted by 20 times by PBS buffer solution, 200 mu L of the diluted sample is added into a 96-well plate, and detection is carried out by a microplate reader at different wavelengths respectively: detecting the green fluorescence intensity of the excitation wavelength of 485nm and the emission wavelength of 528nm, and detecting the red fluorescence intensity of the excitation wavelength of 587nm and the emission wavelength of 610 nm. The fluorescence intensity is shown in fig. 6, the red fluorescence (mCherry) is stronger in the early stage, the green fluorescence (EGFP) is stronger in the later stage, and the fluorescent intensity accords with the process of our esaI/R system and the experimental design concept, so that the next step of application can be carried out.

Example 4: biofilm crystal violet staining experiment

The threonine synthetase thrC (derived from Escherichia coli and having a nucleotide sequence shown in SEQ ID NO.23) catalyzing homoserine to synthesize L-threonine in the L-threonine metabolic pathway is replaced to the position of EGFP gene.

Alternatively, the recombinant plasmid EGFP is cut off by using an enzymatic cleavage site and thrC is ligated by performing one-step cloning with the one-step cloning kit C112 of Novozam, which is similar to the step (3) of example 1, thereby obtaining the strain W1688-P-thrC.

Simultaneously, a strain W1688-thrC containing thrC gene only is constructed (the method is the same as the above, and the used plasmid is an empty plasmid PUC19 without esaI/R system).

(1) Glycerol bacterium WT (original bacterium W1688), modified bacterium W1688-P (the strain containing esaI/R system constructed in example 1), W1688-thrC and W1688-P-thrC were each added in 100. mu.L of each of them to a sterilized 5mL LB liquid medium for overnight culture (ampicillin resistance, 100. mu.g/mL), and activated;

(2) according to the volume ratio of 1: 10, transferring the activated bacterium liquid obtained in the step (1) to 100mL of LB liquid culture medium, and continuously culturing at 37 ℃ and 220rpm until the OD600 value of the bacterium liquid is between 0.8 and 1.2;

(3) measuring the absorbance value of 2mL of bacterial liquid under OD600, and diluting the bacterial liquid by using a sterilized LB liquid culture medium to enable the OD600 of the diluted bacterial liquid to be 1;

(4) adding 200 mu L of diluted bacterial liquid into a 96-well plate, using a liquid culture medium as a reference, and culturing at 37 ℃ for 12h, 24h and 36 h;

(5) pouring out the bacterial liquid in the 96-well plate, taking 200 mu L of 0.01M PBS buffer solution to wash for 2-3 times, and patting dry; adding 200 μ L paraformaldehyde fixing solution, fixing for 15min, pouring off, and air drying. Adding 200 μ L of 1% crystal violet solution into 96-well plate, staining for 10-20min, washing with PBS buffer solution for 2-3 times, and drying; adding 200 μ L glacial acetic acid into 96-well plate to dissolve the biofilm, gently shaking for 40min, measuring OD570nm biofilm yield, and taking average value. The crystal violet staining experiment result is shown in figure 7, which shows that the modified strain W1688-P-thrC biofilm is increased obviously, and the biofilm immobilization fermentation experiment can be carried out.

Example 5: the respective bacteria are used for producing L-threonine by immobilized fermentation

Fermentation medium: 30g/L of glucose, 0.8g/L of sodium chloride, 20g/L of ammonium sulfate, 2g/L of anhydrous potassium dihydrogen phosphate, 0.8g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate pentahydrate, 0.02g/L of ferric sulfate heptahydrate, 10.002g/L of vitamin B, 1g/L of yeast powder and 15g/L of calcium carbonate.

Fermentation: w1688, W1688-P, W1688-thrC, and W1688-P-thrC were removed from the freezer at-80 ℃ and 5mL of LB medium was prepared in tubes, one of which contained 100. mu.g/mL ampicillin in a bottle with an inoculum size of 1%. Culturing in a shaker at 37 deg.C for 12h at 200rpm to obtain bacterial liquid.

The resulting bacterial solution was diluted to OD 0.1 and inoculated into 100mL of a fermentation medium (5 g of cotton fibers cut into squares were placed in the medium in advance), and cultured in a shaker at 37 ℃ for 36 hours at a rotation speed of 200 and 220 rpm.

The detection method comprises the following steps: the L-threonine concentration was measured by high performance liquid chromatography and UV detector (ultraviolet 254nm), using a Sepax AAA ion exclusion column (250X 4.6mm), at 36 ℃ with 0.1mol/L sodium acetate and 80% acetonitrile as mobile phases, at a feed rate of 5. mu.L each time and a running time of 50min, the peak areas of the different samples were determined, and the contents were calculated using the standard solution curve as a reference. The L-threonine content determination experiment result is shown in FIG. 8, the original bacterium yield is 10.5g/L, the modified bacterium yield is 13.5g/L, and the modified bacterium yield is improved by 1.29 times.

The invention provides an escherichia coli genetic engineering bacterium containing an esaI/R quorum sensing system, and a construction method and application thereof, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

<120> method for improving immobilized fermentation of escherichia coli by applying flagellin motA to quorum sensing dynamic regulation and control system

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 606

<212> DNA

<213> esaI

<400> 1

atgatcgtac aaattggtcg gcgcgaagag ttcgataaaa aactgctggg cgagatgcac 60

aagttgcgtg ctcaagtgtt caaggagcgc aaaggctggg acgttagtgt catcgacgag 120

atggaaatcg atggttatga cgcactcagt ccttattaca tgttgatcca ggaagatact 180

cctgaagccc aggttttcgg ttgctggcga attctcgata ccactggccc ctacatgctg 240

aagaacacct tcccggagct tctgcacggc aaggaagcgc cttgctcgcc gcacatctgg 300

gaactcagcc gtttcgccat caactctgga cagaaaggct cgctgggctt ttccgactgt 360

acgctggagg cgatgcgcgc gctggcccgc tacagcctgc agaacgacat ccagacgctg 420

gtgacggtaa ccaccgtagg cgtggagaag atgatgatcc gtgccggcct ggacgtatcg 480

cgcttcggtc cgcacctgaa gatcggcatc gagcgcgcgg tggccttgcg catcgaactc 540

aatgccaaga cccagatcgc gctttacggg ggagtgctgg tggaacagcg actggcggtt 600

tcatga 606

<210> 2

<211> 720

<212> DNA

<213> esaR

<400> 2

atggccttgg ttgacggttt tcttgagctg gaacgctcaa gtggaaaatt ggagtggagc 60

gccatcctgc agaagatggc gagcgacctt ggattctcga agatcctgtt cggcctgttg 120

cctaaggaca gccaggacta cgagaacgcc ttcatcgtcg gcaactaccc ggccgcctgg 180

cgcgagcatt acgaccgggc tggctacgcg cgggtcgacc cgacggtcag tcactgtacc 240

cagagcgtac tgccgatttt ctgggaaccg tccatctacc agacgcgaaa gcagcacgag 300

ttcttcgagg aagcctcggc cgccggcctg gtgtatgggc tgaccatgcc gctgcatggt 360

gctcgcggcg aactcggcgc gctgagcctc agcgtggaag cggaaaaccg ggccgaggcc 420

aaccgtttca tggagtcggt cctgccgacc ctgtggatgc tcaaggacta cgcactgcag 480

agcggtgccg gactggcctt cgaacatccg gtcagcaaac cggtggttct gaccagccgg 540

gagaaggaag tgttgcagtg gtgcgccatc ggcaagacca gttgggagat atcggttatc 600

tgcaactgct cggaagccaa tgtgaacttc catatgggaa atattcggcg gaagttcggt 660

gtgacctccc gccgcgtagc ggccattatg gccgttaatt tgggtcttat tactctctga 720

<210> 3

<211> 184

<212> DNA

<213> PesaS

<400> 3

gctcacaaca gtgtaagcgt atccgttatt gtttgatttt caaggaaaaa agaaaacatt 60

caggctccat gctgcttctt ttacttaacg tggacttaac ctgcactata gtacaggcaa 120

gatgatactt aagagtaact tacaatgaat cattcagagg ttacaatggc ttcagttgtt 180

tagc 184

<210> 4

<211> 102

<212> DNA

<213> PesaR-P

<400> 4

ttgtaacctc tgaatgattc attgtaagtt actcttaagt atcatcttgc ctgtactata 60

gtgcaggtta agtccagcct gtactatagt gcaggtggat cc 102

<210> 5

<211> 717

<212> DNA

<213> mCherry

<400> 5

atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagt ttctagagga catcacctcc cacaacgagg actacaccat cgtggaacag 660

tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaa 717

<210> 6

<211> 720

<212> DNA

<213> EGFP

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 7

<211> 888

<212> DNA

<213> motA

<400> 7

gtgcttatct tattaggtta cctggttgtt ctcggtacag ttttcggcgg ttatttgatg 60

accggtggaa gccttggagc actctatcaa cccgctgaac tggtgattat tgccggtgca 120

gggattgggt cgtttatcgt cggcaataat ggcaaagcga ttaaaggcac gctgaaggcg 180

ctgccgttgc tgtttcgtcg ctccaaatac accaaagcaa tgtatatgga tctgctggct 240

ctgctttatc ggttgatggc gaaatcgcgg cagatgggga tgttttcgct ggaacgtgat 300

attgaaaatc cccgtgagag cgagatcttc gccagctacc cacgcatcct cgcggatagc 360

gtcatgcttg attttatcgt cgattatctg cgcctgatta tcagcggtca catgaacacc 420

ttcgaaatcg aagctctgat ggatgaagag attgagacgc acgaaagcga ggcagaagtc 480

ccggcgaaca gtctggcgct ggtcggggac tcacttccgg cgtttggtat tgttgcggct 540

gtaatggggg tcgttcacgc gttaggttca gccgatcgtc ctgccgccga gctgggtgcg 600

cttatcgcac atgcgatggt ggggactttc ctcggcattt tattggctta cggatttatt 660

tccccattag cgactgtttt acgtcagaaa agcgccgaaa ccagcaaaat gatgcagtgc 720

gtcaaagtca ctctgctttc taatctgaac ggttacgcac cgcctatcgc cgttgagttt 780

ggtcgcaaaa cgctctattc cagcgaacgt ccgtcgttta ttgaactgga agagcatgtg 840

cgtgcggtga aaaatccgca acaacagacg acaaccgagg aagcatga 888

<210> 8

<211> 6944

<212> DNA

<213> pUC19-PJ23119-MCS-esaI/R

<400> 8

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120

cactcattag gcaccccagg cttgacagct agctcagtcc taggtataat actagttttc 180

acacaggaaa cagctatgac catgattacg ccaagcttaa gaaaccaatt gtccatattg 240

catcagacat tgccgtcact gcgtctttta ctggcacttc tcgctaacca aaccggtaac 300

cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa 360

caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg gcgtcacact 420

ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac gctttttatc 480

gcaactctct actgtttctc catgtcgaca tgatcgtaca aattggtcgg cgcgaagagt 540

tcgataaaaa actgctgggc gagatgcaca agttgcgtgc tcaagtgttc aaggagcgca 600

aaggctggga cgttagtgtc atcgacgaga tggaaatcga tggttatgac gcactcagtc 660

cttattacat gttgatccag gaagatactc ctgaagccca ggttttcggt tgctggcgaa 720

ttctcgatac cactggcccc tacatgctga agaacacctt cccggagctt ctgcacggca 780

aggaagcgcc ttgctcgccg cacatctggg aactcagccg tttcgccatc aactctggac 840

agaaaggctc gctgggcttt tccgactgta cgctggaggc gatgcgcgcg ctggcccgct 900

acagcctgca gaacgacatc cagacgctgg tgacggtaac caccgtaggc gtggagaaga 960

tgatgatccg tgccggcctg gacgtatcgc gcttcggtcc gcacctgaag atcggcatcg 1020

agcgcgcggt ggccttgcgc atcgaactca atgccaagac ccagatcgcg ctttacgggg 1080

gagtgctggt ggaacagcga ctggcggttt catgataatg tgagttagct cactcattag 1140

gcaccccagg cttgacagct agctcagtcc taggtataat actagtatgg ccttggttga 1200

cggttttctt gagctggaac gctcaagtgg aaaattggag tggagcgcca tcctgcagaa 1260

gatggcgagc gaccttggat tctcgaagat cctgttcggc ctgttgccta aggacagcca 1320

ggactacgag aacgccttca tcgtcggcaa ctacccggcc gcctggcgcg agcattacga 1380

ccgggctggc tacgcgcggg tcgacccgac ggtcagtcac tgtacccaga gcgtactgcc 1440

gattttctgg gaaccgtcca tctaccagac gcgaaagcag cacgagttct tcgaggaagc 1500

ctcggccgcc ggcctggtgt atgggctgac catgccgctg catggtgctc gcggcgaact 1560

cggcgcgctg agcctcagcg tggaagcgga aaaccgggcc gaggccaacc gtttcatgga 1620

gtcggtcctg ccgaccctgt ggatgctcaa ggactacgca ctgcagagcg gtgccggact 1680

ggccttcgaa catccggtca gcaaaccggt ggttctgacc agccgggaga aggaagtgtt 1740

gcagtggtgc gccatcggca agaccagttg ggagatatcg gttatctgca actgctcgga 1800

agccaatgtg aacttccata tgggaaatat tcggcggaag ttcggtgtga cctcccgccg 1860

cgtagcggcc attatggccg ttaatttggg tcttattact ctctgagcat gcgctcacaa 1920

cagtgtaagc gtatccgtta ttgtttgatt ttcaaggaaa aaagaaaaca ttcaggctcc 1980

atgctgcttc ttttacttaa cgtggactta acctgcacta tagtacaggc aagatgatac 2040

ttaagagtaa cttacaatga atcattcaga ggttacaatg gcttcagttg tttagcgtcg 2100

acgtgcttat cttattaggt tacctggttg ttctcggtac agttttcggc ggttatttga 2160

tgaccggtgg aagccttgga gcactctatc aacccgctga actggtgatt attgccggtg 2220

cagggattgg gtcgtttatc gtcggcaata atggcaaagc gattaaaggc acgctgaagg 2280

cgctgccgtt gctgtttcgt cgctccaaat acaccaaagc aatgtatatg gatctgctgg 2340

ctctgcttta tcggttgatg gcgaaatcgc ggcagatggg gatgttttcg ctggaacgtg 2400

atattgaaaa tccccgtgag agcgagatct tcgccagcta cccacgcatc ctcgcggata 2460

gcgtcatgct tgattttatc gtcgattatc tgcgcctgat tatcagcggt cacatgaaca 2520

ccttcgaaat cgaagctctg atggatgaag agattgagac gcacgaaagc gaggcagaag 2580

tcccggcgaa cagtctggcg ctggtcgggg actcacttcc ggcgtttggt attgttgcgg 2640

ctgtaatggg ggtcgttcac gcgttaggtt cagccgatcg tcctgccgcc gagctgggtg 2700

cgcttatcgc acatgcgatg gtggggactt tcctcggcat tttattggct tacggattta 2760

tttccccatt agcgactgtt ttacgtcaga aaagcgccga aaccagcaaa atgatgcagt 2820

gcgtcaaagt cactctgctt tctaatctga acggttacgc accgcctatc gccgttgagt 2880

ttggtcgcaa aacgctctat tccagcgaac gtccgtcgtt tattgaactg gaagagcatg 2940

tgcgtgcggt gaaaaatccg caacaacaga cgacaaccga ggaagcatga atggtgagca 3000

agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg 3060

agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg 3120

agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg 3180

acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca 3240

tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact 3300

tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca 3360

tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga 3420

agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg 3480

gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga 3540

ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt 3600

ttctagagga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg 3660

ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaattg taacctctga 3720

atgattcatt gtaagttact cttaagtatc atcttgcctg tactatagtg caggttaagt 3780

ccagcctgta ctatagtgca ggtggatccc tcgagatggt gagcaagggc gaggagctgt 3840

tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 3900

gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct 3960

gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg 4020

tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 4080

tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 4140

cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 4200

tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 4260

acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc 4320

gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca 4380

tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga 4440

gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 4500

ggatcactct cggcatggac gagctgtaca agtaagagct cgaattcact ggccgtcgtt 4560

ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 4620

ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 4680

ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 4740

ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 4800

agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 4860

gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 4920

ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 4980

aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 5040

ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 5100

taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 5160

cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 5220

acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 5280

ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 5340

atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 5400

gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 5460

acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 5520

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 5580

accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 5640

ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 5700

acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 5760

gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 5820

tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 5880

ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5940

actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 6000

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 6060

tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 6120

gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 6180

cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 6240

gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 6300

gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 6360

tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 6420

ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 6480

cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 6540

gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 6600

gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 6660

gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 6720

cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 6780

tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 6840

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 6900

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaga 6944

<210> 9

<211> 52

<212> DNA

<213> motA-F

<400> 9

caatggcttc agttgtttag cgtcgacgtg cttatcttat taggttacct gg 52

<210> 10

<211> 47

<212> DNA

<213> motA-R

<400> 10

atcctcctcg cccttgctca ccattcatgc ttcctcggtt gtcgtct 47

<210> 11

<211> 50

<212> DNA

<213> esaI-F

<400> 11

gcaactctct actgtttctc catgtcgaca tgatcgtaca aattggtcgg 50

<210> 12

<211> 65

<212> DNA

<213> esaI-R

<400> 12

gtcaaccaag gccatactag tattatacct aggactgagc tagctgtcaa tcatgaaacc 60

gccag 65

<210> 13

<211> 65

<212> DNA

<213> esaR-F

<400> 13

ctggcggttt catgattgac agctagctca gtcctaggta taatactagt atggccttgg 60

ttgac 65

<210> 14

<211> 37

<212> DNA

<213> esaR-R

<400> 14

gcatgctcag agagtaataa gacccaaatt aacggcc 37

<210> 15

<211> 46

<212> DNA

<213> PesaS-F

<400> 15

tgggtcttat tactctctga gcatgcgctc acaacagtgt aagcgt 46

<210> 16

<211> 47

<212> DNA

<213> PesaS-R

<400> 16

agggtaataa ctcgtttcat gtcgacgcta aacaactgaa gccattg 47

<210> 17

<211> 42

<212> DNA

<213> PesaR-P-F

<400> 17

ctacaacgtc aacatcaagt ttctagagga catcacctcc ca 42

<210> 18

<211> 52

<212> DNA

<213> PesaR-P-R

<400> 18

tctcggcatg gacgagctgt acaagtaact cgagggatcc acctgcacta ta 52

<210> 19

<211> 47

<212> DNA

<213> mcherry-F

<400> 19

gcgtgacttt tgtttatcaa taaatggtga gcaagggcga ggaggat 47

<210> 20

<211> 26

<212> DNA

<213> mcherry-R

<400> 20

tctagattac ttgtacagct cgtcca 26

<210> 21

<211> 47

<212> DNA

<213> EGFP-F

<400> 21

agaagcagcg gatccctcga gatggtgagc aagggcgagg agctgtt 47

<210> 22

<211> 50

<212> DNA

<213> EGFP-R

<400> 22

cgacggccag tgaattcgag ctcttacttg tacagctcgt ccatgccgag 50

<210> 23

<211> 1287

<212> DNA

<213> thrC

<400> 23

atgaaactct acaatctgaa agatcacaac gagcaggtca gctttgcgca agccgtaacc 60

caggggttgg gcaaaaatca ggggctgttt tttccgcacg acctgccgga attcagcctg 120

actgaaattg atgagatgct gaagctggat tttgtcaccc gcagtgcgaa gatcctctcg 180

gcgtttattg gtgatgaaat cccacaggaa atcctggaag agcgcgtgcg cgcggcgttt 240

gccttcccgg ctccggtcgc caatgttgaa agcgatgtcg gttgtctgga attgttccac 300

gggccaacgc tggcatttaa agatttcggc ggtcgcttta tggcacaaat gctgacccat 360

attgcgggtg ataagccagt gaccattctg accgcgacct ccggtgatac cggagcggca 420

gtggctcatg ctttctacgg tttaccgaat gtgaaagtgg ttatcctcta tccacgaggc 480

aaaatcagtc cactgcaaga aaaactgttc tgtacattgg gcggcaatat cgaaactgtt 540

gccatcgacg gcgatttcga tgcctgtcag gcgctggtga agcaggcgtt tgatgatgaa 600

gaactgaaag tggcgctagg gttaaactcg gctaactcga ttaacatcag ccgtttgctg 660

gcgcagattt gctactactt tgaagctgtt gcgcagctgc cgcaggagac gcgcaaccag 720

ctggttgtct cggtgccaag cggaaacttc ggcgatttga cggcgggtct gctggcgaag 780

tcactcggtc tgccggtgaa acgttttatt gctgcgacca acgtgaacga taccgtgcca 840

cgtttcctgc acgacggtca gtggtcaccc aaagcgactc aggcgacgtt atccaacgcg 900

atggacgtga gtcagccgaa caactggccg cgtgtggaag agttgttccg ccgcaaaatc 960

tggcaactga aagagctggg ttatgcagcc gtggatgatg aaaccacgca acagacaatg 1020

cgtgagttaa aagaactggg ctacacttcg gagccgcacg ctgccgtagc ttatcgtgcg 1080

ctgcgtgatc agttgaatcc aggcgaatat ggcttgttcc tcggcaccgc gcatccggcg 1140

aaatttaaag agagcgtgga agcgattctc ggtgaaacgt tggatctgcc aaaagagctg 1200

gcagaacgtg ctgatttacc cttgctttca cataatctgc ccgccgattt tgctgcgttg 1260

cgtaaattga tgatgaatca tcagtaa 1287

Claims

1. An escherichia coli genetic engineering bacterium is characterized in that pUC19-PJ23119-MCS plasmid containing esaI/R system is introduced into escherichia coli W1688 and combined with motor gene motA.

2. The engineered escherichia coli strain of claim 1, wherein the gene contained in the esaI/R system is a transcription regulatory factor having a nucleotide sequence as shown in SEQ ID No.1 and SEQ ID No.2, respectively: esaI, esaR; the nucleotide sequences of the promoter are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4: p is _esaS 、P _esaR-P (ii) a The nucleotide sequences of the reporter genes are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6: red fluorescent protein mCherry, green fluorescent protein EGFP.

3. The engineered escherichia coli strain as claimed in claim 1, wherein the nucleotide sequence of the motor gene motA is represented by SEQ ID No. 7.

4. The method for constructing the Escherichia coli genetically engineered bacterium of any one of claims 1 to 3, comprising the steps of:

5. The method according to claim 4, wherein in step (1), primers for amplifying esaI and esaR genes are esaI-F, esaI-R, esaR-F, esaR-R with nucleotide sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

6. The method of claim 4, wherein in step (1), P is amplified _esaS 、P _esaR-P The primers of the gene are P with nucleotide sequences shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 respectively _esaS -F、P _esaS -R、P _esaR-P -F、P _esaR-P -R。

7. The method of claim 4, wherein in step (1), primers for amplifying mCherry and EGFP genes are mCherry-F, mCherry-R, EGFP-F, EGFP-R with nucleotide sequences shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, respectively.

8. Use of the genetically engineered Escherichia coli of any one of claims 1 to 3 for producing L-threonine.

9. The use of claim 8, wherein threonine synthetase thrC with the nucleotide sequence shown in SEQ ID NO.23 is substituted to the position of EGFP gene in the genetically engineered Escherichia coli, and the genetically engineered Escherichia coli is inoculated into a fermentation medium to produce xylanase through fermentation.

10. The use of claim 9, wherein the fermentation medium comprises 30g/L glucose, 0.8g/L sodium chloride, 20-22g/L ammonium sulfate, 2g/L anhydrous potassium dihydrogen phosphate, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate pentahydrate, 0.02g/L iron sulfate heptahydrate, 10.002g/L vitamin B, 1g/L yeast powder and 15-30g/L calcium carbonate.