CN109576199B

CN109576199B - Dynamic regulation and control system controlled by promoter combination

Info

Publication number: CN109576199B
Application number: CN201811493330.3A
Authority: CN
Inventors: 刘立明; 高聪; 侯建屾; 叶超; 陈修来; 罗秋玲; 刘佳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2020-09-04
Anticipated expiration: 2038-12-07
Also published as: CN109576199A

Abstract

The invention discloses a dynamic regulation and control system controlled by a promoter combination, particularly discloses a dynamic regulation and control system controlled by a promoter combination and application thereof in shikimic acid production, and belongs to the technical field of bioengineering. The invention screens two types of promoters with completely different transcription characteristics, namely a growth-period related promoter and a stationary-period promoter, by a molecular biology means. The characteristic of specific recognition and cutting of specific short peptides by protease is utilized to design and construct a protein abundance regulation and control system without the help of artificial control and exogenous addition of an inducer. The capability of producing shikimic acid without exogenously adding aromatic amino acid and inducer in an inorganic salt culture medium is realized by introducing a dynamic regulation gene circuit of targeted shikimic acid kinase into engineering escherichia coli. The yield of shikimic acid produced by the invention can reach 21.2g/L, and the conversion rate can reach 0.24g/g glucose.

Description

Dynamic regulation and control system controlled by promoter combination

Technical Field

The invention relates to a dynamic regulation and control system controlled by a promoter combination, in particular to a dynamic regulation and control system controlled by a promoter combination and application thereof in shikimic acid production, belonging to the technical field of biological engineering.

Background

The dynamic regulation and control system is a new metabolic flow regulation and control means in the field of metabolic engineering, and is different from static regulation and control, and is mainly characterized in that in the fermentation process, an engineering strain can make corresponding enzyme activity regulation according to fermentation time, physiological state, intracellular metabolite concentration and extracellular environment change, so that the metabolic flow distribution is influenced, and the product production capacity is improved. The dynamic regulation and control system has the advantages that manual regulation and control are not needed in the fermentation process, an inducer is not needed to be added from an external source, and the dynamic regulation and control system has obvious advantages in producing high value-added compounds.

The current dynamic regulation system is mainly controlled by a quorum sensing system, namely a trigger of the system consists of a quorum sensing system of a local source or a heterogeneous source. When the concentration of the thalli reaches a certain concentration, the accumulated micromolecular substances released by the quorum sensing system can act on a receptor of the regulation and control system, so that the regulation and control system can make corresponding actions. For example, in 2017, ApoorvGupta et al, expressed in the dynamic system of Nature biotechnology, is composed of an EsaI quorum sensing system and a target protein C-terminal added degradation tag, when the concentration of bacteria reaches a certain threshold, the target protein is degraded, and the intracellular absolute concentration reaches 0, so that the aims and effects of enzyme activity shutdown and metabolic flow regulation are achieved. The system has the characteristic of independent path and is successfully applied to the production and application of inositol, glucaric acid and shikimic acid. In addition, in 2017, the AND logic gate dynamic regulation system published by Xinyuan He et al in ACSbythentic biology consisted of a quorum sensing system AND a stationary phase sensor protein. When the thallus concentration reaches a certain threshold value, the transcription expression of the target protein is started, thereby realizing the purpose and the effect of regulating and controlling the metabolic flux. When the system is applied to PHB production, the yield of PHB is improved by 1-2 times. In summary, both dynamic regulation systems require the introduction of heterologous quorum sensing systems, however, the introduction of heterologous quorum sensing systems, which often consist of multiple transcriptional regulatory proteins, undoubtedly affects the growth and fermentation performance of the strain, while limiting the amount of expression of pathway enzymes.

In order to construct a more simplified dynamic regulation system, the invention provides a concept of adopting two types of promoter combinations capable of responding to the physiological state of a strain and combining protein degradation. The two promoters have completely different transcription characteristics, when the strain is in a logarithmic growth phase, the transcription activity of a growth phase-associated promoter (abbreviated as GPP) is extremely strong, and can control a large amount of transcription and synthesis of a target protein, while the transcription activity of a stationary phase-associated promoter (abbreviated as SPP) is strongly inhibited, and the transcription and synthesis of the protein are not carried out. When the strain is in a stationary phase, the transcription activity of the growth phase associated promoter is inhibited, the synthesis of the target protein is almost stopped, and on the contrary, the transcription activity of the stationary phase associated promoter is greatly improved, so that the transcription and synthesis of the control protein are promoted. On the basis, a target protein with an N-terminal hidden degradation molecule and a protease with a stable-phase associated promoter responsible for transcriptional control are combined, and a dynamic regulation gene circuit is constructed. When the strain is in logarithmic growth phase, the target protein can start synthesis and accumulate in cells to perform corresponding physiological metabolism function. When the strain enters a stable period, the protease is controlled by the stable period associated promoter to express, the hidden N-terminal degradation molecule of the target protein is promoted to be naked, and then the target protein is degraded, and the corresponding physiological metabolism function of the target protein is closed. However, when different promoters are combined, the transcription initiation time is different, and the effect of regulating gene expression is different in different combinations.

Shikimic acid is a precursor drug for preparing anti-influenza drug tamiflu and is synthesized by the chorismic acid pathway in escherichia coli. The traditional shikimic acid production method is to knock out shikimic acid kinases I and II of strains to block the synthetic flux of shikimic acid to shikimic acid-3-phosphate and realize the accumulation of shikimic acid. However, the downstream products of shikimic acid-3-phosphate contain aromatic amino acids such as phenylalanine, tyrosine and tryptophan which are essential for the growth of E.coli. The direct blocking of the synthesis of shikimic acid-3-phosphate will cause the strain to have growth defects in the inorganic salt culture medium. In order to promote the growth of the strain, the prior art needs to add expensive aromatic amino acid in a culture medium or add an organic nitrogen source such as tryptone, yeast powder and the like which increases the separation difficulty of the product. Therefore, the method for producing shikimic acid in a simple culture medium is provided, and has important significance for industrial preparation of shikimic acid.

Disclosure of Invention

The first purpose of the invention is to provide a genetic engineering bacterium, which contains a growth phase-associated promoter and a stationary phase-associated promoter; the growth phase-associated promoter is connected with a target protein added with an N-terminal cutting short peptide or an N-terminal degradant; the stationary phase associated promoter is linked with protease; the N-terminal cleavage short peptide or N-terminal degradant is recognized by the protease;

the protease comprises TEV, TVMV, SuMMV or HICV;

the growth phase-associated promoter comprises rpsM, rrnB P1, rpsT P2 or rpsJ;

the stationary phase-associated promoter comprises fic, bolA, S4, or S60;

the nucleotide sequences of rpsM, rrnB P1, rpsT P2, rpsJ, fic, bolA, S4 and S60 are respectively shown as SEQ ID NO.1-SEQ ID NO. 8.

In one embodiment of the invention, the target protein is a shikimate kinase.

In one embodiment of the invention, the protease to which the stationary phase-linked promoter is linked is TEV protease.

In one embodiment of the invention, PJ01-GABE-GPP-K and P_SPPTEV is an expression vector.

The PJ01-GABE-GPP-K overexpresses aroG^fbr、tktA、aroB^optPlasmid of aroE and aroK genes, aroG^fbrDAHP (3-deoxy-D-arabino-heptanoate-7-phosphate) synthetase mutant D146N with elimination of product feedback inhibition; aroB^optNamely aroB gene containing the codon optimization of the first 15 amino acids; aroK, i.e. the shikimate kinase I gene aroK containing a TEV protease recognition cleavage site modified together with a phenylalanine degrader.

The construction method of the PJ01-GABE-GPP-K plasmid comprises the following steps:

(1) based on a commercial Plasmid pTargetF (Addgene Plasmid #62226), a T7Te terminator sequence is inserted after an rrnB T1 terminator in a full-Plasmid PCR mode so as to reduce leakage expression; further, a whole plasmid PCR mode is adopted, the sgRNA expression frame is removed, and an engineering plasmid pJ01 only containing a Pj23119 constitutive promoter and double termination is obtained;

(2) respectively amplifying to obtain aroB containing B0034RBS by using Escherichia coli MG1655 genome as template^opt、aroE、aroG^fbrtktA fragment, aroB^optAnd the aroE fragments are inserted into an expression frame of pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-BE plasmid, and aroG is subjected to the same method^fbrInserting the two fragments of tktA into an expression frame of pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-GA plasmid;

(3) taking an Escherichia coli MG1655 genome as a template, amplifying to obtain an aroK fragment containing B0034RBS and N-terminal modification, inserting the aroK fragment into pJ01 plasmid to obtain pJ01-K plasmid, utilizing enzyme cutting sites BglII and SpeI to double-enzyme-cut the pJ01-K, and inserting the same enzyme cutting sites to synthesize a growth-associated promoter sequence to obtain GPP-K plasmid;

(4) adopting a mode of homoplastic enzyme connection (BamHI + BglII + XbaI), respectively assembling pJ01-GA, pJ01-BE and GPP-K plasmids to finally obtain the plasmid PJ 01-GABE-GPP-K.

Said P_SPPThe construction method of the TEV plasmid comprises the following steps: after synthesizing the stationary phase associated promoter gene, B0034RBS and GFP reporter gene are added respectively in a fusion PCR mode, the fused fragment is connected with a vector pTet-1 plasmid to construct and obtain a recombinant plasmid P_SPP-GFP; replacement of the above P with protease TEV Gene fragment_SPPReporter Gene GFP in GFP to obtain plasmid P_SPP-TEV plasmid.

The vector pTet-1 is an engineered vector which is derived from a commercial Plasmid pdCas9-bacteria (Addge Plasmid #44249), a sequence which encodes the dcas9 protein in the pdCas9-bacteria is deleted by adopting a full-Plasmid PCR mode, and the obtained Plasmid is renamed to pTet-1.

The second purpose of the invention is a method for regulating and controlling the gene expression of target protein, which uses growth phase associated promoter to be associated with the target protein expression added with N-terminal cutting short peptide or N-terminal degradation seed, and uses stable phase associated promoter to be associated with the expression of protease; the N-terminal cleavage short peptide or N-terminal degradant is recognized by the protease;

the protease comprises TEV, TVMV, SuMMV or HICV;

the growth phase-associated promoter comprises rpsM, rrnB P1, rpsT P2 or rpsJ;

the stationary phase-associated promoter comprises fic, bolA, S4, or S60;

A third object of the present invention is to provide a method for producing shikimic acid by fermenting the genetically engineered bacterium of claim above, which is hosted in a cell in which shikimic acid kinase gene is knocked out, and the target protein is shikimic acid kinase.

In one embodiment of the present invention, e.coli S4 is used as a host, and the method for constructing e.coli S4 comprises: taking E.coli MG1655 as an original strain, knocking out shikimate kinase I and II genes (aroK, aroL), and replacing a PTS system with a glucose-facilitated protein gene Zmglf derived from Zymomonas mobilis.

In one embodiment of the invention, the fermentation medium comprises NBS mineral salts medium.

In one embodiment of the invention, the fermentation conditions are 35-38 deg.C, 200-₆₀₀Fermenting for 70-75h at 0.04-0.1; or, the fermentation condition is 35-38 ℃, 480-530rpm, the inoculation amount is 5-10%, the ventilation amount is 1-2vvm, and the fermentation is for 90-100 h.

The fourth purpose of the invention is to provide the application of the genetically engineered bacteria in preparing target proteins or in the fields of biology, pharmacy, food or chemical industry.

The fifth purpose of the invention is to provide the application of the method for regulating the expression of the target gene or the method for producing shikimic acid in the preparation of the target protein or the fields of biology, pharmacy, food or chemical industry.

In one embodiment of the invention, the genetically engineered bacteria are PJ01-GABE-GPP-K and P_SPPTEV as expression vector.

In one embodiment of the invention, the shake flask fermentation condition is 35-38 ℃, 200-220rpm, the initial OD is controlled to be 0.04-0.06, and the fermentation time is 70-75 h; the fermentation conditions of the fermentation tank are 35-38 ℃, 480-530rpm, the inoculation amount is 5-10 percent, the ventilation amount is 1-2vvm, and the fermentation is carried out for 90-100 h.

The fourth purpose of the invention is to provide a method for constructing a dynamic regulation gene circuit or the application of the gene engineering bacteria in the fields of biology, pharmacy, food or chemical engineering.

The fifth purpose of the invention is to provide a method for constructing a dynamic regulation gene circuit or the application of the gene engineering bacteria in preparing target protein, wherein the target protein comprises enzyme protein or non-enzyme protein.

A sixth object of the present invention is to provide a use of the method for producing shikimic acid in the fields of biology, pharmacy, food or chemical industry.

Has the advantages that: the invention provides a method for constructing a dynamic regulation gene circuit, which has the advantages that no artificial regulation is needed in the fermentation process, and no aromatic amino acid and inducer are needed to be added from an external source. In addition, the method has simple design, few system elements and low strain growth load. By constructing shikimic acid production engineering strains and introducing dynamic regulation gene lines, shikimic acid with the conversion rate of 0.24g/g glucose can be accumulated in an inorganic salt culture medium, and the method has a good application prospect.

Drawings

FIG. 1: the partial stationary phase correlates with the fluorescent process profile of the promoter.

FIG. 2: partial growth phase correlates with the fluorescent process profile of the promoter.

FIG. 3: plasmid map, a: PJ 01-GABE-GPP-K; b is P_SPP-TEV。

FIG. 4: fluorescence process curve of partial dynamic gene circuit.

FIG. 5: conversion OD of partial dynamic Gene line₆₀₀The value is obtained.

FIG. 6: the genetic engineering of shikimic acid producing strain reforms the target.

Detailed Description

Materials and methods

Plasmid construction is carried out by classical molecular biology means.

Fluorescence process curve measurements were performed using a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, Calif.) with a controlled ambient temperature of 30 ℃.

Seed culture medium: LB culture medium, the ingredients include peptone 10g/L, yeast powder 5g/L, sodium chloride 10 g/L.

Fermentation medium: NBS inorganic salt culture mediumComprises 50g/L, CaCl g of glucose₂·2H₂O15 mg/L, 0.667mL/L of microelement liquid, sterilizing and supplementing MgSO₄·7H₂O 0.25g/L，V_B10.5mg/L, betaine hydrochloride 1 mM. The preparation method of the trace element liquid is FeCl₃·6H₂O 2.4g/L、CoCl₂·6H₂O 0.3g/L、CuCl₂0.15g/L、ZnCl₂·4H₂O0.3g/L、NaMnO₄0.3g/L、H₃BO₃0.075g/L、MnCl₂·4H₂O0.5 g/L, dissolved in 0.1M HCl.

Preparation of a fermentation sample: taking a fermentation liquid sample, centrifuging at 12000rpm for 5min, taking supernatant liquid, diluting, filtering by a 0.22 mu m water system membrane, and taking filtrate as a sample for liquid chromatography analysis.

The content of shikimic acid is measured by using a Diuran high performance liquid chromatograph (equipped with ultraviolet visible detector) and a Berloe AminexHPX-87H (300 × 7.8.8 mm, 9 μ M) chromatographic column with a mobile phase of H with a concentration of 0.005M₂SO₄Filtering the mobile phase with 0.22 μm filter membrane, ultrasonic degassing at flow rate of 0.6mL/min and column temperature of 35 deg.C, and detecting at ultraviolet detection wavelength of 210 nm.

Example 1 promoter Activity evaluation

After the growth period associated promoter gene is synthesized, B0034RBS and reporter gene GFP are respectively added in a fusion PCR mode. And recovering the PCR product, and connecting the PCR product with a vector PJ01 plasmid to construct and obtain a recombinant plasmid PJ 01-GPP-GFP.

After the genes of the associated promoters in the stationary phase are synthesized, B0034RBS and a reporter gene GFP are respectively added in a fusion PCR mode. Recovering the PCR product, connecting with the vector pTet-1 plasmid to construct recombinant plasmid P_SPP-GFP。

The obtained recombinant plasmid PJ01-GPP-GFP was introduced into competent cells E.coli JM109 to obtain a strain containing a growth-associated promoter evaluation plasmid.

The obtained recombinant plasmid P_SPPColi JM109 into competent cells to obtain strains containing stationary phase evaluation plasmids.

The above strains were subjected to continuous fluorescence measurement in LB medium using a SpectraMax M3 microplate reader. The results are shown in FIGS. 1 and 2. As can be seen from FIG. 1, the accumulation of fluorescent protein is very low during the logarithmic growth phase (1-8h) of the strain, while it increases rapidly after the strain has entered the stationary phase (>8 h). The above phenomenon coincides with the stationary phase promoter characteristics. As can be seen from FIG. 2, the accumulated amount of the fluorescent protein continuously increases in the logarithmic growth phase (1-6h) of the strain, and when the strain enters a stable period (>8h), the accumulated amount of the fluorescent protein stabilizes and does not continuously increase. The above phenomenon coincides with growth-associated promoter characteristics.

Example 2 Assembly and detection of dynamically regulated Gene lines

To construct a dynamically regulated gene circuit, the above P was replaced with a protease TEV gene fragment_SPPReporter Gene GFP in GFP to obtain plasmid P_SPP-TEV plasmid. Meanwhile, the reporter gene GFP of the recombinant plasmid PJ01-GPP-GFP is subjected to N-terminal modification, and a TEV protease recognition cleavage site and a phenylalanine degradation molecule are added to obtain the plasmid PJ01-GPP- (teF) GFP. The plasmid P_SPPCo-transforming TEV and PJ01-GPP- (teF) GFP into JM109, screening by using an ampicillin and chloramphenicol double resistant plate, and carrying out PCR verification on a transformant to finally obtain the dynamic control line detection strain containing the double plasmids.

The detection strain is placed in an LB culture medium for culture, and continuous fluorescence measurement is carried out by using a SpectraMax M3 microplate enzyme-labeling instrument. As a result, as shown in FIG. 4, the intracellular fluorescence accumulation tended to increase and decrease as the bacterial culture time increased. The accumulation of fluorescence varied differently under different promoter combinations, e.g., in combinations expressing GFP from the same rrnB P1 promoter, the combination of proteases under the control of the high-intensity stationary phase promoter, e.g., S4 and S60, showed lower peaks of fluorescence accumulation and earlier onset of decline (turnover time). Meanwhile, it can be seen that in the strain (Control) not containing the GFP degradation site, the fluorescent protein did not decrease despite the normal expression of the protease, indicating that the decrease of the fluorescent protein was caused by the protease-mediated degradation of the fluorescent protein.

Example 3 construction of shikimic acid producing strains

Selecting E.coli MG1655 as chassis engineering bacteria, knocking out shikimate kinase I and II (aroK, aroL) of host bacteria, and replacing PTS system of host bacteria with glucose easy protein Zmglf derived from Zymomonas mobilis. The new strain is named S4, and the strain can not grow in NBS inorganic salt culture medium because of blocking shikimic acid path, and can not accumulate shikimic acid.

Example 4 Shake flask fermentation Performance of dynamic Gene lines introduced shikimic acid producing strains

As shown in FIG. 5, with PJ01-GABE-GPP-K and P_SPPTEV is an expression vector, different dynamic gene lines are introduced into E.coli S4 strain, and 12 engineering strains such as DS1-DS12 are obtained.

When the strain is in the logarithmic growth phase, the strain grows normally, PJ01-GABE-GPP-K is expressed, shikimic acid synthesizes shikimic acid-3-phosphate under the action of aroK, shikimic acid can not be accumulated, and the shikimic acid performs the normal physiological metabolism function; when the strain enters a stationary phase, P_SPPTEV expression, TEV protease is expressed under the control of a stationary phase associated promoter, TEV protease promotes the denudation of aroK-linked cryptic N-terminal degradants on PJ01-GABE-GPP-K, and further degrades aroK, shikimic acid closes the corresponding physiological metabolic function, and accumulation is increased, as shown in FIG. 6.

The growth of the strain and the synthesis of the product are detected in an NBS inorganic salt culture medium. The shaking flask fermentation result shows that the DS1-DS4 strain is difficult to effectively grow in NBS inorganic salt medium, the highest bacteria concentration is only 0.89, and the highest shikimic acid accumulation amount is 0.44 g/L. The growth defect of the DS5-DS12 strain is relieved, and the accumulated concentration range reaches OD₆₀₀5.91-6.82, the accumulation amount of shikimic acid reaches 0.44-2.14 g/L. The optimal strain is DS7 of a gene circuit introducing a bolA and rpsT P2 combined promoter, and the fermentation performance of the strain is shikimic acid accumulation of 2.14g/L and OD₆₀₀It was 6.82.

Example 5 fermenter fermentation Performance of dynamic Gene lines introduced into Shikimic acid producing strains

The fermentation performance of the DS7 strain was tested in a 5L fermentor. The temperature was constant at 37 degrees, 500rpm, the initial inoculum size was 5%, the aeration rate was 1vvm, and the fermentation period was 96 h. At the end of fermentation, the shikimic acid accumulation amount reaches 21.2g/L, and the conversion rate reaches 0.24g/g glucose.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A gene engineering bacterium is characterized in that an expression vector containing a growth phase-associated promoter and an expression vector containing a stationary phase-associated promoter are introduced; the growth phase-associated promoter is connected with a target protein added with a protease recognition cleavage site at the N terminal; the stationary phase associated promoter is linked with protease; the protease recognition cleavage site at the N-terminus is recognized by the protease; the genetic engineering bacteria take escherichia coli as a host; the growth phase-associated promoter comprises rrnB P1, rpsT P2 or rpsJ;

the stationary phase-associated promoter comprises fic, bolA, S4, or S60;

the nucleotide sequences of rrnB P1, rpsT P2, rpsJ, fic, bolA, S4 and S60 are shown as SEQ ID NO.2-SEQ ID NO.8 respectively.

2. The genetically engineered bacterium of claim 1, wherein the target protein is a shikimate kinase.

3. The genetically engineered bacterium of claim 2, wherein the protease linked to the stationary phase-associated promoter is TEV protease.

4. The genetically engineered bacterium of claim 1 or 2, further comprising PJ01-GABE-GPP-K and P_SPP-TEV is an expression vector;

(1) based on a commercial plasmid pTargetF, a T7Te terminator sequence is inserted after an rrnB T1 terminator in a full-plasmid PCR mode so as to reduce leakage expression; further, a whole plasmid PCR mode is adopted, the sgRNA expression frame is removed, and an engineering plasmid pJ01 only containing a Pj23119 constitutive promoter and double termination is obtained;

(2) respectively amplifying to obtain aro containing B0034RBS by using Escherichia coli MG1655 genome as template^Bopt、aroE、aroG^fbrtktA fragment, will aro^BoptAnd the aroE fragments are inserted into an expression frame of pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-BE plasmid, and aroG is subjected to the same method^fbrInserting the two fragments of tktA into an expression frame of pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-GA plasmid;

(3) taking an Escherichia coli MG1655 genome as a template, amplifying to obtain an aroK fragment containing B0034RBS and N-terminal modification, inserting the aroK fragment into pJ01 plasmid to obtain pJ01-K plasmid, utilizing enzyme cutting sites BglII and SpeI to double-enzyme-cut the plasmid pJ01-K, and inserting the same enzyme cutting sites to synthesize a growth-associated promoter sequence to obtain GPP-K plasmid;

(4) respectively assembling pJ01-GA, pJ01-BE and GPP-K plasmids by using BamHI, BglII and XbaI in a mode of homoplastic enzyme connection to finally obtain a plasmid PJ 01-GABE-GPP-K;

said P_SPPThe construction method of the TEV plasmid comprises the following steps:

after synthesizing the stationary phase associated promoter gene, B0034RBS and GFP reporter gene are added respectively in a fusion PCR mode, the fused fragment is connected with a vector pTet-1 plasmid to construct and obtain a recombinant plasmid P_SPP-GFP; replacement of the above P with protease TEV Gene fragment_SPPReporter Gene GFP in GFP to obtain plasmid P_SPP-a TEV plasmid; the vector pTet-1 is derived from a commercial plasmid pdCas 9-baceria, the sequence of the pdCas 9-baceria coding for the dcas9 protein is deleted by adopting a full-plasmid PCR mode, and the obtained plasmid is renamed to pTet-1.

5. A method for regulating and controlling gene expression of a target protein is characterized in that a growth phase associated promoter is used for associating the expression of the target protein which is connected with a protease recognition cutting site added with an N tail end, and a stationary phase associated promoter is used for associating the expression of the protease; the protease recognition cleavage site at the N-terminus is recognized by the protease;

the growth phase-associated promoter comprises rrnB P1, rpsT P2 or rpsJ;

the stationary phase-associated promoter comprises fic, bolA, S4, or S60;

6. The method of claim 5, wherein the protease is TEV protease.

7. A method for producing shikimic acid is characterized in that fermentation is carried out by utilizing genetic engineering bacteria, wherein the genetic engineering bacteria take escherichia coli with a shikimic acid kinase gene knocked out as a host; the genetic engineering bacteria introduce an expression vector containing a growth phase associated promoter and an expression vector containing a stationary phase associated promoter; the growth phase-associated promoter is connected with a target protein added with a protease recognition cleavage site at the N terminal; the stationary phase associated promoter is linked with protease; the protease recognition cleavage site at the N-terminus is recognized by the protease; the genetic engineering bacteria take escherichia coli as a host; the growth phase-associated promoter comprises rrnB P1, rpsT P2 or rpsJ; the protease connected with the stationary phase associated promoter is TEV protease; the target protein is shikimate kinase;

the stationary phase-associated promoter comprises fic, bolA, S4, or S60;

8. The method of claim 7, wherein the fermentation medium comprises NBS mineral salts medium.

9. The method as claimed in claim 7 or 8, wherein the fermentation conditions are 35-38 ℃, 200-₆₀₀Fermenting for 70-75h at 0.04-0.1; or, the fermentation condition is 35-38 ℃, 480-530rpm, the inoculation amount is 5-10%, the ventilation amount is 1-2vvm, and the fermentation is for 90-100 h.

10. Use of the method of claim 5 for regulating gene expression of a target protein in the preparation of a protein of interest.