CN110904140B - Protein dynamic expression regulation system and application thereof in shikimic acid production - Google Patents

Abstract

The invention discloses a protein dynamic expression regulation and control system, and belongs to the technical field of bioengineering. The invention designs and constructs a protein dynamic expression regulation and control system without human control and exogenous addition of an inducer by utilizing the characteristics of a growth phase promoter and a degradation label through a molecular biology means. The shikimic acid production genetic engineering bacteria constructed by introducing the dynamic expression regulation system can realize the dynamic regulation of the abundance change of shikimic acid kinase, and the shikimic acid yield can reach 31g/L at most under the condition of not adding an inducer and aromatic amino acid in an inorganic salt culture medium, and is the highest level known and reported under the same condition at present.

Description

Protein dynamic expression regulation system and application thereof in shikimic acid production

Technical Field

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

Background

In order to meet the global demand for sustainable development. Over the past decade, microbial cell factories have become an important tool for the production of high-value compounds (such as biofuels, renewable materials and pharmaceutical intermediates). Metabolically engineering microbial cell factories is an important means of regulating metabolic flow. Researchers have attempted to achieve this goal through both static and dynamic regulation strategies. The use of traditional static regulation strategies such as gene knock-out and gene overexpression may impair cell viability and reduce fermentation parameters of the cell, such as production intensity, yield and yield. In contrast, dynamic regulation strategies are becoming new strategies to increase the efficiency of microbial synthesis. It can balance the endogenous regulation network and artificial synthetic gene network of microbe and realize the uncoupling of cell growth and product synthesis.

Current dynamic regulation is primarily at the transcriptional and post-translational level. Dynamic regulation of transcription levels is widely used in metabolic engineering to control metabolic flux. Based on the response signal, dynamic regulation of transcription levels is divided into three categories: environmental (e.g., temperature, light, and pH) dependent loops, extracellular (e.g., small molecule autoinducers) loops, and intracellular (e.g., cell physiological states and intracellular metabolites) loops. These methods of dynamic regulation of transcription levels have made many advances in altering metabolic flux, reducing cell damage from toxic intermediates, balancing cell growth and chemical synthesis. However, only using transcription level regulation, the regulation response time is long, and the accuracy of metabolic regulation is poor. The other is dynamic regulation of posttranslational levels. The dynamic regulation and control method adopts two strategies, one is that the degradation of target protein is controlled by using a C-terminal degradation label to reduce the flux of an endogenous pathway, and finally the activity of the target protein is reduced by 56% (Metab Eng 2015,28, 104.); another strategy is to use proteases to cleave sequences containing degradation tags, control the expression of the target protein to increase the metabolic flux of the exogenous pathway, and thus achieve state switching between cell growth and PHB production (ACS Synth Biol 2018,7(11), 2686.). However, in both of the above strategies, the target protein is in a state of continuous degradation and continuous synthesis, increasing the energy consumption and load of the cell.

To solve the above disadvantages, a new strategy combining the regulation of transcription level and protein level has recently been proposed. Gupta creates a metabolic pathway independent genetic circuit by combining quorum sensing system QS and protein degradation signature for regulating metabolic flux and effecting chemical synthesis (Nat Biotechnol 2017,35(3), 273.). However, this system requires the introduction of QS containing foreign proteins and is currently validated only at the genomic level. In a recent study (Nat Commun 2019,10(1),3751, CN201811493330.3 also disclose the same), researchers have constructed a dynamic control switch relying on promoter control and protease participation to increase shikimic acid production, but to achieve dynamic regulation, the system requires expression of less soluble viral proteases in prokaryotic cells and fusion of protease recognition sites and degradants to the N-terminus of the target protein, which molecular manipulations may affect the activity and expression level of the target protein. In general, the current dynamic regulation generally needs to be assisted by a transcription factor responding to the metabolite concentration, an effector protein responding to the cell density, a heterologous protease and other heterologous proteins. The design of these systems is complex, and when the above designs are applied, the systems need to be finely optimized, and the workload is large. Therefore, how to design a more compact and effective dynamic expression regulation system is a difficult problem that needs to be solved urgently by those skilled in the art.

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, which contain aromatic amino acids essential for E.coli growth, such as phenylalanine, tyrosine and tryptophan, directly block the synthesis of shikimic acid-3-phosphate, will render the strain growth deficient in mineral salts medium. In order to promote the growth of the strain, the prior art needs to add expensive aromatic amino acid or inducer into the culture medium. Therefore, the method for producing shikimic acid in a simple culture medium is provided, the shikimic acid kinase is controlled to be expressed in the early stage of fermentation, the closing in the later stage of fermentation is realized, the decoupling of cell growth and product synthesis is realized, and the method has important significance for industrial preparation of shikimic acid.

Disclosure of Invention

The invention provides a more simplified and effective dynamic expression regulation and control system, which can realize dynamic regulation and control of the abundance of target protein only by skillfully combining the existing elements (a promoter and a protein degradation label) in an escherichia coli cell, and has important significance for enhancing the robustness of the dynamic expression regulation and control system, reducing the cell load and reducing the experimental workload. Meanwhile, the applicant applies the dynamic expression regulation and control system to the production of shikimic acid, provides a method for producing shikimic acid in a simple culture medium, and realizes the uncoupling of cell growth and product synthesis under the condition of not additionally adding expensive aromatic amino acid or inducer.

It is a first object of the present invention to provide a protein dynamic expression regulatory system which uses a growth phase-associated promoter P in combination in Escherichia coli_GPPAnd acting a C-terminal degradation label SsrA on a target protein to realize dynamic expression of the target protein, wherein the target protein is subjected to a growth period associated promoter P_GPPControl, and contain C terminal degradation label SsrA;

the growth phase-associated promoter P_GPPIncluding P_rpsL、P_rrnA、P_rpsT、P_rrnCOr P_rpsA；

The C-terminal degradation label SsrA comprises LAA, DAS +4, DAS +8 or GSD;

the P is_rpsL、P_rrnA、P_rpsT、P_rrnC、P_rpsAThe nucleotide sequence of (A) is shown in SEQ ID NO.1-SEQ ID NO. 5;

the amino acid sequence of the LAA, the DAS +4, the DAS +8 or the GSD is shown in SEQ ID NO.6-SEQ ID NO. 10.

In a preferred embodiment, the target protein is shikimate kinase I or green fluorescent protein GFP, further preferably shikimate kinase I.

In a specific embodiment, the green fluorescent protein GFP of the present invention is subjected to growth phase promoter P_GPP(P_rrnC,P_rrnA,P_rpsA,P_rpsT,P_rpsL) And controlling, and fusing SsrA degradation labels (LAA, DAS +4, DAS +8 or GSD) with different strengths at the C terminal of the GFP protein, wherein the design schematic diagram of the protein dynamic expression regulation system is shown in figure 1 a. In the logarithmic growth phase of the cells, the promoters in the growth phase have the characteristic of high-level transcription, and GFP has a degradation tag, but the synthesis amount of GFP is larger than the degradation amount, so that GFP can still be accumulated in the cells. When the cells enterAfter the stabilization period, the synthesis rate of GFP is less than the degradation rate of the protein due to the large down-regulation of the transcription activity of the promoter in the growth phase, so that the target protein is not accumulated in the cells. As reflected in the fluorescence progress curve, GFP fluorescence shows a trend of increasing first and then decreasing with time, as shown in figure 1 b.

The second purpose of the invention is to provide a genetic engineering bacterium for producing shikimic acid, wherein the strain takes escherichia coli as host bacteria, and the dynamic expression regulation system is introduced to control the expression of target protein; preferably, the Escherichia coli has knocked out shikimate kinase I gene aroK (SEQ ID No.16) and shikimate kinase II gene aroL (SEQ ID No.17), and the PTS system of the strain is replaced with glucose-facilitated protein gene Zmglf (SEQ ID No. 20); more preferably, the E.coli strain is E.coli MG1655 knocked out shikimate kinase I gene aroK and shikimate kinase II gene aroL and replaced PTS system with glucose-facilitated protein gene Zmglf derived from Zymomonas mobilis, and the strain is named S4.

In a preferred embodiment, the replacement of the PTS system of the strain with the glucose-facilitated protein gene Zmglf means that the gene expression cassette ptshicr of the strain is replaced with the glucose-facilitated protein gene Zmglf using CRISPR/Cas9 technology.

In a preferred embodiment, said gene Zmglf is derived from zymomonas mobilis.

The specific metabolic pathway of the genetically engineered bacterium is shown in figure 2, for example, if the shikimic acid pathway is blocked, the strain cannot grow in an inorganic salt culture medium, and the shikimic acid can not be accumulated, and after a dynamic regulation and control system is introduced, AroK can be normally accumulated in cells in the early stage of fermentation, so that the strain can normally grow in the inorganic salt culture medium, and in the later stage of fermentation, AroK is degraded, and the cells begin to accumulate shikimic acid.

In the above genetic engineering strain, plasmid PJ01-GABE containing overexpression pathway enzyme and dynamic regulation plasmid P15A-P_GPP-aroK-SsrA. Plasmid PJ01-GABE overexpresses aroG encoding a mutant DAHP synthase (D146N) containing an abrogated product feedback inhibition^fbr(SEQ ID NO.19), tktA gene (SEQ ID NO.18), DNA fragment containingaroB codon optimized with the first 15 amino acids^optA gene (SEQ ID NO.21) and an aroE gene (SEQ ID NO. 22). Plasmid P15A-P_GPPThe C terminal of shikimate kinase aroK in aroK-SsrA is fused with degradation label SsrA, the transcription activity is influenced by growth phase promoter P_GPPAnd (5) controlling.

The third purpose of the invention is to provide a construction method of the genetic engineering bacteria, which comprises the following specific steps: (1) constructing genetically engineered plasmids of PJ01-GABE and P15A-P_GPP-aroK-SsrA; (2) transforming the plasmid into escherichia coli; (3) mixed culture to obtain the conjugated shikimic acid producing bacteria.

In a preferred embodiment, the plasmid PJ01-GABE in the step (1) is constructed by the following method:

(1) based on a commercial Plasmid pTargetF (Addgene Plasmid #62226), a T7Te terminator sequence (SEQ ID NO.15) is inserted after an rrnB T1 terminator (SEQ ID NO.12) in a full-Plasmid PCR manner to reduce leaky expression; further, a whole plasmid PCR mode is adopted to remove the sgRNA expression frame, and the expression vector only containing P is obtained_j23119A constitutive promoter and a double-terminated engineering plasmid pJ01(GenBank access number: MK 234843);

(2) the aroB containing B0034RBS (SEQ ID NO.13) was obtained by amplification using E.coli MG1655 genome as template^opt、aroE、aroG^fbrtktA fragment, aroB^optInserting the two segments of aroE into an expression frame of pJ01 in a multi-segment one-step homologous recombination mode to obtain a pJ01-BE plasmid, and carrying out aroG^fbrInserting the two fragments of tktA into an expression frame of a plasmid pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-GA plasmid;

(3) cutting the pJ01-GA plasmid by using BamHI and XbaI restriction enzymes, recovering the vector, cutting the pJ01-BE plasmid by using BgIII and XbaI restriction enzymes, and recovering a fragment; respectively assembling pJ01-GA and pJ01-BE plasmids by adopting a mode of homoplastic enzyme connection, and finally obtaining the plasmid PJ 01-GABE.

In a preferred embodiment, the plasmid P15A-P in said step (1)_GPPThe construction method of the-aroK-SsrA plasmid comprises the following steps:

(1) the used vector pTet-1(GenBank access number: MK234848) is an engineering vector, and a growth phase promoter P is synthesized_GPPSequence, replacement of original P in vector pTet-1_tetPromoter (SEQ ID NO.11), and the obtained plasmid was designated as P15A-P_GPP；

(2) Amplifying a gene aroK by taking an Escherichia coli MG1655 genome as a template, fusing different degradation labels SsrA at the 3' end of the gene aroK, and inserting aroK-SsrA fused with different degradation labels into P15A-P_GPPTo obtain the plasmid P15A-P_GPP-aroK-SsrA。

The fourth purpose of the invention is to provide a method for producing shikimic acid, which takes the genetic engineering bacteria as fermenting microorganism and glucose as carbon source for fermentation.

In specific embodiments, the fermentation medium is M9 mineral salts medium; the fermentation conditions were 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 percent, the ventilation amount is 1-2vvm, and the fermentation is carried out for 90-100 h.

The fifth purpose of the invention is to provide the application of the genetic engineering bacteria or the dynamic expression regulation and control system in the preparation of shikimic acid or shikimic acid-containing products.

The sixth purpose of the invention is to provide the application of the dynamic expression regulation and control system in the preparation of target protein or in the fields of biology, pharmacy, food or chemical industry.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides a brand-new protein dynamic expression regulation and control system, which is simpler than the existing dynamic regulation and control strategy, can realize dynamic control aiming at target protein only by rationally combining an escherichia coli native promoter and a degradation label, does not need to rely on artificial control and exogenous addition of an inducer, and has important significance for enhancing the robustness of the dynamic regulation and control system, reducing cell load and reducing experimental workload.

2. The protein dynamic expression regulation and control system is applied to production of shikimic acid to construct shikimic acid genetic engineering bacteria, normal growth of cells can not be influenced in the early stage of fermentation, and the shikimic acid kinase abundance can be automatically reduced only after the cells enter a stable period, so that the synthesis flux of shikimic acid to shikimic acid-3-phosphate is blocked, and the cells accumulate shikimic acid.

3. The gene engineering bacteria of the invention do not need to add inducer and aromatic amino acid in the fermentation production, thereby reducing the production cost and the downstream purification cost. In an M9 inorganic salt culture medium, the yield of shikimic acid reaches 31g/L by fed-batch fermentation, is obviously improved compared with the prior art, and has better application prospect.

Drawings

FIG. 1: the invention relates to a design schematic diagram of a protein dynamic expression regulation and control system and a fluorescence curve diagram of green fluorescent protein under the control of the protein dynamic expression regulation and control system, wherein, a figure 1a is the design schematic diagram of the protein dynamic expression regulation and control system; FIG. 1b is a graph of the fluorescence process of green fluorescent protein under the control of a protein dynamic expression regulation system.

FIG. 2: the specific metabolic pathway of the genetically engineered bacteria of the present invention is shown in FIG. 2

FIG. 3: relative activity histograms for promoters in 5 growth phases in E.coli in example 1.

FIG. 4: histogram of degradation rates of 5 degraded tags in E.coli in example 2.

FIG. 5: dynamic expression profile of green fluorescent protein for six strains (strain 007, strain 009, strain 016, strain 017, strain 018, strain 020) in example 3.

Detailed Description

The present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

In the following specific examples, unless otherwise specified, the reagents and apparatus used were those commonly used in the art and were obtained commercially; the methods used are conventional in the art, and those skilled in the art can understand how to implement the methods specifically according to the embodiment and achieve the corresponding results.

The reagents adopted by the invention are all common commercial products and can be purchased in the market. The enzymes used in this example (including BamHI restriction enzyme, XbaI restriction enzyme, BgIII restriction enzyme, isocaudara, etc.), DNA Marker and related kits were purchased from Takara Bio Inc., and the primer synthesis and sequencing services used in the examples were performed by Jinzhi science and technology, Inc., Suzhou. The plasmid construction is carried out by adopting a classical molecular biological means, and a homologous recombination kit used in a multi-fragment recombination experiment is purchased from Nanjing NuoZan Biotech Co.

Chassis engineering bacteria E.coli MG1655 mentioned in this example are commercially available from Shanghai Zea leaf Biotech Co., Ltd.

Fluorescence process curve determination: controlling the environment temperature to be 30 ℃, continuously sampling 1mL in the cell culture process, and diluting to the cell density OD₆₀₀The assay was performed using a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CA) ═ 0.3.

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

Fermentation medium: m9 inorganic salt culture medium containing glucose 40g/L and trace element liquid 1mL/L, sterilized and supplemented with MgSO₄·7H₂O is 0.25 g/L. The preparation method of the trace element liquid is FeCl₃·6H₂O 2.4g/L、CoCl₂·6H₂O 0.3g/L、CuCl₂ 0.15 g/L、ZnCl₂·4H₂O 0.3g/L、NaMnO₄ 0.3g/L、H₃BO₃0.075 g/L、MnCl₂·4H₂O0.5 g/L, dissolved in 0.1M HCl.

Preparation of a fermentation sample: taking 2mL of fermentation liquor sample, centrifuging at 12,000rpm for 5min, taking supernatant, diluting by 10 times, filtering by a 0.22 mu m water system membrane, and taking filtrate as a sample for liquid chromatography analysis.

Measurement of shikimic acid content: the DEAN high performance liquid chromatograph (with UV-visible detector and differential detector) adopts Berle Aminex HPX-87H (300 × 7.8mm, 9 μm) chromatographic column with concentrated mobile phaseH at 0.005M₂SO₄Filtering the mobile phase by a filter membrane of 0.22 mu m, and ultrasonically degassing at the flow rate of 0.6mL/min and the column temperature of 35 ℃ under the ultraviolet detection wavelength of 210 nm.

Example 1 promoter P in the middle growth phase of E.coli_GPPIs characterized by

The invention evaluates the promoter P of 5 escherichia coli in the growth period_GPP(P_rrnC，P_rrnA，P_rpsA，P_rpsTAnd P_rpsL) Relative activity of (2). 5 kinds of P_GPPThe nucleotide sequences of the promoters are respectively shown in SEQ ID NO.1-SEQ ID NO. 5. To determine the relative activity of the promoter, the synthesized P was used_GPPThe promoter sequence, B0034RBS sequence, GFP sequence (SEQ ID NO.23) were inserted into Ptet-1 plasmid in order to obtain P15A-P_GPPGFP plasmids.

Further, to reduce experimental error, P is added_{lac UV5}The promoter sequence (SEQ ID NO.14), the B0034RBS sequence, the gene sequence (SEQ ID NO.24) for coding the red fluorescent protein mKate2 are inserted into the P15A-P_GPPIn the GFP plasmid, P15A-P was obtained_GPP-GFP-P_{lac UV5}-mKate 2. In this plasmid, the growth phase promoter P_GPPConstitutive promoter P controlling expression of Green fluorescent protein GFP_{lac UV5}Controlling the expression of the red fluorescent protein.

The serial recombinant plasmid P15A-P constructed above is used_GPP-GFP-P_{lac UV5}Coli JM109 (obtained from Shanghai leaf Biotech Co., Ltd.) was cultured in LB medium at 30 ℃ and the cell density OD of the sample was measured by continuous sampling₆₀₀And fluorescence intensities of green fluorescent protein GFP and red fluorescent protein mKate2, wherein the excitation wavelength for GFP is 488nm, the emission wavelength is 511nm, the excitation wavelength for mKate2 is 588nm, and the emission wavelength is 644 nm. The fluorescence intensity value is defined as the ratio of fluorescence intensity to cell density, and the ratio of fluorescence intensity values of GFP and mKate2 is defined as the activity of the promoter. Further, a constitutive promoter P is specified_{lac UV5}Has an activity of 1 to obtain P_GPPPromotersSee fig. 3 for specific results.

Example 2 characterization of degradation Label Strength

For the determination of the degradation rate of the degradation tag, the ClpP protease gene of the e.coli MG1655 strain was knocked out by using the conventional CRISPR/Cas9 gene editing technology to construct a protease-deficient strain. Meanwhile, the gene coding the original ClpP protease is inserted into the plasmid pTet-1 to construct an inducible protease plasmid. Reporter plasmids were constructed by fusing 5 different degradation tags (LAA, DAS +4, DAS +8, or GSD) to the C-terminus of green fluorescent protein GFP, respectively, and inserting into commercial plasmid PTrcHisA.

The reporter plasmid and the protease plasmid were co-transformed into a protease deficient strain by heat shock transformation and cultured in a 50mL LB system containing 10mM lactose. When cell density OD₆₀₀When 0.6 was reached, ClpP protease expression was induced by 200ng/mL anhydrotetracycline ATC. Sampling 200. mu.L every 0.5h, determining OD₆₀₀And fluorescence intensity, the decrease in fluorescence density per unit time of the cells being defined as the degradation rate. The 5 protein C-terminal degradation tag SsrA was evaluated in the present invention, and the results are shown in FIG. 4. As shown in FIG. 4, compared with the no-tag group without the degradation tag, the degradation rate of the protein C-terminal fused with the degradation tag SsrA is greatly increased.

Example 3 construction and evaluation of protein dynamic expression control System

Construction of P15A-P_GPPGFP-SsrA, the specific steps are as follows:

(a) the used vector pTet-1(GenBank access number: MK234848) is an engineering vector, and a growth phase promoter P is synthesized_GPPSequence, replacement of original P in vector pTet-1_tetPromoter (SEQ ID NO.11), and the obtained plasmid was designated as P15A-P_GPP；

(b) Fusion of different strength degradation tags SsrA (LAA, DAS +4, DAS +8 or GSD) at the 3' end of GFP gene, and insertion of GFP-SsrA fused with different degradation tags SsrA into P15A-P_GPPTo obtain the plasmid P15A-P_GPP-GFP-SsrA。

Further, to reduce the experimentError, will P_{lac UV5}Promoter sequence (SEQ ID NO.14), B0034RBS sequence, gene sequence insertion P15A-P of coding red fluorescent protein mKate2_GPPIn the GFP-SsrA plasmid, P15A-P was obtained_GPP-GFP-SsrA-P_{lac UV5}-mKate 2. In this plasmid, GFP was set to be expressed dynamically and the red fluorescent protein was set to be expressed constitutively. Meanwhile, the series of control plasmids P15A-P containing no SsrA constructed in example 1 was tested_GPP-GFP-P_{lac UV5}-mKate2。

The series of recombinant plasmids obtained above were introduced into competent cells e.coli JM109 by heat shock transformation to obtain a total of 30 evaluated strains as shown in table 1 below.

TABLE 1 construction of strains containing dynamic expression control System

The continuous fluorescence measurements of the above strains were performed in LB medium using a SpectraMax M3 microplate reader, wherein the results of the fluorescence process for representative six strains (007, 009, 016, 017, 018, 020) are shown in Table 2 below, and the specific GFP fluorescence process curves are shown in FIG. 5.

TABLE 2 GFP fluorescence results for target proteins of six strains (007, 009, 016, 017, 018, 020)

In summary, by combining different promoters P_GPPThe degradation label SsrA can realize the effect that the target protein GFP is accumulated in the early growth stage of the cell and is degraded in the later growth stage; containing only promoter P_GPPDynamic regulation of the target protein GFP cannot be realized without adding the degradation tag SsrA.

Example 4 construction of shikimic acid Gene engineering bacteria

Selecting E.coli MG1655 as a chassis engineering bacterium, knocking out shikimate kinase I and II (aroK, aroL) of a host bacterium by adopting a conventional CRISPR/Cas9 gene editing technology, and replacing a PTS system of the host bacterium by using a glucose-facilitated protein Zmglf derived from Zymomonas mobilis. The new strain is named as S4, and the specific construction method refers to a Nat Commun,2019,10(1): 3751.

1) Construction of genetically engineered plasmids PJ01-GABE and P15A-P_GPP-aroK-SsrA；

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

(1) based on a commercial Plasmid pTargetF (Addgene Plasmid #62226), a T7Te terminator sequence (SEQ ID NO.15) is inserted after an rrnB T1 terminator (SEQ ID NO.12) in a full-Plasmid PCR manner to reduce leaky expression; further, a whole plasmid PCR mode is adopted to remove the sgRNA expression frame, and the expression vector only containing P is obtained_j23119A constitutive promoter and a double-terminated engineering plasmid pJ01(GenBank access number: MK 234843);

(2) the aroB containing B0034RBS (SEQ ID NO.13) was obtained by amplification using E.coli MG1655 genome as template^opt(SEQ ID NO.21)、aroE(SEQ ID NO.22)、aroG^fbr(SEQ ID NO.19), tktA (SEQ ID NO.18) fragment, aroB^optInserting the two segments of aroE into an expression frame of pJ01 in a multi-segment one-step homologous recombination mode to obtain a pJ01-BE plasmid, and carrying out aroG^fbrInserting the two fragments of tktA into an expression frame of a plasmid pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-GA plasmid;

(3) cutting the pJ01-GA plasmid by using BamHI and XbaI restriction enzymes, recovering the vector, cutting the pJ01-BE plasmid by using BgIII and XbaI restriction enzymes, and recovering a fragment; respectively assembling pJ01-GA and pJ01-BE plasmids by adopting a mode of homoplastic enzyme connection, and finally obtaining the plasmid PJ 01-GABE.

Plasmid P15A-P_GPPThe construction method of the-aroK-SsrA plasmid comprises the following steps:

(1) the vector pTet-1(GenBank access number: MK234848) used is an engineered vectorSynthesis of growth phase promoter P_GPPSequence, replacement of original P in vector pTet-1_tetPromoter (SEQ ID NO.11), and the obtained plasmid was designated as P15A-P_GPP；

(2) Amplifying a gene aroK by taking an Escherichia coli MG1655 genome as a template, fusing different degradation labels SsrA (LAA, DAS +4, DAS +8, GSD and DAS) at the 3' end of the gene aroK, inserting the aroK-SsrA fused with the different degradation labels into P15A-P_GPPTo obtain the plasmid P15A-P_GPP-aroK-SsrA。

2) Transforming the plasmids PJ01-GABE and p15A-PGPP-aroK-SsrA into escherichia coli, and performing mixed culture to obtain a target gene engineering bacterium subjected to conjugal transfer;

a single colony of the newly activated S4 strain was picked from an LB plate, inoculated into 5mLLB liquid medium, and cultured overnight at 37 ℃. Inoculating the strain suspension into 25mL liquid culture medium at an inoculation ratio of 1%, culturing at 37 deg.C for 3 hr to OD₆₀₀＝0.5。

Transferring the bacterial liquid into a 50mL centrifuge tube, and standing for 10min on ice. Centrifuge at 4000rpm for 5min at 4 ℃, discard the supernatant, resuspend the cells using pre-cooled 0.1M CaCl2 solution, and let stand on ice for 20 min. Centrifuging at 4000rpm for 10min at 4 deg.C, discarding the supernatant, and adding 2mL of pre-cooled 0.1M CaCl₂The solution was resuspended in cells, allowed to stand on ice for 10min, and 0.1mL of competent cells were dispensed into each centrifuge tube to complete the preparation of competent cells of strain S4.

10ng of plasmid PJ01-GABE and plasmid p15A-PGPP-aroK-SsrA were added to each 0.1mL of competent cells, mixed well and left on ice for 30 min. Placing competent cells in the centrifuge tube in 42 deg.C water bath for 90s, rapidly transferring the centrifuge tube to ice bath, and standing for 3 min. Adding 0.8mL of liquid LB culture medium into each tube, resuscitating the mixture for 1h under shaking at 37 ℃, uniformly smearing the mixture on an LB plate containing 100mg/L ampicillin and 30mg/L chloramphenicol at the final concentration, and standing and culturing the mixture at 37 ℃. Details of the obtained strains can be found in table 3 below.

TABLE 3 construction of different shikimic acid producing strains

Strain numbering	PGPP	SsrA	Regulatory plasmids	Pathway enzyme plasmids
					031	PrpsA	LAA	p15A-PrpsA-aroK-LAA	PJ01-GABE
032	PrpsA	DAS+4	p15A-PrpsA-aroK-DAS+4	PJ01-GABE
					033	PrrnA	DAS+8	p15A-PrrnA-aroK-DAS+8	PJ01-GABE
034	PrrnA	GSD	p15A-PrrnA-aroK-GSD	PJ01-GABE
					035	PrrnC	DAS	p15A-PrrnC-aroK-DAS	PJ01-GABE
036	PrrnA	Is free of	p15A-PrrnA-aroK	PJ01-GABE

Example 5 production of shikimic acid from genetically engineered shikimic acid in Shake flasks

The shikimic acid-producing ability of strain 031-036 and strain S4 introduced with the dynamic regulation system prepared in example 4 was tested separately. The seeds were first cultured overnight in LB medium at 37 ℃. Then transferring the seeds into M9 inorganic salt culture medium containing 100mg/L ampicillin and 30mg/L chloramphenicol, and controlling the initial fermentation bacteria concentration OD₆₀₀The fermentation system is a 250mL shaking flask, the working volume is 50mL, the rotation speed is 200rpm, the culture temperature is 37 ℃, and sampling is carried out every 6h to determine the cell density OD600 and the concentration of the extracellular metabolite shikimic acid.

As can be seen from the results, strain S4 could not grow normally and shikimic acid accumulation was not detected, and both of the strain 031-036 cells introduced into the dynamic regulation system could grow normally and shikimic acid accumulation was detected, wherein strain 032 (containing P15A-P)_rpsAThe combination of-aroK-DAS +4 plasmid and PJ01-GABE plasmid) has the highest shikimic acid accumulation, the yield reaches 3.2g/L, and the conversion rate reaches 0.24g/g glucose.

Example 6 production of shikimic acid from genetically engineered shikimic acid in fermentation tank

The strain 032 from example 5 was taken out and subjected to fermentation culture in a 5L fermentor. First, strain 032 was inoculated into 50mL of LB medium containing ampicillin at a final concentration of 100mg/L and chloramphenicol at a final concentration of 30mg/L, and cultured at 37 ℃ at 200rpm for 12 hours. Then, the seed liquid was collected and centrifuged to discard the supernatant. M9 mineral salts medium containing 40g/L glucose was used for resuspension, the initial inoculum size was controlled to 10%, and the inoculum was inoculated into a fermentor containing 2L M9 mineral salts medium. 30% ammonia and 2M hydrochloric acid were automatically fed to maintain the pH at 7. The air flow rate was set to 1.5vvm, the stirring was constantly set at 500rpm, and the culture temperature was controlled at 37 ℃. When the residual glucose is lower than 10g/L, 40g/L glucose is added, 100g glucose is supplemented totally, and the fermentation period is 96 h. When the fermentation is finished, the shikimic acid accumulation amount reaches 31g/L, and the conversion rate reaches 0.22g/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.

Nucleotide or degradation tag amino acid sequence of promoter in growth phase of Table 4

TABLE 5 nucleotide sequences of genes or elements used in the present invention

Sequence listing

<110> Taizhou institute of occupational and technology

<120> protein dynamic expression regulation system and application thereof in shikimic acid production

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 109

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcgtcagact tacggttaag caccccagcc agatggcctg gtgatggcgg gatcgttgta 60

tatttcttga caccttttcg gcatcgccct aaaattcggc gtcctcata 109

<210> 2

<211> 200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgtcaggcg gtgaaacgga tacgcgcaaa gaagttgtct ataccgattg ggagcaggtg 60

gcgaatttcg cccgagaaat cgcccattta accgacaaac cgacgctgaa ataagcataa 120

agaataaaaa atgcgcggtc agaaaattat tttaaatttc ctcttgtcag gccggaataa 180

ctccctataa tgcgccacca 200

<210> 3

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcattgccat ggcgcaaatc acgggaagaa actgaccgcc tgctgcaatt tttatcgcgg 60

aaaagctgta ttcacacccc gcaagctggt agaatcctgc gccatcacta cgtaacgagt 120

<210> 4

<211> 200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cttaaaggca ttacttatct tcctttttct ttttattcct ccttagtatg ccaccaggaa 60

gtgtgattac ggttgcaaaa acggcaaatt gcttgtttta tggcacatta acggggcttt 120

tgctgaaaaa atgcgcggtc agaaaattat tttaaatttc ctcttgtcag gccggaataa 180

ctccctataa tgcgccacca 200

<210> 5

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgaaaatttt ccttgacgcc tcctcggaag aacgtgcgca tcgccgcatg ctacagttgc 60

aggagaaggg ctttagtgtt aactttgagc gccttttggc cgagatcaaa 110

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Ala Asn Asp Glu Asn Tyr Ala Leu Ala Ala

1 5 10

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ala Ala Asn Asp Glu Asn Tyr Ala Asp Ala Ser

1 5 10

<210> 8

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ala Ala Asn Asp Glu Asn Tyr Ser Glu Asn Tyr Ala Asp Ala Ser

1 5 10 15

<210> 9

<211> 34

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ala Ala Ala Ala Asn Asp Glu Asn Tyr Ser Glu Ser Glu Ser Glu Asn

1 5 10 15

Tyr Ala Asp Ala Ser Asn Asp Glu Asn Tyr Ser Glu Asn Tyr Ala Asp

20 25 30

Ala Ser

<210> 10

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Ala Ala Asn Asp Glu Asn Tyr Gly Ser Asn Tyr Ala Asp Ala Ser

1 5 10 15

<210> 11

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

taattcctaa tttttgttga cactctatcg ttgatagagt tattttacca ctccctatca 60

gtgatagaga aaa 73

<210> 12

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtttta 46

<210> 13

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aaagaggaga aa 12

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tttacacttt atgcttccgg ctcgtataat 30

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggctcacctt cgggtgggcc tttctgcg 28

<210> 16

<211> 522

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggcagaga aacgcaatat ctttctggtt gggcctatgg gtgccggaaa aagcactatt 60

gggcgccagt tagctcaaca actcaatatg gaattttacg attccgatca agagattgag 120

aaacgaaccg gagctgatgt gggctgggtt ttcgatttag aaggcgaaga aggcttccgc 180

gatcgcgaag aaaaggtcat caatgagttg accgagaaac agggtattgt gctggctact 240

ggcggcggct ctgtgaaatc ccgtgaaacg cgtaaccgtc tttccgctcg tggcgttgtc 300

gtttatcttg aaacgaccat cgaaaagcaa cttgcacgca cgcagcgtga taaaaaacgc 360

ccgttgctgc acgttgaaac accgccgcgt gaagttctgg aagcgttggc caatgaacgc 420

aatccgctgt atgaagagat tgccgacgtg accattcgta ctgatgatca aagcgctaaa 480

gtggttgcaa accagattat tcacatgctg gaaagcaact aa 522

<210> 17

<211> 525

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgacacaac ctctttttct gatcgggcct cggggctgtg gtaaaacaac ggtcggaatg 60

gcccttgccg attcgcttaa ccgtcggttt gtcgataccg atcagtggtt gcaatcacag 120

ctcaatatga cggtcgcgga gatcgtcgaa agggaagagt gggcgggatt tcgcgccaga 180

gaaacggcgg cgctggaagc ggtaactgcg ccatccaccg ttatcgctac aggcggcggc 240

attattctga cggaatttaa tcgtcacttc atgcaaaata acgggatcgt ggtttatttg 300

tgtgcgccag tatcagtcct ggttaaccga ctgcaagctg caccggaaga agatttacgg 360

ccaaccttaa cgggaaaacc gctgagcgaa gaagttcagg aagtgctgga agaacgcgat 420

gcgctatatc gcgaagttgc gcatattatc atcgacgcaa caaacgaacc cagccaggtg 480

atttctgaaa ttcgcagcgc cctggcacag acgatcaatt gttga 525

<210> 18

<211> 1992

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgtcctcac gtaaagagct tgccaatgct attcgtgcgc tgagcatgga cgcagtacag 60

aaagccaaat ccggtcaccc gggtgcccct atgggtatgg ctgacattgc cgaagtcctg 120

tggcgtgatt tcctgaaaca caacccgcag aatccgtcct gggctgaccg tgaccgcttc 180

gtgctgtcca acggccacgg ctccatgctg atctacagcc tgctgcacct caccggttac 240

gatctgccga tggaagaact gaaaaacttc cgtcagctgc actctaaaac tccgggtcac 300

ccggaagtgg gttacaccgc tggtgtggaa accaccaccg gtccgctggg tcagggtatt 360

gccaacgcag tcggtatggc gattgcagaa aaaacgctgg cggcgcagtt taaccgtccg 420

ggccacgaca ttgtcgacca ctacacctac gccttcatgg gcgacggctg catgatggaa 480

ggcatctccc acgaagtttg ctctctggcg ggtacgctga agctgggtaa actgattgca 540

ttctacgatg acaacggtat ttctatcgat ggtcacgttg aaggctggtt caccgacgac 600

accgcaatgc gtttcgaagc ttacggctgg cacgttattc gcgacatcga cggtcatgac 660

gcggcatcta tcaaacgcgc agtagaagaa gcgcgcgcag tgactgacaa accttccctg 720

ctgatgtgca aaaccatcat cggtttcggt tccccgaaca aagccggtac ccacgactcc 780

cacggtgcgc cgctgggcga cgctgaaatt gccctgaccc gcgaacaact gggctggaaa 840

tatgcgccgt tcgaaatccc gtctgaaatc tatgctcagt gggatgcgaa agaagcaggc 900

caggcgaaag aatccgcatg gaacgagaaa ttcgctgctt acgcgaaagc ttatccgcag 960

gaagccgctg aatttacccg ccgtatgaaa ggcgaaatgc cgtctgactt cgacgctaaa 1020

gcgaaagagt tcatcgctaa actgcaggct aatccggcga aaatcgccag ccgtaaagcg 1080

tctcagaatg ctatcgaagc gttcggtccg ctgttgccgg aattcctcgg cggttctgct 1140

gacctggcgc cgtctaacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200

gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260

atctccctgc acggtggctt cctgccgtac acctccacct tcctgatgtt cgtggaatac 1320

gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380

cacgactcca tcggtctggg cgaagacggc ccgactcacc agccggttga gcaggtcgct 1440

tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500

gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccgt 1560

cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620

tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680

gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740

gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800

ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860

aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920

gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980

gaactgctgt aa 1992

<210> 19

<211> 1053

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60

gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120

aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180

tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240

gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300

acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360

gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420

gcaggtgagt ttctcaatat gatcacccca caatatctcg ctgacctgat gagctggggc 480

gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540

tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600

aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660

gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720

tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780

caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840

gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900

gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960

ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020

ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053

<210> 20

<211> 1422

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgagttctg aaagtagtca gggtctagtc acgcgactag ccctaatcgc tgctataggc 60

ggcttgcttt tcggttacga ttcagcggtt atcgctgcaa tcggtacacc ggttgatatc 120

cattttattg cccctcgtca cctgtctgct acggctgcgg cttccctttc tgggatggtc 180

gttgttgctg ttttggtcgg ttgtgttacc ggttctttgc tgtctggctg gattggtatt 240

cgcttcggtc gtcgcggcgg attgttgatg agttccattt gtttcgtcgc cgccggtttt 300

ggtgctgcgt taaccgaaaa attatttgga accggtggtt cggctttaca aattttttgc 360

tttttccggt ttcttgccgg tttaggtatc ggtgtcgttt caaccttgac cccaacctat 420

attgctgaaa ttgctccgcc agacaaacgt ggtcagatgg tttctggtca gcagatggcc 480

attgtgacgg gtgctttaac cggttatatc tttacctggt tactggctca tttcggttct 540

atcgattggg ttaatgccag tggttggtgc tggtctccgg cttcagaagg cctgatcggt 600

attgccttct tattgctgct gttaaccgca ccggatacgc cgcattggtt ggtgatgaag 660

ggacgtcatt ccgaggctag taaaatcctt gctcgtctgg aaccgcaagc cgatcctaat 720

ctgacgattc aaaagattaa agctggcttt gataaagcca tggacaaaag cagcgcaggt 780

ttgtttgctt ttggtatcac cgttgttttt gccggtgtat ccgttgctgc cttccagcag 840

ttagtcggta ttaacgccgt gctgtattat gcaccgcaga tgttccagaa tttaggtttt 900

ggagctgata cggcattatt gcagaccatc tctatcggtg ttgtgaactt catcttcacc 960

atgattgctt cccgtgttgt tgaccgcttc ggccgtaaac ctctgcttat ttggggtgct 1020

ctcggtatgg ctgcaatgat ggctgtttta ggctgctgtt tctggttcaa agtcggtggt 1080

gttttgcctt tggcttctgt gcttctttat attgcagtct ttggtatgtc atggggccct 1140

gtctgctggg ttgttctgtc agaaatgttc ccgagttcca tcaagggcgc agctatgcct 1200

atcgctgtta ccggacaatg gttagctaat atcttggtta acttcctgtt taaggttgcc 1260

gatggttctc cagcattgaa tcagactttc aaccacggtt tctcctatct cgttttcgca 1320

gcattaagta tcttaggtgg cttgattgtt gctcgcttcg tgccggaaac caaaggtcgg 1380

agcctggatg aaatcgagga gatgtggcgc tcccagaagt ag 1422

<210> 21

<211> 1089

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atggagcgta ttgtcgttac tctcggggaa cgtagttacc caattaccat cgcatctggt 60

ttgtttaatg aaccagcttc attcttaccg ctgaaatcgg gcgagcaggt catgttggtc 120

accaacgaaa ccctggctcc tctgtatctc gataaggtcc gcggcgtact tgaacaggcg 180

ggtgttaacg tcgatagcgt tatcctccct gacggcgagc agtataaaag cctggctgta 240

ctcgataccg tctttacggc gttgttacaa aaaccgcatg gtcgcgatac tacgctggtg 300

gcgcttggcg gcggcgtagt gggcgatctg accggcttcg cggcggcgag ttatcagcgc 360

ggtgtccgtt tcattcaagt cccgacgacg ttactgtcgc aggtcgattc ctccgttggc 420

ggcaaaactg cggtcaacca tcccctcggt aaaaacatga ttggcgcgtt ctaccaacct 480

gcttcagtgg tggtggatct cgactgtctg aaaacgcttc ccccgcgtga gttagcgtcg 540

gggctggcag aagtcatcaa atacggcatt attcttgacg gtgcgttttt taactggctg 600

gaagagaatc tggatgcgtt gttgcgtctg gacggtccgg caatggcgta ctgtattcgc 660

cgttgttgtg aactgaaggc agaagttgtc gccgccgacg agcgcgaaac cgggttacgt 720

gctttactga atctgggaca cacctttggt catgccattg aagctgaaat ggggtatggc 780

aattggttac atggtgaagc ggtcgctgcg ggtatggtga tggcggcgcg gacgtcggaa 840

cgtctcgggc agtttagttc tgccgaaacg cagcgtatta taaccctgct caagcgggct 900

gggttaccgg tcaatgggcc gcgcgaaatg tccgcgcagg cgtatttacc gcatatgctg 960

cgtgacaaga aagtccttgc gggagagatg cgcttaattc ttccgttggc aattggtaag 1020

agtgaagttc gcagcggcgt ttcgcacgag cttgttctta acgccattgc cgattgtcaa 1080

tcagcgtaa 1089

<210> 22

<211> 819

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggaaacct atgctgtttt tggtaatccg atagcccaca gcaaatcgcc attcattcat 60

cagcaatttg ctcagcaact gaatattgaa catccctatg ggcgcgtgtt ggcacccatc 120

aatgatttca tcaacacact gaacgctttc tttagtgctg gtggtaaagg tgcgaatgtg 180

acggtgcctt ttaaagaaga ggcttttgcc agagcggatg agcttactga acgggcagcg 240

ttggctggtg ctgttaatac cctcatgcgg ttagaagatg gacgcctgct gggtgacaat 300

accgatggtg taggcttgtt aagcgatctg gaacgtctgt cttttatccg ccctggttta 360

cgtattctgc ttatcggcgc tggtggagca tctcgcggcg tactactgcc actcctttcc 420

ctggactgtg cggtgacaat aactaatcgg acggtatccc gcgcggaaga gttggctaaa 480

ttgtttgcgc acactggcag tattcaggcg ttgagtatgg acgaactgga aggtcatgag 540

tttgatctca ttattaatgc aacatccagt ggcatcagtg gtgatattcc ggcgatcccg 600

tcatcgctca ttcatccagg catttattgc tatgacatgt tctatcagaa aggaaaaact 660

ccttttctgg catggtgtga gcagcgaggc tcaaagcgta atgctgatgg tttaggaatg 720

ctggtggcac aggcggctca tgcctttctt ctctggcacg gtgttctgcc tgacgtagaa 780

ccagttataa agcaattgca ggaggaattg tccgcgtga 819

<210> 23

<211> 717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac 60

catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg 120

gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc 180

gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt 240

cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg 300

ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag 360

ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa 420

gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt 480

catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt 540

gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta 600

ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc 660

cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccat 717

<210> 24

<211> 699

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgctctcag aattaattaa agaaaatatg cacatgaaat tatatatgga aggtactgtc 60

aacaatcatc atttcaaatg cacatccgaa ggtgaaggta aaccatatga aggcacacaa 120

acaatgcgca tcaaagcagt tgaaggtgga cccctgccct ttgcgtttga cattctcgca 180

acgagcttta tgtacgggtc taaaactttt atcaatcaca cccaaggcat tcctgacttt 240

tttaaacagt cctttcctga aggctttacc tgggaacgtg taacaactta tgaagatggc 300

ggtgtactta cagcaactca agatacgagt ttacaagatg gctgtctgat ttacaatgtt 360

aaaatccgtg gcgtaaattt cccgagtaac ggacccgtaa tgcaaaaaaa aactcttggt 420

tgggaagcat caacagaaac cttatatcct gcggacggtg gcttagaagg acgcgcagac 480

atggcactga aattagttgg aggcggtcat ttaatctgca acctgaaaac aacctatcgt 540

tccaaaaaac ccgctaaaaa ccttaaaatg cctggagtat actatgttga tcgtcgctta 600

gaacgtatta aagaagctga taaagaaacc tacgttgaac aacatgaagt agccgtagcc 660

cgttattgtg accttccgtc gaaattagga catcgttga 699

Claims (10)

1. A system for regulating the dynamic expression of a protein, which is characterized by using a growth phase-associated promoter P in combination in Escherichia coli_GPPAnd the C-terminal degradation tag SsrA acts on the target proteinNow dynamic expression of the target protein, wherein the target protein is subject to a growth phase-associated promoter P_GPPControl, and contain C terminal degradation label SsrA; the growth phase-associated promoter P_GPPIncluding P_rpsL、P_rrnA、P_rpsT、P_rrnCOr P_rpsA(ii) a The C-terminal degradation label SsrA comprises LAA, DAS +4, DAS +8 or GSD; the P is_rpsL、P_rrnA、P_rpsT、P_rrnCOr P_rpsAThe nucleotide sequences of (A) are respectively shown in SEQ ID NO.1-SEQ ID NO. 5; the amino acid sequences of the LAA, the DAS +4, the DAS +8 or the GSD are respectively shown in SEQ ID NO.6-SEQ ID NO. 10.

2. The regulatory system of claim 1, wherein the target protein is shikimate kinase I or green fluorescent protein GFP.

3. The regulatory system of claim 2, wherein the target protein is shikimate kinase I.

4. A genetically engineered bacterium, wherein Escherichia coli is used as a host bacterium, and the expression of a target protein is controlled by introducing the dynamic expression control system according to claim 1.

5. The genetically engineered bacterium of claim 4, wherein said Escherichia coli has been knocked out of shikimate kinase I gene aroK and shikimate kinase II gene aroL and has its PTS system replaced by glucose-facilitated protein gene Zmglf.

6. The genetically engineered bacterium of claim 5, wherein said E.coli is E.coli MG1655 that has the shikimate kinase I gene aroK and shikimate kinase II gene aroL knocked out and has the PTS system replaced with the glucose-labile protein gene Zmglf derived from Zymomonas mobilis.

7. A method for constructing the genetically engineered bacterium according to any one of claims 4 to 6,the method is characterized by comprising the following specific steps: (1) constructing genetically engineered plasmids of PJ01-GABE and P15A-P_GPP-aroK-SsrA; (2) transforming the plasmid into escherichia coli; (3) mixed culture is carried out to obtain the conjugated shikimic acid producing strain, wherein,

the construction method of the plasmid PJ01-GABE in the step (1) comprises the following steps: respectively amplifying to obtain aroB containing B0034RBS by using Escherichia coli MG1655 genome as template^opt、aroE、aroG^fbrtktA fragment, aroB^optInserting the two segments of aroE into an expression frame of pJ01 in a multi-segment one-step homologous recombination mode to obtain a pJ01-BE plasmid, and carrying out aroG^fbrInserting the two fragments of tktA into an expression frame of a plasmid pJ01 in a multi-fragment one-step homologous recombination mode to obtain a pJ01-GA plasmid, cutting the pJ01-GA plasmid by using BamHI and XbaI restriction enzymes, recovering the vector, cutting the pJ01-BE plasmid by using BgIII and XbaI restriction enzymes, and recovering the fragments; respectively assembling pJ01-GA and pJ01-BE plasmids by adopting a mode of homoplastic enzyme connection to finally obtain a plasmid PJ 01-GABE;

the plasmid P15A-P in the step (1)_GPPThe construction method of the aroK-SsrA comprises the following steps: synthetic growth phase promoter P_GPPSequence, replacement of original P in vector pTet-1_tetThe obtained plasmid was named P15A-P_GPPAmplifying a gene aroK by taking an Escherichia coli MG1655 genome as a template, fusing different degradation labels SsrA at the 3' end of the gene aroK, and inserting aroK-SsrA fused with different degradation labels into P15A-P_GPPTo obtain the plasmid P15A-P_GPP-aroK-SsrA。

8. A process for producing shikimic acid, characterized in that the genetically engineered bacterium of any one of claims 4 to 6 is used as a fermenting microorganism and fermentation is carried out with glucose as a carbon source.

9. The method of claim 8, wherein the fermentation medium is M9 mineral salts medium; the fermentation conditions were 35-38 deg.C, 200-₆₀₀Fermenting for 70-75h at 0.04-0.1; or fermentation conditionsAt 35-38 ℃, 480-530rpm, the inoculation amount of 5-10 percent and the ventilation amount of 1-2vvm, and fermenting for 90-100 h.

10. Use of the dynamic expression control system of any one of claims 1 to 3 or the genetically engineered bacteria of any one of claims 4 to 6 in the preparation of shikimic acid or a product containing shikimic acid.

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