Escherichia coli recombinant strain for producing 3-dehydroshikimic acid, construction method and application

Abstract

The invention relates to an Escherichia coli recombinant strain for producing 3-dehydroshikimic acid, a construction method and application, and belongs to the field of enzyme engineering.The Escherichia coli recombinant strain TP06 serves as an original strain, shikimic acid dehydrogenase AroE genes are knocked out, endogenous ppsA, transaldolase talA and tktA genes of Escherichia coli are overexpressed, prerequisite supply of shikimic acid ways is increased, and the yield of 3-dehydroshikimic acid is increased. The synthesis flux of DHS is improved; an aroG mutant aroGFBR from escherichia coli is expressed, and the 3-deoxy-D-arabinoheptanoic acid-7-phosphate synthetase which is subjected to feedback inhibition by aromatic amino acid is relieved by the mutant. Then, a key gene poxB in a byproduct synthesis route is knocked out through a Red homologous recombination gene knockout technology, acetic acid accumulation in the fermentation process is reduced, high-glucose-concentration fermentation is achieved, and the yield of DHS is further increased. And finally, the engineering strain realizes higher DHS production through fed-batch fermentation.

Description

Technical Field

[0001] The present invention belongs to the field of microbial engineering, and specifically relates to a recombinant strain of Escherichia coli producing 3-dehydroshikimic acid, a construction method and application thereof Background Art

[0002] 3-dehydroshikimate (DHS) is an important intermediate in the biosynthetic metabolic pathway of aromatic amino acids in microorganisms, and plays an important role in maintaining normal cell growth and completing metabolic processes. It can be further processed to synthesize a series of important chemical products such as protocatechuate, vanillin, catechol, gallic acid, etc. The use of DHS to synthesize these chemical products can avoid the use of toxic raw materials such as benzene and toluene, and reduce the impact on human body and environment. In addition, DHS is also a very effective antioxidant, and its activity is better than some commercial antioxidants such as gallic acid, propyl gallate, BHQ (Tertbutylhydroquinone), butylatedhydroxytoluene (BHT) and tocopherol (α-tocopherol), and has important application value. In addition, DHS, as a small molecule chiral compound, can also be used as a very potential synthetic intermediate in drug synthesis. Therefore, studying the production of DHS has important application prospects.

[0003] Phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), the precursors required for the biosynthesis of 3-dehydroshikimic acid, are intermediate metabolites of the glycolysis pathway (TCA) and the pentose phosphate pathway (HMP) in the central metabolic pathway, respectively. Glucose outside the Escherichia coli is phosphorylated by the glucose transport system and transported into the cell, and this process requires the participation of PEP.

[0004] The common pathway of aromatic amino acids is also known as the shikimic acid pathway. This pathway starts from the condensation of two major precursor substances, PEP and E4P. The condensation product is 3-deoxy-D-arabinoheptulose-7-phosphate (N-Palmalkyl-1-3-propanediamine, DAHP). The production of DAHP is the first step in the three major common pathways of aromatic amino acids and the first rate-limiting reaction in the 3-dehydroshikimic acid biosynthesis pathway. As the first enzyme in the common pathway of aromatic amino acid biosynthesis, the activity of DAHP synthase determines the amount of cellular carbon that directly enters DHS synthesis. Historically, transcriptional inhibition and feedback inhibition of DAHP synthase by aromatic amino acids have been considered to be the regulatory mechanisms that control the catalytic activity of the enzyme in vivo. 3-Deoxy-D-arabinoheptulose-7-phosphate synthase AroG affects the synthesis of DAHP, and its mutant AroG FBR (Du Lihong. (2024). Research progress on L-tyrosine production by Escherichia coli. Fermentation Science and Technology Newsletter. doi:10.16774 / j.cnki.issn.1674-2214.2024.02.010) Enhanced resistance to feedback inhibition of metabolic end products, aromatic amino acids, can promote the synthesis of DAHP, thereby affecting the production of DHS. The induction of DAHP synthase overexpression is achieved by increasing the addition of isopropyl-β-D-thiogalactoside during the fermentation process. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant strain of Escherichia coli producing 3-dehydroshikimic acid and its application. Xu Qingyang et al. (Directed transformation method of tyrosine production strain and production strain and tyrosine fermentation method, application number 202310280671.7) disclose a modified recombinant strain of Escherichia coli TP06 producing tyrosine, and the present invention is further improved on the basis of the recombinant strain of Escherichia coli TP06. A recombinant strain of Escherichia coli producing 3-dehydroshikimic acid is obtained.

[0006] The present invention is achieved through the following technical solutions:

[0007] A recombinant Escherichia coli strain for producing 3-dehydroshikimic acid is used as a starting strain, wherein the pyruvate dehydrogenase gene (poxB gene), the negative regulatory transcription inhibitor tyrR and the shikimate dehydrogenase AroE gene on the recombinant Escherichia coli strain TP06 are knocked out; and the DAHP synthase gene aroG which resists feedback inhibition is overexpressed. FBR , overexpressing phosphoenolpyruvate synthase ppsA, transketolase tktA and transaldolase talA.

[0008] gene aroG FBRThe base G at position 436 in the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene aroG is mutated to A to form a mutant gene aroG FBR , and insert the pTrc promoter upstream.

[0009] The present invention also provides a method for preparing the recombinant strain, which is as follows:

[0010] Step 1: Using E. coli TP06 as the starting strain, preparing E. coli TP06 competent strain, adding pKD46 plasmid, preparing E. coli TP06-pKD46 strain;

[0011] Step 2: Obtain the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene, referred to as the aroG gene, and mutate the base G at position 436 in the aroG gene to A to form a mutant gene AroG FBR After being put aside, the product was amplified using primers containing restriction sites EcoRI / BamHI. The PCR-purified product was fused with the pTrc99a plasmid that was also double-digested with EcoRI / BamHI, and transformed into E. coli DH5a competent cells. The plasmid DNA was extracted and pTrc::AroG FBR The fragment is amplified and purified before proceeding to the next step.

[0012] The fragment pTrc::AroG FBR Transformed into E. coli TP06-pKD46 competent cell to obtain E. coli engineered strain TP06-DHS01;

[0013] Step 3, using the above-obtained engineered strain TP06-DHS01 as the starting strain, knocking out the negative regulatory transcription inhibitor tyrR to obtain the Escherichia coli engineered strain TP06-DHS02;

[0014] Step 4: Using the engineered strain TP06-DHS02 obtained above as the starting strain, knocking out the shikimate dehydrogenase gene aroE to obtain the Escherichia coli engineered strain TP06-DHS03;

[0015] Step 5, using the above-obtained engineered strain TP06-DHS03 as the starting strain, knocking out the transketolase gene tktA to obtain the Escherichia coli engineered strain TP06-DHS04;

[0016] Step 6: Using the engineered strain TP06-DHS04 obtained above as the starting strain, the transaldolase gene talA was knocked out to obtain the Escherichia coli engineered strain TP06-DHS05.

[0017] Finally, the pyruvate dehydrogenase gene (poxB gene), negative regulatory transcriptional repressor tyrR and shikimate dehydrogenase AroE gene of the recombinant Escherichia coli strain TP06 were knocked out; the anti-feedback inhibition DAHP synthase gene aroG FBR , overexpressing phosphoenolpyruvate synthase ppsA, transketolase tktA and transaldolase talA.

[0018] The present invention also provides an application of the recombinant strain, and the application method is to produce 3-dehydroshikimic acid by fermentation using the recombinant strain.

[0019] As a preferred embodiment, the fermentation process is carried out by fed-batch fermentation.

[0020] A biological preparation, comprising the recombinant strain, and capable of producing 3-dehydroshikimic acid by fermentation.

[0021] The beneficial effects of the present invention compared with the prior art are as follows:

[0022] The present invention uses the recombinant Escherichia coli strain TP06 as the starting strain, knocks out the shikimate dehydrogenase AroE gene, overexpresses the endogenous ppsA, transaldolase talA and tktA genes of Escherichia coli, increases the supply of the prerequisites for the shikimate pathway, and improves the synthesis flux of DHS; expresses the aroG mutant aroG from Escherichia coli FBR , the mutant relieved the 3-deoxy-D-arabinoheptulose-7-phosphate synthase of aromatic amino acid feedback inhibition. Then, the key gene poxB in the byproduct synthesis pathway was knocked out by Red homologous recombination gene knockout technology, which reduced the accumulation of acetic acid during fermentation, achieved high glucose concentration fermentation, and further increased DHS production. Finally, the engineered strain achieved higher DHS production through batch fed fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The biosynthetic pathway for 3-dehydroshikimic acid in Escherichia coli;

[0024] Figure 2 The invention overexpresses the DHAP synthase gene aroG which is resistant to feedback inhibition. FBR Plasmid pTrc99a-aroG FBR Structural diagram;

[0025] Figure 3 The schematic diagram of the structure of the plasmid pTrc99a-tktA for overexpressing the transketolase gene tktA of the present invention;

[0026] Figure 4The schematic diagram of the structure of the plasmid pTrc99a-talA for overexpressing the transaldolase gene talA of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

[0028] Materials and methods involved in the examples:

[0029] 1. Culture medium

[0030] (1) Slant culture medium: glucose 1 g / L, beef extract 10 g / L, yeast powder 5 g / L, peptone 10 g / L, phenylalanine 0.2 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, VB1 20 mg / L, NaCl 5 g / L, agar 20 g / L, pH 7.0-7.2, sterilized at 121°C for 30 min.

[0031] (2) Primary seed culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.

[0032] (3) Fermentation medium: glucose 30 g / L, yeast powder 5 g / L, citric acid monohydrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 1 g / L, sodium chloride 1 g / L, manganese sulfate monohydrate 2 mg / L, ferrous sulfate heptahydrate 2 mg / L, VH 0.1 mg / L, VB1, VB3, VB5, VB 12 0.2mg / L, p-aminobenzoic acid 2mg / L, tryptophan 0.5g / L, tyrosine 0.5g / L, phenylalanine 0.5g / L, metal ion solution 1.5mL / L (ZnSO46g / L, CoCl25g / L, CuSO40.5g / L, sodium molybdate 0.24g / L, boric acid 0.1g / L), calcium carbonate 2%.

[0033] (4) Seed culture:

[0034] Primary seed culture: Pick a single colony from the slant culture medium and inoculate it into the seed culture medium, and culture it in a shaking incubator at 32°C for 12-14 hours;

[0035] Secondary seed culture: Take 1 mL of primary seed solution and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of culture medium. Incubate at 32°C with a reciprocating shaker at 200 rpm / min for about 5-6 hours. OD 600 Control between 12-18.

[0036] (5) Fermentation culture:

[0037] The recombinant strains TP06-DHS01, TP06-DHS02, TP06-DHS03, TP06-DHS04, TP06r-DHS05, and TP06-DHS06 were fermented in 500 mL baffled bottles. The specific fermentation conditions were as follows:

[0038] The seed liquid was inoculated into a 500mL shake flask at a 10% inoculation rate, the fermentation temperature was 32°C, the horizontal shaker speed was 200rpm / min, and the pH was adjusted once every 2h with ammonia water. The amount of ammonia water added was fine-tuned according to the color of the fermentation medium displayed by the pH indicator. During the fermentation process, the residual sugar concentration in the fermentation liquid was measured every 4h. When the glucose in the fermentation medium was lower than 1g / L, glucose was added to a final concentration of 10g / L. The fermentation cycle was 48h.

[0039] (6) Determination of glucose:

[0040] Fermentation broth pretreatment: centrifuge the fermentation broth at 12000r / min for 5min to obtain the supernatant. Dilute to an appropriate multiple and detect the glucose concentration of the fermentation broth by high performance liquid chromatography.

[0041] (7) Determination of 3-dehydroshikimic acid:

[0042] High performance liquid chromatography: Prepare 1g / L DHS solution, dilute to 0.1, 0.2, 0.3, 0.4 and 0.5g / L, use high performance liquid chromatography (HPLC) for detection, obtain the peak time and the peak area corresponding to different concentrations of shikimic acid, use the concentration of DHS solution as the horizontal coordinate and the peak area as the vertical coordinate to draw a standard curve and obtain the linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used. The instrument is Shimadzu high performance liquid chromatograph, and the chromatographic column uses Ultimate XB-C18 liquid chromatography column (4.6*250mm, 5um); the mobile phase is 20% methanol + 0.07% perchloric acid; the flow rate is set to 1mL / min; the detector is a UV detector, the detection wavelength is 278nm, and the column temperature is 40℃.

[0043] Fermentation broth pretreatment: centrifuge the fermentation broth at 12000r / min for 10min and take the supernatant. After diluting the sample to an appropriate multiple, pass the sample through a membrane and detect it using HPLC. Substitute the obtained peak area into the linear regression equation, and the result multiplied by the dilution multiple is the DHS concentration in the fermentation broth.

[0044] High performance liquid chromatography (HPLC) measured the final DHS production in the supernatant of the fermentation broth of the recombinant strains TP06-DHS01, TP06-DHS02, TP06-DHS03, TP06-DHS04 and TP06-DHS05 to be 2.35, 3.36, 4.72, 10.57 and 15.63 g / L, respectively.

[0045] The recombinant Escherichia coli strain TP06 is obtained according to the method of patent "Application No. 202310280671.7, directed transformation method of tyrosine production strain and production strain and tyrosine fermentation method".

[0046] This embodiment provides a recombinant E. coli for producing DHS, and the following improvements are made to the recombinant E. coli strain TP06 using Red recombination technology:

[0047] (1) The poxB gene was knocked out, which reduced the accumulation of acetic acid during the fermentation process and achieved high glucose concentration fermentation;

[0048] (2) Overexpression of the endogenous ppsA, transaldolase talA, and tktA genes of Escherichia coli increased the supply of the precursors for the shikimate pathway and improved the synthesis flux of DHS;

[0049] (4) Expression of aroG from Escherichia coli FBR The mutant relieved the feedback inhibition of 3-deoxy-D-arabinoheptulose-7-phosphate synthase by aromatic amino acids;

[0050] (5) knockout of the negative regulatory transcriptional repressor tyrR;

[0051] (6) Knockout of the AroE gene increased the ability of E. coli to accumulate DHS;

[0052] Finally, a recombinant Escherichia coli with high DHS production was obtained, and the fermentation conditions were optimized to achieve efficient production of DHS.

[0053] In the Escherichia coli, the DHS dehydrogenase gene aroE is knocked out, the negative regulatory transcription inhibitor tyrR is knocked out, and the DAHP synthase gene aroG that resists feedback inhibition is overexpressed. FBR , knocking out the pyruvate dehydrogenase gene poxB, and simultaneously overexpressing phosphoenolpyruvate synthase ppsA, transketolase tktA and transaldolase talA. The numbers of the genes are shown in Table 1.

[0054] Table 1 Gene numbers

[0055]

[0056] 3-deoxy-D-arabinoheptulose-7-phosphate synthase encoding gene aroG FBR The nucleotide sequence is shown in SEQ ID NO.1.

[0057] SEQ ID NO.1 is as follows:

[0058]

[0059] In one embodiment of the present invention, the DAHP synthase encoding gene aroG is overexpressed. FBR talA, tktA and ppsA were expressed by using a multi-copy promoter pTrc (obtained from the commercial plasmid pTrc99a). The schematic diagram of the overexpression plasmid is shown in FIG. Figure 2-4 shown.

[0060] Example 1: Construction of DHS-synthetic recombinant Escherichia coli

[0061] (1) Preparation of engineered strain DHS01: E. coli TP06 strain was cultured at 37°C overnight with shaking, and the culture solution was transferred to 100 mL LB medium at a ratio of 1:100 and cultured with shaking until OD 600 =0.3-0.4, transfer the culture medium to a 50mL centrifuge tube, place on ice for 15-30min, then centrifuge at 4000rpm, 4℃ for 10min, discard the supernatant, resuspend with ice-cold 0.1M CaCl2 solution, place on ice for 20-30min, centrifuge at 4000rpm, 4℃ for 10min, discard the supernatant. Finally, add 1.5mL 0.1MCaCl2 and 80% glycerol, mix well, and dispense into 100ul for use.

[0062] Take E.coli TP06 competent cells, add 1ul pKD46 plasmid (commercial plasmid), mix gently and place on ice for 30 minutes. Heat shock at 42℃ for 20 seconds, then immediately place on ice for 2 minutes, add LB after 2 minutes, and recover at 28℃ for 60 minutes. Take 100-150uL of recovery solution and coat LB solid plate (Amp resistance, 50mg / ml), then invert the plate and culture in a 28℃ incubator for 24-36 hours to obtain E.coli TP06-pKD46.

[0063] Take multiple polyclonal E. coli TP06-pKD46 strains, place them in 3 mL LB (Amp resistance, 50) liquid medium, and culture them at 28°C overnight. Add the overnight bacterial solution to 50 mL LB (Amp resistance, 50) liquid medium at a ratio of 1:100 and culture at 28°C until OD 600 ≈0.2, then arabinose (final concentration 1 mM) was added and cultured at 28°C until OD 600 ≈0.4-0.5. Take out the bacterial solution and place it on ice for 30 minutes, then pour it into a 50mL centrifuge tube, centrifuge at 4000rpm, 4℃ for 10min, and discard the supernatant. Wash the precipitate twice with ice-cold 10% glycerol, and finally add 500uL 10% glycerol to make competent cells and place them on ice for use, and then transform them after 30 minutes.

[0064] The recombinant E. coli strain TP06 was selected as the starting strain for fermentation production of DHS. Figure 1 As shown in the 3-dehydroshikimic acid synthesis pathway, 3-deoxy-D-arabinoheptulose-7-phosphate synthase AroG affects the synthesis of 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP), and its mutant AroG FBR It enhances the resistance to feedback inhibition of the metabolic end products, aromatic amino acids, and can promote the synthesis of DAHP, thereby affecting the production of DHS.

[0065] According to the research of Du Lihong et al. (Du Lihong. (2024). Research progress on L-tyrosine production by Escherichia coli. Fermentation Science and Technology Newsletter. doi:10.16774 / j.cnki.issn.1674-2214.2024.02.010). The mutant AroG was overexpressed using RED recombination technology. FBR Using Escherichia coli DH5α (purchased commercial strain) as a template, the AroG fragment was amplified using primers AroG-down / AroG-up (Table 2), and the base G at position 436 in the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene aroG was mutated to A using mutation primers AroG-F mutation / AroG-R mutation to form the mutant gene AroG FBR The mutant gene AroG FBR The plasmid was double-digested with EcoRI / BamHI and the primers were AroG-F EcorI / AroG-RBamHI. The plasmid was fused with the pTrc99a plasmid (commercial plasmid) which was also double-digested with EcoRI / BamHI to obtain pTrc99a-aroG FBR Plasmids, structures such as Figure 2 As shown, pTrc99a-aroG FBR After the plasmid was transformed into E. coli DH5a competent medium (commercial competent medium), the plasmid was extracted according to the steps described in the plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the primers kana-F / AroG-R BamHI were used to convert pTrc::AroG FBR The fragment is amplified and purified before proceeding to the next step.

[0066] The fragment pTrc::AroG FBRThe Kana resistance gene (commercial plasmid pKD4, obtained by amplification with primers Kana-F / Kana-R) was connected using ClonExpress II One Step CloningKit ligase at 37°C for 2 hours, and the fusion fragment was electroporated into E. coli TP06-pKD46 competent cells. Take 100uL of E. coli TP06-pKD46 competent cells, add 100ng of the fusion fragment, and add up to 10uL. Mix gently and add to the electroporation cup. Then put it into the electroporator (Xinzhi) for electroporation (1800v). After the electroporation, the cells were immediately taken out and 1 mL of SOC liquid culture medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM D-glucose, pH 7.5) was added. The culture medium was transferred into a 1.5 mL centrifuge tube and cultured at 37°C for 1 hour at a speed of 160-170 r. The precipitate was collected by centrifugation and all coated on LB solid culture medium containing Kana (20) and cultured in a 37°C incubator for 16-24 hours. After a single colony was grown, the primers Kana-F / AroG-R BamHI were used for PCR verification and sent for sequencing. The recombinant strain E. coli pTrc::AroG was obtained. FBR ::Kana, and then the recombinant vector E.coli pTrc::AroG FBR ::Kana was prepared into electrocompetent state, transferred into pCP20 plasmid, Kana resistance gene was removed, and engineered strain Escherichia coli was obtained

[0067] TP06-DHS01.

[0068] Table 2 Primer sequences

[0069]

[0070] (2) Preparation of engineered strain DHS02

[0071] The TyrR regulator in Escherichia coli contains 8 transcription units, each of which encodes a protein involved in the synthesis and transport of three aromatic amino acids, and its expression is regulated by the TyrR protein. Knocking out the negative regulatory transcription repressor tyrR relieves the inhibitory effect of the TyrR protein on the key enzymes of the shikimate pathway.

[0072] The engineered strain DHS01 was used as the starting strain, and the electroporation competent TP06-DHS01-pKD46 was prepared using the same method. The primer sequences used are shown in Table 3.

[0073] Using the E. coli DH5a genome as a template, the primer pairs tyrR-up-F / tyrR-up-R and

[0074] tyr-down-F / tyr-down-R amplified the upstream and downstream homology arms of tyrR and purified the fragments; using the purified fragments as templates, the primer pair tyrR-F / tyrR-R was used to amplify the knockout cassette and purify the fragments.

[0075] Kana resistance gene fusion, electroporation into DHS01 competent cells to obtain DHS02ΔtyrR::kan strain. After a single colony grows, PCR verification is performed and sequencing verification is sent. DHS01ΔtyrR::kan strain was prepared according to the above method.

[0076] The cells were electroporated into the pCP20 plasmid, the Kana resistance gene was removed, and plates containing Chl+Apm resistance were coated. The single colonies were amplified by colony PCR using primers tyrR-up-F / tyrR-down-R and sent for sequencing verification to obtain the engineered strain Escherichia coli TP06-DHS02.

[0077] Table 3. Primer sequences

[0078]

[0079] (3) Preparation of engineered strain DHS03

[0080] Shikimate dehydrogenase aroE is responsible for converting DHS into shikimate, so the ability of E. coli to accumulate DHS can be increased by mutating or knocking out aroE. The above-obtained engineered strain E. coli DHS02 was used as the starting strain, and the electroporation competent cells were prepared using the same method. The primer sequences used are shown in Table 4.

[0081] Using the Escherichia coli DH5a genome as a template, the upstream and downstream homologous arms of the gene aroE were amplified using primer pairs up-F / up-R and down-F / down-R, respectively, and the fragments were purified; using the purified fragment as a template, the knockout box was amplified using primer pairs, and the fragments were purified. The fragment was fused with the Kana resistance gene and electrotransformed into the engineering strain Escherichia coli DHS02 competent state to obtain the DHS03-ΔaroE::kana strain. After a single colony is grown, the primer pair Verification-F / Verification-R is used for PCR amplification and sent for sequencing verification. The DHS03-ΔaroE::kan strain was prepared into an electrotransformation competent state according to the above method, transferred into the pCP20 plasmid, the Kana resistance gene was removed, and a plate containing Chl+Apm resistance was coated. The single colony was amplified by colony PCR using primers Verification-F / Verification-R, and sent for sequencing verification to obtain the engineering strain Escherichia coli TP06-DHS03.

[0082] Table 4. Primer sequences

[0083]

[0084] (4) Preparation of engineered strain DHS04

[0085] The transketolase gene tktA in the synthesis pathway is the gene mainly responsible for the synthesis of erythrose-4-phosphate (E4P), which will affect the synthesis of the target product DHS. The primer sequences used are shown in Table 5.

[0086] The transketolase gene tktA used in the present invention is derived from Escherichia coli, and the genomic DNA of Escherichia coli DH5a is extracted; according to the published genome information sequence (YP_026188.1), the verification primer pairs tktA-F / tktA-R are respectively designed, and the genomic DNA of Escherichia coli DH5a is extracted as a template, and the tktA gene is respectively amplified and obtained by using a standard PCR amplification system and procedure. The tktA obtained by PCR amplification is recovered by agarose gel nucleic acid electrophoresis gel cutting, and the expression vector pTrc is double-digested for 2 hours by restriction endonucleases EcoRI and BamHI, and the digestion products are recovered by agarose gel nucleic acid electrophoresis gel, and the sizes of the tktA gene and the linearized plasmid are 1992bp and 2502bp respectively. First, the tktA gene was connected to the linearized pTrc99a plasmid using ClonExpress II One Step CloningKit ligase at 37°C for 2 hours and transformed into Escherichia coli DH5a competent cells. Single colonies were picked to extract DNA for PCR amplification using primers trc-kana-F / tktA-down-R. Positive transformants were selected for sequencing. The expression vector was successfully constructed and the plasmid was named pTrc99a-tktA. The structure is shown in the figure. Figure 3 Then, the DNA of plasmid pTrc99a-tktA was extracted, and the pTrc promoter and tktA fragment were amplified using primers trc-kana-F / tktA-down-R, and then fused with the Kana resistance gene (amplified from the pKD4 plasmid using primers) using Clon Express II One Step Cloning Kit ligase to obtain the fusion fragment pTrc::tktA::Kana.

[0087] The engineered strain TP06-DHS03 obtained in the above step was cultured at 37°C, 220rpm overnight, prepared into a competent state using the above method, and stored at -80°C. Subsequently, the pKD46 plasmid was transformed into a competent state, 100-150uL of the recovery solution was coated on an LB solid plate (Amp resistance, 50), and then the plate was inverted and cultured in a 28°C incubator for 24-36 hours to obtain the E. coli TP06-DHS03-pKD46 strain. The obtained strain was shaken at 28°C until the OD 600=0.3-0.4, add ice-cold 10% glycerol and prepare E. coli TP06-DHS03-pKD46 electroporation competent state.

[0088] The fusion fragment pTrc::ktA::Kana was electroporated into the competent cell of E.coli TP06-DHS03-pKD46. Take 100uL of competent cells of E.coliTP06-DHS03-pKD46, add 100ng of the fusion fragment, and add a maximum of 10uL. Mix gently and add to the electroporation cup. Then put it into the electroporator for electroporation (1800v). Take it out immediately after electroporation, add 1mL of SOC liquid culture medium, and transfer the culture medium into a 1.5mL centrifuge tube, incubate at 37℃ for 1 hour, rotate at 160-170r, collect the precipitate by centrifugation, and spread it all on the LB solid culture medium containing Kana (20), and incubate it in a 37℃ incubator for 16-24 hours. After a single colony was grown, PCR verification was performed and sequencing verification was performed. The sequence was correct, and the E. coli TP06-DHS03::tktA::Kana strain was obtained. The E. coli TP06-DHS03::tktA::Kana strain was prepared into an electroporation competent state according to the above method, transferred into the pCP20 plasmid, the Kana resistance gene was removed, and a plate containing Chl+Apm resistance was coated. The single colony was amplified by colony PCR using primers tktA-up / tktA-down, and the plate was sent for sequencing verification to obtain the engineered strain Escherichia coli TP06-DHS04.

[0089] Table 5 Primer sequences

[0090]

[0091] (5) Preparation of engineered strain DHS05

[0092] The transaldolase gene talA used in this embodiment is derived from Escherichia coli DH5a, and the genomic DNA of Escherichia coli DH5a is extracted; according to the published genome information sequence (NP_414549.1), the verification primer pairs talA-EcoRI-F / talA-BamHI-R are designed respectively, and the genomic DNA of Escherichia coli is extracted as a template, and the talA gene is amplified using a standard PCR amplification system and procedure. The talA obtained by PCR amplification is recovered by agarose gel electrophoresis, and the expression vector pTrc99a is double-digested with restriction endonucleases EcoRI and BamHI for 2h, and the digestion products are recovered by agarose gel electrophoresis. The sizes of the talA gene and the linearized plasmid are 951bp and 2502bp, respectively. First, the talA gene was connected to the linearized pTrc plasmid using Clon Express II One Step Cloning Kit ligase at 37°C for 2 hours and transformed into Escherichia coli DH5a competent cells. Single colony PCR was picked and verified using talA-up-F / talA-down-R. The positive transformants were sequenced and the alignment was correct, proving that the expression vector was successfully constructed. The plasmid was named pTrc99a-talA. The structural diagram is shown in the figure. Figure 4 Then, the plasmid was extracted, and the pTrc promoter and talA fragment were amplified using primers trc-kana-F / talA-R, and then fused with the Kana gene (amplified from the pKD4 plasmid using primers) using ClonExpress II One Step Cloning Kit ligase to obtain the fusion fragment pTrc::talA::Kana.

[0093] The obtained engineered strain TP06-DHS04 was cultured at 37°C, 220rpm overnight, prepared into competent cells using the above method, and stored at -80°C. Subsequently, the pKD46 plasmid was transformed into competent cells, 100-150uL of the recovery solution was coated on LB solid plates (Amp resistance, 50), and then the plates were inverted and cultured in a 28°C incubator for 24-36 hours to obtain the E. coli TP06-DHS04-pKD46 strain. The obtained strain was shaken at 28°C until OD 600 =0.3-0.4, add ice-cold 10% glycerol and prepare E. coli TP06-DHS04-pKD46 electroporation competent state.

[0094] The fusion fragment pTrc::talA::Kana was electroporated into the competent cell of E.coli TP06-DHS04-pKD46. Take 100uL of competent cells of E.coli TP06-DHS04-pKD46, add 100ng of the fusion fragment, and add a maximum of 10uL. Mix gently and add to the electroporation cup. Then put it into the electroporator for electroporation (1800v). Take it out immediately after electroporation, add 1mL of SOC liquid culture medium, and transfer the culture medium into a 1.5mL centrifuge tube, incubate at 37℃ for 1 hour, rotate at 160-170r, collect the precipitate by centrifugation, and spread it all on the LB solid culture medium containing Kana (20), and incubate it in a 37℃ incubator for 16-24 hours. After a single colony grows, PCR amplification is performed and sent for sequencing verification. The sequence is correct, and the E.coli TP06-DHS03-talA::Kana strain is obtained. The E. coli TP06-DHS03-talA::Kana strain was prepared into an electroporation competent state according to the above method, and the pCP20 plasmid was transferred to remove the Kana resistance gene, and the plate containing Chl+Apm resistance was coated. The single colony was amplified by colony PCR and sent for sequencing verification to obtain the engineered strain E. coli TP06-DHS05. The primer sequences used are shown in Table 6.

[0095] Table 6 Primer sequences

[0096]

[0097] Example 2 Fed-batch fermentation of recombinant strain TP06-DHS05 in a fermenter

[0098] The recombinant strain TP06-DHS05 was fermented in a 5L fermenter, and the specific fermentation conditions were as follows:

[0099] Fermentation medium: glucose 30g / L, yeast powder 5g / L, citric acid monohydrate 2g / L, dipotassium hydrogen phosphate 2g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 1g / L, sodium chloride 1g / L, manganese sulfate monohydrate 2mg / L, ferrous sulfate heptahydrate 2mg / L, VH 0.1mg / L, VB1, VB3, VB5, VB 12 0.2mg / L, p-aminobenzoic acid 2mg / L, tryptophan 0.5g / L, tyrosine 0.5g / L, phenylalanine 0.5g / L, metal ion solution 1.5mL / L (ZnSO4 6g / L, CoCl2 5g / L, CuSO4 0.5g / L, sodium molybdate 0.24g / L, boric acid 0.1g / L), sterilized at 115℃ for 15min. After sterilization, install it to the control console, turn on the temperature control, wait until the temperature cools to 37℃, add ammonia water to adjust the pH to 7.0, and prepare for inoculation.

[0100] The shaken recombinant strain TP06-DHS05 seed solution was inoculated into the fermentation tank at an inoculation rate of 10%. During the fermentation process, the temperature automatic control was turned on to maintain the fermentation temperature at 37°C, and ammonia water was used to adjust the pH between 6.95 and 6.75.

[0101] Dissolved oxygen conditions: initial ventilation volume 2vvm, rotation speed 300rpm. During the fermentation process, the dissolved oxygen in the fermentation process was maintained between 20% and 40% by adjusting the ventilation volume and the rotation speed of the stirring paddle.

[0102] During the fermentation process, after the initial glucose in the fermentation medium is lower than 1 g / L, 800 g / L of glucose solution is added to control the glucose concentration below 5 g / L. The fermentation cycle is 48 hours.

[0103] The final DHS yield in the fermentation broth supernatant was measured by high performance liquid chromatography (HPLC) to be 64.4 g / L, and the conversion rate to glucose reached 40%.

Claims

1. A recombinant Escherichia coli strain producing 3-dehydroshikimic acid, characterized in that: The recombinant strain is based on the Escherichia coli recombinant strain TP06, and the pyruvate dehydrogenase gene poxB, the negative regulatory transcription inhibitor tyrR and the shikimate dehydrogenase AroE gene on it are knocked out; the anti-feedback inhibition DAHP synthase gene aroG is overexpressed FBR , overexpressing phosphoenolpyruvate synthase ppsA, transketolase tktA and transaldolase talA. gene aroG FBR The base G at position 436 in the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene aroG is mutated to A to form a mutant gene aroG FBR , and insert the pTrc promoter upstream.

2. The method for preparing the recombinant strain according to claim 1, characterized in that: The method is as follows: Step 1: Using E. coli TP06 as the starting strain, preparing E. coli TP06 competent strain, adding pKD46 plasmid, preparing E. coli TP06-pKD46 strain; Step 2: Obtain the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene, referred to as the aroG gene, and mutate the base G at position 436 in the aroG gene to A to form a mutant gene AroG FBR After being put aside for use, the double-enzyme digestion product was fused with the pTrc99a plasmid that was also double-enzyme digested with EcoRI / BamHI, and transformed into E. coli DH5a competent cells. The plasmid DNA was extracted and pTrc::AroG FBR The fragment is amplified and purified before proceeding to the next step. The fragment pTrc::AroG FBR Fusion to E. coli TP06-pKD46 competent cell to obtain E. coli engineered strain TP06-DHS01; Step 3, using the above-obtained engineered strain TP06-DHS01 as the starting strain, knocking out the negative regulatory transcription inhibitor tyrR to obtain the Escherichia coli engineered strain TP06-DHS02; Step 4: Using the engineered strain TP06-DHS02 obtained above as the starting strain, knocking out the shikimate dehydrogenase gene aroE to obtain the Escherichia coli engineered strain TP06-DHS03; Step 5, using the above-obtained engineered strain TP06-DHS03 as the starting strain, knocking out the transketolase gene tktA to obtain the Escherichia coli engineered strain TP06-DHS04; Step 6: Using the engineered strain TP06-DHS04 obtained above as the starting strain, the transaldolase gene talA was knocked out to obtain the Escherichia coli engineered strain TP06-DHS05.

3. Use of the recombinant strain according to claim 1 or the recombinant strain prepared by the method according to claim 2, characterized in that: The application method is to utilize the recombinant strain to produce 3-dehydroshikimic acid through fermentation.

4. The use according to claim 3, characterized in that: The fermentation was carried out by batch feeding.

5. A biological preparation, comprising the recombinant strain according to claim 1 or the recombinant strain prepared by the method according to claim 2; the biological preparation can produce 3-dehydroshikimic acid by fermentation.

Images