Recombinant Escherichia coli for efficient production of shikimic acid and its application

Abstract

The present invention discloses a recombinant Escherichia coli for efficiently producing shikimic acid and its application. The recombinant Escherichia coli of the present invention is named Escherichia coli FMME-SA07. The present invention first obtains a recombinant Escherichia coli that accumulates shikimic acid by knocking out the encoding genes aroK and aroL of shikimate kinases I and II. Subsequently, 10 key pathway targets for shikimic acid synthesis by Escherichia coli were screened based on the enzyme constraint model ec_iML1515. By transforming these targets, the Escherichia coli metabolic pathway was reconstructed to produce high shikimic acid. Finally, the shikimic acid production intensity was enhanced by pressure response regulation engineering; 126.4 g / L of shikimic acid was produced by fermentation for 48 hours at the fermentor level. The method for producing shikimic acid by fermentation of the present invention is simple and easy to operate, and the culture medium cost is low, and it is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli for efficiently producing shikimic acid and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Shikimic acid, also known as (3R,4S,5R)-(-)-3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid, is composed of a six-carbon cyclic alcohol and three chiral carbon and carboxylic acid functional groups. Its chemical formula is C7H 10 O5. This compound appears as a white powder and is readily soluble in water with a solubility of 180 g / L. It is poorly soluble in chloroform, benzene, and petroleum ether. Its melting point is 185°C to 191°C, with a strong absorption peak near 210 nm. Its optical rotation is -180°, and it has a pungent, sour odor.

[0003] Shikimic acid is a valuable compound and an important precursor in the pharmaceutical and chemical industries. In the pharmaceutical field, its most important value is its use in the synthesis of the anti-influenza drug Tamiflu, which played a significant role during outbreaks of avian and swine flu and possesses extremely high commercial value. Furthermore, shikimic acid can also be used in the chemical industry to synthesize various chiral compounds and aromatic small molecules. Currently, the annual global consumption of shikimic acid has reached 4,000 tons, with sales reaching US$40 million. Traditional methods for producing shikimic acid include plant extraction, chemical synthesis, and microbial fermentation. However, because plant extraction and chemical synthesis methods are subject to raw material limitations, serious pollution problems, and high costs, the unique advantages of microbial methods, such as low cost, environmental friendliness, and high-density fermentation, make them the preferred method for synthesizing shikimic acid.

[0004] Shikimic acid belongs to the shikimate pathway, which is widely present in plants, algae, bacteria, and eukaryotic cells. In Escherichia coli, the shikimate metabolic pathway begins with glucose uptake by the bacterium, followed by glucose production via the glycolysis (EMP) pathway and erythrose 4-phosphate (E4P) via the pentose phosphate pathway (HMP). These two condense to form the common aromatic precursor, 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP). This process is catalyzed by three DAHP synthase isozymes, encoded by aroG, aroF, or aroH, and is subject to feedback inhibition by aromatic amino acids. Shikimic acid is then synthesized using DAHP as a precursor through the sequential steps of 3-dehydroquinate synthase AroB, 3-dehydroquinate dehydratase AroD, and shikimate dehydrogenase AroE. Due to factors such as low metabolic flux in the shikimate pathway, unclear key rate-limiting targets, and product inhibition, the current production of shikimate in E. coli is characterized by low yield, efficiency, and robustness. Therefore, it is necessary to rationally screen and modify target sites to construct a high-shikimate-producing E. coli strain. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a recombinant Escherichia coli for producing shikimic acid. First, the genes encoding shikimate kinases I and II, aroK and aroL, were deleted to obtain a recombinant Escherichia coli that accumulates shikimic acid. Then, the enzyme constraint model ec_iML1515 was used to predict and verify 10 key targets for shikimic acid production: aroG, aroD, aroE, talB, tktA, glyA, ptsH, ptsI, dhaL, and pykF. Metabolic engineering of these targets increased the carbon flow metabolic flux of the shikimic acid pathway. Finally, the target ProV that tolerates high concentrations of shikimic acid was dynamically regulated to enhance the shikimic acid production intensity. Finally, a strain of Escherichia coli with high shikimic acid production was obtained.

[0006] The first object of the present invention is to provide a recombinant Escherichia coli for efficiently producing shikimic acid, wherein the recombinant Escherichia coli is deleted with the shikimate kinase I encoding gene aroK, the shikimate kinase II encoding gene aroL, the phosphate transporter encoding gene ptsH, the phosphoenolpyruvate protein phosphotransferase encoding gene ptsI, and the PEP-dependent dihydroxyacetone kinase encoding gene dhaL; and the growth-coupled promoter P is used to generate the recombinant Escherichia coli. rrnC Dynamically regulate the pyruvate kinase I encoding gene pykF and overexpress the feedback-resistant DAHP synthase encoding gene aroG FBR , aroD encoding gene for 3-dehydroquinate dehydratase, aroE encoding gene for shikimate dehydrogenase, talB encoding gene for transaldolase, tktA encoding gene for transketolase and glyA encoding gene for serine hydroxymethyltransferase, Zmglf encoding gene for glucose-stimulating protein and Zmglk encoding gene for glucokinase of Pseudomonas mobilis.

[0007] Furthermore, the NCBI number of the shikimate kinase I encoding gene aroK is YP_026215.2, the NCBI number of the shikimate kinase II encoding gene aroL is NP_414922.1, the NCBI number of the phosphate transporter encoding gene ptsH is NP_416910.1, the NCBI number of the phosphoenolpyruvate protein phosphotransferase encoding gene ptsI is NP_416911.1, and the NCBI number of the PEP-dependent dihydroxyacetone kinase encoding gene dhaL is NP_415717.1.

[0008] Furthermore, the NCBI number of the pyruvate kinase I encoding gene pykF is NP_416191.1, the NCBI number of the 3-dehydroquinate dehydratase encoding gene aroD is NP_416208.1, the NCBI number of the shikimate dehydrogenase encoding gene aroE is NP_417740.1, the NCBI number of the transaldolase encoding gene talB is NP_414549.1, the NCBI number of the transketolase encoding gene tktA is YP_026188.1, the NCBI number of the serine hydroxymethyltransferase encoding gene glyA is NP_417046.1; the DAHP synthase encoding gene aroG FBR The nucleotide sequence of the gene encoding the glucose-promoting protein of Pseudomonas mobilis is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the glucose-promoting protein of Pseudomonas mobilis Zmglf is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the glucose kinase Zmglk is shown in SEQ ID NO.3.

[0009] Furthermore, overexpression of aroG FBR , aroD and aroE genes were expressed using the strong promoter P J23119 Control aroG separately FBR , aroD and aroE genes were expressed, and overexpression of talB was achieved by using a strong promoter P J23101 Controlling talB expression, overexpression of tktA was achieved by using a strong promoter P J23108 Control of tktA expression and overexpression of glyA were achieved by using the strong promoter P tac Control of glyA expression.

[0010] Furthermore, the recombinant Escherichia coli also includes over-expression of the shikimic acid tolerance gene proV.

[0011] Furthermore, the overexpression of the shikimate tolerance gene proV is achieved by using a stress response promoter P rpoS Regulates the expression of the shikimate tolerance gene proV.

[0012] Furthermore, the NCBI number of the shikimic acid tolerance gene proV is NP_417163.1.

[0013] The second object of the present invention is to provide the use of the recombinant Escherichia coli in the fermentation production of shikimic acid.

[0014] Furthermore, the application is to use the recombinant Escherichia coli to carry out full aerobic fermentation in a fermentation medium to obtain a fermentation liquid containing shikimic acid.

[0015] Furthermore, during the fully aerobic fermentation process, the dissolved oxygen is controlled at above 10%.

[0016] Furthermore, during the fully aerobic fermentation process, when the glucose in the initial culture medium is exhausted, 700-900 g / L of glucose is added, and the glucose concentration is controlled at 8-12 g / L.

[0017] Furthermore, during the fully aerobic fermentation process, the pH is controlled at 6.5-6.8, and the fermentation temperature is controlled at 36-38°C.

[0018] Furthermore, the fermentation medium formula is: glucose 18-22 g / L, yeast powder 8-12 g / L, peptone 4-6 g / L, ferric citrate ammonium 1.4-1.6 g / L, dipotassium hydrogen phosphate 4-6 g / L, magnesium sulfate heptahydrate 0.8-1.2 g / L, tryptophan 0.08-0.12 g / L, tyrosine 0.08-0.12 g / L, phenylalanine 0.08-0.12 g / L, and metal ion solution 0.8-1.2 mL / L.

[0019] The beneficial effects of the present invention are:

[0020] The Escherichia coli FMME-SA07 strain of the present invention can withstand the osmotic stress of high-concentration shikimic acid and has good genetic stability. It can stably produce approximately 120 g / L of shikimic acid even after 12 generations of continuous passage. In a fermenter, the yield of L-tryptophan can reach up to 126.4 g / L. The fermentation process for producing shikimic acid is simple and easy to operate, and the culture medium cost is low, making it suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To verify the ability of screening targets to produce shikimic acid based on enzyme constraint model;

[0022] Figure 2 A pathway map for transforming E. coli to produce shikimic acid based on screening targets;

[0023] Figure 3 This is a diagram for functional validation of the high-concentration shikimic acid stress-responsive promoter;

[0024] Figure 4 Schematic diagram of the effect of ProV overexpression on shikimic acid tolerance in recombinant Escherichia coli;

[0025] Figure 5 OD of E. coli during horizontal fermentation in a fermenter 600 and shikimic acid production change curve. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0027] Materials and methods involved:

[0028] (1) Culture medium:

[0029] Solid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L.

[0030] Seed culture medium: disodium hydrogen phosphate 6.78 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium chloride 0.011 g / L, glucose 4 g / L.

[0031] Fermentation medium: glucose 20 g / L, yeast powder 10 g / L, peptone 5 g / L, ferric citrate ammonium 1.5 g / L, dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 1 g / L, tryptophan 0.1 g / L, tyrosine 0.1 g / L, phenylalanine 0.1 g / L, metal ion solution 1 mL / L.

[0032] (2) Determination of glucose:

[0033] Fermentation broth pretreatment: Centrifuge the fermentation broth at 12,000 rpm for 5 minutes, collect the supernatant, dilute to an appropriate dilution, and measure the glucose concentration of the fermentation broth using an M-100 biosensor analyzer.

[0034] (3) Determination of shikimic acid:

[0035] High-performance liquid chromatography method: Prepare a 1g / L shikimic acid solution and dilute it to 0.1, 0.2, 0.3, 0.4, and 0.5g / L. Use a high-performance liquid chromatograph (HPLC) for detection to obtain the peak time and peak area corresponding to different concentrations of shikimic acid. Use the concentration of the shikimic acid solution as the horizontal axis and the peak area as the vertical axis to draw a standard curve to obtain a linear regression equation. The regression coefficient of the linear regression equation should be above 0.990 before it can be used. The instrument is an Agilent high-performance liquid chromatograph, and the chromatographic column is an Aminex HPX-87H column; the mobile phase is 5mM dilute sulfuric acid; the flow rate is set to 0.6mL / min; the detector is a UV detector with a detection wavelength of 210nm and a column temperature of 55°C.

[0036] Fermentation broth pretreatment: Centrifuge the fermentation broth at 12,000 rpm for 10 minutes and collect the supernatant. After dilution to an appropriate multiple, filter the sample and analyze it using HPLC. Substitute the peak area into the linear regression equation and multiply the result by the dilution multiple to obtain the L-tryptophan concentration in the fermentation broth.

[0037] Example 1: Construction of a shikimic acid production chassis in Escherichia coli

[0038] In Escherichia coli, shikimate is an upstream product of tryptophan. E. coli W3110 was selected as the host for this research. By knocking out the genes encoding shikimate kinases I and II, aroK and aroL, shikimate accumulation was achieved. In shake flask fermentation, recombinant E. coli SA01 produced 0.48 g / L of shikimate.

[0039] In this embodiment, Escherichia coli W3110 was used as the chassis host, and the following specific embodiments were carried out. However, those skilled in the art can infer that this metabolic pathway exists in conventional Escherichia coli, and the transformation according to the present invention can achieve the purpose of the present invention. The host Escherichia coli W3110 cannot limit the scope of protection of the present invention.

[0040] Example 2: Prediction of key targets for shikimic acid synthesis based on the enzyme constraint model ec_iML1515

[0041] (1) Protein requirement analysis and calculation

[0042] Based on the enzyme-constrained model ec_iML1515, previously developed in our laboratory, we calculated the protein requirements for shikimate synthesis, using shikimate as the target product and glucose as the starting carbon source. Twelve potential targets for efficient shikimate synthesis were identified: increasing the expression of seven proteins (GlyA, TalB, TktA, AroG, AroD, AroE, and PfkB) and downregulating the expression of five proteins (PtsH, PtsI, PykF, DhaL, and TpiA) enhanced shikimate synthesis. Based on the metabolic pathway of Escherichia coli, the functional analysis of the above 12 proteins can be divided into four modules: (1) the core pathway module of shikimic acid synthesis: AroG, AroD and AroE; (2) the precursor PEP accumulation module: PtsH, PtsI, PfkB, PykF, DhaL and TpiA; (3) the precursor E4P synthesis module: TktA and TalB; (4) the resource replenishment module: the highest demand protein GlyA, which is a key factor involved in the synthesis of various basic cellular resources such as purine, thymine, methionine, choline, and lipids.

[0043] (2) Verification of predicted target function

[0044] The recombinant E. coli SA01 from Example 1 was used for target function verification. Overexpression of glyA, talB, aroG, aroD, aroE, and tktA increased shikimate production by 87.5%, 57.8%, 112.5%, 39.3%, 48.5%, and 45.2% compared to the original strain, reaching 1, 0.76, 1.02, 0.67, 0.71, and 0.7 g / L, respectively. Deletion of ptsH, ptsI, pykF, and dhaL in SA01 increased shikimate production by 32.5%, 38.2%, 17.5%, and 19.3%, reaching 0.63, 0.66, 0.56, and 0.57 g / L, respectively. Overexpression of pfkB had no significant effect on shikimate production, while deletion of tpiA decreased shikimate production by 28.7% (0.35 g / L). Therefore, we selected glyA, talB, aroG, aroD, aroE, tktA, dhaL, ptsH, ptsI, and pykF as targets for the next step of strain modification. The nucleotide sequences of the modified targets have been published in NCBI: aroK (YP_026215.2), aroL (NP_414922.1), ptsH (NP_416910.1), ptsI (NP_416911.1), dhaL (NP_415717.1), pykF (NP_416191.1), aroD (NP_416208.1), aroE (NP_417740.1), talB (NP_414549.1), tktA (YP_026188.1), glyA (NP_417046.1); aroG FBR The nucleotide sequences of Zmglf and Zmglk are shown in SEQ ID NOs. 1 to 3, respectively.

[0045] Example 3: Bacterial strain transformation

[0046] The recombinant E. coli SA01 strain in Example 1 was selected for transformation. The target sites screened in Example 2 were subjected to rational metabolic engineering transformation to obtain a recombinant E. coli strain that produces high shikimic acid.

[0047] (1) Increase the accumulation of precursors PEP and E4P

[0048] To increase the content of precursor PEP, the genes dhaL, ptsH and ptsI were knocked out, and the glucose-promoting protein gene Zmglf and glucose kinase Zmglk were introduced to avoid the reduction of glucose uptake; on this basis, the pykF native promoter was replaced with the growth-coupled promoter P rrnC, reducing the transcriptional activity of the pykF gene in stable cells, allowing more metabolic flux to enter the shikimate pathway, and obtaining recombinant E. coli SA02. To increase the precursor E4P, tktA and talB need to be overexpressed. Because the model prediction showed a high demand for talB, we introduced a strong promoter P into the genome. J23101 Controlled by talB and the middle promoter P J23108 The recombinant E. coli SA03 was obtained by controlling tktA.

[0049] (2) Strengthening core pathways and resource-supplemented gene expression

[0050] Using a strong promoter P J23119 Control aroG separately FBR , aroD and aroE genes were expressed to strengthen the expression of the shikimate core pathway genes, allowing more metabolic flows to enter the shikimate pathway, and recombinant Escherichia coli SA04 was obtained. tac The promoter controlled the expression of glyA, which synthesized some basic resources to accelerate the synthesis of shikimic acid. Finally, the recombinant Escherichia coli SA05 was obtained.

[0051] The constructed strains are shown in Table 1 below:

[0052] Table 1

[0053] Strain name Features E.coli SA01 E.coli W3110,ΔaroK,ΔaroL E.coli SA02 <![CDATA[E.coli SA01,ΔdhaL,ΔptsH::P trc -glf,ΔptsI::P trc -glk,ΔpykF]]> E.coli SA03 <![CDATA[E.coli SA02,ΔaroK::P J23101 -ofB-P J23108 -tktA]]> E.coli SA04 <![CDATA[E.coli SA03,ΔaroL::P J23119 -aroG FBR -aroD-aroE]]> E.coli SA05 <![CDATA[E.coli SA04,ΔdhaL::P trc -glyA]]> E.coli SA06 <![CDATA[E.coli SA05,ΔykgH::P J23119 -proV]]> E.coli SA07 <![CDATA[E.coli SA05,ΔykgH::P rpoS -proV]]>

[0054] The promoter sequences used above are shown in Table 2:

[0055] Table 2

[0056]

[0057] Example 4: Screening of shikimate-tolerant targets and responsive promoters

[0058] In order to enhance the production intensity of shikimic acid, we are committed to enhancing the tolerance of recombinant Escherichia coli SA05 to high-concentration shikimic acid stress. Using transcriptomics sequencing and flow cytometry to detect cell survival, we screened a target ProV that helps to enhance the shikimic acid tolerance of Escherichia coli. At a concentration of 90g / L shikimic acid, overexpression of proV can increase the survival rate of Escherichia coli from 54% to 87%, while increasing the shikimic acid production by 17.4%. However, overexpression of proV will extend the fermentation time by nearly 8h. To avoid inhibition caused by this metabolic burden, we evaluated the ability of the promoter of the global stress regulator RpoS to respond to shikimic acid stress and found that P rpoS It has significant shikimate response ability. rpoSUnder the control of the promoter, production delays caused by metabolic burden were successfully avoided. The strain was named Escherichia coli FMME-SA07. The sequence of the RpoS promoter is shown in Table 3 below.

[0059] Table 3

[0060]

[0061] Example 5: Shake flask fermentation of recombinant Escherichia coli

[0062] (1) Seed activation and cultivation

[0063] Solid culture medium configuration: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L;

[0064] Solid plate activation: inoculate a loopful of bacterial solution from the storage tube onto a solid plate and incubate at 37°C for 16 hours;

[0065] Shake flask fermentation seed culture medium configuration: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L;

[0066] Seed culture of shake flask fermentation: Pick a well-grown single colony from the plate and inoculate it into a 250mL Erlenmeyer flask filled with 30mL of culture medium. Incubate at 37℃ with a reciprocating shaker at 200rpm / min for about 12 hours until the seed solution is diluted 10 times and the OD 660 The value is between 0.6-0.8.

[0067] (2) Shake flask culture

[0068] The recombinant strain was fermented in a 500 mL Erlenmeyer flask containing 50 mL of shake flask medium. The specific fermentation conditions were as follows:

[0069] Shake flask fermentation medium: glucose 10 g / L, yeast powder 5 g / L, peptone 5 g / L, ferric citrate ammonium 1.5 g / L, dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 1 g / L, tryptophan 0.1 g / L, tyrosine 0.1 g / L, phenylalanine 0.1 g / L, metal ion solution 1 mL / L, pH adjusted to 6.6 with ammonia water.

[0070] Sterilize at 121°C for 15 minutes. Cool to room temperature and prepare for inoculation in a clean bench.

[0071] Inoculation amount: inoculate the prepared shake flask seed solution into the shake flask medium at an inoculation amount of 5%;

[0072] Fermentation temperature and speed: 37°C, speed 200 rpm / min;

[0073] Fermentation pH: Use phenol red as an indicator and use ammonia water to adjust the pH to between 6.6 and 6.7;

[0074] During the fermentation process, the residual sugar was controlled below 10 g / L and the total fermentation time was 72 hours.

[0075] The shikimic acid production indicators during shake flask fermentation of recombinant E. coli are shown in Table 4 below.

[0076] Table 4

[0077] strain Shake flask yield (g / L) E. coli FMME-SA01 0.48 E. coli FMME-SA02 1.95 E. coli FMME-SA03 3.42 E. coli FMME-SA04 5.05 E. coli FMME-SA05 5.73 E. coli FMME-SA06 6.53 E. coli FMME-SA07 6.81

[0078] Example 6: Batch fermentation of recombinant Escherichia coli FMME-SA07

[0079] (1) Seed activation and cultivation

[0080] Solid culture medium configuration: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L;

[0081] Primary seed culture medium configuration: disodium hydrogen phosphate 6.78 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium chloride 0.011 g / L, glucose 4 g / L, tryptophan 10 mg / L, tyrosine 10 mg / L, phenylalanine 10 mg / L;

[0082] Slant activation: inoculate a loopful of strain from the storage tube onto the slant medium, keep the temperature at 36°C, and incubate for 12 hours;

[0083] Seed culture: Pick a well-grown single colony from the plate and inoculate it into a 500mL Erlenmeyer flask containing 50mL of culture medium. Incubate at 37℃ with a reciprocating shaker at 200rpm / min for about 12 hours until the seed solution is diluted 10 times and the OD 660 The value is between 0.6-0.8.

[0084] (2) Fermentation culture

[0085] The recombinant strain was fermented in a 5 L fermenter. The specific fermentation conditions were as follows:

[0086] Fermentation medium: glucose 20 g / L, yeast powder 10 g / L, peptone 5 g / L, ferric citrate ammonium 1.5 g / L, dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 1 g / L, tryptophan 0.1 g / L, tyrosine 0.1 g / L, phenylalanine 0.1 g / L, metal ion solution 1 mL / L.

[0087] Sterilize at 121°C for 15 minutes. After sterilization, install it on the control console and turn on the temperature control. After the temperature cools to 37°C, add ammonia water to adjust the pH to 6.6 and prepare for inoculation.

[0088] Inoculation amount: inoculate the prepared seed solution into the fermentation tank at an inoculation amount of 10%;

[0089] Fermentation temperature: During the fermentation process, turn on the temperature control to maintain the fermentation temperature at 37°C;

[0090] Fermentation pH: Use ammonia to adjust the pH to between 6.6 and 6.7;

[0091] Dissolved oxygen conditions: Initial conditions: ventilation volume 2 vvm, rotation speed 500 rpm;

[0092] During the fermentation process, the dissolved oxygen content is maintained above 10% by adjusting the ventilation volume and the rotation speed of the stirring blade.

[0093] During the fermentation process, after the initial glucose in the fermentation medium was completely consumed, 800 g / L of glucose solution was added in a pulsed manner to control the glucose concentration below 10 g / L.

[0094] The production indicators of shikimic acid produced by recombinant Escherichia coli SA07 are shown in Table 5 below.

[0095] Table 5

[0096] Production indicators Production data Yield ≥120g / L Yield 0.38-0.40g / L glucose Production intensity ≥2.6 g / L / h

[0097] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A recombinant Escherichia coli for efficiently producing shikimic acid, characterized in that: In the recombinant E. coli, the shikimate kinase I encoding gene was deleted. aroK , shikimate kinase II encoding gene aroL , phosphate transporter encoding genes ptsH , phosphoenolpyruvate protein phosphotransferase encoding gene ptsI and PEP-dependent dihydroxyacetone kinase encoding genes dhaL ; Using growth-coupled promoter P rrnC Dynamic regulation of the gene encoding pyruvate kinase I pykF ; and overexpressed the gene encoding the feedback-resistant DAHP synthase aroG FBR , 3-dehydroquinate dehydratase encoding gene aroD , shikimate dehydrogenase encoding gene aroE , transaldolase encoding gene talB , transketolase encoding gene tktA , serine hydroxymethyltransferase encoding gene glyA , Pseudomonas mobilis glucose-stimulating protein encoding gene Zmglf and glucokinase encoding genes Zmglk ; Among them, the gene encoding shikimate kinase I aroK The NCBI accession number is YP_026215.2, and the gene encoding shikimate kinase II aroL The NCBI accession number is NP_414922.1, and the gene encoding the phosphate transporter ptsH The NCBI accession number is NP_416910.1, the gene encoding phosphoenolpyruvate protein phosphotransferase ptsI The NCBI number is NP_416911.1, and the PEP-dependent dihydroxyacetone kinase encoding gene dhaL The NCBI accession number is NP_415717.1, and the gene encoding pyruvate kinase I pykF The NCBI number is NP_416191.1, the gene encoding 3-dehydroquinate dehydratase aroD The NCBI number is NP_416208.1, the shikimate dehydrogenase encoding gene aroE The NCBI accession number is NP_417740.1, the transaldolase encoding gene talB The NCBI number is NP_414549.1, the transketolase encoding gene tktA The NCBI number is YP_026188.1, the gene encoding serine hydroxymethyltransferase glyA The NCBI accession number is NP_417046.1; the DAHP synthase encoding gene aroG FBR The nucleotide sequence of which is shown in SEQ ID NO.1, the gene encoding the glucose-promoting protein of Pseudomonas mobilis Zmglf The nucleotide sequence of the glucokinase encoding gene is shown in SEQ ID NO.

2. Zmglk The nucleotide sequence is shown in SEQ ID NO.

3.

2. The recombinant Escherichia coli according to claim 1, characterized in that Overexpression of the DAHP synthase encoding gene aroG FBR , 3-dehydroquinate dehydratase encoding gene aroD and shikimate dehydrogenase encoding genes aroE By using the strong promoter P J23119 Control expression and overexpress transaldolase encoding gene talB By using a strong promoter P J23101 Controlled expression, overexpression of transketolase encoding gene tktA By using a strong promoter P J23108 Control expression and overexpression of serine hydroxymethyltransferase encoding gene glyA By using a strong promoter P tac Control expression.

3. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli also includes an overexpression of a shikimic acid tolerance gene proV , the shikimate tolerance gene proV The NCBI accession number is NP_417163.

1.

4. The recombinant Escherichia coli according to claim 3, characterized in that The overexpressed shikimate tolerance gene proV By using the stress-responsive promoter P rpoS Regulate expression.

5. Use of the recombinant Escherichia coli according to any one of claims 1 to 4 in the fermentation production of shikimic acid, characterized in that: The application is to use the recombinant Escherichia coli to carry out full aerobic fermentation in a fermentation medium to obtain a fermentation liquid containing shikimic acid.

6. The use according to claim 5, characterized in that During the fully aerobic fermentation process, when the glucose in the initial culture medium is exhausted, 700-900 g / L of glucose is fed, and the glucose concentration is controlled at 8-12 g / L.

7. The use according to claim 5, characterized in that During the fully aerobic fermentation process, the pH is controlled at 6.5-6.8, and the fermentation temperature is controlled at 36-38°C.

8. The use according to claim 5, characterized in that The fermentation medium formula is: 18-22 g / L glucose, 8-12 g / L yeast powder, 4-6 g / L peptone, 1.4-1.6 g / L ferric citrate ammonium, 4-6 g / L dipotassium hydrogen phosphate, 0.8-1.2 g / L magnesium sulfate heptahydrate, 0.08-0.12 g / L tryptophan, 0.08-0.12 g / L tyrosine, 0.08-0.12 g / L phenylalanine, and 0.8-1.2 mL / L metal ion solution.

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