CN117683759A - 3-dehydroshikimate dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid - Google Patents

3-dehydroshikimate dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid Download PDF

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CN117683759A
CN117683759A CN202311752427.2A CN202311752427A CN117683759A CN 117683759 A CN117683759 A CN 117683759A CN 202311752427 A CN202311752427 A CN 202311752427A CN 117683759 A CN117683759 A CN 117683759A
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escherichia coli
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mutant
protocatechuic acid
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刘立明
王明
高聪
陈修来
胡贵鹏
刘佳
魏婉清
宋伟
吴静
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Jiangnan University
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Abstract

The invention discloses a 3-dehydroshikimic acid dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid, and the mutant ApAroz R363A The amino acid sequence of (2) is shown as SEQ ID NO.3, and is obtained by mutating the 363 rd arginine of the starting sequence into alanine; the invention also constructs a recombinant escherichia coli based on the mutant, and specifically: knocking out 3-dehydroshikimic acid in E.coli by blocking the carbon flow from the 3-dehydroshikimic acid flow to downstream shikimic acidThe shikimate dehydrogenase gene aroE successfully constructs a chassis strain with high yield of 3-dehydroshikimate, and introduces a high-activity 3-dehydroshikimate dehydratase mutant ApAroz with resistance to product inhibition R363A And constructing a protocatechuic acid biosynthesis path in the chassis strain, and finally constructing an escherichia coli recombinant strain for efficiently biosynthesis of protocatechuic acid.

Description

3-dehydroshikimate dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a 3-dehydroshikimate dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid.
Background
Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural phenolic acid that is widely found in plants. Researches show that the compound has biological activities of antioxidation, anti-inflammation, antibiosis, antivirus, anti-tumor and the like, and can also be used as a material monomer for synthesizing high-performance polymers and food packaging materials, so that the compound is widely applied to pharmaceutical, health-care food, cosmetics and novel material industries. Currently, PCA production mainly involves plant extraction and chemical synthesis. A common PCA production method is extraction from plants, but its industrial production is limited due to low yield (e.g., 10.81. Mu.g/g extracted from Gordonia axillaris fruits). Chemical PCA synthesis by means of vanillic acid demethylation has several drawbacks, such as the use of environmental contaminants (hydrochloric acid) and the need for high temperature conditions (250 ℃). In this case, the microbial fermentation to produce PCA represents a green, more efficient, sustainable alternative to obtaining this compound, and is therefore of increasing interest to researchers, with a broad market prospect.
The biosynthesis of 3-dehydroshikimate in E.coli starts with glucose and two intermediates phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) are metabolized by the central carbon and converted to 3-dehydroshikimate by 3-deoxy-D-arabinoheptulo-7-phosphate synthase (DAHP), DHQ synthase (aroB) and DHQ dehydratase (aroD). Subsequently, 3-dehydroshikimic acid is catalyzed by heterologous 3-dehydroshikimic acid dehydratase to protocatechuic acid. To date, many groups at home and abroad have engineered different microbial host strains, including E.coli, corynebacterium glutamicum, and Pseudomonas putida, for the production of PCA. However, complicated fermentation processes and low yields limit the commercial production of protocatechuic acid. The escherichia coli has the characteristics of multiple genetic tools, clear genetic information, rapid growth and high carbon absorptivity, is an ideal host for producing protocatechuic acid, but the high-efficiency production of protocatechuic acid in the escherichia coli is limited by the reduction of the 3-dehydroshikimase activity of the PCA product inhibition and the toxicity of the product to cells.
Disclosure of Invention
In order to solve the problems, the invention provides a 3-dehydroshikimic acid dehydratase mutant and an escherichia coli recombinant strain for efficiently biosynthesizing protocatechuic acid, which are based on high-throughput screening work of mutation sites, so as to obtain a mutant for removing the product inhibition of protocatechuic acid to ApAroZ, and the recombinant escherichia coli containing the mutant is subjected to metabolic modification, so that the escherichia coli with high yield of protocatechuic acid is finally obtained, and the fed-batch yield of a 5L fermentation tank reaches 37.02g/L.
The first object of the invention is to provide a 3-dehydroshikimate dehydratase mutant, the amino acid sequence of which is shown in SEQ ID NO.3, and which is obtained by mutating arginine at 363 rd position of the starting sequence into alanine. Specifically, the sequence is as follows:
MNILRLTTLALGLMVSGIAAAQTYVVDRYQDDSNKGSLRWAIEQANANPGEASDILIQAVGKAPYAIKLNSALPEIKAPVKIIGTQWDKTGEYIAIDGSNYIKGEGAKACPGANPEQYGTNVRTMTLPGLVLRDVNNVTLKGLDIHRFCIGVLINRSSNNLIQHNRISNNYGGAGVMLTGDDGQGNPTATTTNNNKVLDNIFQDNGDGLELTRGAAFNLIANNHFVSTKANPEPSQGIEILWGNDNAVVGNKFENYSDGLQINWGKRNYIAYNEMTNNSIGFNMTGDGNILDSNKVHGNRIGVAIRSEKDANAKITLTKNLIWDNGKDIKRCEAGGSCVPDQRLGAIVFAVPALEHAGFVGSAGGGVIVDPSKQQKTCTQPNEQGCNAQPNQGIKAPKLTANKGLVAVEVNGLPNQRYQVEFFSNQNAASKEAEQYLGAITVATDAQGIAKANWKPSVKVASITANITDRFGATSELSSALQTK。
a second object of the present invention is to provide a nucleic acid encoding the above 3-dehydroshikimate dehydratase mutant.
Further, the nucleotide sequence is shown as SEQ ID NO. 1.
It is a third object of the present invention to provide a vector carrying the above nucleic acid.
A fourth object of the present invention is to provide cells expressing the above 3-dehydroshikimate dehydratase mutants.
Further, the cell is a bacterial, fungal, plant cell or animal cell.
A fifth object of the invention is to provide the use of the above-mentioned 3-dehydroshikimate dehydratase mutants, nucleic acids, vectors, cells for the preparation of a protocatechuic acid containing product.
The sixth object of the present invention is to provide a recombinant escherichia coli producing protocatechuic acid, which is obtained by modifying escherichia coli as a host, wherein the modifying comprises: over-expressing a gene encoding the above 3-dehydroshikimate dehydratase mutant.
Further, a strong promoter P is used J23119 Controlling the expression of the 3-dehydroshikimate dehydratase gene.
Further, ligating a pro-lytic tag that includes, but is not limited to, SUMO (SEQ ID NO. 2), in one embodiment of the invention by a ligating peptide (GGGGS), enhances soluble expression of 3-dehydroshikimate dehydratase 3 SUMO and ApAroz R363A And (5) connection.
Further, the retrofitting also includes any of the following: knocking out shikimate kinase I encoding gene aroK, shikimate kinase II encoding gene aroL, phosphotransporter encoding gene ptsH, phosphoenolpyruvate phosphotransferase encoding gene ptsI, PEP-dependent dihydroxyacetone kinase encoding gene dhaL and shikimate dehydrogenase encoding gene aroE; using growth-coupled promoter P rrnC Dynamically regulating and controlling a pyruvic acid kinase I coding gene pykF; and overexpress the gene aroG encoding the anti-feedback inhibition DAHP synthase FBR 3-take-offThe coding genes aroD, the transaldolase coding gene talB, the transketolase coding gene tktA, the serine hydroxymethyltransferase coding gene glyA, the Pseudomonas mobaraensis glucose-promoting protein coding gene Zmglf, the glucokinase coding gene Zmglk and the shikimate tolerance gene proV of the hydroquinic acid dehydratase.
Further, the aroG is overexpressed FBR And aroD Gene by using a strong promoter P J23119 Separately controlling aroG FBR Expression of aroD Gene, overexpression of the talB Gene was achieved by the use of the strong promoter P J23101 Controlling the expression of talB and over-expressing the tktA gene by using a strong promoter P J23108 Controlling tktA expression, over-expressing glyA gene by using strong promoter P tac Controlling glyA expression, over-expressing the proV gene by using the pressure responsive promoter P rpoS Regulating and controlling expression of shikimic acid tolerance gene proV.
Further, NCBI coding for shikimate kinase I gene aroK is YP_026215.2, NCBI coding for shikimate kinase II gene aroL is NP_414922.1, NCBI coding for phosphotransporter gene ptsH is NP_416910.1, NCBI coding for phosphoenolpyruvate phosphotransferase gene ptsI is NP_416911.1, NCBI coding for PEP-dependent dihydroxyacetone kinase gene dhaL is NP_415717.1, and NCBI coding for shikimate dehydrogenase gene aroE is NP_417740.1.
Further, the method comprises the steps of, NCBI number NP_416191.1 for the pyruvic acid kinase I encoding gene pykF, NCBI number NP_416208.1 for the 3-dehydroquinic acid dehydratase encoding gene aroD, NCBI number NP_414549.1 for the transaldolase encoding gene talB, NCBI number YP_026188.1 for the transketolase encoding gene tkTA, NCBI number NP_417046.1 for the serine hydroxymethyltransferase encoding gene glyA, NCBI number NP_417163.1 for the shikimate tolerance gene proV; DAHP synthetase encoding gene aroG FBR The nucleotide sequence of the gene Zmglf is shown as SEQ ID NO.4, the nucleotide sequence of the gene Zmglf is shown as SEQ ID NO.5, and the nucleotide sequence of the gene Zmglk is shown as SEQ ID NO. 6.
Further, growth-coupled promoter P rrnC The nucleotide sequence of (B) is shown as SEQ ID NO.7, and the strong promoter P J23119 The nucleotide sequence of (C) is shown as SEQ ID NO.8, and the strong promoter P J23101 The nucleotide sequence of (B) is shown as SEQ ID NO.9, and the strong promoter P J23108 The nucleotide sequence of (B) is shown as SEQ ID NO.10, and the pressure response promoter P rpoS The nucleotide sequence of (2) is shown as SEQ ID NO. 11.
A seventh object of the present invention is to provide the use of the recombinant E.coli described above for the preparation of a protocatechuic acid-containing product.
Further, the application is that the recombinant escherichia coli is adopted to ferment in a fermentation medium to obtain fermentation liquor containing protocatechuic acid.
Further, in the fermentation process, when the glucose in the initial culture medium is exhausted, 500-800 g/L glucose is fed in, and the glucose concentration is controlled below 5 g/L.
Further, in the fermentation process, the pH is controlled to be 6.5-7.1, the fermentation temperature is controlled to be 36-38 ℃, and the dissolved oxygen is controlled to be 10% -30%.
Further, the formula of the fermentation medium is as follows: 18-22 g/L of glucose, 8-12 g/L of yeast powder, 4-6 g/L of peptone, 1.3-1.7 g/L of ferric citrate amine, 4-6 g/L of dipotassium hydrogen phosphate, 0.8-1.2 g/L of magnesium sulfate heptahydrate, 0.08-0.12 g/L of tryptophan, 0.08-0.12 g/L of tyrosine, 0.08-0.12 g/L of phenylalanine and 0.8-1.2 mL/L of metal ion solution.
The invention has the beneficial effects that:
the invention discovers a mutant which can effectively relieve the product inhibition of protocatechuic acid on ApAroz by carrying out mutation site screening on 3-dehydroshikimate dehydratase, and constructs Escherichia coli FMME-PCA01 of high-yield protocatechuic acid based on the mutant, wherein the 3-dehydroshikimate dehydrogenase gene aroE in escherichia coli is knocked out, and the high-activity 3-dehydroshikimate dehydratase mutant ApAroz with resistance to product inhibition is overexpressed R363A . The recombinant escherichia coli has better genetic stability, can be continuously passed for 12 generations, and can also stably produce about 37.02g/L protocatechuic acid, and the invention is used for fermentation productionThe protocatechuic acid has simple and easy process operation and low cost of culture medium, and is suitable for industrial production.
Drawings
FIG. 1 is a MD simulation graph of a complex of ApAroz-PCA.
FIG. 2 is a MD simulation of alanine mutations at various residues.
FIG. 3 is a diagram showing the production of protocatechuic acid by modifying E.coli based on the target point.
FIG. 4 is a mutant ApAroz overexpressing 3-dehydroshikimate dehydratase R363A Gene plasmid pEM-SUMO-ApAroz R363A A schematic structural diagram.
FIG. 5 is a fermenter fed-batch fermentation result for the construction of engineering strain E.coli FMME-PCA 01.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Sequence information related to the present invention:
SEQ ID NO.1 is 3-dehydroshikimate dehydratase mutant ApAroz R363A Nucleotide sequence of the gene; SEQ ID NO.2 is the nucleotide sequence of the dissolution promoting tag SUMO; SEQ ID NO.3 ApAroz R363A Is a sequence of amino acids of (a).
The invention relates to materials and methods:
(1) 1mL/L of metal ion liquid
Each 1L of culture medium contains 1mL of microelement liquid; microelement liquid: feCl 2 ·6H 2 O 2.4g/L、CoCl 2 ·6H 2 O 0.3g/L、CuCl 2 0.15g/L、ZnCl 2 ·4H 2 O 0.3g/L、NaMnO 4 0.3g/L、H 3 BO 3 0.075g/L、MnCl 2 ·4H 2 O0.495 g/L, dissolved in 0.1M HCl.
(2) Determination of glucose:
pretreatment of fermentation liquor: taking fermentation liquor 12000r/min, centrifuging for 5min, and taking supernatant. Diluting to a proper multiple, and detecting the glucose concentration of the fermentation broth by using an M-100 biosensing analyzer.
(3) Measurement of protocatechuic acid:
high performance liquid chromatography: preparing 1g/L concentration protocatechuic acid solution, diluting to 0.1, 0.2, 0.3, 0.4 and 0.5g/L, detecting by using a High Performance Liquid Chromatograph (HPLC) to obtain peak time and peak areas corresponding to different concentrations of protocatechuic acid, drawing a standard curve by taking the concentration of the protocatechuic acid solution as an abscissa and the peak areas as an ordinate, and obtaining a linear regression equation. The regression coefficient of the linear regression equation should be above 0.990. The instrument is a waters high performance liquid chromatograph, and the chromatographic column adopts a ZORBAX SB-C18 liquid chromatographic column; the mobile phase is 20% methanol+0.07% perchloric acid; the flow rate was set at 1mL/min; the detector is an ultraviolet detector, the detection wavelength is 278nm, and the column temperature is 40 ℃.
Pretreatment of fermentation liquor: taking fermentation liquor 12000r/min, centrifuging for 10min, and taking supernatant. After dilution by a proper multiple, the sample is subjected to membrane treatment, HPLC is used for detection, the obtained peak area is substituted into a linear regression equation, and the obtained result is multiplied by the dilution multiple to obtain the protocatechuic acid concentration in the fermentation broth.
(4) Enzyme activity determination:
activity of AroZ and its mutants were tested using pure enzyme: 200. Mu.L of the reaction system contained 0.1. Mu.M enzyme, 20mM DHS, 75mM HEPES buffer (pH 7.5). The reaction was quenched with 20% methanol containing 0.07% perchloric acid. After 1min of reaction at 37 ℃, the initial conversion rate of PCA was determined by HPLC to calculate the enzyme activity. The activity of 1 unit is defined as the amount of enzyme required to convert to 1. Mu.M PCA per minute. 200. Mu.L reactions were characterized by kinetic parameters using 75mM Hepes (pH 7.5), 0.1. Mu.M enzyme and different concentrations of 3-DHS (0-40 mM). All experiments were performed in 3 replicates.
Example 1: screening of mutation sites
By MD simulation of the complex of ApAroz-PCA, it was found that the conformation of a small peptide chain (amino acid residues 360 to 370) in the loop L5 of the longest ApAroz was significantly changed, resulting in a closed conformation that almost completely blocked the catalytic pocket (FIG. 1). Since there is no significant change in the conformation of the region where L2 interacts with L5 in MD simulation, we next primarily modulate the conformational dynamics of L5 to increase the open flexibility of L5. Based on analysis of the structure of L5, candidate mutant residues selected include S362, R363, V367, I368, V369, D370 in the region of conformational change, and the surrounding bulky amino acids L354, E355, K373, and Q374 that may affect conformational change. To confirm critical residues around the L5 region that increase conformational dynamics, we performed MD simulations for wild-type and alanine substitutions for each candidate residue based on closed conformational structure and evaluated changes in Root Mean Square (RMSF) dynamics of the C atom. The results showed that a significant change in RMSF in the L5 region was observed from the R363A mutant (fig. 2). Therefore, we selected R363A as the best mutant, hopefully impairing product inhibition by conformational changes of L5 by increasing the kinetics of L5.
Example 2: enzyme activity and kinetic parameter determination
To assess the effect of mutations on product inhibition, we constructed the R363A mutant. The results showed that the kinetic and inhibition constants of mutant R363A (K p Value) compared to wild type (0.75 mM), K of mutant R363A p At 1.21mM, 61.33% higher, k cat The value is increased by 2.07 times, k cat /K m The constant value is increased by 19.78%. This suggests that mutant R363A resulted in a significant decrease in product inhibition and an increase in DHS conversion efficiency.
The kinetic parameters described above are shown in table 1 below.
TABLE 1
AroZ K cat (s -1 ) K m (mΜ) K cat /K m (mΜ -1 S -1 ) K p (mΜ)
wt 185.4±4.9 1.0±0.1 191.1±5.0 0.75
R363A 384.6±25.6 1.7±0.4 228.9±15.3 1.21
Example 3: construction of Chassis strains
Coli W3110 was selected as the host for transformation. Knocking out coding genes aroK and aroL of shikimate kinase I and shikimate kinase II, and constructing a recombinant escherichia coli SA01 strain. On the basis, in order to increase the content of the precursor PEP, glucose uptake reduction is avoided by knocking out genes dhaL, ptsH and ptsI and simultaneously introducing glucose promoting protein genes Zmglf and glucokinase Zmglk; replacement of the pykF native promoter with the growth-coupled promoter P rrnC Recombinant E.coli SA02 was obtained. To increase the precursor E4P, a strong promoter P was introduced into the genome J23101 Controlled talB and midpromoter P J23108 Controlled tktA, recombinant e.coli SA03 was obtained. Use of the strong promoter P J23119 Separately controlling aroG FBR aroD gene expression to enhance protocatechuic acid core pathway gene expression, recombinant E.coli SA04 was obtained. Using P tac The promoter controls the expression of glyA, and some basic resources are synthesized to obtain recombinant escherichia coli SA05. To enhance the tolerance of recombinant E.coli SA05, proV was controlled to P rpoS Control of the promoterProduction delays caused by metabolic burden are successfully avoided. This was designated as E.coli (Escherichia coli) FMME-SA07.
The promoter sequences mentioned above are shown in Table 2 below.
TABLE 2
Example 4: construction of protocatechuic acid producing Strain
The E.coli SA07 was used as chassis cell to obtain the mutant strain DHS01 by knocking out aroE gene on the genome of E.coli SA07 strain to block carbon flow from 3-dehydroshikimic acid to downstream shikimic acid. ApAroz is then applied R363A Introducing into strain DHS01 to obtain strain PCA01, fermenting strain PCA01 in a 5L fermenter, wherein the yield, the yield and the production strength of protocatechuic acid reach 37.02g/L, 0.24g/g and 1.32g/L/h respectively in 28 h.
Example 5: fed-batch fermentation of recombinant E.coli (Escherichia coli) FMME-PCA01
(1) Seed activation and culture
Solid medium configuration: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 20g/L of agar powder;
primary seed medium configuration: disodium hydrogen phosphate 6.78g/L, potassium dihydrogen phosphate 3g/L, sodium chloride 0.5g/L, ammonium chloride 1g/L, magnesium sulfate heptahydrate 0.5g/L, calcium chloride 0.011g/L, glucose 4g/L, tryptophan 10mg/L, tyrosine 10mg/L, phenylalanine 10mg/L;
slope activation: inoculating a loop of strain to a slant culture medium from a preservation tube, and culturing for 12 hours at a constant temperature of 36 ℃;
seed culture: picking single colony from the plate, inoculating to 500mL triangular flask containing 50mL culture medium, reciprocating shaking table 200rpm/min, culturing at 37deg.C for about 12 hrAfter 10 times dilution of the seed solution, OD 660 The value is between 0.6 and 0.8.
(2) Fermentation culture
Fermenting and culturing the recombinant strain in a 5L fermentation tank under the following specific fermentation conditions:
fermentation medium: 20g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 1.5g/L of ferric citrate, 5g/L of dipotassium hydrogen phosphate, 1g/L of magnesium sulfate heptahydrate, 0.1g/L of tryptophan, 0.1g/L of tyrosine, 0.1g/L of phenylalanine and 1mL/L of metal ion liquid.
Sterilizing at 121deg.C for 15min. After sterilization, the mixture is arranged on a control console, the temperature is automatically controlled, ammonia water is added to adjust the pH value to 6.6 after the temperature is cooled to 37 ℃, and inoculation is prepared.
Inoculation amount: inoculating the prepared seed liquid into a fermentation tank at an inoculation amount of 10%;
fermentation temperature: in the fermentation process, the starting temperature is automatically controlled, and the fermentation temperature is maintained at 37 ℃;
fermentation pH: ammonia water is used for regulating the pH value to be between 6.6 and 6.7;
dissolved oxygen condition: initial conditions: aeration rate 2vvm, rotational speed 300rpm;
in the fermentation process, dissolved oxygen in the fermentation process is maintained between 10 and 30 percent by adjusting ventilation and rotating speed of a stirring paddle.
In the fermentation process, after the initial glucose in the fermentation culture medium is completely exhausted, 800g/L of glucose solution is fed in a pulse mode, and the glucose concentration is controlled below 5 g/L.
The production indexes of the recombinant escherichia coli PCA01 for fermentation production of protocatechuic acid are shown in the following table.
TABLE 2
Production index Mutant strains Wild strain
Yield of products ≥37g/L ≥28g/L
Yield rate 0.22-0.24g/gglucose 0.19-0.21g/gglucose
Production strength ≥1.30-1.35g/L/h ≥1.00-1.05g/L/h
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. The 3-dehydroshikimate dehydratase mutant is characterized in that the amino acid sequence of the 3-dehydroshikimate dehydratase mutant is shown as SEQ ID NO. 3.
2. A nucleic acid encoding the 3-dehydroshikimate dehydratase mutant of claim 1.
3. A vector carrying the nucleic acid of claim 2.
4. A cell expressing the 3-dehydroshikimate dehydratase mutant of claim 1.
5. Use of the 3-dehydroshikimate dehydratase mutant of claim 1, the nucleic acid of claim 2, the vector of claim 3 or the cell of claim 4 for the preparation of protocatechuic acid.
6. A recombinant escherichia coli producing protocatechuic acid, characterized in that the recombinant escherichia coli producing protocatechuic acid is obtained by taking escherichia coli as a host, and the transformation comprises: overexpression of the gene encoding the 3-dehydroshikimate dehydratase mutant according to claim 1.
7. The recombinant E.coli according to claim 6, wherein the attachment of the pro-lytic tag enhances the soluble expression of 3-dehydroshikimate dehydratase.
8. The recombinant escherichia coli of claim 6, wherein the engineering further comprises any one of: knocking out shikimate kinase I encoding gene aroK, shikimate kinase II encoding gene aroL, phosphotransporter encoding gene ptsH, phosphoenolpyruvate phosphotransferase encoding gene ptsI, PEP-dependent dihydroxyacetone kinase encoding gene dhaL and shikimate dehydrogenase encoding gene aroE; using growth-coupled promoter P rrnC Dynamically regulating and controlling a pyruvic acid kinase I coding gene pykF; overexpression feedback inhibition resistant DAHP synthetase coding gene aroG FBR The coding genes aroD, talB, tktA, glyA, zmglf, zmglk and proV of glucose-promoting protein, respectively.
9. The recombinant escherichia coli of claim 8, wherein aroG is overexpressed FBR And aroD Gene by using a strong promoter P J23119 Separately controlling aroG FBR Expression of aroD Gene, overexpression of the talB Gene was achieved by the use of the strong promoter P J23101 Controlling the expression of talB and over-expressing the tktA gene by using a strong promoter P J23108 Controlling tktA expression, over-expressing glyA gene by using strong promoter P tac Controlling glyA expression, over-expressing the proV gene by using the pressure responsive promoter P rpoS Regulating and controlling expression of shikimic acid tolerance gene proV.
10. Use of a recombinant escherichia coli according to any one of claims 6-9 for the preparation of a protocatechuic acid containing product.
CN202311752427.2A 2023-12-19 2023-12-19 3-dehydroshikimate dehydratase mutant and recombinant escherichia coli for producing protocatechuic acid Pending CN117683759A (en)

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CN113717994A (en) * 2021-09-27 2021-11-30 中国科学院天津工业生物技术研究所 Method for producing protocatechuic acid

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WO2017169399A1 (en) * 2016-03-28 2017-10-05 公益財団法人地球環境産業技術研究機構 Transformant, and method for producing protocatechuic acid or salt thereof using same
CN113717994A (en) * 2021-09-27 2021-11-30 中国科学院天津工业生物技术研究所 Method for producing protocatechuic acid

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