Method for synthesizing shikimic acid from shikimic acid dehydrogenase of plants and microorganisms
By obtaining and optimizing the shikimate dehydrogenase gene and using CRISPR-Cas9 technology to transform Escherichia coli, the problems of insufficient enzyme activity and side reactions in the shikimate synthesis pathway were solved, and efficient shikimate production was achieved, which has important industrial application value.
Patent Information
- Application Number
- CN202510261805.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-10-10
AI Technical Summary
In the existing technology, the enzyme activity in the shikimic acid synthesis pathway is insufficient, the proportion of side reactions is high, product inhibition and imperfect metabolic flow regulation result in low shikimic acid production efficiency in Escherichia coli.
By obtaining the shikimate dehydrogenase gene from plants, optimizing the codons and inserting it into an expression vector, and using CRISPR-Cas9 technology to transform the Escherichia coli gene, an efficient shikimate synthase was constructed, and enzymes with few side reactions and high activity were screened for in vitro catalytic reactions and fermentation cultures.
The invention realizes efficient production of shikimic acid, the enzyme is not inhibited by high concentration products, has strong genetic stability, low production cost, high output and yield, few by-products, and has industrial application value.
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Figure CN120758537A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, in particular to a method for synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms. Background Art
[0002] Shikimic acid is an important natural product widely used in medicine, food, agriculture, and other fields. Traditional shikimic acid production methods rely primarily on extraction from plants, but this method suffers from low yields and resource waste. Therefore, developing an efficient microbial fermentation method to synthesize shikimic acid using genetically engineered Escherichia coli has become a research hotspot.
[0003] In the shikimate synthesis pathway, shikimate dehydrogenase is responsible for catalyzing the reduction of 3-dehydroshikimate to shikimate, making it a key enzyme in shikimate synthesis. However, shikimate dehydrogenase has bidirectional catalytic activity, catalyzing both the production of shikimate and its reverse reaction, oxidizing shikimate to 3-dehydroshikimate. Furthermore, as shikimate accumulates during fermentation, shikimate dehydrogenase is subject to feedback inhibition by shikimate, further limiting the efficiency of shikimate production. For these reasons, when shikimate is produced by fermentation in Escherichia coli, a high proportion of the byproduct 3-dehydroshikimate typically accumulates in the fermentation product.
[0004] Currently, research has attempted to increase shikimic acid production by engineering E. coli metabolic pathways. However, existing technologies still face challenges, such as insufficient enzyme activity in the shikimic acid synthesis pathway, a high proportion of side reactions, product inhibition, and imperfect regulation of metabolic flux. Therefore, how to improve shikimic acid production in E. coli through genetic engineering remains an urgent problem. Summary of the Invention
[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.
[0006] In view of the problems existing in the above-mentioned existing plant shikimate dehydrogenase and the method for synthesizing shikimate by microorganisms, the present invention is proposed.
[0007] Therefore, the purpose of the present invention is to provide a method for synthesizing shikimic acid by using plant shikimate dehydrogenase and microorganisms, in order to solve the problems of "insufficient enzyme activity in the shikimic acid synthesis pathway, high proportion of side reactions, product inhibition and imperfect regulation of metabolic flow, etc.; how to improve the shikimic acid production capacity of Escherichia coli through genetic engineering modification".
[0008] To solve the above technical problems, the present invention provides the following technical solution: a method for synthesizing shikimic acid by using plant shikimate dehydrogenase and microorganisms, characterized by the following steps:
[0009] S1. Obtaining shikimate dehydrogenase gene sequence from plants;
[0010] S2. Codon optimization of genes;
[0011] S3, inserting the optimized shikimate dehydrogenase gene into the expression vector;
[0012] S4, transferring the expression vector into an engineered Escherichia coli bacterium to express shikimate dehydrogenase;
[0013] S5. Through in vitro catalytic reaction, a shikimate synthase with a relatively low side reaction ratio and high activity is screened and obtained. The nucleotide coding sequences thereof are shown in SEQ ID NO.5, SEQ ID NO.8, and SEQ ID NO.9, and are used for synthesizing shikimate.
[0014] As a preferred embodiment of the method for synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms, the in vitro catalytic reaction comprises mixing 3-dehydroshikimic acid, NADP, glucose and shikimate dehydrogenase enzyme solution and reacting at 37°C for 2 hours.
[0015] As a preferred embodiment of the method for synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms of the present invention, the engineered Escherichia coli contains a plant shikimate dehydrogenase gene constructed by gene editing, which is inserted into the AroL site of shikimate kinase or other suitable sites.
[0016] As a preferred embodiment of the method for synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms of the present invention, the shikimate kinase AroL gene is edited by CRISPR-Cas9 technology and inserted into the plant shikimate dehydrogenase gene.
[0017] As a preferred embodiment of the method of synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms of the present invention, a gene editing method for transforming Escherichia coli using CRISPR-Cas9 technology is characterized by comprising the following steps:
[0018] A1. Designing the N20 sequence targeting the shikimate kinase AroL gene or the shikimate kinase AroK gene;
[0019] A2, using PCR to amplify the homology arms containing the plant shikimate dehydrogenase gene;
[0020] A3. Integrate the homology arms with the editing target gene to obtain gene-edited Escherichia coli.
[0021] As a preferred embodiment of the method for synthesizing shikimic acid by using the plant shikimate dehydrogenase and microorganisms of the present invention, the engineered Escherichia coli is cultured in a suitable culture medium through shake flask fermentation or tank fermentation to synthesize shikimic acid.
[0022] As a preferred embodiment of the method for synthesizing shikimic acid by using the plant shikimate dehydrogenase and microorganisms of the present invention, the culture medium comprises M9 salt solution, glucose, a nitrogen source, a phosphorus source and appropriate trace elements, and the pH is adjusted to a suitable range for cultivation.
[0023] As a preferred embodiment of the method for synthesizing shikimic acid using the plant shikimate dehydrogenase and microorganisms of the present invention, the engineered Escherichia coli bacteria further includes a weakened shikimate kinase AroK gene, wherein the start codon of the AroK gene is changed from ATG to TTG or CTG to increase the yield of shikimic acid synthesis.
[0024] Beneficial effects of the present invention:
[0025] The present invention overexpresses plant-derived shikimate dehydrogenase in Escherichia coli for the first time. The resulting enzyme exhibits minimal side effects, resistance to inhibition by high-concentration products, and high activity. This enzyme was utilized through metabolic engineering to produce a recombinant strain for shikimate production. This strain, which does not utilize a plasmid, exhibits strong genetic stability, requires no additional aromatic amino acids in the fermentation medium, offers low production costs, high yields, and minimal metabolic byproducts, demonstrating its significant industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:
[0027] Figure 1 Schematic diagram of fermentation results. DETAILED DESCRIPTION
[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.
[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0030] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.
[0031] Example 1
[0032] Derived from the synthesis of plant shikimate dehydrogenase gene;
[0033] The sequences of 3-dehydroquinate dehydratase / shikimate dehydrogenase from different plant sources were retrieved from the GenBank database, and the accession numbers were: XP_010649799.1, AAW65140.1, KDO74990.1, BBL52471.1, BBL52472.1, AAS90325.1, NP001234051.1, XP010031275.2,
[0034] NP_187286.1, KAI5572505.1, XP_004289250.1, XP_004288087.1. Based on the codon preference of E. coli and the principle of avoiding repetitive sequences, the gene sequence was codon-optimized using codon optimization software without changing the amino acid sequence. The optimized sequences are shown in SEQ ID NOs. 1-12, encoding enzymes E1-E12, respectively. The genes were synthesized at BGI and inserted into expression vectors such as pET-21a(+) via NdeI and XhoI to generate expression plasmids.
[0035] Example 2
[0036] Expression of plant shikimate dehydrogenase gene in Escherichia coli and preparation of enzyme solution;
[0037] The expression plasmid synthesized in Example 1 was transformed into BL21(DE3) cells, and a single colony was selected and inoculated into 5 mL of LB liquid medium containing 100 mg / L ampicillin and cultured overnight at 37°C. A 1% inoculum was then transferred to 0.5 mL of LB liquid medium (2L flask) and cultured at 37°C and 220 rpm until the OD600 of the culture medium was approximately 0.6-0.5. The shaker temperature was then adjusted to 30°C, and IPTG was added to a final concentration of 1 mM. The culture was continued for 16-18 hours. The cells were collected by centrifugation, resuspended in PBS to a wet weight of 100 g / L, and disrupted by sonication before use in in vitro catalysis.
[0038] Example 3 Determination and screening of plant-derived shikimate dehydrogenase activity
[0039] Determination of shikimate synthase activity (3-dehydroshikimate to shikimate): 1 mL reaction system contains 1 g / L 3-dehydroshikimate, 0.1 mL crushed enzyme solution, 0.1 mL glucose dehydrogenase enzyme solution, a final concentration of 1 mM NADP, and 10 g / L glucose. The pH is adjusted to 6.5 with NaOH, and the reaction is incubated on a shaker at 37°C, 200 rpm for 1 h. The shikimate and 3-dehydroshikimate contents are determined by HPLC, respectively.
[0040] Side reaction (shikimic acid to 3-dehydroshikimic acid) to activity determination: 1 mL reaction system contains 1 g / L shikimic acid, 0.1 mL crushed enzyme solution, 0.1 mL glucose dehydrogenase enzyme solution, a final concentration of 1 mM NADP, and 10 g / L glucose. The pH is adjusted to 6.5 with NaOH. Place on a shaker at 37°C, 200 rpm for 1 hour, and then determine the shikimic acid and 3-dehydroshikimic acid contents by HPLC.
[0041] The activity test results are shown in the table below. The side reactions of E5, E8, and E9 accounted for ≤15%, indicating that they have application potential in the microbial production of shikimic acid.
[0042] Enzyme number E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 Shikimate synthase activity 1.77 0.25 1.84 1.84 1.69 1.77 1.85 1.86 1.75 1.61 1.86 1.24 Side reaction activity 0.52 1.11 0.32 0.43 0.25 0.38 0.35 0.24 0.18 0.82 0.43 0.30 Relative proportion 29％ 225％ 17％ 23％ 15％ 21％ 19％ 13％ 10％ 51％ 23％ 24％
[0043] Example 4
[0044] Derived from plant shikimate dehydrogenase gene in vitro catalysis;
[0045] To 100 mL of fermentation broth containing 50 g / L 3-dehydroshikimic acid, add 0.1-1 mL of crushed E5, E8, or E9 enzyme solution, 0.5-5 mL of glucose dehydrogenase solution, a final concentration of 0.1-1 mM NADP, and 10-110 g / L glucose. Adjust the pH to 6.5 with NaOH and incubate on a shaker at 37°C, 200 rpm, for 2 hours. Shikimic acid and 3-dehydroshikimic acid concentrations were determined by HPLC. The results are shown in the following table:
[0046] Enzyme number E5 E8 E9 Shikimic acid concentration (g / L) 46 49.5 48.2 DHS concentration (g / L) 4.1 0.8 2.2
[0047] Example 5
[0048] The plant shikimate dehydrogenase gene was used to construct a shikimate-producing Escherichia coli engineered bacterium;
[0049] In the following examples, the CRISPR-Cas9 system was used to edit the genome of Escherichia coli. For details, please refer to Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system, Appl Environ Microbiol 2015 Apr; 81(7): 2506-2514. The N20 sequence was designed using the online website http: / / crispor.org.
[0050] 1) The plant shikimate dehydrogenase gene is inserted into the shikimate kinase AroL site;
[0051] The following takes the insertion of SEQ ID NO.1 into AroL as an example to exemplify the process of inserting the plant shikimate dehydrogenase gene into the shikimate kinase AroL site. Based on the shikimate kinase AroL gene sequence of Escherichia coli DH5a, the N20 sequence was designed. Using SEQ ID NO.13 and SEQ ID NO.14 as primers and pTarget as a template, E. coli was amplified and transformed by PCR to prepare the pTarget-aroL plasmid. Based on the upstream and downstream sequences of the designed N20 sequence, primers SEQ ID NO.15-16 and SEQ ID NO.17-18 were designed to amplify the upstream and downstream homology arms, and two primers SEQ ID NO.19 and SEQ ID NO.20 were designed to amplify the SDH gene containing a constitutive promoter. Using the three amplified fragments as templates and SEQ ID NO.15 and SEQ ID NO.18 as primers, PCR amplification was performed to obtain a homology template containing the upstream and downstream homology arms and the SDH expression cassette. The pTarget-aroL plasmid and the SDH homology template were transformed into E. coli DH5a containing the pECCas9 plasmid by electroporation, and the insertion of SDH was verified by PCR to obtain the strain SA01.
[0052] 2) weakening the shikimate kinase AroK gene;
[0053] AroK is attenuated by changing the start codon of AroK gene from ATG to TTG or CTG. The process of attenuating AroK (the start codon is changed from ATG to TTG) is exemplarily introduced as follows. According to the sequence of shikimic acid kinase AroK gene of E. coli DH5a, N20 sequence is designed, and pTarget is used as a template to prepare pTarget-aroK plasmid by PCR amplification and transformation of E. coli; primers SEQ ID NO. 23-26 are designed according to the upstream and downstream sequences of the designed N20 sequence for amplifying the upstream and downstream homologous arms; the two amplified fragments are used as templates, and SEQ ID NO. 23 and SEQ ID NO. 26 are used as primers to obtain a homologous template containing the upstream and downstream homologous arms and the start codon of AroK changed to TTG by PCR amplification; the pTarget-aroL plasmid and the above homologous template are transformed into SA01 containing pECCas9 plasmid by electroporation, and strain SA02 is obtained after PCR verification.
[0054] Example 6
[0055] Shake flask fermentation of shikimic acid synthesis E. coli engineering strain;
[0056] Single colonies are picked from the plates of SA01 and SA02 respectively into a shaking tube containing 3 mL LB liquid medium, and cultured at 37°C, 200 rpm overnight until the OD is 4. Then the strains are transferred into 250 mL shake flasks containing 50 mL M9 fermentation medium at a 10% inoculation amount. The culture is performed at 30°C, 200 rpm for 72 h, and the results are shown in the following table.
[0057]
[0058] Example 7
[0059] Top tank fermentation of shikimic acid synthesis E. coli engineering strain;
[0060] The components and contents of the seed medium and the fed-batch fermentation medium mentioned in the present application are as follows, and the corresponding adjustment can be made according to the needs.
[0061] Seed medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L.
[0062] Fermentation medium: glucose 10-30 g / L, potassium phosphate dibasic 1-10 g / L, magnesium sulfate 1-4 g / L, ammonium sulfate 1-5 g / L, ferrous sulfate 0.15-0.05 g / L, citric acid 1-4 g / L, yeast extract 1-5 g / L.
[0063] Feed medium: 500 g / L glucose solution.
[0064] First, the gallic acid-producing strain SA02 was inoculated into 10 mL of LB medium for primary seed culture overnight. Then, the primary seed solution was inoculated into a suitable shake flask containing 0.5 L of fermentation medium at a volume fraction of 1%. The culture was continued for 6-8 hours, and then 10% was inoculated into a 4 L fermenter for fermentation culture. The process parameters for fermenter culture were: temperature 30-37 ° C, ventilation volume 3-5 m 3 / h, maintaining the dissolved oxygen level at approximately 30%, and adding 30% (w / v) aqueous ammonia to adjust the fermentation broth pH to 6.5-7.0. Feeding was initiated when glucose in the fermentation broth was completely consumed. The fermentation supernatant was sampled and shikimic acid concentration was determined by high-performance liquid chromatography. After 56 hours of fermentation, the shikimic acid concentration reached a maximum of 80 g / L.
[0065] SEQ ID NO. 1 to SEQ ID NO. 26 are shown below;
[0066]
[0067]
[0068]
[0069]
[0070]
[0071] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.
Claims
1. A method for synthesizing shikimic acid using plant shikimate dehydrogenase and microorganisms, characterized by the following steps: S1. Obtaining shikimate dehydrogenase gene sequence from plants; S2. Codon optimization of genes; S3, inserting the optimized shikimate dehydrogenase gene into the expression vector; S4, transferring the expression vector into an engineered Escherichia coli bacterium to express shikimate dehydrogenase; S5. Through in vitro catalytic reaction, a shikimate synthase with a relatively low side reaction ratio and high activity is screened and obtained. The nucleotide coding sequences thereof are shown in SEQ ID NO.5, SEQ ID NO.8, and SEQ ID NO.9, and are used for synthesizing shikimate.
2. The method for synthesizing shikimic acid by using a plant shikimate dehydrogenase and a microorganism according to claim 1, characterized in that: The in vitro catalytic reaction includes mixing 3-dehydroshikimic acid, NADP, glucose and shikimate dehydrogenase enzyme solution and reacting at 37° C. for 2 hours.
3. The method for synthesizing shikimic acid using a plant shikimate dehydrogenase and a microorganism according to claim 2, characterized in that: The engineered Escherichia coli contains a plant shikimate dehydrogenase gene constructed by gene editing, and the gene is inserted into the shikimate kinase AroL site or other suitable sites.
4. The method for synthesizing shikimic acid using a plant shikimate dehydrogenase and a microorganism according to claim 3, characterized in that: The shikimate kinase AroL gene was edited using CRISPR-Cas9 technology and inserted into the plant shikimate dehydrogenase gene.
5. The method for synthesizing shikimic acid by using plant shikimate dehydrogenase and microorganisms according to claim 4, wherein the gene editing method of transforming Escherichia coli using CRISPR-Cas9 technology is used. Its characteristics include the following steps: A1. Designing the N20 sequence targeting the shikimate kinase AroL gene or the shikimate kinase AroK gene; A2, using PCR to amplify the homology arms containing the plant shikimate dehydrogenase gene; A3. Integrate the homology arms with the editing target gene to obtain gene-edited Escherichia coli.
6. The method for synthesizing shikimic acid using a plant shikimate dehydrogenase and a microorganism according to claim 1, characterized in that: The engineered Escherichia coli is cultured in a suitable culture medium through shake flask fermentation or tank fermentation to synthesize shikimic acid.
7. The method for synthesizing shikimic acid using a plant shikimate dehydrogenase and a microorganism according to claim 6, characterized in that: The culture medium contains M9 salt solution, glucose, a nitrogen source, a phosphorus source and appropriate trace elements, and the pH is adjusted to a suitable range for cultivation.
8. The method for synthesizing shikimic acid using a plant shikimate dehydrogenase and a microorganism according to claim 3, characterized in that: The engineered E. coli bacteria also include a weakened shikimate kinase AroK gene, wherein the start codon of the AroK gene is changed from ATG to TTG or CTG to increase the yield of shikimate synthesis.