CN116926089A - Method for producing shikimic acid by whole cell catalysis - Google Patents

Method for producing shikimic acid by whole cell catalysis Download PDF

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CN116926089A
CN116926089A CN202210354152.6A CN202210354152A CN116926089A CN 116926089 A CN116926089 A CN 116926089A CN 202210354152 A CN202210354152 A CN 202210354152A CN 116926089 A CN116926089 A CN 116926089A
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aroe
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王钦宏
吴凤礼
王晓霜
宋国田
张媛媛
彭彦峰
陈五九
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses a method for producing shikimic acid by whole cell catalysis. The invention uses genetically engineered recombinant Escherichia coli (Escherichia coli) which coexpresses shikimate dehydrogenase AroE and glucose dehydrogenase GDH as a whole-cell catalyst, uses 3-dehydroshikimate fermentation liquor as a substrate and uses glucose as a hydrogen donor to construct a circulating catalytic system coupling 3-dehydroshikimate conversion and NADPH regeneration. The catalytic system can efficiently convert 3-dehydroshikimic acid into shikimic acid without adding NADPH. The method for producing shikimic acid by whole cell catalysis has the characteristics of simple operation, low cost, no need of adding auxiliary factors and high synthesis efficiency, and has good industrialized application prospect.

Description

Method for producing shikimic acid by whole cell catalysis
Technical Field
The invention belongs to the technical field of bioengineering, relates to a method for producing shikimic acid by catalyzing 3-dehydroshikimic acid by genetically engineered recombinant escherichia coli, and in particular relates to a method for producing shikimic acid by catalyzing whole cells of shikimate dehydrogenase coupled glucose dehydrogenase.
Background
Shikimic acid (Shikimic acid), also known as 3,4,5-Trihydroxy-1-cyclohexene-1-carboxylic acid (3, 4, 5-Trihydroxy-1-cyclohexene-1-carboxilic acid), is white needle-like crystal, is easily dissolved in water, is slightly dissolved in organic solvents such as ethanol and diethyl ether, and has a melting point of 185-187 ℃. Shikimic acid is a key intermediate in the biological aromatic amino acid biosynthesis pathway. In addition, shikimic acid is an important precursor for synthesis of various antiviral and anticancer drugs as a six-membered cyclic compound having three chiral carbon atoms, and can be used as a synthetic parent nucleus of Oseltamivir (trade name Tamiflu) which is an anti-influenza specific drug.
Shikimic acid is widely present in many plants, where the shikimic acid content in mature fruits (e.g. star anise) of the genus Illicium (Illicium sp.) is most abundant, up to 17% of the dry weight of the fruit. Therefore, plant extraction has been the main production of shikimic acid (CN 200910039670.3, CN 20110136556. X, CN 201910344805.0). Plant extraction has a number of disadvantages, such as limited raw material seasons and regions, low extraction rate, difficult control of the production process, strong damage to the natural environment, etc., and is unfavorable for continuous industrial production. The traditional plant extraction production mode is gradually difficult to meet the increasing market demand of shikimic acid.
Chemical synthesis shikimic acid and analogues thereof can be synthesized by chemical catalysis with quinic acid (CN 99106395.3), triacetylshikimic acid (CN 200910199827.9), crotonaldehyde (CN 201210094437.7) and the like as precursors. However, it is difficult to apply to large-scale industrial production because it is limited by the high price of the synthesis precursor, the severe catalytic reaction conditions, the difficulty in treating chemical wastes, and the like.
Shikimic acid pathway is widely present in cells of plants, algae, fungi and bacteria, and 3-deoxy-D-arabinoheptulose 7-phosphate (DAHP) is produced by condensation using phosphoenolpyruvate (PEP) in glycolytic pathway and erythrose 4-phosphate (E4P) in pentose phosphate pathway as precursors. DAHP is subjected to 6-step catalytic reaction in shikimic acid way to generate branched acid, and important aromatic compounds such as phenylalanine, tyrosine, tryptophan, folic acid, vitamin K2 and the like are further synthesized.
At present, scholars at home and abroad perform metabolic engineering transformation on the central metabolic pathway of microbial cells and the shikimic acid pathway by a genetic engineering means, and a series of genetic engineering strains for producing shikimic acid are constructed. In 2003, the university of michigan state and the roche company cooperate to use escherichia coli RB791 as a host cell, and aroB homologous recombination is used to replace serA, so as to promote DAHP to synthesize 3-dehydroquinic acid; promoting shikimic acid accumulation by inactivating aroL and aroK; the glucose transport pathway is reconstructed by replacing the PTS system key gene ptsH, ptsI, crr of the escherichia coli by glf-glk of zymomonas mobilis (Zymomonas mobilis); overexpression of aroF Using plasmid FBR tktA, aroE, serA, enhancing shikimate pathway metabolic flux; the nutrient medium with 15g/L yeast extract added is used, and a 10L fermenter is used for feed fermentation for 60h to accumulate 87g/L shikimic acid (Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol Prog,2003,19 (3): 808-814.). In addition to the need for yeast extract, the production process requires maintenance of a relatively high concentration of glucose throughout the fermentation process to facilitate glucose transport into the cell, resulting in accumulation of large amounts of acetic acid in the fermentation broth. In 2006, E.coli W3110 was used as a host cell by the national academy of sciences Shanghai life sciences Yang, and recombinant plasmid pSUFEBP was used to overexpress key gene aroF, aroE, aroB, ppsA, tktA by knocking out aroL and aroK, and the fermentation was continued for 128 hours with continuous feed by using a 5L fermenter, and shikimic acid yield was 39.3g/L (CN 200610030985.8). The process has long culture time and low shikimic acid synthesis rate and yield. In 2020, dongguan city Dongyang photo-biological synthetic medicine Co., ltd Liu Cuicui and the like utilize Escherichia coli BL21 (DE 3) as host cells and CRISPR The aroK, aroL, ptsH, ptsI, crr, lacI gene is knocked out by Cas9 technology, and aroB, aroD, aroE, aroF, tktA, ppsA is over-expressed by pET28a vector and T7 promoter, so as to obtain recombinant engineering bacterium ESA-pSA-14. The recombinant strain is continuously fed and fermented for about 70-80 hours in a nutrient-rich culture medium rich in glucose and organic nitrogen sources, and the shikimic acid yield reaches 90.0g/L in a fermentation system of a 50L tank; in a fermentation system of a 500L tank, the shikimic acid yield reaches 84.6g/L; in a fermentation system with 8000L tank, shikimic acid yield reaches 73.4g/L (CN 201911327349.5). The process has high shikimic acid yield, but needs to be cultivated at a lower temperature of 30 ℃ for a longer fermentation time, and needs to supplement a large amount of organic nitrogen sources.
Biological enzyme catalysis is also a highly efficient biosynthesis method. In 2006, the university of Japanese mountain Osao ADACHI and the like take quinic acid as a precursor, and quinic acid dehydrogenase and 3-dehydroquinic acid dehydratase are utilized to convert quinic acid into 3-dehydroshikimic acid, wherein the conversion rate is up to 77%; the shikimate dehydrogenase and glucose dehydrogenase are then coupled to catalyze the synthesis of 3-dehydroshikimate with a catalytic efficiency approaching 100% in a short time (High Shikimate Production from Quinate with Two Enzymatic Systems of Acetic Acid bacteria biosci. Biotechnol. Biochem.,2006,70 (10): 2579-2582). In 2010, the study group adopted an enzyme immobilization technology to immobilize the two groups of catalytic enzymes on two polymeric materials respectively, further improving the conversion rate of quinic acid to shikimic acid (Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate dehydrogenase. Biosci. Biotechnol. Biochem.,2010:74 (12), 2438-2444). The production process has complicated enzyme preparation and catalysis process due to high quinic acid price, and requires addition of cofactor NADP + Therefore, it is difficult to realize large-scale industrial production.
3-dehydroshikimic acid is a direct precursor of biosynthesized shikimic acid and is directly converted into shikimic acid by an asymmetric reduction reaction. The applicant is in the early stage of research group to construct the recombinant engineering strain WJ060 of the escherichia coli for high-yield 3-dehydroshikimic acid through a series of metabolic engineering transformation. The strain takes glucose as a carbon source, and is fermented for 52 hours by a 5L tank in an inorganic salt culture medium without adding any organic nitrogen source and aromatic amino acid, and the yield of 3-dehydroshikimic acid reaches 94.4g/L (CN 201711002831.2). This work can provide a high concentration of the synthesis precursor 3-dehydroshikimic acid for the catalytic synthesis of shikimic acid.
Disclosure of Invention
The invention provides a novel production method of biologically synthesized shikimic acid, which is characterized in that 3-dehydroshikimic acid produced by a fermentation method is converted into shikimic acid in a whole-cell catalysis mode, so that the high-efficiency production from low-cost glucose to high-added-value shikimic acid is realized.
The invention provides a system for producing shikimic acid by whole cell catalysis, which comprises 3-dehydroshikimic acid, glucose and recombinant bacteria co-expressing glucose dehydrogenase GDH and shikimate dehydrogenase AroE.
Wherein, in the system, the concentration of the 3-dehydroshikimic acid is 1-120g/L; the concentration of glucose is 1-200g/L; the cell amount of the recombinant bacteria is 0.1-100OD 600 。
Wherein the glucose dehydrogenase comprises an amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 or SEQ ID NO. 10; the shikimate dehydrogenase comprises an amino acid sequence shown as SEQ ID NO. 12.
The recombinant bacterium is escherichia coli containing a recombinant vector, wherein the recombinant vector comprises any one of a nucleotide sequence shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9 and a nucleotide sequence shown in SEQ ID NO. 11.
Wherein the recombinant vector is as shown in any one of the following A1) to A7):
a1 A recombinant vector pETDuet-Bmgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 1, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a2 A recombinant vector pETDuet-Tagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 3, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a3 A recombinant vector pETDuet-Lstdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 5, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
A4 A recombinant vector pETDuet-Bsgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 7, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a5 A recombinant vector pETDuet-Bagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 9, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a6 Recombinant vector pRSFDuet-Bmgdh-aroE, replacing the sequence between BamHI and XhoI sites of pRSFDuet-1 vector with the sequence between BamHI and XhoI sites of recombinant vector A1), and leaving the other sequences unchanged;
a7 Recombinant vector pRSFDuet-Bmgdh-aroE-serA, and the sequence shown in SEQ ID NO. 13 was inserted into the XhoI site of recombinant vector A6) and the other sequences were kept unchanged.
Wherein the recombinant bacterium is any one of the following recombinant bacterium:
b1 Recombinant E.coli BL21 (DE 3)/pETDuet-Bmgdh-aroE, and transferring the recombinant vector pETDuet-Bmgdh-aroE into recombinant strain of E.coli BL21 (DE 3);
B2 Recombinant E.coli BL21 (DE 3)/pETDuet-Tagdh-aroE, and transferring the recombinant vector pETDuet-Tagdh-aroE into the recombinant strain obtained by the E.coli BL21 (DE 3);
b3 Recombinant E.coli BL21 (DE 3)/pETDuet-Lstdh-aroE, and transferring the recombinant vector pETDuet-Lstdh-aroE into E.coli BL21 (DE 3);
b4 Recombinant E.coli BL21 (DE 3)/pETDuet-Bsgdh-aroE, and transferring the recombinant vector pETDuet-Bsgdh-aroE into E.coli BL21 (DE 3);
b5 Recombinant E.coli BL21 (DE 3)/pETDuet-Bagdh-aroE, and transferring the recombinant vector pETDuet-Bagdh-aroE into the recombinant strain obtained by E.coli BL21 (DE 3);
b6 Recombinant E.coli BL21 (DE 3)/pRSFDuet-Bmgdh-aroE, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE into E.coli BL21 (DE 3);
b7 Recombinant E.coli BL21 (DE 3) ΔserA/pRSFDuet-Bmgdh-aroE-serA, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE-serA into E.coli BL21 (DE 3) ΔserA, wherein E.coli BL21 (DE 3) ΔserA is a strain in which the D-3-phosphoglycerate dehydrogenase encoding gene serA on the genome of E.coli BL21 (DE 3) is knocked out.
The invention also claims the recombinant vector, which is shown in any one of the following A1) -A7):
A1 A recombinant vector pETDuet-Bmgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 1, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a2 A recombinant vector pETDuet-Tagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 3, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a3 A recombinant vector pETDuet-Lstdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 5, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a4 A recombinant vector pETDuet-Bsgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 7, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
A5 A recombinant vector pETDuet-Bagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 9, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a6 Recombinant vector pRSFDuet-Bmgdh-aroE, replacing the sequence between BamHI and XhoI sites of pRSFDuet-1 vector with the sequence between BamHI and XhoI sites of recombinant vector A1), and leaving the other sequences unchanged;
a7 Recombinant vector pRSFDuet-Bmgdh-aroE-serA, and the sequence shown in SEQ ID NO. 13 was inserted into the XhoI site of recombinant vector A6) and the other sequences were kept unchanged.
The invention also claims the recombinant bacteria, wherein the recombinant bacteria are any one of the following recombinant bacteria:
b1 Recombinant E.coli BL21 (DE 3)/pETDuet-Bmgdh-aroE, and transferring the recombinant vector pETDuet-Bmgdh-aroE into recombinant strain of E.coli BL21 (DE 3);
b2 Recombinant E.coli BL21 (DE 3)/pETDuet-Tagdh-aroE, and transferring the recombinant vector pETDuet-Tagdh-aroE into the recombinant strain obtained by the E.coli BL21 (DE 3);
b3 Recombinant E.coli BL21 (DE 3)/pETDuet-Lstdh-aroE, and transferring the recombinant vector pETDuet-Lstdh-aroE into E.coli BL21 (DE 3);
B4 Recombinant E.coli BL21 (DE 3)/pETDuet-Bsgdh-aroE, and transferring the recombinant vector pETDuet-Bsgdh-aroE into E.coli BL21 (DE 3);
b5 Recombinant E.coli BL21 (DE 3)/pETDuet-Bagdh-aroE, and transferring the recombinant vector pETDuet-Bagdh-aroE into the recombinant strain obtained by E.coli BL21 (DE 3);
b6 Recombinant E.coli BL21 (DE 3)/pRSFDuet-Bmgdh-aroE, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE into E.coli BL21 (DE 3);
b7 Recombinant E.coli BL21 (DE 3) ΔserA/pRSFDuet-Bmgdh-aroE-serA, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE-serA into E.coli BL21 (DE 3) ΔserA, wherein E.coli BL21 (DE 3) ΔserA is a strain in which the D-3-phosphoglycerate dehydrogenase encoding gene serA on the genome of E.coli BL21 (DE 3) is knocked out. .
The application of the system, the recombinant vector and the recombinant bacteria in preparing shikimic acid or downstream products containing shikimic acid is also within the protection scope of the invention.
The invention also claims a method for producing shikimic acid by whole cell catalysis, which uses the recombinant bacterium of claim 8 to produce shikimic acid by using 3-dehydroshikimic acid fermentation liquor as a substrate.
Wherein the pH of the whole cell catalyzed reaction is 5.0-9.0.
Wherein the whole cell catalyzed reaction temperature is 30-40 ℃.
Wherein the whole cell catalyzed reaction time is 0.5-24h.
Wherein, no NADP+ and/or NADPH is added in the production process.
Wherein isopropyl-beta-D-thiogalactoside (IPTG) or lactose is adopted to induce the expression of glucose dehydrogenase gene gdh and shikimate dehydrogenase gene aroE, so as to obtain the whole cell catalyst.
The invention has the advantages that the invention provides a method for biosynthesis of shikimic acid, which utilizes a genetically engineered recombinant escherichia coli co-expressing shikimic acid dehydrogenase and glucose dehydrogenase as a whole cell catalyst, takes 3-dehydroshikimic acid fermentation liquor as a substrate and glucose as a hydrogen donor to construct a whole cell circulation catalytic system coupling 3-dehydroshikimic acid conversion and NADPH regeneration. The catalytic system can efficiently convert 3-dehydroshikimic acid into shikimic acid without adding cofactor. The method for producing shikimic acid by whole cell catalysis has the advantages of simple and efficient catalysis process, low cost and easy obtainment of raw materials, no need of adding auxiliary factors, and good industrialized application prospect.
Drawings
FIG. 1 is a schematic diagram of the whole cell catalyzed synthesis of shikimic acid from 3-dehydroshikimic acid.
FIG. 2 is a pETDuet-Bmgdh-aroE plasmid map of an embodiment of the invention.
FIG. 3 is a map of pRSFDuet-Bmgdh-aroE plasmid of an embodiment of the present invention.
FIG. 4 is a map of pRSFDuet-Bmgdh-aroE-serA plasmid of an example of the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The technical scheme adopted by the invention is as follows:
1. PCR amplification reaction System and conditions
(1) When constructing the vector and preparing the homologous recombination fragment, amplification was performed using TaKaRa' high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase. The general amplification system comprises: 5 XPrimeSTAR Buffer (Mg) 2+ Plus) 10. Mu.l, dNTP mix (2.5 mM each) 4. Mu.l, upstream primer (10. Mu.M) 1. Mu.l, downstream primer (10. Mu.M) 1. Mu.l, template 1. Mu.l, primeSTAR HS DNA Polymerase (2.5U/. Mu.l) 0.5. Mu.l, sterile water 32.5. Mu.l, and total volume 50. Mu.l. The general amplification procedure is as follows: (1) 3min at 95 ℃; (2) step (2) was repeated 30 times at 98℃for 10sec,55℃for 15sec, and 72℃for 1 kb/min; (3) and at 72℃for 5min.
(2) When verifying whether the constructed vector and the knockout transformant were properly sequenced and sampled, amplification was performed using 2 XEs Taq Master mix (Dye) from century Biotechnology Co. The general amplification system comprises: 2 XEs Taq Master mix (Dye) 12.5. Mu.l, upstream primer (10. Mu.M) 1. Mu.l, downstream primer (10. Mu.M) 1. Mu.l, template 0.5. Mu.l, sterile water 10. Mu.l, total volume 25. Mu.l. The general amplification procedure is as follows: (1) 94 ℃ for 3min; (2) repeating step (2) 30 times at 94℃for 30sec,55℃for 30sec, and 72℃for 2 kb/min; (3) and at 72℃for 5min. The PCR products were sent to Beijing engine biotechnology Co.
2. Restriction enzyme double-enzyme reaction system and condition
When constructing the expression vector, restriction enzymes of New England Biolabs (NEB) company are used to cleave the vector and the target fragment, respectively. The general enzyme digestion reaction system comprises: 10 XCutSmart Buffer 3. Mu.L, 500-1000ng of vector or target fragment, 0.5. Mu.L of endonuclease A (20U/. Mu.L), 0.5. Mu.L of endonuclease B (20U/. Mu.L) and the addition of sterile water to 30. Mu.L. The general enzyme digestion reaction condition is 1-2h at 37 ℃.
3. T4 DNA ligase ligation reaction System and conditions
When the vector is ligated to the desired fragment or when the vector is circularized, ligation is performed using T4 DNA ligase from Thermo Scientific. The general enzyme-linked reaction system comprises: 10×T DNA Ligase Buffer 2.mu.L, linearized vector 20-100ng, fragment of interest 20-100ng (vector circularization no this is done) T4 DNA Ligase (5U/. Mu.L) 0.5. Mu.L, supplemented with sterile water to 20. Mu.L. The general enzyme-linked reaction condition is 0.5-1h at 22 ℃.
4. Chemical transformation of E.coli
(1) Preparation of competent cells: e.coli DH5 alpha or BL (DE 3) single colony is selected and inoculated in 3mL of liquid LB culture medium, and cultured for 8-12h at 37 ℃ and 250rpm to be used as seed liquid; inoculating 100 μl of seed solution into 50mL of liquid LB medium, culturing at 37deg.C and 250rpm to OD 600 About 0.3 to about 0.4; ice bath for 15min, and transferring the bacterial liquid into a precooled 50mL centrifuge tube; centrifuge 2000g for 5min, discard supernatant, use 15mL pre-chilled 100mM CaCl 2 Resuspension of the cells with the solution, repeating this step once; 2000g was centrifuged for 5min and the supernatant discarded, and 2mL of pre-chilled 10% (v/v) glycerol-100 mM CaCl2 solution was usedThe resuspended cells are competent cells.
(2) Conversion: adding 5-10 mu L of enzyme-linked product into 50 mu L of competent cells, gently mixing, and ice-bathing for 30min; heat shock at 42deg.C for 45s, ice-bath for 2min, adding 500 μl LB liquid medium, and resuscitating and culturing at 37deg.C at 250rpm for 40-60min; centrifuging at 8000rpm for 2min, removing most of the supernatant, coating the residual bacterial liquid on solid LB medium with corresponding resistance (100 mug/mL ampicillin or 50 mug/mL kanamycin or 50 mug/mL spectinomycin), and culturing at 37 ℃ overnight; several monoclonals are selected for colony PCR verification and sample feeding sequencing, and the clones with correct sequencing are selected for plasmid extraction.
The LB culture medium comprises 10g/L of tryptone and 5g/L, naCl g/L of yeast extract.
5. Construction of glucose dehydrogenase expression vector
Since the reaction catalyzing 3-dehydroshikimic acid to synthesize shikimic acid requires the participation of NADPH cofactor, whereas glucose dehydrogenase uses glucose as hydrogen donor, oxidized NA (D) P + The NAD (P) H converted into reduced form provides reducing power for the synthesis reaction of shikimic acid. To screen for efficient glucose dehydrogenases, 5 glucose dehydrogenase genes gdh from different species were screened from NCBI database, bmgdh from Bacillus megaterium WSH-002, tagdh from Thermoplasma acidophilum, lstdh from Lysinibacillus sphaericus G, bstdh from Bacillus subtilis 9902, bagdh from Bacillus amyloliquefaciens DSM 7, respectively, with corresponding NCBI accession numbers NC_017138.1REGION:complex (4157260.. 4158045), AL445065.1REGION:284009..285094, FJ908710.1, EF626962.1, NC_014551.1REGION:404084..404869, respectively. The genes are subjected to codon optimization according to the codon preference of escherichia coli and comprise nucleotide sequences shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9. The above 5 genes were synthesized in general biosystems (Anhui) and were committed to be ligated between BamHI and PstI cleavage sites on pETDuet-1 vector (novagen, 71146-3), to give the following recombinant vectors:
The recombinant vector pETDuet-Bmgdh is obtained by replacing the sequence between the BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 1 and keeping other sequences unchanged;
the recombinant vector pETDuet-Tagdh is obtained by replacing the sequence between the BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 3 and keeping other sequences unchanged;
the recombinant vector pETDuet-Lstdh is obtained by replacing the sequence between the BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 5 and keeping other sequences unchanged;
the recombinant vector pETDuet-Bsgdh is obtained by replacing the sequence between the BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 7 and keeping other sequences unchanged;
the recombinant vector pETDuet-Bagdh is obtained by replacing the sequence between the BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 9 and keeping other sequences unchanged.
6. Construction of shikimate dehydrogenase expression vector
Shikimate dehydrogenase catalyzes the synthesis of shikimate from 3-dehydroshikimate. The NCBI database is searched for the shikimate dehydrogenase gene aroE of Escherichia coli, which contains the nucleotide sequence shown as SEQ ID NO. 11 and has the accession number NC-000913.3 REGION:complex (3430020.. 3430838). Designing a primer aroE-NdeI-F/aroE-XhoI-R, amplifying an aroE full-length sequence by taking escherichia coli str.K-12 substre.MG1655 genome DNA as a template, and connecting the amplified aroE full-length sequence between NdeI and XhoI sites on the vector obtained in the step 5 after double digestion by NdeI and XhoI to construct gdh and aroE coexpression vectors respectively:
The recombinant vector pETDuet-Bmgdh-aroE is obtained by replacing the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector with the sequence shown in SEQ ID NO. 1, replacing the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keeping other sequences unchanged;
the recombinant vector pETDuet-Tagdh-aroE is obtained by replacing the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 3, replacing the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keeping other sequences unchanged;
the recombinant vector pETDuet-Lstdh-aroE is obtained by replacing the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector with the sequence shown in SEQ ID NO. 5, replacing the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keeping other sequences unchanged;
the recombinant vector pETDuet-Bsgdh-aroE is obtained by replacing the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector with the sequence shown in SEQ ID NO. 7, replacing the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keeping other sequences unchanged;
The recombinant vector pETDuet-Bagdh-aroE is obtained by replacing the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector with the sequence shown in SEQ ID NO. 9, replacing the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keeping other sequences unchanged;
recombinant vector pRSFDuet-Bmgdh-aroE, replace the sequence between BamHI and PstI cleavage sites of pRSFDuet-1 vector (novagen, 71341-3) with the sequence shown in SEQ ID NO. 1, replace the sequence between NdeI and XhoI cleavage sites with the sequence shown in SEQ ID NO. 11, and keep other sequences unchanged;
wherein, the plasmid map of pETDuet-Bmgdh-aroE is shown in FIG. 2, and the plasmid map of pRSFDuet-Bmgdh-aroE is shown in FIG. 3.
7. Construction of D-3-phosphoglycerate dehydrogenase expression vector
D-3-phosphoglycerate dehydrogenase participates in the first catalytic reaction in the L-serine synthesis pathway. The encoding gene serA of the escherichia coli D-3-phosphoglycerate dehydrogenase is searched in NCBI database, and comprises a nucleotide sequence shown as SEQ ID NO. 13, wherein the encoded amino acid sequence is shown as SEQ ID NO. 14, and the accession number is CP001509.3REGION:complex (2888036.. 2889453). Primers serA-HR-F/serA-HR-R were designed, the full-length sequence of serA was amplified using the genomic DNA of E.coli BL21 (DE 3) as a template, and ligated to pRSFDuet-Bmgdh-aroE vector following XhoI site to construct gdh, aroE, serA co-expression vector pRSFDuet-Bmgdh-aroE-serA, whose plasmid map is shown in FIG. 4.
8. D-3-phosphoglycerate dehydrogenase gene knockout
(1) Designing a primer: the serA on the E.coli BL21 (DE 3) genome was knocked out using the CRISPR-Cas9 technology reported in the literature (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl. Environ. Microbiol.,2015, 81:2506-2514.). According to the CP001509.3 genome sequence, two pairs of primers serA-D-1F/serA-D-1R and serA-D-2F/serA-D-2R for amplifying homologous recombination fragments used for knocking out serA and an N20-sgRNA expression vector amplification primer serA-N20-F are designed.
(2) Preparation of homologous recombination fragments: amplifying a left homologous arm serA-F by using escherichia coli BL21 (DE 3) genome DNA as a template and a serA-D-1F/serA-D-1R primer, and amplifying a right homologous arm serA-R by using a serA-D-2F/serA-D-2R primer; the homologous recombination fragments serA-FR are formed by using two homologous arm fragments serA-F and serA-R as templates and using serA-D-1F/serA-D-2R primers to connect the serA-F and the serA-R through fusion PCR amplification technology.
(3) Construction of an N20-sgRNA expression vector: amplifying the full-length sequence of pTarget-serA-N20 by using the pTarget F plasmid as a template and a serA-N20-F/pTarget F-R primer; the pTarget-serA-N20 fragment was digested with SpeI and circularized from the ligation to form the expression vector pTarget-serA-N20.
(4) Preparation of competent cells: e.coli BL21 (DE 3) single colony carrying pCas plasmid is selected and inoculated in 3mL of liquid LB culture medium containing 50 mug/mL kanamycin, and cultured for 12-16h at 30 ℃ and 250rpm to be used as seed liquid; 200. Mu.L of seed solution was inoculated into 30mL of liquid LB medium containing 3%L-arabinose (w/v) and 50. Mu.g/mL kanamycin, and cultured at 30℃at 250rpm to OD 600 About 0.5 to about 0.6; ice bath for 15min, transferring the bacterial liquid into a precooled 50mL centrifuge tube, centrifuging for 5min at 2000g, and discarding the supernatant; re-suspending the bacterial pellet with 10mL pre-chilled 10% glycerol (v/v), centrifuging for 5min at 5000g, discarding the supernatant, and repeating this step twice; the cell pellet was resuspended with 300. Mu.L of pre-chilled 10% glycerol (v/v) to give electrotransformation competent cells.
(5) Electric conversion and verification: 100ng of pTarget-serA-N20 plasmid and 400ng of homologous recombination fragment serA-FR were mixed with 50. Mu.L of competent cells, added to a 2mm electric beaker and ice-incubated for 5min; wiping the electric rotating cup, and then placing the electric rotating cup into an electroporation apparatus for transformation under the condition of 2.5kV; immediately adding 1mL of precooled liquid LB culture medium into the electric rotating cup, and carrying out ice bath for 5min; transferring the bacterial liquid into a 2mL sterile centrifuge tube, and culturing for 2h at 30 ℃ and 250 rpm; centrifuging at 8000rpm for 1min, discarding most supernatant, coating the rest bacterial liquid on solid LB culture medium containing 50 μg/mL kanamycin and 50 μg/mL spectinomycin, and culturing at 30deg.C until colony is grown; and (3) selecting a plurality of monoclonal antibodies to perform colony PCR verification and sample feeding sequencing, wherein the primers are serA-D-1F/serA-D-2R, and the clone with correct sequencing is the serA knockout strain.
(6) Plasmid elimination: when the pTargetF-serA-N20 plasmid is eliminated, inoculating cells into a liquid LB culture medium containing 50 mug/mL kanamycin and 0.5mM IPTG, culturing for 8-16h at 30 ℃ at 250rpm, streaking on a solid LB culture medium containing 50 mug/mL kanamycin, culturing at 30 ℃ until colonies grow out, and picking out colonies sensitive to spectinomycin, namely the strains which succeed in eliminating the pTargetF-serA-N20 plasmid; when pCas plasmid is eliminated, cells are inoculated in a liquid LB culture medium, cultured for 8-12h at 37 ℃ and 250rpm, streaked on a solid LB culture medium, cultured until bacterial colonies grow out at 37 ℃, and bacterial colonies sensitive to kanamycin are selected to obtain the bacterial strain which is successful in eliminating pCas plasmid.
TABLE 1 primer sequences for PCR amplification
9. Shake flask culture of genetically engineered recombinant strains
According to the classical recombinant escherichia coli culture and induction expression scheme, selecting a single colony of the recombinant escherichia coli, inoculating the single colony into 3mL of LB culture medium containing 100 mug/mL ampicillin or 50 mug/mL kanamycin, and culturing at 37 ℃ for 10-12h at 250rpm to obtain seed liquid; the seed solution was transferred to 150mL of LB medium containing 100. Mu.g/mL ampicillin or 50. Mu.g/mL kanamycin at an inoculum size of 0.5%, and cultured at 37℃at 250rpm to OD 600 Reaching 0.4-0.8, adding IPTG with the final concentration of 0.1-1.0mM, and inducing expression for 6-24h at the temperature of 16-37 ℃ and 220 rpm; After the induction expression is finished, centrifuging at 8000rpm for 5min, and collecting cells to obtain the whole cell catalyst.
Wherein reference is made to cell OD upon induction of expression 600 The IPTG addition concentration, the induction temperature and the induction time can be correspondingly adjusted according to the actual growth condition. For example:
cell OD 600 The induction is started when the temperature reaches 0.4-0.8, and the specific time is 0.4 or 0.5 or 0.6 or 0.7 or 0.8;
IPTG is added at a concentration of 0.1 to 1.0mM, specifically 0.1mM or 0.2mM or 0.4mM or 0.6mM or 0.8mM or 1.0mM, etc.;
the induction temperature is 16-37deg.C, specifically 16 deg.C or 20 deg.C or 25 deg.C or 30 deg.C or 34 deg.C or 37 deg.C, etc.;
the induction time is 6-24h, and can be specifically 6h or 8h or 12h or 16h or 20h or 24h, etc.;
10. 5L tank batch fed-batch fermentation culture of genetically engineered recombinant strain
(1) Culturing test tube seeds: a single colony of recombinant E.coli was picked and inoculated into 3mL of LB medium containing 100. Mu.g/mL ampicillin or 50. Mu.g/mL kanamycin, and cultured at 37℃at 250rpm for 10-12 hours.
(2) Shake flask seed culture: the tube seed solution was transferred to 200mL of LB medium containing 100. Mu.g/mL ampicillin or 50. Mu.g/mL kanamycin at an inoculum size of 0.5%, and cultured at 37℃for 10-12 hours at 250 rpm.
(3) Culturing in a 5L fermentation tank: transferring the shake flask seed liquid into 2.3L fermentation tank culture medium containing 100 μg/mL ampicillin or 50 μg/mL kanamycin or without antibiotics according to 8% inoculation amount, wherein the initial fermentation temperature is 37 ℃, the aeration rate is 1vvm, the rotation speed of stirring pulp is gradually increased in the fermentation process to maintain the dissolved oxygen in the fermentation liquid to be more than 30%, and the fermentation pH value is controlled to be 7.0 by 25% ammonia water; waiting for cell OD 600 When the fermentation temperature reaches 20-50, adjusting the fermentation temperature to 22-37 ℃, adding 0.1-1.0mM IPTG, and inducing expression for 6-16h; when dissolved oxygen rises in the fermentation process, feeding is started, and the feeding speed of a feeding culture medium is controlled, so that the concentration of glycerol in the culture medium is always lower than 5g/L; after the induction expression is finished, the whole cell catalyst is obtained by centrifuging at 6000rpm for 20min and collecting cells.
Among them, the mentioned seed culture medium is LB medium, including tryptone 10g/L, yeast extract 5g/L, naCl g/L.
Wherein the dosage of each component in the fermentation tank culture medium is adjusted according to actual requirement, and comprises glucose 2-10g/L, glycerol 2-20g/L, yeast extract 5-25g/L, tryptone 0-20g/L, K 2 HPO 4 ·3H 2 O2-10g/L、NaH 2 PO 4 ·2H 2 O 1-5g/L、NaCl 2-10g/L、(NH 4 ) 2 SO 4 2-10g/L, citric acid monohydrate 1-5g/L, mgSO 4 ·7H 2 O 0.2-2g/L、FeSO 4 ·7H 2 O 0.05-0.5g/L。
For example: the glucose content in the culture conditions is 2-10g/L, and specifically can be 2g/L, or 4g/L, or 6g/L, or 8g/L, or 10g/L, etc.; the glycerol content is 2-20g/L, and can be specifically 2g/L, 4g/L, 8g/L, 12g/L, 16g/L, 20g/L, etc.; the yeast extract content is 5-25g/L, specifically 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, etc.; the tryptone content is 0-20g/L, and can be specifically 0g/L or 5g/L or 10g/L or 15g/L or 20g/L, etc.; k (K) 2 HPO 4 ·3H 2 The O content is 2-10g/L, and can be specifically 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, etc.; naH (NaH) 2 PO 4 ·2H 2 The O content is 1-5g/L, and can be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, etc.; the NaCl content is 2-10g/L, and can be specifically 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, etc.; (NH) 4 ) 2 SO 4 The content is 2-10g/L, and can be specifically 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, etc.; the citric acid monohydrate content is 1-5g/L, and can be 1g/L or 2g/L or 3g/L or 4g/L or 5 g/L; mgSO (MgSO) 4 ·7H 2 The O content is 0.2-2g/L, and can be specifically 0.2g/L or 0.4g/L or 0.8g/L or 1.2g/L or 1.6g/L or 2.0g/L, etc.; feSO 4 ·7H 2 The O content is 0.05-0.5g/L, and can be specifically 0.05g/L or 0.1g/L or 0.2g/L or 0.3g/L or 0.4g/L or 0.5g/L, etc.
Wherein, the dosage of each component of the feed medium of the fermentation tank is correspondingly adjusted according to actual needs, and comprises 100-600g/L of glycerin, 50-150g/L of yeast extract and 50-200g/L of tryptone. For example: the glycerol content is 100-600g/L, and can be 100g/L, 200g/L, 300g/L, 400g/L, 500g/L or 600g/L; the yeast extract content is 50-150g/L, specifically 50g/L or 75g/L or 100g/L or 125g/L or 150g/L; the tryptone content is 50-200g/L, and can be specifically 50g/L or 75g/L or 100g/L or 125g/L or 150g/L or 175g/L or 200g/L.
Wherein reference is made to cell OD upon induction of expression 600 The IPTG addition concentration, the induction temperature and the induction time can be correspondingly adjusted according to the actual growth condition. For example: cell OD 600 The induction is started when reaching 20-50, and can be specifically 20 or 25 or 30 or 35 or 40 or 45 or 50, etc.; IPTG is added at a concentration of 0.1 to 1.0mM, specifically 0.1mM or 0.2mM or 0.4mM or 0.6mM or 0.8mM or 1.0mM, etc.; the induction temperature is 22-37deg.C, specifically 22 deg.C or 25 deg.C or 28 deg.C or 30 deg.C or 34 deg.C or 37 deg.C, etc.; the induction time is 6-18h, and can be specifically 6h or 8h or 10h or 12h or 14h or 16h or 18h, etc.
11. Obtaining 3-dehydroshikimic acid fermentation liquor
The escherichia coli recombinant strain WJ060 (with the strain preservation number of CGMCC No. 14602) disclosed in the patent (CN 201711002831.2) and a fermentation production process thereof are utilized, in an inorganic salt culture medium taking glucose as a carbon source, a 5L tank is utilized for fed-batch fermentation to obtain a fermentation liquor containing high-concentration 3-dehydroshikimic acid, and the fermentation liquor is used as a 3-dehydroshikimic acid substrate of a whole-cell catalytic reaction.
12. Whole cell catalytic system
(1) Low substrate concentration catalysis: the whole cell catalytic reaction of 3-dehydroshikimic acid with low concentration is carried out in a 50mL centrifuge tube, 100mM sodium phosphate buffer (pH 5.0-9.0), the concentration of 3-dehydroshikimic acid is 100mM, the concentration of glucose is 150mM, and the concentration of the whole cell catalyst is 0.1-20OD 600 The reaction system is 10mL, the reaction temperature is 30-40 ℃, the reaction time is 0.5-24h, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by using a high performance liquid chromatograph.
Wherein, the concentration of the whole cell catalyst, the reaction temperature, the reaction pH and the reaction time can be correspondingly adjusted according to the actual catalysis condition. For example: the concentration of the whole cell catalyst is 0.1-20OD 600 Specifically, it may be 0.1 or 0.5 or 1 or 3 or 5 or 10 or 15 or 20, etc.; the reaction temperature is 30-40deg.C, specifically 30 deg.C or 34 deg.C or 37 deg.C or 40 deg.C, etc.; the reaction pH is 5.0-9.0, and can be specifically 5.0 or 6.0 or 7.0 or 8.0 or 9.0, etc.; the reaction time is 0.5-24h, and can be specifically 0.5h or 1.5h or 3h or 6h or 9h or 12h or 24h, etc.
(2) High substrate concentration catalysis: the high-concentration 3-dehydroshikimic acid whole cell catalytic reaction is carried out in a 5L fermentation tank, the concentration of the 3-dehydroshikimic acid is 40-120g/L, the concentration of dextrose monohydrate is 50-200g/L, and the concentration of the whole cell catalyst is 10-100OD 600 Controlling the reaction pH to 5.0-9.0 with 10M NaOH at 30-40 deg.C, the reaction time to 0.5-24h, the aeration rate to 0-1vvm and the reaction volume to 1-4L, and measuring the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid by high performance liquid chromatograph.
Wherein, the concentration of the 3-dehydroshikimic acid, the concentration of the dextrose monohydrate, the concentration of the whole cell catalyst, the reaction temperature, the reaction pH, the reaction time, the ventilation and the reaction volume can be correspondingly adjusted according to the actual catalysis condition. For example:
the concentration of the 3-dehydroshikimic acid is 40-120g/L, and can be specifically 40g/L or 60g/L or 80g/L or 100g/L or 120g/L, etc.;
the concentration of the dextrose monohydrate is 50-200g/L, and can be particularly 50g/L or 75g/L or 100g/L or 125g/L or 150g/L or 175g/L or 200g/L or the like;
the concentration of the whole cell catalyst is 10-100OD 600 Specifically, 10 or 20 or 40 or 60 or 80 or 100, etc.;
the reaction temperature is 30-40deg.C, specifically 30 deg.C or 34 deg.C or 37 deg.C or 40 deg.C, etc.;
the reaction pH is 5.0-9.0, and can be specifically 5.0 or 6.0 or 7.0 or 8.0 or 9.0, etc.;
the reaction time is 0.5-24h, and can be specifically 0.5h or 1.5h or 3h or 6h or 9h or 12h or 18h or 24h, etc.;
the ventilation amount is 0-1 vm, specifically 0 vm or 0.2 vm or 0.4 vm or 0.6 vm or 0.8 vm or 1 vm, etc.;
the reaction volume is 1 to 4L, specifically 1L or 2L or 3L or 4L, etc.
13. Detection analysis of samples
3-dehydroshikimic acid, shikimic acid and glycyrrhizic acid in fermentation liquidQuantitative analysis of oil: centrifuging the fermentation broth sample at 12000rpm for 10min, diluting the supernatant with distilled water for a certain multiple, and filtering with 0.22 μm water system microporous filter membrane; adopting an Agilent1200 high performance liquid chromatograph, and providing a VWD ultraviolet detector and a RID differential refraction detector, wherein a chromatographic Column is a Phenomenex Rezex RFQ-Fast Acid H+ (8%) (LC Column 100X 7.8 mm); the chromatographic conditions were mobile phase 5mM H 2 SO 4 The sample amount of the aqueous solution is 5 mu L, the flow rate is 0.6mL/min, the column temperature is 55 ℃, and the detection wavelength is 210nm.
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is described in detail below with reference to the embodiments. It should be noted that the specific embodiments described herein are for the purpose of illustrating the invention only and are not to be construed as limiting the invention.
Example 1
Screening for efficient glucose dehydrogenase
Glucose dehydrogenase expression strain construction: for screening for high efficiency glucose dehydrogenases, 5 glucose dehydrogenase genes from different species were selected from NCBI database, bmgdh from Bacillus megaterium WSH-002, tagdh from Thermoplasma acidophilum, lsgdh from Lysinibacillus sphaericus G10, bsgdh from Bacillus subtilis 9902, bagdh from Bacillus amyloliquefaciens DSM 7, respectively. These genes were ligated between BamHI and PstI sites on pETDuet-1 vector, respectively, and transferred into E.coli BL21 (DE 3) to obtain the corresponding recombinant strain.
Induction of expression: the seed solution of the recombinant strain was transferred to 150mL of LB medium containing 100. Mu.g/mL ampicillin at an inoculum size of 0.5%, and cultured at 37℃at 250rpm to OD 600 Reaching 0.6, adding IPTG with the final concentration of 0.2mM, and carrying out induction expression for 10 hours at the temperature of 28 ℃; after the induction expression is finished, centrifuging at 8000rpm for 5min, and collecting cells for enzyme activity detection.
And (3) enzyme activity detection: adding the extracting solution according to the specification of glucose dehydrogenase activity detection kit produced by Beijing box manufacturing technology Co., ltd, ultrasonically crushing cells for 5min, ultrasonically treating with 20% power for 3s, centrifuging at intervals of 7s at a temperature of 8000g for 10min, and taking the supernatant for enzyme activity detection. Definition of enzyme activity: catalytic production of 1nmol NADH per ten thousand cells per minute is defined as one unit of enzyme activity. As a result, as shown in Table 2, among the 5 candidate glucose dehydrogenases, bmGDH derived from Bacillus megaterium WSH-002 had the highest enzymatic activity.
TABLE 2 comparison of enzyme Activity of different glucose dehydrogenases
Recombinant bacterium Protein name Enzyme Activity U/10 4 cells
E.coli BL21(DE3)/pETDuet-Bmgdh BmGDH 7.907
E.coli BL21(DE3)/pETDuet-Tagdh TaGDH 0.004
E.coli BL21(DE3)/pETDuet-Lsgdh LsGDH 5.284
E.coli BL21(DE3)/pETDuet-Bsgdh BsGDH 6.532
E.coli BL21(DE3)/pETDuet-Bagdh BaGDH 3.867
Example 2
Optimal glucose dehydrogenase-shikimate dehydrogenase combination screening
Recombinant strain construction and induction expression: in order to screen the optimal catalytic combination of glucose dehydrogenase-shikimate dehydrogenase, combining shikimate dehydrogenase genes aroE derived from escherichia coli with the 5 glucose dehydrogenase genes gdh expression vectors in example 1 respectively, connecting the genes aroE with NdeI and XhoI sites on the pETDuet-gdh vector, and transferring the genes aroE into escherichia coli BL21 (DE 3) to obtain corresponding recombinant strains; whole cell catalysts co-expressed with aroE from different sources of gdh were obtained according to the inducible expression method of example 1.
Whole cell catalysis: 100mM sodium phosphate buffer (pH 7.0), 100mM 3-dehydroshikimic acid, 150mM glucose, 20OD were added sequentially to a 50mL centrifuge tube 600 The whole-cell catalyst, the reaction system is 10mL, the reaction temperature is 37 ℃, the reaction time is 3h, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by using a high performance liquid chromatograph. The results are shown in Table 3, with the highest shikimic acid yield of BmGDH-AroE, up to 92.98% relative to the molar conversion of 3-dehydroshikimic acid, among the 5 candidate combinations.
TABLE 3 comparison of the Whole-cell catalytic Effect of different GDH-AroE combinations
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Recombinant bacteria E.coli BL21 (DE 3)/pETDuet-Bmgdh-aroE with optimal catalytic effect combination BmGDH-aroE are named as WSA01.
Example 3
Whole cell catalyst dosage analysis
Whole cell catalyst acquisition: to test the whole cell catalytic ability of the WSA01 recombinant strain, thereby determining the amount of the whole cell catalyst used in the catalytic reaction, the WSA01 whole cell catalyst was obtained according to the inducible expression method of example 1.
Whole cell catalysis: sequentially adding 100mM sodium phosphate buffer (pH 7.0), 100mM 3-dehydroshikimic acid, 150mM glucose, and 0.1-20OD into 50mL centrifuge tube 600 The whole-cell catalyst, the reaction system is 10mL, the reaction temperature is 37 ℃, the reaction time is 3h, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by using a high performance liquid chromatograph. The results are shown in Table 4, wherein the shikimic acid yield gradually increases with the increase of the WSA01 whole cell catalyst amount; 5OD 600 The whole-cell catalyst is catalyzed for 3 hours, and the molar conversion rate of the substrate 3-dehydroshikimic acid can reach more than 80 percent.
TABLE 4 comparison of catalytic Effect with different amounts of Whole cell catalyst
WSA01 Whole cell catalyst usage (OD 600 ) Shikimic acid content (mM) Conversion (%)
0.1 0.45 0.45
0.5 7.23 7.23
1 15.19 15.19
3 73.30 73.30
5 83.22 83.22
10 86.25 86.25
15 94.35 94.35
20 95.15 95.15
Example 4
Whole cell catalytic reaction pH analysis
Testing WSA01 whole cell catalyzed optimum reaction pH: 100mM sodium phosphate buffer (pH 5.0 or 6.0 or 7.0 or 8.0 or 9.0), 100mM 3-dehydroshikimic acid, 150mM glucose, 3OD were added sequentially to 50mL centrifuge tubes 600 The whole-cell catalyst, the reaction system is 10mL, the reaction temperature is 37 ℃, the reaction time is 3h, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by using a high performance liquid chromatograph. The results are shown in Table 5, wherein the shikimic acid yield increases and decreases with increasing pH of the catalytic reaction; shikimic acid yield and conversion were highest at pH 7.0. WSA01 whole cell catalyzed optimum reaction pH was 7.0.
TABLE 5 comparison of Whole cell catalytic Effect under different pH conditions
Whole cell catalytic reaction pH Shikimic acid content (mM) Conversion (%)
5.0 2.54 2.54
6.0 12.89 12.89
7.0 22.68 22.68
8.0 17.02 17.02
9.0 4.23 4.23
Example 5
Whole cell catalytic reaction temperature analysis
Testing the WSA01 whole cell catalytic optimum reaction temperature: 100mM sodium phosphate buffer (pH 7.0), 100mM 3-dehydroshikimic acid, 150mM glucose, 3OD were added sequentially to a 50mL centrifuge tube 600 The whole cell catalyst, 10mL of reaction system, the reaction temperature of 30 ℃ or 34 ℃ or 37 ℃ or 40 ℃ and the reaction time of 3h are measured by using a high performance liquid chromatograph. The results are shown in Table 6Showing that as the catalytic reaction temperature increases, shikimic acid yield increases and then decreases; the shikimic acid yield and conversion were highest at 34 ℃. The optimal reaction temperature for WSA01 whole cell catalysis is 34 ℃.
TABLE 6 comparison of Whole cell catalytic Effect at different temperatures
Whole cell catalytic reaction temperature (. Degree. C.) Shikimic acid content (mM) Conversion (%)
30 22.81 22.81
34 27.55 27.55
37 26.01 26.01
40 22.99 22.99
Example 6
Whole cell catalytic effect analysis of different genetically engineered recombinant strains
The pRSFDuet-1 vector was obtained by replacing the sequence between BamHI and PstI cleavage sites with Bmgdh and the sequence between NdeI and XhoI cleavage sites with aroE, and the plasmid map was shown in FIG. 3. pRSFDuet-Bmgdh-aroE was transferred into E.coli BL21 (DE 3) to obtain recombinant strain E.coli BL21 (DE 3)/pRSFDuet-Bmgdh-aroE, which was designated WSA02.
The D-3-phosphoglycerate dehydrogenase encoding gene serA on the genome of E.coli BL21 (DE 3) was knocked out using the CRISPR-Cas9 technology reported in the literature (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl. Environ. Microbiol.,2015, 81:2506-2514.), resulting in cells exhibiting an L-serine auxotrophic growth phenotype in mineral salts media, forming a novel protein expression host E.coli BL21 (DE 3) ΔserA. Ligating the serA gene to pRSFDuet-Bmgdh-aroE vector behind XhoI site to form pRSFDuet-Bmgdh-aroE-serA expression vector with plasmid map shown in FIG. 4; then it was transferred into a new host strain E.coli BL21 (DE 3) ΔserA to obtain a recombinant strain E.coli BL21 (DE 3) ΔserA/pRSFDuet-Bmgdh-aroE-serA, which was designated WSA03.
Carrying out fed-batch fermentation on WSA01, WSA02 and WSA03 respectively by using a 5L tank, and inducing protein expression by using IPTG to obtain a corresponding whole-cell catalyst, wherein the specific fed-batch fermentation process comprises the following steps:
WSA01 fermentation: selecting WSA01 single colony, inoculating the single colony into 3mL of LB medium containing 100 mug/mL ampicillin, and culturing at 37 ℃ for 12 hours at 250rpm to obtain test tube seed liquid; transferring the test tube seed liquid into 200mL LB culture medium containing 100 mug/mL ampicillin according to 0.5% inoculum size, and culturing at 37 ℃ for 12h at 250rpm to obtain shake flask seed liquid; transferring the shake flask seed liquid into 2.3L of fermentation tank culture medium containing 100 μg/mL ampicillin according to 8% inoculation amount, wherein the initial fermentation temperature is 37 ℃, the ventilation rate is 1vvm, the rotation speed of stirring slurry is gradually increased in the fermentation process to maintain the dissolved oxygen in the fermentation liquid above 30%, and the fermentation pH value is controlled to 7.0 by using 25% ammonia water; when OD is 600 When 29 is reached, the fermentation temperature is adjusted to 30 ℃, 0.5mM IPTG and 50 mug/mL ampicillin are added, and the induction and the expression are carried out for 14 hours; when dissolved oxygen rises in the fermentation process, feeding is started, the feeding speed of the feeding culture medium is controlled, and the glycerol concentration is kept to be lower than 5g/L all the time.
WSA02 fermentation: WSA02 single colony is selected and inoculated in 3mL LB culture medium containing 50 mug/mL kanamycin, and cultured for 12 hours at 37 ℃ at 250rpm to serve as test tube seed liquid; the test tube seed liquid is transferred into 200mL LB culture medium containing 50 mug/mL kanamycin according to the inoculation amount of 0.5 percent, and is cultured for 12 hours at 37 ℃ and 250rpm to be used as shake flask seed liquid; transferring the shake flask seed liquid into 2.3L fermentation tank culture medium containing 50 μg/mL kanamycin according to 8% inoculation amount, wherein the initial fermentation temperature is 37 ℃, the ventilation rate is 1vvm, the rotation speed of stirring pulp is gradually increased in the fermentation process to maintain dissolved oxygen in the fermentation liquid above 30%, and the fermentation pH value is controlled to 7.0 by 25% ammonia water; when OD is 600 When 27 is reached, the fermentation temperature is adjusted to 30 ℃, 0.5mM IPTG is added, and the induction expression is carried out for 14.5 hours; when dissolved oxygen rises in the fermentation process, feeding is started, the feeding speed of the feeding culture medium is controlled, and the glycerol concentration is kept to be lower than 5g/L all the time.
WSA03 fermentation: selecting WSA03 single colony, inoculating the single colony into 3mL LB culture medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ for 12 hours at 250rpm to obtain test tube seed liquid; the test tube seed liquid is transferred into 200mL LB culture medium containing 50 mug/mL kanamycin according to the inoculation amount of 0.5 percent, and is cultured for 12 hours at 37 ℃ and 250rpm to be used as shake flask seed liquid; transferring the shake flask seed liquid into 2.3L fermentation tank culture medium (without adding kanamycin) according to 8% inoculation amount, wherein the initial fermentation temperature is 37 ℃, the ventilation rate is 1vvm, the rotation speed of stirring pulp is gradually increased in the fermentation process to maintain the dissolved oxygen in the fermentation liquid above 30%, and the fermentation pH value is controlled to 7.0 by using 25% ammonia water; when OD is 600 When 25 is reached, the fermentation temperature is adjusted to 30 ℃, 0.5mM IPTG is added, and the induction expression is carried out for 10 hours; when dissolved oxygen rises in the fermentation process, feeding is started, the feeding speed of the feeding culture medium is controlled, and the glycerol concentration is kept to be lower than 5g/L all the time.
Fermentation tank culture medium components: glucose 2g/L, glycerol 10g/L, yeast extract 10g/L, tryptone 16g/L, K 2 HPO 4 ·3H 2 O 4g/L、NaH 2 PO 4 ·2H 2 O 2g/L、NaCl 3g/L、(NH 4 ) 2 SO 4 2.5g/L, citric acid monohydrate 2g/L, mgSO 4 ·7H 2 O 0.5g/L、FeSO 4 ·7H 2 O 0.3g/L。
The feed medium comprises the following components: glycerol 600g/L, yeast extract 125g/L, tryptone 175g/L.
Whole cell catalysis: 100mM sodium phosphate buffer (pH 7.0), 100mM 3-dehydroshikimic acid, 150mM glucose, 5OD were added sequentially to a 50mL centrifuge tube 600 The whole-cell catalyst (WSA 01, WSA02 or WSA 03) has a reaction system of 10mL, a reaction temperature of 34 ℃ and a reaction time of 1.5h, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by using a high performance liquid chromatograph.
As a result, the results are shown in Table 7, WSA01 was fermented for 21 hours, OD 600 Up to 106.8, whole cell catalysis for 1.5h, shikimic acid content up to 59.04mM, relative to molar conversion of 3-dehydroshikimic acid 59.04%; WSA02 fermentation for 22h, OD 600 Up to 100.4, whole cell catalysis for 1.5h, shikimic acid content up to 92.79mM, relative to molar conversion of 3-dehydroshikimic acid 92.79%; WSA03 fermentation for 19h, OD 600 Up to 72.1, whole cell catalysis for 1.5h, shikimic acid content up to 90.48mM, relative to the molar conversion of 3-dehydroshikimic acid of 90.48%. The WSA02 and WSA03 have better catalytic effects, and the stable inheritance of plasmids and better protein expression effects can be maintained without adding kanamycin in a WSA03 feed fermentation medium, so that the subsequent processing difficulty and environmental pollution caused by using antibiotics are avoided.
TABLE 7 comparison of the growth and Whole cell catalytic Effect of different recombinant bacteria
Recombinant strains Fermentation time (h) Fermentation OD 600 Shikimic acid content (mM) Conversion (%)
WSA01 21 106.8 59.04 59.04
WSA02 22 100.4 92.79 92.79
WSA03 19 72.1 90.48 90.48
Example 7
Analysis of catalytic effect of high concentration substrate
The WSA03 whole cell is used for catalyzing high-concentration 3-dehydroshikimic acid to synthesize shikimic acid. The catalytic reaction is carried out in a 5L fermentation tank, the concentration of 3-dehydroshikimic acid in the fermentation liquid is 90.49g/L (the fermentation liquid contains thalli), 3L of fermentation liquid is added, the adding amount of glucose monohydrate is 343.77g, the dosage of whole cell catalyst is 69.45g (the final concentration is 20 OD) 600 The wet weight of the thalli is 23.15 g/L), the reaction temperature is 34 ℃, 10M NaOH is used for controlling the pH value to 7.0 in the catalytic process, the reaction time is 3 hours, the ventilation rate is 0vvm, the initial volume is about 3.25L, and the content of 3-dehydroshikimic acid and shikimic acid in the reaction liquid is measured by a high performance liquid chromatograph.
Results: since glucose, whole-cell catalyst and 10M NaOH are required to be added in the catalytic reaction, the final volume after 3h of catalysis is 3.40L, the 3-dehydroshikimic acid residue in the reaction liquid is 2.15g/L (containing thalli), the shikimic acid yield is 79.55g/L (containing thalli), and the molar conversion rate is 98.49 percent relative to the 3-dehydroshikimic acid. The catalytic reaction time is prolonged, and the conversion rate of the 3-dehydroshikimic acid can be further improved.
The above-mentioned novel production method for synthesizing shikimic acid by using whole cell catalysis 3-dehydroshikimic acid established by using shikimate dehydrogenase and glucose dehydrogenase, construction and fermentation culture method of genetic engineering recombinant strain, whole cell catalysis effect analysis and the like are only preferred embodiments of the present application, and are not intended to limit the present application. Any modification, equivalent replacement, improvement optimization and the like which are made on the basis of the technical principle of the application are included in the protection scope of the application.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> a method for producing shikimic acid by whole cell catalysis
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atgtacaccg atctgaaagg caaagttgtg gcaattaccg gtgcaagcag tggtctgggc 60
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gcagatccgg ttcagaaaaa agatgttgaa agcatgattc cgatgggcta tattggtgaa 660
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Met Tyr Thr Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ser
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Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Val
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Asn Pro Val Glu Ser His Lys Met Pro Leu Lys Asp Trp Asn Lys Val
100 105 110
Ile Asn Thr Asn Leu Thr Gly Ala Phe Leu Gly Cys Arg Glu Ala Ile
115 120 125
Lys Tyr Tyr Val Glu Asn Asp Ile Gln Gly Asn Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Met Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
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Ala Pro Lys Arg Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Val Gln Lys Lys Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Val Trp Leu Ala Ser Lys Glu Ser Ser Tyr Val Thr
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Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
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Gln Ala Gly Arg Gly
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<210> 11
<211> 819
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atggaaacct atgctgtttt tggtaatccg atagcccaca gcaaatcgcc attcattcat 60
cagcaatttg ctcagcaact gaatattgaa catccctatg ggcgcgtgtt ggcacccatc 120
aatgatttca tcaacacact gaacgctttc tttagtgctg gtggtaaagg tgcgaatgtg 180
acggtgcctt ttaaagaaga ggcttttgcc agagcggatg agcttactga acgggcagcg 240
ttggctggtg ctgttaatac cctcatgcgg ttagaagatg gacgcctgct gggtgacaat 300
accgatggtg taggcttgtt aagcgatctg gaacgtctgt cttttatccg ccctggttta 360
cgtattctgc ttatcggcgc tggtggagca tctcgcggcg tactactgcc actcctttcc 420
ctggactgtg cggtgacaat aactaatcgg acggtatccc gcgcggaaga gttggctaaa 480
ttgtttgcgc acactggcag tattcaggcg ttgagtatgg acgaactgga aggtcatgag 540
tttgatctca ttattaatgc aacatccagt ggcatcagtg gtgatattcc ggcgatcccg 600
tcatcgctca ttcatccagg catttattgc tatgacatgt tctatcagaa aggaaaaact 660
ccttttctgg catggtgtga gcagcgaggc tcaaagcgta atgctgatgg tttaggaatg 720
ctggtggcac aggcggctca tgcctttctt ctctggcacg gtgttctgcc tgacgtagaa 780
ccagttataa agcaattgca ggaggaattg tccgcgtaa 819
<210> 12
<211> 272
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Met Glu Thr Tyr Ala Val Phe Gly Asn Pro Ile Ala His Ser Lys Ser
1 5 10 15
Pro Phe Ile His Gln Gln Phe Ala Gln Gln Leu Asn Ile Glu His Pro
20 25 30
Tyr Gly Arg Val Leu Ala Pro Ile Asn Asp Phe Ile Asn Thr Leu Asn
35 40 45
Ala Phe Phe Ser Ala Gly Gly Lys Gly Ala Asn Val Thr Val Pro Phe
50 55 60
Lys Glu Glu Ala Phe Ala Arg Ala Asp Glu Leu Thr Glu Arg Ala Ala
65 70 75 80
Leu Ala Gly Ala Val Asn Thr Leu Met Arg Leu Glu Asp Gly Arg Leu
85 90 95
Leu Gly Asp Asn Thr Asp Gly Val Gly Leu Leu Ser Asp Leu Glu Arg
100 105 110
Leu Ser Phe Ile Arg Pro Gly Leu Arg Ile Leu Leu Ile Gly Ala Gly
115 120 125
Gly Ala Ser Arg Gly Val Leu Leu Pro Leu Leu Ser Leu Asp Cys Ala
130 135 140
Val Thr Ile Thr Asn Arg Thr Val Ser Arg Ala Glu Glu Leu Ala Lys
145 150 155 160
Leu Phe Ala His Thr Gly Ser Ile Gln Ala Leu Ser Met Asp Glu Leu
165 170 175
Glu Gly His Glu Phe Asp Leu Ile Ile Asn Ala Thr Ser Ser Gly Ile
180 185 190
Ser Gly Asp Ile Pro Ala Ile Pro Ser Ser Leu Ile His Pro Gly Ile
195 200 205
Tyr Cys Tyr Asp Met Phe Tyr Gln Lys Gly Lys Thr Pro Phe Leu Ala
210 215 220
Trp Cys Glu Gln Arg Gly Ser Lys Arg Asn Ala Asp Gly Leu Gly Met
225 230 235 240
Leu Val Ala Gln Ala Ala His Ala Phe Leu Leu Trp His Gly Val Leu
245 250 255
Pro Asp Val Glu Pro Val Ile Lys Gln Leu Gln Glu Glu Leu Ser Ala
260 265 270
<210> 13
<211> 1233
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atggcaaagg tatcgctgga gaaagacaag attaagtttc tgctggtaga aggcgtgcac 60
caaaaggcgc tggaaagcct tcgtgcagct ggttacacca acatcgaatt tcacaaaggc 120
gcgctggatg atgaacaatt aaaagaatcc atccgcgatg cccacttcat cggcctgcga 180
tcccgtaccc atctgactga agacgtgatc aacgccgcag aaaaactggt cgctattggc 240
tgtttctgta tcggaacaaa ccaggttgat ctggatgcgg cggcaaagcg cgggatcccg 300
gtatttaacg caccgttctc aaatacgcgc tctgttgcgg agctggtgat tggcgaactg 360
ctgctgctat tgcgcggcgt gccggaagcc aatgctaaag cgcaccgtgg cgtgtggaac 420
aaactggcgg cgggttcttt tgaagcgcgc ggcaaaaagc tgggtatcat cggctacggt 480
catattggta cgcaattggg cattctggct gaatcgctgg gaatgtatgt ttacttttat 540
gatattgaaa ataaactgcc gctgggcaac gccactcagg tacagcatct ttctgacctg 600
ctgaatatga gcgatgtggt gagtctgcat gtaccagaga atccgtccac caaaaatatg 660
atgggcgcga aagaaatttc actaatgaag cccggctcgc tgctgattaa tgcttcgcgc 720
ggtactgtgg tggatattcc ggcgctgtgt gatgcgctgg cgagcaaaca tctggcgggg 780
gcggcaatcg acgtattccc gacggaaccg gcgaccaata gcgatccatt tacctctccg 840
ctgtgtgaat tcgacaacgt ccttctgacg ccacacattg gcggttcgac tcaggaagcg 900
caggagaata tcggcctgga agttgcgggt aaattgatca agtattctga caatggctca 960
acgctctctg cggtgaactt cccggaagtc tcgctgccac tgcacggtgg gcgtcgtctg 1020
atgcacatcc acgaaaaccg tccgggcgtg ctaactgcgc tgaacaaaat cttcgccgag 1080
cagggcgtca acatcgccgc gcaatatctg caaacttccg cccagatggg ttatgtggtt 1140
attgatattg aagccgacga agacgttgcc gaaaaagcgc tgcaggcaat gaaagctatt 1200
ccgggtacca ttcgcgcccg tctgctgtac taa 1233
<210> 14
<211> 410
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Met Ala Lys Val Ser Leu Glu Lys Asp Lys Ile Lys Phe Leu Leu Val
1 5 10 15
Glu Gly Val His Gln Lys Ala Leu Glu Ser Leu Arg Ala Ala Gly Tyr
20 25 30
Thr Asn Ile Glu Phe His Lys Gly Ala Leu Asp Asp Glu Gln Leu Lys
35 40 45
Glu Ser Ile Arg Asp Ala His Phe Ile Gly Leu Arg Ser Arg Thr His
50 55 60
Leu Thr Glu Asp Val Ile Asn Ala Ala Glu Lys Leu Val Ala Ile Gly
65 70 75 80
Cys Phe Cys Ile Gly Thr Asn Gln Val Asp Leu Asp Ala Ala Ala Lys
85 90 95
Arg Gly Ile Pro Val Phe Asn Ala Pro Phe Ser Asn Thr Arg Ser Val
100 105 110
Ala Glu Leu Val Ile Gly Glu Leu Leu Leu Leu Leu Arg Gly Val Pro
115 120 125
Glu Ala Asn Ala Lys Ala His Arg Gly Val Trp Asn Lys Leu Ala Ala
130 135 140
Gly Ser Phe Glu Ala Arg Gly Lys Lys Leu Gly Ile Ile Gly Tyr Gly
145 150 155 160
His Ile Gly Thr Gln Leu Gly Ile Leu Ala Glu Ser Leu Gly Met Tyr
165 170 175
Val Tyr Phe Tyr Asp Ile Glu Asn Lys Leu Pro Leu Gly Asn Ala Thr
180 185 190
Gln Val Gln His Leu Ser Asp Leu Leu Asn Met Ser Asp Val Val Ser
195 200 205
Leu His Val Pro Glu Asn Pro Ser Thr Lys Asn Met Met Gly Ala Lys
210 215 220
Glu Ile Ser Leu Met Lys Pro Gly Ser Leu Leu Ile Asn Ala Ser Arg
225 230 235 240
Gly Thr Val Val Asp Ile Pro Ala Leu Cys Asp Ala Leu Ala Ser Lys
245 250 255
His Leu Ala Gly Ala Ala Ile Asp Val Phe Pro Thr Glu Pro Ala Thr
260 265 270
Asn Ser Asp Pro Phe Thr Ser Pro Leu Cys Glu Phe Asp Asn Val Leu
275 280 285
Leu Thr Pro His Ile Gly Gly Ser Thr Gln Glu Ala Gln Glu Asn Ile
290 295 300
Gly Leu Glu Val Ala Gly Lys Leu Ile Lys Tyr Ser Asp Asn Gly Ser
305 310 315 320
Thr Leu Ser Ala Val Asn Phe Pro Glu Val Ser Leu Pro Leu His Gly
325 330 335
Gly Arg Arg Leu Met His Ile His Glu Asn Arg Pro Gly Val Leu Thr
340 345 350
Ala Leu Asn Lys Ile Phe Ala Glu Gln Gly Val Asn Ile Ala Ala Gln
355 360 365
Tyr Leu Gln Thr Ser Ala Gln Met Gly Tyr Val Val Ile Asp Ile Glu
370 375 380
Ala Asp Glu Asp Val Ala Glu Lys Ala Leu Gln Ala Met Lys Ala Ile
385 390 395 400
Pro Gly Thr Ile Arg Ala Arg Leu Leu Tyr
405 410
<210> 15
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gtcgttgcat atggaaacct atgctgtttt tgg 33
<210> 16
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
cagtctcgag ttacgcggac aattcctcct g 31
<210> 17
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ggaggaattg tccgcgtaac tcgagcctgg ctattgtcga ttgctc 46
<210> 18
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gcagcggttt ctttaccaga ttagtacagc agacgggcg 39
<210> 19
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
cctggctatt gtcgattgct c 21
<210> 20
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ttacccaatc ctgtcttttg aaatg 25
<210> 21
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tcaaaagaca ggattgggta attccccttc tctgaaaatc aac 43
<210> 22
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gttacagccc catgctgcc 19
<210> 23
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ctgactagtt ctgttgcgga gctggtgatg ttttagagct agaaatagc 49
<210> 24
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
atgactagta ttatacctag gactgagc 28

Claims (15)

1. A system for producing shikimic acid by whole cell catalysis, which is characterized by comprising 3-dehydroshikimic acid, glucose and recombinant bacteria simultaneously expressing glucose dehydrogenase GDH and shikimate dehydrogenase aroE.
2. The system according to claim 1, wherein the concentration of 3-dehydroshikimic acid in the system is 1-120g/L; the concentration of glucose is 1-200g/L; the cell amount of the recombinant bacteria is 0.1-100OD 600 。
3. The system of claim 1, wherein the glucose dehydrogenase comprises an amino acid sequence as set forth in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8 or SEQ ID No. 10; the shikimate dehydrogenase comprises an amino acid sequence shown as SEQ ID NO. 12.
4. The system according to claim 3, wherein the recombinant bacterium is Escherichia coli comprising a recombinant vector, and the recombinant vector comprises any one of the nucleotide sequences shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11.
5. The system of claim 4, wherein the recombinant vector is as set forth in any one of the following A1) -A7):
A1 A recombinant vector pETDuet-Bmgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 1, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a2 A recombinant vector pETDuet-Tagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 3, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a3 A recombinant vector pETDuet-Lstdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 5, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a4 A recombinant vector pETDuet-Bsgdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 7, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
A5 A recombinant vector pETDuet-Bagdh-aroE, wherein the sequence between BamHI and PstI cleavage sites of the pETDuet-1 vector is replaced by the sequence shown in SEQ ID NO. 9, and the sequence between NdeI and XhoI cleavage sites is replaced by the sequence shown in SEQ ID NO. 11, and other sequences are kept unchanged;
a6 Recombinant vector pRSFDuet-Bmgdh-aroE, replacing the sequence between BamHI and XhoI sites of pRSFDuet-1 vector with the sequence between BamHI and XhoI sites of recombinant vector A1), and leaving the other sequences unchanged;
a7 Recombinant vector pRSFDuet-Bmgdh-aroE-serA, and the sequence shown in SEQ ID NO. 13 was inserted into the XhoI site of recombinant vector A6) and the other sequences were kept unchanged.
6. The system of claim 5, wherein the recombinant bacterium is any one of the following:
b1 Recombinant E.coli BL21 (DE 3)/pETDuet-Bmgdh-aroE, and transferring the recombinant vector pETDuet-Bmgdh-aroE into recombinant strain of E.coli BL21 (DE 3);
b2 Recombinant E.coli BL21 (DE 3)/pETDuet-Tagdh-aroE, and transferring the recombinant vector pETDuet-Tagdh-aroE into the recombinant strain obtained by the E.coli BL21 (DE 3);
B3 Recombinant E.coli BL21 (DE 3)/pETDuet-Lstdh-aroE, and transferring the recombinant vector pETDuet-Lstdh-aroE into E.coli BL21 (DE 3);
b4 Recombinant E.coli BL21 (DE 3)/pETDuet-Bsgdh-aroE, and transferring the recombinant vector pETDuet-Bsgdh-aroE into E.coli BL21 (DE 3);
b5 Recombinant E.coli BL21 (DE 3)/pETDuet-Bagdh-aroE, and transferring the recombinant vector pETDuet-Bagdh-aroE into the recombinant strain obtained by E.coli BL21 (DE 3);
b6 Recombinant E.coli BL21 (DE 3)/pRSFDuet-Bmgdh-aroE, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE into E.coli BL21 (DE 3);
b7 Recombinant E.coli BL21 (DE 3) ΔserA/pRSFDuet-Bmgdh-aroE-serA, and transferring the recombinant vector pRSFDuet-Bmgdh-aroE-serA into E.coli BL21 (DE 3) ΔserA, wherein E.coli BL21 (DE 3) ΔserA is a strain in which the D-3-phosphoglycerate dehydrogenase encoding gene serA on the genome of E.coli BL21 (DE 3) is knocked out.
7. The recombinant vector of claim 6.
8. The recombinant bacterium according to any one of claims 1 to 5.
9. Use of the system according to any one of claims 1-6, the recombinant vector according to claim 7, the recombinant bacterium according to claim 8 for the preparation of shikimic acid or a downstream shikimic acid containing product.
10. A method for producing shikimic acid by whole cell catalysis, which is characterized in that the recombinant bacterium of claim 8 is used for producing shikimic acid by whole cell catalysis by taking 3-dehydroshikimic acid fermentation liquor as a substrate.
11. The method of claim 10, wherein the whole cell catalyzed reaction has a pH of 5.0 to 9.0.
12. The method of claim 10, wherein the whole cell catalyzed reaction temperature is 30-40 ℃.
13. The method of claim 10, wherein the whole cell catalyzed reaction time is 0.5 to 24 hours.
14. The method according to claim 10, wherein nadp+ and/or NADPH is not added during the production process.
15. The method according to any one of claims 10-14, wherein the whole cell catalyst is obtained by inducing the expression of the glucose dehydrogenase gene and the shikimate dehydrogenase gene with isopropyl- β -D-thiogalactoside (IPTG) or lactose.
CN202210354152.6A 2022-04-06 2022-04-06 Method for producing shikimic acid by whole cell catalysis Pending CN116926089A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117736959A (en) * 2024-01-26 2024-03-22 湖北大学 Engineering strain of zymomonas mobilis, preparation method and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117736959A (en) * 2024-01-26 2024-03-22 湖北大学 Engineering strain of zymomonas mobilis, preparation method and application
CN117736959B (en) * 2024-01-26 2024-05-14 湖北大学 Engineering strain of zymomonas mobilis, preparation method and application

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