CN101134958A - Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby - Google Patents
Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby Download PDFInfo
- Publication number
- CN101134958A CN101134958A CNA2006101126657A CN200610112665A CN101134958A CN 101134958 A CN101134958 A CN 101134958A CN A2006101126657 A CNA2006101126657 A CN A2006101126657A CN 200610112665 A CN200610112665 A CN 200610112665A CN 101134958 A CN101134958 A CN 101134958A
- Authority
- CN
- China
- Prior art keywords
- shikimic acid
- gene
- polygene
- box
- ptacarofarobaroekan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses biosynthesis process of preparing shikimic acid and the recombinant colibacillus therefor. The biosynthesis process of preparing shikimic acid includes constituting vector including the polygene kit of shikimic acid metabolizing pathway rate-limiting enzyme, replacing the polygene kit for shiA gene, knocking out shikimate kinase isozyme with Red recombinant system, fermenting recombinant engineering bacteria to obtain shikimic acid, purifying shikimic acid and other steps. The process has high shikimic acid expressing yield and lowered shikimic acid producing cost, and can provide material for preparing Oseltamivir phosphate as influenza preventing and treating medicine.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to and a kind ofly prepare the method for shikimic acid, also relate to employed engineering bacteria in this method by biosynthesis technology.
Background technology
In recent years, bird flu has been proved to be can the direct infection mankind, broke with tremendous force the case fatality rate height.According to statistics, people surplus bird flu has infected 100, patient death wherein over half.By in October, 2005, the popular of H5N1 virus successively appearred in a countries and regions surplus Cambodia, the China etc. ten, still spreading so far.At present, national governments are all responding actively the big area outburst that bird flu may occur in the mankind.The influenza virus expert thinks that a new flu outbreak is inevitable, may be in the near future.In the discussion of on July 4th, 2005 by World Health Organization's hosting, scientists points out that bird flu is still dangerous, take precautions against it and be very popular in the mankind.
Severe day by day along with the bird flu situation, Tamiflu receives the concern of various countries as the main medicine of prevention and the bird flu of treatment H5N1 type, only United States Government's about 100,000,000 person-portions of plan deposit Tamiflu time for this reason just.The Tamiflu main active ingredient is an oseltamivir, and shikimic acid (shikimic acid) is the starting raw material of oseltamivir, and the production of shikimic acid is at present mainly extracted from the seed of Magnoliacea plant star anise by chemical extraction method.Because each state is all laying in this anti-avian influenza medicine of Tamiflu as much as possible, supply falls short of demand to cause shikimic acid, bring the shortage of starting material star anise thus, so chemical method exists the limited serious drawback of starting material, especially when flu outbreak and harvest are not good enough.
In addition, shikimic acid still is many alkaloids, arylamine acid, indole derivatives and chiral drug synthetic key precursor, its drug derivative major part still is in the experimental phase, have two: one development prospect that relatively receives publicity, antibiotic antitumor action, had in 1987 and mention Japanese scholar in the report and find that a kind of compound of shikimic acid is to hela cell strain (HeLacells) and Ai Xili ascites carcinoma Ehrlichascitescarcinoma) the obvious suppression effect arranged, and can prolong the survival time of the mouse of inoculation leukemia cell L1210, and toxicity is relatively low, and to indicate restraining effect mainly be because this kind shikimic acid compound and sulfohydrate react; Another kind of compound has certain antagonistic action to trichophyton mentagrophytes.And there is report (people such as Sun Kuailin) synthetic shikimic acid derivative to have effect (Sun Kuailin with the similar vitro inhibition leukemia cell L1210 of ambomycin, Li Runsun, Lei Xinghan. the synthetic and bioactivity research of some shikimic acid derivatives. Acta Pharmaceutica Sinica, 1990, (1)).The 2nd, to the effect of cardiovascular systems, discoveries such as the Sun Jianning of Beijing University of Chinese Medicine, shikimic acid and derivative thereof have the effect (Sun Wenyan of antithrombotic formation and anticoagulant, Sun Jianning, Liu Zhenquan, yellow low-priced English, Zhang Wenbo. the inflammation mechanism pre-test " Chinese Pharmaceutical Journal " 2005,40 (9): 678～680 that iso propylidene shikimic acid Chinese People's Anti-Japanese Military and Political College mouse is cerebral ischemia re-pouring injured; Sun Wenyan, Sun Jianning, Liu Zhenquan, yellow low-priced English, Zhang Wenbo. iso propylidene shikimic acid is to arteria cerebri media ischemia-reperfusion rat provide protection " Beijing University of Chinese Medicine's journal " 2005,28 (3): 43～47; ).
Increasingly serious along with the bird flu situation guarantees to produce the required raw-material abundance of Tamiflu the needs that are international situation.The traditional processing technology complexity of shikimic acid, and raw material influenced by natural cause bigger, the plant extract mode that breaks traditions, the mode that adopts the bioengineered strain of environmental protection, cheapness, stable yield to produce shikimic acid is an international trend.Shikimic acid pathway is seen Fig. 1, is to be present in one of important pathways metabolism in microorganism, fungi and the plant, but is not present in the Mammals.Five important enzymes are arranged from glucose to the shikimic acid synthetic mesophase, be respectively 3-deoxidation-D-Arab-heptanone-7-phosphate synthase (3-deoxy-7-phosphoheptulonate synthase is called for short the DAHP synthetic enzyme), 3-dehydroquinic acid synthetic enzyme (3-dehydroquinate synthase), 3-dehydroquinate dehydratase (3-dehydroquinatedehydratase), shikimate dehydrogenase (shikimate dehydrogenase) and shikimate kinase isozyme (shikimate kinase isozyme).Respectively by aroF, aroB, aroD, aroE, aroK and aroL coding, wherein aroK and aroL all encode the shikimate kinase isozyme it shikimic acid is converted into 3-phosphoric acid shikimic acid (shikimate-3-phosphate) (1.Dansette, P.and R.Azerad, Theshikimate pathway.I.Preparation of 3-3H, 4-3H and 2,4-3H2 D-shikimic acid.Biochimie, 1973.55 (5): p.583-9; 2.Dansette, P.and R.Azerad, The shikimatepathway:II.Stereospecificity of hydrogen transfer catalyzed byNADPH-dehydroshikimate reductase of E.coli.Biochimie, 1974.56 (5): p.751-5; 3.Le Marechal, P.and R.Azerad, The shikimate pathway.III.3-dehydroquinatesynthetase of E.coli.Mechanistic studies by kinetic isotope effect.Biochimie, 1976.58 (9): p.1123-8; 4.Le Marechal, P.and R.Azerad, The shikimatepathway.IV.3-dehydroquinate synthetase of E coli.Substrate analogs andinhibitors.Biochimie, 1976.58 (9): p.1145-8; 5.Ife, R.J., et al., The shikimatepathway.PartV.Chorismic acid and chorismate mutase.J Chem Soc[Perkin1], 1976 (16): p.1776-83.).In intestinal bacteria and Bacillus subtilus, above-mentioned several genes are not chain.But in shikimic acid pathway, shikimic acid is intermediate product just also, therefore, utilize the synthetic shikimic acid of shikimic acid pathway just must destroy the effect of shikimate kinase isozyme, and shikimic acid can be assembled in microbe in a large number.
At present, existing abroad two patents of producing shikimic acid by intestinal bacteria (E.coli), Bacillus subtilus (bacillus subtilis).Roche Holding Ag has taken the lead in adopting intestinal bacteria that conversion of glucose is become shikimic acid with Germany one tame biotech company cooperation.Roche Holding Ag reveals that they produce the extraction of main raw material shikimic acid 2/3 from star anise of Tamiflu, and 1/3 utilizes biotechnology to obtain from intestinal bacteria.Another patent of the U.S. is to utilize Bacillus subtilus to produce shikimic acid, and these two kinds of methods play the same tune on different musical instruments, and transformation efficiency is also comparatively similar.The at present domestic report that utilizes the synthetic shikimic acid of biotechnology of not seeing as yet.In the five step reactions from glucose to the shikimic acid, its relevant katalaze enzyme is catalysis substrate conversion separately effectively, thereby makes its output that has reduced shikimic acid, simultaneously, has also influenced degree of purity of production.The rate-limiting enzyme of shikimic acid pathway is: DHQ synthetic enzyme (aroB) in two patents of the U.S., all is to utilize the plasmid vector that has the rate-limiting enzyme of encoding to import the carbon stream that recipient bacterium is improved shikimic acid.Yet the gene on the multiple copied plasmid vector may cause the expression of enzyme to exceed the initial requirement that improves the rate-limiting enzyme influence, causes the burden in the cellular metabolism, thereby reduces the growth velocity of cell and the synthesis rate and the output of shikimic acid.Many problems certainly will will be brought in the large-scale industrial production afterwards.In addition, former studies shows, whole regulatory gene shiA mutant strain can be than glycogen concentration (the Yang H of wild strain 20 times of poly collection in cell, Liu M Y, Romeo T.Coordinate genetic regulation of glycogen catabolism andbiosynthesis in Escherichia coli via the ShiA gene product.J Bacteriol, 1996,178:1012～1017).Therefore, the present invention knocks out the precursor that the shiA gene will improve synthetic shikimic acid in the bacterium effectively, improves the transformation efficiency of shikimic acid greatly.
Have following some advantage by the synthetic shikimic acid of biotechnology: (1) is not subjected to the restriction of material of vegetable origin production cycle and output: star anise is the fruit of lily magnolia plant star anise, the annual fruit phase is spring in autumn to next year, production cycle is long, output is limited, especially runs into the popular and fruit of flu outbreak when in poor harvest; The synthetic required raw material of biotechnology is bacterium, glucose etc., and these materials can obtain at any time, are not subject to seasonal restrictions.(2) protection environment; (3) with low cost: as, to reduce production costs about 2/3 by the synthetic shikimic acid of biotechnology relatively from the star anise extraction.Therefore be trend of the times by the synthetic shikimic acid of genetic engineering technique.
Utilize biotechnology to have important economical, societal benefits from the synthetic shikimic acid of intestinal bacteria, face today that bird flu threatens in the whole world, it is particularly important that its Research Significance more seems.
Summary of the invention
In order to improve in the method for utilizing the synthetic shikimic acid of genetic engineering technique at present the shikimic acid synthesis rate and to yield poorly, the defective that purity is low the invention discloses a kind of method of utilizing biosynthesis technology to prepare shikimic acid.
Preparation method of the present invention comprises the steps:
1. utilize genetic engineering technique to make up the PtacaroFaroBaroEkan polygene box of shikimic acid pathways metabolism rate-limiting enzyme.Make up PtacaroFaroBaroEkan polygene box strategy as shown in Figure 2.Comprise the steps:
(1) pcr amplification gene aroF, aroB, aroE, Ptac, shiA, kan;
(2) difference construction recombination plasmid pUC-aroA, pUC-shiA, pET22b-aroB, pET22b-aroC, pET22b-kan;
(3) make up PtacaroFaroBaroEkan polygene box: three gene aroF, aroB, aroE are cut by enzyme and be linked in sequence, before three genes, add Ptac I strong promoter, add the kan resistant gene behind three genes.
Ptac I strong promoter is the hybrid promoter from trp promotor and 1ac promotor that DeBoer etc. makes up, because the consistence of its-35 sequence and Pribnow box, so expression of exogenous gene is efficiently in controlling intestinal bacteria.It is reported that its starting efficiency exceeds 11 times more than than 1acUV5 promotor.Kan resistant gene in the box gene is for the ease of the screening of gene knockout and gene replacement back bacterial strain on the one hand, has also given the selective pressure of bacterial strain after gene knockout and gene are replaced on the other hand, makes it be not easy to take place reverse mutation.In addition, the kan gene is finished and can be utilized the FLP recombinase system that can discern the FRT site and eliminate.
2. utilize the Red recombination system to knock out shikimate kinase isozyme aroK and aroL in the bacterium.The Red recombination system is the new technology that developed recently gets up, and can directly utilize the PCR linear fragment that contains homology arm, replaces out the goal gene in the homology arm.Red is the recombinase that a kind of lambda particles phage is responsible for homologous recombination, by exo, beta and gam genomic constitution, Gam suppresses the RecBCD exonuclease of host bacterium, make the external source linear DNA be unlikely to be degraded immediately, Exo and Bet guiding linear fragment with homologous region reorganization takes place replaces, and the more traditional homologous recombination method of efficient is high tens times.AroK and the aroL shikimate kinase isozyme of all encoding will impel shikimic acid in intracellular gathering.Therefore, comprise the deletion mutantion that the plasmid of Red recombination system comes intestinal bacteria are carried out aroK and aroL gene by structure, improved intestinal bacteria are standby.
3. knock out the shiA gene in aroK and the aroL disappearance bacterium, and knock in PtacaroFaroBaroEkan polygene box.Knock in PtacaroFaroBaroEkan polygene box and adopt pulsed electrical conversion polygene box linear fragment method to carry out, knock out the shiA gene simultaneously.Carry out the screening that gene is replaced bacterial strain, because the insertion of box gene, ruined while of shiA gene, the integrity of having preserved box gene.The present invention knocks out with gene by southern blot test identified gene and replaces bacterial strain.
4. utilize ferment this recombinant bacterial strain of genetic engineering technique to obtain shikimic acid.By the shake flask fermentation shikimic acid and carry out the evaluation of transformation efficiency, explore best fermentation condition and transformation efficiency.In the present invention, used engineering bacteria is bacteriums such as intestinal bacteria, Bacillus subtilus, and wherein intestinal bacteria are preferred.Transformation efficiency reaches more than 40%.
5. separate, purifying shikimic acid.Separation and purification shikimic acid method used in the present invention is ion exchange chromatography and recrystallization method.
The invention also discloses the recombinant bacterial strain in preparation shikimic acid method, this project bacterium has knocked out aroK and aroL gene and shiA gene, comprises PtacaroFaroBaroEkan polygene box.Wherein PtacaroFaroBaroEkan polygene box is made up of rate-limiting enzyme aroF, aroB, aroE and the resistant gene kan of strong promoter Ptac, shikimic acid route of synthesis.
Recombinant bacterial strain disclosed in this invention is bacteriums such as intestinal bacteria, Bacillus subtilus, preferred intestinal bacteria.
Shikimic acid is a kind of monomeric compound, and molecular formula is: C
7H
10O
5, structural formula as shown in fig. 1, relative molecular mass is 174.15, formal name used at school is 3,4,5-trihydroxy--1-tetrahydrobenzene-1-carboxylic acid is the white fine powder, and gas is little, and it is sad to distinguish the flavor of, soluble in water, be insoluble in chloroform, benzene and sherwood oil, fusing point is 185 ℃～187 ℃, specific rotation is-180 °.
The present invention is by making up box gene PtacaroFaroBaroEkan, and at forward and backward strong promoter and the resistance screening gene of adding respectively of box gene, the Red recombination system that utilization depends on lambda particles phage knocks out metabolic regulation gene shiA, and replace with box gene PtacaroFaroBaroEkan, thereby make the shikimic acid route of synthesis reach optimization, thereby increased intracellular effective sugared concentration, releasing is to the negative regulation of PEP synthetic enzyme, in addition, we also will knock out shikimate kinase isozyme aroK and aroL, make that the shikimic acid in the intestinal bacteria is able to assemble in cell.
In the shikimic acid pathways metabolism, carbon is stored regulatory factor, and (carbon storage regulator A, disappearance ShiA) can increase the synthetic of glyconeogenesis and glycogen, reduces glycolysis-.In addition, ShiA also regulates and control to participate in directly phosphoenolpyruvic acid (phosphoenolpyruvate, PEP) activity level of metabolic 3 enzymes: pyruvate kinase (PykF) is just regulated and control, and PEP carboxylase (PckA) and PEP synthetic enzyme are then by negative regulation.In addition, the enzyme of some non-direct PEP of influence is also regulated and control by ShiA, thereby knocking out of shiA gene can make intracellular PEP increase greatly, for the biosynthesizing of shikimic acid provides a large amount of precursors.
The carbon that the present invention uses the Red recombination system that relies on phage to knock out in the intestinal bacteria is stored regulatory factor shiA, and replacing with shikimic acid pathway rate-limiting enzyme box gene---strong promoter+aroFaroBaroE gene+screening-gene is optimized intestinal bacteria shikimic acid synthesis system, working method is simply accurate, obviously is better than the research that two in the U.S. utilizes intestinal bacteria and Bacillus subtilus growth shikimic acid respectively from designing.
The present invention is as follows with respect to the innovative point of external two patents:
(1) stores regulatory factor by knocking out colibacillary carbon, and replace making shikimic acid pathway reach optimization with box gene PtacaroFaroBaroEkan;
(2) the Red recombination system that has used development in recent years to depend on lambda particles phage rapidly carries out gene knockout, knocks in and replaces e. coli chromosomal dna.This method can effectively utilize linear DNA fragment as the target practice molecule, do not need restriction enzyme and dna ligase, operating process simply, accurately and fast, economy, shortened the time that makes up targeting vector greatly;
(3) method of box gene replacement has avoided utilizing carrier to import the limitation of multiple copied rate-limiting enzyme genetic method, has alleviated the burden of bacterial metabolism, has improved the growth velocity of bacterium and the synthesis rate and the output of shikimic acid.
Intestinal bacteria are present biology laboratory engineering bacterias commonly used.The Red recombination system that relies on lambda particles phage is the gene knockout method that newly-developed gets up.The present invention successfully expresses shikimic acid at the intestinal bacteria camber at home first, is the host bacterium with intestinal bacteria, carries out temperature-induced expression, and expression amount is more than 40%.
The present invention is by making up PtacaroFaroBaroEkan polygene box and replacing whole regulatory gene shiA, improved the synthetic expression of rate-limiting enzyme in intestinal bacteria of shikimic acid, the catalysis conversion of substrate separately effectively, reduced extrachromosomal DNA as much as possible, avoided the burden in the cellular metabolism, the disappearance of shiA can increase the synthetic of glyconeogenesis and glycogen simultaneously, reduces glycolysis-, intracellular PEP is increased greatly, for the biosynthesizing of shikimic acid provides a large amount of precursors.The disappearance of shikimate kinase isozyme aroK and aroL has promoted the gathering of shikimic acid in the intestinal bacteria kytoplasm.
The present invention is based on the purpose that improves shikimic acid biosynthesizing output and reduce market value, design construction the polygene box of forming by shikimic acid anabolism rate-limiting enzyme, and replace whole regulatory gene shiA, disappearance is fallen the shikimate kinase isozyme simultaneously, make the intestinal bacteria after the reorganization can efficiently express shikimic acid in vivo, transformation efficiency can reach 40-50%, for the mass preparation shikimic acid has been created condition.
The present invention expresses shikimic acid for China utilizes the recombination bacillus coli height first, for with the shikimic acid be raw material medicine for example the production cost of Tamiflu etc. possible economic environmental protection approach is provided, China is as the maximum country of world population, when reply bird flu outburst, need Tamiflu deposit enormous amount, so the production of Tamiflu production starting material-shikimic acid is extremely urgent.The present invention has huge market and economic worth, and can greatly improve the adaptibility to response of China to control bird flu and other influenzas.
Description of drawings
Fig. 1 is the shikimic acid pathways metabolism.A is the DAHP synthetic enzyme; B is 3-dehydroquinic acid synthetic enzyme (3-dehydroquinate synthase); C is that 3-dehydroquinate dehydratase (3-dehydroquinatedehydratase) D is shikimate dehydrogenase (shikimate dehydrogenase); E is shikimate kinase isozyme (shikimate kinase isozyme).
Fig. 2 is the structure synoptic diagram of PtacaroFaroBaroEkan polygene box.Wherein pUC-:pUC19 carrier and goal gene; PET-:pET22b carrier and goal gene; Ptac: strong promoter Ptac; Kan: resistance screening gene; X:Xba I; S:Sal I; E:EcoR I; Ba:BamH I; Bg:Bgl II
Embodiment
The structure of embodiment one shikimic acid expression strain
(1) pcr amplification goal gene aroF, aroB, aroE, Ptac, shiA, kan; With the full DNA of E.coli31884 is template, with corresponding oligonucleotide primer amplifying target genes; Synthetic Ptac complete sequence, and, use corresponding primer amplification as template.
Ptac I strong promoter expression of exogenous gene in the control intestinal bacteria is efficiently.It is reported that its starting efficiency exceeds 11 times more than than 1acUV5 promotor.Kan resistant gene in the box gene is for the ease of the screening of gene knockout and gene replacement back bacterial strain on the one hand, has also given the selective pressure of bacterial strain after gene knockout and gene are replaced on the other hand, makes it be not easy to take place reverse mutation.In addition, the kan gene is finished and can be utilized the FLP recombinase system that can discern the FRT site and eliminate.
(2) difference construction recombination plasmid pUC-aroA, pUC-shiA, pET22b-aroB, pET22b-aroC, pET22b-kan.
(3) make up PtacaroFaroBaroEkan polygene box: three rate-limiting enzyme gene aroF, aroB, the aroE of this box gene coding shikimic acid route of synthesis, the intestinal bacteria 3-deoxidation-D-Arab-heptanone-7-phosphate synthase of encoding respectively, 3-dehydroquinic acid synthetic enzyme, 3-shikimate dehydrogenase.Add Ptac I strong promoter before three genes, add the kan resistant gene behind three genes, as Fig. 2.
(4) utilize restriction enzyme site to destroy the pUC19shiA gene, and insert PtacaroFaroBaroEkan polygene box.
(5) make up the plasmid pKD46 that comprises the Red recombination system, it is converted among the E.coli31884, knock out wherein shikimate kinase isozyme genes aroK and aroL, to promote the gathering of shikimic acid in the intestinal bacteria kytoplasm, improved intestinal bacteria are standby.
(6) normal condition is carried out pulsed electrical conversion polygene box linear fragment.
Embodiment two shikimic acid expression and purifications
(1) shikimic acid is expressed: shake flask fermentation, purifying shikimic acid and evaluation transformation efficiency;
Conditions of flask fermentation: temperature: 37 ℃; PH:6.5-7.5; Time 12h;
Be inoculated on the fermention medium;
(2) shikimic acid purifying: adopt ion exchange chromatography that the shikimic acid of expressing is separated, then further by the recrystallization method purifying shikimic acid.
Claims (9)
1. method of utilizing biosynthesis technology to prepare shikimic acid mainly may further comprise the steps:
(1) utilize genetic engineering technique to make up the PtacaroFaroBaroEkan polygene box of shikimic acid pathways metabolism rate-limiting enzyme;
(2) utilize the Red recombination system to knock out shikimate kinase isozyme aroK and aroL in the bacterium;
(3) knock out shiA gene in the bacterium, and knock in PtacaroFaroBaroEkan polygene box;
(4) utilize ferment this recombinant bacterial strain of genetic engineering technique to obtain shikimic acid;
(5) separation, purifying shikimic acid.
2. according to the described method of claim 1, wherein the construction process of PtacaroFaroBaroEkan polygene box comprises the steps:
(1) pcr amplification gene aroF, aroB, aroE, Ptac, shiA, kan;
(2) difference construction recombination plasmid pUC-aroF, pUC-shiA, pET22b-aroB, pET22b-aroE, pET22b-kan;
(3) make up PtacaroFaroBaroEkan polygene box: three gene aroF, aroB, aroE are cut by enzyme and be linked in sequence, before three genes, add Ptac I strong promoter, add the kan resistant gene behind three genes.
3. according to the described method of claim 1, wherein bacterium is intestinal bacteria or Bacillus subtilus.
4. according to the method for claim 1, wherein utilize the Red recombination system to knock out aroK and aroL, make up the engineering strain of aroK, aroL disappearance, step is as follows: 1. the primer that is used for homologous recombination: 5 ' end is the homology arm of aroK gene both sides, 3 ' end in like manner designs aroL chloramphenicol resistance gene primer for the primer of the chloramphenicol resistance gene that is used to increase; The chloramphenicol resistance gene in pcr amplification band FRT site; 2. above linear fragment is changed in the bacterial strain of expressing the Red recombinase; 3. select the chlorampenicol resistant transformant; 4.FLP recombinase is eliminated chloramphenicol resistance gene.
5. according to the described method of claim 1, wherein knock in PtacaroFaroBaroEkan polygene box and adopt pulsed electrical to transform polygene box linear fragment method.
6. according to the described method of claim 1, wherein the separation and purification shikimic acid adopts ion exchange chromatography and recrystallization method.
7. the recombinant bacterial strain in the described method of claim 1, it is characterized in that this project bacterium has knocked out aroK and aroL gene and shiA gene, comprise the PtacaroFaroBaroEkan polygene box that rate-limiting enzyme aroF, aroB, aroE and resistant gene kan by strong promoter Ptac, shikimic acid route of synthesis form.
8. according to the described recombinant bacterial strain of claim 7, it is characterized in that this project bacterium is intestinal bacteria or Bacillus subtilus.
9. according to the described recombinant bacterial strain of claim 7, it is characterized in that this project bacterium is intestinal bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101126657A CN101134958A (en) | 2006-08-29 | 2006-08-29 | Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101126657A CN101134958A (en) | 2006-08-29 | 2006-08-29 | Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101134958A true CN101134958A (en) | 2008-03-05 |
Family
ID=39159282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006101126657A Pending CN101134958A (en) | 2006-08-29 | 2006-08-29 | Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101134958A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930556A (en) * | 2011-09-07 | 2014-07-16 | 国立大学法人信州大学 | Method for producing useful metabolite from filamentous fungus |
| CN107619817A (en) * | 2017-10-24 | 2018-01-23 | 中国科学院天津工业生物技术研究所 | Produce 3 dehydroshikimate E. coli recombinant stains and its construction method and application |
| CN109423468A (en) * | 2017-08-24 | 2019-03-05 | 中国科学院微生物研究所 | The method for improving the compound in aromatic amino acid biosynthesis pathway and its derivative yield |
| CN111057672A (en) * | 2019-12-20 | 2020-04-24 | 东莞市东阳光生物合成药有限公司 | Recombinant strain and application thereof |
-
2006
- 2006-08-29 CN CNA2006101126657A patent/CN101134958A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930556A (en) * | 2011-09-07 | 2014-07-16 | 国立大学法人信州大学 | Method for producing useful metabolite from filamentous fungus |
| CN103930556B (en) * | 2011-09-07 | 2016-03-30 | 国立大学法人信州大学 | From the method for thread fungus production desirable metabolites |
| US9834796B2 (en) | 2011-09-07 | 2017-12-05 | Shinshu University | Method for producing useful metabolite from filamentous fungus |
| CN109423468A (en) * | 2017-08-24 | 2019-03-05 | 中国科学院微生物研究所 | The method for improving the compound in aromatic amino acid biosynthesis pathway and its derivative yield |
| CN107619817A (en) * | 2017-10-24 | 2018-01-23 | 中国科学院天津工业生物技术研究所 | Produce 3 dehydroshikimate E. coli recombinant stains and its construction method and application |
| CN107619817B (en) * | 2017-10-24 | 2021-02-02 | 中国科学院天津工业生物技术研究所 | Escherichia coli recombinant strain for producing 3-dehydroshikimic acid and construction method and application thereof |
| CN111057672A (en) * | 2019-12-20 | 2020-04-24 | 东莞市东阳光生物合成药有限公司 | Recombinant strain and application thereof |
| CN111057672B (en) * | 2019-12-20 | 2022-02-15 | 宜昌东阳光生化制药有限公司 | Recombinant strain and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Convergent engineering of syntrophic Escherichia coli coculture for efficient production of glycosides | |
| CN103602627B (en) | Novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as construction method and application thereof | |
| CN107586794B (en) | Method for producing tyrosol and hydroxytyrosol by heterogenous metabolic pathway | |
| CN103509726B (en) | Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium | |
| CN104059872A (en) | High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof | |
| CN103409485A (en) | Method for improving adenosine fermentation output through feeding organic nitrogen source | |
| CN101173308A (en) | Method for ferment for producing adenomethionine with genetic engineering bacterium | |
| CN112592843B (en) | Recombinant yarrowia lipolytica for producing alpha-lupinene and construction method and application thereof | |
| CN101134958A (en) | Method for producing shikimic acid by biological engineering invoice method expression and engineering bacterium constructed thereby | |
| CN104561134A (en) | Method for preparing 1-3-propanediol by microbiological fermentation method | |
| CN101085998A (en) | Method for preparing shikimic acid using biosynthesis technology and engineered bacteria | |
| Zhang et al. | Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila | |
| CN104762242B (en) | The production bacterium of mevalonic acid and the method for producing mevalonic acid | |
| Christen et al. | Evaluation of four Candida utilis strains for biomass, acetic acid and ethyl acetate production from ethanol | |
| CN104560856A (en) | Escherichia coli for aerobically synthesizing vitamin B12 as well as construction and application of escherichia coli | |
| CN103820506B (en) | A kind of method of gene recombination bacterium fermentation preparation of cozymase Q 10 | |
| CN102936577A (en) | Escherichia coli engineering bacterium for synthesizing pinocembrin by using glucose as substrate and application thereof | |
| CN1182249C (en) | Method of adding reducing agent from extraneous sources for accelerating thallus to synthesize 1,3-propylene glycol | |
| KR101758910B1 (en) | Recombinant Microorganisms Producing Butanol and Method for Preparing Butanol Using the Same | |
| CN103484417A (en) | Gluconobacter oxydans improving 2-KLG fermentation yield and application thereof | |
| Ye et al. | Engineering coenzyme A-dependent pathway from Clostridium saccharobutylicum in Escherichia coli for butanol production | |
| CN107099558B (en) | Novel method for simultaneously biosynthesizing 1, 3-propylene glycol and acetoin | |
| CN111560340B (en) | Synechocystis genetically engineered bacterium for biosynthesis of inositol, and construction method and application thereof | |
| Das et al. | Current knowledge on cyanobacterial biobutanol production: advances, challenges, and prospects | |
| CN103937733A (en) | Genetic engineering strain utilize sucrose to produce succinic acid from and method for production of succinic acid by fermenting the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20080305 |