CN104120119B - A kind of 3-dehydroquinate synthase mutant and gene thereof and application - Google Patents
A kind of 3-dehydroquinate synthase mutant and gene thereof and application Download PDFInfo
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- CN104120119B CN104120119B CN201310157144.3A CN201310157144A CN104120119B CN 104120119 B CN104120119 B CN 104120119B CN 201310157144 A CN201310157144 A CN 201310157144A CN 104120119 B CN104120119 B CN 104120119B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03004—3-Dehydroquinate synthase (4.2.3.4)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
Abstract
The invention discloses a kind of 3-dehydroquinate synthase mutant and gene thereof and application.Does is this 3-dehydroquinate synthase mutant by SEQ in such as sequence table? ID? the 153rd, the 175th, the 177th of the protein of aminoacid sequence shown in No:1 and the 336th amino acids through being substituted by an amino acid and have 3-dehydroquinate synthase activity by its derivative protein.Restructuring 3-dehydroquinate synthase of the present invention significantly improves the activity of the original enzyme that sets out, and greatly can improve the output of shikimic acid in shikimic acid pathway.
Description
Technical field
The invention belongs to genetically engineered and technical field of enzyme engineering, be specifically related to a kind of 3-dehydroquinate synthase mutant and gene thereof and application.
Background technology
Shikimic acid (Shikimicacid, SA), chemistry SA by name.It is the precursor of synthetic aromatic amino acid, alkaloid, indole derivatives and chiral drug, himself has analgesia, anti-inflammatory and antithrombotic effect.
The preparation method of shikimic acid mainly comprises: plant extraction method, chemical synthesis and biological synthesis process.Plant extraction method is mainly separated and obtains shikimic acid from the fruit of star anise.Although the shikimic acid content in star anise is up to more than 10%, but star anise belongs to the small-sized seeds in Magnoliaceae east, only be distributed in global only a few area, its growth is comparatively large by the impact of the physical environments such as temperature, humidity and soil, causes the output of plant extraction method cannot meet the demand day by day increased far away.Though chemosynthesis shikimic acid has number of ways, all complex process and productive rate is not high, the productive rate as Diels-Alder and inverse Diels-Alder reaction synthesis method all only has about 15%.Biological synthesis process mainly with the recombinant bacterium obtained through classic mutagenesis and molecular breeding for producing bacterial strain, with glucose etc. for raw material, synthesize shikimic acid by fermentation culture.3-dehydroquinate synthase is one of key enzyme of shikimic acid route of synthesis in microbe, and the height of its enzymic activity is closely related with shikimic acid output.
Protein molecular orientation evolvement technology is the New Policy of protein engineering, mainly utilizes molecular biology method to transform the gene of enzyme in vitro, produce gene diversity, then in conjunction with sensitive triage techniques, desirable mutant is obtained rapidly, the valuable non-natural enzyme of final orthoselection.Its core technology is mutant library constructing technology and high-throughput screening method.The growing demand of shikimic acid all can not be met based on current plant extraction method and chemical synthesis, and biological synthesis process output is also in dissatisfactory situation, it may be necessary the biosynthetic pathway of genetic engineering means to shikimic acid to study and transform, to reach the object of high yield shikimic acid.
Summary of the invention
Technical problem to be solved by this invention is active not high enough and cause the defect that shikimic acid output is not high in order to overcome in existing biological synthesis process 3-dehydroquinate synthase, provides a kind of 3-dehydroquinate synthase mutant and gene thereof and application.
The 3-dehydroquinate synthase of fallibility round pcr to intestinal bacteria (Escherichiacoli) W3110 is utilized to carry out directional transformation in the present invention, and high flux screening is carried out in the gene function covering of utilization to auxotroph host, and then investigate the expression of mutant gene and measure mutant enzyme activity, the final mutant obtaining enzymic activity and significantly improve.
One of technical scheme provided by the invention is: a kind of protein of separation, and the protein of described separation is by such as SEQ ID No: the 153rd, the 175th, the 177th of the protein of aminoacid sequence shown in 1 and the 336th amino acids through being substituted by an amino acid and have 3-dehydroquinate synthase activity by its derivative protein.
In the present invention, the protein of described separation has 3-dehydroquinate synthase activity, and it is restructuring 3-dehydroquinate synthase.Preferably, the protein of described separation is for by as SEQ ID No: the methionine(Met) of the 153rd of the protein of aminoacid sequence shown in 1 sports Threonine (M153T); In described aminoacid sequence, the proline(Pro) of the 175th sports Threonine (P175T); In described aminoacid sequence, the glutamic acid mutation of the 177th is glycine (E177G); In described aminoacid sequence, the leucine of the 336th sports the protein of Serine (L336S) gained.
In the present invention, the preparation method of the protein of described separation is the customary preparation methods of this area.Described preparation method is preferably: be separated from the naturally occurring protein of occurring in nature and obtain, and is separated and obtains or obtained by artificial sequence synthetic protein from this protein expression transformant recombinant expressed.Described preparation method is more preferably: by the cloned nucleic acid molecule of code for said proteins in recombinant vectors, gained recombinant vectors is transformed in transformant, obtaining recombinant expressed transformant, by cultivating the recombinant expressed transformant of gained, separation and purification can obtain described protein.
Two of technical scheme provided by the invention is: a kind of nucleic acid of separation, and described nucleic acid is the nucleic acid molecule of protein of above-mentioned separation of encoding.
The preparation method of wherein said nucleic acid is the preparation method of this area routine, described preparation method preferably comprises: the nucleic acid molecule extracting the protein of the above-mentioned separation of naturally occurring coding from occurring in nature, the nucleic acid molecule of protein of the above-mentioned separation of coding is obtained by gene clone technology, or the nucleic acid molecule of protein of the above-mentioned separation that obtains encoding by the method that artificial complete sequence synthesizes.
As is known to the person skilled in the art: the base sequence of the nucleic acid molecule of the protein of above-mentioned separation of encoding suitably can be introduced replacement, disappearance, change, inserts or increase the homologue providing a polynucleotide.The homologue of described polynucleotide can be replaced, lack or increase by one or more bases of the base sequence of the nucleic acid molecule of the protein to this separation of coding and obtain within the scope of maintenance enzymic activity.
Three of technical scheme provided by the invention is: a kind of recombinant expression vector comprising above-mentioned nucleic acid.
Wherein said recombinant expression vector obtains by this area ordinary method, that is: nucleic acid molecule of the present invention is connected to structure on various expression vector and forms.Described expression vector is the various carriers of this area routine.Described carrier preferably comprises: various plasmid, clay, phage or virus vector etc., the plasmid pETDuet-1 of the preferred Novagen company of carrier of the present invention.
Four of technical scheme provided by the invention is: a kind of recombinant expressed transformant comprising above-mentioned recombinant expression vector.
The preparation method of wherein said recombinant expressed transformant is preferably: be converted into by above-mentioned recombinant expression vector in host microorganism obtained.Described host microorganism is preferably the various host microorganisms of this area routine, as long as can meet, above-mentioned recombinant expression vector is stably copied voluntarily, and the gene of the protein of separation described in entrained coding can by effective expression.Wherein said host microorganism is preferably: intestinal bacteria (E.coli), is more preferably e. coli bl21 (DE3).Aforementioned recombinant expression plasmid is converted in E.coliBL21 (DE3), the preferred engineering strain of the present invention.Wherein said method for transformation is this area conventional transformation methods, is preferably chemical transformation, heat shock method or electric robin.
Five of technical scheme provided by the invention is: a kind of preparation method of 3-dehydroquinate synthase of recombinating, and comprising following steps: cultivate above-mentioned recombinant expressed transformant, obtains restructuring 3-dehydroquinate synthase from culture.
Wherein said preparation method is preferably: above-mentioned recombination bacillus coli is seeded to LB substratum, and after 37 DEG C of shaking culture are spent the night, the inoculum size by 1% is seeded in LB substratum, and 37 DEG C are cultured to OD
600when value reaches 0.6, adding IPTG to final concentration is 0.5mM, after inducing culture 4h, by bacterium liquid centrifugal 5min under 12000r/min, abandons supernatant and obtains thalline, then use 10mM phospho-glycerol salts solution (containing 0.25mMCoCl
2, 0.5mMNAD
+, pH6.6) in the resuspended thalline of the ratio of 1.5ml/g thalline, bacteria suspension is ultrasonication in ice bath, at 12000r/min4 DEG C, centrifugal 20min, gets supernatant, supernatant liquor with 10mM potassium phosphate solution (containing 0.25mMCoCl
2, pH6.6) and after the 24h that dialyses at 4 DEG C, the restructuring 3-dehydroquinate synthase of high expression can be obtained.
Six of technical scheme provided by the invention is: the protein of above-mentioned separation or above-mentioned recombinant expressed transformant are producing the application in shikimic acid.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
Along with the continuous outburst of influenza virus sub-strain, as the main raw material of synthesis anti influenza specifics " Tamiflu ", shikimic acid becomes that supply falls short of demand.Restructuring 3-dehydroquinate synthase of the present invention has the activity of 3-dehydroquinate synthase, is the key enzyme in biosynthesizing shikimic acid pathway.Restructuring 3-dehydroquinate synthase of the present invention significantly improves the activity of the original enzyme that sets out, and its enzymic activity comparatively wild-type 3-dehydroquinate synthase improves 30%, thus greatly can improve the output of shikimic acid in shikimic acid biosynthetic pathway.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, characteristic sum beneficial effect of the present invention is described.
Fig. 1 is 3-dehydroquinate synthase (aroB) genetically deficient and the impact of covering on thalli growth on M9 solid plate, wherein, and 1: e. coli bl21 (DE3); 2 ~ 4:aroB Gene Deletion e. coli bl21 (DE3); 5 ~ 8: utilize plasmid pETDuet-aroB to carry out the aroB Gene Deletion e. coli bl21 (DE3) of gene function covering.
Fig. 2 is the SDS-PAGE electrophorogram of aroB Gene Deletion escherichia coli BL21(DE3) expression aroB gene mutation body, M: protein molecular weight standard (wide); 1 ~ 6: the aroB Gene Deletion e. coli bl21 (DE3) containing plasmid pETDuet-aroB; 7:aroB Gene Deletion e. coli bl21 (DE3).
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
The present invention is to intestinal bacteria (EscherichiacoliW3110) 3-dehydroquinate synthase gene (this Gene Name: aroB, its accession number in GenBank is 12930302) carry out orthogenesis, fallibility round pcr is utilized to build 3-dehydroquinate synthase gene mutation body library, and with 3-dehydroquinate synthase auxotrophic strain for host is screened its gene mutation body library by gene function covering, and then investigate gene expression amount and the enzymic activity of well-grown mutant, the mutant that final acquisition one strain enzymic activity significantly improves.
In following embodiment, the primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
The structure of the mutant library of embodiment 13-dehydroquinate synthase gene
(1) primer of fallibility pcr amplification 3-dehydroquinate synthase gene is designed for, upstream primer B1(is as shown in SEQIDNo:2) sequence be 5 '-AAAACCATGGAGAGGATTGTCGTTACTCTCGG-3 ', downstream primer B2(is as shown in SEQIDNo:3) sequence be 5 '-GACAAAGCTTACGCTGATTGACAATCGGCAAT-3 ', respectively containing NcoI and HindIII restriction enzyme site.
(2) with intestinal bacteria W3110(purchased from E.coliGeneticStockCenter, YaleUniversity, i.e. Yale University intestinal bacteria preservation center) genome be template, primer B1 and B2 is utilized to carry out pcr amplification under the catalysis of Taq DNA polymerase, obtain 3-dehydroquinate synthase gene fragment, sepharose reclaims the gene fragment that first round fallibility PCR obtains, and carries out second with it for template and take turns fallibility PCR.PCR reaction system (20 μ l) is as follows: Taq DNA polymerase (5U) 0.2 μ l, chromogene template 0.5 μ l, Mg
2+(25mM) 4 μ l, Mn
2+(5mM) 0.4 μ l, dATP(10mM) 0.4 μ l, dTTP(10mM) 2 μ l, dGTP(10mM) 0.4 μ l, dCTP(10mM) 2 μ l, upstream primer B1(20 μM) 0.5 μ l, primer B2(20 μM) 0.5 μ l, 10 × PCR damping fluid is not (containing Mg
2+) 2 μ l, ddH
2o7.1 μ l, above reagent is all purchased from Shanghai Jierui Biology Engineering Co., Ltd.The program of amplification is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40s, 58 DEG C of annealing 40s, 72 DEG C extend 1min, totally 35 circulations; 72 DEG C extend 10min again.
(3) with NcoI and HindIII to through the 3-dehydroquinate synthase gene fragment of two-wheeled fallibility pcr amplification and carrier pETDuet-1(Novagen company) carry out double digestion, agarose gel electrophoresis reclaims object fragment, utilize T4 ligase enzyme (TaKaRa company) in 16 DEG C of connections of spending the night, connect product conversion 3-dehydroquinate synthase gene defection type bacterial strain (building process is shown in embodiment 2) and the LB agar plate coated containing 100mg/mL penbritin, cultivate 12 ~ 16h for 37 DEG C, after single bacterium colony grows, flat board is put in 4 DEG C of preservations for subsequent use.
The screening in embodiment 23-dehydroquinate synthase gene mutation body library
(1) structure of 3-dehydroquinate synthase gene defection type bacterial strain
To the plasmid pKD46(of Red recombinase be expressed purchased from Yale University's intestinal bacteria preservation center, this plasmid information is at document BabaT, AraT, HasegawaM, etal.ConstructionofEscherichiacoliK-12in-frame, disclosure is had in single-geneknockoutmutants:theKeiocollectionMolSystBiol2 006:2-8) import E.coliBL21 (DE3), the LB solid plate containing 100mg/mL penbritin screens transformant (culture temperature is 30 DEG C).Transformant is inoculated LB liquid nutrient medium, shaking culture under 30 DEG C of 200r/min, as thalline OD
600add 1mML-pectinose abduction delivering Red recombinase when reaching 0.2, induction time is no less than 1h, OD
600gather in the crops thalline when being about 0.6, prepare competent cell.Electrotransformation is utilized the fragment being used for homologous recombination to be imported in competent cell.Fragment for homologous recombination is with BR1(5 '-GCGGCTCTGTGAAATCCC-3 ') and BR2(5 '-GTTGTTGACCATCCGTTGC-3 ') be primer (sequence of BR1 and BR2 is respectively as shown in SEQIDNo:4 and SEQIDNo:5), with intestinal bacteria 3-dehydroquinate synthase mutant strain E.coliJW3352-1(purchased from Yale University's intestinal bacteria preservation center, this bacterial strain information is at document BabaT, AraT, HasegawaM, etal.ConstructionofEscherichiacoliK-12in-frame, have disclosure in single-geneknockoutmutants:theKeiocollectionMolSystBiol2 006:2-8) genome be template obtain containing the PCR fragment of kalamycin resistance.Then, be rapidly in the competent cell after electric turning and add 1mlLB substratum, heat up 42 DEG C after 30 DEG C of cultivation 1h and continue cultivation 1h, get the LB solid plate of bacterium liquid coating containing 25mg/mL kantlex after appropriate cultivation.Next day, by dibbling test and Selection, there is kalamycin resistance but not there is the transformant of amicillin resistance, with the disappearance of the method validation 3-dehydroquinate synthase gene of bacterium colony PCR, and checking order.Sequencing result shows the success of 3-dehydroquinate synthase gene defection type strain construction.
(2) screening in 3-dehydroquinate synthase gene mutation body library
3-dehydroquinate synthase genetically deficient will cause Host Strains to become auxotrophic strain, therefore, can on minimum medium, with this auxotrophic strain for host, by 3-dehydroquinate synthase gene function covering experiment, mutant library is screened, thus obtains the mutant (Fig. 1) still with enzyme function.By all single bacterium colony dibbling M9 solid medium (minimum medium) obtained in embodiment 1, quiescent culture 24h at 37 DEG C, is chosen at the bacterium colony that M9 solid medium can grow.M9 substratum (1L): 5 × M9 salts solution 200ml, 20% glucose 20ml, 1MMgSO
42ml, 1MCaCl
20.1ml, agar 15g.5 × M9 salts solution (1L): Sodium phosphate dibasic (Shi Ershui) 75g, dipotassium hydrogen phosphate 15g, sodium-chlor 2.5g, ammonium chloride 5g, with deionized water constant volume to 1L, regulate pH to 7.4, autoclaving 20min is for subsequent use.
The expression of embodiment 33-dehydroquinate synthase gene mutation body
Being seeded to by the mutants which had of M9 cultured on solid medium in embodiment 2 is equipped with in the 250ml triangular flask of 20ml seed culture medium, and 37 DEG C of shaking culture are seeded in fresh LB by 1% inoculum size after spending the night, and 37 DEG C are cultured to OD
600when being about 0.6, adding IPTG to final concentration is 1mM, and by bacterium liquid centrifugal 5min under 12000r/min after continuation inducing culture 4h, abandon supernatant, thalline is used for SDS-PAGE and detects.As shown in Figure 2, compared with No. 7 Host Strains, 1 ~ No. 6 bacterial strain have expressed the target protein of 38kDa to result, and its expression amount is higher.
The enzyme activity determination of embodiment 43-dehydroquinate synthase gene mutation body
Reference literature (FrostJW, BenderJL, KadonagaJT, etal.DehydroquinatesynthetasefromEscherichiacoli:purific ation, cloning, andconstructionofoverproducersoftheenzyme.Biochemistry, 1984,23 (19): 4470-4475.) method in carries out enzyme activity determination.Thalline is collected, with 10mM phospho-glycerol salts solution (containing 0.25mMCoCl according to the method in embodiment 3
2, 0.5mMNAD
+, pH6.6) and in the resuspended thalline of the ratio of 1.5ml/g thalline, bacteria suspension is ultrasonication in ice bath, and at 12000r/min4 DEG C, centrifugal 20min, gets supernatant.Supernatant liquor with 10mM potassium phosphate solution (containing 0.25mMCoCl
2, pH6.6) and be crude enzyme liquid, for enzyme activity determination after the 24h that dialyses at 4 DEG C.Containing 100 μMs of potassium phosphate buffers (pH7.4) in 2mL enzyme activity determination system, 0.5 μM of DAHP, 0.2 μM of CoCl
2, 0.02 μM of NAD
+and appropriate crude enzyme liquid, temperature of reaction is 37 DEG C, and (reference is from document SrinivasanPR, RothschildJ, SprinsonDB.Theenzymicconversionof3-deoxy-D-arabino-heptu losonicacid7-phosphateto5-dehydroquinate [J] .JBiolChem, 1963,238 (10): 3176-3182).The reaction solution of reaction 0min and 30min respectively gets 0.5mL, adds 0.2mL10% trichoroacetic acid(TCA) respectively with protein precipitation, centrifuging and taking supernatant, adds nitrite ion 2mL, measures the absorbancy at 548nm place, calculated difference.Enzyme activity unit: under the above-described reaction conditions, makes DAHP reduce by the enzyme amount of 1 μM in 1min in reaction system.
Enzyme activity determination result shows that the enzymic activity of No. 3 mutant improves 30% compared with the enzymic activity of wild-type.By this mutant inoculation LB liquid nutrient medium, incubated overnight under 37 DEG C of 200r/min, extract plasmid, with upstream primer P1(5 '-ATGCGTCCGGCGTAGA-3 ') and downstream primer P2(5 '-TGCTAGTTATTGCTCAGCGG-3 ') carry out check order (sequence of P1 and P2 is respectively as shown in SEQIDNo:6 and SEQIDNo:7), result shows that its mutational site is M153T, P175T, E177G and L336S.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. the protein be separated, it is characterized in that, the protein of described separation is by such as SEQ ID No: the 153rd, the 175th, the 177th of the protein of aminoacid sequence shown in 1 and the 336th amino acids through being substituted by an amino acid and have 3-dehydroquinate synthase activity by its derivative protein, in the aminoacid sequence of described protein, the methionine(Met) of the 153rd sports Threonine; In the aminoacid sequence of described protein, the proline(Pro) of the 175th sports Threonine; In the aminoacid sequence of described protein, the glutamic acid mutation of the 177th is glycine; In the aminoacid sequence of described protein, the leucine of the 336th sports Serine.
2. the nucleic acid be separated, is characterized in that, described nucleic acid is the nucleic acid molecule of protein as claimed in claim 1 of encoding.
3. one kind comprises the recombinant expression vector of nucleic acid as claimed in claim 2.
4. one kind comprises the recombinant expressed transformant of recombinant expression vector as claimed in claim 3.
5. recombinate the preparation method of 3-dehydroquinate synthase, it is characterized in that, described preparation method comprises the steps: to cultivate recombinant expressed transformant as claimed in claim 4, obtains restructuring 3-dehydroquinate synthase from culture.
6. protein or the application of recombinant expressed transformant in production shikimic acid as claimed in claim 4 as claimed in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0874053A2 (en) * | 1997-04-22 | 1998-10-28 | Smithkline Beecham Corporation | 3-Dehydroquinate synthase (aroB) |
| EP1038968A2 (en) * | 1999-03-25 | 2000-09-27 | Ajinomoto Co., Inc. | Method for producing shikimic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6472190B1 (en) * | 2000-03-16 | 2002-10-29 | Board Of Trustees Operating Michigan State Univerisity | Biocatalytic synthesis of galloid organics |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0874053A2 (en) * | 1997-04-22 | 1998-10-28 | Smithkline Beecham Corporation | 3-Dehydroquinate synthase (aroB) |
| EP1038968A2 (en) * | 1999-03-25 | 2000-09-27 | Ajinomoto Co., Inc. | Method for producing shikimic acid |
Non-Patent Citations (2)
| Title |
|---|
| Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein;ALISON PARK et al.;《Protein Science》;20041231;全文 * |
| Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori;S. Bereswill et al.;《Med Microbiol Immunol》;19971231;全文 * |
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