CN102250874A - Recombinant Escherichia coli for producing shikimic acid and construction method thereof - Google Patents
Recombinant Escherichia coli for producing shikimic acid and construction method thereof Download PDFInfo
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Abstract
The invention relates to a recombinant Escherichia coli for producing shikimic acid and a construction method thereof. The invention relates to a new strategy for constructing a shikimic acid production strain, and firstly discovers that the yield of the shikimic acid can be improved by knocking out ydiB gene. By knocking out a pts HIcrr operon, the ydiB gene, shiA gene and aroL gene or aroL gene and aroK gene, and integrating T7RNA polymerase on a chromosome, a modified host bacteria DHPYASA-T7 is obtained; and a recombinant low-copy expression plasmid pAOC-TGEFB containing relative key enzyme genes such as aroB, aroE, aroF and glk and tktA in the shikimic acid path of a T7 promotor is automatically constructed, then the recombinant expression plasmid is transplanted into the modified host bacteria to obtain the shikimic acid production strain DHPYAS-T7. The shikimic acid production strain DHPYAS-T7 obtained by using the construction method disclosed by the invention can realize the effective accumulation of shikimic acid in the shake flask production, thereby laying the foundation for the industrial production of shikimic acid.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of shikimic acid prepared bacterial strain and construction process thereof.
Background technology
Shikimic acid (shikimic acid) is that the path of biosynthesizing die aromatischen Aminosaeuren is the important intermediate of shikimic acid pathway, the physiological function of having reported has: come anticoagulant by influencing arachidonic acid metabolism, thereby can suppress artery and vein thrombus and cerebral thrombosis; Have anti-inflammatory, analgesic activity; Also can be used as antiviral and intermediate cancer therapy drug, pharmaceutical use or the like is widely arranged.Its chemical name is 3,4,5-trihydroxy--1-tetrahydrobenzene-1-carboxylic acid, and chemical structural formula is as follows:
At present the specific medicament in order to treatment H5N1 type avian influenza virus is Ro 64-0796/002 (oseltamivir phosphate), and commodity be called Tamiflu, and shikimic acid anti-avian influenza medicine " Tamiflu " synthetic key chirality starting raw material just.Produce the Magnoliacea plant---anise that required shikimic acid major part comes from Guangxi China at present, but this kind complex manufacturing, yield is lower, and raw material is subject to effects limit such as season, weather, it is required to be difficult to satisfy scale operation, Switzerland Roche Holding Ag has the patent right that this kind Tamiflu is produced, and costs an arm and a leg.
Therefore, the transformation of microorganism die aromatischen Aminosaeuren metabolism stream had bright prospects to improve shikimic acid output.Starting material that microorganism growth is required such as glucose etc. can obtain at any time, and its reproduction speed is fast, and metabolic capacity is strong, so the cycle is short, and the production process environmental pollution is little, and is with low cost, has very big advantage than plant extract.Thereby have very important economical, societal benefits so make it the mass production shikimic acid by biological means improvement project bacterium.
Summary of the invention
The objective of the invention is to intestinal bacteria CCM (central carbon metabolism) and shikimic acid pathway be transformed, thereby a kind of production bacterial strain of new shikimic acid is provided by engineered method.
Another object of the present invention is to provide the construction process of described shikimic acid prepared bacterial strain.
The construction process of shikimic acid prepared bacterial strain of the present invention may further comprise the steps:
(1), makes up the intestinal bacteria DHPYASA-T7 that knocks out ptsHIcrr operon, ydiB, shiA, aroL and aroK gene and integrate t7 rna polymerase.
(2), make up the recombinant expression plasmid pAOC-TGEFB of key gene tktA, glk, aroE, aroF and aroB in the shikimic acid pathways metabolism that contains the T7 promotor.
(3), recombinant expression plasmid pAOC-TGEFB is transformed the production bacterial strain DHPYAS-T7 that transforms bacterial strain DHPYASA-T7 acquisition shikimic acid.
Bacterial strain of the present invention is compared with having patent both at home and abroad now, and the innovation part is following 2 points:
(1), knocks out ptsHIcrr operon gene, the supply that improves shikimic acid pathway precursor PEP." phenomenon is checked in the resolvent metabolism " removed in ptsHIcrr gene knockout or sudden change, can more effectively utilize the complex medium hydrolysate to keep thalli growth, the phosphate that deletion mycopremna dependence GalP of PTS system and Glk provide with ATP carries out the glucose phosphorylation transhipment, this glucose transport system improves the utilization ratio of PEP in the born of the same parents, helps increasing carbon and flows to into the die aromatischen Aminosaeuren approach;
(2), knock out the ydiB gene and improve shikimic acid output.YdiB is a kind of bifunctional enzyme (quinic acid/shikimate dehydrogenase), be closely related with quinic acid (QA) metabolism, bacterial strain lacks the quinate dehydrogenase activity behind the ydiB gene knockout, the synthetic QA approach of three dehydroquinic acids (DHQ) is obstructed, by product QA output descends, and DHQ obtains accumulation and synthetic a large amount of three dehydrogenation shikimic acids (DHS) under dehydroquinate dehydratase catalysis; And the shikimate dehydrogenase activity can be compensated by the expression of crossing of aroE gene, and the DHS of high density efficiently synthesizes shikimic acid as the substrate of shikimate dehydrogenase, and purpose product output significantly improves;
The constructed shikimic acid prepared bacterial strain DHPYAS-T7 of the present invention has submitted on 03 03rd, 2011 and has been positioned at Pekinese China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.4631.
The engineering strain DHPYAS-T7 that utilizes the present invention to obtain ferments, and can effectively obtain the accumulation of purpose product shikimic acid, thereby lay a good foundation for the biosynthetic industrialization of shikimic acid.
Description of drawings
Fig. 1 is a ptsHIcrr gene knockout strategy, and wherein L is a ptsHIcrr upstream homologous gene in the intestinal bacteria, and R is a ptsHIcrr downstream homologous gene.
Fig. 2 shows that for the PCR that DH5 α Δ ptsHIcrr knocks out bacterium DHP identifies the ptsHIcrr gene has successfully been knocked out; Wherein M is DNA marker; 1,2 is wild-type e. coli DH5 α; 3 knock out bacterium for ptsHIcrr.
Fig. 3 is a pET-28a-YLR/DH5 α positive bacteria qualification result; Wherein M is DNA Marker, and 11-20 carries out the result that PCR identifies for the different single bacterium colonies of intestinal bacteria after transforming through pET-28a-YLR, and the result shows all positive band of 11-14,16,19.
Fig. 4 is the positive bacterium colony qualification result of the ydiB gene knockout of DHP bacterium; M is DNA Marker, and 1-10 is to induce and knocks out that single bacterium colony of picking carries out the result that PCR identifies behind the ydiB gene, and the result shows all negative band of 1-8,10, and No. 9 positive bands illustrates that the ydiB gene knockout successfully in No. 9.
Fig. 5 is a pET-28a-ALR/DH5 α positive bacteria qualification result; Wherein M is DNA Marker, and 1-10 carries out the result that PCR identifies for the different single bacterium colonies of intestinal bacteria after transforming through pET-28a-ALR, and the result shows 10 negative bands, all positive band of 1-9.
Fig. 6 is the aroL gene knockout positive bacteria qualification result of DHPY bacterium; M is DNA Marker, and 1-10 induces to knock out that single bacterium colony of picking carries out the result that PCR identifies behind the aroL gene, and the result shows 1,3-10 is all negative, and No. 2 positive bands of band illustrates that the aroL gene knockout successfully in No. 2.
Fig. 7 is that the T7RNP of DHPYA bacterium integrates qualification result; M is DNA Marker among the figure, and 1-5 is the result of the bacterial strain of integration T7RNP through the PCR evaluation, and the result shows all positive band, and the T7RNP successful integration is described.
Fig. 8 is a DHPYA-T7 bacterial strain shiA gene knockout positive bacteria qualification result; M is DNA Marker, the negative contrast of DH5 α bacterium, 1-9 number, be for 11-20 number to induce and knock out that single bacterium colony of picking carries out the result that PCR identifies behind the shiA gene, the result does not show except No. 5 bacterium not grow and causes not having the band that all the other all positive bands illustrate the success of shiA gene knockout.
Fig. 9 is a DHPYAA-T7 bacterial strain aroK gene knockout positive bacteria qualification result; M is DNA Marker, the negative contrast of DH5 α bacterium, and 1-9 number, be for 11-20 number to induce and knock out that single bacterium colony of picking carries out the result that PCR identifies behind the aroK gene, the result shows all positive band, illustrates that the aroK gene knockout is successfully.
Figure 10 is a pET3b-TGEFB plasmid joining method.
Figure 11 makes up the output contrast of the reorganization bacterium JDL02/pTrc-aroG-tktA of bacterial strain DHPYAS-T7 and domestic literature report high level expression shikimic acid for this experiment.
Embodiment
The intestinal bacteria DHPYASA-T7 that embodiment 1, structure knock out ptsHIcrr, ydiB, aroL, aroK, shiA gene and integrate t7 rna polymerase
Make up intestinal bacteria DHP 1.1 knock out the ptsHIcrr operon
1.1.1 plasmid and bacterial strain: low copy helper plasmid pKDS (makes up voluntarily, contains lambda particles phage recombinase and the I-Sce I restriction endonuclease temperature sensitive type replicon of going back to the nest; Exo, bet, gam and the restriction endonuclease of going back to the nest all are subjected to the pectinose promoter regulation); High copy target practice plasmid pC is a fundamental construction with pET-28a (+); Bacillus coli DH 5 alpha.
1.1.2 main agents and instrument: restriction enzyme, DNA marker are available from precious biotechnology (Dalian) company limited; Glucose detection test kit and acetate detection kit are respectively available from middle north control bio tech ltd, the Boehringer-Mannheim company of giving birth to; Paraxin, kantlex are available from AMRESCO company; Electroporation SystemECM 399 is available from HARVARD APPARATUS company, and other are the analytical pure chemical reagent.
1.1.3 substratum and culture condition: LB substratum (g/L): peptone 10, yeast powder 5, sodium-chlor 10; LBG substratum (g/L): peptone 10, yeast powder 5, sodium-chlor 10, glucose 10; FMG substratum (g/L): K
2HPO
43H
2O7, NaH
2PO
32H
2O3, NaCl 2.5, yeast powder 12, peptone 15, other adds 0.5g MgSO
47H
2O and 2g glucose; It is to revise a little to obtain Na on the M9 medium base that M9 modifies substratum (g/L)
2HPO
47H
2O64, KH
2PO
415, NaCl2.5, NH
4Cl 5.0, and other adds 2ml 1mol/l MgSO
4With 100 μ l 1mol/L CaCl
2, add final concentration again and be 0.2% glucose and 0.1% yeast powder.Paraxin, kantlex working concentration are respectively 50 μ g/ml and 50 μ g/ml.
1.1.4 primer: band EcoR I, Xho I restriction enzyme site among primer C1 and the C4.PtsHIcrr operon target practice DNA linear fragment obtains with overlapping PCR, thus primer C3 and C2 partial sequence reverse complemental, primers designed C5/C6.Synthetic and the relevant order-checking of primer is finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Primer sequence is as follows:
C1: see sequence 1 C2 in the sequence table: see sequence 2 in the sequence table
C3: see sequence 3 C4 in the sequence table: see sequence 4 in the sequence table
C5: see sequence 5 C6 in the sequence table: see sequence 6 in the sequence table
1.1.5PCR the preparation of target practice fragment and target practice plasmid: with DH5 α genome is template, is that primer carries out pcr amplification and obtains ptsHIcrr operon left side homology arm with C1/C2; C3/C4 is that the primer PCR amplification obtains the right homology arm of ptsHIcrr operon, is common template with the left and right sides homology arm that reclaims respectively again, is that primer carries out the linearity target practice dna fragmentation C1 that overlapping PCR obtains the ptsHIcrr gene with C1/C4.Target practice fragment C1 and pET carrier be through EcoR I, Xho I double digestion, steps such as the connection plasmid pC that obtains practicing shooting.
1.1.6ptsHIcrr operon knocks out fast and identifies: (1) electricity transforms: getting 1 μ l target practice plasmid and 7 μ l helper plasmid pKDS to 50 μ l electricity changes competent cell and mixing, behind the ice bath mixture is transferred in the 1mm electric shock cup of precooling, 1.8KV electric shock transforms, electric shock is finished rapidly adding 1ml SOC substratum to 30 ℃ of back, cultivate behind the 220r/min vibration 1h back coating card that, the mould two anti-plates of chlorine, the screening positive transformant, final obtain " corotation bacterium liquid ".(2) induce and knock out: get 500 μ l " corotation bacterium " contain the mould resistance of chlorine in 50ml FMG liquid nutrient medium, 30 ℃, 220r/min were cultured to OD at about 0.2 o'clock, add final concentration and be 0.2% L-arabinose and induce 4h, induce and get 100 μ l after the end and induce bacterium liquid to be diluted to 10
-4, get dilution bacterium liquid (10 again
-4) the mould resistance plate of 100 μ l coating chlorine, 30 ℃ of cultivations.See gene knockout policy map 1.(3) knocking out the positive bacterium colony PCR of bacterium identifies and separation: with single bacterium colony in the mould plate of above-mentioned chlorine is template, is that primer carries out bacterium colony PCR to identify positive reorganization bacterium colony with C5/C6, sees Fig. 2; The positive bacterium colony that will contain helper plasmid is inoculated in liquid LB, 42 ℃ of about 12h of cultivation to reject helper plasmid pKDS, subsequently with bacterium liquid coating nonreactive plate, after 37 ℃ of cultivations at random in the picking nonreactive plate 20 single bacterium be inoculated in the chlorampenicol resistant plate, behind 37 ℃ of cultivation 12h, can not promptly be to knock out bacterium at the single bacterium colony on the pairing nonreactive plate of single bacterium colony of chlorampenicol resistant plate growth.
1.2 with the DHP bacterial strain is that receptor knockout removes the gene constructed intestinal bacteria DHPY of ydiB bacterial strain
1.2.1 homology arm makes up
With DH5 α genome is template, primer YL5/YL3, and YR5/YR3, pcr amplification through 1% agarose gel electrophoresis, reclaims about 1000bp band, is YL, YR.With YL, YR is a template then, primer YL5/YR3, and pcr amplification through 1% agarose gel electrophoresis, reclaims about 2000bp band, is YLR.
Required primer sequence:
YL5: see sequence 7 YL3 in the sequence table: see sequence 8 in the sequence table
YR5: see sequence 9 YR3 in the sequence table: see sequence 10 in the sequence table
1.2.2 target practice plasmid construction
With EcoRI/BamHI double digestion pET28a and ALR, enzyme is cut product and is reclaimed, and connects.Get DH5 α competent cell, after melting, get 50 μ L on ice, add 10 μ L and connect product, mixing gently, ice bath 10min, 42 ℃ of water-bath thermal shock 90s, ice bath 2min adds 500 μ L nonreactive LB liquid nutrient mediums, 37 ℃, 220rpm, 1h gets 200 those resistant panel of μ L bacterium liquid card-coating, 37 ℃ of overnight incubation.With single bacterium colony on the flat board is template, and T7/T7t is that primer carries out PCR, through 1% agarose gel electrophoresis, and the positive band of 2500bp band, as Fig. 3, getting 14, No. 19 bacterium liquid sample presentation sequence verification is the purpose fragment.
1.2.3 corotation bacterium liquid preparation
DH5aPTS-(DHP) electricity changes the competence preparation: picking list bacterium colony is to the 5mLLB liquid nutrient medium, and 37 ℃ of overnight incubation are got 500 μ L bacterium liquid and are seeded in the 50mL LB liquid nutrient medium, and 37 ℃ are cultured to the about 0.6-0.7 of OD600.Bacterium liquid places 20-30min on ice, fully after the cooling, goes in the 50mL centrifuge tube of precooling, and 4 ℃, 6000rpm, centrifugal 10min carefully abandons supernatant.The ddH that 50mL is ice-cold
2The O re-suspended cell, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant.The 10% glycerine re-suspended cell that 30mL is ice-cold, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant, repeats this step.0.5mL 10% ice-cold glycerine re-suspended cell, 1.5mL EP manages packing, every pipe 100 μ L.
Electric shock transforms: getting 50 μ L DHP electricity changes competence, adds 2 μ L target practice plasmid pET28a-YLR, 7 μ L helper plasmid SN3, ice bath 2-3min, said mixture carefully is transferred in the 1mm electric shock cup of precooling, is fixed to electroporation, adjust voltage to 1.8kv, electric shock transforms, rapidly 1mL SOC substratum is added in the electric revolving cup after finishing, carefully blow and beat mixing, be transferred to 1.5mL EP pipe, 30 ℃ of shaking culture 75min, coating Kan Cm is two anti-dull and stereotyped.
Picking list bacterium colony is corotation bacterium liquid to the two anti-LB liquid nutrient mediums of Kan Cm.
1.2.4ydiB gene inducedly knock out and identify
Get 50mL Cm LB liquid nutrient medium, add 250 μ L, 20% glucose, 500 μ L corotation bacterium liquid, 30 ℃ add 500 μ L, 20% pectinose when being cultured to OD6000.552 and induce and knock out, 30 ℃ of 220rpm induce 4h, get bacterium liquid dilution coated plate, 30 ℃ of cultivations overnight.With single bacterium colony on the flat board is template, and YJ1/YJR is a primer, carries out PCR, through 1% agarose gel electrophoresis, and as Fig. 4, the positive band of 1500bp band, No. 9 are positive.
Required primer sequence:
YJ1: see sequence 11 YJ2 in the sequence table: see sequence 12 in the sequence table
YJL: see sequence 13 YJR in the sequence table: see sequence 14 in the sequence table
Get the dilution of positive bacteria liquid and be coated with the Cm flat board, 20 single bacterium colonies of picking identify that the PCR system is the same, and PCR result is all positive, get a pipe and continue diluted passage, identify all positive once more.Get 50 μ L bacterium liquid and be seeded to 42 ℃ of cultivations of nonreactive LB liquid nutrient medium to reject the SN3 plasmid.Dilution is coated with the nonreactive flat board behind the 12h, and 20 correspondences of single bacterium colony mark are chosen to the Cm flat board 30 ℃ of cultivations on should flat board, the plasmid that is of not growing on the Cm flat board is rejected bacterial strain, get respective number bacterium colony reservation bacterial classification on the nonreactive, the ydiB that is DHP knocks out bacterial classification, is designated as DHPY.After obtaining DHPY and knocking out bacterial classification, be primer, carry out EasyTaqPCR with YJ1/YJ2, the order-checking of PCR stoste sample presentation, sequencing result shows that ydiB knocks out successfully.
1.3DHPY middle aroL gene knockout makes up the DHPYA bacterial strain
1.3.1 homology arm makes up
With DH5 α genome is template, primer AL5/AL3, and AR5/AR3 carries out pcr amplification, through 1% agarose gel electrophoresis, reclaims about 1000bp band, is AL, AR.With AL, AR is a template, and primer AL5/AR3 carries out pcr amplification again, through 1% agarose gel electrophoresis, reclaims about 2000bp band, is ALR.
Required primer sequence:
AL5: see sequence 15 AL3 in the sequence table: see sequence 16 in the sequence table
AR5: see sequence 17 AR3 in the sequence table: see sequence 18 in the sequence table
1.3.2 target practice plasmid construction
Through EcoRI/HindIII double digestion pET28a and ALR, enzyme is cut product and is reclaimed, and connects.Get DH5 α competent cell then, after melting, get 50 μ L on ice, add 10 μ L and connect product, mixing gently, ice bath 10min, 42 ℃ of water-bath thermal shock 90s, ice bath 2min adds 500 μ L nonreactive LB liquid nutrient mediums, 37 ℃, 220rpm, 1h gets 200 those resistant panel of μ L bacterium liquid card-coating, 37 ℃ of overnight incubation.With single bacterium colony on the flat board is template, and T7/T7t is a primer, carries out PCR, and through 1% agarose gel electrophoresis, as Fig. 5, the positive band of 2500bp band is got 4, No. 5 bacterium liquid sample presentation order-checkings, and sequencing result shows correct.
1.3.3 corotation bacterium liquid preparation
The DHPY electricity changes the competence preparation: picking list bacterium colony is to 5mL LB liquid nutrient medium, and 37 ℃ of overnight incubation are got 500 μ L bacterium liquid and are seeded in the 50mL LB liquid nutrient medium, and 37 ℃ are cultured to OD
600About 0.6-0.7.Bacterium liquid places 20-30min on ice, fully after the cooling, goes in the 50mL centrifuge tube of precooling, and 4 ℃, 6000rpm, centrifugal 10min carefully abandons supernatant.The ddH that 50mL is ice-cold
2The O re-suspended cell, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant.The 10% glycerine re-suspended cell that 30mL is ice-cold, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant, repeats this step.0.5mL 10% ice-cold glycerine re-suspended cell, 1.5mL EP manages packing, every pipe 100 μ L.
Electric shock transforms: getting 50 μ L DHPY electricity changes competence, adds 2 μ L target practice plasmid pET28a-ALR, 7 μ L helper plasmid SN3, ice bath 2-3min, said mixture carefully is transferred in the 1mm electric shock cup of precooling, is fixed to electroporation, adjust voltage to 1.8kv, electric shock transforms, rapidly 1mL SOC substratum is added in the electric revolving cup after finishing, carefully blow and beat mixing, be transferred to 1.5mL EP pipe, 30 ℃ of shaking culture 75min, coating Kan Cm is two anti-dull and stereotyped.
Picking list bacterium colony is corotation bacterium liquid to the two anti-LB liquid nutrient mediums of Kan Cm.
1.3.4aroL gene inducedly knock out and identify
Get 50mL Cm LB liquid nutrient medium, add 250 μ L, 20% glucose, 500 μ L corotation bacterium liquid, 30 ℃ are cultured to OD
600Added 500 μ L, 20% pectinose at about 0.5 o'clock and induce and knock out, 30 ℃ of 220rpm induce 4h, get bacterium liquid dilution coated plate, 30 ℃ of cultivations overnight.With single bacterium colony on the flat board is template, and JL/J2 is a primer, carries out PCR, through 1% agarose gel electrophoresis, and as Fig. 6, the positive band of 1500bp band, No. 2 are positive.
Required primer sequence:
J1: see sequence 19 J2 in the sequence table: see sequence 20 in the sequence table
JL: see sequence 21 JR in the sequence table: see sequence 22 in the sequence table
Get the dilution of positive bacteria liquid and be coated with the Cm flat board, 20 single bacterium colonies of picking identify that the PCR system is the same, and PCR result is all positive, get a pipe and continue diluted passage, identify all positive once more.Get 50 μ L bacterium liquid and be seeded to 42 ℃ of cultivations of nonreactive LB liquid nutrient medium to reject the SN3 plasmid.Dilution is coated with the nonreactive flat board behind the 12h, and 20 correspondences of single bacterium colony mark are chosen to the Cm flat board 30 ℃ of cultivations on should flat board, the plasmid that is of not growing on the Cm flat board is rejected bacterial strain, get respective number bacterium colony reservation bacterial classification on the nonreactive, the aroL that is DHPY knocks out bacterial classification, is designated as DHPYA.With DHPYA bacterium liquid is template, and LJ1/LJ2 is a primer, carries out EasyTaqPCR, the order-checking of PCR stoste sample presentation, and sequencing result shows that aroL knocks out successfully.
Make up the host DHPYA-T7 that controls the abduction delivering t7 rna polymerase by pectinose 1.4 in DHPYA karyomit(e), integrate t7 rna polymerase (T7RNP) fragment
1.4.1 homology arm and target practice fragment make up
With BL21 (AI) is template, and pcr amplification comprises left and right sides homology arm and t7 rna polymerase gene and tetracycline gene, the about 5000bp of size, and required primer sequence:
AT5: see sequence 23 in the sequence table
AT3: see sequence 24 in the sequence table
1.4.2 electric shock transforms
The SN3/DHPYA electricity changes the competence preparation: picking list bacterium colony is to 5mL LB liquid nutrient medium, and 30 ℃ of overnight incubation are got 500 μ L bacterium liquid and are seeded in the 50mL Cm LB liquid nutrient medium, and 30 ℃ are cultured to OD
600Added final concentration 0.4%L-pectinose abduction delivering 40min at about 0.3 o'clock.Bacterium liquid places 20-30min on ice, fully after the cooling, goes in the 50mL centrifuge tube of precooling, and 4 ℃, 6000rpm, centrifugal 10min carefully abandons supernatant.The ddH that 50mL is ice-cold
2The O re-suspended cell, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant.The 10% glycerine re-suspended cell that 30mL is ice-cold, 4 ℃, 6000rpm, centrifugal 15min carefully abandons supernatant, repeats this step.The 10% glycerine re-suspended cell that 150 μ L are ice-cold, 1.5mLEP manages packing, every pipe 100 μ L.
Electric shock transforms immediately: getting 50 μ L electricity changes competence, add 7 μ L target practice fragment ice bath 2-3min, said mixture carefully is transferred in the 1mm electric shock cup of precooling, is fixed to electroporation, adjust voltage to 1.8kv, electric shock transforms, rapidly 1mL SOC substratum is added in the electric revolving cup after finishing, carefully blow and beat mixing, be transferred to 1.5mL EP pipe, 30 ℃ of shaking culture 75min, coating Tet flat board.
Identify 1.4.3 integrate
Required primer sequence:
Z1: see sequence 25 in the sequence table
Z2: see sequence 26 in the sequence table
Primer Z1/Z2 identifies that as Fig. 7, the positive band of the about 5000bp of PCR product is got positive strain and rejected plasmid for 42 ℃.
1.5 on DHPYA-T7 karyomit(e), knock out the gene constructed DHPYAA-T7 of shiA
1.5.1 the structure of target practice plasmid PKSMN-LR
Required sequence:
S1: see sequence 27 S2 in the sequence table: see sequence 28 in the sequence table
S3: see sequence 29 S4 in the sequence table: see sequence 30 in the sequence table
Use primer S1 and S2 respectively, primer S3 and S4, the primerSTAR enzyme, pcr amplification goes out shiA gene left side homology arm L fragment and right homology arm R fragment from template; To use primer S1 and S4, the primerSTAR enzyme is a template with L and R fragment again, and overlapping pcr amplification goes out the LR fragment, overlapping fragments LR is handled plasmid PKSMN NotI/MluI double digestion with the T4DNA polysaccharase; The fragment handled and the plasmid of double digestion are connected with quick ligase enzyme; Transform the connection product to competent cell, PCR checking positive colony is delivered order-checking, finishes the structure of plasmid PKSMN-LR.
1.5.2 single stage method directly knocks out the shiA gene
Required sequence:
S5: see sequence 31S6 in the sequence table: see sequence 32 in the sequence table
With target practice plasmid PKSMN-LR and pKOBEGA-SEC cotransformation DHPYA-T7 host competent cell, with the two anti-screenings of Amp/Kan, the mono-clonal on the two anti-plates of picking is inoculated in the two anti-liquid LB substratum of 5ml Amp/Kan and cultivates OD
600Be about 0.3-0.4, add final concentration 0.2%L-pectinose to induce the carrying out of target practice plasmid cleavage and reorganization; After inducing 5h, with bacterium liquid dilution 10
-7, get the LB flat board that 100ul coats the Tet resistance, 37 ℃ of incubated overnight; 40 mono-clonals of picking carry out PCR checking recon with homology arm outer primer S5 and S6, and the result has 7 positive recombinants, with 7 positive recombinant point plates in the LB of Kan resistance flat board, except No. 40 positive recombinants, other several not long on the Kan plate; Get one of 6 positive colony of residue, line Tet resistance LB flat board, the cultivation of going down to posterity, 37 ℃ of incubated overnight; 20 mono-clonals of picking with homology arm outer primer S5 and S6 bacterium liquid PCR checking recon, are got positive colony, continue to line Tet resistance LB flat board, and the cultivation of going down to posterity is cultivated more than the 12h for 42 ℃; And then 20 mono-clonals of picking, with homology arm outer primer S5 and S6 bacterium liquid PCR checking recon, the result is as shown in Figure 8.With No. 16 bacterium liquid PCR products, deliver order-checking, sequencing result shows that shiA successfully knocks out; With No. 16 bacterium liquid dilutions 10
-7, coat nonreactive LB flat board, 37 ℃ of incubated overnight; 20 single bacterium colony point plates on the picking nonreactive LB flat board are in Amp resistance LB flat board, 30 ℃ of incubated overnight; 20 single bacterium colonies are not long on the Amp plate, and then helper plasmid pKOBENGA-SEC rejects totally, preserve the degerming of shiA clpp gene, name DHPYAA-T7.
1.6 on DHPYAA-T7 karyomit(e), knock out the gene constructed DHPYASA-T7 of aroK
1.6.1 the structure of target practice plasmid PKSMN-LR
Required sequence:
Aro1: see sequence 33 Aro2 in the sequence table: see sequence 34 in the sequence table
Aro3: see sequence 35 Aro4 in the sequence table: see sequence 36 in the sequence table
Use primer Aro1 and Aro2 respectively, primer Aro3 and Aro4, the primerSTAR enzyme, pcr amplification goes out aroK gene left side homology arm L fragment and right homology arm R fragment from template; To use primer Aro1 and Aro4, the primerSTAR enzyme is a template with L and R fragment again, and overlapping pcr amplification goes out the LR fragment, overlapping fragments LR is handled plasmid PKSMN NotI/MluI double digestion with the T4DNA polysaccharase; The fragment handled and the plasmid of double digestion are connected with quick ligase enzyme; Transform the connection product to competent cell, PCR checking positive colony is delivered order-checking, finishes the structure of plasmid PKSMN-LR.
1.6.2 single stage method directly knocks out the aroK gene
Required sequence:
Aro5: see sequence 37 Aro6 in the sequence table: see sequence 38 in the sequence table
With target practice plasmid PKSMN-LR and pKOBEGA-SEC cotransformation DHPYAS-T7 host competent cell, the two anti-screenings of Amp/Kan; Mono-clonal on the two anti-plates of picking is inoculated in the two anti-liquid LB substratum of 5ml Amp/Kan and cultivates OD
600Be about 0.3-0.4, add 20%L-pectinose to final concentration 0.2% and induce the carrying out of target practice plasmid cleavage and reorganization; After inducing 5h, with bacterium liquid dilution 10
-7, get the LB flat board that 100ul coats the Tet resistance, 37 ℃ of incubated overnight; 40 mono-clonals of picking, with homology arm outer primer Aro5 and Aro6 bacterium liquid PCR checking recon, the result has 3 positive recombinants, and 3 positive recombinant point plates are in the LB of Kan resistance flat board, not long on the Kan plate, illustrates that then the target practice plasmid cleavage is clean; Get one of them positive colony, line Tet resistance LB flat board, the cultivation of going down to posterity, 37 ℃ of incubated overnight; 20 mono-clonals of picking with homology arm outer primer Aro5 and Aro6 bacterium liquid PCR checking recon, are got one of them positive colony, continue to line Tet resistance LB flat board, and the cultivation of going down to posterity is cultivated more than the 12h for 42 ℃; 20 mono-clonals of picking, with homology arm outer primer Aro5 and Aro6 bacterium liquid PCR checking recon, the result with No. 1 bacterium liquid PCR product, delivers order-checking as shown in Figure 9, and sequencing result shows that aroK successfully knocks out, afterwards, with No. 1 bacterium liquid dilution 10
-7, coat nonreactive LB flat board, 37 ℃ of incubated overnight, 20 single bacterium colony point plates on the picking nonreactive LB flat board are in Amp resistance LB flat board, 30 ℃ of incubated overnight; 20 single bacterium colonies are not long on the Amp plate, and then helper plasmid pKOBENGA-SEC rejects totally, preserve the degerming of aroK clpp gene, name DHPYASA-T7.
TktA, glk, aroE, aroF, aroB carrier that embodiment 2, structure are expressed by the control of T7 promotor
2.1 high copy number plasmid pET-TGEFB with the pET vector construction
2.1.1 site-directed point mutation and plasmid construction
The design primer carries out same sense mutation, eliminates the common restriction enzyme site of part that exists in tktA, glk, the aroF gene.After order-checking is correct, obtain pET3b-tktAt, pET22b-glkt, pET22b-AaroFft:tktAt are connected to the pET3b carrier after with Nde I/BamH I double digestion; Glkt is connected to the pET22b carrier with Xho I/BamH I double digestion; AroF is connected to the pET22b carrier with Xho I/BamHI double digestion; AroE is connected to the pET3b carrier with Nde I/BamH I double digestion; AroB is connected to the pET28a carrier with NcoI/BamH I double digestion.After order-checking is correct, obtain pET3b-tktAt, pET22b-glkt, pET22b-AaroFft, pET3b-aroE, pET28a-aroB plasmid.
2.1.2 plasmid splicing
The enzyme that carries out plasmid is cut connection as shown in Figure 8, and enzyme is cut to connect and is ordinary method, and is the same.Cut connection by enzyme and obtain the pET3b-TGEFB plasmid.
2.2 with the low copy of pACYC vector construction plasmid pAOC-TGEFB
The pAOC plasmid: being masterplate amplification P15Aori and CmR fragment with pACYCDeut is connected with top SphI/EcoRI endonuclease bamhi (about 500bp), enzyme is cut product and is reclaimed, reclaim pAOC fragment and segmental quick connection of TGEFB, be converted into DH5 α, primer identifies that correct back upgrading grain promptly obtains pAOC-TGEFB.
The acquisition of embodiment 3, shikimic acid expression strain DHPYAS-T7
The bacterial strain DHPYASA-T7 that embodiment 1 is obtained makes competent cell, changes the recombinant plasmid pAOC-TGEFB that obtains among the embodiment 2 over to described competent cell with chemical process then, thereby obtains shikimic acid prepared bacterial strain.
More than the shikimic acid expression strain DHPYAS-T7 of Huo Deing has been committed on March 3rd, 2011 and has been positioned at Pekinese China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.4631.
4.1 substratum and culture condition
LB substratum (g/L): peptone 10, yeast powder 5, sodium-chlor 10;
FMG substratum (g/L): K
2HPO
43H
2O 7, NaH
2PO
32H
2O 3, and NaCl 2.5, yeast powder 15, and peptone 10, other adds 0.5g MgSO
47H
2O and 25g glucose;
Paraxin working concentration: 25mg/L; Tsiklomitsin working concentration: 10mg/L.
4.2 cultural method
With the reorganization of the intestinal bacteria among the embodiment 1 bacterium DHPYAS-T7, picking list colony inoculation is in the test tube that contains the two anti-LB liquid nutrient mediums of 3ml, 37 ℃ of incubated overnight 8h are inoculated in the 250ml that contains 50ml FMG substratum by 1% inoculum size then and shake in the bottle, cultivate 6～8 hours to OD
600When being about 0.6 left and right sides, adding final concentration and be 0.2% L-arabinose induces, continue to cultivate 48 hours, allow shikimic acid give full expression to accumulation, reorganization bacterium DHPYAS-T7 accumulation volume behind shake-flask culture 48h has reached maximum 920mg/L, as Fig. 9, produce bacterium JDL02/pTrc-aroG-tktA with the shikimic acid of people such as domestic Yang Jie report and shake in the bottle rate ratio, be the latter's (94.33mg/L) 9.8 times.
Claims (11)
1. construction process of producing the recombination bacillus coli of shikimic acid, its feature at first is to have knocked out the ydiB gene.
2. according to claim 1, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to have knocked out the ptsHIcrr operon.
3. as described in the claim 1-2, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to knock out the aroL gene.
4. as described in the claim 1-3, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to knock out the aroK gene.
5. as described in the claim 1-4, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to knock out the shiA gene.
6. as described in the claim 1-5, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to have integrated on its karyomit(e) the t7 rna polymerase gene by the L-arabinose regulating and expressing.
7. as described in the claim 1-6, this kind produced the construction process of shikimic acid recombination bacillus coli, and its feature also is to have transformed in its thalline recombinant expressed low copy plasmid pAOC-TGEFB.
8. as described in the claim 7, recombinant expression plasmid pAOC-TGEFB includes key gene tktA, glk, aroE, aroF and the aroB in the shikimic acid pathways metabolism.
9. as described in the claim 8, it is characterized in that contained tktA, glk, aroE, aroF and the aroB gene of described recombinant expressed low copy plasmid pAOC-TGEFB all has the T7 promotor.
10. as described in the claim 1-7, make up the recombinant escherichia coli strain that obtains to produce shikimic acid, it is characterized in that its preserving number in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation is: CGMCC No.4631 with this method.
11., it is characterized in that this project bacterium is a bacillus coli DH 5 alpha as recombinant bacterial strain as described in the claim 10.
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| CN112779201A (en) * | 2021-01-14 | 2021-05-11 | 宜昌东阳光生化制药有限公司 | Recombinant microorganism, application thereof and method for preparing shikimic acid and oseltamivir |
| CN112779201B (en) * | 2021-01-14 | 2023-05-09 | 宜昌东阳光生化制药有限公司 | Recombinant microorganism and application thereof, and method for preparing shikimic acid and oseltamivir |
| CN114591880A (en) * | 2022-03-16 | 2022-06-07 | 江南大学 | Construction and application of escherichia coli capable of accumulating shikimic acid |
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