CN102304539B - Construction method of genetic engineering strain for producing shikimic acid - Google Patents
Construction method of genetic engineering strain for producing shikimic acid Download PDFInfo
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Abstract
The invention discloses a construction method of a genetic engineering strain for producing shikimic acid. The method comprises the following steps of: 1, constructing an escherichia coli strain of which the aroA gene is knocked out; 2, constructing a recombinant expression plasmid pAR63 containing key enzyme genes aroGFBR, aroE, aroB, aroD, tktA and ppsA in the metabolic pathway of the shikimic acid, so that the genes are subjected to transcriptional control of a tryptophan promoter Ptrp; and 3, transferring the recombinant expression plasmid pAR63 into the escherichia coli strain of which the aroA gene is knocked out to obtain a production strain for expressing the shikimic acid. In the method, the aim of interrupting the metabolism of the shikimic acid is fulfilled by the knock-out of the single gene aroA, a small number of genes are knocked out, the operating difficulty is low, and the expression of the genes is in the state of starting and stopping automatically and is started automatically in the middle and late period without the induction of inducers, so the possibility of adding toxic substances into a culture medium is reduced, and the shikimic acid has high yield.
Description
Technical field
The invention belongs to the genetically engineered field, relate in particular to the construction process that engineering strain is used in a kind of shikimic acid production, this bacterial strain can be used for the fermentative production shikimic acid.
Background technology
Shikimic acid (Shikimic acid, Shikimate), chemical name [3R-(3 α, 4 α, 5 β)]-3,4,5-trihydroxy--1-tetrahydrobenzene-1-carboxylic acid, being the intermediate product of organism intracellular metabolite process, also is the raw material of synthetic many alkaloids, aromatic amino acid and indole derivatives, chiral drug (such as antiviral drug), extensively is present in plant and the microorganism.
Shikimic acid is by affecting arachidonic acid metabolism, and anticoagulant suppresses the formation of artery and vein thrombus and cerebral thrombosis, and shikimic acid not only has anti-inflammatory, analgesic activity, can also be as antiviral and cancer therapy drug intermediate.Some derivatives of shikimic acid also have certain drug effect.
Take shikimic acid as raw material synthetic " Tamiflu " be world health organisation recommendations prevent and treat the high pathogenic avian influenza medicine, also be that the bird flu medicine is effectively treated in present unique confirmation.Since bird flu broke out, the world sharply enlarged the demand of " Tamiflu ".The Chinese government and enterprise be all in the deposit and the production that step up respectively to carry out the anti-avian influenza medicine, but because producing main raw material---the deficiency of shikimic acid output of " Tamiflu ", seriously limited the production of " Tamiflu ".
At present, the preparation method of shikimic acid mainly contains three kinds, is respectively plant tissue extraction method, chemical synthesis and microbe fermentation method.Shikimic acid is present in higher plant and the microorganism in a large number, contains shikimic acid 8%～13% such as octagonal fruit, is that all kinds of shikimic acid content of having studied in the floristics are the highest, also is the most frequently used raw material of suitability for industrialized production shikimic acid.But because Illicium lanceolalum really contains highly toxic substance, its volatile oil can cause paralysis, need to can meet the requirement of environmental protection through strict processing when extracting shikimic acid, and production cost is higher.Have the advantages such as simple and convenient, that technical limitation is less although extract shikimic acid from plant, and mostly present shikimic acid is to extract from the plant tissues such as star anise also, the method is limited by raw material output, can't produce in a large number.Along with the raising of star anise price, the shikimic acid cost also rises thereupon in addition.Chemical synthesis process is organic and toxic reagent because of more use, can not be widely used in shikimic acid production.Microbe fermentation method easily continues suitability for industrialized production because of the less restriction that is subject to raw material sources, and environmental pollution is less, and is with low cost, is applied to gradually shikimic acid production.
The shikimic acid circulation extensively is present in the microorganism, also can extract shikimic acid by microbial fermentation.Conversion of glucose is die aromatischen Aminosaeuren (L-Phe, 1B and L-Trp) in intestinal bacteria, and shikimic acid is intermediate product.Bacillus coli communis can accumulate shikimic acid after transforming.Biotechnology center cooperation of Roche Holding Ag 2005 and Germany by to a kind of harmless intestinal bacteria excess supply glucose, forces bacterium to discharge shikimic acid.At present, the shikimic acid of Roche Holding Ag 2/3rds is taken from star anise, and remaining 1/3rd are provided by fermentation method.
In self metabolism of organism, shikimic acid is difficult to a large amount of accumulation.Shikimic acid is as intermediate metabolites, in case syntheticly just be used to particularly aromatic molecule synthetic of other biomolecules by meeting.Thereby biological primary metabolism must be broken just and may accumulate shikimic acid.With the synthetic involved enzyme of shikimic acid 3-deoxidation-D-Arab-synthetic (3-deoxy-7-phosphohep tulonate synthase of reward-7-phosphoric acid in heptan is arranged, be called for short the DAHP synthetic enzyme, catalysis DAHP's is synthetic), 3-dehydroquinate synthase (3-dehydroq uinate synthase, catalysis is by the reaction of DAHP to the 3-dehydroquinic acid), 3-dehydroquinate dehydratase (3-dehydroquinate dehydratase, the dehydration of catalysis 3-dehydroquinic acid generates the dehydrogenation shikimic acid), shikimate dehydrogenase (shikimate dehydrogenase, the catalytic dehydrogenation shikimic acid is to the transformation of shikimic acid, this reaction is reversible) and shikimate kinase isozyme (shikimate kinase isozyme, catalysis shikimic acid phosphoric acid turns to the 3-phosphoric acid shikimic acid).They in intestinal bacteria respectively by
AroF(
AroG,
AroH),
AroB,
AroD,
AroE,
AroK(
AroL) coding, wherein
AroF,
AroGWith
AroHEqual encoding D AHP synthetic enzyme (
AroFBe subjected to the tyrosine feedback inhibition,
AroGBe subjected to the phenylalanine feedback inhibition,
AroHBe subjected to the tryptophane feedback inhibition),
AroKWith
AroLThe shikimate kinase isozyme of all encoding, it is converted into 3-phosphoric acid shikimic acid (shikimate-3-phosphate) with shikimic acid.Owing to synthesizing the catalysis that relevant enzyme is not sufficient to accumulate in a large number shikimic acid with shikimic acid, therefore shikimate kinase catalysis shikimic acid is produced bacterial strain and must be carried out the transformation of relevant enzyme to be fit to synthesizing in a large number of shikimic acid to the conversion of other compound.
At present, the Production by Microorganism Fermentation about shikimic acid has relevant report at home and abroad.Yurgis etc. are by transforming to produce shikimic acid to subtilis, this bacterial strain by inactivation shikimate kinase gene (
AroI) or 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS, but catalysis shikimic acid-3-phosphoric acid is to the conversion of chorismic acid) gene, import the genes such as shikimate dehydrogenase and DHAP synthetic enzyme, can produce approximately 20g/L of shikimic acid.John W. Frost etc. are by carrying out genetic modification improving shikimic acid output to intestinal bacteria, these bacterial strains comprise the gene synthetic relevant with shikimic acid as
AroF,
AroE,
SerA,
TktA,
PpsAWith
AroDDeng, so that shikimic acid output reaches about 70g/L.John W. Frost to 2-keto-3-deoxygalactonic acid zymohexase (KDPGal aldolase) gene carry out molecular evolution obtain a kind of can catalysis pyruvic acid and the synthetic DAHP of E4P, compare with the DAHP synthetic enzyme, can save a large amount of PEP for cell, further improve shikimic acid output.Wang Li is with the shikimic acid synthesis related gene
AroE,
AroFWith
AroBImport in the genome of E.coli, knock out simultaneously the shikimate kinase gene (
AroLWith
AroK) and
ShiA, adopt this bacterial strain to ferment, can produce shikimic acid 8.3g/L.In addition, also there is the report that adopts citric acid bacillus to carry out the shikimic acid fermentation in Japan.
Trp promoter is initial transcribing expeditiously.Trp promoter be subject to aporepressor TrpR(by
TrpRGenes encoding) regulation and control, when the tryptophane capacity, TrpR is combined into the startup that activity form checks trp promoter with tryptophane; When tryptophane was deficient, TrpR was inactive form without the tryptophane combination, can't check the startup of promotor, was subjected to the gene transcriptional start of trp promoter control.Because above-mentioned this characteristics, trp promoter is widely used in expression and the Enzyme Production of recombinant protein.
The metabolism of shikimic acid is interrupted in existing invention mostly by knocking out gene, and imports effective accumulation that shikimic acid anabolism gene is realized shikimic acid, and these class methods need to knock out more gene from host cell, operate relatively loaded down with trivial details.
Summary of the invention
The object of the present invention is to provide a kind of shikimic acid production construction process of engineering strain, it is few that the method knocks out gene, and the bacterial strain of structure can effectively interrupt the shikimic acid metabolism and realize a large amount of accumulation of shikimic acid.
For achieving the above object, the present invention adopts following technical scheme:
A kind of shikimic acid production construction process of engineering strain, it may further comprise the steps:
1. make up
AroAThe coli strain of gene knockout;
Utilize the Red system constructing
AroAThe coli strain W3110(of gene knockout
AroA).The inventive method is used intestinal bacteria
E. coliBacterial strain W3110, but bacterial strain is not limited only to one kind of W3110.The Red system is the technology that is used for genetic manipulation of new development, utilizes recombinant technology can knock out easily, knock in or mutator gene, and is at present multiplex in intestinal bacteria.
AroAGenes encoding EPSPS, catalysis shikimic acid-3-phosphoric acid and PEP reaction generate 5-enol acetone shikimic acid-3-phosphoric acid.Knock out
AroAGene can cause bacterium disappearance 5-enol acetone shikimic acid-3-phosphate synthase, and metabolism stream interrupts at shikimic acid-3-phosphoric acid, thereby is beneficial to accumulation shikimic acid and shikimic acid-3-phosphoric acid.
2. make up the key gene that contains in the shikimic acid pathways metabolism
AroG FBR (enzyme of this genes encoding is not subjected to the phenylalanine feedback inhibition),
AroE,
AroB,
AroD,
TktAWith
PpsARecombinant expression plasmid pAR63, and these genes all are in trp promoter
P Trp To transcribe control lower;
Recombinant expression plasmid pAR63 is by successively with gene
PpsA,
TktA,
AroB,
AroD,
AroEWith
AroG FBR Being connected to the pAR plasmid obtains.Wherein, gene
AroG,
AroE,
AroD,
AroB,
TktAWith
PpsAAll derive from intestinal bacteria, the plasmid that sets out of recombinant expression plasmid pAR63 is pACYC184.
3. recombinant expression plasmid pAR63 is changed over to and knock out
AroAThe coli strain W3110(of gene
AroA), the production bacterial strain of shikimic acid is expressed in acquisition.The production bacterial strain can add glucose mode dissolved oxygen controlled fermentation by stream and prepare shikimic acid.
Concrete, 1. step comprises:
A, obtain by pcr amplification
AroAHomology is replaced sequence D aroA;
B, plasmid pKD46 is imported coli strain W3110;
C, induce step b gained positive strain to produce the required enzyme of homologous recombination, the results thalline prepares competent cell;
D, the homology replacement sequence D aroA that transforms step a acquisition by electric shock import the competent cell that step c obtains, and obtain
AroAThe bacterial strain that gene is replaced;
The resistant gene fragment of e, use pCP20 plasmid removal process d obtained strains obtains
AroAGene knock-out bacterial strain.
Concrete, 2. step comprises:
A, make up based on plasmid pACYC184 and to comprise
TrpRThe transformation carrier pAR of gene can express a certain amount of TrpR, is used for checking on the carrier
P Trp Promotor;
B, the anti-feedback inhibition of structure
AroG FBR Gene, and before gene, add trp promoter
P Trp
C, clone
AroBWith
AroEGene, and be its interpolation ribosome bind site RBS;
D, clone gene
TktA,
PpsAWith
AroD, and
TktABefore add trp promoter
P Trp ,
PpsAThe back adds that transcription terminator (use by transcription terminator
TerExpression);
E, with each gene according to
P Trp ,
AroG FBR ,
AroE,
AroB,
AroD,
P Trp ,
TktA,
PpsAWith being linked in sequence to the pAR plasmid of transcription terminator, consist of the pAR63 plasmid.
Contain trp promoter aporepressor gene among the described recombinant expression plasmid pAR63
TrpR, and
TrpRWith self promotor, can independently express.
Contained among the described recombinant expression plasmid pAR63
PpsAThe gene downstream sequence is the Transcription Termination subsequence; Contained among the described recombinant expression plasmid pAR63
TktAGene upstream sequence is the tryptophan promoter subsequence; Contained among the described recombinant expression plasmid pAR63
AroBWith
AroEGene upstream sequence all contains ribosome bind site (RBS) sequence; Contained among the described recombinant expression plasmid pAR63
AroDGene is with self RBS sequence; Contained among the described recombinant expression plasmid pAR63
AroG FBR Gene upstream sequence is the tryptophan promoter subsequence,
AroG FBR Be by
AroG180 site rite-directed mutagenesises of gene are that Phe obtains.Described Transcription Termination subsequence is
RrnBThe Transcription Termination subsequence.
The present invention knocks out the EPSPS gene in the bacterium, make up to import simultaneously contain trp promoter (
P Trp ) carrier of rate-limiting enzyme gene of control, reduce the katabolism of shikimic acid, automatically open related gene expression at suitable growth period, body burden before increasing is accelerated building-up reactions, thereby promotes that the raw material Efficient Conversion is shikimic acid.
Be compared with existing technology, the innovative point of the inventive method is as follows:
(1) by knocking out
AroASingle-gene realizes interrupting the purpose of shikimic acid metabolism, need to knock out gene few, with dual-gene
AroL/KKnockout technique is compared operation easier and is reduced, and the shikimic acid metabolism flows to shikimic acid-3-phosphoric acid, reduces shikimic acid to the feedback inhibition of the upstream enzymes such as shikimate dehydrogenase, reduces the intermediate product content in the shikimic acid building-up process, improves shikimic acid output.The reaction final product is mainly shikimic acid and shikimic acid-3-phosphoric acid, can change shikimic acid into by acidifying and heat treated.
(2) imported the shikimic acid synthesis related gene, and be in
P Trp Under the regulation and control, the fermentation initial stage, contain abundant amino acid in the substratum, genetic expression is in the state of checking, and is conducive to the thalline accumulation.Owing to shikimic acid can't enter the die aromatischen Aminosaeuren anabolism, the synthetic bacterial strain of shikimic acid is the tryptophane defective type, can't carry out the production of tryptophane, and concentration reduces after tryptophane consumption, and each gene progressively begins to express, and a large amount of of catalysis shikimic acid synthesize.
(3) because shikimic acid can't enter the die aromatischen Aminosaeuren anabolism, the synthetic bacterial strain of shikimic acid is the tryptophane defective type, can't carry out the production of tryptophane, and genetic expression is passed through
P Trp Regulating and expressing, and the tryptophane coupling in the substratum in the fermenting process of shikimic acid synthesis bacterium, natural controlling gene are expressed has the time difference opposite sex, has simplified the operation in the production technique.
(4) expression of gene is in the auto-switch state, automatically starts in the middle and later periods, need not inducing of inductor, has reduced the possibility of adding toxic substance in the substratum.
Description of drawings
Fig. 1 is
AroAHomology replace sequence D aroA PCR product the agarose gel electrophoresis result and
AroAKnock-out bacterial strain W3110(
AroA) the PCR qualification result; Wherein DaroA length is 1596bp, and W3110 bacterial strain PCR fragment should be 1626bp, W3110(
Kan R AroA) bacterial strain PCR fragment should be 1838bp, the bacterial strain W3110(after kalamycin resistance gene is eliminated
AroA) the PCR fragment should be 445bp.The result show DaroA length normal and
AroABe knocked, the resistant gene fragment is also eliminated.Swimming lane M:1kb Marker among the figure; Swimming lane 1:DaroA; Swimming lane 2: bacterial strain W3110; Swimming lane 3: bacterial strain W3110(
Kan R AroA); Swimming lane 4-5: bacterial strain W3110(
AroA);
Fig. 2 is PCR product and plasmid enzyme restriction qualification result in the pAR plasmid construction process; Swimming lane M:1kb Marker wherein; Swimming lane 1:
TrpRSequence PCR product; Swimming lane 2:pACYC184 plasmid PCR product; Swimming lane 3: plasmid pAR's
HinDIII single endonuclease digestion collection of illustrative plates; Swimming lane 4: plasmid pAR's
HinDIII and
PstI double digestion collection of illustrative plates;
Fig. 3 is that PCR product and enzyme are cut qualification result in the plasmid pAR01 building process; Swimming lane M:1kb Marker among the figure; Swimming lane 1:
PpsAThe gene PCR product; Swimming lane 2: terminator sequence PCR product; Swimming lane 3:
PpsABe connected the PCR product with the terminator sequence; Swimming lane 4: plasmid pAR01's
BamHI single endonuclease digestion result; Swimming lane 5: plasmid pAR01's
BamHI and
PstI double digestion result;
Fig. 4 is that PCR product and plasmid pAR03 enzyme are cut qualification result in the plasmid pAR03 building process; Swimming lane M1:100 bp Marker among the figure; Swimming lane M2:1kb Marker; Swimming lane 1:
P Trp Sequence PCR product; Swimming lane 2:
TktAThe gene PCR product; Swimming lane 3:
P Trp With
TktAGene connects the PCR product; Swimming lane 4: plasmid pAR03's
BamHI single endonuclease digestion result; Swimming lane 5: plasmid pAR03's
BamHI and
PspOMI double digestion result;
Fig. 5 is that PCR product and plasmid pAR17 enzyme are cut qualification result in the plasmid pAR17 building process; Swimming lane M:1kb Marker among the figure; Swimming lane 1:
AroBThe gene PCR product; Swimming lane 2:
AroDThe gene PCR product; Swimming lane 3:
AroBWith
AroDThe gene PCR product; Swimming lane 4: plasmid pAR17's
BamHI single endonuclease digestion result; Swimming lane 5: plasmid pAR17's
BamHI and
EagI double digestion result;
Fig. 6 is that PCR product and plasmid pAR31 enzyme are cut qualification result in the plasmid pAR31 building process; Swimming lane M:1kb Marker among the figure; Swimming lane 1:
AroEThe gene PCR product; Swimming lane 2: interpolation RBS sequence
AroEThe gene PCR product; Swimming lane 3: plasmid pAR31's
BamHI single endonuclease digestion result; Swimming lane 4: plasmid pAR31's
BamHI and
SacI double digestion result;
Fig. 7 is that PCR product and plasmid pAR63 enzyme are cut qualification result in the plasmid pAR63 building process; Swimming lane M1:100bp Marker among the figure; Swimming lane 1:
P Trp Sequence PCR product; Swimming lane 2:
AroG(first half) PCR product; Swimming lane 3:
AroG(latter half) PCR product; Swimming lane 4:
P Trp -aroG(first half) PCR product; Swimming lane 5:
P Trp -aroG FBR The PCR product; Swimming lane 6:pAR63's
BamHI single endonuclease digestion product; Swimming lane 7:pAR63's
BamHI and
SphI double digestion product; Swimming lane M2:1kb Marker;
Fig. 8 is structure and the structural representation of recombinant plasmid pAR63;
Fig. 9 be in the fermenting process of fermentative Production shikimic acid among the embodiment 1 cell concentration and shikimic acid content with the variation of fermentation time.
Embodiment
The present invention is described in detail by the following examples, but protection scope of the present invention is not limited to this.The used LB liquid nutrient medium of embodiment consists of: 1% Tryptones, 1% sodium-chlor and 0.5% yeast extract; The LB solid medium consists of: 1% Tryptones, 1% sodium-chlor, 0.5% yeast extract and 1.5% agar.The processes such as the preparation of mentioning among the embodiment or conversion if no special instructions, then adopt the ordinary method of this area all can realize, therefore repeat no more.
A kind of shikimic acid production construction process of engineering strain, it may further comprise the steps:
One, makes up
AroAThe coli strain W3110(of gene knockout
AroA);
The principle of Red recombinant technology is that two sections dna fragmentations that contain the goal gene homologous sequence are imported host bacteria, and under the effect of recombinase, thereby the goal gene fragment is replaced the work that knocks out of finishing goal gene by other fragment.
) a, obtain by pcr amplification
AroAHomology is replaced sequence D aroA;
PKD4(is provided by Shanghai Inst. of Life Science, CAS take plasmid) be template, utilize the primer aroAF(to see sequence 1 in the sequence table) and aroAR(see sequence 2 in the sequence table), among the pcr amplification plasmid pKD4
Kan R Gene.5 ' end of homologous recombination primer is goal gene both sides homologous sequence, and 3 ' end is resistant gene both sides sequence, and primer sequence is as follows:
AF：5’-atggaatccctgacgttacaacccatcgctcgtgtcgatggcactattaagtgtaggctggagctgcttc-3’
AR：5’-tcaggctgcctggctaatccgcgccagctgctcgaaataatccggaaatgatgggaattagccatggtcc-3’
The pcr amplification condition is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 1596bp, and agarose gel electrophoresis reclaims this fragment and is DaroA, sees Fig. 1.Gained PCR fragment both sides are
AroAThe dna homolog fragment, middle portion is that resistant gene of card.The PCR fragment is carried out behind the agarose gel electrophoresis purifying for subsequent use.
) b, plasmid pKD46 is imported coli strain W3110;
With reference to the molecular cloning experiment guide third edition, the chemoreception attitude cell of preparation W3110, and provided by Shanghai Inst. of Life Science, CAS with plasmid pKD46() transform W3110 bacterial strain competent cell, coat on the LB solid medium flat board that contains 100 μ g/mL penbritins (Amp) behind 30 ℃ of cultivation 2h, 30 ℃ of incubated overnight, the transformant that can grow is the intestinal bacteria W3110 bacterial strain that contains the pKD46 plasmid, selects positive transformant and saves backup.
) c, induce step b gained positive strain to produce the required enzyme of homologous recombination, the results thalline prepares competent cell;
The positive strain that step b is obtained is inoculated in the 5mL ammonia benzyl resistance LB liquid nutrient medium (containing 100 μ g/mL Amp) in 30 ℃, 200r/min incubated overnight, inoculate next day in 1mL to the 100mL ammonia benzyl resistance LB liquid nutrient medium (containing 100 μ g/mL Amp), 30 ℃, 200r/min are cultured to cell concentration OD
600=0.25 o'clock, adding L-arabinose to final concentration was 30mmol/L, is induced to OD
600=0.6, with reference to the molecular cloning experiment guide third edition, harvested cell preparation electric shock transformed competence colibacillus cell.
) d, transform the homology that step a is obtained by electric shock and replace sequence D aroA and import the competent cell that step c obtains, obtain
AroAThe bacterial strain that gene is replaced;
The PCR product D aroA that gets 5 μ L step a gained adds in the competent cell that 50 μ L step c make, and places in the electric shock cup that changes 2mm after 5 minutes over to shock by electricity (the electric shock condition is 200 Ω, 25 μ F, voltage 2.5kV, electric shock time 4-5ms) on ice.Add in the 950 μ L LB liquid nutrient mediums after the electric shock, 150r/min cultivates 2h, the centrifugal 1min of 12000 * g for 37 ℃, discard upper strata 800 μ L supernatant liquors, be applied to dull and stereotyped (containing 10 μ g/mL kantlex) the upper 37 ℃ of incubated overnight of that resistance of card LB solid medium after remaining cell is resuspended gently.The transformant that can grow is and knocks out
AroAThe bacterial strain of gene is selected positive transformant and is saved backup, and is designated as W3110(
Kan R AroA) bacterial strain.
) e, use the resistant gene fragment of pCP20 plasmid removal process d obtained strains, obtain
AroAGene knock-out bacterial strain.
Plasmid pCP20(is provided by Shanghai Inst. of Life Science, CAS) change in the positive colony of above-mentioned steps d acquisition, 30 ℃ of cultivations, choose the clone who has simultaneously that resistance of card and ammonia benzyl resistance, be inoculated in the LB liquid nutrient medium, 30 ℃, 200r/min cultivates 8h, then temperature is brought up to 42 ℃, this moment FLP recombinase (Flipase recombination enzyme, the Flippases recombinase) begins to express, catalysis FRT(Flp recombination target, Flippases recombination site) elimination of resistant gene fragment between the site, plasmid pCP20 loses gradually owing to stopping to copy.Dull and stereotyped with coating the LB solid medium after the dilution of bacterium liquid, select single colony inoculation on that resistance of card LB solid medium dull and stereotyped (containing 10 μ g/mL kantlex), the bacterium colony that can't grow is the bacterial strain that resistant gene is eliminated.
AroAGene knock-out bacterial strain is with W3110(
AroA) expression.
Obtained strains (comprises W3110(
Kan R AroA) and W3110(
AroA)) see sequence 3 in the sequence table take DF(respectively) and DR(see sequence 4 in the sequence table) identify as primer carries out PCR, the W3110 bacterial strain is contrast.Primer sequence is as follows: DF:5 '-tcatggttgagttcgaacgccgtc-3 '; DR:5 '-gtgggtattttgacaccctgtcag-3 ';
The pcr amplification condition is: 95 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.Be different take different strains as the dna fragmentation length that template was obtained, can judge whether thus from genome, to knock out
AroAGene the results are shown in Figure 1, judges that therefore the last bacterial strain that obtains is
E. coliW3110(
AroA), save backup.
Two, make up the key gene that contains in the shikimic acid pathways metabolism
AroG FBR (this gene is not subjected to the phenylalanine feedback inhibition),
AroE,
AroB,
AroD,
TktAWith
PpsARecombinant expression plasmid pAR63, it makes up and structural representation is seen Fig. 8;
2.1 the structure of pAR plasmid
trpRF：5’-gcgctgcaggtcggatccacgtcgttactgatccgcac-3’；
trpRR：5’-gggaagcttccacgtcttatcaggcctac-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 480bp, the results are shown in Figure 2.
pAF：5’-gcgctgcagggcctcaggcatttgagaag-3’；
pAR：5’-ttaaagcttatcgatgataagctgtc-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 2min15s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2197bp, the results are shown in Figure 2.
Above-mentioned two kinds of dna fragmentations are used respectively
HinDIII and
PstCarry out gel behind the I double digestion and reclaim, then use the T4 dna ligase to connect two fragments.Connect product and transform Top10 competent cell (available from the auspicious bio tech ltd of Nanjing Sai Hong), screen positive strain at the LB solid medium flat board that contains 20 μ g/mL paraxin, the gained plasmid is the pAR plasmid.
Gained pAR plasmid employing single endonuclease digestion (
HinDIII) and double digestion (
HinDIII and
PstI) identify, agarose gel electrophoresis figure sees Fig. 2.
Will
PpsAGene imports the pAR plasmid
Adopt ppsAF(to see sequence table sequence 9) and ppsAR(see sequence table sequence 10) be primer, bacterial strain W3110 genome is that template is carried out pcr amplification and obtained fragment 1, reclaims for subsequent use.Primer sequence is as follows:
ppsAF：5’-catggatccctcgggcccatgtgtttctcaaaccgttc-3’；
ppsAR：5’-gccgccaggcaaattctgtttttatttcttcagttcagcc-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 2min30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2460bp, the results are shown in Figure 3.
Adopt TerF(to see sequence table sequence 11) and TerR(see sequence table sequence 12) be primer, the W3110 genome is that template is carried out pcr amplification and obtained fragment 2, reclaims for subsequent use.Primer sequence is as follows:
TerF：5’-ggctgaactgaagaaataaaaacagaatttgcctggcggc-3’；
TerR：5’-gcgctgcagaagagtttgtagaaacgcaaaaaggccatc-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 40s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 382bp, the results are shown in Figure 3.
In 20 μ L PCR reaction systems (reaction system contains 0.5U Taq archaeal dna polymerase, 1 * Taq dna polymerase buffer liquid, 1mmol dNTPs), do not add primer, add fragment 1 obtained above and fragment 2 each 2 μ L, carry out circulating reaction 10 times.Then add primer ppsAF and TerR to concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 3, reclaim for subsequent use.
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 45s, 72 ℃ are extended 3min, circulate altogether 10 times; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 3min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2803bp, the results are shown in Figure 3.
With
BamHI and
PstThen I double digestion plasmid pAR and dna fragmentation 3 connect two fragments with the T4 dna ligase, transform the Top10 competent cell, screen positive colony, and the gained plasmid is pAR01.
Gained plasmid pAR01 carry out respectively single endonuclease digestion (
BamHI) and double digestion identify (
BamHI and
PstI), qualification result is seen Fig. 3.
Will
TktAGene imports the pAR01 plasmid
Adopt PtrpF(to see sequence table sequence 13) and PtrpR(see sequence table sequence 14) be primer, bacterial strain W3110 genome is that template is carried out pcr amplification and obtained fragment 1, reclaims for subsequent use.Primer sequence is as follows:
PtrpF：5’-catggatccctccggccgcggttctggcaaatattc-3’；
PtrpR：5’-gcaagctctttacgtgaggacattgtcgataccctttttacg-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 132bp, the results are shown in Figure 4.
Adopt tktAF(to see sequence table sequence 15) and tktAR(see sequence table sequence 16) be primer, the W3110 genome is that template is carried out pcr amplification and obtained fragment 2, reclaims for subsequent use.Primer sequence is as follows:
tktAF：5’-cgtaaaaagggtatcgacaatgtcctcacgtaaagagcttgc-3’；
tktAR：5’-ctcgggcccttacagcagttcttttgctttcgcaac-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2019bp, the results are shown in Figure 4.
In 20 μ L PCR reaction systems (reaction system contains 0.5U Taq archaeal dna polymerase, 1 * Taq dna polymerase buffer liquid, 1mmol dNTPs), do not add primer, add above-mentioned two kinds of fragments 1 and fragment 2 each 2 μ L, carry out circulating reaction 10 times.Then add primer PtrpF and tktAR, carry out circulating reaction 30 times, obtain dna fragmentation 3, reclaim for subsequent use.
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 10 times; 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 2min30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2110bp, the results are shown in Figure 4.
With
BamHI and
PspThen OMI double digestion plasmid pAR01 and dna fragmentation 3 connect two fragments with the T4 dna ligase, connect product and transform the Top10 competent cell, screen positive colony, and the gained plasmid is pAR03.
Gained pAR03 plasmid adopt respectively single endonuclease digestion (
BamHI) and double digestion (
BamHI and
PspOMI) identify, the results are shown in Figure 4.
Will
AroBWith
AroDGene imports the pAR03 plasmid
See sequence table sequence 17 take aroBF() and aroBR(see sequence table sequence 18) be that primer, W3110 genome are that template is carried out PCR, obtain fragment 1, for subsequent use after gel reclaims.Primer sequence is as follows:
aroBF：5’-gcgccatggagaggattgtcgttact-3’；
aroBR：5’-tgcgctagcttgttgttacgctgattgac-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min15s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 1108bp, the results are shown in Figure 5.
With
NcoI and
NheI is endonuclease bamhi 1 and pET28a+ plasmid (available from Novagen company) respectively, connects two fragments with the T4 dna ligase, obtains plasmid pET28a-
AroB
See sequence table sequence 19 take aroDF() and aroDR(see sequence table sequence 20) be that primer, W3110 genome are that template is carried out PCR, obtain fragment 2, for subsequent use after gel reclaims.Primer sequence is as follows:
aroDF：5’-cgggctagcgtaacagggtgatcatgag-3’；
aroDR：5’-catcggccgcctgatgcttcgtatttacg-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 1260bp, the results are shown in Figure 5.
With
NheI and
EagI is endonuclease bamhi 2 and plasmid pET28a-aroB respectively, connects two fragments with the T4 dna ligase, obtains plasmid pET28a-aroB-aroD.
See sequence table sequence 21 take aroBFL() and aroDR(see sequence table sequence 20) be that primer, pET28a-aroB-aroD are that template is carried out PCR, obtain fragment 3, it is for subsequent use that gel reclaims.Primer sequence is as follows:
aroBFL：5’-ctgggatcccatgagctcttaactttaagaaggag-3’；
aroDR：5’-catcggccgcctgatgcttcgtatttacg-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 2406bp, the results are shown in Figure 5.
With
BamHI and
EagI is endonuclease bamhi 3 and plasmid pAR03 respectively, connects two fragments with the T4 dna ligase, obtains plasmid plasmid pAR17.
The gained plasmid adopt respectively single endonuclease digestion (
BamHI) and double digestion (
BamHI and
EagI) identify, the results are shown in Figure 5.
Will
AroEGene imports the pAR17 plasmid
See sequence table sequence 22 take aroEF() and aroER(see sequence table sequence 23) be that primer, W3110 genome are template, carry out PCR and obtain fragment 1, it is for subsequent use that gel reclaims.Primer sequence is as follows:
aroEF：5’-ctggccatggaaacctatgctg-3’；
aroER：5’-cgcgagctcacgcggacaattcctc-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 49 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 831bp, the results are shown in Figure 6.
With
NcoI and
SacI is endonuclease bamhi 1 and pET28a+ plasmid respectively, connects two fragments with the T4 dna ligase, obtains plasmid pET28a-
AroE
See the sequence table sequence 24 take aroEFL() and aroERR(see sequence table sequence 25) be primer, pET28a-
AroECarry out PCR for template, obtain fragment 2, gel reclaims for subsequent use.Primer sequence is as follows:
aroEFL：5’-ctgggatcccatgcatgcttaactttaagaaggag-3’；
aroERR：5’-acggagctcacgcggacaattcctcctgcaattgc-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 867bp, the results are shown in Figure 6.
With
BamHI and
SacI is endonuclease bamhi 2 and plasmid pAR17 respectively, connects two fragments with the T4 dna ligase, obtains plasmid pAR31.
Gained plasmid pAR31 adopt respectively single endonuclease digestion (
BamHI) and double digestion (
BamHI and
SacI) identify, the results are shown in Figure 6.
Make up
AroG FBR Gene also imports the pAR31 plasmid
See sequence table sequence 26 take PtrpF1() and PtrpR1(see sequence table sequence 27) be that primer, W3110 genome are template, carry out PCR and obtain fragment 1, it is for subsequent use that gel reclaims.Primer sequence is as follows:
PtrpF1：5’-catggatcccggttctggcaaatattctgaaatgag-3’；
PtrpR1：5’-cgtcgttctgataattcattgtcgataccctttttac-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 119bp, the results are shown in Figure 7.
See sequence table sequence 28 take aroGLF() and aroGLR(see sequence table sequence 29) be that primer, W3110 genome are template, carry out PCR and obtain fragment 2, it is for subsequent use that gel reclaims.Primer sequence is as follows:
aroGLF：5’-gtaaaaagggtatcgacaatgaattatcagaacgacg-3’；
aroGLR：5’-atttttgaagccgaccggacaaaaaagccctgatgccagttcg-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 581bp, the results are shown in Figure 7.
See sequence table sequence 30 take aroGRF() and aroGRR(see sequence table sequence 31) be that primer, W3110 genome are template, carry out PCR and obtain fragment 3, it is for subsequent use that gel reclaims.Primer sequence is as follows:
aroGRF：5’-cgaactggcatcagggcttttttgtccggtcggcttcaaaaat-3’；
aroGRR：5’-cgcgcatgcttacccgcgacgcgcttttactgcat-3’；
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 543bp, the results are shown in Figure 7.
In 20 μ L PCR reaction systems (reaction system contains 0.5U Taq archaeal dna polymerase, 1 * Taq dna polymerase buffer liquid, 1mmol dNTPs), do not add primer, add above-mentioned fragment 1 and fragment 2 each 2 μ L, carry out circulating reaction 10 times.Then add primer PtrpF1 and aroGLR to final concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 4, reclaim for subsequent use.
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 10 times; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 661bp, the results are shown in Figure 7.
In 20 μ L PCR reaction systems (reaction system contains 0.5U Taq archaeal dna polymerase, 1 * Taq dna polymerase buffer liquid, 1mmol dNTPs), do not add primer, add above-mentioned fragment 3 and fragment 4 each 2 μ L, carry out circulating reaction 10 times.Then add primer PtrpF1 and aroGRR to final concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 5, reclaim for subsequent use.
The pcr amplification condition is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 10 times; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 90s, circulate altogether 30 times.Obtaining length after PCR finishes is the dna fragmentation of 1162bp, the results are shown in Figure 7.
With
BamHI and
SphI is endonuclease bamhi 5 and plasmid pAR31 respectively, connects two fragments with the T4 dna ligase, obtains plasmid pAR63.Plasmid pAR63 is the carrier that finally successfully constructs.
Gained plasmid pAR63 adopt respectively single endonuclease digestion (
BamHI) and double digestion (
BamHI and
SphI) identify, the results are shown in Figure 7.
Three, recombinant expression plasmid pAR63 is changed over to knock out
AroAThe coli strain W3110(of gene
AroA), the production bacterial strain of shikimic acid is expressed in acquisition.The production bacterial strain can add glucose mode dissolved oxygen controlled fermentation by stream and prepare shikimic acid.
(1) makes up the purpose engineering strain
The method that reference molecule cloning experimentation guide third edition chemical method prepares intestinal bacteria competence and conversion foreign gene changes plasmid pAR63 over to bacterial strain
E. coliW3110(
AroA), obtain the purpose engineering strain
E. coliW3110(
AroA) pAR63.
(2) fermentative Production shikimic acid
With the purpose engineering strain that successfully constructs
E. coliW3110(
AroA) pAR63 carries out slant activation, is inoculated in the LB liquid nutrient medium, 37 ℃, 220r/min is cultured to OD
600During ≈ 2, be inoculated in the 5L fermentor tank fermentor tank charging 2.4L.Fermention medium consists of: glucose 15g, yeast extract 1g, K
2HPO
48g, Citric acid monohydrate Food grade 2.5g, L-Phe 0.7g, L-Tyr 0.7g, L-Trp 0.7g, MgSO
40.24g, H
3BO
30.0247g, ZnSO
47H
2O 0.0029g, CuSO
45H
2O 0.0025g, MnCl4H
2O 0.0158g, P-hydroxybenzoic acid 0.010g, para-amino benzoic acid 0.010g, 2,3-resorcylic acid 0.010g.37 ℃ of leavening temperatures, initial mixing speed 50r/min, initial air flow 1:0.1(is fermentating liquid volume: the sterile air amount that per minute passes into=10).Beginning is controlled at 30% by the control rotating speed with dissolved oxygen first, when rotating speed reaches the highest 800r/min, by adjusting air flow control dissolved oxygen 20%, when air flow reaches 1:1, adds 60% glucose control dissolved oxygen at 10%-15% by stream.
Every the 3h sampling, detect shikimic acid content and cell concentration in the fermented liquid in the fermenting process.The shikimic acid detection method of content: it is in 80 ℃ of heating 20min after 2.0 that fermented liquid is transferred to the pH value with sulfuric acid, then according to spectrophotometry shikimic acid content, be specially: the fermented liquid that the aforementioned processing that takes a morsel is crossed is centrifugal, after the suitable dilution of centrifugal gained supernatant liquor, get the supernatant liquor that 1mL diluted, adding 1mL contains the aqueous solution of 0.5% Periodic acid and 0.5% sodium metaperiodate, behind 37 ℃ of reaction 30min, adding 2mL contains the aqueous solution termination reaction of 3g/L S-WAT and 0.6mol/L sodium hydroxide, reads reaction solution at the light absorption value at 382nm place in 1h.Utilize simultaneously shikimic acid standard substance production standard curve, the reference standard curve can obtain the corresponding shikimic acid content of light absorption value.Cell concentration adopts turbidimetry to measure light absorption value at 600nm wavelength place and detects.
During fermentation 52h, shikimic acid content reaches 43g/L in the fermented liquid.Cell concentration and shikimic acid content are seen Fig. 9 with the variation of fermentation time in the fermenting process.
With the purpose engineering strain that successfully constructs
E. coliW3110(
AroA) pAR63 carries out slant activation, is inoculated in the LB substratum, 37 ℃, 220r/min is cultured to OD
600During ≈ 2, be inoculated in the 5L fermentor tank, fermentor tank charging 2.4L, fermention medium is: glucose 15g, yeast extract 1g, K
2HPO
48g, Citric acid monohydrate Food grade 2.5g, L-Phe 0.7g, L-Tyr 0.7g, L-Trp 0.7g, MgSO
40.24g, H
3BO
30.0247g, ZnSO
47H
2O 0.0029g, CuSO
45H
2O 0.0025g, MnCl4H
2O 0.0158g, P-hydroxybenzoic acid 0.010g, para-amino benzoic acid 0.010g, 2,3-resorcylic acid 0.010g.37 ℃ of leavening temperatures, initial mixing speed 50r/min, initial air flow 1:0.1.Begin by the control rotating speed dissolved oxygen to be controlled at 30%, when rotating speed reaches the highest 800r/min, by adjusting air flow control dissolved oxygen 20%, when air flow reaches 1:1, add the control of 60% glucose equally at 10%-15% by stream.
Detect shikimic acid content and cell concentration (detection method is the same) every the 3h sampling.During fermentation 60h, shikimic acid content reaches 54g/L in the fermented liquid.
With the purpose engineering strain that successfully constructs
E. coliW3110(
AroA) pAR63 carries out slant activation, is inoculated in the LB substratum, 37 ℃, 220r/min is cultured to OD
600During ≈ 2, be inoculated in the 5L fermentor tank, fermentor tank charging 2.4L, fermention medium is glucose 15g, yeast extract 1g, K
2HPO
48g, Citric acid monohydrate Food grade 2.5g, L-Phe 0.7g, L-Tyr 0.7g, L-Trp 0.7g, MgSO
40.24g, H
3BO
30.0247g, ZnSO
47H
2O 0.0029g, CuSO
45H
2O 0.0025g, MnCl4H
2O 0.0158g, P-hydroxybenzoic acid 0.010g, para-amino benzoic acid 0.010g, 2,3-resorcylic acid 0.010g.37 ℃ of leavening temperatures, initial mixing speed 50r/min, initial air flow 1:0.1.Begin by the control rotating speed dissolved oxygen to be controlled at 30%, when rotating speed reaches the highest 800r/min, by adjusting air flow control dissolved oxygen 20%, when air flow reaches 1:1, add the control of 60% glucose equally at 10%-15% by stream.
Detect shikimic acid content and cell concentration (detection method is the same) every the 3h sampling.During fermentation 46h, shikimic acid content reaches 36g/L in the fermented liquid.
SEQUENCE LISTING
＜110〉Henan becomes Bioceuticals Inc. the Meng
＜120〉a kind of shikimic acid production construction process of engineering strain
<130>
<160> 31
<170> PatentIn version 3.4
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<212> DNA
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atggaatccc tgacgttaca acccatcgct cgtgtcgatg gcactattaa gtgtaggctg 60
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<212> DNA
＜213〉artificial sequence
<400> 2
tcaggctgcc tggctaatcc gcgccagctg ctcgaaataa tccggaaatg atgggaatta 60
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tcatggttga gttcgaacgc cgtc 24
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gtgggtattt tgacaccctg tcag 24
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gcgctgcagg tcggatccac gtcgttactg atccgcac 38
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gggaagcttc cacgtcttat caggcctac 29
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gcgctgcagg gcctcaggca tttgagaag 29
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ttaaagctta tcgatgataa gctgtc 26
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catggatccc tcgggcccat gtgtttctca aaccgttc 38
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gccgccaggc aaattctgtt tttatttctt cagttcagcc 40
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ggctgaactg aagaaataaa aacagaattt gcctggcggc 40
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gcgctgcaga agagtttgta gaaacgcaaa aaggccatc 39
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catggatccc tccggccgcg gttctggcaa atattc 36
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gcaagctctt tacgtgagga cattgtcgat acccttttta cg 42
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cgtaaaaagg gtatcgacaa tgtcctcacg taaagagctt gc 42
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ctcgggccct tacagcagtt cttttgcttt cgcaac 36
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gcgccatgga gaggattgtc gttact 26
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tgcgctagct tgttgttacg ctgattgac 29
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cgggctagcg taacagggtg atcatgag 28
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catcggccgc ctgatgcttc gtatttacg 29
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ctgggatccc atgagctctt aactttaaga aggag 35
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ctggccatgg aaacctatgc tg 22
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cgcgagctca cgcggacaat tcctc 25
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ctgggatccc atgcatgctt aactttaaga aggag 35
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acggagctca cgcggacaat tcctcctgca attgc 35
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catggatccc ggttctggca aatattctga aatgag 36
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cgtcgttctg ataattcatt gtcgataccc tttttac 37
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gtaaaaaggg tatcgacaat gaattatcag aacgacg 37
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atttttgaag ccgaccggac aaaaaagccc tgatgccagt tcg 43
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cgaactggca tcagggcttt tttgtccggt cggcttcaaa aat 43
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cgcgcatgct tacccgcgac gcgcttttac tgcat 35
Claims (4)
1. the construction process of a shikimic acid production usefulness engineering strain is characterized in that, may further comprise the steps:
1. make up
AroAThe coli strain of gene knockout; 2. make up the key gene that contains in the shikimic acid pathways metabolism
AroG FBR ,
AroE,
AroB,
AroD,
TktAWith
PpsARecombinant expression plasmid pAR63; 3. recombinant expression plasmid pAR63 is changed over to and knock out
AroAThe coli strain of gene, the production bacterial strain of shikimic acid is expressed in acquisition;
Wherein, 1. step comprises: a, obtain by pcr amplification
AroAHomology is replaced sequence D aroA, take plasmid pKD4 as template, utilizes primer aroAF and aroAR, among the pcr amplification plasmid pKD4
Kan R Gene; Gained PCR fragment both sides are
AroAThe dna homolog fragment, middle portion is that resistant gene of card; Described primer aroAF and aroAR see respectively the sequence 1 and 2 in the sequence table;
B, plasmid pKD46 is imported coli strain W3110;
C, induce step b gained positive strain to produce the required enzyme of homologous recombination, the results thalline prepares competent cell;
D, the homology replacement sequence D aroA that transforms step a acquisition by electric shock import the competent cell that step c obtains, and obtain
AroAThe bacterial strain that gene is replaced;
The resistant gene fragment of e, use pCP20 plasmid removal process d obtained strains obtains
AroAGene knock-out bacterial strain;
2. step comprises: a, make up based on plasmid pACYC184 and to comprise
TrpRThe transformation carrier pAR of gene;
B, the anti-feedback inhibition of structure
AroG FBR Gene, and before gene, add trp promoter
P Trp
C, clone
AroBWith
AroEGene, and be its interpolation ribosome bind site RBS;
D, clone gene
TktA,
PpsAWith
AroD, and
TktABefore add trp promoter
P Trp ,
PpsAThe back adds transcription terminator;
E, with each gene according to
P Trp ,
AroG FBR ,
AroE,
AroB,
AroD,
P Trp ,
TktA,
PpsAWith being linked in sequence to the pAR plasmid of transcription terminator, consist of the pAR63 plasmid.
2. shikimic acid production is characterized in that with the construction process of engineering strain as claimed in claim 1, contains trp promoter aporepressor gene among the described recombinant expression plasmid pAR63
TrpR, and
TrpRWith self promotor.
3. shikimic acid production is characterized in that with the construction process of engineering strain as claimed in claim 1, and is contained among the described recombinant expression plasmid pAR63
PpsAThe gene downstream sequence is the Transcription Termination subsequence; Contained among the described recombinant expression plasmid pAR63
TktAGene upstream sequence is the tryptophan promoter subsequence; Contained among the described recombinant expression plasmid pAR63
AroBWith
AroEGene upstream sequence all contains the RBS sequence; Contained among the described recombinant expression plasmid pAR63
AroDGene is with self RBS sequence; Contained among the described recombinant expression plasmid pAR63
AroG FBR Gene upstream sequence is the tryptophan promoter subsequence.
4. shikimic acid production is characterized in that with the construction process of engineering strain as claimed in claim 3, and described Transcription Termination subsequence is
RrnBThe Transcription Termination subsequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110248675 CN102304539B (en) | 2011-08-26 | 2011-08-26 | Construction method of genetic engineering strain for producing shikimic acid |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541483A (en) * | 2017-10-24 | 2018-01-05 | 中国科学院天津工业生物技术研究所 | Produce levodopa E. coli recombinant stain and its construction method and application |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994439A (en) * | 2012-12-12 | 2013-03-27 | 江南大学 | Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof |
| CN107446870B (en) * | 2016-11-23 | 2021-06-29 | 中国科学院微生物研究所 | Preparation method of high-yield shikimic acid mutant |
| CN109423468B (en) * | 2017-08-24 | 2022-12-20 | 中国科学院微生物研究所 | Method for increasing yield of compound and derivative thereof in aromatic amino acid biosynthesis pathway |
| CN109423504B (en) * | 2017-08-24 | 2022-10-04 | 廊坊梅花生物技术开发有限公司 | Strain for producing L-tryptophan and application thereof |
| CN109576199B (en) * | 2018-12-07 | 2020-09-04 | 江南大学 | Dynamic regulation and control system controlled by promoter combination |
| 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 |
| CN110904140B (en) * | 2019-12-20 | 2021-05-07 | 台州职业技术学院 | Protein dynamic expression regulation system and application thereof in shikimic acid production |
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| EP1038968A3 (en) * | 1999-03-25 | 2002-10-02 | Ajinomoto Co., Inc. | Method for producing shikimic acid |
| CN101085998A (en) * | 2006-06-07 | 2007-12-12 | 汪莉 | Method for preparing shikimic acid using biosynthesis technology and engineered bacteria |
| CN101139566A (en) * | 2006-09-08 | 2008-03-12 | 中国科学院上海生命科学研究院 | Shikimic acid prepared bacterial strain and constructing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541483A (en) * | 2017-10-24 | 2018-01-05 | 中国科学院天津工业生物技术研究所 | Produce levodopa E. coli recombinant stain and its construction method and application |
| 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 |
| CN107541483B (en) * | 2017-10-24 | 2020-12-01 | 中国科学院天津工业生物技术研究所 | Escherichia coli recombinant strain for producing levodopa and construction method and application thereof |
| 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 |
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