CN102304539A - 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 of a kind of shikimic acid production with engineering strain, this bacterial strain can be used for the fermentative prodn 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 vegeto-alkalis, aromatic amino acid and indole derivatives, chiral drug (like antiviral drug), extensively is present in plant and the mikrobe.
Shikimic acid is through influencing 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 midbody.Some verivates of shikimic acid also have certain drug effect.
With the shikimic acid be 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 to carry out the anti-avian influenza medicine respectively, but because of 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 mikrobe in a large number, contains shikimic acid 8%～13% like 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 pass through strict processing when extracting shikimic acid and can reach environmental requirement, and production cost is higher.Have advantages such as simple and convenient, that technical limitation is less though from plant, extract shikimic acid, and mostly present shikimic acid is from plant tissues such as star anise, to extract also, this method is limited by raw material output, can't mass production.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 continues suitability for industrialized production easily because of the less restriction that receives raw material sources, and environmental pollution is less, and is with low cost, is applied to shikimic acid production gradually.
The shikimic acid circulation extensively is present in the mikrobe, also can extract shikimic acid through microbial fermentation.Conversion of glucose is die aromatischen Aminosaeuren (L-phenylalanine(Phe), L-Methionin and a L-tryptophane) in intestinal bacteria, and shikimic acid is an intermediate product.Bacillus coli communis can accumulate shikimic acid after transforming.Roche Holding Ag 2005 and Germany's one tame biotechnology center cooperation through to a kind of harmless intestinal bacteria excess supply glucose, force 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 quilt be broken just and possibly accumulated 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-dehydroquinic acid synthetic enzyme (3-dehydroq uinate synthase; Catalysis is by the reaction of DAHP to the 3-dehydroquinic acid), 3-dehydroquinate dehydratase (3-dehydroquinate dehydratase; Catalysis 3-dehydroquinic acid dehydration generation dehydrogenation shikimic acid), shikimate dehydrogenase (shikimate dehydrogenase; The catalytic dehydrogenation shikimic acid is to the transformation of shikimic acid, and 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 (
AroFReceive the tyrosine feedback inhibition,
AroGReceive the phenylalanine(Phe) feedback inhibition,
AroHReceive 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 be not sufficient to accumulate in a large number the catalysis of shikimic acid with the synthetic relevant enzyme of shikimic acid, shikimate kinase catalysis shikimic acid is to other conversion of compounds, therefore produces transformation that bacterial strain must carry out relevant enzyme to be fit to a large amount of synthetic of shikimic acid.
At present, about the existing at home and abroad relevant report of the Production by Microorganism Fermentation of shikimic acid.Yurgis etc. are through transforming subtilis producing shikimic acid, this bacterial strain through inactivation shikimate kinase gene (
AroI) or 5-enol pyruvoyl shikimic acid-3-phosphate synthase (EPSPS, but catalysis shikimic acid-3-phosphoric acid is to the conversion of chorismic acid) gene, import genes such as shikimate dehydrogenase and DHAP synthetic enzyme, can produce the about 20g/L of shikimic acid.John W. Frost etc. are through 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, make shikimic acid output reach about 70g/L.John W. Frost carries out molecular evolution to 2-keto-3-deoxygalactonic acid zymohexase (KDPGal aldolase) gene and obtains a kind ofly can to synthesize DAHP with E4P by the catalysis pyruvic acid; 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 bacillus coli gene group, 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 can initially expeditiously be transcribed.Trp promoter receive 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 did not have tryptophane and combines, and is inactive form, can't check the startup of promotor, receives the gene transcriptional start of trp promoter control.Because above-mentioned this characteristics, trp promoter are widely used in the synthetic of Recombinant Protein Expression and enzyme.
Existing invention is interrupted the metabolism of shikimic acid through knocking out gene mostly, and imports effective accumulation that shikimic acid anabolism gene is realized shikimic acid, and these class methods need knock out more gene from host cell, and it is loaded down with trivial details relatively to operate.
Summary of the invention
The object of the present invention is to provide the construction process of a kind of shikimic acid production with engineering strain, it is few that this method knocks out gene, and the bacterial strain of structure can effectively interrupt shikimic acid metabolism and a large amount of accumulation that realize shikimic acid.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
A kind of shikimic acid production is with the construction process of engineering strain, and 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, is used for intestinal bacteria at present more.
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 and contain the key gene in the shikimic acid pathways metabolism
AroG FBR (enzyme of this genes encoding does not receive the phenylalanine(Phe) feedback inhibition),
AroE,
AroB,
AroD,
TktAWith
PpsARecombinant expression plasmid pAR63, and these genes all are in trp promoter
P Trp Transcribe control down;
Recombinant expression plasmid pAR63 is through 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 control fermentation through stream and prepare shikimic acid.
Concrete, 1. step comprises:
A, obtain through pcr amplification
AroAHomology replacement 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 through 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 to check 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 of transcription terminator, constitute the pAR63 plasmid to the pAR plasmid.
Contain trp promoter aporepressor gene among the said recombinant expression plasmid pAR63
TrpR, and
TrpRHave self promotor, can independently express.
Contained among the said recombinant expression plasmid pAR63
PpsAThe gene downstream sequence is the Transcription Termination subsequence; Contained among the said recombinant expression plasmid pAR63
TktAGene upstream sequence is the tryptophan promoter subsequence; Contained among the said recombinant expression plasmid pAR63
AroBWith
AroEGene upstream sequence all contains ribosome bind site (RBS) sequence; Contained among the said recombinant expression plasmid pAR63
AroDGene has self RBS sequence; Contained among the said 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.Said Transcription Termination subsequence does
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, open related gene expression automatically at suitable growth period, body burden before increasing is quickened building-up reactions, thereby promotes that raw material efficiently is converted into shikimic acid.
Compare with prior art, the innovative point of the inventive method is following:
(1) through knocking out
AroASingle-gene realize to interrupt the metabolic purpose of shikimic acid, and it is few to knock out gene, 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 the feedback inhibition of shikimic acid to upper reaches enzymes such as shikimate dehydrogenases, 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 through 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 rich in amino acid in the substratum, genetic expression is in the state of checking, and helps the thalline accumulation.Because shikimic acid can't get into 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, treat tryptophane consumption after concentration reduce, each gene progressively begins to express, catalysis shikimic acid a large amount of synthetic.
(3) because shikimic acid can't get into 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 Tryptophane coupling in the fermenting process of regulating and expressing and shikimic acid synthesis bacterium in the substratum, 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, starts automatically in the middle and later periods, need not inducing of inductor, has reduced the possibility of in substratum, adding toxic substance.
Description of drawings
Fig. 1 does
AroAThe agarose gel electrophoresis result of homology replacement sequence D aroA PCR product with
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
AroAKnocked out, 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 a 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 with
PstI double digestion collection of illustrative plates;
Fig. 3 cuts qualification result for PCR product in the plasmid pAR01 building process and enzyme; 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 with
PstI double digestion result;
Fig. 4 cuts qualification result for PCR product in the plasmid pAR03 building process and plasmid pAR03 enzyme; 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 with
PspOMI double digestion result;
Fig. 5 cuts qualification result for PCR product in the plasmid pAR17 building process and plasmid pAR17 enzyme; 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 with
EagI double digestion result;
Fig. 6 cuts qualification result for PCR product in the plasmid pAR31 building process and plasmid pAR31 enzyme; 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 with
SacI double digestion result;
Fig. 7 cuts qualification result for PCR product in the plasmid pAR63 building process and plasmid pAR63 enzyme; 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 with
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
Below through embodiment the present invention is elaborated, but protection scope of the present invention is not limited thereto.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.Processes such as preparation of being mentioned among the embodiment or conversion like no specified otherwise, then adopt the ordinary method of this area all can realize, so repeat no more.
A kind of shikimic acid production is with the construction process of engineering strain, and 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 target gene fragment is accomplished the work that knocks out of goal gene by other fragment replacement.
) a,Obtain through pcr amplification
AroAHomology replacement sequence D aroA;
With plasmid pKD4 (being provided by Shanghai Inst. of Life Science, CAS) is template, utilizes primer aroAF (seeing sequence 1 in the sequence table) and aroAR (seeing 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 following:
AF：5’-atggaatccctgacgttacaacccatcgctcgtgtcgatggcactattaagtgtaggctggagctgcttc-3’
AR：5’-tcaggctgcctggctaatccgcgccagctgctcgaaataatccggaaatgatgggaattagccatggtcc-3’
The pcr amplification condition is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.It is the dna fragmentation of 1596bp that PCR finishes back acquisition length, and agarose gel electrophoresis reclaims this fragment and is DaroA, sees Fig. 1.Gained PCR fragment both sides do
AroAThe dna homolog fragment, middle portion is that resistant gene of card.The PCR fragment is carried out behind the agarose gel electrophoresis purifying 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, coat on the LB solid medium flat board that contains 100 μ g/mL penbritins (Amp) 30 ℃ of incubated overnight behind 30 ℃ of cultivation 2h with plasmid pKD46 (providing) conversion W3110 bacterial strain competent cell by Shanghai Inst. of Life Science, CAS; The transformant that can grow is the intestinal bacteria W3110 bacterial strain that contains the pKD46 plasmid, selects positive transformant and preserves subsequent use.
) 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 replacement sequence D aroA that step a is obtained through electric shock 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.The electric shock back adds in the 950 μ L LB liquid nutrient mediums; 150r/min cultivates 2h, the centrifugal 1min of 12000 * g for 37 ℃; Discard upper strata 800 μ L supernatants, be applied to dull and stereotyped (containing 10 μ g/mL kantlex) the last 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, select positive transformant preserve subsequent use, be 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 (being provided by Shanghai Inst. of Life Science, CAS) is changed in the positive colony of above-mentioned steps d acquisition, the clone who has that resistance of card and ammonia benzyl resistance is simultaneously chosen in 30 ℃ of cultivations; Be inoculated in the LB liquid nutrient medium, 30 ℃, 200r/min cultivates 8h; Then temperature is brought up to 42 ℃; FLP recombinase this moment (Flipase recombination enzyme, Flippases recombinase) begins to express catalysis FRT (Flp recombination target; The Flippases recombination site) the segmental elimination of resistant gene between the site, plasmid pCP20 loses owing to stop to duplicate gradually.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 with W3110 (
AroA) expression.
Obtained strains (comprise W3110 (
Kan R AroA) and W3110 (
AroA)) be that primer carries out the PCR evaluation with DF (seeing sequence 3 in the sequence table) and DR (seeing sequence 4 in the sequence table) respectively, the W3110 bacterial strain is contrast.Primer sequence is following: DF:5 '-tcatggttgagttcgaacgccgtc-3 '; DR:5 '-gtgggtattttgacaccctgtcag-3 ';
The pcr amplification condition is: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.With the different strains is that the dna fragmentation length that template obtained is different, can judge whether thus from genome, to knock out
AroAGene, the result sees Fig. 1, judges that therefore the last bacterial strain that obtains is
E. coliW3110 (
AroA), preserve subsequent use.
Two, structure contains the key gene in the shikimic acid pathways metabolism AroG FBR (this gene does not receive the phenylalanine(Phe) feedback inhibition),
AroE,
AroB,
AroD,
TktAWith
PpsA Recombinant expression plasmid pAR63, it makes up and structural representation is seen Fig. 8;
2.1 the structure of pAR plasmid
With trpRF (sequence 5 in the sequence table) and trpRR (sequence 6 in the sequence table) is primer, and bacterial strain W3110 genome is that template is carried out pcr amplification
TrpRComplete sequence, it is subsequent use that gel reclaims the back, and primer sequence is following:
trpRF：5’-gcgctgcaggtcggatccacgtcgttactgatccgcac-3’；
trpRR：5’-gggaagcttccacgtcttatcaggcctac-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.It is the dna fragmentation of 480bp that PCR finishes back acquisition length, and the result sees Fig. 2.
With pAF (sequence 7 in the sequence table) and pAR (sequence 8 in the sequence table) is primer, and plasmid pACYC184 (being provided by Shanghai Inst. of Life Science, CAS) carries out pcr amplification plasmid fragment for template, and it is subsequent use that gel reclaims the back.Primer sequence is following:
pAF：5’-gcgctgcagggcctcaggcatttgagaag-3’；
pAR：5’-ttaaagcttatcgatgataagctgtc-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 2min15s, circulate altogether 30 times.It is the dna fragmentation of 2197bp that PCR finishes back acquisition length, and the result sees Fig. 2.
Above-mentioned two kinds of dna fragmentations are used respectively
HinDIII with
PstCarry out gel behind the I double digestion and reclaim, use the T4 dna ligase to connect two fragments then.Connect product and transform Top10 competent cell (available from the auspicious bio tech ltd of Nanjing Sai Hong), on the LB solid medium flat board that contains 20 μ g/mL paraxin, screen positive strain, the gained plasmid is the pAR plasmid.
Gained pAR plasmid employing single endonuclease digestion (
HinDIII) and double digestion (
HinDIII with
PstI) identify that agarose gel electrophoresis figure sees Fig. 2.
Will
PpsAGene imports the pAR plasmid
Adopting ppsAF (seeing sequence table sequence 9) and ppsAR (seeing sequence table sequence 10) is primer, and bacterial strain W3110 genome is that template is carried out pcr amplification and obtained fragment 1, reclaims subsequent use.Primer sequence is following:
ppsAF：5’-catggatccctcgggcccatgtgtttctcaaaccgttc-3’；
ppsAR：5’-gccgccaggcaaattctgtttttatttcttcagttcagcc-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 2min30s, circulate altogether 30 times.It is the dna fragmentation of 2460bp that PCR finishes back acquisition length, and the result sees Fig. 3.
Adopting TerF (seeing sequence table sequence 11) and TerR (seeing sequence table sequence 12) is primer, and the W3110 genome is that template is carried out pcr amplification and obtained fragment 2, reclaims subsequent use.Primer sequence is following:
TerF：5’-ggctgaactgaagaaataaaaacagaatttgcctggcggc-3’；
TerR：5’-gcgctgcagaagagtttgtagaaacgcaaaaaggccatc-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 40s, circulate altogether 30 times.It is the dna fragmentation of 382bp that PCR finishes back acquisition length, and the result sees Fig. 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 the above-mentioned fragment that obtains 1 and fragment 2 each 2 μ L, carry out circulating reaction 10 times.Add then primer ppsAF and TerR to concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 3, reclaim subsequent use.
The pcr amplification condition is: 94 ℃ of preparatory sex change 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.It is the dna fragmentation of 2803bp that PCR finishes back acquisition length, and the result sees Fig. 3.
Use
BamHI with
PstI double digestion plasmid pAR and dna fragmentation 3 connect two fragments with the T4 dna ligase then, 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 with
PstI), qualification result is seen Fig. 3.
Will
TktAGene imports the pAR01 plasmid
Adopting PtrpF (seeing sequence table sequence 13) and PtrpR (seeing sequence table sequence 14) is primer, and bacterial strain W3110 genome is that template is carried out pcr amplification and obtained fragment 1, reclaims subsequent use.Primer sequence is following:
PtrpF：5’-catggatccctccggccgcggttctggcaaatattc-3’；
PtrpR：5’-gcaagctctttacgtgaggacattgtcgataccctttttacg-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.It is the dna fragmentation of 132bp that PCR finishes back acquisition length, and the result sees Fig. 4.
Adopting tktAF (seeing sequence table sequence 15) and tktAR (seeing sequence table sequence 16) is primer, and the W3110 genome is that template is carried out pcr amplification and obtained fragment 2, reclaims subsequent use.Primer sequence is following:
tktAF：5’-cgtaaaaagggtatcgacaatgtcctcacgtaaagagcttgc-3’；
tktAR：5’-ctcgggcccttacagcagttcttttgctttcgcaac-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate altogether 30 times.It is the dna fragmentation of 2019bp that PCR finishes back acquisition length, and the result sees Fig. 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.Add primer PtrpF and tktAR then, carry out circulating reaction 30 times, obtain dna fragmentation 3, reclaim subsequent use.
The pcr amplification condition is: 94 ℃ of preparatory sex change 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.It is the dna fragmentation of 2110bp that PCR finishes back acquisition length, and the result sees Fig. 4.
With
BamHI with
PspOMI double digestion plasmid pAR01 and dna fragmentation 3 connect two fragments with the T4 dna ligase then, 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 with
PspOMI) identify that the result sees Fig. 4.
Will
AroBWith
AroDGene imports the pAR03 plasmid
With aroBF (seeing sequence table sequence 17) and aroBR (seeing sequence table sequence 18) is primer, and the W3110 genome is that template is carried out PCR, obtains fragment 1, and it is subsequent use that gel reclaims the back.Primer sequence is following:
aroBF：5’-gcgccatggagaggattgtcgttact-3’；
aroBR：5’-tgcgctagcttgttgttacgctgattgac-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min15s, circulate altogether 30 times.It is the dna fragmentation of 1108bp that PCR finishes back acquisition length, and the result sees Fig. 5.
With
NcoI with
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
With aroDF (seeing sequence table sequence 19) and aroDR (seeing sequence table sequence 20) is primer, and the W3110 genome is that template is carried out PCR, obtains fragment 2, and it is subsequent use that gel reclaims the back.Primer sequence is following:
aroDF：5’-cgggctagcgtaacagggtgatcatgag-3’；
aroDR：5’-catcggccgcctgatgcttcgtatttacg-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min30s, circulate altogether 30 times.It is the dna fragmentation of 1260bp that PCR finishes back acquisition length, and the result sees Fig. 5.
With
NheI with
EagI is endonuclease bamhi 2 and plasmid pET28a-aroB respectively, connects two fragments with the T4 dna ligase, obtains plasmid pET28a-aroB-aroD.
With aroBFL (seeing sequence table sequence 21) and aroDR (seeing sequence table sequence 20) is primer, and pET28a-aroB-aroD is that template is carried out PCR, obtains fragment 3, and gel reclaims subsequent use.Primer sequence is following:
aroBFL：5’-ctgggatcccatgagctcttaactttaagaaggag-3’；
aroDR：5’-catcggccgcctgatgcttcgtatttacg-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min30s, circulate altogether 30 times.It is the dna fragmentation of 2406bp that PCR finishes back acquisition length, and the result sees Fig. 5.
With
BamHI with
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 with
EagI) identify that the result sees Fig. 5.
Will
AroEGene imports the pAR17 plasmid
With aroEF (seeing sequence table sequence 22) and aroER (seeing sequence table sequence 23) is primer, and the W3110 genome is a template, carries out PCR and obtains fragment 1, and gel reclaims subsequent use.Primer sequence is following:
aroEF：5’-ctggccatggaaacctatgctg-3’；
aroER：5’-cgcgagctcacgcggacaattcctc-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 49 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.It is the dna fragmentation of 831bp that PCR finishes back acquisition length, and the result sees Fig. 6.
With
NcoI with
SacI is endonuclease bamhi 1 and pET28a+ plasmid respectively, connects two fragments with the T4 dna ligase, obtains plasmid pET28a-
AroE
With aroEFL (seeing the sequence table sequence 24) and aroERR (seeing sequence table sequence 25) is primer, pET28a-
AroECarry out PCR for template, obtain fragment 2, gel reclaims subsequent use.Primer sequence is following:
aroEFL：5’-ctgggatcccatgcatgcttaactttaagaaggag-3’；
aroERR：5’-acggagctcacgcggacaattcctcctgcaattgc-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.It is the dna fragmentation of 867bp that PCR finishes back acquisition length, and the result sees Fig. 6.
With
BamHI with
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 with
SacI) identify that the result sees Fig. 6.
Make up
AroG
FBR
Gene also imports the pAR31 plasmid
With PtrpF1 (seeing sequence table sequence 26) and PtrpR1 (seeing sequence table sequence 27) is primer, and the W3110 genome is a template, carries out PCR and obtains fragment 1, and gel reclaims subsequent use.Primer sequence is following:
PtrpF1：5’-catggatcccggttctggcaaatattctgaaatgag-3’；
PtrpR1：5’-cgtcgttctgataattcattgtcgataccctttttac-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 30 times.It is the dna fragmentation of 119bp that PCR finishes back acquisition length, and the result sees Fig. 7.
With aroGLF (seeing sequence table sequence 28) and aroGLR (seeing sequence table sequence 29) is primer, and the W3110 genome is a template, carries out PCR and obtains fragment 2, and gel reclaims subsequent use.Primer sequence is following:
aroGLF：5’-gtaaaaagggtatcgacaatgaattatcagaacgacg-3’；
aroGLR：5’-atttttgaagccgaccggacaaaaaagccctgatgccagttcg-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.It is the dna fragmentation of 581bp that PCR finishes back acquisition length, and the result sees Fig. 7.
With aroGRF (seeing sequence table sequence 30) and aroGRR (seeing sequence table sequence 31) is primer, and the W3110 genome is a template, carries out PCR and obtains fragment 3, and gel reclaims subsequent use.Primer sequence is following:
aroGRF：5’-cgaactggcatcagggcttttttgtccggtcggcttcaaaaat-3’；
aroGRR：5’-cgcgcatgcttacccgcgacgcgcttttactgcat-3’；
The pcr amplification condition is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate altogether 30 times.It is the dna fragmentation of 543bp that PCR finishes back acquisition length, and the result sees Fig. 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.Add then primer PtrpF1 and aroGLR to final concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 4, reclaim subsequent use.
The pcr amplification condition is: 94 ℃ of preparatory sex change 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.It is the dna fragmentation of 661bp that PCR finishes back acquisition length, and the result sees Fig. 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.Add then primer PtrpF1 and aroGRR to final concentration be 0.5 μ mol, carry out circulating reaction 30 times, obtain dna fragmentation 5, reclaim subsequent use.
The pcr amplification condition is: 94 ℃ of preparatory sex change 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.It is the dna fragmentation of 1162bp that PCR finishes back acquisition length, and the result sees Fig. 7.
Use
BamHI with
SphI is endonuclease bamhi 5 and plasmid pAR31 respectively, connects two fragments with the T4 dna ligase, obtains plasmid pAR63.Plasmid pAR63 is the final successful carrier that makes up.
Gained plasmid pAR63 adopt respectively single endonuclease digestion (
BamHI) and double digestion (
BamHI with
SphI) identify that the result sees Fig. 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 control fermentation through stream and prepare shikimic acid.
(1) makes up the purpose engineering strain
Reference molecule cloning experimentation guide third edition chemical method prepares the intestinal bacteria competence and changes plasmid pAR63 over to bacterial strain with the method that transforms foreign gene
E. coliW3110 (
AroA), obtain the purpose engineering strain
E. coliW3110 (
AroA) pAR63.
(2) fermentative Production shikimic acid
With making up successful purpose engineering strain
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, PHB 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 a fermentating liquid volume: sterile air amount=10 that PM feeds).Beginning earlier is controlled at 30% through the control rotating speed with dissolved oxygen, when rotating speed reaches the highest 800r/min, 20%, when air flow reaches 1:1, adds 60% glucose control dissolved oxygen at 10%-15% through stream through adjustment air flow control dissolved oxygen.
Every in the fermenting process at a distance from the 3h sampling, detect shikimic acid content and cell concentration in the fermented liquid.The shikimic acid detection method of content: using sulfuric acid to transfer to the pH value fermented liquid is that 2.0 backs are in 80 ℃ of heating 20min; Detect shikimic acid content according to spectrophotometry then; Be specially: the fermented liquid that the aforementioned processing that takes a morsel is crossed is centrifugal, after centrifugal gained supernatant is suitably diluted, gets the supernatant that 1mL diluted; Adding 1mL contains the aqueous solution of 0.5% Periodic acid 99 and 0.5% sodium metaperiodate; Behind 37 ℃ of reaction 30min, add the aqueous solution termination reaction that 2mL contains 3g/L S-WAT and 0.6mol/L sodium hydroxide, in 1h, read the light absorption value of reaction solution at the 382nm place.Utilize shikimic acid standard substance production standard curve simultaneously, the reference standard curve can obtain the pairing shikimic acid content of light absorption value.Cell concentration adopts turbidimetry to measure light absorption value in the 600nm wavelength 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 making up successful purpose engineering strain
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, PHB 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 dissolved oxygen to be controlled at 30%, when rotating speed reaches the highest 800r/min, 20%, when air flow reaches 1:1, add the control of 60% glucose equally at 10%-15% through stream through adjustment air flow control dissolved oxygen through the control rotating speed.
Every at a distance from 3h sampling detection shikimic acid content and cell concentration (detection method is the same).During fermentation 60h, shikimic acid content reaches 54g/L in the fermented liquid.
With making up successful purpose engineering strain
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, PHB 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 dissolved oxygen to be controlled at 30%, when rotating speed reaches the highest 800r/min, 20%, when air flow reaches 1:1, add the control of 60% glucose equally at 10%-15% through stream through adjustment air flow control dissolved oxygen through the control rotating speed.
Every at a distance from 3h sampling detection shikimic acid content and cell concentration (detection method is the same).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 is with the construction process of engineering strain
<130>
<160> 31
<170> PatentIn?version?3.4
<210> 1
<211> 70
<212> DNA
< 213>artificial sequence
<400> 1
atggaatccc?tgacgttaca?acccatcgct?cgtgtcgatg?gcactattaa?gtgtaggctg 60
<210> 2
<211> 70
<212> DNA
< 213>artificial sequence
<400> 2
tcaggctgcc?tggctaatcc?gcgccagctg?ctcgaaataa?tccggaaatg?atgggaatta 60
<210> 3
<211> 24
<212> DNA
< 213>artificial sequence
<400> 3
tcatggttga?gttcgaacgc?cgtc 24
<210> 4
<211> 24
<212> DNA
< 213>artificial sequence
<400> 4
gtgggtattt?tgacaccctg?tcag 24
<210> 5
<211> 38
<212> DNA
< 213>artificial sequence
<400> 5
gcgctgcagg?tcggatccac?gtcgttactg?atccgcac 38
<210> 6
<211> 29
<212> DNA
< 213>artificial sequence
<400> 6
gggaagcttc?cacgtcttat?caggcctac 29
<210> 7
<211> 29
<212> DNA
< 213>artificial sequence
<400> 7
gcgctgcagg?gcctcaggca?tttgagaag 29
<210> 8
<211> 26
<212> DNA
< 213>artificial sequence
<400> 8
ttaaagctta?tcgatgataa?gctgtc 26
<210> 9
<211> 38
<212> DNA
< 213>artificial sequence
<400> 9
catggatccc?tcgggcccat?gtgtttctca?aaccgttc 38
<210> 10
<211> 40
<212> DNA
< 213>artificial sequence
<400> 10
gccgccaggc?aaattctgtt?tttatttctt?cagttcagcc 40
<210> 11
<211> 40
<212> DNA
< 213>artificial sequence
<400> 11
ggctgaactg?aagaaataaa?aacagaattt?gcctggcggc 40
<210> 12
<211> 39
<212> DNA
< 213>artificial sequence
<400> 12
gcgctgcaga?agagtttgta?gaaacgcaaa?aaggccatc 39
<210> 13
<211> 36
<212> DNA
< 213>artificial sequence
<400> 13
catggatccc?tccggccgcg?gttctggcaa?atattc 36
<210> 14
<211> 42
<212> DNA
< 213>artificial sequence
<400> 14
gcaagctctt?tacgtgagga?cattgtcgat?acccttttta?cg 42
<210> 15
<211> 42
<212> DNA
< 213>artificial sequence
<400> 15
cgtaaaaagg?gtatcgacaa?tgtcctcacg?taaagagctt?gc 42
<210> 16
<211> 36
<212> DNA
< 213>artificial sequence
<400> 16
ctcgggccct?tacagcagtt?cttttgcttt?cgcaac 36
<210> 17
<211> 26
<212> DNA
< 213>artificial sequence
<400> 17
gcgccatgga?gaggattgtc?gttact 26
<210> 18
<211> 29
<212> DNA
< 213>artificial sequence
<400> 18
tgcgctagct?tgttgttacg?ctgattgac 29
<210> 19
<211> 28
<212> DNA
< 213>artificial sequence
<400> 19
cgggctagcg?taacagggtg?atcatgag 28
<210> 20
<211> 29
<212> DNA
< 213>artificial sequence
<400> 20
catcggccgc?ctgatgcttc?gtatttacg 29
<210> 21
<211> 35
<212> DNA
< 213>artificial sequence
<400> 21
ctgggatccc?atgagctctt?aactttaaga?aggag 35
<210> 22
<211> 22
<212> DNA
< 213>artificial sequence
<400> 22
ctggccatgg?aaacctatgc?tg 22
<210> 23
<211> 25
<212> DNA
< 213>artificial sequence
<400> 23
cgcgagctca?cgcggacaat?tcctc 25
<210> 24
<211> 35
<212> DNA
< 213>artificial sequence
<400> 24
ctgggatccc?atgcatgctt?aactttaaga?aggag 35
<210> 25
<211> 35
<212> DNA
< 213>artificial sequence
<400> 25
acggagctca?cgcggacaat?tcctcctgca?attgc 35
<210> 26
<211> 36
<212> DNA
< 213>artificial sequence
<400> 26
catggatccc?ggttctggca?aatattctga?aatgag 36
<210> 27
<211> 37
<212> DNA
< 213>artificial sequence
<400> 27
cgtcgttctg?ataattcatt?gtcgataccc?tttttac 37
<210> 28
<211> 37
<212> DNA
< 213>artificial sequence
<400> 28
gtaaaaaggg?tatcgacaat?gaattatcag?aacgacg 37
<210> 29
<211> 43
<212> DNA
< 213>artificial sequence
<400> 29
atttttgaag?ccgaccggac?aaaaaagccc?tgatgccagt?tcg 43
<210> 30
<211> 43
<212> DNA
< 213>artificial sequence
<400> 30
cgaactggca?tcagggcttt?tttgtccggt?cggcttcaaa?aat 43
<210> 31
<211> 35
<212> DNA
< 213>artificial sequence
<400> 31
cgcgcatgct?tacccgcgac?gcgcttttac?tgcat 35
Claims (6)
1. a shikimic acid production is characterized in that with the construction process of engineering strain, may further comprise the steps:
1. make up
AroAThe coli strain of gene knockout; 2. make up and contain the key gene 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.
2. shikimic acid production is characterized in that with the construction process of engineering strain 1. step comprises according to claim 1: a, obtain through pcr amplification
AroAHomology replacement 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 through 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.
3. shikimic acid production is characterized in that with the construction process of engineering strain 2. step comprises according to claim 1:
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 of transcription terminator, constitute the pAR63 plasmid to the pAR plasmid.
4. like the construction process of the said shikimic acid production of claim 3, it is characterized in that, contain trp promoter aporepressor gene among the said recombinant expression plasmid pAR63 with engineering strain
TrpR, and
TrpRHave self promotor.
5. like the construction process of the said shikimic acid production of claim 3, it is characterized in that with engineering strain, contained among the said recombinant expression plasmid pAR63
PpsAThe gene downstream sequence is the Transcription Termination subsequence; Contained among the said recombinant expression plasmid pAR63
TktAGene upstream sequence is the tryptophan promoter subsequence; Contained among the said recombinant expression plasmid pAR63
AroBWith
AroEGene upstream sequence all contains the RBS sequence; Contained among the said recombinant expression plasmid pAR63
AroDGene has self RBS sequence; Contained among the said 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.
6. like the construction process of the said shikimic acid production of claim 5, it is characterized in that said Transcription Termination subsequence does with engineering strain
RrnBThe Transcription Termination subsequence.
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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 |
| CN104911137A (en) * | 2015-04-14 | 2015-09-16 | 中国科学院微生物研究所 | Shikimic acid-producing corynebacterium glutamicum, and construction method and application thereof |
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| CN109423468A (en) * | 2017-08-24 | 2019-03-05 | 中国科学院微生物研究所 | The method for improving the compound in aromatic amino acid biosynthesis pathway and its derivative yield |
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| CN110904140A (en) * | 2019-12-20 | 2020-03-24 | 台州职业技术学院 | Protein dynamic expression regulation system and application thereof in shikimic acid production |
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| 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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| 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 |
| CN104911137A (en) * | 2015-04-14 | 2015-09-16 | 中国科学院微生物研究所 | Shikimic acid-producing corynebacterium glutamicum, and construction method and application thereof |
| CN107446870A (en) * | 2016-11-23 | 2017-12-08 | 中国科学院微生物研究所 | A kind of preparation method of high yield thick grass mutant polynucleotides |
| 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 |
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| CN109423468A (en) * | 2017-08-24 | 2019-03-05 | 中国科学院微生物研究所 | The method for improving the compound in aromatic amino acid biosynthesis pathway and its derivative yield |
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| CN111057672A (en) * | 2019-12-20 | 2020-04-24 | 东莞市东阳光生物合成药有限公司 | Recombinant strain and application thereof |
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