CN101892185A - Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof - Google Patents
Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof Download PDFInfo
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Abstract
This invention discloses a genetically engineered strain of streptomyces coeruleorubidus highly producing epi-daunorubicin, which is a genetically engineered strain to block dnmV gene and express aveBIV (avermectins keto-reductase) by inserting a first foreign gene containing aveBIV gene in the dnmV gene of a wild strain of the streptomyces coeruleorubidus or changing the dnmV gene of the wild strain of the streptomyces coeruleorubidus into the first foreign gene containing aveBIV gene. A second foreign gene is further integrated in the genome of the genetically engineered strain, and the first and second foreign genes are chain segments of a promoter and the aveBIV gene. The constructed genetically engineered strain does not contain any resistance marker and is convenient for further genetic engineering construction. The yield of epi-daunorubicin is preferably 70mg/L and is far greater than that of the epi-daunorubicin made by existing methods; through continuous passage, the yield of epi-daunorubicin is very stable with good repeatability.
Description
Technical field
The invention belongs to gene engineering technology field, genetic engineering bacterium of particularly a kind of sky blue light red streptomycete of high yield EPIDNR and preparation method thereof and used recombinant vectors and transformant.
Background technology
Daunorubicin and be that raw material synthetic Zorubicin (doxorubin), pidorubicin (epirubicin) are crucial clinically at present antitumor anthracycline antibioticss with it.The cardiac toxic and the bone marrow toxicity of pidorubicin are lower than Zorubicin, and antineoplastic effect is equal or higher.EPIDNR (epi-daunorubicin) is the important as precursors of industrial production pidorubicin, and it only is that with the difference of daunorubicin desoxy sugar C4 position hydroxyl configuration is different in the molecule.From the daunorubicin to the EPIDNR, the reaction process of multistep conditional request harshness need be passed through by chemical synthesis, and environmental pollution can be caused.If adopt the microbial fermentation processes direct production to save cost greatly, raise the efficiency.If want the generation bacterium of daunorubicin be transformed into can be directly the bacterial strain of synthetic EPIDNR in vivo, need to change soften a step in the biosynthetic process of brown sugar amine of TDP-L-.Wherein dnmV is a TDP-6-desoxy sugar C4 keto reductase, and it has determined hydroxyl configuration reaction in desoxy sugar C4 position in the bacterial strain molecule, makes it generate daunorubicin.A Wei rhzomorph keto reductase is ave BIV just in time opposite with stereoselectivity dnmV.Professor Hutchinson of winconsin university is with the aveBIV gene, import daunorubicin and produce bacterium (ripple match streptomycete), and the generation of the gentle brown sugar amine of interruption, the engineering bacteria that makes up produces EPIDNR (Krishnamurthy Madduri, Jonathan Kennedy, Giovanni Rivola, et al.Production of the antitumor drug epirubicin (4 '-epidoxorubicin) and its precursorby a genetically engineered strain of Streptomyces peucetius[J] .NatureBiotechnology, 1998,16:69-74).And the applicant is before in producing daunorubicin streptomyces coelicolor SIPI-1482, interrupted gentle brown sugar amine biosynthesis gene dnmV, inserted the resistant gene of aveBIV gene and A Baila mycin (having another name called apramycin), made up EPIDNR and produced bacterium (Chinese patent application 200610146614.6).It is lower that the engineering bacteria that this method makes up produces EPIDNR output, verifies that through us its fermentation yield is no more than 20mg/L, and unstable, continuous passage more than 2 times fermentation unit tangible reduction is just arranged.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly not high at the genetic engineering bacterium EPIDNR output of the sky blue light red streptomycete of existing production EPIDNR, the deficiency that genetic stability is low, a kind of sky blue light red streptomyces gene engineering bacteria of production EPIDNR of improvement is provided, this bacterium EPIDNR output is much higher than existing EPIDNR produces bacterium, and it is highly stable to go down to posterity.The present invention also provides the preparation method of genetic engineering bacterium of sky blue light red streptomycete of this production EPIDNR and used recombinant vectors and transformant.
The inventor finds that through scrutinizing with repetition test the external insertion gene in the genetic engineering bacterium of the sky blue light red streptomycete of production EPIDNR comprises antibiotics resistance gene and aveBIV gene.Antibiotics resistance gene has the length of 2kb, is selection markers, and the aveBIV gene is to make sky blue light red streptomycete produce the gene of EPIDNR, and external insertion gene fragment is excessive, has influenced the genetic stability of engineering bacteria and the output of EPIDNR.Therefore, the inventor changes into alien gene only and is made up of aveBIV gene and promotor thereof, does not add antibiotics resistance gene, reduces the length of alien gene greatly.On this basis, the present invention also finds, change the integrated plasmid that contains promotor and aveBIV gene over to by conjugal transfer, make erythromycin promotor and aveBIV gene random integration on whole genome, the erythromycin promotor that doubled and aveBIV gene, thus can screen the mutant strain that EPIDNR output further increases.The inventor finds that also the exchange arm length that increases the homologous recombination double exchange in the preparation of genetic engineering bacterium can increase the frequency of double exchange greatly, and the workload of screening double exchange reduces greatly, thereby has finished the present invention fast.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: the genetic engineering bacterium of a kind of sky blue light red streptomycete of production EPIDNR, it is a kind of first foreign gene that comprises the aveBIV gene that inserts or be replaced in the dnmV of sky blue light red streptomycete wild strain gene, the dnmV gene is blocked and expresses the genetic engineering bacterium of aveBIV (Avrmectin keto reductase), wherein, also integrating second foreign gene in the genome of this genetic engineering bacterium, described first foreign gene and second foreign gene all are the chain fragments of promotor and aveBIV gene.
According to the present invention, described promotor is the erythromycin promotor preferably, and the nucleotide sequence of better described promotor is the nucleotide sequence shown in the 1525th～2198 of SEQ ID NO.1 in the sequence table.Described aveBIV gene claims Avrmectin keto reductase gene again.The nucleotide sequence of described aveBIV gene is preferably shown in the 2199th～3492 of SEQ ID NO.1 in the sequence table.Described dnmV gene claims thymidine bisphosphate-6-desoxy sugar C4 keto reductase gene again.
According to the present invention, described first foreign gene is inserted in the dnmV gene of sky blue light red streptomycete wild strain, perhaps with the dnmV gene in dna fragmentation displacement.The dnmV gene order of described sky blue light red streptomycete wild strain can be whole dnmV gene by alien gene metathetical part maximum length, and preferably the nucleotide fragments between the 273rd of the dnmV gene the～675 is replaced.Therefore, the dnmV gene is blocked and can not expresses.And first foreign gene that inserts, promptly the chain fragment of promotor and aveBIV gene can be expressed, thereby makes this sky blue light red streptomycete of having been transformed can produce EPIDNR.
According to the present invention, described second exogenous origin gene integrator is in the genome of sky blue light red streptomyces gene engineering bacteria of the present invention.Integration position in genome is in genomic any position, and only otherwise influence the biosynthesizing of bacterial strain itself, situation that can the high yield EPIDNR is all within protection scope of the present invention.
According to the present invention, described sky blue light red streptomycete wild strain is preferable selects domestic industry bacterial strain---sky blue light red streptomycete (Streptomyces coeruleorubidus) SIPI-1482 for use.
The present invention has made up a kind of recombinant shuttle plasmid earlier, its contain by in sky blue light red streptomyces gene group with the upstream gene of dnmV gene linkage or its fragment, dnmV gene 5 ' end fragment, promotor, aveBIV gene, dnmV gene 3 ' end fragment and in sky blue light red streptomyces gene group with the upstream gene or the chain successively dna fragmentation of forming of its fragment of dnmV gene linkage.In the sky blue light red streptomycete of wild-type, the chain successively expression of gene dnmZ, dnmU, dnmV, dnmJ and dnmI, dnmZ and dnmU are in the upstream of dnmV, and dnmJ and dnmI are in the downstream of dnmV, and the nucleotide sequence of this linked gene is (GenBank accession number: AF006633) openly.What the recombinant fragment that contains in the recombinant shuttle vector of the present invention was preferable then is the sequence of inserting or be replaced as by part dnmV gene order promotor and aveBIV gene in the dnmV of above-mentioned chain sequence gene order.Can be whole dnmV gene by the length maximum of constant series in the dnmV gene, preferably dnmV gene intermediary nucleotide sequence.That is to say, the recombinant DNA sequence that recombinant shuttle vector of the present invention contains preferably by dnmZ gene or its fragment, dnmU gene, in sky blue light red streptomyces gene group with the dnmV gene fragment of dnmU gene linkage, promotor, aveBIV gene, in sky blue light red streptomyces gene group with dnmV gene fragment, dnmJ gene and dnmI gene or the chain successively dna fragmentation of forming of its fragment of dnmJ gene linkage.Promotor described in the present invention is the erythromycin promotor preferably.Preferable, the recombinant fragment that contains in the described recombinant shuttle vector, the dna fragmentation of promptly described chain composition successively, its nucleotide sequence is shown in SEQ ID NO.1 in the sequence table.Wherein the 1st～617 nucleotide sequence is described dnmZ gene fragment; The 618th～1251 nucleotide sequence is described dnmU gene; The 1252nd～1524 nucleotide sequence be described in sky blue light red streptomyces gene group with the dnmV gene fragment of dnmU gene linkage; The 1525th～2198 nucleotide sequence is described erythromycin promotor; The 2199th～3492 nucleotide sequence is described aveBIV gene; The 3493rd～3741 Nucleotide be described in sky blue light red streptomyces gene group with the dnmV gene fragment of dnmJ gene linkage; The 3742nd～4855 Nucleotide is described dnmJ gene; The 4856th～5028 Nucleotide is described dnmI gene.
The recombinant shuttle plasmid that the present invention makes up can be used for and the double exchange of sky blue light red streptomyces gene group generation homology, thereby obtains the genetic engineering bacterium of the sky blue light red streptomycete of production EPIDNR.Wherein, in this recombinant shuttle vector, described dnmZ, dnmU and with the chain segmental part of dnmV of dnmU be the left arm that the homologous recombination double exchange takes place; With the part of chain dnmV fragment, dnmJ and the dnmI of dnmJ be the right arm that the homologous recombination double exchange takes place.The part of promotor and aveBIV gene is the alien gene that imports when the reorganization double exchange takes place.The length of described left arm can be 500～2000bp, preferably 1525bp, i.e. sequence shown in the 1st～1524 of the SEQ ID NO.1 in the sequence table.The length of described right arm can be 500～2000bp, preferably 1536bp, i.e. sequence shown in the 3293rd～5028 of the SEQ IDNO.1 in the sequence table.
According to the present invention, the skeleton of described recombinant shuttle plasmid is plasmid pKC1139 preferably.
With described recombinant shuttle plasmid transformed host cell, obtain transformant.Described host cell is intestinal bacteria ET12567 preferably.This transformant and sky blue light red streptomycete are engaged, select homology double exchange zygote.The preferably sky blue light red streptomycete SIPI-1482 of described sky blue light red streptomycete.This homology double exchange zygote is the sky blue light red streptomycete mutant strain that has inserted the aveBIV gene in the dnmV gene.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: a kind of recombinant vectors, it contains the chain fragment of promotor and aveBIV gene.According to the present invention, the carrier framework of described recombinant vectors is plasmid pSET152 preferably.Described promotor is erythromycin promotor (ErmP) preferably.The chain segmental nucleotide sequence of described promotor and aveBIV gene preferable shown in the 1525th～3492 of SEQ ID NO.1 in the sequence table.
The present invention solves the problems of the technologies described above three of the technical scheme that adopted: a kind of transformant, it contains described recombinant vectors.According to the present invention, the host cell of described transformant is intestinal bacteria ET12567 preferably.
The present invention solves the problems of the technologies described above four of the technical scheme that adopted: the method for the genetic engineering bacterium of a kind of sky blue light red streptomycete for preparing the production EPIDNR, wherein, comprise aforesaid transformant is engaged with the sky blue light red streptomycete that produces EPIDNR, select zygote.With A Baila mycin screening, what can grow is exactly that plasmid has been incorporated into zygote on the genome.According to the present invention, the sky blue light red streptomycete of described product EPIDNR has preferably inserted the sky blue light red streptomycete mutant strain of aveBIV gene in the dnmV gene in the genome.The zygote of gained is the genetic engineering bacterium of the sky blue light red streptomycete of production EPIDNR of the present invention, is integrating 2 foreign genes in its genome, and these two foreign genes all are the chain fragments of promotor and aveBIV gene.The preferably sky blue light red streptomycete of the sky blue light red streptomycete of described product daunorubicin (Streptomyces coeruleorubidus) SIPI-1482.
The genetic engineering bacterium of sky blue light red streptomycete of the present invention can be used for the production EPIDNR.Therefore, the present invention also provide a kind of prepare EPIDNR method, comprise the genetic engineering bacterium of cultivating the above-mentioned sky blue light red streptomycete of the present invention, from culture, obtain EPIDNR.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art, beneficial effect of the present invention is as follows: the present invention will produce the sky blue light red streptomycete of daunorubicin and transform, knocked out part dnmV gene wherein on the one hand, the aveBIV gene is gone in displacement, has obtained the engineering bacteria of the EPIDNR of production by the gene substitution method; On the other hand, by complementary method at random, another aveBIV gene integration is gone in the genome, change the aveBIV gene over to thereby influence under the biosynthetic situation of bacterial strain itself 2 times, thereby make the output of EPIDNR that a very big raising arranged again at bottom line.The engineering bacteria that the present invention is constructed does not contain any resistance marker, easily is used for further genetic engineering modified.The engineering bacteria that the present invention is constructed, EPIDNR output is far longer than existing method, preferably can reach 70mg/L.The engineering bacteria that the present invention is constructed, by continuous passage, EPIDNR output is highly stable, good reproducibility.In preparation method of the present invention, increased the length of double exchange both arms.In Chinese patent application 200610146614.6 disclosed methods, the length of exchange both arms is 700bp, and the length of exchange both arms is 1500bp in the preferred version of the present invention, has increased the double exchange odds, accelerates the zygosporic screening of double exchange.The present invention adopts PCR to screen mutant strain, solved the insertion resistant gene as selection markers (for example A Baila mycin resistant gene), insert the excessive problem of bringing that influences the upstream and downstream gene transcription of alien gene fragment, thereby the base and the coded amino acid at the two ends of the dnmV gene that maintenance is remaining are constant, reduce the influence of interrupting dnmV and inserting the gene of aveBIV gene pairs upstream and downstream to greatest extent, increase The stability of strain greatly, improved EPIDNR output.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is the construction of recombinant plasmid synoptic diagram that contains the aveBIV gene.
Fig. 2 is the structure synoptic diagram that fixed point is inserted plasmid.
Fig. 3 is the construction strategy synoptic diagram that EPIDNR is produced bacterium.
Fig. 4 is the electrophorogram of PCR checking.M, marker; 1, the double exchange genotype; 2, the npr gene type.
Fig. 5 is that the HPLC of fermented liquid that produces the engineering bacteria of EPIDNR analyzes collection of illustrative plates.
Fig. 6 is the electrophorogram of PCR checking.M，marker；1，ErmP+aveBIV。
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the embodiment is meant the temperature of the operation room of testing, and is generally 25 ℃.
Employed toolenzyme and dna molecular amount mark are all available from precious biotech firm (Takara), and the concrete reaction conditions and the method for use are all with reference to catalogue
Employed glue reclaims test kit available from vast Tyke, Beijing biological gene technology limited liability company, and using method is with reference to catalogue.
Intestinal bacteria E.coli DH5 α, ET12567 are available from Shanghai ancient cooking vessel state biotechnology limited liability company.
Sky blue light red streptomycete SIPI-1482 can obtain from Shanghai Institute of Pharmaceutical Industry.
The clone of embodiment 1 double exchange left and right arms and aveBIV sequence
In sky blue light red streptomycete SIPI-1482, dnmZ and dnmU are successively in the upstream of dnmV, dnmJ and dnmI are successively in the downstream of dnmV, the present invention gets dnmZ, dmnU and adds the left arm of a part of dmnV as double exchange, and dnmI, dnmJ add a part of dmnV and make up the double exchange plasmid as the right arm of double exchange.At first according to the sky blue light red streptomycete SIPI-1482 corresponding sequence delivered (GenBank accession number: AF006633) design primer:
The left arm upstream primer is 5 '-AAAAAGCTTCTGGGACGGAATGGGC-3 ',
The left arm downstream primer is 5 '-AAATTCGAATCGCACCAGTCGCAGATG-3 ';
The right arm upstream primer is 5 '-AAATCTAGAGGGCTGGTCGTCAACATC-3 ',
The right arm downstream primer is 5 '-AAAGGATCCCATCAAACTCCGCAAGACAT-3 '.
Above-mentioned primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.Total genome with sky blue light red streptomycete SIPI-1482 is a template.Use the primer star high-fidelity DNA polymerase of precious biotech firm to carry out segmental clone.The PCR condition is: 98 ℃, and 10s, 66 ℃, 15s, 72 ℃, 2min.Behind the sample electrophoresis, reclaim the purpose band on the PCR product.The fragment that reclaims is connected with plasmid, and left arm PCR product is linked plasmid pSP72 (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), inserts site HindIII and XbaI, called after pSL-02-3-1 and order-checking; Right arm PCR product is linked plasmid pSP72, inserts site XbaI and EcoRV, called after pSL-R15, and order-checking.Corresponding sequence comparison among left arm sequence and the GenBank accession number AF006633 is 97.4% homology, and the corresponding sequence comparison among right arm sequence and the numbering AF006633 is 99.3% homology.
On PubMed, find the gene order of aveBIV, design corresponding primer: upstream primer: 5 '-AAAAGATCTGCACGGGTCAACGGGTAA-3 ', downstream primer: 5 '-AAAAGATCTTGATGTCGAGCGGCTACG-3 '.Total DNA with Avid kyowamycin S.avermitilis (available from ATCC company) is a template, obtains the gene of aveBIV with the amplification of primer star high-fidelity DNA polymerase.The aveBIV gene is connected into plasmid pSP72, and connection site BglII and BglII obtain plasmid pSL-02-20-1.
The genome of producing bacterial strain S.erythraea HL 3168E3 (available from ATCC company) with erythromycin is a template, upstream primer 5 '-AAATCTAGAAGCCCGACCCGAGCA-3 ', downstream primer 5 '-AAAAAGCTTTCCGGAGGTCGCACC-3 ', carry out PCR, be connected behind the gained PCR product purification on the plasmid pGEM-3zf (giving birth to the worker) available from Shanghai, obtain plasmid pSL-HM-312, promptly contain the recombinant vectors of erythromycin promotor.With restriction enzyme EcoRI and BamHI from plasmid pSL-HM-312 cut the erythromycin promotor, the erythromycin promotor is connected into plasmid pSET152 (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) obtains pSL-02-18-1, inserting the site is EcoRI and BamHI.Be connected into pSL-02-18-1 with BglII from the aveBIV gene forward that pSL-02-20-1 cuts, this plasmid is the pSET152 random integration plasmid that inserts erythromycin promotor and aveBIV structure gene, called after pSL-02-22-1.Design and synthesize the PCR primer, upstream primer is 5 '-AAATCTAGAGGTACCAGCCCGACCCGAGC-3 ', and downstream primer is 5 '-AAATCTAGATGATGTCGAGCGGCTACG-3 '.With plasmid pSL-02-22-1 is template, amplify the dna fragmentation (ErmEP+aveBIV gene) of erythromycin promotor and aveBIV gene linkage, link plasmid pSP72, insert site XbaI and XbaI, obtain recombinant plasmid, called after pSL-02-26-1, order-checking, the nucleotide sequence of gained aveBIV gene is seen in the sequence table shown in the 2199th～3492 of the SEQ ID NO.1, with in the ncbi database the aveBIV gene order (the sequence accession number: AB032523) Bi Dui homology is 100%, and the plasmid construction process is seen Fig. 1.
Structure, conjugal transfer that embodiment 2 double exchanges fixed point is inserted plasmid
Right arm among the embodiment 1 is linked plasmid pKC1139 (available from company of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), inserts site XbaI and EcoRV, obtains plasmid, called after pSL-02-7-1.The left arm forward is linked plasmid pSL-02-7-1, inserts site HindIII and XbaI, obtains plasmid, called after pSL-02-12-1.Cutting out ErmEP+aveBIV gene forward in the pSL-02-26-1 plasmid of employing restriction enzyme XbaI from embodiment 1 links among the pSL-02-12-1, this plasmid is double exchange and knocks out dmnV, and fixed point is inserted the EPIDNR expression integrated plasmid of aveBIV simultaneously.Transform large intestine DH5 α with this plasmid, transfer transformant and cultivate in LB, the extraction plasmid carries out enzyme and cuts the checking with PCR, finally is built into the double exchange fixed point and inserts plasmid, called after pSL-02-30-1.Construction strategy is seen Fig. 2.
Cultivate in 50ml TSB substratum from an amount of thalline of sky blue light red streptomycete SIPI-1482 inclined-plane picking and to reach logarithmic phase in about 56 hours, 1% (v/v) inoculum size is transferred, and cultivation made thalline reach the logarithmic growth later stage in about 48 hours in 50ml TSB substratum, the centrifugal supernatant that goes obtains mycelium, mycelium washs 2 (4000rpm with the LB liquid nutrient medium of 20ml, 10min, 4 ℃), be resuspended at last in the 20ml LB substratum, stand-by.With pSL-02-30-1 transformed competence colibacillus intestinal bacteria ET12567, choose transformant to being equipped with in the 4ml LB substratum small test tube of [containing A Baila mycin (Am) 100 μ g/ml, kantlex (Kn) 25 μ g/ml, paraxin (Cm) 100 μ g/ml], 37 ℃ of shaking culture 12 hours.Intestinal bacteria ET12567 is inoculated in the 250ml triangular flask that 50ml LB substratum is housed, 37 ℃ of shaking culture made bacterium liquid OD value between 0.4～0.6 about 4 hours, bacterium liquid were moved into the aseptic plastic centrifuge tube of 50ml, centrifugal (4000rpm, 10min, 4 ℃), remove supernatant, thalline washs 2 (4000rpm with the 20mlLB substratum, 10min, 4 ℃), be resuspended at last in 1～2ml LB substratum.With this colibacillus liquid and stand-by sky blue light red streptomycete SIPI-1482 mycelium before with 1: 1 (resuspended liquid volume ratio) mixing in the EP pipe.Mixed bacteria liquid is coated with the MS flat board, and with being coated with fully mixing bacterium liquid of rod, 30 ℃ of thermostat containers are cultivated.Cultivate after 20 hours, take out flat board, be coated with microbiotic, consumption is the mixed solution that per 10 flat boards are coated with 10ml distilled water, 100 μ l Am and 400 μ l Nalidixic Acids (NC), cultivates in 30 ℃ of thermostat containers again.Zygote appears after cultivating a week.EPIDNR produces the building process of bacterium and sees Fig. 3.
The screening of embodiment 3 double exchange engineering bacterias
The zygote of picking embodiment 2 gained (contains the granulated glass sphere that adds 2～5 millimeters of 2～3 diameters in Am50 μ g/ml, the test tube) in the small test tube that 4ml TSB substratum is housed, 30 ℃ of shaking culture.Bacterium liquid shake dense after, get 1 μ l bacterium liquid and be diluted in the 1ml sterilized water, get 100 μ l behind the mixing and be coated with the Gause I flat board that contains Am 50 μ g/ml, 37 ℃ of cultivations.Because the pKC1139 plasmid is the temperature sensitive type plasmid, only be incorporated on the karyomit(e) and could survive, the homologous recombination exchange had taken place in all integrons that come out.After 4 to 5 days, the picking integron is cultivated in the TSB of antibiotic-free substratum, and continuous passage impels it that double exchange takes place.Get the 3rd generation bacterium liquid 1 μ l, be diluted in the sterilized water of 10ml, get 100 μ l behind the mixing and be coated with Gause I flat board (non-resistant), 30 ℃ of cultivations.After 4 to 5 days, single bacterium colony occurs, the same single bacterium colony of picking is cultivated having on resistance and the nonresistant flat board respectively, the dull and stereotyped growth of screening non-resistant, and the bacterium colony that resistant panel is not grown obtains 30 bacterium colonies.In the dull and stereotyped growth of non-resistant and the clone that resistant panel is not grown is exactly that double exchange or regressive clone are taken place.Picking colony is cultivated in liquid TSB substratum, then its total DNA of extracting.The corresponding primer of design on left and right arms, upstream primer: 5 '-ACGGGTGGGACGTGCAC-3 ', downstream primer: 5 '-CACATGCTGTGGACGGAG-3 ' carries out the PCR checking.The electrophorogram of PCR product is seen Fig. 4.Amplifying the segmental of 800bp is the wild strain genome of answer type, and amplifying the segmental of 2400bp is double exchange mutant strain genome.We screen 6 double exchange mutant strains from 30 bacterium colonies, the double exchange occurrence probability is very high.
Embodiment 4 produces the fermentation checking of the engineering bacteria of EPIDNR
Can amplify the 30 ℃ of cultivations of the segmental double exchange mutant strain of 2400bp among the picking embodiment 3, cultivate after 15 days, be inoculated in seed culture fluid (starch 2.0, Semen Maydis powder 2.0, glucose 1.0, wheat bran 1.0, seitan powder 1.0, MgSO in the Gause I inclined-plane
40.1, K
2HP0
40.1 (g/100ml)).Seed culture fluid is cultivated after 2 days and is transferred in fermention medium (Semen Maydis powder 7.0, glucose 1.0, wheat bran 1.0, seitan powder 0.75, K
2HP0
40.01, (NH
4)
2SO
40.1), inoculum size 10% (v/v).30 ℃, 220r/m cultivated after 5 days puts bottle, fermented liquid with oxalic acid adjust pH to 5 about, the acetone mixing that adds 2 times of volumes again soaked more than 5 hours, got supernatant after centrifugal and was HPLC and analyzes.The HPLC condition, moving phase: the acetonitrile of 0.1% (v/v) formic acid: the aqueous solution of 0.1% (v/v) formic acid=72: 28, flow velocity: 1ml/min, column temperature: 35 ℃, detect wavelength: 254nm, sample size: 5 μ l, analytical column: Agilent C18.The HPLC analytical results is seen Fig. 5, as seen: by the engineering bacteria that the double exchange fixed point is inserted, do not had the daunorubicin component in the tunning, and the concentration of EPIDNR is about 45mg/L.
Embodiment 5aveBIV integrated plasmid changes the gentle mutant strain of table of aveBIV displacement dnmV over to
(1) with random integration plasmid pSL-02-22-1 transformed into escherichia coli ET12567 competent cell, choose transformant to 4ml LB (Am100 μ g/ml, Kn25 μ g/ml, Cm100 μ g/ml) 37 ℃ of shaking culture are 12 hours in the small test tube, transfer in the 250ml of 50ml LB triangular flask by the inoculum size of 2% (v/v) then, 37 ℃ of shaking culture are about 4 hours, make bacterium liquid OD value between 0.4～0.6, bacterium liquid is moved into the aseptic plastic centrifuge tube of 50ml, centrifugal (4000rpm, 10min, 4 ℃), remove supernatant, thalline washs 2 times (4000rpm, 10min, 4 ℃) with 20ml LB, be resuspended at last among 1～2ml LB, standby.
(2) among the picking embodiment 3 gained can amplify the segmental double exchange mutant strain of 2400bp in the Gause I inclined-plane, cultivate after 15 days for 30 ℃, cultivate in 50ml TSB from an amount of thalline of inclined-plane picking and to reach logarithmic phase in about 56 hours, transfer to cultivate by the inoculum size of 1% (v/v) and made bacterium liquid reach the logarithmic growth later stage in about 48 hours in 50ml TSB, the centrifugal supernatant that goes obtains mycelium, mycelium 2 (4000rpm of LB liquid scrubbing of 20ml, 10min, 4 ℃), be resuspended in 20ml LB at last, standby.
(3) the standby mycelium of gained is pressed 1: 1 mixing in the EP pipe of resuspended liquid volume ratio in colibacillus liquid that gained in the step (1) is standby and the step (2).Mixed bacteria liquid is coated with the MS flat board, and with being coated with fully mixing bacterium liquid of rod, 30 ℃ of thermostat containers are cultivated.Cultivate after 20 hours, take out flat board, be coated with microbiotic (Am50 μ g/ml, NC (Nalidixic Acid) 50 μ g/ml), cultivate in 30 ℃ of thermostat containers again.Cultivate the bacterium colony that grows after the week and be zygote.NC is anticolibacillary, so the gained bacterium colony can not be the colibacillary transformant of plasmid pSL-02-22-1 random integration, and the double exchange mutant strain can not be survived under the Am resistance, so just obtained the purpose zygote through the screening of Am and NC.
Extracting and the genotypic checking of the total DNA of embodiment 6 mutant strains
Picking zygote (Am 50 μ g/ml) in liquid TSB cultivates carries out the resistance checking, and 30 ℃, 220r/m cultivates after 2 days and filters out the bacterial strain that can grow.Get the above-mentioned bacterium liquid 1.5ml that can in resistance TSB, cultivate, the centrifugal supernatant that goes, behind the resuspended mycelium of 500 μ l STE, the centrifugal 2min of 8000r/min, outwell supernatant after repeated washing once.500 μ l STE suspended bacteria filaments add N,O-Diacetylmuramidase, and final concentration is 2mg/L (N,O-Diacetylmuramidase is a solid, can add earlier among the STE, is made into 2mg/L), and 37 ℃, water-bath 30min.Add 250 μ l then, the SDS of 2% (w/v) shakes even.The phenol that adds 250 μ l: chloroform (volume ratio 1: 1) shakes even, 15000r/min, and 10min gets supernatant (boundary adularescent denatured protein, because whole supernatant is more sticky, the denatured protein of need ining succession sucks together).Repeat previous step and do not have obvious sticky phenomenon to supernatant rapid 7～8 times.Then use and the isopyknic chloroform extracting of supernatant phenol, pipette supernatant, add the 3M NaAC of 1/10 volume, add the equal-volume Virahol, put upside down mixing back and forth, room temperature is placed 30min, the centrifugal 5min of 12000r/min.Use 70% (v/v) washing with alcohol once to volatilize in room temperature at last, the white solid that obtains is total DNA, and it is dissolved in the distilled water of 50 μ l.
As template, utilize universal primer M13-47:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ' and RV-M:5 '-GAGCGGATAACAATTTCACACAGG-3 ' to carry out PCR with this total DNA.The PCR program: 95 ℃, 3min; 94 ℃, 1min; 55 ℃, 1min; 72 ℃, 1min (step 2 is to 30 circulations of step 4); Last 72 ℃, 2min.PCR product electrophorogram is seen Fig. 6.As seen the PCR product is the fragment about about 2KB, is exactly the size of ErmP+aveBIV, shows that ErmP+aveBIV has changed integrated plasmid over to.
Embodiment 7 produces the fermentation checking of the engineering bacteria of EPIDNR
The picking zygote is in the Gause I inclined-plane that contains 50mg/L A Baila mycin, cultivate after 15 days for 30 ℃, the an amount of thalline of picking is cultivated in seed liquor, 30 ℃, 220r/m cultivates after 2 days and transfers in fermention medium 30 ℃, 220r/m cultivated after 5 days puts bottle, to pH5, the acetone mixing that adds 2 times of volumes again soaked 5 hours fermented liquid with the oxalic acid adjust pH, got supernatant after centrifugal and was HPLC and analyzes.The HPLC condition, moving phase: the acetonitrile of 0.1% (v/v) formic acid: the aqueous solution of 0.1% (v/v) formic acid=72: 28, flow velocity: 1ml/min, column temperature: 35 ℃, detect wavelength: 254nm, sample size: 5 μ l, analytical column: Agilent C18.The HPLC analytical results shows: all do not had daunorubicin in all engineering bacteria list bacterium colony tunnings by insertion of double exchange fixed point and multiplication aveBIV, and the concentration of EPIDNR increases all, is up to about 70mg/L.
Sequence table
＜110〉Shanghai Institute of Pharmaceutical Industry
＜120〉sky blue light red streptomyces gene engineering bacteria of product EPIDNR and preparation method thereof
<130>P4-091166C
<160>1
<170>PatentIn?version?3.4
<210>1
<211>5028
<212>DNA
<213>Artificial
<220>
＜223〉recombinant DNA
<220>
<221>misc_feature
<222>(62)..(62)
<223>n?is?a，c，g，or?t
<220>
<221>promoter
<222>(1525)..(2198)
＜223〉erythromycin promotor
<220>
<221>gene
<222>(2199)..(3492)
＜223〉aveBIV gene
<400>1
ctgggacgga?atgggcatgc?ggtcgtcggg?cagtgtggac?atcgtcttcg?acggctgtcc 60
gntggaccgg?gaccgggtgc?tgccgcgacg?gcgagccggg?cgtcgcgacg?acgcggcgct 120
cgccgggcag?acgtcagctc?gtacgcatgc?tggctatcac?tcggctacgc?cgaggcgctc 180
gaggatcgcc?ctcacggaac?tgcgcaggcg?cggcggggcg?ccggccgggg?tgcggaccac 240
ggtggccgag?atagacgccc?ggctgttcgc?gctgcacacg?gcggtggcga?gcgcgttgac 300
caccgccgac?cggctcgccg?acgacctcag?cggcgacctc?gccgcccgcg?gccgcgccat 360
gatgacgccc?ttccagtacg?ccaagctgct?cgtcaaccgg?cactcggtgg?gcgtggtgga 420
cgactgcctg?atgctcgtgg?gaggggccgg?atacagcaac?tcccacccgc?tggcgcgcct 480
gtaccgcgat?gtccgggcgg?gcgggttcat?gcatccatac?aacttcacgg?acggcgtcga 540
ctacctgagc?gaagtggcgt?tgggccgatg?agcggcgggc?gacgcgggac?cgggatccct 600
ggagtcggag?gacgtggatg?aaggcgcggg?aactggcggt?gcagggggcg?tacacgttcg 660
agccggaggt?gtttccggac?gagcgggggc?tgttcgtctc?accgttccgg?gaggatgcgt 720
tcacggccgc?ggtcggccac?cccctcttcc?cggtgcggca?gacgaatcac?agccggtcgc 780
ggcgcggggt?ggtgcggggc?gtgcactaca?ccgtgacgcc?gccgggctcg?gccaagtacg 840
tgtactgcgc?ccgtggcagg?tcgctggaca?tcgtcgtcga?cgtgcgggtc?ggctccccga 900
cgtacggccg?gtgggacgcc?gtcgagctgg?agccgcgtga?gttccgggcg?gtgtacttcc 960
cggtcggggt?ggggcacgcc?ttcgtggccc?tggaggacga?caccgtcatg?tcgtacatgc 1020
tgtccgggga?gtacgtgcag?gccaacgaac?tcgccgtgtc?ggtgctggac?ccggccctcg 1080
ggctgcccgt?gccgggtgac?ctggagcctc?tgctgtccgg?ccgggaccgg?gctgcaccgc 1140
ccctggagca?ggcccgggcc?gccgggaccc?ttccggagta?cgccgcatgc?cgggcggtcg 1200
agtcggagct?gtggccgccg?gccgggtcac?gcggagacgg?gtgaggcgga?catgcgggtc 1260
gtggttctgg?gggcgacggg?cagcgtcggt?cggcaggtgt?gtgcggcgta?ccaggcgcac 1320
gggtgggacg?tgcacggggt?ggcccgccgc?ccggcgccgc?acctgagcgg?gtgcgggttc 1380
acggagctgg?acctcgcggc?cgccgcgcct?gggcggatcg?ccacggtgct?gggtgatctt 1440
ccggcggacg?tcgtggtcaa?cgcggcgggc?ggctggggcg?acaccgagga?ggagatgacg 1500
tactcgcatc?tgcgactggt?gcgagaattc?ggtaccagcc?cgacccgagc?acgcgccggc 1560
acgcctggtc?gatgtcggac?cggagttcga?ggtacgcggc?ttgcaggtcc?aggaagggga 1620
cgtccatgcg?agtgtccgtt?cgagtggcgg?cttgcgcccg?atgctagtcg?cggttgatcg 1680
gcgatcgcag?gtgcacgcgg?tcgatcttga?cggctggcga?gaggtgcggg?gaggatctga 1740
ccgacgcggt?ccacacgtgg?caccgcgatg?ctgttgtggg?cacaatcgtg?ccggttggta 1800
ggatctgcag?ccaagctctg?gaccgccccc?gactcctgac?cgccggccat?ccgtgaccac 1860
cgctcagcac?gaaggagaac?agaccgtggc?agccgccgcc?aagatcgcca?tgtccagtgt 1920
ggcgcccagt?caccggcagg?ggggcagcac?ccgcgccctg?ctgacgccgt?cgtcggtggg 1980
tgcgacctcc?ggagagctcg?gtacccgggg?atctgcacgg?gtcaacgggt?aaagccgaga 2040
gccggttccc?cccgcgagca?cgattccctt?catgtcggac?tccccgcagt?cgacgttata 2100
tatctctgcc?gtctgcccga?cggtaccaag?tggcggaaaa?cgcaccagga?attcgagcgc 2160
cgctaggggg?aagggctcaa?gaagataggg?gccaccagat?ggggcggttt?tcggtgtgcc 2220
cgccccggcc?gaccggaata?ctgaagagca?tgctgacgac?tgggatgtgc?gaccgaccgc 2280
tggtcgtcgt?actcggagcc?tccggctata?tcgggtcggc?cgtcgcggcg?gaactcgccc 2340
ggtggccggt?cctgttgcgg?ctggtggccc?ggcgaccggg?cgtcgttccg?ccgggcggcg 2400
ccgcggagac?cgagacgcgt?acggccgacc?tgacggcggc?gagcgaggtc?gccctcgccg 2460
tgacggacgc?cgacgtggtg?atccacctgg?tcgcgcgcct?cacccaggga?gcggcatggc 2520
gggcggcgga?gagcgatccg?gtggccgagc?gggtgaacgt?cggggtgatg?cacgacgtcg 2580
tcgcggccct?gcggtccggg?cgccgcgccg?ggccgccccc?ggtggtggtg?ttcgccgggt 2640
cggtctacca?ggtgggccgc?ccgggtcggg?tcgacggcag?tgagccggac?gagcccgtga 2700
cggcctatgc?ccgtcagaaa?ctcgacgccg?aacggacgtt?gaagtccgcc?acggtcgagg 2760
gtgtcctgcg?ggggatctcg?ctgcggctgc?ccaccgtcta?cggcgcgggg?ccgggcccgc 2820
agggcaacgg?cgtcgtgcag?gcgatggtgc?tccgggcgct?cgccgacgag?gccctcaccg 2880
tgtggaacgg?aagcgtggtg?gagcgtgacc?tggtgcatgt?ggaggatgtc?gcgcaggcct 2940
tcgtgagctg?cctggcgcac?gcggatgcgc?tcgccgggcg?gcactggctg?ctcggcagcg 3000
gtcgtcctgt?gaccgtcccg?cacctcttcg?gtgccatcgc?cgccggcgtg?tccgcccgca 3060
ccgggcgccc?cgcggtgccc?gtgaccgcgg?tggaccctcc?ggcgatggcg?acggcggcgg 3120
acttccacgg?gaccgtcgtc?gactcctcgg?cgttccgcgc?ggtcaccggg?tggcggccgc 3180
ggctgtcgct?tcaggagggc?ctggaccaca?tggtggcggc?ttacgtgtag?cgccggggtg 3240
gcggccgggc?ccgggcggtg?acggcccgga?tccgggtcgg?ccgtcacagc?ttctcgtcga 3300
gggcgcggct?cgcgcggtac?tccggcaaca?tgccgcgtcg?cagggcctgc?tggagagtcg 3360
gcgcgcgccg?gtcgcgctcg?gagaggatcg?gtgcccgccc?gaggtggtgg?ccgaggggca 3420
gggcgaggtc?cggatcctcg?ggcgagaggg?cgtgttcgtt?ctgcggaacg?tagccgctcg 3480
acatcaagat?ctgggctggt?cgtcaacatc?gggcgcgggg?acgccgtgcc?gatcggtgat 3540
ctggtcggct?ggctgctgga?ggccgccgcc?ttcccggagg?accgggtcga?ccgccgggag 3600
gcgccggtgc?ggagcaaggg?cggcgactgg?acccggctgg?acatcgggcg?ggcccggcgg 3660
ttgctgtcct?gggcgccgcg?catcggcctg?cgggactccg?tccacagcat?gtggcggacc 3720
gcgcacggcg?ccccggccta?gctcacaggc?ttccgacgac?ctcccgcacc?gcgtcgatca 3780
ccttctcctg?cgcgtcggga?cgcagcgagg?ggtacatcgg?cagggagaag?atctcgccgg 3840
ccagccgttc?ggtcaccggc?aggtcgccgg?ggccgtagcc?gaggtgggcg?aatccgctca 3900
tggtgtggac?gggccagggg?tagctgatgt?tgagatggat?gtcgtaggcg?gtgagcgcct 3960
ccaggatgcg?gtcgcgttcg?gggtgacgca?ccacgtacac?gtagtagacg?tggtcgttgc 4020
cctcggcgat?cgtgggcagc?accaggccgt?cgaggtcacc?gagtccctcc?tcgtagcggc 4080
gggcgacggc?ccggcggccc?tcgacatagg?cgtcgaggcg?gcgcagtttc?cggcgcagga 4140
tctcggcctg?cacctcgtcg?agcctgctgt?tgtgcccggg?ggtgtccacg?acgtagtagc 4200
gctcccccat?gccgtagtag?cgcaaccgcc?gcagccgccg?gtcgacttcc?gcgtccggcg 4260
tgacgacggc?gccgccgtca?ccgtaggcac?cgaggacctt?ggtcgggtag?aaggagaagg 4320
ccgccgcatg?cccctgggtg?ccgaccagcc?gtccgtgacg?gcgggcaccg?tgggcctggg 4380
cgcagtcctc?cagcaccttc?aggtcgtgct?cggcggcgag?ttcgagcacg?ggggtcatgt 4440
ccacgctctg?tccgtagagg?tggacgggca?gcaggcaccg?ggtgcgcggg?ccgatcaccg 4500
accggagccg?gccggtgtcc?atgaggtagt?tctcctcgtg?cacgtcgacg?aagacggggg 4560
tggcgcccac?ggcgtcgatg?gccaccacgg?tgggggccgc?ggtgttggag?accgtcacga 4620
cctcgtcccc?cgggccgatg?ccgagcgccc?gcaggccgag?gaccagcgcg?ttggtgccgt 4680
tgtcgacgcc?cgtgcagtac?ggcagtccgt?gataagcggc?gaactcctcc?tcgaaagagc 4740
ggacgctggt?cccgagaata?agctggccgg?actcgaatac?ggtttccacc?gcatcgagaa 4800
tgtcggcgcg?ttcttcccgg?tattcattga?ggtattgcca?gacgtaggtg?gacacgtgac 4860
tccttgtcgg?ggcgcggtca?ggcaagcgcg?acggacgcgg?ctgccgggac?ttccggaccg 4920
ttccggtcga?tgccggggtc?ggcccgcaga?atcgcgtgct?ggaggtgctg?gagacgggcg 4980
gacggttcca?cccccagctc?gttcaccaat?gtcttgcgga?gtttgatg 5028
Claims (11)
1. genetic engineering bacterium of producing the sky blue light red streptomycete of EPIDNR, it is a kind of first foreign gene that comprises the aveBIV gene that inserts or be replaced in the dnmV of sky blue light red streptomycete wild strain gene, the dnmV gene is blocked and expresses the genetic engineering bacterium of aveBIV, it is characterized in that, also integrating second foreign gene in the genome of this genetic engineering bacterium, described first foreign gene and second foreign gene all are the chain fragments of promotor and aveBIV gene.
2. genetic engineering bacterium as claimed in claim 1, it is characterized in that, the nucleotide sequence of described promotor is the nucleotide sequence shown in the 1525th～2198 of SEQ ID NO.1 in the sequence table, and the nucleotide sequence of described aveBIV gene is shown in as SEQ ID NO.1 in the sequence table the 2199th～3492.
3. genetic engineering bacterium as claimed in claim 1 is characterized in that, the nucleotide fragments between the 273rd～675 of the dnmV gene of described sky blue light red streptomycete wild strain is replaced by described first foreign gene.
4. genetic engineering bacterium as claimed in claim 1 is characterized in that, the integration position of described second foreign gene in genome is in genomic any position.
5. genetic engineering bacterium as claimed in claim 1 is characterized in that, described sky blue light red streptomycete wild strain is sky blue light red streptomycete Streptomyces coeruleorubidus SIPI-1482.
6. recombinant vectors, it contains the chain fragment of promotor and aveBIV gene.
7. recombinant vectors as claimed in claim 6, it is characterized in that, the carrier framework of described recombinant vectors is plasmid pSET152, and the chain segmental nucleotide sequence of described promotor and aveBIV gene is shown in the 1525th～3492 of SEQ ID NO.1 in the sequence table.
8. a transformant is characterized in that, it contains each described recombinant vectors of claim 6～7.
9. transformant as claimed in claim 8 is characterized in that, the host cell of described transformant is intestinal bacteria ET12567.
10. the method for the genetic engineering bacterium of a sky blue light red streptomycete for preparing the production EPIDNR is characterized in that, comprises transformant as claimed in claim 8 is engaged with the sky blue light red streptomycete that produces EPIDNR, selects zygote.
11. one kind prepare EPIDNR method, it is characterized in that, comprise the genetic engineering bacterium of cultivating as each described sky blue light red streptomycete of claim 1～5, from culture, obtain EPIDNR.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104122348A (en) * | 2014-08-06 | 2014-10-29 | 山东蓝星东大化工有限责任公司 | Preparation method of high-viscosity standard solution for quantification of headspace gas chromatography external standard method |
| CN107541481A (en) * | 2016-06-23 | 2018-01-05 | 上海医药工业研究院 | A kind of genetic engineering bacterium for producing Epi-ADM and its application |
| CN112080454A (en) * | 2020-09-01 | 2020-12-15 | 浙江大学 | Engineering streptomycete for producing daunorubicin and construction method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104122348A (en) * | 2014-08-06 | 2014-10-29 | 山东蓝星东大化工有限责任公司 | Preparation method of high-viscosity standard solution for quantification of headspace gas chromatography external standard method |
| CN107541481A (en) * | 2016-06-23 | 2018-01-05 | 上海医药工业研究院 | A kind of genetic engineering bacterium for producing Epi-ADM and its application |
| CN107541481B (en) * | 2016-06-23 | 2021-07-02 | 上海医药工业研究院 | Genetic engineering bacterium for producing epirubicin and application thereof |
| CN112080454A (en) * | 2020-09-01 | 2020-12-15 | 浙江大学 | Engineering streptomycete for producing daunorubicin and construction method thereof |
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