CN102453708B - Method for increasing yield of streptomyces coeruleorubidus daunorubicin - Google Patents
Method for increasing yield of streptomyces coeruleorubidus daunorubicin Download PDFInfo
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Abstract
The invention discloses a method for increasing the yield of streptomyces coeruleorubidus daunorubicin, which comprises the following steps: 1) constructing a dauW gene knockout plasmid which contains a dauW gene homologous recombinant left arm, a marker gene, a dauW gene homologous recombinant right arm; 2) performing conjugative transfer of the dauW gene knockout plasmid obtained in step 1) into streptomyces coeruleorubidus, preparing dauW gene knockout engineering bacteria of the streptomyces coeruleorubidus; 3) culturing the dauW gene knockout engineering bacteria of the streptomyces coeruleorubidus obtained in step 2), separating daunorubicin from the culture. The invention also provides the dauW gene blocked-mutant bacteria of streptomyces coeruleorubidus and a preparation method thereof. The dauW gene is knockout; the expression of the dauW gene is completely blocked; the yield of the obtained streptomyces coeruleorubidus daunorubicin is greatly increased, and is higher than the yield when the dauW gene is inserted and not completely blocked; and thus a more direct and effective approach is provided for the increase of daunorubicin yield.
Description
Technical field
The invention belongs to biological technical field, dauW gene disruption mutant bacteria of a kind of particularly method that improves streptomyces coeruleorubidus daunorubicin output, and this streptomyces coeruleorubidus and preparation method thereof.
Background technology
Daunorubicin (Daunorubicin, DNR) is important anthracycline antibiotics, using daunorubicin as precursor can semi-synthetic Dx, a series of line antitumor drugs clinically such as epirubicin, pirarubicin.What the biosynthetic pathway of daunorubicin had been studied at present is comparatively clear, and draws accordingly the structure and function distribution plan of daunorubicin biological synthesis gene cluster.In research before, this laboratory produces clone bacterium streptomyces coeruleorubidus SIPI-1482 genome from domestic daunorubicin and obtains gene dauW, and insert inactivation plasmid pYG770 by structure, carrying out homologous recombination single cross changes, dauW in streptomyces coeruleorubidus SIPI-1482 is inserted to inactivation, found that dauW inserts that the output of daunorubicin of inactivation mutant strain is the highest has increased by 6 times.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for daunorubicin output of a kind of raising streptomyces coeruleorubidus (Streptomyces coeruleorubidus) and the streptomyces coeruleorubidus mutant strain that use therein dauW gene is blocked completely.
The present invention solves the problems of the technologies described above one of adopted technical scheme: the method for the daunorubicin output of a kind of raising streptomyces coeruleorubidus (Streptomyces coeruleorubidus), comprises the following steps:
1) build dauW gene knockout plasmid, it contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm;
2) by step 1) the dauW gene knockout plasmid conjugal transfer of gained enters streptomyces coeruleorubidus, prepares dauW gene knockout (knockout) engineering bacteria of streptomyces coeruleorubidus;
3) culturing step 2) the dauW gene knockout engineering bacteria of the streptomyces coeruleorubidus of gained, separated daunorubicin from culture.
In the present invention, step 1) described dauW gene knockout plasmid contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm.The length of described dauW homologous recombination left arm is 1～3kb preferably, preferred 2kb, and its sequence preference is the sequence shown in the 208th to the 2334th of Genbank database login M73758.The length of described dauW homologous recombination right arm is 1～3kb preferably, preferred 2kb, and its sequence preference is the sequence shown in the 140th to the 2218th of Genbank database login AF048833.Described marker gene can be the marker gene of this area routine, as antibiotics resistance gene or fluorescent mark gene etc.The preferred apramycin resistance gene apr of described antibiotics resistance gene, its sequence preference is the sequence shown in the 2866th to the 3681st of Genbank database login AY072040.The plasmid skeleton of described dauW gene knockout plasmid can be intestinal bacteria-streptomycete shuttle plasmid of this area routine, preferred plasmid pJTU870.
In the present invention, step 2) described streptomyces coeruleorubidus (Streptomyces coeruleorubidus) is the conventional microorganism for the production of daunorubicin in this area, preferred streptomyces coeruleorubidus (Streptomyces coeruleorubidus) SIPI-1482, more preferably daunorubicin Producing Strain Dau27.By containing in steps 1) intestinal bacteria and the streptomyces coeruleorubidus of gained dauW gene knockout plasmid cultivate altogether, after both engage, described dauW gene knockout plasmid enters into the dauW gene generation homologous recombination of streptomyces coeruleorubidus and streptomyces coeruleorubidus genome, selects the dauW gene knockout engineering bacteria that homology double exchange obtains streptomyces coeruleorubidus.
In the present invention, step 3) method of the dauW gene knockout engineering bacteria of described cultivation streptomyces coeruleorubidus, and the method for separated daunorubicin is all the ordinary method of this area from culture.
The present invention finds, dauW gene in streptomyces coeruleorubidus genome is knocked, the expression of dauW gene is blocked completely, then the output of the streptomyces coeruleorubidus daunorubicin of gained but improves greatly, the streptomyces coeruleorubidus output that is inserted into non-blocking-up completely than dauW gene is also high, therefore for improving the output of daunorubicin, provides more direct, an effective approach.
The present invention solves the problems of the technologies described above two of adopted technical scheme: the dauW gene disruption mutant bacteria of a kind of streptomyces coeruleorubidus (Streptomyces coeruleorubidus), described dauW gene is knocked.
In the present invention, described dauW gene is preferably labeled Gene Replacement and knocks out.The method that described dauW gene is knocked can be the ordinary method of this area, as passed through homologous recombination double exchange by dauW gene knockout.Described streptomyces coeruleorubidus (Streptomyces coeruleorubidus) is the conventional microorganism for the production of daunorubicin in this area, preferred streptomyces coeruleorubidus (Streptomyces coeruleorubidus) SIPI-1482, more preferably daunorubicin Producing Strain Dau27.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of method of preparing the dauW gene disruption mutant bacteria of streptomyces coeruleorubidus as above (Streptomyces coeruleorubidus), comprises the following steps:
A) build dauW gene knockout plasmid, it contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm;
B), by a) the dauW gene knockout plasmid transformed host cell of gained of step, obtain transformant;
C) using streptomyces coeruleorubidus (Streptomyces coeruleorubidus) as acceptor, with step b) described transformant carries out conjugal transfer, selects homologous recombination double exchange, i.e. the dauW gene disruption mutant bacteria of streptomyces coeruleorubidus.
In the present invention, a) described dauW gene knockout plasmid is as mentioned above for step.Step b) host cell described in is intestinal bacteria preferably.Step c) acceptor described in is streptomyces coeruleorubidus spore preferably, and after the pre-sprouting of spore, as acceptor, pre-sprouting condition is preferably 50 ℃ of heat shocks 10 minutes.
In the present invention, above-mentioned optimum condition can arbitrary combination on the basis that meets this area general knowledge, obtains the preferred embodiments of the invention.
The present invention is except special instruction, and per-cent used is all mass percent.
The raw material that the present invention is used or reagent except special instruction, equal commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows: by homologous recombination double cross, bring the dauW gene knocking out completely in daunorubicin production bacterium streptomyces coeruleorubidus, adopt antibiotics resistance gene to replace original dauW gene in genome, investigate its function and study its impact on daunorubicin output, find that daunorubicin output improves greatly, can improve more than 8 times, and in daunorubicin Producing Strain, carry out technique operation, the daunorubicin output increase rate of dauW blocked mutant is also more than 7 times, the streptomyces coeruleorubidus output that is inserted into non-blocking-up completely than dauW gene is also high, thereby for improve the output of daunorubicin provide one more direct, effective approach.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, feature of the present invention and beneficial effect are described.
Fig. 1 pcr amplification drrAdrrB and dnrXdpsY gene.A：drrAdrrB?S+drrAdrrB?AS；B：dnrXdpsY?S+?dnrXdpsY?AS。M:DL2000; 1:SIPI-1482 is as template; 2:H
2o is as template.
PCR and the enzyme of Fig. 2 plasmid pYG255-1 are cut checking.A: enzyme is cut checking.M：λDNA/HindIII；1：pJTU870/EcoRI；2：pYG255-1/EcoRI；3：pYG255-1/EcoRI+BamHI。B:PCR checking.M:DL2000; 1:SIPI-1482 is as template; 2:pYG255-1 is as template.
PCR and the enzyme of Fig. 3 plasmid pYG255 are cut checking.A: enzyme is cut checking.M：λDNA/HindIII；1：pYG255/EcoRI+BglII；2：pYG255/EcoRI。B:PCR checking.M:DL2000; 1:SIPI-1482 is as template; 2:pYG255 is as template.
The structure of Fig. 4 plasmid pYG255.
Fig. 5 homologous recombination schematic diagram.
Fig. 6 PCR checking SIPI-1482-pYG255 mutant strain.A:Apr1+Apr2 is as primer.M:DL2000; 1:pYG255 is as template; 2:SIPI-1482-pYG255 genomic dna is as template; 3:SIPI-1482-pYG255 (1) is as template; 4:SIPI-1482 genomic dna is as template; 5:H
2o is as template.B:P255 (2)+, P255 (2) is as primer.M:DL2000; 1:pYG255 is as template; 2:SIPI-1482 genomic dna is as template; 3:SIPI-1482-pYG255 (1) is as template; 4:SIPI-1482-pYG255 genomic dna is as template; 5:H
2o is as template.
Set out bacterium SIPI-1482 and mutant strain SIPI-1482-W2 of Fig. 7 produces anti-HPLC figure.A: daunorubicin (DNR) standard substance; B:SIPI-1482; C mutant strain SIPI-1482-pYG255.
Fig. 8 Dau27-pYG255PCR checking.A:Apr1+Apr2 is template; B:P255 (2)+, P255 (2)-be template.M:DL2000; 1:H
2o is template; 2:Dau-27 genomic dna is template; 3:Dau27--pYG255 is template; 4:pYG255 is template.
The representative HPLC analysis chart of Fig. 9 Dau27-pYG255 tunning.A: standard substance DNR; B:Dau-27; C: mutant strain Dau27-pYG2553#.
Embodiment
With embodiment, further illustrate the present invention below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the present invention refers to the temperature of the operation room of testing, and is generally 25 ℃.
The amplification of 1.dauW double exchange arm fragment
Bacterial classification: streptomyces coeruleorubidus Streptomyces coeruleorubidus SIPI-1482, Ke Cong Shanghai Institute of Pharmaceutical Industry obtains.
Substratum: YMB liquid nutrient medium, YEME liquid nutrient medium (Anthony Hopwood. streptomycete genetic manipulation laboratory manual [M], first version, Changsha: Hunan science tech publishing house, 1988.).
Main solution and damping fluid: TSE, and TEL (Anthony Hopwood. streptomycete genetic manipulation laboratory manual [M], first version, Changsha: Hunan science tech publishing house, 1988.), saturated phenol chloroform.
Method:
First extract streptomyces coeruleorubidus SIPI-1482 genomic dna.Fresh slant pore is inoculated in the 250ml triangular flask that 20ml YMB liquid nutrient medium is housed to the about 48h of shaking table shaking culture under 30 ℃, 230r/min.With 10% inoculum size (v/v), aforesaid liquid seed access is equipped with in the 250ml triangular flask of 20ml YEME liquid nutrient medium to the about 48h of shaking table shaking culture under 30 ℃, 220r/min.Nutrient solution is transferred in the centrifuge tube of 50ml to centrifugal collection mycelium (Hitachi CR21G, rotor R 20A2,10,000r/min, 10min), centrifugal supernatant discarded after the sterilized water washing of proper volume.In centrifuge tube, add 5ml to contain the TSE solution of 5mg/ml N,O-Diacetylmuramidase, vortex vibrating dispersion thalline is placed on 37 ℃ of water bath heat preservation to solution and becomes sticky thick.Add Proteinase K and 250 μ l10% (wt) SDS of 50 μ l 20mg/ml to be respectively 200 μ g/ml and 0.5% (wt) to final concentration, in 55 ℃ of water-baths, be incubated 30min, every 10min, mix once therebetween.After at room temperature cooling, add the saturated phenol/chloroform of equal-volume (TNE) extracting secondary, make substantially free of impurities between two-phase interface, and then with chloroform extracting once.Add isopyknic Virahol, with the aseptic dropper of envelope mouth, in interface, slowly stir, DNA is wrapped on dropper, and with 70% (v/v) ice-cold ethanolic soln washing, at room temperature dry.TEL solution dissolving DNA by proper volume.After DNA sample is divided in Eppendorf pipe-20 ℃ of preservations.
DauW upstream region of gene has self resistant gene drrA and drrB gene, and there are dnrX, dpsY gene in downstream, and the gene fragment of PCR clone dauW gene upstream and downstream, as two sufficiently long exchange arms, is brought and blocked dauW gene completely by homologous recombination double cross.
The primer sequence of table 1 amplification drrAdrrB and dnrXdpsY gene
The SIPI-1482 genomic dna that the aforesaid method of take extracts carries out PCR, annealing temperature n=60 ℃ as template.
Result:
PCR result as shown in Figure 1, obtains respectively the band of two 2kb left and right, conforms to theory.
The structure of interstitial granules during 2.dauW blocks completely
Bacterial classification: bacillus coli DH 5 alpha (purchased from Shanghai Sheng Gong Bioisystech Co., Ltd).
Plasmid: pJTU870, for blocking the structure of interstitial granules pYG255-1 completely, contain apramycin resistance and thiostrepton resistance, for intestinal bacteria-streptomycete shuttle plasmid, take from the Bai Linquan of Shanghai Communications University professor, contact address: the No.1954, Hua Shan Road, Shanghai City Bai Linquan of Shanghai Communications University Life Sciences professor, postcode: 200030, phone: 86-21-62932418 E-mail:bailq sjtu.edu.cn.
Main solution and damping fluid: Solution I, Solution II, Solution III (Pehanorm Brooker J, Ritchie EF not, Manny A Disi T. molecular cloning experiment guide [M], second edition, Beijing: Science Press, 1992.).
Method:
Adopt the GeneClean II test kit of BIO101 company, reclaim the PCR fragment that above amplification obtains.DNA running gel is placed under long-wave ultra violet lamp, cuts out the gel containing required DNA fragmentation, pack an Eppendorf centrifuge tube of weighing in advance into, weigh up the weight of gel.Add the long-pending 6mol/LNaI of triploid (0.1g gel adds 300 μ l), 45 ℃ of water bath heat preservation 5min.Add 5 μ l Glassmilk, in vortex mixer, mix.Be placed in ice bath and keep 10min, and every a centrifuge tube of 1～2min vibration, DNA is adsorbed as far as possible by Glassmilk.Centrifugal (desk centrifuge, 13,200r/min, 5s) precipitates Glassmilk, abandoning supernatant.New Wash damping fluid New Wash damping fluid (the 50mmol/L NaCl that adds the freezing preservation of 200 μ l (20 ℃), 2.5mmol/L (pH7.5) EDTA, 10mmol/L (pH7.5) Tris, 50% (v/v) ethanol), in vortex mixer suspension Glassmilk, centrifugal (desk centrifuge, 13,200r/min, 5s) washing.This process needs in triplicate, and last washing should be blotted as far as possible and be remained in the pipe New Wash damping fluid at the end.Add 5～10 μ l sterile distilled waters, vibration suspends Glassmilk, places 45 ℃ of water bath heat preservation 5min, and the DNA of absorption is desorbed, and centrifugal (desk centrifuge, 13,200r/min, 5s), moves into a new centrifuge tube by elutriant.This process needs in triplicate, merges three times elutriant.The Glassmilk that centrifugal (desk centrifuge, 13,200r/min, 5s) precipitation may be brought into, moves into a new centrifuge tube by elutriant.
EcoR1+Bgl II double digestion for the drrAdrrB gene fragment that recovery is obtained, plasmid pJTU870, with after EcoRI and BamHI double digestion, carries out fragment connection.
Preparation E.coli DH5 α competent cell, the liquid nutrient medium of preserving bacterium liquid inoculation 2ml LB with flat board list bacterium colony or the Freezing Glycerine of fresh growth, spends the night in 37 ℃ of shaking culture.The 250ml triangular flask of the liquid nutrient medium of 20ml LB is housed with 1% inoculum size inoculation, in 37 ℃ of shaking culture 1.5h left and right, controls OD
6000.3～0.5.Bacterium liquid is moved in the aseptic Beckman plastic centrifuge tube of 50ml to ice bath 10min.Centrifugal (Hitachi CR21G, rotor R 20A2,6,000r/min, 4 ℃, 10min, lower same) recovery thalline.Abandon supernatant, and centrifuge tube is inverted, the trace nutrient solution of final residual is flow to end.Bacterial sediment is suspended in to the 0.1mol/L CaCl of 10ml ice precooling
2in solution, thalline centrifugal recovery after washing.By thalline Eddy diffusion in the 0.1mol/L CaCl of 10ml ice precooling
2in solution, ice bath 30min.Centrifugal, abandon supernatant, and centrifuge tube is inverted, the solution of final residual is flow to end.Thalline is suspended in to the ice-cold 0.1mol/L CaCl of 800 μ l
2in solution, be placed in 4 ℃ of preservations.Competent cell within fresh preparation and 4 ℃ of Refrigerator store 48h all can be directly used in conversion.
Connect product and be converted into E.coli DH5 α, DNA is connected in the Eppendorf centrifuge tube that contains 100 μ l E.coli DH5 α competent cells that product (volume=10 μ l, DNA=50ng) adds ice precooling to finger tapping centrifuge tube bottom, carefully mix ice bath 30min.By the centrifuge tube that contains transfering DNA and competent cell heat shock 90s in 42 ℃ of waters bath with thermostatic control, do not shake centrifuge tube therebetween.Fast centrifuge tube is placed in to ice bath and keeps 5min.Add 800 μ l LB liquid nutrient mediums, put upside down and mix, 37 ℃, 230rpm vibration incubation 1h, makes cell proliferation.Transformant is coated to the LB planar surface that contains penbritin (100mg/ml) and apramycin (50 μ g/ml).After liquid is absorbed, in 37 ℃, is inverted and cultivates (being no more than 16h), screening positive clone.
By single colony inoculation of grow overnight on flat board, in the 2ml LB liquid nutrient medium that contains penbritin (100 μ g/ml) and apramycin (50 μ g/ml), 37 ℃ of shaking culture are spent the night.Draw 1.5ml bacterium liquid in Eppendorf centrifuge tube, centrifugal (desk centrifuge, 12,000r/min, 1min), abandons supernatant, and vacuum blots residual liquid.To precipitate Eddy diffusion in 100 μ l Solution I, on vortex mixer, vibration disperses thalline, is placed in ice bath 10min.Add the freshly prepared Solution II of 200 μ l, cover tightly after lid, put upside down pipe and make for several times Solution II fully contact with thalline, so as lysis, ice bath 3～5min.The Solution III that adds 150 μ l precoolings, acutely shakes pipe and makes it fully mixed, ice bath 3～5min.Centrifugal (desk centrifuge, 12,000r/min, 10min), moves to another centrifuge tube by supernatant liquor, adds 0.6 times of volume Virahol, and room temperature is placed 15min.Centrifugal (desk centrifuge, 12,000r/min, 10min) abandons supernatant, and precipitation is placed in vacuum drier dry by 70% ice-cold washing with alcohol.Finally add 20 μ l TE dissolution precipitations, plasmid solution is 4 ℃ or-20 ℃ of preservations.
Result: the middle interstitial granules that extraction is obtained carries out PCR and restriction enzyme digestion and electrophoresis is identified, result is as Fig. 2.100 times of the plasmid of take dilutions are template pcr amplification, obtain setting out bacterium SIPI-1482 as the identical band of template, about 2kb with take; EcoRI single endonuclease digestion obtains the wall scroll band of about 9.2kb, and BamHI+EcoRI double digestion has obtained the Insert Fragment band of about 1.75kb and the carrier strap of about 9.6kb.Enzyme is cut result and is conformed to theory, proves that plasmid is correct, called after pYG255-1.
3.dauW blocks the structure of plasmid completely
Bacterial classification: bacillus coli DH 5 alpha, purchased from Shanghai Sheng Gong Bioisystech Co., Ltd.
Plasmid: pYG255-1, pYG813 (still jade-like stone .2006. daunorubicin produces bacterium dnmV gene disruption mutant strain and function replacement research. Shanghai Institute of Pharmaceutical Industry's Ph D dissertation .), complete apramycin resistance gene contained.
Method: the same.
EcoRI+ClaI double digestion pYG813 obtains complete apr gene fragment, sheet segment DNA apr (about 1.5kb) is reclaimed in rubber tapping, ClaI+Hind III double digestion PCR reclaims the dnrXdpsY fragment obtaining, interstitial granules PYG255-1 in EcoRI+Hind III double digestion, these three fragments are connected, transform.Fig. 4 is shown in by the structure schematic diagram of plasmid pYG255.
Result: the plasmid that extraction is obtained carries out PCR and restriction enzyme digestion and electrophoresis is identified, result is as Fig. 3.100 times of the plasmid of take dilutions are template pcr amplification dnrXdpsY gene, obtain setting out bacterium SIPI-1482 as the identical band of template with take, and about 2.0kb, conforms to theory.BglII+EcoRI double digestion has obtained the big or small fragment that is respectively 2.0kb and 12.9kb, and (seeing Fig. 3) conforms to theory.Proof plasmid is correct, called after pYG255.
4.dauW blocks plasmid conjugal transfer completely to streptomycete SIPI-1482
Bacterial classification: intestinal bacteria ET12567 (reference MacNeil DJ, et al, Gene, 1992,111:61), streptomyces coeruleorubidus SIPI-1482 (daunorubicin produces bacterium).
Plasmid: pYG255.
Substratum: MS (Kieser T, Bibb M.Practical Streptomyces Genetics[J] .Norwich:The John Innes Foundation, 2000.).
To verify above correct pYG255 plasmid, transform intestinal bacteria ET12567 (pUZ8002), on LB+Apr flat board, screen to obtain transformant ET12567/pYG255.ET12567/pYG255 is inoculated in to 2ml LB liquid nutrient medium (containing paraxin 34 μ g/ml, kantlex 25 μ g/ml), and 37 ℃ of shaking culture are spent the night.1: the 100 fresh LB of inoculation (containing paraxin 34 μ g/ml, kantlex 25 μ g/ml) 20ml, is cultured to OD value 0.4～0.6 for good.With the centrifugal supernatant (Hitachi CR21G, rotor R 20A2,10,000r/min, 10min) that goes of 50ml centrifuge tube, the fresh LB washed cell twice of 10ml, finally use 0.1 times of volume LB substratum (2ml) to suspend.Scraping streptomycete spore prepares approximately 10
8/ ml spore suspension, centrifuged deposit is used 2 * YT substratum instead and is suspended, and gets 500 μ l heat shock ten minutes in 50 ℃ of water-baths.Get 500 μ l and be added in the spore suspension after 500 μ l heat shocks containing the ET12567 (pUZ8002) of recombinant plasmid, mix and jog.The most of supernatant of centrifugal removal, MS is dull and stereotyped for the coating of suspension residual liquid, is inverted for 28 ℃ and cultivates.The aqueous solution of the nalidixic acid that contains 0.5mg at dull and stereotyped upper berth 1ml after 16～20h and 50 μ g apramycins, after liquid is absorbed, continues at 28 ℃ and is inverted cultivation.Picking zygote list bacterium colony is enrichment culture on fresh MS+Apr+ nalidixic acid flat board.
The screening of the complete blocked mutant of 5.dauW and checking
Method:
Choosing the zygote that comparatively fast grows spore carries out temperature-induced screening and goes down to posterity: collect spore, prepare spore suspension, with approximately 100 spores of every culture dish, coat G1+Apr flat board (No. 1 substratum of Gao Shi, the apramycin that this flat board contains 50 μ g/ml substratum), cultivate 48～72h for 28 ℃, after observation has small colonies to grow, go to 39 ℃ and carry out temperature-induced.Within approximately 9 days, find afterwards to have grown a large amount of spores under 39 ℃ of cultivations.Because plasmid pYG255 contains responsive to temperature type replicon, can not be independently duplicated higher than 34 ℃, therefore the bacterium colony of 39 ℃ of growths should be after plasmid enters thalline by being integrated into karyomit(e) with the exchange of homology arm, follow chromosomally copy, transcript and expression and make this regeneration bacterium colony there is antibiotics resistance.
The transformant SIPI-1482-pYG255 (1) that the generation single cross obtaining after temperature control induction is changed continues to go down to posterity, and makes it that double exchange further occur, and its spore of scraping is in the dull and stereotyped 28 ℃ of cultivations of going down to posterity of fresh G1+Apr.Reaching rear picking list bacterium colony coating of F6 generation G1+Apr dull and stereotyped, there is the bacterial strain SIPI-1482-pYG255 of homologous recombination double exchange in screening, and the genotype of homologous recombination double exchange is shown in Fig. 5.
DauW for our desired acquisition replaces blocked mutant completely, the single cross of take is in theory changed mutant strain SIPI-1482-pYG255 (1) genome as can the increase Apr band of 533bp of template, and fragment and the Apr containing complete dauW of 1.9kb have replaced the 1.7kb fragment after dauW gene; And take SIPI-1482-pYG255 genomic dna as the template Apr band that should increase, the dauW fragment 1.7kb having replaced with Apr, obtain some strain dauW blocked mutants, selecting wherein two strains extracts genomic dnas and carries out PCR the result and see Fig. 6, as seen from the figure, the PCR result of SIPI-1482-pYG255 conforms to prediction, really for having there is the bacterial strain of homologous recombination double exchange.
Primer sequence and the character of the amplification Apr gene of table 2 PCR
Primer sequence and the character of table 3 PCR checking SIPI-1482-pYG255 mutant strain
6.dauW the fermentation analysis of blocked mutant SIPI-1482-pYG255 completely
Bacterial classification: streptomycete SIPI-1482, the complete blocked mutant SIPI-1482-pYG255 of dauW.
Substratum: seed and fermention medium (Jiang Shichun, Bai Hua, Tao Zhengli. the research [J] that the ion implantation daunorubicin of nitrogen produces bacterium mutagenicity high-yield bacterial strain. Chinese microbiotic magazine, 2000,25 (6): 3.).
Method:
Fresh slant pore is inoculated in the 250ml triangular flask that 20ml seed culture medium is housed, at 28 ℃, 230r/min shaking table shaking culture 2d.Then cultured seed liquor is inoculated in the 250ml triangular flask that 20ml fermention medium is housed to 28 ℃, 230r/min shaking table shaking culture 5d with 10% (v/v) inoculum size.Stop, after fermentation, 1ml fermented liquid is regulated to pH to 1.2～1.5 with saturated oxalic acid solution, 50 ℃ of insulation 1h, shake several times therebetween.Add isopyknic acetone, centrifugation after extracting half an hour, gets supernatant.With HPLC methods analyst tunning, take corresponding reference substance as contrast simultaneously.Before sample introduction, sample is through inclined to one side fluorine membrane filtration.HPLC analysis condition is:
HPLC instrument: Waters 2487 Dual λ Absoubance Detector
Waters?1525Binary?HPLC?pump
Waters?717plus?Autosampler
Chromatographic column: the Symmetry C of Waters company
18(5 μ m, 4.6 * 150mm)
Column temperature: 35 ℃
Moving phase: methyl alcohol: water: acetic acid: triethylamine (56: 44: 2.5: 0.5), aperture
The inclined to one side fluorine membrane filtration of 0.22 μ m
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Sample size: 20 μ l
Result:
SIPI-1482 starting strain and mutant strain SIPI-1482-pYG255 are not fermenting containing in antibiotic substratum respectively, and HPLC detects tunning, sees Fig. 7.As seen from Figure 7, SIPI-1482-pYG255 has compared very big difference with SIPI-1482, the about 41min rhodomycinon of retention time peak can not be detected, and the output of daunorubicin increases greatly, is almost nearly 8 times of bacterium SIPI-1482 of setting out.
7. genetic stability analysis
After SIPI-1482-pYG255 is gone down to posterity, every generation slant pore is carried out to fermenting experiment.Continued to reach 3 generations, result shows that F1 to F3 is more or less the same for daunorubicin output, totally there is no significant difference, has good stability.
8.dauW blocks plasmid conjugal transfer completely to daunorubicin Producing Strain Dau27
Bacterial classification: intestinal bacteria ET12567, streptomyces coeruleorubidus Dau27 (daunorubicin Producing Strain), Ke Cong Shanghai Institute of Pharmaceutical Industry obtains.
Plasmid: pYG255.
Substratum: MS (Kieser T, Bibb M.Practical Streptomyces Genetics[J] .Norwich:The John Innes Foundation, 2000.).
Utilize intestinal bacteria-streptomycete, in conjunction with shifting, pYG-255 is proceeded to Dau27, method is with 4.
There is the mutant strain Dau27-pYG255 of homologous recombination double exchange in screening, method is with 5.
DauW for desired acquisition replaces blocked mutant completely, take Dau27-pYG255 genomic dna as the template apr band that should increase, with the approximately 1.7kb fragment after the dauW having replaced with apr, and take starting strain Dau-27 genomic dna, it is template, should increase and obtain the about 1.9kb of fragment that comprises dauW gene, the results are shown in Figure 8.As seen from the figure, the PCR result of Dau27-pYG255 conforms to prediction, really for having there is the bacterial strain of homologous recombination double exchange.
The fermentation analysis of the complete blocked mutant Dau27-pYG255 of 9.dauW
Method: with 6.
Result: Dau27 starting strain and mutant strain Dau27-pYG255 are not fermenting containing in antibiotic substratum respectively, and HPLC detects tunning, sees Fig. 9.As seen from Figure 9, dauW blocks recombinant bacterium Dau27-pYG255 completely and compares with the bacterium Dau27 that sets out, and mutant strain DNR output improves obviously, and its production peak can reach 7 times of the bacterium Dau27 that sets out.
Claims (4)
1. improve a method for the daunorubicin output of streptomyces coeruleorubidus (Streptomyces coeruleorubidus), it is characterized in that, comprise the following steps:
1) build dauW gene knockout plasmid, it contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm, the sequence of described dauW homologous recombination left arm is the sequence shown in the 208th to the 2334th of Genbank database login M73758, the sequence of described dauW homologous recombination right arm is the sequence shown in the 140th to the 2218th of Genbank database login AF048833, and described marker gene is apramycin resistance gene apr; The plasmid skeleton of described dauW gene knockout plasmid is intestinal bacteria-streptomycete shuttle plasmid pJTU870;
2) the dauW gene knockout plasmid conjugal transfer of step 1) gained is entered to streptomyces coeruleorubidus (Streptomyces coeruleorubidus) SIPI-1482, prepare the dauW gene knockout engineering bacteria of streptomyces coeruleorubidus;
3) culturing step 2) the dauW gene knockout engineering bacteria of the streptomyces coeruleorubidus of gained, separated daunorubicin from culture.
2. a method of preparing the dauW gene disruption mutant bacteria of streptomyces coeruleorubidus (Streptomyces coeruleorubidus), is characterized in that, comprises the following steps:
A) build dauW gene knockout plasmid, it contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm, the sequence of described dauW homologous recombination left arm is the sequence shown in the 208th to the 2334th of Genbank database login M73758, the sequence of described dauW homologous recombination right arm is the sequence shown in the 140th to the 2218th of Genbank database login AF048833, and described marker gene is apramycin resistance gene apr; The plasmid skeleton of described dauW gene knockout plasmid is intestinal bacteria-streptomycete shuttle plasmid pJTU870;
B), by the dauW gene knockout plasmid transformed host cell intestinal bacteria of step a) gained, obtain transformant;
C) using streptomyces coeruleorubidus (Streptomyces coeruleorubidus) SIPI-1482 as acceptor, carry out conjugal transfer with the transformant described in step b), select homologous recombination double exchange, i.e. the dauW gene disruption mutant bacteria of streptomyces coeruleorubidus.
3. method according to claim 2, is characterized in that, the acceptor described in step c) is streptomyces coeruleorubidus SIPI-1482 spore, spore is pre-sprout after as acceptor, pre-sprouting condition is 50 ℃ of heat shocks 10 minutes.
4. a dauW gene knockout plasmid, it is characterized in that, it contains dauW homologous recombination left arm, marker gene and dauW homologous recombination right arm, the sequence of described dauW homologous recombination left arm is the sequence shown in the 208th to the 2334th of Genbank database login M73758, the sequence of described dauW homologous recombination right arm is the sequence shown in the 140th to the 2218th of Genbank database login AF048833, described marker gene is apramycin resistance gene apr, the plasmid skeleton of described dauW gene knockout plasmid is intestinal bacteria-streptomycete shuttle plasmid pJTU870.
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