CN102010846B - Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof - Google Patents
Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a gene blocking mutant for streptomyces coeruleorubidus and a preparation method thereof. In the gene blocking mutant for streptomyces coeruleorubidus, a dauW gene is blocked. In the technical scheme, the dauW gene in streptomyces coeruleorubidus is blocked through homologous recombination exchange, the yield of daunorubicin of streptomyces coeruleorubidus is greatly improved after the dauW gene is blocked and has increased by five times, and the effect is much better than that of blocking other genes, e.g. dnrX, dnrH and the like, the yield of which only can be increased by three times. Therefore, the blocking of the dauW gene substantially improves the fermentation unit of daunorubicin.
Description
Technical field
The invention belongs to biological technical field, particularly gene disruption mutant bacteria of a kind of sky blue light red streptomycete and preparation method thereof.
Background technology
Daunorubicin (Daunorubicin; DNR) reaching with it is that raw material synthetic Zorubicin (Doxorubicin), pidorubicin (Epirubicin) are important clinically anthracene nucleus antineoplastic antibiotics; As the antitumor drug of a line, be mainly used in the treatment of multiple solid tumor and acute leukemia.The microbial fermentation processes direct production daunorubicins that adopt in the industry more.What the biosynthetic pathway of daunorubicin had been studied at present is comparatively clear, and draws the 26S Proteasome Structure and Function distribution plan of daunorubicin biological synthesis gene cluster in view of the above.People analyze and transform the 26S Proteasome Structure and Function of these genes widely, in the hope of further improving the output of the generation bacterium of daunorubicin.Hu He, Shang Ke, recklessly good again etc. (clone of the dauW gene of sky blue light red streptomycete SIPI-1482 and the expression [J] in intestinal bacteria. Chinese biological engineering magazine; 2007; 27 (12): 26-30.) clone has obtained one section sequence between drrB and the dnrX from the sky blue light red streptomyces gene group of domestic daunorubicin production bacterium; Should be dauW and submit GenBank (accession number EF523565) to by new unnamed gene; Through sequential analysis and comparison, and attempt its expression in intestinal bacteria, tentatively investigated the relation between dauW gene and the daunorubicin resistance.But it is on the knees of the gods how to utilize the dauW gene to improve daunorubicin output.
Summary of the invention
Therefore; The technical problem that the present invention will solve is exactly the deficiency that yields poorly to existing sky blue light red streptomycete daunorubicin; Gene disruption mutant bacteria of a kind of sky blue light red streptomycete and preparation method thereof is provided, and the daunorubicin output of the gene disruption mutant bacteria of this sky blue light red streptomycete obtains bigger raising.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: the gene disruption mutant bacteria of a kind of sky blue light red streptomycete (Streptomyces coeruleorubidus), the gene that is blocked are the dauW genes.
Among the present invention, the preferably sky blue light red streptomycete of described sky blue light red streptomycete (Streptomyces coeruleorubidus) (Streptomyces coeruleorubidus) SIPI-1482.What described dau W gene was preferable is inserted and blocks by foreign gene.Described foreign gene is intestinal bacteria-streptomycete shuttle plasmid preferably, and that better is intestinal bacteria-streptomycete shuttle plasmid pKC 1139.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: the method for the gene disruption mutant bacteria of a kind of aforesaid arbitrary sky blue light red streptomycete of preparation (Streptomyces coeruleorubidus) may further comprise the steps:
1) carrier is gone in the dauW gene clone, obtain to contain the recombinant expression vector of dauW gene;
2) will contain the recombinant expression vector transformed host cell of dauW gene, obtain transformant;
3) with sky blue light red streptomycete (Streptomyces coeruleorubidus) as acceptor, with step 2) described transformant carries out conjugal transfer, obtains zygote, i.e. the dauW gene disruption mutant bacteria of sky blue light red streptomycete.
Among the present invention, the described carrier of step 1) is intestinal bacteria-streptomycete shuttle plasmid pKC1139 preferably.Step 2) preferably intestinal bacteria of described host cell, that better is intestinal bacteria ET12567.The preferably sky blue light red streptomycete spore of the described acceptor of step 3), spore are sprouted the back in advance as acceptor, and spore is sprouted general available 25～65 ℃ in advance, and the time is 5～20 minutes, and better is 50 ℃ of heat shocks 10 minutes.The preferably sky blue light red streptomycete SIPI-1482 of described sky blue light red streptomycete.
The present invention solves the problems of the technologies described above three of the technical scheme that adopted: a kind of method for preparing daunorubicin; Comprise the genetic engineering bacterium of cultivating the sky blue light red streptomycete that produces daunorubicin; From culture, separate daunorubicin; Wherein, the genetic engineering bacterium of the sky blue light red streptomycete of described product daunorubicin is the gene disruption mutant bacteria of aforesaid arbitrary sky blue light red streptomycete.
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 following: the present invention exchanges the dauW gene of blocking in the sky blue light red streptomycete through homologous recombination, investigates its function and studies its influence to daunorubicin output.The scheme that improves the daunorubicin output of sky blue light red streptomycete through genetic engineering means has occurred a lot; The present invention is first with the dauW gene disruption in the sky blue light red streptomycete; After finding that the dauW gene is blocked; The output of the daunorubicin of sky blue light red streptomycete has obtained great raising, reaches 5 times more than, and is more far better than the barrier effect to some other gene.Such as: to dnrX, dnrH etc. all can only improve 3 times.Thus it is clear that,, increased substantially the fermentation unit of daunorubicin to the blocking-up of dauW.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 is the PCR product electrophorogram of pcr amplification dauW portion gene.M:DL2000; 1:H
2O is a template; The 2:SIPI-1482 genome is a template.
Fig. 2 is that the enzyme that reaches of plasmid pYG770 is cut checking and PCR checking.A. enzyme is cut checking.M：λDNA/HindIII+DL2000；1：pYG766/EcoRI；2：pYG766/BamHI+HindIII。The B.PCR checking.M:DL2000; 1:H
2O is a template; The 2:SIPI-1482 genome is a template; 3:pYG770 is a template.
Fig. 3 is the building process synoptic diagram of plasmid pYG770.
Fig. 4 is the homologous recombination synoptic diagram.
Fig. 5 is the PCR checking of mutant strain.M:DL2000; 1:H
2O is a template; The 2:SIPI-1482 genomic dna is a template; The 3:pYG770 plasmid is a template; The 4:770-1 genomic dna is a template; The 5:770-2 genomic dna is a template.
Fig. 6 is the HPLC figure of bacterium and mutant strain W2 tunning of setting out.A:SIPI-1482; B: mutant strain W2; C: daunorubicin standard substance.
Fig. 7 is the recon different sub is produced the daunorubicin ability between generation comparison.
Fig. 8 is the PCR checking of mutant strain.M:DL2000; 1:H
2O is a template; The 3:pYG770 plasmid is a template; The 4:W2-F5 genomic dna is a template; The 5:W2-F10 genomic dna is a template.
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 that makes an experiment, and is generally 25 ℃.
Embodiment 1dauW portion gene fragment amplification
Bacterial classification: daunorubicin produces the sky blue light red streptomycete of bacterium (Streptomyces coeruleorubidus) SIPI-1482, can obtain from Shanghai Institute of Pharmaceutical Industry.
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, TEL (Anthony Hopwood streptomycete genetic manipulation laboratory manual [M], first version, Changsha: Hunan science tech publishing house, 1988), saturated phenol/chloroform.
Method:
Extract sky blue light red streptomyces gene group DNA
Fresh slant pore is inoculated in the 250ml triangular flask that 20ml YMB liquid nutrient medium is housed the about 48h of shaking table shaking culture under 30 ℃, 230r/min.With 10% (v/v) inoculum size the aforesaid liquid seed is inserted and to be equipped with in the 250ml triangular flask of 20ml YEME liquid nutrient medium, the about 48h of shaking table shaking culture under 30 ℃, 220r/min.Nutrient solution is transferred in the centrifuge tube of 50ml, and centrifugal collection mycelium (Hitachi CR21G, rotor R 20A2,10,000r/min, 10min), through the centrifugal supernatant discarded in sterilized water washing back of proper volume.In centrifuge tube, add the TSE solution that 5ml contains the 5mg/ml N,O-Diacetylmuramidase, vortex vibrating dispersion thalline is placed on 37 ℃ of water bath heat preservation to solution becomes thickness.The Proteinase K and 250 μ l 10% (w/v) SDS that add 50 μ l20mg/ml are respectively 200 μ g/ml and 0.5% (w/v) to final concentration, in 55 ℃ of water-baths, are incubated 30min, and every therebetween separated 10min mixes once.At room temperature cooling back adds the saturated phenol/chloroform of equal-volume (TNE) extracting secondary, makes substantially free of impurities between the two-phase interface, and then with the chloroform extracting once.Add isopyknic Virahol, slowly stirring at the interface, DNA is wrapped on the dropper with the aseptic dropper that seals mouth, and with the ice-cold ethanolic soln washing of 70% (v/v), at room temperature dry.TEL solution dissolving DNA with 500 μ l.The DNA sample is divided in the Eppendorf pipe back-20 ℃ of preservations.
According to the dauW sequence (the Genbank accession number: EF523565), with a pair of primer, primer sequence and character such as the table 1 that has added HindIII, BamHI restriction enzyme site respectively of center segmented design.To amplify the fragment of 1022bp in theory.
The primer sequence and the character of table 1. amplification dauW portion gene
Sky blue light red streptomyces gene group DNA with said extracted is that template is carried out PCR, annealing temperature n=58 ℃.
The result: amplification obtains 1kb left and right sides band, sees Fig. 1, conforms to theory.
The structure of embodiment 2dauW blocking-up plasmid
Bacterial classification: bacillus coli DH 5 alpha, give birth to worker Bioisystech Co., Ltd available from Shanghai.
Plasmid: pKC1139, intestinal bacteria-streptomycete shuttle plasmid, the apramycin resistance all has selective action to intestinal bacteria and streptomycete; The streptomycete replicon is a temperature sensitive type, and temperature is higher than 34 ℃ can not carry out self-replicating (reference Bierman M, Logan R; O ' Brien K, Seno ET, Nagaraja Rao R; Schoner BE; Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia coli to Streptomyces spp.Gene, 1992,116:43-49).
Main solution and damping fluid: Solution I, Solution II, Solution III (Sa nurse 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.The DNA running gel is placed under the long-wave ultra violet lamp, cut out the gel that contains required dna fragmentation, the Eppendorf centrifuge tube of packing into and weighing in advance weighs up the weight of gel.Add the long-pending 6mol/L NaI (the 0.1g gel adds 300 μ l) of triploid, 45 ℃ of water bath heat preservation 5min.Add 5 μ l Glassmilk, in vortex mixer mixing.Place ice bath to keep 10min, and, DNA is adsorbed as far as possible by Glassmilk whenever at a distance from centrifuge tube of 1～2min vibration.Centrifugal (desk centrifuge, 13,200r/min, 5s) deposition Glassmilk, abandoning supernatant.The New Wash damping fluid (50mmol/L NaCl, 2.5mmol/L (pH7.5) EDTA, 10mmol/L (pH7.5) Tris that add the freezing preservation of 200 μ l (20 ℃); 50% (v/v) ethanol), in vortex mixer suspension Glassmilk, centrifugal (desk centrifuge; 13,200r/min, 5s) washing.This process needs triplicate, and last washing should be blotted as far as possible and remained in the pipe NewWash 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 that makes absorption is by desorb, centrifugal (desk centrifuge, 13,200r/min, 5s), with new centrifuge tube of elutriant immigration.This process needs triplicate, merges three times elutriant.Centrifugal (desk centrifuge, 13,200r/min 5s) precipitates the Glassmilk that possibly bring into, and elutriant is moved into a new centrifuge tube.
The dauW portion gene fragment that recovery obtains is used the HindIII+BamHI double digestion, be connected with the HindIII+BamHI double digestion fragment of plasmid pKC1139.
Preparation E.coli DH5 α competent cell
With dull and stereotyped single bacterium colony of fresh growth or the liquid nutrient medium of the freezing preservation of glycerine bacterium liquid inoculation 2ml LB, spend the night in 37 ℃ of shaking culture.With the inoculation of the inoculum size of 1% (v/v) the 250ml triangular flask of the liquid nutrient medium of 20ml LB is housed, in about 37 ℃ of shaking culture 1.5h, control OD
6000.3～0.5.Bacterium liquid is moved in the aseptic Beckman plastic centrifuge tube of 50ml ice bath 10min.Centrifugal (HitachiCR21G, rotor R 20A2,6,000r/min, 4 ℃, 10min, down together) the 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 the 0.1mol/L CaCl of 10ml ice precooling
2In the solution, thalline is centrifugal recovery after washing.Thalline is suspended in again the 0.1mol/L CaCl of 10ml ice precooling
2In the 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 the ice-cold 0.1mol/L CaCl of 800 μ l
2In the solution, place 4 ℃ of preservations.The competent cell that prepared fresh and 4 ℃ of refrigerators are preserved within the 48h all can directly be used for transforming.
Connect product Transformed E .coli DH5 α
Above-mentioned DNA is connected product, and (volume=10 μ l DNA=50ng) adds in the Eppendorf centrifuge tube that contains 100 μ lE.coli DH5 α competent cells of ice precooling, finger tapping centrifuge tube bottom, careful mixing, ice bath 30min.To contain centrifuge tube heat shock 90s in 42 ℃ of waters bath with thermostatic control of transfer DNA and competent cell, not shake centrifuge tube therebetween.Place ice bath to keep 5min centrifuge tube fast.Add 800 μ l LB liquid nutrient mediums, put upside down mixing, 37 ℃, 230rpm vibration incubation 1h makes cell proliferation.Transformant is coated the LB planar surface that contains apramycin (50mg/ml).After liquid is absorbed, be inverted cultivation (being no more than 16h) in 37 ℃.
The extracting plasmid
To contain in the LB liquid nutrient medium of 50 μ g apramycin/ml substratum in 2ml at single colony inoculation of grow overnight on the flat board, 37 ℃ of shaking culture are spent the night.Draw 1.5ml bacterium liquid in the Eppendorf centrifuge tube, centrifugal (desk centrifuge, 12,000r/min 1min), abandons supernatant, and vacuum blots residual liquid.To precipitate and be suspended in 100 μ l Solution I again, vibration disperses thalline on the vortex mixer, places ice bath 10min.Add the freshly prepared Solution II of 200 μ l, cover tight lid after, put upside down pipe Solution II fully contacted with thalline so that 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 with supernatant, adds 0.6 times of volume Virahol, and room temperature is placed 15min.Centrifugal (desk centrifuge, 12,000r/min 10min) abandons supernatant, and deposition is placed in the vacuum drier dry with the ice-cold washing with alcohol of 70% (v/v).Add 20 μ l TE dissolution precipitations at last, plasmid solution is 4 ℃ or-20 ℃ of preservations.
The result: the plasmid that extraction is obtained carries out PCR and restriction enzyme digestion and electrophoresis evaluation, result such as Fig. 2.With plasmid solution dilution is the template pcr amplification for 100 times, obtains and be the identical band of template with the bacterium that sets out about 1kb; The EcoRI single endonuclease digestion obtains the wall scroll band of about 7.5kb, and the BamHI+HindIII double digestion has obtained the carrier ribbon of insertion fragment band He the about 6.5kb of about 1kb.Enzyme is cut the result and is conformed to theory, proves that plasmid is correct, called after pYG770 (plasmid construction such as Fig. 3).
The conjugal transfer of embodiment 3dauW blocking-up plasmid is to sky blue light red streptomycete SIPI-1482
Bacterial classification: intestinal bacteria ET12567 (can buy, article No.: BAA-525) by the ATCC place; Sky blue light red streptomycete SIPI-1482.
Plasmid: pYG770.
Substratum: MS (Kieser T, Bibb M.Practical Streptomyces Genetics [J] .Norwich:The John Innes Foundation, 2000.).
Method:
The pYG770 plasmid that above checking is correct, transformed into escherichia coli ET12567, on the LB+Apr flat board, screen transformant ET 12567/pYG770.Transformant ET 12567/pYG770 is inoculated in 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: 100 inoculation fresh LB (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 50ml centrifuge tube centrifugal go supernatant (Hitachi CR21G, rotor R 20A2,10,000r/min, 10min), the fresh LB washed cell twice of 10ml uses 0.1 times of volume LB substratum (2ml) to suspend at last.Scrape and get sky blue light red streptomycete SIPI-1482 spore and prepare concentration about 10
8The spore suspension of individual/milliliter, centrifuged deposit are used 2 * YT [2 * YT substratum (g/L): Tryptones 16g, NaCl 5g, yeast extract 10g, pH 7.0] substratum instead and are suspended, and get 500 μ l heat shock 10 minutes in 50 ℃ of water-baths.The intestinal bacteria ET12567 that contains recombinant plasmid that gets 500 μ l is added in the spore suspension after the 500 μ l heat shocks, mixes 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.Contain the nalidixic acid of 0.5mg and the aqueous solution of 50 μ g apramycins at dull and stereotyped upper berth 1ml behind 16～20h, after liquid is absorbed, continues at 28 ℃ and be inverted cultivation.Picking zygote list bacterium colony enrichment culture on freshly prepared MS flat board, this MS flat board contain (1mg nalidixic acid and 50 μ g apramycins)/(ml substratum).
The screening and the checking of embodiment 4dauW blocked mutant
Method:
The zygote of choosing the above-mentioned gained that comparatively fast grows spore carries out temperature-induced screening and the collection spore that goes down to posterity; The preparation spore suspension; Coat the G1 flat board with about 100 spores of every petridish; This G1 flat board contains the apramycin of 50 μ g/ (ml substratum), cultivates 48～72h for 28 ℃, goes to 39 ℃ after observation has small colonies to grow and carries out temperature-induced.Find after about 9 days to have grown a large amount of spores under 39 ℃ of cultivations.Because plasmid pYG770 contains the responsive to temperature type replicon in pKC1139 source; Be higher than 34 ℃ promptly can not be independently duplicated; So at the bacterium colony of 39 ℃ of growths should be to be integrated into karyomit(e) through the exchange with homology arm after plasmid gets into thalline, follow THE REPLICATION OF CHROMOSOME, transcript and expression and make this regeneration bacterium colony have antibiotics resistance.
Select two strain dauW blocked mutants and be numbered 770-1,770-2 extracts genomic dna and carries out the PCR checking.Design primer P770+, P770-, wherein P770+ is positioned at 5 ' end of dauW gene, and P770-is positioned at the downstream of dauW portion gene on the carrier, and primer sequence and character are seen table 2.
The primer sequence and the character of table 2.PCR checking mutant strain
Homologous recombination synoptic diagram such as Fig. 4.After having only plasmid integration that single cross takes place to the genome to change, be that primer, mutant strain genome are the template pcr amplification, just can obtain in theory 1, the positive band of 383bp, and the Apr gene of about 500bp that can increase with P770+, P770-.
The result: the electrophoresis result of PCR product is seen Fig. 5, and PCR result conforms to theory, chooses mutant strain 770-1 called after W2.
The fermentation analysis of embodiment 5dauW blocked mutant W2
Bacterial classification: sky blue light red streptomycete SIPI-1482, dauW blocked mutant W2.
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 the 20ml seed culture medium is housed, 28 ℃, 230r/min shaking table shaking culture 2 days.Then cultured seed liquid is inoculated in the 250ml triangular flask that the 20ml fermention medium is housed 28 ℃, 230r/min shaking table shaking culture 5 days with 10% (v/v) inoculum size.Stop after the fermentation 1ml fermented liquid being regulated pH to 1.2～1.5,50 ℃ insulation 1 hour with saturated oxalic acid solution, shake several times therebetween.Add isopyknic acetone, supernatant is got in spinning after extracting half a hour.Can use HPLC methods analyst tunning, be contrast with corresponding reference substance simultaneously.Sample is through inclined to one side fluorine membrane filtration before the sample introduction.The HPLC analysis condition is:
HPLC appearance: Waters 2487Dual 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: acetate: triethylamine (56: 44: 2.5: 0.5), the aperture
0.22 the inclined to one side fluorine membrane filtration of μ m
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Sample size: 20 μ l
The result: sky blue light red streptomycete SIPI-1482 starting strain and mutant strain W2 ferment in not containing antibiotic substratum respectively, and HPLC detects tunning, sees Fig. 6.
Can find out that by HPLC figure W2 compares very big difference with SIPI-1482: the output of (1) mutant strain W2 daunorubicin increases greatly, almost is nearly 5 times of bacterium SIPI-1482 of setting out.This explanation destroys the dauW gene through the homologous recombination exchange, has increased substantially the fermentation unit of sky blue light red streptomycete daunorubicin.(2) W2 fermentation produce among the SIPI-1482 almost can not the about 6.6min of detected RT and 7.0min about two products, two peaks overlap to some extent, integral area is respectively 31.6%, 25.6% of a daunorubicin, this two product is a unknown product.
W2 is prepared spore liquid, and suitably the dilution coating contains the G1 flat board of 50 μ g/ (ml substratum) apramycin, picking list bacterium colony fermentation at random.Repeat the experiment of several batch fermentations, find that the daunorubicin mean yield of W2 is 192.0 ± 61.6 (n=6) μ g/ml, and the bacterial strain slant pore enriches, is pewter, the substratum color is dark red, and fermented liquid also is scarlet.These have all explained the high yield of gentle red product mycin, and inclined-plane or shake-flask culture just can know that output is very high as long as observe form.
Embodiment 6 genetic stability analyses
Method: after W2 gone down to posterity each is carried out fermenting experiment for slant pore.Continued to reach 10 generations, extract F5, F10 genomic dna respectively and carry out the PCR checking.
Result: F1 to F3 is more or less the same for daunorubicin output, does not totally have significant difference result such as table 3 with shown in Figure 7.Continued to reach 10 generations, its genotype does not change, and is as shown in Figure 8.Picking list bacterium colony carries out fermenting experiment at random, and daunorubicin output descends about 50%.
Table 3. mutant strain W2 different sub is produced the comparison of daunorubicin ability between generation
Claims (7)
1. the gene disruption mutant bacteria of a sky blue light red streptomycete (Streptomyces coeruleorubidus) SIPI-1482 is characterized in that the gene that is blocked is the dauW gene, and described dauW gene is inserted and blocks by foreign gene.
2. gene disruption mutant bacteria according to claim 1 is characterized in that, described foreign gene is intestinal bacteria-streptomycete shuttle plasmid.
3. gene disruption mutant bacteria according to claim 2 is characterized in that, described intestinal bacteria-streptomycete shuttle plasmid is pKC1139.
4. a method for preparing like the gene disruption mutant bacteria of each described sky blue light red streptomycete (Streptomyces coeruleorubidus) SIPI-1482 of claim 1～3 is characterized in that, may further comprise the steps:
1) carrier intestinal bacteria-streptomycete shuttle plasmid pKC1139 is gone in the dauW gene clone, obtain to contain the recombinant expression vector of dauW gene;
2) will contain the recombinant expression vector transformed host cell of dauW gene, obtain transformant;
3) with sky blue light red streptomycete (Streptomyces coeruleorubidus) SIPI-1482 as acceptor, with step 2) described transformant carries out conjugal transfer, obtains zygote, i.e. the dauW gene disruption mutant bacteria of sky blue light red streptomycete.
5. method according to claim 4 is characterized in that step 2) described host cell is intestinal bacteria.
6. method according to claim 4 is characterized in that, the described acceptor of step 3) is sky blue light red streptomycete spore, and spore is sprouted the back in advance as acceptor, and sprouting condition is 50 ℃ of heat shocks 10 minutes in advance.
7. method for preparing daunorubicin; Comprise the genetic engineering bacterium of cultivating the sky blue light red streptomycete that produces daunorubicin; From culture, separate daunorubicin; It is characterized in that the genetic engineering bacterium of the sky blue light red streptomycete of described product daunorubicin is the gene disruption mutant bacteria of each described sky blue light red streptomycete of claim 1～3.
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