CN103881951B - SCO6974 genetically deficient streptomyces coelicolor and the application in raising antibiotic yield thereof - Google Patents
SCO6974 genetically deficient streptomyces coelicolor and the application in raising antibiotic yield thereof Download PDFInfo
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- 241000187432 Streptomyces coelicolor Species 0.000 title claims abstract description 31
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- VTIKDEXOEJDMJP-UHFFFAOYSA-N Actinorhodine Natural products CC1OC(CC(=O)O)CC2=C1C(=O)c3c(O)c(cc(O)c3C2=O)c4cc(O)c5C(=O)C6=C(C(C)OC(CC(=O)O)C6)C(=O)c5c4O VTIKDEXOEJDMJP-UHFFFAOYSA-N 0.000 claims abstract description 25
- VTIKDEXOEJDMJP-WYUUTHIRSA-N actinorhodin Chemical compound C([C@@H](CC(O)=O)O[C@@H]1C)C(C(C2=C(O)C=3)=O)=C1C(=O)C2=C(O)C=3C(C(=C1C2=O)O)=CC(O)=C1C(=O)C1=C2[C@@H](C)O[C@H](CC(O)=O)C1 VTIKDEXOEJDMJP-WYUUTHIRSA-N 0.000 claims abstract description 25
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Abstract
SCO6974 genetically deficient streptomyces coelicolor of the present invention and the application in raising antibiotic yield thereof.The invention provides a kind of method building recombinant bacterium, comprise the steps: the SCO6974 gene knocked out in streptomyces coelicolor genome, obtain recombinant bacterium; The nucleotides sequence of described SCO6974 gene is classified as the sequence 1 in sequence table.Experiment of the present invention proves, the present invention utilizes PCR-Targeting technique construction SCO6974 gene knockout engineering strain, detect it and microbiotic produces relation, result is compared with the wild type strain do not knocked out, and the amount that SCO6974 gene knockout engineering strain produces actinorhodin (ACT) and calcium dependence microbiotic (CDA) is large.Illustrate that this gene produces relevant with microbiotic.
Description
Technical field
The present invention relates to biological technical field, particularly relate to SCO6974 genetically deficient streptomyces coelicolor and the application in raising antibiotic yield thereof.
Background technology
Streptomycete (Streptomyces) is a kind of microorganism having using value.So far as is known, about 2/3rds for medicine natural antibiotics and more than totally 9000 plant tool biologically active substance and all produced by streptomycete, they have vital role in antitumor, antimycotic, parasiticide, immunosuppression etc.The type strain that streptomyces coelicolor is studied as streptomycete, its researching value and using value high.As the type strain of microbiotic metabolic regulation research, comparatively deep to the research of streptomyces coelicolor, it can produce actinorhodin (ACT), undecylprodigiosin (RED), calcium dependence microbiotic (CDA), methylenomycin (Mmy).ACT belongs to the heterochromatic alkane quinone of the benzo (benzoisochromanequinone in polyketone, BIQ) microbiotic of class, there is anti-microbial activity, cultivate under condition of different pH, ACT can show distinct colors, contributes to the multiple-effect regulation and control studying and directly observe streptomyces coelicolor secondary metabolism process.CDA is a kind of microbiotic of lipopeptid class, has anti-microbial activity.
ACT chemical structural formula is as shown in the formula shown in 1:
Formula 1;
The chemical structural formula of CDA is as shown in Equation 2:
Formula 2.
Summary of the invention
The object of this invention is to provide SCO6974 genetically deficient streptomyces coelicolor and the application in raising antibiotic yield thereof.
The invention provides the method building recombinant bacterium, comprise the steps: the SCO6974 gene knocked out in streptomyces coelicolor genome, obtain recombinant bacterium;
The nucleotides sequence of described SCO6974 gene is classified as the sequence 1 in sequence table.
In aforesaid method, described in knock out the SCO6974 gene in streptomyces coelicolor genome and realized by the method for homologous recombination.
In aforesaid method, the method for described homologous recombination comprises the steps:
1) setting out in bacterium by importing containing the fragment knocking out SCO6974 gene in advance containing SCO6974 gene cosmid, obtaining middle bacterium, extracting the plasmid of described middle bacterium, obtain the cosmid of SCO6974 genetically deficient;
2) by the cosmid conjugal transfer of SCO6974 genetically deficient in streptomyces coelicolor, obtain the recombinant bacterium of SCO6974 genetically deficient.
In aforesaid method, described in set out bacterium according to the method preparation comprised the steps: by the cosmid importing E. coli BW25113/pIJ790 containing SCO6974 gene, obtain the bacterium that sets out;
The cosmid of described SCO6974 genetically deficient passes through E.coliET12567 (pUZ8002) conjugal transfer in streptomyces coelicolor.
In aforesaid method, the nucleotides sequence of the described fragment containing knocking out SCO6974 gene is in advance classified as the sequence 3 in sequence table;
Described streptomyces coelicolor is Streptomyces coelicolor M145.
The recombinant bacterium prepared by aforesaid method is also the scope of protection of the invention.
The material of inactivation SCO6974 gene or above-mentioned recombinant bacterium are also the scope of protection of the invention improving the application in antibiotic yield.
In above-mentioned application, the material of described inactivation SCO6974 gene is the fragment containing knocking out SCO6974 gene in advance, and the nucleotides sequence of described fragment is classified as the sequence 3 in sequence table;
Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
The application of SCO6974 gene in regulation and control antibiotic yield is also the scope of protection of the invention; The nucleotides sequence of described SCO6974 gene is classified as the sequence 1 in sequence table; Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
Above-mentioned regulation and control antibiotic yield is for improving antibiotic yield.
Present invention also offers a kind of DNA fragmentation, its nucleotides sequence is classified as the sequence 3 in sequence table.
Experiment of the present invention proves, the present invention utilizes PCR-Targeting technique construction SCO6974 gene knockout engineering strain, detect it and microbiotic produces relation, result is compared with the wild type strain do not knocked out, and the amount that SCO6974 gene knockout engineering strain produces actinorhodin (ACT) and calcium dependence microbiotic (CDA) is large.Illustrate that this gene produces relevant with microbiotic.
Accompanying drawing explanation
Fig. 1 is that SCO6974 gene knockout engineering bacteria builds sketch and PCR proof diagram
Fig. 2 is SCO6974 gene knockout engineering bacteria actinorhodin (ACT) output increased design sketch
Fig. 3 is that SCO6974 gene knockout engineering bacteria calcium relies on microbiotic (CDA) output increased design sketch
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, culture medium prescription is as follows:
LB culture medium prescription:
Microbial culture tryptone (bacto-tryptone) 10g
Bacto-yeast extract (bacto-yeastextract) 5g
NaCl10g
Add suitable quantity of water to dissolve, then be settled to 1000ml, solid LB media adds the agar of 1.2%
2 × YT culture medium prescription:
Microbial culture tryptone (bacto-tryptone) 16g
Bacto-yeast extract (bacto-yeastextract) 10g
NaCl5g
Add suitable quantity of water to dissolve, regulate pH to 7.0, adding distil water is to 1000ml.
MS substratum:
YEME liquid nutrient medium
Following sterilized solution is added before using:
MgCl
2·6H
2O(2.5M)2ml
Glucose(50%)20ml
Glycine(20%)25ml
Note: the thalline transformed if not preparation, can not add Glycine; Be 20% for cultivating its sucrose concentration of YEME of streptomyces ansochromogenes.
R
2yE substratum
Be dissolved in water, then be settled to 800ml, every bottle of packing 160ml also adds agar 3.6g
Following sterilized solution is added before using:
DNA substratum
NaCl5g
Be dissolved in water, then be settled to 1000ml,
Every bottle of packing 200ml also adds DifcoNutrientAgar4.6g.
In following embodiment, the nucleotides sequence of SCO6974 gene is classified as sequence 1, and the aminoacid sequence of the albumen of its coding is sequence 2.
Embodiment 1, utilize PCR-Targeting technique construction SCO6974 gene knockout engineering bacteria
One, the structure of SCO6974 gene knockout engineering bacteria
1, the fragment knocking out SCO6974 gene in advance obtains
(the recognition site FRT sequence of transfer replication initiation site oriT and FLP recombinase in this sequence, is also comprised according to the sequence of spectinomycin resistance gene in the sequence of streptomyces coelicolor SCO6974 gene and plasmid pIJ778, oriT is convenient to the Conjugative tiansfer of recombinant plasmid below) design the primer pair of long a pair 59nt and 58nt, be made up of primer 1 and primer 2, wherein primer 1 comprises the FRT sequence of the spectinomycin resistance gene upstream of 20nt, and the sequence of one of SCO6974 upstream region of gene section of 39nt, primer 2 comprises the complementary sequence of the FRT sequence in the spectinomycin resistance gene downstream of 19nt and one section of 39nt of SCO6974 downstream of gene, two primer sequences are as follows:
Primer 1
5'CACCCAGTCAGTCCCCTACGCAAGGGAGGTACGTCCATGATTCCGGGGATCCGT CGACC3'(sequence 1)
Primer 2:
5'CGGACGCGGTCCACCGGGTCCGGCGCGGTGAACCGCTCATGTAGGCTGGAGCTG CTTC3'(sequence 2)
With the plasmid pIJ778(Ghatge containing spectinomycin resistance gene, M.S., Palaniappan, N., Alhamadsheh, M.M., DiBari, J., andReynolds, K.A. (2009) ApplicationofaNewlyIdentifiedandCharacterized18-O-Acyltr ansferaseinChemoenzymaticSynthesisofSelectedNaturalandNo nnaturalBioactiveDerivativesofPhoslactomycins.Appliedand environmentalmicrobiology75, 3469-3476, the public is from Institute of Microorganism, Academia Sinica) be template, primer 1 and primer 2 is utilized to carry out pcr amplification.
Obtain the PCR primer of 1490bp, for knocking out the fragment of SCO6974 gene in advance.
Through order-checking, the nucleotides sequence knocking out the fragment of SCO6974 gene is in advance classified as the sequence 3 in sequence table, it is containing spectinomycin resistance gene (sequence 3 is from 5 ' end 139-931 position Nucleotide), and two ends are respectively with the homologous fragment of the SCO6974 gene upstream and downstream of 39nt.
2, the cosmid containing SCO6974 gene turns into E.coliBW25113/pIJ790
Inoculation E.coliBW25113/pIJ790(GustB, ChallisGL, FowlerK, KieserT, ChaterKF (2003) PCRtargetedStreptomycesgenereplacementidentifiesaprotein domainneededforbiosynthesisofthesesquiterpenesoilodorgeo smin.ProcNatlAcadSciUSA100:1541-1546, the public obtains from Institute of Microorganism, Academia Sinica) 10ul is in 10ml containing the LB liquid medium of 25ug/ml paraxin, and 28 DEG C of shaking culture are spent the night.Get the bacterium liquid that 100ul shakes and be inoculated in 10ml containing 20mMMgSO
4, 25ug/ml paraxin SOB substratum in, 28 DEG C, 200rpm shaking culture 3-4h to OD
600to about 0.4.By thalline at 4 DEG C, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 10ml precooling, and latter 4 DEG C of piping and druming mixing, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 5ml precooling.Latter 4 DEG C of piping and druming mixing, the centrifugal 5min of 4000rpm, outwells supernatant, add 10% glycerine of 100ul precooling, draw 50ul after piping and druming mixing in electric revolving cup, add 100ng and contain Cosmid::SCO6974(name of product Cosmid::SCO6974, can purchased from TheJohnInnescentre), mixing.Be positioned in electroporation apparatus by electric revolving cup, 200 Ω, 25uF, 2.5KV, electricity turns 4ms.Electricity adds the LB of 1ml precooling immediately after turning, 28 DEG C of oscillation incubation 1h.Bacterium liquid is uniformly coated on containing 50ug/ml ammonia benzyl, on 25mg/ml paraxin LB solid medium, 28 DEG C of grow overnight, the single bacterium colony obtained is the E.coliBW25113/pIJ790 proceeding to SCO6974cosmid, called after E.coliBW25113/pIJ790/SCO6974cosmid.
3, the acquisition of the cosmid of SCO6974 genetically deficient
Get above-mentioned 2 E.coliBW25113/pIJ790/SCO6974cosmid bacterium liquid 100ul obtained that shaking culture spends the night to be inoculated in 10ml and to contain 50ug/ml ammonia benzyl, in the LB liquid medium of 25mg/ml paraxin.Add the 1ML-pectinose solution of 100ul, 28 DEG C, 3-4h to OD600 is to about 0.4 for 200rpm shaking culture.By thalline at 4 DEG C, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 10ml precooling, and latter 4 DEG C of piping and druming mixing, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 5ml precooling.Latter 4 DEG C of piping and druming mixing, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 100ul precooling, draws 50ul after piping and druming mixing in electric revolving cup, adds the PCR primer (knocking out the fragment of SCO6974 gene in advance) of above-mentioned 1 preparation of 100ng, mixing.Be positioned in electroporation apparatus by electric revolving cup, 200 Ω, 25uF, 2.5KV, electricity turns 4ms.Electricity adds the LB of 1ml precooling immediately after turning, 28 DEG C of oscillation incubation 1h.By bacterium liquid all be coated on containing 50ug/ml ammonia benzyl, on 25mg/ml spectinomycin LB solid medium, 37 DEG C of grow overnight.
Picking growth single bacterium colony, extract plasmid with the little extraction reagent kit of plasmid (TIANGEN), be cosmid(cosmid:: △ SCO6974 of SCO6974 genetically deficient); PIJ790 decomposes when temperature raises.
4, the structure of SCO6974 genetically deficient streptomyces coelicolor
Cosmid(cosmid:: the △ SCO6974 by SCO6974 genetically deficient) pass through E.coliET12567/pUZ8002(Sioud, S., Aigle, B., Karray-Rebai, I., Smaoui, S., Bejar, S., andMellouli, L. (2009) IntegrativegenecloningandexpressionsystemforStreptomyces sp.US24andStreptomycessp.TN58bioactivemoleculeproducings trains.Journalofbiomedicine & biotechnology2009, 464986, the public obtains from Institute of Microorganism, Academia Sinica) conjugal transfer enters Streptomyces coelicolor M145 (Pan, Y.Y., Lu, C., Dong, H.L., Yu, L.J., Liu, G., andTan, H.R. (2013) DisruptionofrimP-SC, encodingaribosomeassemblycofactor, markedlyenhancestheproductionofseveralantibioticsinStrep tomycescoelicolor.MicrobCellFact12, the public obtains from Institute of Microorganism, Academia Sinica), obtain SCO6974 genetically deficient streptomyces coelicolor SCO6974DM, concrete structure is as follows:
The cosmid of 3ulSCO6974 genetically deficient is joined in E.coliET12567 (pUZ8002) competent cell, place 30min on ice, place ice bath 2min on ice after 42 DEG C of heat shock 60s immediately, add 500ulLB liquid nutrient medium, 37 DEG C of oscillation incubation 1h.Getting the thalline that 100ul hatches is coated on the LB solid medium containing 100ug/ml spectinomycin, 100ug/ml kantlex, 25ug/ml paraxin, 15ug/ml tetracycline, be inverted overnight incubation for 37 DEG C, picking list bacterium colony is in the 10mlLB liquid nutrient medium test tube containing 100ug/ml spectinomycin, 100ug/ml kantlex, 25ug/ml paraxin, 15ug/ml tetracycline, and 37 DEG C have been cultured to obvious vaporific generation.3000rpm collected by centrifugation thalline, with equal-volume LB(non-resistant) wash thalline 2 ~ 3 times, collected by centrifugation thalline, supernatant discarded, with 0.5ml2 × YT suspension thalline, obtain the Bacillus coli cells of the cosmid containing SCO6974 genetically deficient, for subsequent use.
By Streptomyces coelicolor M145 streptomycete spore, be suspended in 0.5ml2 × YT liquid, 50 DEG C of water-bath effects 10 minutes, take out and be cooled to room temperature.
By the Bacillus coli cells of the cosmid containing SCO6974 genetically deficient and streptomycete spore, simple mixing, be applied to dull and stereotyped 28 DEG C of MS and cultivate 16-18 hour, wait coating 30ul30mg/ml nalidixic acid, 15ul200mg/ml spectinomycin, cultivate 3 days for 28 DEG C, the single bacterium colony grown is SCO6974 gene knockout engineering bacteria (SCO6974 gene is by spectinomycin gene substitution), called after SCO6974DM.
Through order-checking, SCO6974DM is the SCO6974 gene in disappearance Streptomyces coelicolor M145 genome, is specially and SCO6974 gene is replaced with spectinomycin gene.
Two, SCO6974 gene knockout engineering bacteria qualification
At SCO6974 gene internal design primer 3 and primer 4, at SCO6974 gene two ends design primer 5 and primer 6, PCR checking is carried out to the genome extracted.With gene internal primer 3 and primer 4, can not amplify product in mutant strain, in the middle of wild strain, theoretical size is 341bp; Gene two ends primer 5 and primer 6 theoretical size in mutant strain is 2.2kb, and in wild strain, theoretical size is 1.6kb(Figure 1A).
Primer 3:5'GCTGGTCGACAAGGGGCT3'
Primer 4:5'CGCGCATCATCCGGTACA3'
Primer 5:5'CTGATGAACCCGACCGAGAT3'
Primer 6:5'CGGTCTCGGTGTGTCCCC3'
The genomic dna extracting SCO6974DM, as template, uses primer 3 and primer 4 to carry out pcr amplification as primer pair (PCR1), primer 5 and primer 6 as primer pair (PCR2) respectively.With Streptomyces coelicolor M145 (WT) in contrast, NC is not for add any template in contrast.
Result as shown in Figure 1B, can be found out, DM primer 3 and primer 4 carry out pcr amplification, do not have amplified production, and carry out pcr amplification with primer 5 and primer 6, obtain the product of 1.6kb;
WT primer 3 and primer 4 carry out pcr amplification, obtain 341bp amplified production, and carry out pcr amplification with primer 5 and primer 6, obtain the product of 2.2kb.
Three, complemented strain builds
XbaI enzyme cutting site is all contained) at SCO6974 gene two ends design primer 7/ primer 8(5 ' end.
With Streptomyces coelicolor M145 genomic dna for template, under high-fidelity enzyme (KOD-FX) effect, carry out pcr amplification with primer 7 and primer 8, obtain the PCR primer (SCO6974 gene, nucleotides sequence is classified as the sequence 1 in sequence table) of 815bp.
The PCR primer of 815bp is inserted in pEASY-Blunt carrier, obtains intermediate carrier
pEASY-Blunt-SCO6974。
To pEASY-Blunt-SCO6974 XbaI enzyme cutting, the endonuclease bamhi obtained and the carrier pSET152::rrnFp(Pan cut through same enzyme, Y.Y., Lu, C., Dong, H.L., Yu, L.J., Liu, G., andTan, H.R. (2013) DisruptionofrimP-SC, encodingaribosomeassemblycofactor, markedlyenhancestheproductionofseveralantibioticsinStrep tomycescoelicolor.MicrobCellFact12, the public is from Institute of Microorganism, Academia Sinica) connect, obtain recombinant vectors.
Recombinant vectors is sent to order-checking, the carrier of this recombinant vectors of result for obtaining between the XbaI enzyme cutting site of the SCO6974 gene insertion vector pSET152-rrnf shown in sequence in sequence table 1, called after pSET152-rrnf-SCO6974.
Recombinant vectors pSET152-rrnf-SCO6974 and above-mentioned two SCO6974DM prepared is carried out conjugal transfer, containing sulfuric acid aburamycin resistant gene in pSET152-rrnf, therefore can be verified by sulfuric acid Ah cloth bleomycin resistance, collect resistant strain.
Extract the genomic dna of resistant strain, carry out PCR checking with primer 7/ primer 8, what result obtained 815bp is positive strain, and being SCO6974 gene complementation Strain Designation is SCO6974CM.
Primer 7:TCTAGAGCAAGGGAGGTACGTCCAT
Primer 8:TCTAGATCAGTTCCTGACCAGCAGTTG.
Embodiment 2, SCO6974 gene knockout engineering bacteria DM are producing the application in microbiotic
1, the mensuration of the actinorhodin ACT output of SCO6974 gene knockout engineering bacteria DM
Obtain spore with MS slat chain conveyor, at R2YE dull and stereotyped upper berth glassine paper, inoculate 1 × 10 respectively
7the M145 wild type strain of individual spore, SCO6974DM and SCO6974CM.28 DEG C of cultivations, get off the streptomycete microorganism collection on glassine paper when 2d, 3d, 4d, 5d, 6d, 7d respectively, and each time point is often planted bacterial strain and collected 6 dull and stereotyped thalline.Wherein be collected in the 1.5ml centrifuge tube having taken weight (being called that the beginning measures) respectively with 3 dull and stereotyped thalline, then in 37 DEG C of baking ovens, 48h is dried, make its complete drying, then take weight (being called whole amount), with measuring the dry weight (in grams) deducting and begin to measure thalline eventually.First other 3 dull and stereotyped thalline are collected in 1.5ml centrifuge tube, then add the KOH solution of 1.5ml1Mol/L, fully vibrate, place 2 hours, centrifugal 3 minutes of 12000rpm, get supernatant spectrophotometer and measure its absorption value A at 640nm place
640.The molar extinction coefficient of actinorhodin is 25320L/Mol/cm, and the method for calculation of every gram of dry weight thalline generation actinorhodin are A
640÷ 25320 × 1.5 ÷ dry weight, unit is mMol/g.
Result as shown in Figure 2,
M145 is 0.01574mMol/g, 0.06033mMol/g, 0.05982mMol/g, 0.06478mMol/g, 0.06199mMol/g, 0.06161mMol/g in the ACT output of 2d, 3d, 4d, 5d, 6d, 7d.
SCO6974DM is 0.02477mMol/g, 0.08632mMol/g, 0.08034mMol/g, 0.086mMol/g, 0.09037mMol/g, 0.08581mMol/g in the ACT output of 2d, 3d, 4d, 5d, 6d, 7d.
SCO6974CM is 0.00139mMol/g, 0.06169mMol/g, 0.06049mMol/g, 0.06626mMol/g, 0.07065mMol/g, 0.06532mMol/g in the ACT output of 2d, 3d, 4d, 5d, 6d, 7d.
Show, SCO6974 gene knockout can improve the actinorhodin ACT output of streptomycete.
2, the calcium of SCO6974 gene knockout engineering bacteria relies on the mensuration of microbiotic CDA output
It is dull and stereotyped that plain substratum DNA is produced in preparation, inoculates 1 × 10 respectively
7the M145 wild type strain (WT) of individual spore and SCO6974DM, cultivate 7 days, often kind of every day collects one flat plate, be positioned over-70 DEG C of refrigerator and cooled and freeze preservation, collected completely until 7 days, flat board is taken out from-70 DEG C of refrigerators, thaw, the nutrient solution after thawing is mixed, draws 1ml for subsequent use.Preparation LB solid medium (agar concentration is 0.8%) and interpolation final concentration are 1.2mMCaNO
3lB solid medium (agar concentration is 0.8%).Medium sterilization, is cooled to 50 DEG C of inoculations streptococcus aureus (CGMCCNo.0910) after heating and melting.The flat board being connected to streptococcus aureus punches, in hole, adds the nutrient solution 50ul of collection, just put overnight incubation for 37 DEG C.
Result as shown in Figure 3, when not adding CaNO in substratum
3(Ca
-) time, wild strain and mutant strain do not have the generation of inhibition zone substantially, namely do not have the generation of CDA (inhibition zone minimum during 3d-5d is produced by ACT); When adding CaNO in substratum
3(Ca afterwards
+), wild strain and mutant strain have the generation of inhibition zone, and obviously large than wild strain of the inhibition zone of mutant strain, and it is obviously many than wild strain that this represents CDA that mutant strain produces.
Show, the calcium that SCO6974 gene knockout can improve streptomycete relies on microbiotic CDA output.
Claims (9)
1. build a method for recombinant bacterium, comprise the steps: the SCO6974 gene knocked out in streptomyces coelicolor genome, obtain recombinant bacterium;
The nucleotides sequence of described SCO6974 gene is classified as the sequence 1 in sequence table.
2. method according to claim 1, is characterized in that: described in knock out the SCO6974 gene in streptomyces coelicolor genome and realized by the method for homologous recombination.
3. method according to claim 2, is characterized in that: the method for described homologous recombination comprises the steps:
1) setting out in bacterium by importing containing the fragment knocking out SCO6974 gene in advance containing SCO6974 gene cosmid, obtaining middle bacterium, extracting the plasmid of described middle bacterium, obtain the cosmid of SCO6974 genetically deficient;
2) by the cosmid conjugal transfer of SCO6974 genetically deficient in streptomyces coelicolor, obtain the recombinant bacterium of SCO6974 genetically deficient.
4. method according to claim 3, is characterized in that:
The described bacterium that sets out is according to the method preparation comprised the steps: imported in E. coli BW25113/pIJ790 by the cosmid containing SCO6974 gene, obtain the bacterium that sets out;
The cosmid of described SCO6974 genetically deficient passes through E.coliET12567 conjugal transfer in streptomyces coelicolor.
5. the method according to claim 3 or 4, is characterized in that:
The nucleotides sequence of the described fragment containing knocking out SCO6974 gene is in advance classified as the sequence 3 in sequence table;
Described streptomyces coelicolor is Streptomyces coelicolor M145.
6. the recombinant bacterium prepared by method described in claim 1-5.
7. the streptomyces coelicolor of inactivation SCO6974 gene or recombinant bacterium according to claim 6 are improving the application in antibiotic yield; The material of described inactivation SCO6974 gene is the fragment containing knocking out SCO6974 gene in advance, and the nucleotides sequence of described fragment is classified as the sequence 3 in sequence table;
Described microbiotic is that actinorhodin and/or calcium rely on microbiotic.
8. the application of inactivation SCO6974 gene in regulation and control streptomyces coelicolor antibiotic yield, the nucleotides sequence of described SCO6974 gene is classified as the sequence 1 in sequence table; Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
9. a DNA fragmentation, its nucleotides sequence is classified as the sequence 3 in sequence table.
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