CN103881951A - SCO6974 gene deleted streptomyces coelicolor and application thereof to yield increment of antibiotics - Google Patents
SCO6974 gene deleted streptomyces coelicolor and application thereof to yield increment of antibiotics Download PDFInfo
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- CN103881951A CN103881951A CN201410116480.8A CN201410116480A CN103881951A CN 103881951 A CN103881951 A CN 103881951A CN 201410116480 A CN201410116480 A CN 201410116480A CN 103881951 A CN103881951 A CN 103881951A
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Images
Abstract
The invention discloses SCO6974 gene deleted streptomyces coelicolor and an application thereof to yield increment of antibiotics, and provides a method for reconstructing a recombinant bacterium. The method comprises the following step of knocking out an SCO6974 gene in a streptomyces coelicolor genome to obtain a recombinant bacterium, wherein the nucleotide sequence of the SCO6974 gene is a sequence 1 as shown in a sequence table. According to experiments, an engineered strain in which the SCO6974 gene is knocked out is constructed by using a PCR (Polymerase Chain Reaction)-Targeting technology, the relation between the gene and the generation of antibiotics is detected, and the result shows that the quantities of actinorhodin (ACT) and calcium dependent antibiotics (CDA) generated by the engineered strain in which the SCO6974 gene is knocked out are higher than the quantities of ACT and CDA generated by a wild strain in which the SCO6974 gene is not knocked out, so that the gene is proved to be related to the generation of the antibiotics.
Description
Technical field
The present invention relates to biological technical field, relate in particular to SCO6974 genetically deficient streptomyces coelicolor and the application in raising microbiotic output thereof.
Background technology
Streptomycete (Streptomyces) is a kind of microorganism that has using value.So far as is known, approximately 2/3rds for medical natural antibiotics and more than totally 9000 plant tool biologically active substance and all produced by streptomycete, and they have vital role at aspects such as antitumor, antimycotic, parasiticide, immunosuppression.Streptomyces coelicolor is as the type strain of streptomycete research, and its researching value and using value are 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, under condition of different pH, cultivate, ACT can show distinct colors, contributes to research and directly observes the multiple-effect regulation and control of 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 microbiotic output thereof.
The invention provides the method that builds recombinant bacterium, comprise the steps: to knock out the SCO6974 gene in streptomyces coelicolor genome, obtain recombinant bacterium;
The nucleotides sequence of described SCO6974 gene is classified the sequence 1 in sequence table as.
In aforesaid method, described in knock out SCO6974 gene in streptomyces coelicolor genome and realize by the method for homologous recombination.
In aforesaid method, the method for described homologous recombination comprises the steps:
1) import and contain the setting out in bacterium of SCO6974 gene cosmid containing the fragment that knocks out in advance SCO6974 gene, bacterium in the middle of obtaining, extract described in the middle of the plasmid of 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 comprising the steps: the cosmid that contains SCO6974 gene is imported in intestinal bacteria E.coli BW25113/pIJ790, obtains the bacterium that sets out;
The cosmid of described SCO6974 genetically deficient passes through E.coli ET12567 (pUZ8002) conjugal transfer in streptomyces coelicolor.
In aforesaid method, described in contain the fragment that knocks out in advance SCO6974 gene nucleotides sequence classify the sequence 3 in sequence table as;
Described streptomyces coelicolor is Streptomyces coelicolor M145.
The recombinant bacterium of being 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 in the application improving in microbiotic output.
In above-mentioned application, the material of described inactivation SCO6974 gene is to contain the fragment that knocks out in advance SCO6974 gene, and the nucleotides sequence of described fragment is classified the sequence 3 in sequence table as;
Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
The application of SCO6974 gene in regulation and control microbiotic output is also the scope of protection of the invention; The nucleotides sequence of described SCO6974 gene is classified the sequence 1 in sequence table as; Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
Above-mentioned regulation and control microbiotic output is for improving microbiotic output.
The present invention also provides a kind of DNA fragmentation, and its nucleotides sequence is classified the sequence 3 in sequence table as.
Of the present invention experimental results show that, the present invention has utilized PCR-Targeting technique construction SCO6974 gene knockout engineering strain, detect itself and microbiotic and produce relation, result is compared with the wild type strain not knocking 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.
Brief description of the drawings
Fig. 1 is that SCO6974 gene knockout engineering bacteria builds sketch and PCR proof diagram
Fig. 2 is that SCO6974 gene knockout engineering bacteria actinorhodin (ACT) output improves design sketch
Fig. 3 is that SCO6974 gene knockout engineering bacteria calcium relies on microbiotic (CDA) output raising design sketch
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In following embodiment, culture medium prescription is as follows:
LB culture medium prescription:
Tryptone for microbial culture (bacto-tryptone) 10g
Yeast extract for microbial culture (bacto-yeast extract) 5g
NaCl10g
Add suitable quantity of water and dissolve, then be settled to 1000ml, solid LB substratum adds 1.2% agar
2 × YT culture medium prescription:
Tryptone for microbial culture (bacto-tryptone) 16g
Yeast extract for microbial culture (bacto-yeast extract) 10g
NaCl 5g
Add suitable quantity of water and dissolve, regulate pH to 7.0, adding distil water is to 1000ml.
MS substratum:
YEME liquid nutrient medium
Before use, add following sterilized solution:
MgCl
2·6H
2O(2.5M) 2ml
Glucose(50%) 20ml
Glycine(20%) 25ml
Note: transform the thalline of use if not preparation, can not add Glycine; Be 20% for its sucrose concentration of YEME of cultivating streptomyces ansochromogenes.
R
2yE substratum
Be dissolved in water, then be settled to 800ml, every bottle of packing 160ml also adds agar 3.6g
Before use, add following sterilized solution:
DNA substratum
NaCl 5g
Be dissolved in water, then be settled to 1000ml,
Every bottle of packing 200ml also adds Difco Nutrient Agar4.6g.
In following embodiment, the nucleotides sequence of SCO6974 gene is classified sequence 1 as, 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, knock out in advance the fragment acquisition of SCO6974 gene
(in this sequence, also comprise the recognition site FRT sequence of transfer replication initiation site oriT and FLP recombinase according to the sequence of spectinomycin resistance gene in the sequence of streptomyces coelicolor SCO6974 gene and plasmid pIJ778, the combination that oriT is convenient to recombinant plasmid is below shifted) design the primer pair of a pair of long 59nt and 58nt, formed by primer 1 and primer 2, the FRT sequence of the spectinomycin resistance gene upstream that wherein primer 1 comprises 20nt, and the sequence of one section of 39nt of SCO6974 upstream region of gene, the complementary sequence of one section of 39nt in the FRT sequence in the spectinomycin resistance gene downstream that primer 2 comprises 19nt and SCO6974 gene downstream, 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 that contains spectinomycin resistance gene, M.S., Palaniappan, N., Alhamadsheh, M.M., DiBari, J., and Reynolds, K.A. (2009) Application of a Newly Identified and Characterized18-O-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins.Applied and environmental microbiology75, 3469-3476, the public is from Institute of Microorganism, Academia Sinica) be template, utilize primer 1 and primer 2 to carry out pcr amplification.
Obtain the PCR product of 1490bp, for knocking out in advance the fragment of SCO6974 gene.
Through order-checking, the nucleotides sequence that knocks out in advance the fragment of SCO6974 gene is classified the sequence 3 in sequence table as, 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, turn the BW25113/pIJ790 into E.coli containing the cosmid of SCO6974 gene
Inoculation E.coli BW25113/pIJ790(Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.Proc Natl Acad Sci U S A100:1541-1546, the public obtains from Institute of Microorganism, Academia Sinica) 10ul in 10ml containing the liquid LB substratum of 25ug/ml paraxin, 28 DEG C of shaking culture are spent the night.Get the bacterium liquid that 100ul shakes and be inoculated in 10ml containing 20mM MgSO
4, 25ug/ml paraxin SOB substratum in, 28 DEG C, 200rpm shaking culture 3-4h to OD
600to 0.4 left and right.By thalline, at 4 DEG C, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 10ml precooling, and piping and druming mixes latter 4 DEG C, and the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 5ml precooling.Piping and druming mixes latter 4 DEG C, the centrifugal 5min of 4000rpm, outwell supernatant, add 10% glycerine of 100ul precooling, piping and druming mixes rear absorption 50ul in electric revolving cup, add 100ng to contain Cosmid::SCO6974(name of product Cosmid::SCO6974, can be purchased from The John Innes centre), mix.Electric revolving cup is positioned in electroporation apparatus, 200 Ω, 25uF, 2.5KV, electricity turns 4ms.After turning, electricity adds immediately the LB of 1ml precooling, 28 DEG C of oscillation incubation 1h.Bacterium liquid is uniformly coated on and contains 50ug/ml ammonia benzyl, on 25mg/ml paraxin LB solid medium, 28 DEG C of grow overnight, the single bacterium colony obtaining is the E.coli BW25113/pIJ790 that proceeds to SCO6974cosmid, called after E.coli BW25113/pIJ790/SCO6974cosmid.
3, the acquisition of the cosmid of SCO6974 genetically deficient
Get above-mentioned 2 E.coli BW25113/pIJ790/SCO6974cosmid bacterium liquid 100ul that obtain that shaking culture spends the night and be inoculated in 10ml and contain 50ug/ml ammonia benzyl, in the liquid LB substratum of 25mg/ml paraxin.Add the 1M L-arabinose solution of 100ul, 28 DEG C, 200rpm shaking culture 3-4h to OD600 is to 0.4 left and right.By thalline, at 4 DEG C, the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 10ml precooling, and piping and druming mixes latter 4 DEG C, and the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 5ml precooling.Piping and druming mixes latter 4 DEG C, and the centrifugal 5min of 4000rpm, outwells supernatant, adds 10% glycerine of 100ul precooling, and piping and druming mixes rear absorption 50ul in electric revolving cup, adds the PCR product (knocking out in advance the fragment of SCO6974 gene) of above-mentioned 1 preparation of 100ng, mixes.Electric revolving cup is positioned in electroporation apparatus, 200 Ω, 25uF, 2.5KV, electricity turns 4ms.After turning, electricity adds immediately the LB of 1ml precooling, 28 DEG C of oscillation incubation 1h.Bacterium liquid is all contained to 50ug/ml ammonia benzyl with being coated on, 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 the cosmid(cosmid: of SCO6974 genetically deficient: △ SCO6974); PIJ790 decomposes when temperature raises.
4, the structure of SCO6974 genetically deficient streptomyces coelicolor
Cosmid(cosmid: by SCO6974 genetically deficient: △ SCO6974) by E.coli ET12567/pUZ8002(Sioud, S., Aigle, B., Karray-Rebai, I., Smaoui, S., Bejar, S., and Mellouli, L. (2009) Integrative gene cloning and expression system for Streptomyces sp.US24and Streptomyces sp.TN58bioactive molecule producing strains.Journal of biomedicine & 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., and Tan, H.R. (2013) Disruption of rimP-SC, encoding a ribosome assembly cofactor, markedly enhances the production of several antibiotics in Streptomyces coelicolor.Microb Cell Fact12, the public obtains from Institute of Microorganism, Academia Sinica), obtain SCO6974 genetically deficient streptomyces coelicolor SCO6974DM, concrete structure is as follows:
The cosmid of 3ul SCO6974 genetically deficient is joined in E.coli ET12567 (pUZ8002) competent cell, place 30min on ice, after 42 DEG C of heat shock 60s, place immediately ice bath 2min on ice, add 500ul LB liquid nutrient medium, 37 DEG C of oscillation incubation 1h.Getting the thalline that 100ul hatches is coated on the LB solid medium that contains 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 10ml LB liquid nutrient medium test tube that contains 100ug/ml spectinomycin, 100ug/ml kantlex, 25ug/ml paraxin, 15ug/ml tetracycline, and 37 DEG C have been cultured to obvious vaporific generation.The centrifugal collection thalline of 3000rpm, with equal-volume LB(non-resistant) wash thalline 2～3 times, centrifugal collection thalline, supernatant discarded, with 0.5ml2 × YT suspension thalline, obtains the Bacillus coli cells of the cosmid that contains 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 are cooled to room temperature.
By the Bacillus coli cells of the cosmid that contains SCO6974 genetically deficient and streptomycete spore, simply mix, 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 growing 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, and being specially SCO6974 Gene Replacement is spectinomycin gene.
Two, SCO6974 gene knockout engineering bacteria qualification
At SCO6974 gene indoor design primer 3 and primer 4, at SCO6974 gene two ends design primer 5 and primer 6, the genome extracting is carried out to PCR checking.With the inner primer 3 of gene and primer 4, in mutant strain, can not amplify product, 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'
Extract the genomic dna of SCO6974DM as template, use respectively primer 3 and primer 4 to carry out pcr amplification as primer pair (PCR1), primer 5 and primer 6 as primer pair (PCR2).With Streptomyces coelicolor M145 (WT) in contrast, NC is not for adding any template in contrast.
Result as shown in Figure 1B, can find out, DM carries out pcr amplification with primer 3 and primer 4, there is no amplified production, and carries out pcr amplification with primer 5 and primer 6, obtains the product of 1.6kb;
WT carries out pcr amplification with primer 3 and primer 4, obtains 341bp amplified production, and carries out pcr amplification with primer 5 and primer 6, obtains the product of 2.2kb.
Three, complementary strain construction
All contain XbaI enzyme cutting site at SCO6974 gene two ends design primer 7/ primer 8(5 ' end).
Taking Streptomyces coelicolor M145 genomic dna as template, under high-fidelity enzyme (KOD-FX) effect, carry out pcr amplification with primer 7 and primer 8, obtain the PCR product (SCO6974 gene, nucleotides sequence is classified the sequence 1 in sequence table as) of 815bp.
The PCR product of 815bp is inserted in pEASY-Blunt carrier, obtain intermediate carrier
pEASY-Blunt-SCO6974。
To pEASY-Blunt-SCO6974 XbaI enzyme cutting, the endonuclease bamhi obtaining and the carrier pSET152::rrnFp(Pan cutting through same enzyme, Y.Y., Lu, C., Dong, H.L., Yu, L.J., Liu, G., and Tan, H.R. (2013) Disruption of rimP-SC, encoding a ribosome assembly cofactor, markedly enhances the production of several antibiotics in Streptomyces coelicolor.Microb Cell Fact12, the public is from Institute of Microorganism, Academia Sinica) connect, obtain recombinant vectors.
Recombinant vectors is sent to order-checking, and this recombinant vectors of result is the carrier 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.
The SCO6974DM of recombinant vectors pSET152-rrnf-SCO6974 and above-mentioned two preparations is carried out to conjugal transfer, pSET152-rrnf contains sulfuric acid aburamycin resistant gene, therefore can verify 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, result obtains the positive bacterial strain of 815bp, is SCO6974 gene complementation bacterial strain called after SCO6974CM.
Primer 7:TCTAGAGCAAGGGAGGTACGTCCAT
Primer 8:TCTAGATCAGTTCCTGACCAGCAGTTG.
Embodiment 2, SCO6974 gene knockout engineering bacteria DM are in the application of producing in microbiotic
1, the mensuration of the actinorhodin ACT output of SCO6974 gene knockout engineering bacteria DM
Obtain spore with dull and stereotyped cultivation of MS, at the dull and stereotyped upper berth of R2YE glassine paper, inoculate respectively 1 × 10
7m145 wild type strain, SCO6974DM and the SCO6974CM of individual spore.28 DEG C of cultivations, respectively at 2d, 3d, 4d, 5d, 6d, when 7d gets off the streptomycete microorganism collection on glassine paper, and every kind of bacterial strain of each time point is collected 6 dull and stereotyped thalline.Wherein be collected in respectively and taken in the weight 1.5ml centrifuge tube of (being called beginning amount) with 3 dull and stereotyped thalline, then in 37 DEG C of baking ovens, dry 48h, make its complete drying, then take weight (being called whole amount), deduct with measuring eventually the dry weight (in grams) that begins to measure thalline.First other 3 dull and stereotyped thalline are collected in 1.5ml centrifuge tube, then add the KOH solution of 1.5ml1Mol/L, and fully vibration, places 2 hours, and centrifugal 3 minutes of 12000rpm gets supernatant spectrophotometer and measures 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 weights, unit is mMol/g.
Result as shown in Figure 2,
M145 is at 2d, 3d, and 4d, 5d, 6d, the ACT output of 7d is 0.01574mMol/g, 0.06033mMol/g, 0.05982mMol/g, 0.06478mMol/g, 0.06199mMol/g, 0.06161mMol/g.
SCO6974DM is at 2d, 3d, and 4d, 5d, 6d, the ACT output of 7d is 0.02477mMol/g, 0.08632mMol/g, 0.08034mMol/g, 0.086mMol/g, 0.09037mMol/g, 0.08581mMol/g.
SCO6974CM is at 2d, 3d, and 4d, 5d, 6d, the ACT output of 7d is 0.00139mMol/g, 0.06169mMol/g, 0.06049mMol/g, 0.06626mMol/g, 0.07065mMol/g, 0.06532mMol/g.
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
Plain substratum DNA flat board is produced in preparation, inoculates respectively 1 × 10
7m145 wild type strain (WT) and the SCO6974DM of individual spore, cultivate 7 days, collects one flat plate every kind of every day, be positioned over-70 DEG C of refrigerator and cooled and freeze preservation, after 7 days collect completely, flat board is taken out from-70 DEG C of refrigerators, thaw, the nutrient solution after thawing is mixed, draw 1ml for subsequent use.Preparation LB solid medium (agar concentration is 0.8%) and interpolation final concentration are 1.2mM CaNO
3lB solid medium (agar concentration is 0.8%).Medium sterilization, is cooled to 50 DEG C of inoculation streptococcus aureuses (CGMCC No.0910) after heating and melting.On the flat board that is connected to streptococcus aureus, punch, in hole, add the nutrient solution 50ul of collection, just putting 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, there is no the generation (when 3d-5d, minimum inhibition zone is produced by ACT) of CDA; 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 this CDA that represents that mutant strain produces is obviously many than wild strain.
Show, the calcium that SCO6974 gene knockout can improve streptomycete relies on microbiotic CDA output.
Claims (10)
1. build a method for recombinant bacterium, comprise the steps: to knock out the SCO6974 gene in streptomyces coelicolor genome, obtain recombinant bacterium;
The nucleotides sequence of described SCO6974 gene is classified the sequence 1 in sequence table as.
2. method according to claim 1, is characterized in that: described in knock out SCO6974 gene in streptomyces coelicolor genome and realize 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) import and contain the setting out in bacterium of SCO6974 gene cosmid containing the fragment that knocks out in advance SCO6974 gene, bacterium in the middle of obtaining, extract described in the middle of the plasmid of 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 prepared according to the method comprising the steps: the cosmid that contains SCO6974 gene is imported in intestinal bacteria E.coli BW25113/pIJ790, obtain the bacterium that sets out;
The cosmid of described SCO6974 genetically deficient passes through E.coli ET12567 (pUZ8002) conjugal transfer in streptomyces coelicolor.
5. according to the method described in claim 3 or 4, it is characterized in that:
The described nucleotides sequence that contains the fragment that knocks out in advance SCO6974 gene is classified the sequence 3 in sequence table as;
Described streptomyces coelicolor is Streptomyces coelicolor M145.
6. the recombinant bacterium of being prepared by method described in claim 1-5.
7. the material of inactivation SCO6974 gene or recombinant bacterium claimed in claim 6 are in the application improving in microbiotic output.
8. application according to claim 7, is characterized in that: the material of described inactivation SCO6974 gene is to contain the fragment that knocks out in advance SCO6974 gene, and the nucleotides sequence of described fragment is classified the sequence 3 in sequence table as;
Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
The application of 9.SCO6974 gene in regulation and control microbiotic output, the nucleotides sequence of described SCO6974 gene is classified the sequence 1 in sequence table as; Described microbiotic is specially actinorhodin and/or calcium relies on microbiotic.
10. a DNA fragmentation, its nucleotides sequence is classified the sequence 3 in sequence table as.
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| CN109880863A (en) * | 2019-01-25 | 2019-06-14 | 南开大学 | The method for improving yield of streptomycete antibiotic as additive using nano material |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105985924A (en) * | 2015-01-27 | 2016-10-05 | 浙江震元制药有限公司 | Streptomyces chattanovgensis being subjected to gene deletion modification and application thereof |
| CN105985924B (en) * | 2015-01-27 | 2019-05-21 | 浙江震元制药有限公司 | A kind of streptomyces chatanoogensis and its application through gene delection transformation |
| CN111019965A (en) * | 2018-10-10 | 2020-04-17 | 中国科学院微生物研究所 | Engineering bacterium for genetic modification of neomycin biosynthesis gene cluster and application thereof |
| CN111019965B (en) * | 2018-10-10 | 2022-05-13 | 中国科学院微生物研究所 | Engineering bacterium for genetic modification of neomycin biosynthesis gene cluster and application thereof |
| CN109504690A (en) * | 2019-01-16 | 2019-03-22 | 河南省商业科学研究所有限责任公司 | Participate in stress, signal and the transcriptional modulatory gene of regulation streptomycete ACT yield |
| CN109504690B (en) * | 2019-01-16 | 2022-06-21 | 河南省商业科学研究所有限责任公司 | Stress, signal and transcription regulation gene involved in regulating and controlling yield of streptomycete ACT |
| CN109880863A (en) * | 2019-01-25 | 2019-06-14 | 南开大学 | The method for improving yield of streptomycete antibiotic as additive using nano material |
| CN109880863B (en) * | 2019-01-25 | 2022-07-22 | 南开大学 | Method for improving yield of streptomycete antibiotics by using nano material as additive |
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