CN101392229A - Engineering strain for directly producing gernebcin and use thereof - Google Patents
Engineering strain for directly producing gernebcin and use thereof Download PDFInfo
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- CN101392229A CN101392229A CNA2008100114721A CN200810011472A CN101392229A CN 101392229 A CN101392229 A CN 101392229A CN A2008100114721 A CNA2008100114721 A CN A2008100114721A CN 200810011472 A CN200810011472 A CN 200810011472A CN 101392229 A CN101392229 A CN 101392229A
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- tobramycin
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Abstract
The invention pertains to the field of medical technology and relates to engineering bacteria directly producing tobramycin and the application thereof, which mainly damages carboxamide transferase gene in bacteria produced by the tobramycin, therefore the strain does not produce carboxamide tobramycin any more but tobramycin. The invention, by the method of deleting inactivation within the frame, breaks tacA gene in Streptomyces tenebrarius, comprising the composition of the tacA gene breaking plasmid, the breaking of the transformation of plasmid pSPU303 into Streptomyces tenebrarius H6, the screening of double exchange strains, the detection of fermentation products and the identification of new compositions. The method of deleting inactivation within the frame respectively provides two segments with the upper reach molecular weight and the lower reach molecular weight of the tacA gene not less than 500bp for the PCR, both segments are connected to pIJ2925 simultaneously, one end of the segment is connected to resistance gene of antibiotics that is expressible in the Streptomyces tenebrarius such as erythromycin resistance gene ermE, and three gene segments are connected to shuttle vector pHZ132 by using Bg1II Enzyme cutting. The engineering bacteria directly producing tobramycin and the application thereof can simplify the production technology and reduce the production cost, thus facilitating the quality control of the products.
Description
Technical field
The invention belongs to the microbiotic pharmacy field, relate to a kind of engineering bacteria and application thereof of direct generation tobramycin, specifically, the present invention utilizes the molecular biology operative technique, blocking-up streptomyces tenebrarius (Streptomyces tenebrarius) biosynthesis gene tacA, obtain directly to produce the engineering strain of tobramycin, can in the microbiotic pharmacy, use.
Background technology
Streptomyces tenebrarius (Streptomyces tenebrarius) is that the researchist of U.S. Li Lai company in 1967 separates from soil and obtains, and this bacterial strain can produce one group of microbiotic, and wherein 2,4,5 ' is major constituent.Component 2 be apramycin (Apramycin, Am), component 4 is 6 "-O-carboxamide kanendomycin (6 "-O-Carbamoylkanamycin B, CKB), component 5 ' is 6 "-O-carboxamide tobramycin (6 "-O-Carbamoyltobramycin, CTB).Component 4,5 ' forms kanendomycin and tobramycin respectively behind pyrohydrolysis.
Tobramycin is a kind of microbiotic of broad-spectrum high efficacy, external various bacteria there is anti-microbial activity, particularly some is effective to the drug-fast pseudomonas aeruginosa of gentamicin, and the ear of tobramycin, renal toxicity are relatively low, are the antimicrobial drugs of widespread use clinically.
The scientific effort personnel mainly concentrate on aspects such as conventional selection by mutation and optimization production technology to the research of streptomyces tenebrarius and obtain some progress for many years.The streptomyces gene engineering research that rise the 1980s obtains significant progress at the aspects such as clone, output raising, component improvement and hybrid antibiotic production of microbiotic biosynthesis gene.At molecular level, Li Tianbai etc. are cloned into one section 50Kb zone, prove wherein to contain to participate in tobramycin synthetic dTDP-glucose-4,6-dehydratase.Madan Kumar Kharel etc. utilizes the method for homology hybridization to be cloned into one section 13.8Kb carboxamide tobramycin biological synthesis gene cluster, has proved the wherein gene function of tbmA, tbmB, infers the biosynthetic pathway of carboxamide tobramycin.
At present, the production method of tobramycin is to separate to obtain the carboxamide tobramycin from the streptomyces tenebrarius fermented liquid, and pyrohydrolysis obtains tobramycin under alkaline condition again.The shortcoming of this method is that impurity is many, and product yield is low, the production cost height.
Along with the development of Protocols in Molecular Biology, make that utilizing engineered method to transform the microbiotic component becomes possibility.Madan Kumar Kharel etc. is cloned into tobramycin partial synthesis gene cluster in the streptomyces tenebrarius, and Blastn finds that wherein gene TacA is a transcarbamylase, may be with 6 " the position carbamyl is synthetic relevant.
Summary of the invention
The objective of the invention is by method the tacA inactivation, acquisition can directly produce the engineering bacteria of tobramycin, this bacterial strain is registered preservation on March 20th, 2008 at China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2409.
The present invention mainly comprises following step by tacA gene in the method blocking-up streptomyces tenebrarius of deletion inactivation in the framework:
The structure of A, tacA gene disruption plasmid;
B, blocking-up plasmid transform streptomyces tenebrarius;
The screening of C, transformant;
The evaluation of D, new component tobramycin.
The method of deletion inactivation is that PCR obtains tacA gene upstream and downstream molecular weight respectively and is not less than two fragments of 500bp in the framework, be connected to both among the pIJ2925 simultaneously, be connected into the antibiotics resistance gene such as the erythromycin resistance gene ermE that in streptomyces tenebrarius, can express toward a segmental end again, utilize the BglII enzyme to cut three gene fragments are linked among the shuttle vectors pHZ132.
Wherein transform be with E.coliET12657 (pUZ8002) mediation in conjunction with transfer method.Screening is for screening erythromycin resistance (erm earlier
R) bacterial strain, after going down to posterity, no medicine therefrom screens erythromycin-sensitive (erm again
S) bacterial strain, therefrom screen the transformant that produces new component.Being accredited as ferment filtrate adopts thin-layer chromatography TLC, new component to adopt MS to analyze and HPLC-ELSD.
The present invention had not only simplified production technique, but also had reduced production cost, also was more convenient for carrying out simultaneously the quality control of product.
Bacterial strain of the present invention is registered preservation on March 20th, 2008 in China Committee for Culture Collection of Microorganisms common micro-organisms center (Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), its called after streptomyces tenebrarius (Streptomyces tenebrarius) of classifying, deposit number is CGMCC No.2409.
Description of drawings
Fig. 1 is tacA Gene Double exchange principle and restriction enzyme site synoptic diagram
Fig. 2 is that genetic engineering bacterium fermentation product components TLC analyzes
1 is the tobramycin standard substance, and 2 is streptomyces tenebrarius H6 (pSPU303-3), and 3 is wild type strain streptomyces tenebrarius H6.A is the carboxamide tobramycin, and B is a tobramycin, and C is an apramycin.
Fig. 3 is the HPLC-ELSD collection of illustrative plates of tobramycin standard substance and new component
A is the tobramycin standard substance, and B is new component
Fig. 4 is the mass spectroscopy collection of illustrative plates of new component
Embodiment
Embodiment 1:
The present invention mainly comprises following key step:
1. make up tacA gene disruption plasmid
The tobramycin biological synthesis gene cluster sequence of delivering according to Madan Kumar Kharel etc. (GenBank Accession Number AJ579650), at two pairs of primers of tacA gene upstream and downstream design, be that template is carried out two fragment PCR 1-2 of pcr amplification acquisition and PCR3-4 respectively with the total DNA of streptomyces tenebrarius.Two fragments are connected product called after Δ tacA, and it has deleted 111 bases of tacA gene 5 ' end, inner 260 bases and 88 bases of 3 ' end.Δ tacA linked among the carrier pIJ2925 obtain pSPU301.KpnI site in the pSPU301 is connected into erythromycin resistance gene ermE (pXZ1 cuts back to close about 1.6Kb size fragment through the KpnI enzyme) and obtains recombinant plasmid pSPU302 again, utilize the BglII enzyme to cut at last Δ tacB+ermE is connected to BamHI site among the carrier pHZ132, obtain blocking-up plasmid pSPU303.
2. blocking-up plasmid pSPU303 transforms streptomyces tenebrarius H6
To block plasmid pSPU303 and change among the E.coli ET12567 (pUZ8002), obtain donor bacterium E.coli ET12567 (pUZ8002, pSPU303).The method that shifts by combination transforms streptomyces tenebrarius H6 spore then, covers with the erythromycin and pyridine acid (final concentration is respectively 100 μ g/mL and the 50 μ g/mL) aqueous solution behind 28 ℃ of cultivation 20h, and 28 ℃ of continuation are cultivated and grown transformant in 5-6 days, with the erm that is obtained
RTransformant is at the R that contains erythromycin (200 μ g/mL) and PPA (50 μ g/mL)
242 ℃ of sectional streaks are cultivated on the YE substratum, and 48h grows bacterium colony because the blocking-up plasmid can not duplicate at 42 ℃, can only be incorporated on the karyomit(e) by the homology exchange after zygote could grow, show the erythromycin resistance.Single cross has taken place and has changed integration in the proof zygote.Provoke a strain called after streptomyces tenebrarius H6 (pSPU303) at random.
3. screening double exchange bacterial strain and tunning detect
With erm
RTransformant no medicine close on the V flat board pass the three generations continuously after, picking list bacterium colony dibbling respectively closes the dull and stereotyped and pastille of V to no medicine and closes V (erythromycin 100 μ g/mL) flat board, screens 32 erm of acquisition from 544 strains
SBacterial strain.Called after streptomyces tenebrarius H6 (pSPU303-1) is to H6 (pSPU303-32) respectively.These 32 bacterial strains are fermented, filter and collect fermented liquid, filtrate is by silica gel G F
254Thin-layer chromatography, biological developing find that all bacterial strains all can normally produce apramycin, but streptomyces tenebrarius H6 (pSPU303-3) no longer produces 6 "-O-carboxamide tobramycin, and produce the close new component of Rf value of a Rf value and tobramycin.
4. new component is identified
New component has following several steps in the extraction fermented liquid:
A, slightly carry
After the acidifying respectively of fermented liquid dilution back, alkalization remove albumen, with 732 resin Static Adsorption 6～8 hours.Then 3% ammoniacal liquor with 6 times of amounts carries out desorb, when effluent liquid reaches pH9.0 when above, is connected in series on 711 resin columns of suitable 732 resins, 1/5 volume, collects tree and takes off liquid, 1/8～1/10 of simmer down to original volume.
B, separation
Concentrated solution (pH7.0～7.5) is gone up the D151 resin and is carried out dynamic adsorption.After the washing respectively the ammoniacal liquor with 0.15mol/L and 0.3mol/L carry out wash-out, elution flow rate 1/100～1/150/ minute with phospho-wolframic acid control terminal point, is collected the ammoniacal liquor elutriant of 0.3mol/L.Sulfuric acid is transferred pH to 7.0～7.5, the D152 dynamic adsorption, after the salt-free water washing respectively the ammoniacal liquor with 0.1mol/L and 0.15mol/L carry out wash-out, elution flow rate 1/100～1/150/ minute is with phospho-wolframic acid control terminal point, the ammoniacal liquor elutriant of collection 0.15mol/L.
C, refining, crystallization
The ammoniacal liquor elutriant of 0.15mol/L is concentrated, transfer to pH5.5～6.0, go up the 732 resin column dynamic adsorption of having handled well then, salt-free water washing with sulfuric acid.Carry out wash-out with 3% ammoniacal liquor again, collect stripping liquid 2-3 and doubly measure.Be concentrated into 100,000 units per ml afterwards, add 0.5% (w/v) gac, 50～60 ℃ are stirred decolouring 1 hour, and suction filtration gets destainer.Destainer is concentrated into 200,000 units per ml, obtains concentrated solution.
Under agitation, the ethanol that slowly is added dropwise to 10 times of amounts in concentrated solution carries out crystallization, and this process need 3～4 hours separates with whizzer afterwards, and 85% ethanolic soln drip washing promptly gets wet finished product.
With the wet finished product vacuum-drying that obtains, more than the vacuum tightness 500mmHg, 60 ℃ of temperature dry 6 hours, get product.
Finished product is used for HPLC-ELSD and analyzes (Fig. 3) and mass spectrum (Fig. 4).Liquid-phase condition: moving phase 0.2mol/L trifluoroacetic acid solution: methyl alcohol (95:5), flow velocity are 1.0mL/min, and detector is SofTA200s ELSD, and drift tube temperature is 105 ℃, and the mist pipe temperature is 40 ℃, chromatographic column Dikma C
18(5 μ m, 200 * 4.6mm).
Embodiment 2: utilize the tobramycin genetic engineering bacterium to produce tobramycin
Tobramycin genetic engineering bacterium provided by the invention can be directly used in production, and bacterial classification extracts tobramycin after fermenting, as antibacterials.
1. the shake flask fermentation of tobramycin genetic engineering bacterium
Seed culture medium: raw soya bean powder 10g, glucose 10g, peptone 3g, yeast powder 1g, Semen Maydis powder 5g, CaCO
3(lightweight) 1g adds tap water to 1L.
Fermention medium: Zulkovsky starch 20, raw soya bean powder 20g, glucose 10g, NH
4Cl 5g, CaCO
3(lightweight) 5g, MgSO
44g, FeSO
40.05g, ZnSO
40.03g, MnCl
20.3g soya-bean oil 0.6mL/40mL adds tap water to 1L.
The genetic engineering bacterium streptomyces tenebrarius H6 (pSPU303-3) that step 3 in the example 1 is obtained carries out shake flask fermentation.Producing the abundant single bacterium colony of spore with the dilution-plate method separation earlier before the fermentation transfers in synthetic V inclined-plane, cultivated 7 days for 37 ℃, dig piece and be inoculated in seed culture medium (loading amount is the 20mL/250mL triangular flask), 37 ℃ of shaking tables are cultivated 18h (rotating speed is 180rpm, eccentricity 4.0cm).In fermention medium (loading amount is the 40mL/250mL triangular flask), 37 ℃ of shaking tables are cultivated 120h (rotating speed is 180rpm, eccentricity 4.0cm) by 10% inoculum size transferred species.
2. the HPLC-ELSD that sends out product pure analyzes
1) sample extraction purifying: press the tobramycin component in the step 4 intermediate ion exchange process extraction fermented liquid in the example 1, omitted high-temperature alkaline hydrolysing step in the traditional technology, improved the rate of recovery, reduced production cost.
2) HPLC-ELSD analysis condition: Dikma C
18Reverse post (5 μ m, 200 * 4.6mm), 25 ℃ of column temperatures, moving phase is the 0.2mol/L trifluoroacetic acid solution: methyl alcohol (95:5), and flow velocity is 1.0mL/min, advances volume 20 μ l, detector is SofTA 200s ELSD, and drift tube temperature is 105 ℃, and the mist pipe temperature is 40 ℃.
3) standard substance: the tobramycin standard substance are available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The HPLC-ELSD analytical results shows that genetic engineering bacterium directly produces tobramycin as shown in Figure 3.
Claims (6)
1, a kind of engineering bacteria of direct generation tobramycin is characterized in that: by tacA gene in the method blocking-up streptomyces tenebrarius of deletion inactivation in the framework, mainly may further comprise the steps:
The structure of A, tacA gene disruption plasmid;
B, blocking-up plasmid transform streptomyces tenebrarius;
The screening of C, transformant;
The evaluation of D, new component tobramycin.
2, the engineering bacteria of a kind of direct generation tobramycin according to claim 1, the method that it is characterized in that deletion inactivation in the described framework is, PCR obtains tacA gene upstream and downstream molecular weight respectively and is not less than two fragments of 500bp, be connected to both among the pIJ2925 simultaneously, be connected into the antibiotics resistance gene such as the erythromycin resistance gene ermE that in streptomyces tenebrarius, can express toward a segmental end again, utilize the BglII enzyme to cut three gene fragments are linked among the shuttle vectors pHZ132.
3, the engineering bacteria of a kind of direct generation tobramycin according to claim 1, it is characterized in that described conversion be with E.coliET12657 (pUZ8002) mediation in conjunction with transfer method.
4, the engineering bacteria of a kind of direct generation tobramycin according to claim 1 is characterized in that described screening for screening the erythromycin resistant strain earlier, therefrom screens the erythromycin-sensitive bacterial strain again after no medicine goes down to posterity, and therefrom screens the transformant that produces new component.
5, the engineering bacteria of a kind of direct generation tobramycin according to claim 1 is characterized in that the described ferment filtrate that is accredited as adopts thin-layer chromatography TLC, new component to adopt HPLC-ELSD to analyze and the MS analysis.
6, the application of a kind of engineering bacteria of direct generation tobramycin in the preparation antibacterials.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810011472 CN101392229B (en) | 2008-05-20 | 2008-05-20 | Engineering strain for directly producing gernebcin and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810011472 CN101392229B (en) | 2008-05-20 | 2008-05-20 | Engineering strain for directly producing gernebcin and use thereof |
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| CN101392229A true CN101392229A (en) | 2009-03-25 |
| CN101392229B CN101392229B (en) | 2013-05-15 |
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| CN 200810011472 Expired - Fee Related CN101392229B (en) | 2008-05-20 | 2008-05-20 | Engineering strain for directly producing gernebcin and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373174A (en) * | 2011-10-28 | 2012-03-14 | 福州大学 | Engineering bacterium for generating carbamoyl tobramycin and application thereof |
| CN102586165A (en) * | 2012-02-17 | 2012-07-18 | 福州大学 | Engineering bacterium for producing apramycin and application of engineering bacterium |
| CN103614330A (en) * | 2013-11-30 | 2014-03-05 | 福州市鼓楼区荣德生物科技有限公司 | Kanamycin B yielding engineering strain and construction and application thereof |
| CN103740628A (en) * | 2013-11-30 | 2014-04-23 | 福州市鼓楼区荣德生物科技有限公司 | Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium |
-
2008
- 2008-05-20 CN CN 200810011472 patent/CN101392229B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373174A (en) * | 2011-10-28 | 2012-03-14 | 福州大学 | Engineering bacterium for generating carbamoyl tobramycin and application thereof |
| CN102373174B (en) * | 2011-10-28 | 2013-01-16 | 福州大学 | Engineering bacterium for generating carbamoyl tobramycin and application thereof |
| CN102586165A (en) * | 2012-02-17 | 2012-07-18 | 福州大学 | Engineering bacterium for producing apramycin and application of engineering bacterium |
| CN102586165B (en) * | 2012-02-17 | 2014-03-12 | 福州大学 | Engineering bacterium for producing apramycin and application of engineering bacterium |
| CN103614330A (en) * | 2013-11-30 | 2014-03-05 | 福州市鼓楼区荣德生物科技有限公司 | Kanamycin B yielding engineering strain and construction and application thereof |
| CN103740628A (en) * | 2013-11-30 | 2014-04-23 | 福州市鼓楼区荣德生物科技有限公司 | Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium |
| CN103614330B (en) * | 2013-11-30 | 2016-07-13 | 福州市鼓楼区荣德生物科技有限公司 | Produce bekanamycin engineering bacteria and structure thereof and application |
| CN103740628B (en) * | 2013-11-30 | 2016-08-17 | 福州市鼓楼区荣德生物科技有限公司 | Tobramycin engineering bacteria and structure thereof and application are produced in direct fermentation |
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| Publication number | Publication date |
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| CN101392229B (en) | 2013-05-15 |
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