CN101392229B - 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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- CN101392229B CN101392229B CN 200810011472 CN200810011472A CN101392229B CN 101392229 B CN101392229 B CN 101392229B CN 200810011472 CN200810011472 CN 200810011472 CN 200810011472 A CN200810011472 A CN 200810011472A CN 101392229 B CN101392229 B CN 101392229B
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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 use in the microbiotic pharmacy.
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 is apramycin (Apramycin, Am), component 4 is 6 "-O-carboxamide kanendomycin (6 "-O-Carbamoylkanamycin B, CKB), component 5 ' is 6 "-O-carboxamide tobramycin (6 "-O-Carbamoyl tobramycin, CTB).Component 4,5 ' forms respectively kanendomycin and tobramycin after 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 pseudomonas aeruginosa of gentamicin resistance, and the ear of tobramycin, renal toxicity are relatively low, are the antimicrobial drugs of widespread use clinically.
Scientific Research Workers mainly concentrates on the aspects such as conventional selection by mutation and optimized production process to the research of streptomyces tenebrarius and obtains 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 the synthetic dTDP-Glc-4 of tobramycin, 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, then pyrohydrolysis obtains tobramycin under alkaline condition.The shortcoming of the method is that impurity is many, and product yield is low, and production cost is high.
Along with the development of Protocols in Molecular Biology, make and utilize engineered method transformation microbiotic component to become possibility.Madan Kumar Kharel etc. is cloned into tobramycin partial synthesis gene cluster in streptomyces tenebrarius, and Blastn finds that wherein gene TacA is transcarbamylase, may be with 6 " the position carbamyl is synthetic relevant.
Summary of the invention
The objective of the invention is by the method to the tacA inactivation, acquisition can directly produce the engineering bacteria of tobramycin, this bacterial strain on March 20th, 2008 in the registration preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, 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 framework:
The structure of A, tacA gene disruption plasmid;
B, blocking-up Plasmid Transformation streptomyces tenebrarius;
The screening of C, transformant;
The evaluation of D, new component tobramycin.
The method of deletion inactivation is that PCR obtains respectively tacA gene upstream and downstream molecular weight and is not less than two fragments of 500bp in framework, be connected to simultaneously both in pIJ2925, an end toward fragment is connected into antibiotics resistance gene such as the erythromycin resistance gene ermE that can express in streptomyces tenebrarius again, utilizes the BglII enzyme to cut three gene fragments are linked in shuttle vectors pHZ132.
Wherein transform be with E.coliET12657 (pUZ8002) mediation in conjunction with transfer method.Screening is for first screening erythromycin resistance (erm
R) bacterial strain, therefrom screen again erythromycin-sensitive (erm after going down to posterity without medicine
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 has been registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center (Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica) on March 20th, 2008, its Classification And Nomenclature is streptomyces tenebrarius (Streptomyces tenebrarius), and deposit number is CGMCC No.2409.
Description of drawings
Fig. 1 is tacA Gene Double exchange principle and restriction enzyme site schematic 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 tobramycin, and C is 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. build tacA gene disruption plasmid
The tobramycin biological synthesis gene cluster sequence (GenBank Accession Number AJ579650) of delivering according to Madan Kumar Kharel etc., at two pairs of primers of tacA gene upstream and downstream design, carry out pcr amplification take the total DNA of streptomyces tenebrarius as template and obtain two fragment PCR 1-2 and PCR3-4 respectively.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 is linked in carrier pIJ2925 obtain pSPU301.KpnI site in the pSPU301 is connected into erythromycin resistance gene ermE (pXZ1 cuts back to close approximately 1.6Kb size fragment through the KpnI enzyme) and obtains recombinant plasmid pSPU302 again, utilize at last the BglII enzyme to cut Δ tacB+ermE is connected to BamHI site in carrier pHZ132, obtain blocking-up plasmid pSPU303.
2. blocking-up plasmid pSPU303 transforms streptomyces tenebrarius H6
To block plasmid pSPU303 and change in E.coliET12567 (pUZ8002), obtain donor bacterium E.coliET12567 (pUZ8002, pSPU303).Then the method that shifts by combination transforms streptomyces tenebrarius H6 spore, cover with erythromycin and pyridine acid (final concentration is respectively 100 μ g/mL and the 50 μ g/mL) aqueous solution after 28 ℃ of cultivation 20h, 28 ℃ of continuation are cultivated and were grown transformant in 5-6 days, with the erm that obtains
RTransformant is at the R that contains erythromycin (200 μ g/mL) and PPA (50 μ g/mL)
2On the YE substratum, 42 ℃ of sectional streaks are cultivated, and 48h grows bacterium colony, because the blocking-up plasmid can not copy at 42 ℃, can only exchanges by homology and are incorporated on karyomit(e) afterwards zygote and could grow, and show the erythromycin resistance.Single cross has occured and has changed integration in the proof zygote.Provoke at random a strain called after streptomyces tenebrarius H6 (pSPU303).
3. screening double exchange bacterial strain and tunning detect
With erm
RTransformant close without medicine on the V flat board pass the three generations continuously after, picking list bacterium colony dibbling respectively closes V (erythromycin 100 μ g/mL) flat board to close the dull and stereotyped and pastille of V without medicine, screens 32 erm of acquisition from 544 strains
SBacterial strain.Called after streptomyces tenebrarius H6 (pSPU 303-1) 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, 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
In the extraction fermented liquid, new component has following several step:
A, slightly carry
Acidifying respectively after fermented liquid dilution, alkalization be except after 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 pH 9.0 when above, is connected in series on 711 resin columns of suitable 732 resin 1/5 volumes, collects tree and takes off liquid, 1/8～1/10 of simmer down to original volume.
B, separation
The upper D151 resin of concentrated solution (pH7.0～7.5) carries out dynamic adsorption.After 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 is controlled terminal point with phospho-wolframic acid, collects the ammoniacal liquor elutriant of 0.3mol/L.Sulfuric acid is transferred pH to 7.0～7.5, the D152 dynamic adsorption, after 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 controlled terminal point with phospho-wolframic acid, the ammoniacal liquor elutriant of collection 0.15mol/L.
C, refining, crystallization
The ammoniacal liquor elutriant of 0.15mol/L is concentrated, transfer to pH 5.5～6.0 with sulfuric acid, then go up the 732 resin column dynamic adsorption of having handled well, salt-free water washing.Carry out wash-out with 3% ammoniacal liquor again, collect stripping liquid 2-3 and doubly measure.Be concentrated into afterwards 100,000 unit/ml, add 0.5% (w/v) gac, 50～60 ℃ are stirred decolouring 1 hour, and suction filtration gets destainer.Destainer is concentrated into 200,000 unit/ml, obtains concentrated solution.
Under agitation, the ethanol that slowly is added dropwise to 10 times of amounts in the concentrated solution carries out crystallization, and this process need 3～4 hours separates with whizzer afterwards, and 85% ethanolic soln drip washing namely gets wet finished product.
With the wet finished product vacuum-drying that obtains, more than vacuum tightness 500mmHg, temperature 60 C dry 6 hours, gets 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 fermentation, 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
4Cl5g, 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 example 1 is obtained carries out shake flask fermentation.First producing the abundant single bacterium colony of spore with the dilution-plate method separation before 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 of tunning analyzes
1) sample extraction purifying: press the tobramycin component in step 4 intermediate ion exchange process extraction fermented liquid in example 1, omitted high-temperature alkaline hydrolysing step in 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 (4)
1. an engineering bacteria that directly produces tobramycin, is characterized in that: in the method blocking-up streptomyces tenebrarius by deletion inactivation in framework
TacAGene mainly comprises the following steps:
A
, tacAThe structure of gene disruption plasmid;
B, blocking-up Plasmid Transformation streptomyces tenebrarius;
The screening of C, transformant;
The evaluation of D, new component tobramycin;
In described framework, the method for deletion inactivation is: PCR obtains respectively
TacAGene upstream and downstream molecular weight is not less than two fragments of 500bp, exists respectively
TacATwo pairs of primers of gene upstream and downstream design carry out pcr amplification and obtain two fragment PCR 1-2 and PCR3-4 take the total DNA of streptomyces tenebrarius as template, two fragments are connected the product called after
△ tacA,It has been deleted
TacAInner 260 bases of gene are connected to both in pIJ2925 simultaneously, then are connected into toward an end of fragment the erythromycin resistance gene that can express in streptomyces tenebrarius
ErmE, utilize
BglThe II enzyme is cut three gene fragments is linked in shuttle vectors pHZ132;
Described screening therefrom screens the erythromycin-sensitive bacterial strain again for first screening the erythromycin resistant strain after going down to posterity without medicine, therefrom screen the transformant that produces new component.
2. the engineering bacteria of a kind of direct generation tobramycin according to claim 1, it is characterized in that described conversion be with
E.coliET12567 mediation in conjunction with transfer method.
3. 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.
4. the application of engineering bacteria claimed in claim 1 in direct fermentation generation tobramycin.
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| Application Number | Priority Date | Filing Date | Title |
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| 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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| Publication Number | Publication Date |
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| CN101392229A CN101392229A (en) | 2009-03-25 |
| CN101392229B true CN101392229B (en) | 2013-05-15 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373174B (en) * | 2011-10-28 | 2013-01-16 | 福州大学 | Engineering bacterium for generating carbamoyl tobramycin and application thereof |
| CN102586165B (en) * | 2012-02-17 | 2014-03-12 | 福州大学 | Engineering bacterium for producing apramycin and application of engineering bacterium |
| 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 |
| 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 |
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2008
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Non-Patent Citations (3)
| Title |
|---|
| 杨钧 等.黑暗链霉菌中tbmA基因的功能研究.《微生物学杂志》.2007,第27卷(第4期),31-34. * |
| 毕见州 等.利用基因工程技术选育氨甲酰妥布霉素高含量菌种.《沈阳药科大学学报》.2010,第27卷(第9期),751-758. * |
| 王普宏.利用基因突变技术进行安普霉素单组分产生菌的选育.《中国优秀硕士学位论文全文数据库工程科技I辑》.2007,摘要，说明书23-25页，30页. * |
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