CN103740628A - Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium - Google Patents

Abstract

The invention discloses an engineered bacterium for producing tobramycin by direct fermentation and construction and application of the engineered bacterium. By utilizing genetic engineering technology, octose synthatase gene aprK in apramycin biosynthesis gene cluster of streptomyces tenebrarius apramycinis subjected to knocking-out in frame, and further carbamoyl transferase gene tobZ in apramycin biosynthesis gene cluster is knocked out, so that the engineered bacterium (S.tenebrarius318 (delta aprK + delta tobZ)), of which the fermentation metabolite does no generate apramycin any longer and mainly generates tobramycin, is obtained. Tobramycin is directly produced by the engineered bacterium through fermentation, so that a conventional production process of preparing tobramycin from carbamoyltobramycin is avoided, the production flow is substantially simplified, the production cost is reduced, pollution is reduced, the product quality is improved, and the engineered bacterium has vita practical significance and social significance on industrialized production.

Description

Tobramycin engineering bacteria and structure and application are produced in direct fermentation

Technical field

The invention belongs to genetically engineered and microbial fermentation technology field, relate to engineering bacteria and structure and application that fermentation method directly produces tobramycin, it is the further upgrading of last patent " carboxamide tobramycin engineering bacteria and application thereof are produced in a strain " (201110333833.6), having realized engineering bacterium fermentation end product terminates on tobramycin, and fermenting process does not produce apramycin and kanendomycin equally, fermentating metabolism product main ingredient is target product tobramycin, has solved the gordian technique bottleneck in production process.

Background technology

Streptomyces tenebrarius is that the important aminoglycoside antibiotics of a strain produces bacterium, its compoiste fermented thing is generically and collectively referred to as nebramycin, main ingredient is apramycin, carboxamide tobramycin and a small amount of carboxamide kanendomycin, very complicated [the Irina Borodina of metabolic regulation, Charlotte Scho ¨ ller, Anna Eliasson, and Jens Nielsen. Metabolic Network Analysis of streptomyces tenebrarius, a streptomycesspecies with an Active Entner-Doudoroff Pathway. Applied and environmental microbiology, May 2005, p. 2294 – 2302].Apramycin is the very unique aminoglycoside antibiotics of a kind of structure, contains a pungent disaccharides structure of rare dicyclo in its molecule, is difficult for by the passivation of aminoglycoside antibiotics deactivating enzyme institute, in livestock and poultry cultivation field, is used widely.Tobramycin is a kind of widely used aminoglycoside antibiotics clinically, is mainly used in the severe infections that treatment is caused by Gram-negative bacteria, is the choice drug for the treatment of the septicemia being caused by Pseudomonas aeruginosa.In addition, tobramycin also has the effect that in inhibition of gene expression process, mRNA translated early stopping, and translated early stopping, be major cause [the Altamura N that causes cystic fibrosis equimolecular inherited disease, Castaldo R, Finotti A, et al. Tobramycin is a suppressor of premature termination codons [J]. Journal of Cystic Fibrosis, 2013], tobramycin is expected to become the newtype drug of such genetic diseases for the treatment of.Therefore, the biosynthesizing of research tobramycin, constantly promotes production technology level, reduces production costs, and reduces environmental pollution, improves the quality of products, and solves the gordian technique difficulty of manufacturing processed, has important practical significance, social effect and industrialization meaning.

All cannot adopt at present fermentation method direct production tobramycin both at home and abroad, fermentation method direct production tobramycin has become an international science difficult problem, and a gordian technique bottleneck in Ye Shi tobramycin manufacturing enterprise is the Root of Clark Clematis that the technology upgrading of production whole process is regenerated.Current, tobramycin preparation is all that in first nebramycin mixture from fermented liquid, separation obtains carboxamide tobramycin, then is hydrolyzed under alkaline hot conditions, after removal carbamyl, obtains tobramycin.Because nebramycin mixture component is many, chemical structure is close, physico-chemical property is similar, the biosynthetic pathway complexity that interweaves, therefore, utilizes streptomyces tenebrarius to produce carboxamide tobramycin, there is other complicated component, particularly apramycin and carboxamide kanendomycin etc., not only can reduce the output of carboxamide tobramycin, and the extraction of returning carboxamide tobramycin makes troubles.In addition, in the hydrolysis of carboxamide tobramycin, prepare the complicated [Hong Wenrong of tobramycin process, Guo Yanghao, Shi Xianai etc. characteristic research is refined in tobramycin chemical extraction, China Medicine University's journal, 2000,31 (4), p304-308], tobramycin also may further be hydrolyzed formation Ni Bo and draw amine and the mould amine of Ka Na; Kanendomycin may further be hydrolyzed and form card that mould amine and neomycin A, artificially manufactures secondary by product.Kanendomycin, Ni Bo draw amine, block that mould amine and neomycin A is four kinds of inevitable impurity in tobramycin production process.Therefore, utilize existing method to produce tobramycin, extraction and purification process complexity, unstable product quality, the production cycle is long, production cost is high.Therefore, if can realize producing the improvement of bacterial strain, direct fermentation tobramycin, above these difficulties will be readily solved one by one.

For a long time, Upgrading to streptomyces tenebrarius, be summed up and mainly contain two aspects: adopt the classical breeding methods such as selection by mutation, rational selection to carry out strain improvement to it on the one hand, to obtaining the high yield strain excellent of apramycin or carboxamide tobramycin; Be that its fermenting process and extraction and purification process are optimized to control on the other hand, to improving constantly the quality product of apramycin or carboxamide tobramycin, reduce production costs.Although this two aspect has all obtained certain progress, but Genetic Mechanisms, regulation mechanism to the synthetic secondary metabolite of streptomyces tenebrarius are known little about it, therefore, finally reducing tobramycin production cost, reduce environmental pollution, simplify production technique, the substantive aspect such as improve the quality of products, does not almost obviously break through.In recent years, along with the molecular biological development of streptomycete, about microbiotic biological synthesis gene cluster clone in streptomyces tenebrarius, biosynthesis gene function is illustrated and the progressively expansion of biosynthetic pathway supposition etc., for by modern biotechnology reconstruct streptomyces tenebrarius, obtain directed synthetic specific components new approaches are provided.Utilize genetic engineering technique and microorganism molecular genetic technique etc., artificial high grade project bacterium [the W. Hong and S. Yan. Engineering with important industry meaning that creates streptomyces tenebrariusto synthesize single component of carbamoyl tobramycin. Letters in Applied Microbiology, 2012, April, p1-7], make it directed synthetic microbiotic or the new antibiotic derivative with significant application value and established necessary basis.

Gene in streptomyces tenebrarius apramycin biological synthesis gene cluster aprK, the pungent disaccharides synthetic enzyme of may encoding, is the key gene that in apramycin biosynthetic process, pungent disaccharides structure forms; In tobramycin biological synthesis gene cluster tobZgene possibility encoding carbamoyl phosphate transferring enzyme, is converted into carboxamide tobramycin by tobramycin in catalysis tobramycin biosynthetic process.

Therefore, based on this, if can utilize modern molecular biology technique, block the biosynthesizing of other component, reach directed synthetic tobramycin, further tobramycin is not modified into again to carboxamide tobramycin, by the technological difficulties that fundamentally solve in the industrialization of long-term puzzlement tobramycin, realize high yield, low consumption, reduces discharging high-quality and clean target of modernization, play the good result of achieving many things at one stroke, produce great economic benefit and social benefit.Important content of the present invention, just around this theme and a difficult problem, has satisfactorily realized this long-cherished wish of dreaming of.

Summary of the invention

The object of the invention is to break through the traditional processing technology that utilizes the hydrolysis of carboxamide tobramycin to prepare tobramycin, high yield, single-component tobramycin genetic engineering bacterium and construction process thereof are provided.And this bacterial strain is applied to suitability for industrialized production, by fermentation method direct production tobramycin.Thereby simplify tobramycin production process, reduce production costs, improve the quality of products, once and for all solves the gordian technique difficult problem in industrialization.

The present invention utilizes genetic engineering technique, knocks out the pungent disaccharides synthase gene of apramycin in streptomyces tenebrarius aprKwith the mould carbamyl phosphate transferase gene of appropriate cloth tobZ, the biosynthesizing of blocking-up apramycin and carboxamide tobramycin, obtaining metabolic end product is the genetic engineering bacterium of tobramycin.

The present invention relates to streptomyces tenebrarius Tt-49( s.tenebrariustt-49) chromosomal DNA (apramycin biological synthesis gene cluster: GenBank accession No.:AJ629123) be optimized, make the key gene of synthetic pungent disaccharides aprKdNA disappearance, lost original function.Thereby the biosynthesizing of apramycin is blocked, and the biosynthetic common intermediate of apramycin and carboxamide tobramycin flows to synthetic ammonia formyl tobramycin, obtains high yield carboxamide tobramycin engineering bacteria s.tenebrarius316 (△ aprK).Then on the basis of high yield carboxamide tobramycin engineering bacteria, further knock out carbamyl phosphate transferase gene tobZ(GenBank accession No.:AJ810851), makes the biosynthesizing of carboxamide tobramycin terminate in tobramycin site, accumulates a large amount of tobramycins, thereby has obtained the synthetic tobramycin engineering bacteria of high yield and high quality.This bacterial strain called after streptomyces tenebrariuskZ318 (△ aprK+ △ tobZ), in the registration preservation of in September, 2013 27 China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No. 8285, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.

The present invention mainly comprises following step:

1) structure of recombinant plasmid pBK5, for knocking out gene aprK(SEQ ID No.1);

The structure of recombinant plasmid pBK5, take temperature sensitive type shuttle plasmid pKC1139 as basis, passes through pcr amplification aprKthe fragment of each about 2000bp of upstream and downstream, as homology exchange arm, is inserted into both in pKC1139 multiple clone site, then inserts erythromycin resistance gene as selection markers in exchange arm outside.

2) recombinant plasmid pBK5 transforms streptomyces tenebrarius Tt-49;

3) deactivation aprKthe screening of mutant strain and evaluation;

4) deactivation aprKmutant strain tunning is analyzed;

5) structure of recombinant plasmid pBZ5, for knocking out gene tobZ(SEQ ID No.2);

The structure of recombinant plasmid pBZ5 adopts the construction process identical with pBK5, and homology exchange arm is gene tobZthe sequence of the about 2000bp in two ends.

6) recombinant plasmid pBZ5 transforms aprKblocked mutant;

7) deactivation tobZthe screening of mutant strain and evaluation;

8) deactivation tobZmutant strain tunning is analyzed.

After treatment, spent ion exchange resin method is extracted microbiotic to fermented liquid, then adopts TLC, biological developing and HPLC-MS to analyze microbiotic component.

Wherein, recombinant plasmid transformed streptomyces tenebrarius, all adopt with e.colithe conjugal transfer method of ET12657 (pUZ8002) mediation.Recombinant plasmid transformed e.colieT12657 imports streptomyces tenebrarius by conjugal transfer, then under erythromycin pressure, screens, and the homology segment obtaining on recombinant plasmid and streptomyces tenebrarius karyomit(e) restructuring occurs and is incorporated into the single cross changing-over zygote on karyomit(e).

Wherein, adopt PCR method to differentiate blocked mutant.By single cross changing-over zygote separated single bacterium colony after lax going down to posterity on inclined-plane, photocopy being to adding the resistant panel of erythromycin and not adding in antibiotic ordinary flat, obtains growing in ordinary flat and the erythromycin-sensitive type (Ery that do not grow in resistant panel ^s) bacterial strain, then, according to homologous recombination pattern layout specific primer, utilize PCR method to Ery ^sbacterial strain carries out screening verification.Obtain gene knockout mutant strain.

The present invention transforms streptomyces tenebrarius by genetic engineering technique, and synthesizing of blocking-up apramycin, prevents that tobramycin is further derivatized to carboxamide tobramycin, and biosynthetic metabolism ends at tobramycin, has obtained engineering bacteria streptomyces tenebrariuskZ318 (△ aprK+ △ tobZ).This project bacterium can be used for large scale fermentation direct production tobramycin, do not exist the hydrolysis of carboxamide tobramycin to prepare the process of tobramycin, thereby simplified tobramycin production technique, shorten the production cycle, reduce production costs, prevent that secondary from producing by product, reduce environmental pollution, improve the quality of products, thoroughly solved the gordian technique difficulty in industrialization, played the good result of achieving many things at one stroke.

Accompanying drawing explanation

Fig. 1 carboxamide tobramycin and tobramycin structural formula.

Fig. 2 recombinant plasmid pBK5 builds schema.

Fig. 3 recombinant plasmid pBZ5 builds schema.

Fig. 4 starting strain s.tenebrariusthe HPLC-MS of Tt-49 tunning analyzes collection of illustrative plates.

Fig. 5 aprKblocked mutant s.tenebrariusthe HPLC-MS of 316 tunnings analyzes collection of illustrative plates.

Fig. 6 tobZblocked mutant s.tenebrariusthe HPLC-MS of KZ318 tunning analyzes collection of illustrative plates.

Embodiment

embodiment 1: the structure of recombinant plasmid pBK5

With s. tenebrariustt-49 chromosomal DNA is template, utilizes the upstream exchange arm BK1 of primer PK1/PK2 amplification 1984bp, and this fragment comprises aprKpartial sequence and upstream fragment thereof, PCR product is used ecoRi and xbai enzyme is cut, and is connected on the pKC1139 carrier of cutting through same enzyme enzyme, obtains middle interstitial granules pBK3.Then take Tt-49 chromosomal DNA as template, utilize the downstream exchange arm BK2 of primer PK3/PK4 amplification 2050bp, this fragment comprises aprKpartial sequence and downstream fragment thereof, PCR product warp xbai with hindIII enzyme is connected to the pBK3 cutting through same enzyme enzyme after cutting upper, obtains middle interstitial granules pBK4.Finally use ecoRi enzyme is cut the plasmid pAGe containing erythromycin resistance gene, the fragment that reclaims 1746bp be inserted into through ecoRthe pBK4 that I enzyme is cut and dephosphorylation is processed is upper, obtains recombinant plasmid pBK5.The related Host Strains of above plasmid construction is e. colidH5 α.PBK5 is carried out to enzyme and cut checking and order-checking, result is all coincide with theoretical prediction, proves that recombinant plasmid pBK5 builds correct, and the structure flow process of recombinant plasmid is shown in Fig. 2.

The primer that embodiment 1 is related:

PK1:5 '-CCGGAATTCCAACCAGGTCCCCGTCTACC-3 ' (SEQ ID No.3), band ecorI restriction enzyme site;

PK2:5 '-CTAGTCTAGAAGGAGGTCGAGGCGGTCAAC-3 ' (SEQ ID No.4), band xbai restriction enzyme site;

PK3:5 '-CTAGTCTAGACTTCGTGGGAGCTGACACTGG-3 ' (SEQ ID No.5), band xbai restriction enzyme site;

PK4:5 '-CCCAAGCTT ACCGAGTTCCTCAGGACGCT-3 ' (SEQ ID No.6), band hind III restriction enzyme site.

embodiment 2: recombinant plasmid pBK5 transforms s.tenebrariustt-49

Recombinant plasmid pBZ5 is transformed e.colieT12567(pUZ8002), obtain the donor bacterium containing recombinant plasmid e.colieT12567(pUZ8002, pBK5), after incubated overnight, be forwarded to 30ml and add corresponding microbiotic (kantlex 25 μ g/ml, paraxin 25 μ g/ml, apramycin 50 μ g/ml) in LB substratum, cultivate 2-3 h and make thalline enter logarithmic phase, 8000 rpm are centrifugal, and 5 min collect thalline, with the fresh LB washing of equal-volume, remove remaining microbiotic 2 times, be suspended in appropriate LB substratum standby.Meanwhile, scraping is appropriate ripe plentiful s. tenebrariustt-49 slant pore is suspended from 2 * YT substratum, and 50 ℃ of heat shock 10 min, are cooled to room temperature.Spore suspension and intestinal bacteria suspension equal proportion are mixed, after 10 times of gradient dilutions, coat MS flat board, be then placed in 37 ℃ and cultivate after 18-24 h, with covering containing erythromycin (50 μ g/ml) and the aqueous solution of Nalidixic Acid (25 μ g/ml), continue to cultivate 3-4 days, zygote waiting grows.The zygote growing on MS flat board is that bacterial strain is changed in single cross.

embodiment 3: aprKthe screening of blocked mutant and checking

Single cross is changed to prominent bacterial strain switching slant medium, lax cultivate separated single bacterium colony after 5 generations, photocopy being to adding the resistant panel of erythromycin and not adding in antibiotic ordinary flat, screens that 7 strains are grown in ordinary flat and the erythromycin-sensitive type (Ery that do not grow in resistant panel after cultivation ^s) bacterial strain.These Ery ^sbacterial strain may be aprKblocked mutant, also possibility reverse mutation strain, for finishing screen is chosen aprKblocked mutant, carries out screening verification by PCR method.

Random picking 3 strain Ery ^sbacterial strain, is numbered respectively K1, K2, K3.Extract chromosomal DNA, utilize primer P5/P6 to carry out PCR evaluation.According to homologous recombination model, aprKblocked mutant can increase and obtain the fragment of about 1022bp, and reverse mutation strain can be increased and be obtained the fragment of about 1415bp.From electrophoresis result, K1, K2 is reverse mutation strain, K3 is blocked mutant.In order further to confirm that K3 is exactly aprKthe double exchange mutant strain of successfully being blocked, utilize PK7/PK8 and PK9/PK10 to carry out PCR check analysis, wherein PK7/PK8 covers BK1 upstream gene group sequence, BK1 full sequence and BK2 partial sequence, and PK9/PK10 covers BK2 downstream gene group sequence, BK2 full sequence and BK1 partial sequence.In theory, can the increase target stripe of 2454bp of PK7/PK8, can the increase target stripe of 2532 bp of PK9/PK10.Electrophoresis result demonstration, K3 genomic dna has all obtained and desired value product of the same size after PK7/PK8 and PK9/PK10 amplification, reclaims PCR product and also carries out DNA sequencing checking, and result is consistent with prediction.Therefore K3 is aprKthe double exchange mutant strain of successfully being blocked, by its called after s. tenebrarius316 (△ aprK).

The related primer of embodiment 3 is as follows:

PK5: 5′-CCTGGTCGACGAGTCGAAGA-3′（SEQ ID No.7）;

PK6: 5′-GTCTACCAACCGATGTCGCG-3′（SEQ ID No.8）;

PK7: 5′-AGCAACGAGATCGACGCGGA-3′（SEQ ID No.9）;

PK8: 5′-TGGCTGGAGGAGAACTACGG-3′（SEQ ID No.10）;

PK9: 5′-GTGGTACTGCTCGACGCTGATC-3′（SEQ ID No.11）;

PK10: 5′-GCTGATGTTCCGCGGTATCC-3′（SEQ ID No.12）.

embodiment 4: engineering bacteria s. tenebrarius316 (△ aprK) tunning is analyzed

1. streptomyces tenebrarius shake flask fermentation technique

Will aprKblocked mutant s. tenebrarius316 are transferred to slant medium, put 37 ℃ and cultivate 3～4 days; After spore maturation is plentiful, the slant pore that takes 1cm * 1cm is seeded to seed culture medium, and 37 ℃, 320rpm shaking culture 16 ~ 18h, makes thalline in logarithmic phase; Cultivate the inoculum size (v/v) by 10% after 16～18 hours and be inoculated in the triangular flask that 50ml fermention medium is housed, 37 ℃, 320 rpm shaking table shaking culture 4 ~ 5 days, simultaneously with starting strain s. tenebrariustt-49 compares.

Slant medium (g/L): sucrose 30.0, peptone 5.0, KCl 0.5, FeSO ₄.7H ₂o 0.01, K ₂hPO ₄1.0, MgSO ₄0.5, agar 20.0, pH nature.

Seed culture medium (g/L): glucose 5.0, W-Gum 10.0, soybean cake powder 15.0, KCl 1.0, MgSO ₄5.0, KH ₂pO ₄0.5, CaCl ₂0.25, pH nature.

Fermention medium (g/L): glucose 15.0, Semen Maydis powder 20.0, soybean cake powder 35.0, W-Gum 20.0, fish meal 6.0, dried silkworm chrysalis meal 7.0, soya-bean oil 10.0, amylase 0.5, (NH4) ₂sO ₄7.0, MgSO ₄11.0, CaCO ₃7.0, ZnSO ₄0.1, pH 7.0 ~ 7.2.

2. the extraction of tunning is separated

After fermentation stops, add vitriol oil acidifying to regulate pH to 1.5 ~ 2.0, standing 30 min, then regulate fermented liquid pH to 6.0 ~ 6.5 with NaOH.Drop into 732-NH ₄ ⁺resin Static Adsorption 2 hours, resin injected volume calculates by 50,000 u/ml left and right.Collect absorption saturated resin, clean with tap water rinsing, until without floating mycelium.After saturated resin dress post, with 0.1% ammoniacal liquor, wash, when effluent liquid pH reaches 9.0 when above, use 5.0% ammoniacal liquor instead and carry out wash-out, collection elutriant, ammonia volume is 8 ~ 10 times of saturated resin volume, elution time is controlled at 8 ~ 10 h.Collect containing antibiotic desorbed solution, heating is concentrated, with the vitriol oil, adjusts pH to 6.0, and then, through ethanol precipitation, decon, obtains microbiotic, finally by the salt-free water dissolution of 1ml, for HPLC-MS, detects.

3. tunning detects

Adopt HPLC-MS tunning to be carried out to analyzing and testing, liquid-phase condition: chromatographic column Agilent SB-C18 post; Moving phase is 0.2 mol/L trifluoroacetic acid aqueous solution: methyl alcohol (95:5); Flow velocity is 0.6 ul/min; 30 ℃ of column temperatures, sample size 1ul.

As can be seen from Figure 4, starting strain s. tenebrariusmain containing apramycin (molecular weight 540.3), carboxamide tobramycin (molecular weight 511.3) and a small amount of tobramycin (molecular weight 468.3) in Tt-49 tunning.And s. tenebrariusin 316 tunnings, can't detect apramycin (Fig. 5), main ingredient carboxamide tobramycin also has a small amount of tobramycin simultaneously.Show aprKafter being knocked, the biosynthetic pathway of apramycin is thoroughly blocked, and the ratio of carboxamide tobramycin s.tenebrariustt-49 has obvious lifting.

embodiment 5: the structure of recombinant plasmid pBZ5

With s. tenebrarius316 chromosomal DNAs are template, utilize primer PZ1/PZ2 amplification gene tobZupstream 2022bp sequence is as upstream homology exchange arm BZ1, and PCR product is used ecoRi and xbai enzyme is cut, and is connected on the PZC1139 carrier of cutting through same enzyme enzyme, obtains middle interstitial granules pBZ3.Then with s. tenebrarius316 chromosomal DNAs are template, utilize primer PZ3/PZ4 amplification gene tobZthe sequence of downstream 2050bp is as downstream homology exchange arm BZ2, PCR product warp xbai and hindIII enzyme is connected to the pBZ3 cutting through same enzyme enzyme after cutting upper, obtains middle interstitial granules pBZ4.Finally ecoRi enzyme is cut the plasmid pAGe containing erythromycin resistance gene, the fragment that reclaims 1746bp be connected to through ecoRthe pBZ4 that I enzyme is cut and dephosphorylation is processed is upper, obtains recombinant plasmid pBZ5.Plasmid construction host used be e. colidH5 α.For the exactness of checking pBZ5, it to be carried out to enzyme and cut and check order, result is all coincide with prediction, illustrates that recombinant plasmid pBZ5 builds correct, and the structure flow process of recombinant plasmid is shown in Fig. 3.

The related primer of embodiment 5 is as follows:

PZ1:5 '-CCGGAATTCAGGTGCCGACGAGGTTCT-3 ' (SEQ ID No.13), band ecorI restriction enzyme site;

PZ2:5 '-CTAGTCTAGATAGGCCACCACGCGTCAA-3 ' (SEQ ID No.14), band xbai restriction enzyme site;

PZ3:5 '-CTAGTCTAGATGTCTCTACCTCCGATGGGGAAG-3 ' (SEQ ID No.15), band xbai restriction enzyme site;

PZ4:5 '-CCCAAGCTTCTGCGTTGTCCACTGTGGA-3 ' (SEQ ID No.16), band hind III restriction enzyme site.

embodiment 6: recombinant plasmid pBZ5 transforms s.tenebrarius316 and tobZthe screening of blocked mutant

Recombinant plasmid pBZ5 transforms e.coliafter ET12567 (pUZ8002), by conjugal transfer, recombinant plasmid is imported s.tenebrariusin 316 thalline, obtain recombinant plasmid and be incorporated into s.tenebrariusmutant strain is changed in single cross on 316 karyomit(e)s.Single cross is changed to bacterial strain switching inclined-plane, laxly cultivate separated single bacterium colony after 5 generations, and photocopy is to the resistant panel containing 50 μ g/ml erythromycin with do not add in antibiotic ordinary flat.On erythromycin flat board, do not grow and in corresponding ordinary flat the Ery of normal growth ^stype bacterium colony, may be tobZblocked mutant or reverse mutation strain.

Random choose 2 strain Ery ^stype bacterial strain, is numbered respectively Z1, Z2, and extraction chromosomal DNA is template, utilizes double exchange screening primer PZ5/PZ6 to carry out PCR detection.According to homologous recombination model, blocked mutant can increase and obtain the fragment of about 794bp, and reverse mutation strain or starting strain can increase and obtain the fragment of about 2507bp.From the known Z1 of electrophoresis result, be reverse mutation strain, Z2 is blocked mutant.For further confirming that Z2 is tobZthe double exchange mutant strain of successfully being blocked, utilizes PZ7/PZ8 and PZ9/PZ10 to carry out PCR check analysis.In theory, can the increase target stripe of 2606bp of PZ7/PZ8, can the increase target stripe of 2545 bp of PZ9/PZ10.Electrophoresis result demonstration, Z2 genomic dna has all obtained and desired value product of the same size after PZ7/PZ8 and PZ9/PZ10 amplification, reclaims PCR product and also checks order, and result is identical with prediction.Therefore Z2 is tobZthe double exchange mutant strain of successfully being blocked, by its called after s. tenebrariuskZ318 (△ aprK+ △ tobZ).

The related primer of embodiment 6 is as follows:

PZ5: 5′-TTGTAGGCG GCGAAGTCCCT-3′（SEQ ID No.17）;

PZ6: 5′-TGCCTTGGTGAGGATGTCGG-3′（SEQ ID No.18）;

PZ7: 5′-ACCGACATCCTCACCAAGGC-3′（SEQ ID No.19）;

PZ8: 5′-TGGCTGGAGGAGAACTACGG-3′（SEQ ID No.20）;

PZ9: 5′-TTGTAGGCGGCGAAGTCCCT-3′（SEQ ID No.21）;

PZ10: 5′-GTCTCCAAGGGACTGGCCAA-3′（SEQ ID No.22）.

embodiment 7: engineering bacteria s. tenebrariuskZ318 tunning is identified

Adopt the method identical with embodiment 4, to engineering bacteria s. tenebrariuskZ318 (△ aprK+ △ tobZ) ferment, and tunning is carried out to HPLC-MS detection.Detected result as shown in Figure 6, with starting strain and s. tenebrarius316 (△ aprK) compare engineering bacteria s. tenebrariuskZ318 (△ aprK+ △ tobZ) resynthesis apramycin not, main ingredient is tobramycin, and a small amount of carboxamide tobramycin and carboxamide kanendomycin.By saturated resin post is suitably washed, remove a small amount of carboxamide tobramycin and carboxamide kanendomycin, just can obtain high purity tobramycin.More than engineering bacterium fermentation unit can reach 3000ug/mL, meet industrialization needs completely.

The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

SEQUENCE LISTING

Rong De bio tech ltd, Gulou District, <110> Fuzhou City

Tobramycin engineering bacteria and structure and application are produced in <120> direct fermentation

<130> 2013

<160> 22

<170> PatentIn version 3.3

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cgagtactcg tcccagtcct cggggtcacg ggcctgcggg tcgatcaggt tcaggcggta

1080

gccgtccgag tcgaacgcga aggcggacag cgtctcgtcc actgtggtcc cgtgcgcggc

1140

cagtcccatg agcttgccgg gattgtcccc gccgagcccc gtgtactcgc tcacgtgctc

1200

gtagaagaac cccagggacc acgcgccggg gaccgtgtcg agcacggtga tcttgccgcc

1260

ctcggcgtgg gcgagggtca cgcactcctc ctcgccctgg ccgtccacga tgagcaccgc

1320

gccacggtcc tcgccggaga agtagtaggc gctcgccgcg tgggccaggt ggtgctggac

1380

gaagtgcagc ggtggatcgg tgcgccgggg gaacacgtcc ctgggcaaca ggatgtccag

1440

cgcctcgctg tcggagtgcg gccagccgcc cagtcgctcg cggtacatcg ccggcaggtc

1500

ccagccgaac gcgaccgcgt cgaggtcgtc cacggtcagg ccggcctggg cgaggcagaa

1560

cgccgccgcc tgcaccgggg ccgtgttgta cccgtgcttc ttccgtgtga accgctcctc

1620

ctcggcgaag gccgcgatcc ggccgtcgac gagcagggcc gccgacgcgt cgtggaagtc

1680

gcggggccag ccgttcagcc cgaggacacg cat

1713

<210> 3

<211> 29

<212> DNA

<213> artificial sequence

<400> 3

ccggaattcc aaccaggtcc ccgtctacc

29

<210> 4

<211> 30

<212> DNA

<213> artificial sequence

<400> 4

ctagtctaga aggaggtcga ggcggtcaac

30

<210> 5

<211> 31

<212> DNA

<213> artificial sequence

<400> 5

ctagtctaga cttcgtggga gctgacactg g

31

<210> 6

<211> 29

<212> DNA

<213> artificial sequence

<400> 6

cccaagctta ccgagttcct caggacgct

29

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<400> 7

cctggtcgac gagtcgaaga

20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

gtctaccaac cgatgtcgcg

20

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<400> 9

agcaacgaga tcgacgcgga

20

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<400> 10

tggctggagg agaactacgg

20

<210> 11

<211> 22

<212> DNA

<213> artificial sequence

<400> 11

gtggtactgc tcgacgctga tc

22

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<400> 12

gctgatgttc cgcggtatcc

20

<210> 13

<211> 27

<212> DNA

<213> artificial sequence

<400> 13

ccggaattca ggtgccgacg aggttct

27

<210> 14

<211> 28

<212> DNA

<213> artificial sequence

<400> 14

ctagtctaga taggccacca cgcgtcaa

28

<210> 15

<211> 33

<212> DNA

<213> artificial sequence

<400> 15

ctagtctaga tgtctctacc tccgatgggg aag

33

<210> 16

<211> 28

<212> DNA

<213> artificial sequence

<400> 16

cccaagcttc tgcgttgtcc actgtgga

28

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<400> 17

ttgtaggcgg cgaagtccct

20

<210> 18

<211> 20

<212> DNA

<213> artificial sequence

<400> 18

tgccttggtg aggatgtcgg

20

<210> 19

<211> 20

<212> DNA

<213> artificial sequence

<400> 19

accgacatcc tcaccaaggc

20

<210> 20

<211> 20

<212> DNA

<213> artificial sequence

<400> 20

tggctggagg agaactacgg

20

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<400> 21

ttgtaggcgg cgaagtccct

20

<210> 22

<211> 20

<212> DNA

<213> artificial sequence

<400> 22

gtctccaagg gactggccaa

20

Claims (8)

1. tobramycin engineering bacteria is produced in direct fermentation, it is characterized in that: the biosynthesizing of this bacterial strain apramycin and carboxamide tobramycin is all blocked, and mainly synthesizes tobramycin, and this bacterial strain is named as streptomyces tenebrarius318 (△ aprK+ △ tobZ), its deposit number is CGMCC 8285, depositary institution: common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on September 25th, 2013.

2. tobramycin engineering bacteria is produced in direct fermentation according to claim 1, it is characterized in that: in wild type strain, knock out the octose synthase gene in apramycin biological synthesis gene cluster aprK.

3. according to the direct fermentation described in claim 1,2, produce tobramycin engineering bacteria, it is characterized in that: knocking out aprKbasis on further knock out the carbamyl phosphate transferase gene in tobramycin biological synthesis gene cluster tobZ.

4. tobramycin engineering bacteria is produced in direct fermentation according to claim 2, it is characterized in that: described wild type strain is streptomyces tenebrarius, and this bacterial strain mainly produces apramycin only having a small amount of carboxamide tobramycin and carboxamide kanendomycin.

5. tobramycin engineering bacteria is produced in direct fermentation according to claim 2, it is characterized in that: knock out aprKthe conservative property sequence of gene.

6. tobramycin engineering bacteria is produced in direct fermentation according to claim 3, it is characterized in that: knock out tobZthe conservative property sequence of gene.

7. the construction process of tobramycin engineering bacteria is produced in direct fermentation as claimed in claim 1, and its feature mainly comprises the following steps:

(1) knock out aprKthe structure of genophore;

(2) knock out aprKgenophore imports streptomyces tenebrarius;

(3) knock out aprKthe screening of transgenation strain and checking;

(4) knock out aprKgenetic engineering bacterium tunning is analyzed;

(5) knock out tobZthe structure of genophore;

(6) knock out tobZgenophore imports streptomyces tenebrarius;

(7) knock out tobZthe screening of transgenation strain and checking;

(8) knock out tobZgenetic engineering bacterium tunning is analyzed.

8. the application of tobramycin engineering bacteria in tobramycin is produced produced in the direct fermentation as described in claim 1 ~ 7.

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