CN103642744A - Gentamycin A2 biosynthesis engineering bacterium, and construction and application thereof - Google Patents

Abstract

The invention discloses a gentamycin A2 biosynthesis engineering bacterium, and construction and application thereof. The strain mainly produces gentamycin A2, and deletes a genS2 sequence in a gentamycin biosynthesis gene. The construction method comprises the following steps: construction of a shuttle vector pFD405 required for knocking out a genS2 gene, conversion of Micromonospora purpurea through the shuttle vector pFD405, screening of an inactivated genS2 gene double-exchange mutant strain, and analysis of double-exchange mutant strain fermentation metabolites. The gentamycin A2 biosynthesis engineering bacterium can be used for producing gentamycin A2. According to the invention, the constructed engineering bacterium is free of any resistance mark; and the gentamycin A2 yield is stable through serial passage.

Description

Gentamicin A2 biosynthesizing engineering bacteria and structure thereof and application

Technical field

The invention belongs to microbiological pharmacy field, be specifically related to main product gentamicin A2 engineering bacteria and structure thereof and application, can be used for microbiotic manufacture.

Background technology

Micromonospora is a genus energy synthetic antibiotic, and has clinical value, will produce the microorganism of very big economic benefit.In more than the 60 kind of micromonospora of successively reporting, there is kind more than 40 to produce microbiotic, comprising Macrolide, aminoglycoside, AMSA and anthracycline etc.Micromonospora not only can produce the microbiotic that streptomycete produces, also produce gentamicin (Gentamicin), sisomicin (Sisomicin), astromicin (Astromivin) etc., there is the great potential that produces significant secondary meta-bolites, be day by day subject to people's attention.

First Weinstein in 1963 etc. find within 1969 years, to be used to by gentamicin clinical from micromonospora fermented liquid.The independent Broad spectrum antibiotics of succeeding in developing of gentamicin Ye Shi China, nineteen sixty-eight goes into operation, the product-feed whole world.Gentamicin (Gentamicin, GM) can by purple-red single-spore bacteria ( micromonospora purpurea) and micromonospora echinospora ( micromonospora echinospora) etc. microorganism through fermentation, biosynthesizing and obtain, belongs to aminoglycoside antibiotics, and multiple gram-negative bacteria and positive bacteria are had to stronger anti-microbial effect, is specially adapted to treat the infection being caused by Pseudomonas aeruginosa.

Gentamicin has a lot of components, structurally very similar, by three different monosaccharide compositions, is respectively deep red brown sugar amine, 2-deoxystreptamine and garamine.According to the difference of molecular structure, can be divided into gentamicinC, Gentamicin A, gentamicinB and gentamicinX isofamily.The gentamicin main ingredient that known micromonospora produces is gentamicinC family, and its constitutional features is by glycosidic link, to connect deep red brown sugar amine and garamine on 4,6 of 2-deoxystreptamine.Different because of the methylation on deep red brown sugar amine C6, formed C1, C2, C2a, tetra-components of C1a.Except gentamicinC, Gentamicin A, gentamicinB and gentamicinX family etc. or antiprotozoal good medicine.Current research is found: aminoglycoside antibiotics also has potential antitumor and antiviral [Henna Kim, Mi-Kyung Lee, Junsang Ko, Chin-Ju Park, Meehyein Kim, Yujeong Jeong, Sungwoo Hong, Gabriele Varani, Byong-Seok Choi. Aminoglycoside antibiotics bind to the influenza A virus RNA promoter. Mol. BioSyst., 2012,8, p2857 – 2859; Maruthi Chittapragada, Sarah Roberts and Young Wan Ham. Aminoglycosides:Molecular Insights on the Recognition of RNA and Aminoglycoside Mimics. Perspectives in Medicinal Chemistry 2009,3, p 21 – 37.] function, further to synthesize the important as precursors of antiviral and antitumor drug, be a rare class prodrug, application prospect is very wide.

But based on Gentamicin A, gentamicinB and gentamicinX family are the biosynthetic intermediates of gentamicin, obtain the engineering bacteria of these intermediates of main product, very difficult, although scientist is through the effort of decades both at home and abroad, but still fail so far to make a breakthrough, cause the exploitation of this class medicine to fail all the time to realize.

Gentamicin A ₂a member as Gentamicin A family, is not only a good antiprotozoal drug, is also further to develop the important as precursors of antiviral and antitumor drug, and it has a extensive future, and will produce great economic benefit.But fail so far to obtain microorganism and the manufacturing technology that realizes industrialization.

Complete on the basis of domestic gentamicin biosynthesis gene library construction (Genbank accession No.:JQ975418), along with the theory of aminoglycoside antibiotics biosynthesis gene function is inferred [Sung Ryeol Park, Je Won Park, Yeon Hee Ban, Jae Kyung Sohng, Yeo Joon Yoon. 2-Deoxystreptamine-containing aminoglycoside antibiotics:Recent advances in the characterization and manipulation of their biosynthetic pathways. Nat. Prod. Rep., 2013, 30, p11-20, Fumitaka Kudo, Tadashi Eguchi. Biosynthetic genes for aminoglycoside antibiotics. The Journal of Antibiotics, 2009, 62, p471 – 481.] and experimental results show that progressively expansion [Wenrong Hong, Lingbin Yan. Identification of gntK, a gene required for the methylation of purpurosamine C-6 ' in gentamicin biosynthesis. J. Gen. Appl. Microbiol., 2012, 58, 349-356.], design creates the engineering bacteria of biosynthesizing gentamicin series compound, become gradually possibility, for researching and developing important amino-oligosacchride series prodrug, established solid foundation.According to bioinformatics technique analysis, genS2/gntF gene encoding production with s.kanamyceticusin kanS2gene product has very high homology. kanS2the biosynthesizing of catalysis kantlex, makes kanamycin A change kanendomycin into, so infer that genS2/gntF is responsible for catalysis 3 " the amination gene of position.

The required micro-gentamicin A2 of scientific research is that separation obtains from the fermented liquid of gentamicin, its preparation method comprises microorganism fermentation, fermented liquid is obtained to filtrate after filtration, filtrate is concentrated through decolouring, concentrated solution macroporous resin adsorption, the saturated resin after absorption carries out desorb with the ethanol-ammoniacal liquor of three kinds of different ratioss, and stripping liquid is through concentrated, absorption and chromatographic separation, finally just can obtain micro-target product: gentamicin A2 again.This preparation method; complex process; operational difficulty; condition is difficult to control, and cost is high, and yield is low; the target product finally obtaining; therefore its purity still not necessarily reaches the required requirement of standard, is not suitable for large-scale production, causes both at home and abroad so far cannot realizing industrialization preparing on gentamicin series compound.

If can obtain gentamicin A2, produce bacterium, can as manufacturing common microbiotic, prepare gentamicin A2, above all difficult problems will be readily solved.In a word, screen or build significant particular type engineering bacteria, focus, difficult point and the emphasis of Cheng Liao various countries scientist research, become technological core and the commanding elevation of development of new amino-oligosacchride medicine.Obtain gentamicin family row engineering bacteria, also just mean to pest-resistant, the antiviral and antitumor amino-oligosacchride class of development of new medicine and stepped a crucial step.The method of finding gentamicin series engineering bacterium mainly contains classical induced mutation breeding method and genetic engineering breeding method.The birth of modern biotechnology, the exploitation of microorganism molecular genetic technique, for artificial constructed specific engineering bacteria is laid a good foundation, is also the brand-new research direction that obtained gentamicin Novel series drug provision.

Summary of the invention

The object of the present invention is to provide unique main product gentamicin A2 engineering bacteria, the construction process of this engineering strain, and the application of engineering bacteria.

The present invention, by micromonospora molecular genetic technique, knocks out on gentamicin biological synthesis gene cluster genS2/gntFgene (SEQ ID No.1), has obtained the engineering bacteria of main product gentamicin A2.This project bacterium genetic background is clear, is difficult for occurring reverse mutation.

Concrete technical scheme of the present invention is as follows:

Gentamicin A2 biosynthesizing engineering bacteria main product gentamicin A2.Main product Gentamicin A ₂engineering bacteria called after micromonospora purpureagS1219( △ genS), deposit number: CGMCC 8284, 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 27th, 2013.

This bacterial strain has been deleted in gentamicin biosynthesis gene genS2sequence, what comprise its function of forfeiture should have sequence.

Wherein, for building the bacterial strain of genetic engineering bacterium, be all generation bacterium that produce gentamicin, comprise micromonospora.

The present invention is by deleting in deep red micromonospora in framework genS2/gntFgene, mainly comprises the following steps:

(1) knock out genS2/gntFthe structure of gene shuttle vectors

Adopt genetic engineering technique, build recombinant shuttle vector pFD405, contain and lacked 650bp conserved sequence genS2/gntFgene and upstream and downstream partial sequence thereof.

By deleting deactivation in framework, build blocking-up plasmid, the method adopts PCR to obtain respectively genS2gene upstream and downstream sequence 2154 bp and 1937bp, delete genS2the conserved sequence of the inner 650bp of gene, is connected to pMD-19T by two fragments upper simultaneously, then passes through xbaI/EcoR Iafter double digestion, these two fragments are connected to shuttle vectors pJTU412, then pass through ecor I enzyme is cut apramycin resistance gene is connected to two exchange arm outsides.

The recombinant vectors building, is to contain intestinal bacteria and streptomycete replication orgin, and the rear disappearance of deletion part conserved sequence genS2the temperature sensitive type plasmid of gene.

(2) shuttle vectors transforms deep red micromonospora

Described recombinant shuttle vector pFD405 is transformed to purple-red single-spore bacteria, obtain transformant.Described transformant contains and knocks out genS2the recombinant vectors of gene, its host cell is intestinal bacteria ET12567.Described method for transformation is conjugal transfer, starting strain be gentamicin produce bacterium ( micromonospora purpureacGMCC No.5245).

(3) deactivation genS2the screening of Gene Double exchange blocking-up strain

Deactivation genS2the method of gene, comprises gene disruption, deletes and Gene Replacement.

According to resistance marker screening double exchange mutant strain, to obtained targeted mutagenesis strain, extract chromosomal DNA, pcr amplification, electrophoresis detection, then carries out determined dna sequence to PCR product, by bioinformatics technique analysis, final definite engineering bacteria screening is aimed strain.

(4) fermentating metabolism product analysis

By the fermented liquid of engineering bacteria, through acid-alkali treatment and resin absorption, desorb, after removal of impurities, analyzes through methods such as HPLC-MS, and the structure of determining engineering bacteria meta-bolites is gentamicin A2 (Fig. 1).

Gentamicin A2 biosynthesizing engineering bacteria can be in producing gentamicin A2 application.Comprise containing and knock out genS2the transformant of gene and the engaging of deep red micromonospora of producing gentamicin, select zygote.

Core technology of the present invention is the important step of having illustrated in gentamicin biosynthetic pathway, infer and proved purple-red single-spore bacteria ( micromonospora purpurea) biosynthesizing structure gene genS( gntF, function 1173bp), the novel micromonospora molecular genetics operation system by set up, adopts unique molecular genetic technique, permanent deactivation genSthe function of gene, accurately blocked the critical sites of gentamicin biosynthetic metabolism stream, obtain main product gentamicin A2 engineering bacteria, solved first a science difficult problem of utilizing engineering bacteria direct fermentation to produce gentamicin A2, fundamentally solved " having " and " nothing " this key issue.

The present invention has reached and has possessed the requirement that gentamicin A2 is prepared in mass-producing, has a extensive future, and will produce great economic benefit and social benefit, has established the good basis of prodrug for synthesizing drug-resistance bacteria medicine and antiviral.

Accompanying drawing explanation

Fig. 1 gentamicin A2 chemical structure

Fig. 2 gentamicin A2 biosynthetic metabolism approach

Fig. 3 pFD405 shuttle vectors builds schema

Fig. 4 engineering bacterium fermentation meta-bolites HPLC-MS analytical results, wherein peak 1 is gentamicin A2.

Embodiment

Embodiment 1: the structure of shuttle vectors pFD405

According to bioinformatics technique, infer genSgene may be to be responsible in catalysis gentamicin biosynthetic pathway gentamicin A2 to the gene (Fig. 2) of Gentamicin A, deactivation genSgene, is expected to block the synthetic of the follow-up meta-bolites of gentamicin A2, accumulation gentamicin A2.According to purple-red single-spore bacteria ( micromonospora purpurea) in the DNA sequence dna (Genbank accession No.:JQ975418) of gentamicin biological synthesis gene cluster, design knocks out genSgene strategy [Wenrong Hong, Lingbin Yan. Identification of gntK, a gene required for the methylation of purpurosamine C-6 ' in gentamicin biosynthesis. J. Gen. Appl. Microbiol., 2012,58,349-356; Yan Lingbin, Hong Wenrong, the local records iron of fine quality, envelope is established an army. the structure of purple-red single-spore bacteria G1008 conjugal transfer system. Chinese microbiotic magazine, 2011,36 (12), p1-5], shuttle vectors builds flow process and sees Fig. 3.Design primer is as follows:

P1 5 – GAATTCACGAGTTCACCGAGCCGTTC-3 ' (SEQ ID No.2), with ecorI restriction enzyme site;

P2 5 – CTCGAGCAGTTGTCGTCGGAGATGTC-3 ' (SEQ ID No.3), with xhoi restriction enzyme site;

The upstream exchange arm S21 of amplification 2154pb fragment;

P3 5 – CTCGAGTACGAGCTGCTCTTCCGGTT-3 ' (SEQ ID No.4), with xhoi restriction enzyme site;

P4 5 – CGGAATTCCAGCGTTGGCAATAATCATCAGC-3 ' (SEQ ID No.5), with xbai restriction enzyme site;

The downstream exchange arm S22 of amplification 1937bp fragment.

Deep red micromonospora CGMCC 5245 genomic dnas of take are template, by primer P15/P25 amplification 2154bp(S21) and 1937bp(S22) two exchange arms.PCR reaction conditions is: (1) 95 ℃ of denaturation 5min; (2) 94 ℃ * 60 s that unwind; Annealing 60 ℃ * 60 s; Extend 72 ℃ * 2min; 30 circulations; (3) 72 ℃ * 10 min.By PCR product S 21 and S22 fragment electrophoretic separation and recovery, through TA-, be cloned into pMD-19 above, transform intestinal bacteria DH-5 α competent cell, screening positive clone, obtains recombinant plasmid pFD401 and pFD402; PFD401 warp xhoi and xbaafter I double digestion, be connected with the S22 fragment (1937bp) of the pFD402 processing through same enzyme.Connect the competent cell that product transforms intestinal bacteria DH-5 α, screening positive clone, obtains recombinant plasmid pFD403; PFD403 process ecorI/ xbaafter I double digestion, reclaim S21-S22 fragment, and be connected with the pJTU412 carrier of processing through same enzyme, connect the competent cell that product transforms intestinal bacteria DH-5 α, screening positive clone, obtains recombinant plasmid pFD404; Last pFD104's ecorI site, inserts apramycin resistance gene, and transforms the competent cell of intestinal bacteria DH-5 α, and screening positive clone, obtains recombinant plasmid pFD405.Shuttle vectors pFD405 builds complete.

Embodiment 2: shuttle vectors confession-recipient bacterium conjugal transfer operation

Shuttle vectors pFD405 is imported e.colieT12567(pUZ8002), obtain donor bacterium e.colieT12567(pUZ8002/pFD405).Will e.colieT12567(pUZ8002/pFD405) single bacterium colony, be inoculated in 3ml LB substratum (containing 25 μ g/ml kantlex, paraxin 25 μ g/ml, apramycin 50 μ g/ml) in, 37 ℃ of overnight incubation, by 10% transferred species, to 50ml, contain in three kinds of antibiotic LB substratum, cultivate 2.5-3.0h for 37 ℃, centrifugation thalline, by fresh LB substratum washed twice, finally thalline is suspended in about 2ml LB substratum or sterilized water, OD value is 0.4-0.6.

The preparation of monospore suspension:

Fresh purple-red single-spore bacteria spore being suspended in to the TES(pH 8.0 of 5mL 0.05mol/L) in damping fluid, concuss is to break up spore;

A. spore suspension is placed in to 37 ℃ of water-bath heat shock 1 min;

B. after being cooled to room temperature, add isopyknic pre-germination medium, mix;

C. in 37 ℃ of shaking table concussions, cultivate 2 hours;

D. centrifugal (8,000 rpm, 5min), collects spore and is resuspended in the sterilized water of appropriate (1ml);

E. on vortex oscillation device, break up spore.

Donor bacterium 0.5 ml handling well is mixed in eppendorf pipe with deep red micromonospora monospore suspension 0.5ml, and centrifugation, removes supernatant liquor, by a small amount of raffinate, is coated on micromonospora plate culture medium after Eddy diffusion.Cultivate 20 hours for 37 ℃, with 800 μ l, containing the aqueous solution of apramycin (concentration degree 50 μ g/ml) and nalidixic acid (final concentration 25 μ g/ml), cover.37 ℃ are continued to cultivate 3-5d, grow single bacterium colony on flat board, may be single cross changing-over zygote.

Embodiment 3: the screening of single double exchange mutant strain

The zygote that embodiment 2 screenings are obtained is on the flat board that contains Nalidixic Acid and apramycin, 37 ℃ of cultured continuously are more than 2 generations, then extract one by one chromosomal DNA, as template, use primer P5/P6(P5:5 '-ACCGTCATCGAGACCCGCAA-3 ＇ (SEQ ID No.6); P6:5 '-ATCCACACCGCCTC GTCGAA-3 ＇ (SEQ ID No.7)) carry out pcr amplification and agarose gel electrophoresis and detect, will obtain the two objective bands of 1055 bp and 523 bp, illustrate that mutant strain single cross has occurred and changed, by its called after micromonospora purpureagDS1219(is called for short GDS1219); Homology single cross is changed to mutant strain (GDS1219) dibbling to relax and cultivates 7 generations, then separated single bacterium colony to not containing on antibiotic inclined-plane at 37 ℃.Single bacterium colony difference photocopy of having grown is arrived containing apramycin (50 μ g/ml) and containing on the flat board of apramycin.On apramycin flat board, do not grow, and may be for there is homology double exchange or reverse mutation bacterial strain in single bacterium colony of normal growth on non-resistant flat board.Extract its chromosomal DNA, as template, with primer P5/P6, carry out PCR and agarose gel electrophoresis detection, what obtain 1055 bp wall scroll bands is reverse mutation bacterial strain; What obtain 523 bp wall scroll bands is double exchange aimed strain.A strain aimed strain called after wherein m. purpureagS1219(is called for short GS1219; GK1101 △ genS2).Karyomit(e) with GS1219 is made template, with primer P5/P6, carries out PCR and agarose gel electrophoresis detection, obtains the wall scroll band of 523bp, and its PCR product is checked order, and result conforms to prediction, proves that homology double exchange has occurred GS1219 really, has knocked out genSgene, has destroyed its function.

Embodiment 4: the analysis of fermentation and product

(1) shake flask fermentation of deep red micromonospora genetic engineering bacterium

Slant medium (%): 1.5 grams, wheat bran, Zulkovsky starch 0.75, asparagine 0.002, MgSO ₄6H ₂o 0.05, KNO ₃1.0, K ₂hPO ₄3H ₂o 0.03, and NaCl 0.05, CaCO ₃0.1, agar 18, pH7.0.

Seed culture medium (%): glucose 1.0g, starch 2.0g, soybean cake powder 1.5g, Semen Maydis powder 1.0g, peptone 0.5g, ammonium sulfate 0.1g, CaCO ₃0.2g, pH7.2.

Fermention medium (%): starch 4.0g, soybean cake powder 2.5g, Semen Maydis powder 1.0g, peptone 0.5g, CoCl ₂0.001g, ammonium sulfate 0.1g, CaCO ₃0.5g, pH 7.2.

Deep red micromonospora is transferred on slant medium, after 37 ℃ of constant temperature culture 9d, digs piece and be inoculated in the 250 mL triangular flasks that 50 mL seed culture mediums are housed, in 35 ℃, 290rpm shaking table shaking culture 25-30 hour.Cultured seed liquor is transferred in the 1000 mL triangular flasks that 150mL fermention medium is housed with 10% inoculum size (v/v), and in 35 ℃, 280 rpm shaking table oscillation and fermentation cultivation 124 hours, more than fermentation unit 620ug/mL, can meet industrialized requirement.

(2) meta-bolites preparation

Fermented liquid adds the vitriol oil and is adjusted to pH1.5, stirs 20min, in the centrifugal 15min of 12000rpm, gets supernatant liquor, with NaOH, neutralizes pH7.0, adds 732-NH ₄ ⁺resin absorption is spent the night; Reclaim the saturated resin after absorption, pack ion exchange column into, distilled water flushing is clean, then through 0.3% NH ₄till OH washs effluent liquid pH8.0 left and right, finally use the 4%NH of 10 times of amount of resin ₄oH wash-out, obtains the elutriant containing gentamicin mixture, and heating, except ammonia, is transferred to pH6.0 with sulfuric acid, controls microbiotic final concentration in 1000u/mL left and right, for HPLC-MS, analyzes.

(5) meta-bolites structural analysis

Adopt HPLC-MS analytical method, determine the molecular weight of meta-bolites, in conjunction with known gentamicin pathways metabolism, determine the structure of meta-bolites.HPLC-MS condition: Electrospray ion trap mass spectrometry, 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 1 uL.

HPLC-MS analytical results is shown in Fig. 4.Peak 2 molecular weight are 455(456-1=455), coincide with the molecular weight of gentamicin A2, in conjunction with the known gentamicin biosynthetic pathway of Fig. 2, prove that genetic engineering bacterium mainly produces gentamicin A2, has good anti-microbial activity.

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

<120> gentamicin A2 biosynthesizing engineering bacteria and structure and application

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Claims (13)

1. gentamicin A2 biosynthesizing engineering bacteria, is characterized in that: this bacterial strain main product Gentamicin A ₂, called after micromonospora purpureagS1219(△ genS), deposit number: CGMCC 8284, 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. according to the gentamicin A2 biosynthesizing engineering bacteria described in claim 1, it is characterized in that: this bacterial strain has been deleted in gentamicin biosynthesis gene genS2sequence, what comprise its function of forfeiture should have sequence.

3. according to the gentamicin A2 biosynthesizing engineering bacteria described in claim 1,2, it is characterized in that: build the bacterial strain of genetic engineering bacterium for producing all generation bacterium of gentamicin, comprise micromonospora.

4. a construction process for the gentamicin A2 biosynthesizing engineering bacteria as described in claim 1 ~ 3, is characterized in that: mainly comprise the following steps:

(1) knock out genS2the structure of the required shuttle vectors pFD405 of gene;

(2) shuttle vectors pFD405 transforms deep red micromonospora;

(3) deactivation genS2the screening of Gene Double exchange mutant strain;

(4) analysis of double exchange mutant strain fermentating metabolism product.

5. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 4, is characterized in that: described step (1) builds blocking-up plasmid by deleting deactivation in framework, and the method adopts PCR to obtain respectively genS2gene upstream and downstream sequence 2154 bp and 1937bp, delete genS2the conserved sequence of the inner 650bp of gene, is connected to pMD-19T by two fragments upper simultaneously, then passes through xbaI/EcoR Iafter double digestion, these two fragments are connected to shuttle vectors pJTU412, then pass through ecoR Ienzyme is cut apramycin resistance gene is connected to two exchange arm outsides.

6. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 4, is characterized in that: the recombinant vectors that described step (1) builds is to contain intestinal bacteria and streptomycete replication orgin, and the rear disappearance of deletion part conserved sequence genS2the temperature sensitive type plasmid of gene.

7. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 4, is characterized in that: described step (3) deactivation △ genS2the method of gene, comprises gene disruption, deletes and Gene Replacement.

8. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 4, it is characterized in that: the screening of described step (3) double exchange blocking-up strain is according to resistant phenotype, screening homology double exchange blocked mutant, PCR checking is carried out in the DNA double exchange that knockout mutant strain is occurred on gene level, thereby screens the transformant that component changes.

9. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 4, is characterized in that: its tunning of described step (4) and parental plant comparison, through HPLC-MS, analyze, and determine the variation that fermentating metabolism product structure occurs.

10. the construction process of gentamicin A2 biosynthesizing engineering bacteria according to claim 8, is characterized in that: described transformant contains and knocks out genS2the recombinant vectors of gene, its host cell is intestinal bacteria ET12567.

The application of 11. gentamicin A2 biosynthesizing engineering bacterias as claimed in claim 1 in producing gentamicin A2.

12. according to the application of gentamicin A2 biosynthesizing engineering bacteria described in claim 11, it is characterized in that: comprise and knocking out containing genS2the transformant of gene and the engaging of deep red micromonospora of producing gentamicin, select zygote.

13. application of gentamicin A2 biosynthesizing engineering bacteria as claimed in claim 1 in preparing gentamicin A2.

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