CN102363759A - Engineering bacteria for producing gentamicin C1a and application thereof - Google Patents
Engineering bacteria for producing gentamicin C1a and application thereof Download PDFInfo
- Publication number
- CN102363759A CN102363759A CN2011103315349A CN201110331534A CN102363759A CN 102363759 A CN102363759 A CN 102363759A CN 2011103315349 A CN2011103315349 A CN 2011103315349A CN 201110331534 A CN201110331534 A CN 201110331534A CN 102363759 A CN102363759 A CN 102363759A
- Authority
- CN
- China
- Prior art keywords
- engineering bacteria
- bacteria
- gentamicin
- spore
- cultivated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
- C12P19/48—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin
- C12P19/485—Having two saccharide radicals bound through only oxygen to non-adjacent ring carbons of the cyclohexyl radical, e.g. gentamycin, kanamycin, sisomycin, verdamycin, mutamycin, tobramycin, nebramycin, antibiotics 66-40B, 66-40D, XK-62-2, 66-40, G-418, G-52
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/29—Micromonospora
- C12R2001/31—Micromonospora purpurea ; Micromonospora echinospora; Micromonospora rhodorangea
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of medicines and relates to engineering bacteria which are used for producing gentamicin C1a and have a function of deactivating gntK and application of the engineering bacteria. The engineering bacteria are micromonospora purpurea GK1101 which were registered and collected in China General Microbiological Culture Collection Center (CGMCC) in September 13, 2011 and have the collection number of CGMCC No.5245. The engineering bacteria are applied to preparation of antibacterial medicines. The engineering bacteria for producing the gentamicin C1a are obtained; the production process is simplified; production cost is reduced; and quality control over the products is facilitated.
Description
Technical field
The invention belongs to the microbiotic pharmacy field, relate to a kind of structure and application thereof of engineering bacteria, specifically, the present invention relates to a strain and produce the Gentamicin C1a engineering bacteria, be applied to the microbiotic manufacturing.
Background technology
Micromonospora can produce abundant secondary metabolite, particularly aminoglycoside antibiotics, like qingfengmeisu qiong, sisomicin and Astromicin etc.These micromonosporas distribute peculiar, and poor growth and cell wall structure are special, so progress is slow.In addition, owing to lack general genetic manipulation system, micromonospora genetic manipulation difficulty makes its molecular biological research is also lagged behind streptomycete greatly.
Micromonospora is that qingfengmeisu qiong produces bacterium, and qingfengmeisu qiong is an aminoglycoside antibiotics, has used nearly half a century clinically, is the essential drugs of the anti-infective indispensability of the China and even the whole world.Qingfengmeisu qiong belongs to the polycomponent meta-bolites, and the main ingredient that plays anti-microbial effect is a C family mixture: C1, C2 and C1a.It is very abundant that qingfengmeisu qiong produces the developable component of bacterium meta-bolites, is the precursor of synthetic drug-resistance bacteria medicine Etimicin (Etimicin) like Gentamicin C1a; GentamicinB is the parent that further synthesizes the drug-resistance bacteria medicine isepamicin; Qingfengmeisu qiong A, B, X further develop antiprotozoal good medicine; Current research finds that aminoglycoside antibiotics also has effects such as anti-HIV.Therefore such rare medicinal mikrobe is furtherd investigate, the particularly research of molecular genetics aspect has very big meaning.
Although people nearly 50 years to the research of micromonospora and qingfengmeisu qiong have also obtained some gratifying achievements, great breakthrough is not arranged always.In the research and development process in penicillium mould year surplus 50, tire and improved nearly ten thousand times, and qingfengmeisu qiong has only improved tens times, difference is great disparity very.The streptomyces gene engineering that rise the 1980s obtained certain progress at the aspects such as clone, output raising, component improvement and hybrid antibiotic production of microbiotic biosynthesis gene, but the progress of application in micromonospora is slow.
Etimicin is novel semi-synthetic qingfengmeisu qiong, is microbiotic one kind new medicine of China's original creation, applies for a patent multinational, and its intellecture property has obtained effective protection, and demand is big, is the clinical choice drug of this type microbiotic.
But because the parent of synthetic Etimicin is Gentamicin C1a (Fig. 1).And Gentamicin C1a need separate from qingfengmeisu qiong produces the meta-bolites of bacterium and just can obtain.Unfortunately, up to the present, the meta-bolites that all qingfengmeisu qiongs produce bacterium is a mixture, mainly comprises gentamicinC1, C2 and C1a etc.These mixture chemical structures are close, and physico-chemical property is similar, therefrom separate very difficulty of single-component Gentamicin C1a; Supply falls short of demand to cause Gentamicin C1a, and production cost is high, and the chromatographic separation cycle is long; Yield is low, and the material consumption is big, seriously polluted; Extraction and purification process is complicated, and unstable product quality, occurs at any time disturbing with the by product of Gentamicin C1a structural similitude; Having caused great uncertainty for the Etimicin production quality control, had a strong impact on stability, security and the validity of medicine, is a science difficult problem that presses for solution.
Produce bacterium if can obtain the specificity Gentamicin C1a, then above all problems will be readily solved, and the economic benefit that it brought, social benefit and environmental benefit etc. are extremely huge, the more important thing is to cleaner production, and safe medication provides reliable guarantee.
Summary of the invention
The object of the present invention is to provide a strain to produce the engineering bacteria of Gentamicin C1a.
Engineering bacillus is deactivation
GntKThe purple-red single-spore bacteria of function (
Micromonospora purpurea) the GK1101 bacterial strain, register preservation on September 13rd, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No. 5245.
The fermentation process of described engineering bacteria, specific as follows: as after producing the abundant single bacterium colony of spore with the dilution-plate method separation earlier before the bacterial strain GK1101 fermentation, to transfer again in slant medium; Cultivated 10 days for 37 ℃, dig piece and be inoculated in seed culture medium, 37 ℃ of shaking tables are cultivated 28 h; Rotating speed is 280rpm; Transfer in fermention medium by 10% inoculum size then, 37 ℃ of shaking tables are cultivated 124 h, and rotating speed is 280rpm; Said seed culture medium: glucose 0.1-1.0%, W-Gum 1.0-2.0%, Semen Maydis powder 0.5-2.5%, peptone 0.1-0.8%, soybean cake powder 0.5-1.5%, KNO
30.05-0.10%, CaCO
30.1-0.5%, pH6.5-7.5, fermention medium: W-Gum 3.0-6.0%, Semen Maydis powder 1.0-2.0%, peptone 0.1-0.8%, soybean cake powder 1.0-4.0 %, KNO
30.01-0.10%, (NH
4)
2SO
40.1-0.5%, CaCO
30.1-0.5%, glycase 0.001-0.025%, pH6.5-7.5.
The application of described engineering bacteria in the preparation antibacterials.
The screening of bacterial strain GK1101 mainly comprises following step:
A.
GntKThe structure of gene substitution plasmid;
B. replace plasmid and transform purple-red single-spore bacteria;
C. the screening of transformant;
D. the evaluation of engineering bacteria component.
The method of gene substitution is a pcr amplification
GntKFragment about each 2000 bp of upstream and downstream is as the homology exchange arm; And erythromycin resistance gene inserted between two exchange arms as selection markers; These three fragments are connected on shuttle vectors such as the pKC1139 simultaneously the screening link after the apramycin resistant gene on the carrier pKC1139 also can be used in addition.
Wherein transform be with
E.coliThe combination transfer method of ET12657 (pUZ8002) mediation.Process contains apramycin resistance (Am simultaneously for screening earlier
R) and Oxacyclotetradecane,erythromycin deriv resistance (ermE
R) bacterial strain, after lax the cultivation more therefrom screening contain Oxacyclotetradecane,erythromycin deriv resistance (ermE
R) but to the responsive (Am of apramycin
S) bacterial strain.The fermented liquid component detects and adopts thin-layer chromatography (TLC), component accurately to measure the HPLC-MS method that adopts.
The present invention has obtained the engineering bacteria that a strain produces Gentamicin C1a, engineering bacillus be purple-red single-spore bacteria (
Micromonospora purpurea) the GK1101 bacterial strain, register preservation on September 13rd, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center, it is called for short CGMCC, and the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No. 5245.Engineering bacteria of the present invention is used to produce Gentamicin C1a, has simplified production technique greatly, and has reduced production costs, and reduces environmental pollution, has improved quality product simultaneously.Reached the target of the Gentamicin C1a that can be mass-produced.
Description of drawings
Fig. 1 is the qingfengmeisu qiong chemical structural formula.
Fig. 2 is a recombinant plasmid pFD306 collection of illustrative plates.
Fig. 3 does
GntKGene function homology exchange deactivation (knocking out) synoptic diagram.
I: parental plant genome position; II: change with the single cross of karyomit(e) KB1 side; III: change with the single cross of karyomit(e) KB2 side; The reverse mutation of IV karyomit(e); V: with: the karyomit(e) double exchange.
Fig. 4 is the TLC comparative analysis result of parental plant purple-red single-spore bacteria S-1212 and engineering bacteria purple-red single-spore bacteria GK1101 meta-bolites; Wherein A is an engineering bacteria purple-red single-spore bacteria GK1101 meta-bolites; B is a parental plant purple-red single-spore bacteria S-1212 meta-bolites.
Fig. 5 is the HPLC-MS collection of illustrative plates of parental plant purple-red single-spore bacteria S-1212 meta-bolites.Wherein peak 1 correspondence of total ion current collection of illustrative plates is Gentamicin C1a, molecular weight 450.2; Peak 2, corresponding is gentamicinC2, molecular weight 464.3; Peak 3, that corresponding is gentamicinC2 b, molecular weight 464.3; Peak 4, that corresponding is gentamicinC2 a, molecular weight 464.3; Peak 5, corresponding is gentamicinC1, molecular weight 478.3.
Fig. 6 is the HPLC-MS collection of illustrative plates of engineering bacteria purple-red single-spore bacteria GK1101 meta-bolites.Wherein peak 15 correspondences of total ion current collection of illustrative plates is Gentamicin C1a, molecular weight 450.2; Peak 20, that corresponding is gentamicinC2 b, molecular weight 464.3; Do not detect the vestige of gentamicinC1, C2 and C2a component.
Embodiment
Embodiment 1:
The present invention includes following key step:
1. make up
GntKThe gene substitution plasmid
According to qingfengmeisu qiong biological synthesis gene cluster (can with reference to GenBank Accession Number AY524043), exist respectively
GntKTwo couples of primer LB1 of gene (SEQ.NO.1) upstream and downstream design and LB2 and LB3 and LB4, with starting strain S-1212 (Xiaolan ZHOU, Huang Jianzhong,, Shi Bihong, Shi Qiaoqin. influence purple-red single-spore bacteria (
Micromonospora purpurea) research of several main factors of S-1212 spore germination. Fujian Normal University's journal (natural science edition); Vol.18 No.1, chromosomal DNA p72-75) (CTAB method) is a template, carries out PCR respectively; Amplification obtains the dna sequence dna at gntK two ends, as the homology exchange arm; Upper reaches exchange arm is called KB1 (LB1/LB2), and length is 2076 bp; The downstream exchange arm is called KB2 (LB3/LB4), and length is 2044bp.With plasmid pAGe is template (Zhu Biyin, Hong Wenrong, Yan Shaode; Zhu Baoquan, Lin Yushuan, envelope is established an army. the structure of streptomyces tenebrarius dna homology recombination system. biotechnology circular 2011 (4); 162-166.); E1 and E2 are primer, amplification resistance screening marker gene ermE, and length is 1711bp (Fig.2).KB1, KB2 and ermE through pMD19-T (TA) clone, obtain positive plasmid, successively called after pFD301, pFD302 and pFD303 respectively.PFD303 cuts through Spe I/Xba I enzyme, reclaims 1711 bp fragments, carries out enzyme with the pFD301 that cut through Xba I enzyme (4758 bp) and connects, and transforms and screening, obtains positive plasmid, called after pFD304; Cut pFD302 with Xba I enzyme, reclaim the 2048bp fragment, carry out enzyme with the pFD304 that cut through Xba I enzyme (6463 bp) and connect, screening obtains positive plasmid, called after pFD305; Cut pFD305 with Bgl II enzyme, reclaim 5801 bp fragments, carry out enzyme with the pKC1139 that cut through BamH I enzyme (6500 bp) and connect,, finally obtain positive plasmid, called after pFD306 (Fig. 2) through screening.
The primer that table 1 embodiment is related
a?Restriction?enzyme?site?are?indicated?by?single?underlines?and?box
2. displacement plasmid pFD306 transforms purple-red single-spore bacteria S-1212
To replace plasmid pFD306 changes over to
E.coliAmong the ET12567 (pUZ8002), obtain the donor bacterium
E.coliET12567 (pUZ8002, pFD306).Method through conjugal transfer transforms purple-red single-spore bacteria S-1212 spore then; Cover with the aqueous solution that contains Oxacyclotetradecane,erythromycin deriv and Nalidixic Acid (final concentration is 25 μ g/mL) behind 33 ℃ of cultivation 20 h; 37 ℃ of continuation are cultivated and were grown transformant in 5 days, with the ermE that is obtained
RThe transformant point is connected on the plate culture medium that contains Oxacyclotetradecane,erythromycin deriv (25-50 μ g/mL), apramycin (25-50 μ g/mL) and Nalidixic Acid (25 μ g/mL) to be placed 37 ℃ and cultivates.Because displacement plasmid pFD306 contains temperature sensitive type and duplicates promotor; When culture temperature surpassed 34 ℃, this free plasmid can not self-replacation in streptomycete, and the zygote that has only the exchange of plasmid pFD306 homology to be incorporated on the purple-red single-spore bacteria karyomit(e) could be grown; Show Oxacyclotetradecane,erythromycin deriv and apramycin resistance; Spore well-grown behind the 8d tentatively is judged as single cross and changes bacterial strain, and long good spore is transferred on the slant medium.Further extract that chromosomal DNA carries out the PCR checking and the PCR product checked order and confirmed that all single cross changes insertion.Obtain a strain single cross and change bacterial strain GK1008 (pFD306).
3. the qualitative detection that changes of double exchange bacterial strain screening and fermentating metabolism product
With single cross change bacterial strain GK1008 (pFD306) do not contain on the antibiotic inclined-plane passed for 3 generations continuously after; Carry out separation and purification; Picking list bacterium colony respectively dibbling to the flat board that contains Oxacyclotetradecane,erythromycin deriv resistance (Oxacyclotetradecane,erythromycin deriv 50 μ g/mL) with contain on the flat board of apramycin resistance (apramycin 50 μ g/mL), therefrom screen responsive and bacterial strain that have the Oxacyclotetradecane,erythromycin deriv resistance to apramycin.Select a wherein strain, extract its chromosomal DNA, design 3 couples of primers (P1/P2, P3/P4 and P5/P6 as template; See table 1 and Fig. 3) carry out PCR checking, and order-checking, confirm as the double exchange bacterial strain after, called after purple-red single-spore bacteria GK1101.This bacterial strain fermented (see embodiment 2 step 1), filter to collect fermented liquid.Filtrating is detected through the silica GF254 thin-layer chromatography, and the result finds that its staple is C1a, contains a small amount of C2b (Fig. 4 A), does not see C1, C2 and C2a component (Fig. 4 B); Filtrating is through HPLC-MS qualitative detection (Fig. 6); Compare with the meta-bolites (Fig. 5) of parental plant purple-red single-spore bacteria S-1212; The result is that staple is C1a (peak 1), and a small amount of C2b (peak 3) does not see the vestige of C1 (peak 5), C2 (peak 2) and C2a (epi-C2 peak 4) component.
The HPLC-MS of component analyzes (Fig. 5 and Fig. 6): the electron spray(ES) ion trap mass spectrometry, and C18 leans on; Moving phase: 0.2M trifluoroacetic acid aqueous solution: methyl alcohol (95:5); Flow velocity 0.6 ul/min column temperature: 30 ℃, sample size 1uL.
Embodiment 2: the preparation of purple-red single-spore bacteria GK1101 meta-bolites C1a
Provided by the invention
GntKDeactivation engineering bacteria purple-red single-spore bacteria GK1101 can directly be used to make Gentamicin C1a.The preparation of Gentamicin C1a as follows (Hong Wenrong. qingfengmeisu qiong zymotechnique optimizing. Chinese Journal of Pharmaceuticals. 1994,25 (1) .p1-3):
1. the fermentation culture of purple-red single-spore bacteria GK1101 bacterial strain
Seed culture medium: glucose 0.1%, W-Gum 1.0%, Semen Maydis powder 1.5%, peptone 0.2%, soybean cake powder 1.0% KNO3 0.05%, CaCO3 0.5%, pH7.0.
Fermention medium: W-Gum 6.0%, Semen Maydis powder 1.0%, peptone 0.4%, soybean cake powder 2.0 %, KNO
30.01%, (NH
4)
2SO
40.1%, CaCO
30.5%, glycase 0.025%, pH7.5.
Shake flask fermentation: the purple-red single-spore bacteria GK1101 that step 3 in the instance 1 is obtained ferments.Produce the abundant single bacterium colony of spore with the dilution-plate method separation earlier before the fermentation and transfer in slant medium again, cultivated 10 days for 37 ℃, dig piece and be inoculated in seed culture medium (loading amount is the 50mL/250mL triangular flask), 37 ℃ of shaking tables are cultivated 28 h (rotating speed is 280rpm).Transfer in fermention medium (loading amount is the 50mL/250mL triangular flask) by 10% inoculum size then, 37 ℃ of shaking tables are cultivated 124 h (rotating speed is 280rpm).
Enlarge fermentation: 20000 liters of fermentor tank productions, 180 rev/mins of mixing speed, air flow 1:0.5-1.0 (M
3/ M
3Min), substratum, culture temperature, the inoculum size ratio, fermentation time is equal to shake flask fermentation.
2 meta-bolitess extract refining
A, slightly carry
Fermented liquid after the expansion fermentation adds hydrochloric acid after adding the dilution of 10% tap water, is acidified to pH2.0-3.0, fully stirs 4 hours; Adjust back to pH5.0-6.5 with sodium hydroxide solution then, drop into 732NH
4 +Resin Static Adsorption 6-8 hour.The resin injected volume calculates about by 50,000 u/mL.Collect the absorption saturated resin, with tap water rinsing totally (do not have floating mycelium till).Saturated resin dress post, with the 0.5M HCl solution pickling saturated resin of twice saturated resin volume, extremely neutral with the vaal water washing again; Use 0.01% ammoniacal liquor then instead and carry out alkali cleaning,, be connected in series on isopyknic 711 resin columns when effluent reaches pH 9.0 when above; Use 4% ammoniacal liquor instead and carry out wash-out; Ammonia volume is 8-10 a times of saturated resin volume, and elution time was controlled at 8-10 hour, collects elutriant.
B, refining and crystallization
Elutriant through thin film concentration to about 25-30 ten thousand u/mL (C1a content reaches more than 90%); Transfer to the pH5.5-6.0. activated carbon decolorizing with the vitriol oil and reach more than 92%, under agitation, slowly in liquid concentrator, be added dropwise to 95% above ethanol to transparence; Carry out crystallization; 3-4 hours time, afterwards through solid-liquid separation, 85% ethanolic soln drip washing promptly gets wet finished product.Wet finished product promptly gets GT C1a finished product through vacuum-drying (more than the vacuum tightness 500mmHg, temperature 600C, dry 6 hours), and yield is more than 80%.
< 110>University of Fuzhou; Changzhou FangYuan Pharmaceutical Co., Ltd
The engineering bacteria and the application thereof of Gentamicin C1a produced in < 120>one strains
<160> 13
<170> PatentIn?version?3.3
<210> 1
<211> 1917
<212> DNA
< 213>purple-red single-spore bacteria (Micromonospora purpurea)
<400> 1
tcagtgggaa?accgcctcgg?cggtcaccag?ttcggcctcc?cggcggttct?cgtcgagcag 60
ctcgacgaag?cggcgctgga?tccggccgat?ccgctcgtca?ccggtgggcc?ggaccatgaa 120
cgagcaccct?tcggcatctc?cggccgtctg?cgctccgacc?tgcgcgctga?cctgcggctg 180
accgctgtac?agcgcgatgg?ccgcagcgat?catgtcaccg?gcgaagcggc?agaactggtc 240
ggcgccggcc?cccttgtcgg?cgtccgcgac?gtgctgcatc?acgcagacgc?aacccagcgg 300
gtgccccagt?gccagcagtc?cgtcggcgga?ctcccggatc?gcccgcacgg?tggccggcag 360
ttcctccggg?ctctccgggt?cgagctgcgc?catgctgtgc?tcgaccagac?gtgcgccggc 420
ctccgccgcc?gagaccggcc?cctgctcgcg?gcgctccgcc?ccctcaagga?cgtacttcgg 480
ccacagcgcc?atggcgaggg?tccccaccca?ggccgtgtag?agctcgtccg?cggccaggac 540
accgttggcg?aaggcgaagt?tgaactggtc?gaacagggcg?gggtcggtca?gcgggtcgac 600
gatgccccgg?gtcagacagg?tctggtagtc?cggggagccg?tagatcgggt?agaacgggtt 660
ggtccagaac?tcgaccccgg?ccagcaccag?gttgaccgcg?tcctgcacga?tcgcgtcgag 720
gctctggtcg?ggcagcccga?cgatgatcgc?ggcgctcgcc?gtcagcccga?acttctcgaa 780
cagggcgacc?ccggcggaga?ccttgtccag?cgaggtgaac?cccttgcgca?tcttgcgcag 840
ccgggccgcg?tcggtggtct?ccagcccgag?gaagaggtcg?tcgaagccgg?ccgagaccat 900
gctctccacc?aggtcctcgg?tgagcttcac?gacggtcatc?ccgttcggca?ggctgagccg 960
gacgtcgagc?ttacggcgga?cgatctcctg?gcagatggcg?tgcacccgct?cgatgtcgaa 1020
ggtgaagttg?tcgtcctcca?ccaggaagcg?gcgcaccccg?tgcacgttga?cgtagtgctc 1080
gatctcgtcg?accacgcgct?gcgggtcacg?ggcccggaac?tgcttgccga?ccgtggcgtg 1140
caccgtgcag?aacgagcacg?agaacgggca?gccccggctg?gtgatcaggg?tgaccgcgtt 1200
gccgtaccgg?tcgaagtcga?gctggtcggc?ggccggcggc?gcgagggagt?cgaggtcggc 1260
gacgaagggc?gcacggggac?ggatgtgcgg?cgtgccgggc?gtggcgcact?cgcagagccc 1320
ctcgccacag?cggaaggcca?ccccgagcag?ctcggtcagg?ggccgcccgg?tggcgaacgc 1380
gtccagcagc?gcgaccgtgg?tcacctcggc?ctcaccgagc?atcaccgcgt?cgacctcggg 1440
cacctcgagc?acgtgcggga?aggcgacggt?gccgtgctga?ccgccaacga?tcaccttcgc 1500
gttcggcagg?acgcgcttcg?ccatccgggc?cagctcgtag?gcgctctcgt?agtagggggt 1560
gaacatgcag?gagatgccga?cgacgtccgc?ctcgctggcg?gcgagggcag?cctcgatgcg 1620
ctccgtccgg?gcgccccagt?gcatgatgtg?ccgccagcgc?gggtggcgct?ccagcttggc 1680
ccgctcgatc?gtggtcagct?ccgcgtccgg?gacgacgtgg?ttgtcctcgg?tgtaggcgag 1740
ggagtcgaaa?atctccgtct?catggcccgc?ggcgcgcagc?gctgacgata?cgtaggcgag 1800
cccgatcgga?acgtgacgtc?cgatttccac?catgcatccg?cggatcggcg?gcttgacgag 1860
gaaaaccttc?acttgattac?cttccgtaac?agaaggcgct?gccaccagcg?cgttcat 1917
<210> 2
<211> 30
<212> DNA
< 213>artificial sequence
<400> 2
aagcttagat?ctatctcccg?cataccgtcc 30
<210> 3
<211> 29
<212> DNA
< 213>artificial sequence
<400> 3
tctagatgtg?gccgaagtac?gtccttgag 29
<210> 4
<211> 25
<212> DNA
< 213>artificial sequence
<400> 4
tctagaatcg?tggtcagctc?cgcgt 25
<210> 5
<211> 36
<212> DNA
< 213>artificial sequence
<400> 5
gaattcagat?ctgcaggccg?acttcgacct?cttccg 36
<210> 6
<211> 27
<212> DNA
< 213>artificial sequence
<400> 6
actagtcaga?aggtcgaact?cgcaccg 27
<210> 7
<211> 24
<212> DNA
< 213>artificial sequence
<400> 7
tctagagtac?cagcccgacc?cgag 24
<210> 8
<211> 20
<212> DNA
< 213>artificial sequence
<400> 8
tgctgtgctc?gaccagacgt 20
<210> 9
<211> 21
<212> DNA
< 213>artificial sequence
<400> 9
gagattttcg?actccctcgc?c 21
<210> 10
<211> 20
<212> DNA
< 213>artificial sequence
<400> 10
accgtatcga?acatcagggc 20
<210> 11
<211> 20
<212> DNA
< 213>artificial sequence
<400> 11
atcagaggtt?gatgtcggcc 20
<210> 12
<211> 19
<212> DNA
< 213>artificial sequence
<400> 12
atcaaccgcg?actagcatc 19
<210> 13
<211> 19
<212> DNA
< 213>artificial sequence
<400> 13
cagctaacga?aagggacca 19
Claims (3)
1. the Gentamicin C1a engineering bacteria is produced in a strain, it is characterized in that said engineering bacteria is deactivation
GntKThe purple-red single-spore bacteria of function (
Micromonospora purpurea) GK1101, register preservation on September 13rd, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No. 5245.
2. the fermentation process of an engineering bacteria as claimed in claim 1 is characterized in that, after elder generation produces the abundant single bacterium colony of spore with the dilution-plate method separation before the bacterial strain GK1101 fermentation; Transfer again in slant medium, cultivated 10 days, and dug piece and be inoculated in seed culture medium for 37 ℃; 37 ℃ of shaking tables are cultivated 28 h, and rotating speed is 280rpm, transfers in fermention medium by 10% inoculum size then; 37 ℃ of shaking tables are cultivated 124 h, and rotating speed is 280rpm; Said seed culture medium: glucose 0.1-1.0%, W-Gum 1.0-2.0%, Semen Maydis powder 0.5-2.5%, peptone 0.1-0.8%, soybean cake powder 0.5-1.5%, KNO
30.05-0.10%, CaCO
30.1-0.5%, pH6.5-7.5, fermention medium: W-Gum 3.0-6.0%, Semen Maydis powder 1.0-2.0%, peptone 0.1-0.8%, soybean cake powder 1.0-4.0 %, KNO
30.01-0.10%, (NH
4)
2SO
40.1-0.5%, CaCO
30.1-0.5%, glycase 0.001-0.025%, pH6.5-7.5.
3. the application of engineering bacteria as claimed in claim 1 in the preparation antibacterials.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110331534A CN102363759B (en) | 2011-10-27 | 2011-10-27 | Engineering bacteria for producing gentamicin C1a and application thereof |
| PCT/CN2011/084415 WO2013060073A1 (en) | 2011-10-27 | 2011-12-22 | Engineering bacteria producing gentamicin cla and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110331534A CN102363759B (en) | 2011-10-27 | 2011-10-27 | Engineering bacteria for producing gentamicin C1a and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102363759A true CN102363759A (en) | 2012-02-29 |
| CN102363759B CN102363759B (en) | 2012-10-17 |
Family
ID=45690353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110331534A Active CN102363759B (en) | 2011-10-27 | 2011-10-27 | Engineering bacteria for producing gentamicin C1a and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN102363759B (en) |
| WO (1) | WO2013060073A1 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617666A (en) * | 2012-03-07 | 2012-08-01 | 广州牌牌生物科技有限公司 | Process for making sisomicin content and micronomicin content of gentamicin sulphate consistent with standards in Chinese pharmacopoeia 2010 |
| CN103642744A (en) * | 2013-11-30 | 2014-03-19 | 福州市鼓楼区荣德生物科技有限公司 | Gentamycin A2 biosynthesis engineering bacterium, and construction and application thereof |
| CN103740627A (en) * | 2013-11-30 | 2014-04-23 | 福州市鼓楼区荣德生物科技有限公司 | Gentamycin JI-20B gene engineering bacterium, and construction and application thereof |
| CN103805544A (en) * | 2014-01-26 | 2014-05-21 | 福州大学 | Engineering bacterium producing gentamicin A and application thereof |
| CN103820362A (en) * | 2014-01-23 | 2014-05-28 | 福州大学 | Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof |
| CN103865840A (en) * | 2014-01-23 | 2014-06-18 | 福州大学 | Engineering strain for producing G418 single component and application thereof |
| CN105200002A (en) * | 2015-10-27 | 2015-12-30 | 福州市鼓楼区荣德生物科技有限公司 | Sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof |
| CN104004681B (en) * | 2014-06-06 | 2016-07-13 | 福州市鼓楼区荣德生物科技有限公司 | A kind of product gentamicinC2 a engineering bacteria and application thereof |
| CN107541503A (en) * | 2016-06-23 | 2018-01-05 | 武汉大学 | A kind of transmethylase GenL and its encoding gene genL and application |
| CN108486014A (en) * | 2018-03-30 | 2018-09-04 | 丽珠集团福州福兴医药有限公司 | A kind of cultural method improving micromonospora production capacity |
| CN113403237A (en) * | 2021-07-27 | 2021-09-17 | 青岛安惠仕生物制药有限公司 | Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54160795A (en) * | 1978-06-09 | 1979-12-19 | Kyowa Hakko Kogyo Co Ltd | Preparation of gentamicin c/a |
| CN1130686A (en) * | 1995-03-08 | 1996-09-11 | 管玉霞 | Method for quickly producing gentamicin |
| JP2004180638A (en) * | 2002-12-06 | 2004-07-02 | Meiji Seika Kaisha Ltd | Gentamicin biosynthesis gene |
| CN1557958A (en) * | 2004-02-12 | 2004-12-29 | 窦德献 | Gentamicin, preparation method and uses thereof |
| CN1560262A (en) * | 2004-02-17 | 2005-01-05 | 窦德献 | Ferment process of anti-feedback inhibition and application in producing antibiotics thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1383903B1 (en) * | 2001-04-04 | 2008-09-24 | Genencor International, Inc. | Methods for the production of ascorbic acid intermediates in host cells |
-
2011
- 2011-10-27 CN CN201110331534A patent/CN102363759B/en active Active
- 2011-12-22 WO PCT/CN2011/084415 patent/WO2013060073A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54160795A (en) * | 1978-06-09 | 1979-12-19 | Kyowa Hakko Kogyo Co Ltd | Preparation of gentamicin c/a |
| CN1130686A (en) * | 1995-03-08 | 1996-09-11 | 管玉霞 | Method for quickly producing gentamicin |
| JP2004180638A (en) * | 2002-12-06 | 2004-07-02 | Meiji Seika Kaisha Ltd | Gentamicin biosynthesis gene |
| CN1557958A (en) * | 2004-02-12 | 2004-12-29 | 窦德献 | Gentamicin, preparation method and uses thereof |
| CN1560262A (en) * | 2004-02-17 | 2005-01-05 | 窦德献 | Ferment process of anti-feedback inhibition and application in producing antibiotics thereof |
Non-Patent Citations (5)
| Title |
|---|
| 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 19791231 B. K. LEE et al Biosynthetic pathway leading to gentamicin C2b. 第589-591页 1-3 第16卷, 第5期 * |
| 《Biochemical and Biophysical Research Communications》 20080603 Jin-Yong Kim et al Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis 第730-734页 1-3 第372卷, * |
| 《THE JOURNAL OF ANTIBIOTICS》 20040731 JAMIE UNWIN et al Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex 第436-445页 1-3 第57卷, 第7期 * |
| 《中国抗生素杂志》 19970228 赵敏 等 庆大霉素C_1a_单组份产生菌_棘孢小单孢菌JIM_202的筛选和研究 第12-15页 1-3 第22卷, 第1期 * |
| 《中国抗生素杂志》 19981231 范铭琦 等 庆大霉素C_1a_高产菌株的推理育种的研究 第410-414页 1-3 第23卷, 第6期 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617666A (en) * | 2012-03-07 | 2012-08-01 | 广州牌牌生物科技有限公司 | Process for making sisomicin content and micronomicin content of gentamicin sulphate consistent with standards in Chinese pharmacopoeia 2010 |
| CN103642744A (en) * | 2013-11-30 | 2014-03-19 | 福州市鼓楼区荣德生物科技有限公司 | Gentamycin A2 biosynthesis engineering bacterium, and construction and application thereof |
| CN103740627A (en) * | 2013-11-30 | 2014-04-23 | 福州市鼓楼区荣德生物科技有限公司 | Gentamycin JI-20B gene engineering bacterium, and construction and application thereof |
| CN103865840B (en) * | 2014-01-23 | 2016-03-30 | 福州大学 | A kind of engineering bacteria and application thereof of producing G418 single component |
| CN103820362A (en) * | 2014-01-23 | 2014-05-28 | 福州大学 | Construction of biosynthesis gentamicin X2 engineering bacteria and application thereof |
| CN103865840A (en) * | 2014-01-23 | 2014-06-18 | 福州大学 | Engineering strain for producing G418 single component and application thereof |
| CN103820362B (en) * | 2014-01-23 | 2016-03-30 | 福州大学 | A kind of structure of biosynthesizing gentamicinX 2 engineering bacteria and application thereof |
| CN103805544A (en) * | 2014-01-26 | 2014-05-21 | 福州大学 | Engineering bacterium producing gentamicin A and application thereof |
| CN104004681B (en) * | 2014-06-06 | 2016-07-13 | 福州市鼓楼区荣德生物科技有限公司 | A kind of product gentamicinC2 a engineering bacteria and application thereof |
| CN105200002A (en) * | 2015-10-27 | 2015-12-30 | 福州市鼓楼区荣德生物科技有限公司 | Sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof |
| CN105200002B (en) * | 2015-10-27 | 2019-03-15 | 福州市鼓楼区荣德生物科技有限公司 | Produce the small monospore engineering bacteria of sisomicin magneta colour and its building and application |
| CN107541503A (en) * | 2016-06-23 | 2018-01-05 | 武汉大学 | A kind of transmethylase GenL and its encoding gene genL and application |
| CN107541503B (en) * | 2016-06-23 | 2020-04-24 | 武汉大学 | Methyltransferase GenL, coding gene genL thereof and application |
| CN108486014A (en) * | 2018-03-30 | 2018-09-04 | 丽珠集团福州福兴医药有限公司 | A kind of cultural method improving micromonospora production capacity |
| CN113403237A (en) * | 2021-07-27 | 2021-09-17 | 青岛安惠仕生物制药有限公司 | Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof |
| CN113403237B (en) * | 2021-07-27 | 2021-12-28 | 青岛安惠仕生物制药有限公司 | Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102363759B (en) | 2012-10-17 |
| WO2013060073A1 (en) | 2013-05-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102363759B (en) | Engineering bacteria for producing gentamicin C1a and application thereof | |
| CN106635869B (en) | A method of producing surfactin using bacillus amyloliquefaciens | |
| CN102433364B (en) | Process for preparing rapamycin by using microbial fermentation method | |
| CN102586146B (en) | Engineering bacteria for generating gentamicin C1a and constructing method of engineering bacteria | |
| CN108753627A (en) | A kind of marine aspergillus source oxa- anthraquinone analog compound and preparation method thereof and the application in preparing antitumor agent | |
| CN105200002B (en) | Produce the small monospore engineering bacteria of sisomicin magneta colour and its building and application | |
| CN101748093B (en) | Streptomyces spectabilis SpLE-16 and application thereof to production of spectinomycin | |
| CN104974100A (en) | Phenazine compounds originated from lysobacter antibioticus OH13 and preparation method and application thereof | |
| CN102586165B (en) | Engineering bacterium for producing apramycin and application of engineering bacterium | |
| CN101392229B (en) | Engineering strain for directly producing gernebcin and use thereof | |
| CN102618473B (en) | Mutant strain for producing sisomicin | |
| CN103820362B (en) | A kind of structure of biosynthesizing gentamicinX 2 engineering bacteria and application thereof | |
| CN110358718A (en) | The building and its application of main product Gentamicin C1a engineering bacteria | |
| CN102373174A (en) | Engineering bacterium for generating carbamoyl tobramycin and application thereof | |
| CN103805544A (en) | Engineering bacterium producing gentamicin A and application thereof | |
| CN109053638A (en) | A kind of method for extraction and purification of fumidil | |
| CN103642744A (en) | Gentamycin A2 biosynthesis engineering bacterium, and construction and application thereof | |
| CN103074356B (en) | Vector for knocking out streptomycete gene as well as constructing method and application of same | |
| CN104004681B (en) | A kind of product gentamicinC2 a engineering bacteria and application thereof | |
| CN100506993C (en) | Portamycin fermentation process | |
| CN102993168B (en) | Streptonigrin analog and preparation method thereof, purposes | |
| CN103865840B (en) | A kind of engineering bacteria and application thereof of producing G418 single component | |
| CN105237544B (en) | Xantholipin B and its production and use | |
| CN103614330A (en) | Kanamycin B yielding engineering strain and construction and application thereof | |
| CN102242073A (en) | Method for preparing daptomycin by fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |