CN102618473A - Mutant strain for producing sisomicin - Google Patents
Mutant strain for producing sisomicin Download PDFInfo
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- CN102618473A CN102618473A CN201210099020XA CN201210099020A CN102618473A CN 102618473 A CN102618473 A CN 102618473A CN 201210099020X A CN201210099020X A CN 201210099020XA CN 201210099020 A CN201210099020 A CN 201210099020A CN 102618473 A CN102618473 A CN 102618473A
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Abstract
The invention relates to a mutant strain for producing sisomicin, which is applied to antibiotic manufacture and belongs to the field of antibiotic pharmacy. The mutant strain is micromonosporainyoensis HP388 registered and preserved in a general microorganism center of the Chinese microscobial preservation management committee on March 19, 2012, and the preservation number is CGMCCNo.5921. The mutant strain HP388 has the most remarkable advantages of high fermentation unit, stable production process, convenience in control, low oxygen consumption, low cost, low pollution and fine product quality.
Description
Technical field
The invention belongs to the microbiotic pharmacy field, relate to a strain and produce the sisomicin mutant strain, be applied to the microbiotic manufacturing.
Background technology
Sisomicin is mainly used in treatment by the severe infections due to the gram negative bacilli of sensitivity, like newborn infant's Sepsis, septicemia, central nervous system infection (comprising meningitis), urinary tract genital system infection, respiratory tract infection, gastrointestinal tract infection, peritonitis, biliary tract infection, skin or bone infection due to Pseudomonas aeruginosa, proteus (indoles is positive and negative), escherichia coli, klebsiella spp, enterobacter, Serratia and the citrobacter genus etc., otitis media, sinusitis paranasal sinusitis, soft tissue infection, listeriosis etc.
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.Therefore, the sisomicin that obtain stable high yield produces bacterium, and is extremely difficult.Owing to lack general genetic manipulation system, micromonospora genetic manipulation difficulty makes its molecular biological research is also lagged behind streptomycete greatly.
According to the knob micromonospora is that sisomicin produces bacterium.Sisomicin belongs to aminoglycoside antibiotics, has used nearly half a century clinically, is the essential drugs of China and even the anti-infective indispensability in the whole world.Sisomicin produces Pseudomonas polycomponent meta-bolites, but sisomicin is its main ingredient, and its constructional feature is to contain a two key in the molecule.Sisomicin (Sisomicin) is the parent that further synthesizes drug-resistance bacteria medicine netilmicin (Netilmicin); 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, the research of biosynthesis gene, the research that the secondary metabolite production level improves etc. all have meaning.
Although people nearly 50 years to the research of micromonospora and sisomicin 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 sisomicin only improved tens times in 40 years, difference is great disparity very.The streptomyces cell engineering that rise the 1980s; The streptomyces gene engineering; Obtained certain progress at the aspects such as clone, output raising, component improvement and hybrid antibiotic research of microbiotic biosynthesis gene, but the progress of application in micromonospora is slow.
Summary of the invention
The object of the present invention is to provide a plant height to produce the mutant strain of sisomicin.
Mutant strain according to the invention be according to the knob micromonospora (
Micromonospora inyoensis) HP 388, register preservation on March 19th, 2012 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.5921.
The present invention also provides the fermentation process of the mutant strain of described product sisomicin, according to the knob micromonospora (
Micromonospora inyoensis) isolate single bacterium colony with dilution-plate method earlier before the HP388 fermentation after, transfer again in slant medium, cultivated 2-5 days for 35-40 ℃; Inoculum size with 4-6% is inoculated in seed culture medium again, and 35-40 ℃ of shaking table cultivated 18-48 h, and rotating speed is 260-300rpm, transfers in fermention medium by the 8-12% inoculum size then, and 35-40 ℃ of shaking table cultivated 4-5 days, and rotating speed is 260-300rpm; Said slant medium: by weight percentage, Zulkovsky starch 1.0-2.0%, wheat bran 1.0-2.0%, MgSO
46H
2O 0.01-0.05%, KNO
30.1-0.5%, K
2HPO
43H
2O 0.01-0.05%, NaCl 0.01-0.09%, CaCO
30.1-0.50%, agar 1.8-2.2%, surplus is a water, pH7-8; Seed culture medium: by weight percentage, glucose 0.1-1.0%, starch 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%, surplus is a water, pH6.5-7.5, fermention medium: by weight percentage, starch 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%, surplus is a water, pH6.5-7.5.
The present invention also provides described mutant strain HP388 application in the preparation antibacterials.
The present invention continues screening repeatedly and tames through following, has finally obtained according to knob micromonospora HP388: the acclimation and screening of bacterial strain HP388, need a plurality of circulations, and each circulation mainly comprises following process:
Starting strain T125 (according to knob micromonospora Chengjiang mutation T125) → separation and purification → mycelium culture → protoplastis preparation → protoplastis merges → anti-self meta-bolites (sisomicin) gradient screening → mutant strain productivity measurement → gain mutant bacterial strain,
Obtain bacterial strain HP388 through repeated screening, amplify, obtain the sisomicin product through substratum and culture condition optimization → lab scale → pilot scale → industriallization.
Cultural characteristic according to knob micromonospora HP388 (CGMCC No.5921): this bacterial strain inclined-plane outward appearance is amaranth.Add gradient self meta-bolites sisomicin, concentration is controlled at about 50 ~ 1000 u/mL, can suppress the generation of its pigment, after domestication, finally becomes the bacterial strain that does not produce pigment, is faint yellow on the inclined-plane.
The present invention adopts the industrial micro breeding method, to carrying out seed selection and screening according to knob micromonospora T125, obtains the mutant strain of sisomicin fermentation stability high yield.This bacterial strain has been protected finish in China Committee for Culture Collection of Microorganisms's common micro-organisms center registration on March 19th, 2012, 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.5921.
The notable feature of mutant strain HP388 is that fermentation unit is high, and stable processing technique is convenient to control, and oxygen-consumption is little, and cost is low, pollute little, good product quality.
Description of drawings
Fig. 1 is according to knob micromonospora HP388 sisomicin biological synthesis gene cluster.
Fig. 2 is for to cut effect electrophoresis detection figure according to knob micromonospora 388 chromosomal DNAs and DNA enzyme; Wherein M: λ-
EcoT14 marker; 1:HP1102 karyomit(e); 2: the HP1102 karyomit(e) after enzyme is cut/
SauThe 3A I; 3: double digestion Supercos-1/
NheI/
BamThe H I; 4: single endonuclease digestion Supercos-1/
NheI.
Fig. 3 clones sub-PCR screening for sisomicin biological synthesis gene cluster target and confirms figure; M:DL5000 marker wherein; 1: the clone through the M1/M2 primer amplification is confirmed is sub; 2: the clone through the S1/S2 primer amplification is confirmed is sub; 3: the clone through the C1/C2 primer amplification is confirmed is sub.
Fig. 4 is the HPLC analytical results of 120301 batches of sisomicins.
Fig. 5 is the HPLC analytical results of 120302 batches of sisomicins.
Fig. 6 is the HPLC analytical results of 120303 batches of sisomicins.
Embodiment
Embodiment 1:
The acclimation and screening of bacterial strain HP388 of the present invention needs 10 more than the circulation at least, and each circulation mainly comprises following process:
Take out and send out the cultured inclined-plane of bacterial strain T125 (according to knob micromonospora Chengjiang mutation T125), add the 3ml sterilized water, lightly spore is scraped with inoculating needle; Take out spore suspension; Fully concussion makes to be dispersed into monospore on vibrator, carries out gradient dilution (10 then
0, 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7), spread plate after the uviolizing (10,20,30,40,50 seconds), in 37 ℃ of cultivations, separates single bacterium colony.When single colony growth (25 days) after can distinguish its characteristic, the bacterium colony of picking different shape characteristic carries out submerged fermentation and cultivates (6 days), and fermentation is finished, and detects fermentation unit, preserves the high person of unit, supplies next step use.
Get above quality strains, cultivate mycelia, the preparation protoplastis; Adopt single parent's deactivation self protoplastis fusion method (self protoplastis merges, with portion fire extinguishing back preparation protoplastis, the portion preparation protoplastis of living of not putting out a fire; Two parts of mixing then, put out a fire self protoplastis of promptly so-called single parent merges) carry out protoplastis and merge spread plate; In 37 ℃ cultivate long good bacterium colony after (25 days), scrape spore, as above prepare monospore suspension; Gradient dilution, spread plate is with the sisomicin solution covering of different gradients; Make that final concentration reaches 100,200,300 successively, ┉ ┉ 1000 μ g/ml (this process be called for short anti-self meta-bolites gradient screening and culturing), cultivate down in 37 ℃, until growing single bacterium colony; Get high resistance bacterium colony and carry out that the inclined-plane is lax to be cultivated, prepare single bacterium colony then, carry out anti-self meta-bolites gradient once more and cultivate 3 times repeatedly.Carry out single bacterium colony again and separate, the bacterial strain of different shape characteristic is carried out productivity measurement, get the high person of unit of fermentation preserve subsequent use, so far, first loop ends, and get into next circulation.
Again prepare monospore suspension; Carry out ultraviolet mutagenesis, single parent's fire fighting method self protoplastis merges, anti-self meta-bolites gradient screening; Productivity measurement; So moving in circles improves constantly the fermentation level of sisomicin, finally confirms as according to knob micromonospora HP388, and continues to produce the optimization of fermentation.
Mutant strain according to knob micromonospora HP388 through substratum and culture condition optimization → lab scale → pilot scale → industriallization amplification → preparation sisomicin product; Fermentation unit is stable; Can satisfy industrialization production; The highest fermentation unit can reach more than 1600 u/mL, than the highest fermentation unit of starting strain 197 ug/mL only, has improved more than 8 times.
Embodiment 2The acquisition and the determined dna sequence thereof of mutant strain HP388 sisomicin biological synthesis gene cluster
1 material
1.1 bacterial classification and plasmid be according to knob micromonospora HP388,
E.coliDH5 α and cosmid vector Supercos-1 (giving birth to the worker) available from Shanghai;
E.coliLE392 is available from Stratagene company.
1.2 reagent restriction enzyme, Proteinase K, calf intestine alkaline phosphatase (CIAP enzyme), T4 dna ligase and Taq enzyme are all available from TAKARA company; N,O-Diacetylmuramidase is given birth to the worker available from Shanghai; Phage packaging albumen extract (Gigapack III XL packaging extract) is available from Stratagene company.
1.3 substratum bacteria culture medium LB adds penbritin (final concentration is 100 μ g/ml) and kantlex (final concentration is 25 μ g/ml) as required; According to knob micromonospora substratum.
The library construction method
(a) according to the structure of knob micromonospora gene library: cosmid vector Supercos-1 is with restriction enzyme
NheI complete digestion and dephosphorylation obtain the single band (Fig. 2) of a treaty 7.9kb.Again with restriction enzyme
BamThe complete digestion of H I, electrophoresis detection obtain two fragments (Fig. 2) of 1.1kb and 6.8kb, reclaim this two fragments, and cryopreservation is subsequent use;
(b) extract completely according to knob micromonospora HP388 chromosomal DNA, electrophoresis detection obtains a single band that is distributed in about 100kb, reaches and satisfies the requirement that makes up the genome cosmid library.Utilize the restriction enzyme of 4 Nucleotide of identification
SauThe chromosomal DNA that the 3A I obtains extraction carries out part digestion, and the control size is between about 30 ~ 40kb (Fig. 2), and the dna fragmentation that is produced has the sticky end that is complementary with carrier, reclaims through postdigestive chromosomal DNA, and cryopreservation is subsequent use.
(c) with the mixed about (a)/(b)=1/5, with T
4The dna ligase connection of spending the night is got 5 μ l then and is connected product (about 1 μ g), carries out external the packing with phage packaging albumen, and infects
E.coliLE392 coats on the LB flat board that contains kantlex (final concentration is 25 μ g/ml) at last, and 37 ℃ of overnight cultures have obtained about 12500 transformants.
The biological synthesis gene cluster screening
Screening according to knob micromonospora sisomicin biological synthesis gene cluster: FJ160413 and the AJ628149 gene cluster announced with GenBank are template, design three couples of screening probe primer M (M1/M2), S (S1/S2) and C (C1/C2):
The primer that table 1 exemplifying embodiment is related
Gradient dilution is carried out in library to obtaining, and extent of dilution is respectively 10
-3, 10
-4, 10
-5, 10
-6, 10
-7With 10
-8Library with after the dilution is a template, utilizes probe primer: M1/M2, S1/S2 and C1/C2 to carry out the pcr amplification screening respectively.M1/M2 PCR cycling condition is: 95 ℃, and 5min; 95 ℃, 45S; 52.7 ℃, 1min; 72 ℃, 3min; 72 ℃, 10min, totally 30 circulations; S1/S2 PCR cycling condition is: 94 ℃, and 5min; 95 ℃, 1min; 59 ℃, 1min; 72 ℃, 1min; 72 ℃, 10min, totally 30 circulations; C1/C2 PCR cycling condition is: 94 ℃, and 5min; 95 ℃, 1min; 58 ℃, 1min; 72 ℃, 1min; 72 ℃, 10min, totally 30 circulations.Obtain band in line (Fig. 3) after the amplification.Get the greatest dilution that can amplify target stripe in the PCR operation: 10
-7Bacterium liquid, coat on the LB flat board that 3 wares contain kantlex (25 μ g/ml) 37 ℃ of overnight cultures.Obtain about 150 transformants, about 50 of every ware.Behind the replica plating, adopt the PCR method that single bacterium colony is carried out once more PCR and identify, until clone's that obtains containing sisomicin biological synthesis gene cluster (HP388).
The HP388 biological synthesis gene cluster: the sisomicin biological synthesis gene cluster of clone on the son entrusted the order-checking of TaKaRa company, and length overall is 51885bp, is JF431003 (as shown in Figure 1) in the registration number of GenBank.The homology of JF431003bp gene cluster and FJ160413 gene cluster is very high, but bigger difference is arranged.Variation has in various degree taken place in the dna sequence dna of HP388 biological synthesis gene cluster, but the compositional model of ORF is similar with starting strain, and significantly improving and optimize of HP388 biosynthesizing sisomicin ability is described, and is relevant with the variation of gene.
Embodiment 3:Preparation according to knob micromonospora HP388 meta-bolites sisomicin (sisomicin)
Stable high yield engineering bacteria provided by the invention directly is used to produce sisomicin according to knob micromonospora HP388, as antibacterials and synthetic netilmicin.
1. according to the fermentation culture of knob micromonospora HP388 bacterial strain
Slant medium (%): by weight percentage, Zulkovsky starch 1.5%, wheat bran 1.5%, MgSO
46H
2O 0.03%, KNO
30.3%, K
2HPO
43H
2O 0.03%, and NaCl 0.05%, CaCO
30.4%, agar 2%, pH7.5.
Seed culture medium: glucose 0.1%, ground melon starch 1.0%, Semen Maydis powder 1.5%, peptone 0.2%, soybean cake powder 1.0%, KNO
30.05%, CaCO
30.5%, pH7.0.
Fermention medium: ground melon starch 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.
Fermentation according to knob micromonospora HP388.Earlier isolate fine list bacterium colony with dilution-plate method before the fermentation, and transfer in slant medium, 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).Plant in fermention medium (loading amount is the 50mL/250mL triangular flask) by the commentaries on classics of 10% inoculum size, 37 ℃ of shaking tables are cultivated 124 h (rotating speed is 280rpm).
50000 liters of fermentor tank productions, 180 rev/mins of mixing speed, air flow 1:0.2 ~ 1.0 (M
3/ M
3Min), substratum, culture temperature, the inoculum size ratio, fermentation times etc. are analogous to shake flask fermentation.
2 meta-bolitess extract refining
A, slightly carry
After acidifying respectively after the fermented liquid dilution, alkalization make protein denaturation, with 732 resin Static Adsorption 6 ~ 8 hours.Collect the absorption saturated resin, with 0.5M HCl solution pickling saturated resin, again with the vaal water washing to neutral, then carry out alkali cleaning with 0.01% ammoniacal liquor, when effluent reaches pH 9.0 when above, be connected in series on isopyknic 711 resin columns the collection elutriant.
B, refining, crystallization
Elutriant, transfers to pH5.5 ~ 6.0. activated carbon decolorizing with the vitriol oil and reaches more than 92%, under agitation to transparence to about 300000ug/mL through thin film concentration; Slowly in liquid concentrator, drip 95% above ethanol; Carry out crystallization, 10-14 hours time, separate through whizzer afterwards; 85% ethanolic soln drip washing promptly gets wet finished product.Wet finished product vacuum-drying (more than the vacuum tightness 500mmHg, temperature 600C, dry 6 hours) promptly gets the Sissomicin sulfate finished product, and yield is more than 80%; And the highest fermentation unit of starting strain 197 ug/mL only, yield is less than 20%.Production cost is high, and quality product is difficult to guarantee.
3. product analysis
Carry out according to Pharmacopoeia of People's Republic of China version in 2010, meet statutory standards.Attach 3 parts of examining reports, table 3 (HPLC Fig. 4), table 4 (HPLC Fig. 5), table 5 (HPLC Fig. 6).
Table 3 Sissomicin sulfate examining report book 1
Table 4 Sissomicin sulfate examining report book 2
Table 5 Sissomicin sulfate examining report book 3
Reference:
Ge Xiangbin, Wang Yuhong, Jiang Guixiang, Liang Yu. the seed selection of resisting high-concentration qingfengmeisu qiong bacterial classification, Chinese microbiotic magazine (2002), 27 (05) 311-312.
Liao Aifang. qingfengmeisu qiong produces bacterium 23-18 Study on mutagenesis breeding, Chinese microbiotic magazine (2002), 27 (04): 199-201.
Long Shihong, Deng Bin. the ultraviolet mutagenesis qingfengmeisu qiong produces the research of bacterium breeding and fermentation condition. the pedagogical higher junior college's journal (2002) in Chenzhou, 23 (05): 120-122.
Xu Zuoying utilizes anti-self metabolite characteristic to carry out the Sisomicin strain improvement, Sichuan Teachers University journal (natural science edition) (1997), and 20 (5): 68-72,
Wen-Rong?Hong,?Mei?Ge,?Zhi-Hong?Zeng?et?al.?Molecular?cloning?and?sequence?analysis?of?the?sisomicin?biosynthetic?gene?cluster?from?Micromonospora?inyoensis.?Biotechnol?Lett?(2009)?31:449–455。
< 110>University of Fuzhou, Zhejiang Zhenyuan Pharmaceutical Co., Ltd
The sisomicin mutant strain is produced in < 120>one strains
<160> 6
<170> PatentIn?version?3.3
<210> 1
<211> 18
<212> DNA
< 213>artificial sequence
<400> 1
atgggcggca?tgcacgtg 18
<210> 2
<211> 21
<212> DNA
< 213>artificial sequence
<400> 2
tcaggactcc?tccatgaggg?a?21
<210> 3
<211> 20
<212> DNA
< 213>artificial sequence
<400> 3
tccaaagagg?gtgggatacc 20
<210> 4
<211> 20
<212> DNA
< 213>artificial sequence
<400> 4
aagctgtgat?cccagggtgt 20
<210> 5
<211> 19
<212> DNA
< 213>artificial sequence
<400> 5
ctccgcacct?ccagtcgcc 19
<210> 6
<211> 19
<212> DNA
< 213>artificial sequence
<400> 6
cggtcagcgt?cttcctgcc 19
Claims (3)
1. the mutant strain of sisomicin is produced in a strain, it is characterized in that, said mutant strain be according to the knob micromonospora (
Micromonospora inyoensis) HP 388, register preservation on March 19th, 2012 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.5921.
2. the fermentation process of the mutant strain of a product sisomicin as claimed in claim 1 is characterized in that, according to the knob micromonospora (
Micromonospora inyoensis) isolate single bacterium colony with dilution-plate method earlier before the HP388 fermentation after, transfer again in slant medium, cultivated 2-5 days for 35-40 ℃; Inoculum size with 4-6% is inoculated in seed culture medium again, and 35-40 ℃ of shaking table cultivated 18-48 h, and rotating speed is 260-300rpm, transfers in fermention medium by the 8-12% inoculum size then, and 35-40 ℃ of shaking table cultivated 4-5 days, and rotating speed is 260-300rpm; Said slant medium: by weight percentage, Zulkovsky starch 1.0-2.0%, wheat bran 1.0-2.0%, MgSO
46H
2O 0.01-0.05%, KNO
30.1-0.5%, K
2HPO
43H
2O 0.01-0.05%, NaCl 0.01-0.09%, CaCO
30.1-0.50%, agar 1.8-2.2%, surplus is a water, pH7-8; Seed culture medium: by weight percentage, glucose 0.1-1.0%, starch 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%, surplus is a water, pH6.5-7.5, fermention medium: by weight percentage, starch 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%, surplus is a water, pH6.5-7.5.
3. the application of the mutant strain of product sisomicin as claimed in claim 1 in the preparation antibacterials.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978133A (en) * | 2012-11-16 | 2013-03-20 | 中国科学院南海海洋研究所 | Micromonospora Rosaria and method for preparing a plurality of antibiotics by Micromonospora Rosaria |
| CN111893154A (en) * | 2020-08-14 | 2020-11-06 | 卓和药业集团有限公司 | Method for producing sisomicin |
| CN112899199A (en) * | 2021-03-01 | 2021-06-04 | 中国医学科学院医药生物技术研究所 | Micromonospora TMD166 and application thereof |
-
2012
- 2012-04-06 CN CN 201210099020 patent/CN102618473B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
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| GOLDBERG SL 等: "伊尼奥小单胞菌产生的西索米星抗性基因克隆及其特性", 《国外医药抗生素分册》 * |
| 廖爱芳等: "西索米星产生菌小单胞菌的诱变育种研究", 《中国抗生素杂质》 * |
| 朱坚屏等: "提高西索米星产生菌产量的遗传育种", 《中国抗生素杂质》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978133A (en) * | 2012-11-16 | 2013-03-20 | 中国科学院南海海洋研究所 | Micromonospora Rosaria and method for preparing a plurality of antibiotics by Micromonospora Rosaria |
| CN102978133B (en) * | 2012-11-16 | 2014-08-06 | 中国科学院南海海洋研究所 | Micromonospora Rosaria and method for preparing a plurality of antibiotics by Micromonospora Rosaria |
| CN111893154A (en) * | 2020-08-14 | 2020-11-06 | 卓和药业集团有限公司 | Method for producing sisomicin |
| CN112899199A (en) * | 2021-03-01 | 2021-06-04 | 中国医学科学院医药生物技术研究所 | Micromonospora TMD166 and application thereof |
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| CN102618473B (en) | 2013-04-03 |
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