CN103937694B - The bacterial strain of high expression level daptomycin and screening method thereof - Google Patents
The bacterial strain of high expression level daptomycin and screening method thereof Download PDFInfo
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Abstract
The present invention relates to bacterial strain and the screening method thereof of high expression level daptomycin, belong to production of antibiotics bacterial strain and screening field thereof.Does is the Classification And Nomenclature of bacterial strain Streptomyces roseosporus (Streptomyces? roseosporus), being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, does is deposit number respectively CGMCC? NO.6899, NO.7014 and NO.7015.Present invention also offers a kind of screening method of bacterial strain of high expression level daptomycin, ultraviolet mutagenesis combination antibiotic resistance screening is adopted to increase the sudden change probability of bacterial strain, the bacterial strain of the high expression level daptomycin obtained produces the output of daptomycin all at more than 59.86mg/L, can up to 79.44mg/L.The present invention can improve daptomycin production level quickly and efficiently, is of great practical significance.
Description
Technical field
The present invention relates to bacterial strain and the screening method thereof of high expression level daptomycin, belong to production of antibiotics bacterial strain and screening field thereof.
Background technology
Daptomycin is N-phthalidyl in the last of the ten Heavenly stems derivative of A21978, is produced by Streptomyces roseosporus metabolism.The fatty acid chain of the cyclic lipopeptide that daptomycin molecule is made up of 13 amino acid and ten carbon formed.When A-21978C generation deacylation sloughs N-terminal linking group, A-21978C core can be obtained, and the N-terminal of A21978C core can obtain daptomycin (structural formula is as follows) again with after capric acid generation acylation.
Daptomycin structural formula
In the past in four more than ten years, in anti-infectives field, represent the microbial medicine that brand new classification enters clinical application, having only in order to naphthalene azoles amine is the azoles gastral cavity ketone antibiotic of representative, and is the Cyclic lipopeptide antibiotic of representative with daptomycin.But just there is the report of crossing drug resistant bacterium in sharp naphthalene azoles amine listing, the listing of daptomycin, has enriched existing microbiotic structured sort less than 1 year.Daptomycin novel structure, mechanism of action is special, it has most gram-positive microorganism kills activity fast, comprise MRSA, VRSA, VISA and vancomycin-resistant enterococcus (VRE) etc., it can form ionic channel on gram-positive bacteria cell film, destroy membrane potential, upset cytolemma to amino acid whose transhipment, the biosynthesizing hindering bacteria cell wall peptidoglycan and cell wall acid esters, thus kill bacterium.The cyclic lipopeptide structure of its uniqueness and mechanism of action, make it can not be subject to, from the impact of cross resistance caused by other microbiotic, having wide market outlook.In September, 2003, because patient is to the active demand of new antibiotic, U.S. FDA is used for the treatment of the concurrency skin and skin structure infection that are caused by some Gram-positive sensitive strains, as abscess, surgery cut infection and skin ulcer through quick trial program approval injection daptomycin (trade(brand)name Cubicin).In succession have approved again daptomycin afterwards and be used for the treatment of the microbemia and right side endocarditis that S. aureus L-forms causes.The application method of daptomycin is convenient, and toxic side effect is little, becomes the best substitute of " pathogenic bacteria last line of defense---vancomycin " clinically.
At present, it is semi-synthetic etc. that the preparation method of daptomycin mainly contains natural fermented method, precursor revulsion, chemosynthesis, enzyme process, due to the distinctive cyclic lipopeptide structure of daptomycin, and containing special acid composition, thus fermentable produces daptomycin or method comparatively conventional at present.Streptomyces roseosporus (ATCC31568) is as the original strain producing daptomycin, its throughput is lower, cannot meet the need of market, the currently reported biological method of combination that utilizes is carried out directional transformation to daptomycin structure or is realized the heterogenous expression of its whole synthetic gene bunch, although these methods are with clearly defined objective, but complicated operation, only rest on laboratory level at present, industrial production cannot be put into, so utilize traditional method to produce bacterium to daptomycin carry out strain improvement, promote daptomycin production level, be of great practical significance.
In microbiotic bacterial classification Breeding Process, antibiotics resistance screening is one of main method.Due to production of antibiotics gene and the arrangement of resistant gene cluster, and sudden change altogether easily occurs, these sudden changes directly can improve the throughput of production of antibiotics bacterium.Therefore the dependency utilizing antibiotics generated bacterium to produce between anti-ability and its resistant mutation is screened, and can reduce the blindness of microbiotic Producing Strain screening to a great extent, improve the screening efficiency to object bacterium.
Ultraviolet mutagenesis technology is one of method commonly used in microbial strains Breeding Process, but, use ultraviolet mutagenesis to there is no report in conjunction with the method for Rifampin/gentamicin to improve daptomycin production level.
Summary of the invention
In order to improve the production level of producing daptomycin, the invention provides the bacterial strain of three kinds of high expression level daptomycins, the daptomycin that output is up to 79.44mg/L can be obtained.
A kind of bacterial strain Ge-27 of high expression level daptomycin, for the production of daptomycin, the Classification And Nomenclature of this bacterial strain Ge-27 is Streptomyces roseosporus (Streptomycesroseosporus), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on November 30th, 2012, address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.6899.This bacterial classification can obtain the daptomycin that output reaches 79.44mg/L.
The bacterial strain RG-4 of another kind of high expression level daptomycin, for the production of daptomycin, the Classification And Nomenclature of this bacterial strain RG-4 is Streptomyces roseosporus (Streptomycesroseosporus), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 18th, 2012, address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.7014.This bacterial classification can obtain the daptomycin that output reaches 77.87mg/L.
The bacterial strain Ri-5 of another high expression level daptomycin, for the production of daptomycin, the Classification And Nomenclature of this bacterial strain Ri-5 is Streptomyces roseosporus (Streptomycesroseosporus), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 18th, 2012, address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCCNO.7015.This bacterial classification can obtain the daptomycin that output reaches 75.07mg/L.
Present invention also offers a kind of screening method for screening high expression level daptomycin bacterial strain, utilizing the method, efficiently can filter out the bacterial strain of high expression level daptomycin, thus promote daptomycin production level.The method fast and effeciently can improve the production level of daptomycin.
A screening method for the bacterial strain of high expression level daptomycin, comprises the steps:
(1) screening has the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance;
(2) from described, there is the bacterial strain screening high expression level daptomycin the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance.
In step (1), the starting strain that screening has the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance can be Streptomyces roseosporus ATCC31586, buy in American Type Culture Collecti (ATCC), be numbered StreptomycesroseosporusATCC31586, this bacterial strain is the original strain producing daptomycin.
Preferably, screening has the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance is screen from the Streptomyces roseosporus bacterial strain through mutagenic treatment.By mutagenesis, increase the chance obtaining high expression level bacterial strain.
Utilize aforesaid method, the bacterial strain of the high expression level daptomycin obtained produces the output of daptomycin at about more than 59.86mg/L.
Bacterial strain Ge-27, RG-4, Ri-5 of the above-mentioned high expression level daptomycin of the present invention also can be used as starting strain, utilize method of the present invention screening to obtain output further higher, namely higher than the mutant strain of about 79.44mg/L.
Mutagenesis and antibiotics resistance screening are carried out to starting strain (original strain) Streptomyces roseosporus (ATCC31568) producing daptomycin, comprises the steps:
1) Streptomyces roseosporus spore suspension or the Streptomyces roseosporus spore suspension after mutagenic treatment are coated the solid plate containing Rifampin and/or gentamicin, cultivate, filter out the Streptomyces roseosporus mutant strain of anti-Rifampin and/or gentamicin;
2) select the Streptomyces roseosporus mutant strain list bacterium colony of anti-Rifampin and/or gentamicin respectively, carry out shake flask fermentation and detect daptomycin content, screening the bacterial strain increased than starting strain throughput, obtaining the bacterial strain of high expression level daptomycin.
In step 1), described Streptomyces roseosporus spore suspension preparation method comprises: (a) collects spore with physiological saline, and gained spore liquid is diluted 100 times and poured into and be equipped with in the sterilizing triangular flask of granulated glass sphere; (b) 30 DEG C, 200rpm vibrates 20min, makes spore disperse activation; C () filters through sterilized non-fat cotton, obtained monospore suspension 10
5-10
6individual/mL.
Specifically, in ripe Spore cultivation ware (90mm) of Streptomyces roseosporus, add 5 ~ 6mL physiological saline, scrape spore, (concentration is generally 10 to gained spore liquid
9-10
10individual/mL) with after normal saline dilution 100 times, pour into and be equipped with in the sterilizing triangular flask of granulated glass sphere, in 30 DEG C, 200rpm vibrates 20 ~ 40min, make spore disperse activation, filter through sterilized non-fat cotton, obtained monospore suspension, with blood counting chamber counting, spore concentration is made to be 10
5-10
6individual/mL.
In one embodiment, be about 0.4 to about 1.8 μ g/mL for screening the Concentration of Rifampicin of the Streptomyces roseosporus bacterial strain with rifampicin resistance.Namely the concentration of the Rifampin applied during preliminary screening on solid plate is 0.4 ~ 1.8 μ g/mL, and screening determines that its minimal inhibitory concentration (MIC) is 1.4 μ g/mL.
In another embodiment, be about 0.4 to about 1.1 μ g/mL for screening the gentamicin concentration of the Streptomyces roseosporus bacterial strain with gentamicin resistance.Namely the gentamicin used during preliminary screening concentration on solid plate is 0.4 ~ 1.1 μ g/mL, determines that its minimal inhibitory concentration (MIC) is 1.0 μ g/mL.
Another preferred embodiment in, be about 0.1 to about 1.0 μ g/mL for screening the gentamicin concentration of the Streptomyces roseosporus bacterial strain with gentamicin and rifampicin resistance, simultaneously Concentration of Rifampicin is about 0.14 to 1.4 μ g/mL.Namely Rifampin used during preliminary screening and the concentration of these two kinds of antibiotic combinations of gentamicin on solid plate are 0.1 × MIC ~ 1.0 × MIC, and namely gentamicin concentration is about 0.1 to about 1.0 μ g/mL, and Concentration of Rifampicin is about 0.14 to 1.4 μ g/mL simultaneously.Determine that its minimal inhibitory concentration is 0.6 × MIC, namely the while of Rifampin 0.84 μ g/mL, gentamicin is 0.6 μ g/mL.
Described solid plate is Gause I substratum.
In step 1), described mutagenic treatment is ultraviolet mutagenesis: the Streptomyces roseosporus spore suspension getting the above-mentioned preparation of 5 ~ 6mL irradiates (power 30W in sterile petri dish medium ultraviolet, distance 30cm), irradiation time is 20s ~ 70s, spread plate after spore suspension after process dilutes 10 times, 5d(days is cultivated in 30 DEG C of darkrooms) ~ 7d(days).
In step 2) in, select the Streptomyces roseosporus mutant strain list bacterium colony of anti-Rifampin and/or gentamicin respectively, described single bacterium colony is that spore is plentiful, single bacterium colony that bacterium colony is larger; Described shake flask fermentation is that the single bacterium colony selected is moved to seed culture medium, is forwarded to fermention medium after amplification culture; The detection method of described detection daptomycin content is for first using HPLC(high performance liquid chromatography) external standard method detection, then verify further with Odontothrips loti; Screen the bacterial strain higher than starting strain throughput.Utilize the method, more fast and effeciently can obtain the object bacterial strain that output is higher.
In order to effectively promote daptomycin production level in the present invention, shake flask fermentation be first picking individual colonies to seed culture medium, be forwarded to fermention medium after amplification culture.Wherein seed culture medium is: analysis for soybean powder 0.5%, yeast powder 0.5%, calglucon 1%, KCl0.02%, MgSO
40.02%, defoamer 0.03%, pH7.0; Fermention medium is: analysis for soybean powder 2.2%, Fe (NH
4)
2sO
40.66%, dextrin 4.125%, molasses 0.275%, pH7.0.
The present invention is in order to accurately detect daptomycin content in fermented liquid, step 2) in detection method be first use HPLC external standard method to detect, then use Odontothrips loti to carry out Bactericidal test to fermented liquid to verify further, the indicator that wherein Bactericidal test uses is subtilis (BS168).
The dependency that the present invention utilizes antibiotics generated bacterium to produce between anti-ability and its resistant mutation is screened, and filters out the Streptomyces roseosporus mutant strain of anti-Rifampin and/or gentamicin; Decrease the blindness of microbiotic Producing Strain screening to a great extent, improve the screening efficiency to object bacterium; Use ultraviolet mutagenesis to improve daptomycin production level in conjunction with the method for Rifampin/gentamicin simultaneously.
Utilize the inventive method efficiently can filter out the bacterial strain of high expression level daptomycin, screen the bacterial strain obtained and include but not limited to Ge-27.The resistant strain shake flask fermentation production peak screened is 79.44mg/L, improves 42.2% than starting strain.Present method can improve daptomycin production level quickly and efficiently, is of great practical significance.
Accompanying drawing explanation
Fig. 1 is the graph of a relation of the ultraviolet mutagenesis dosage of Streptomyces roseosporus and lethality rate, positive mutation rate.
Fig. 2 is the starting strain and Ge-27 fermented liquid bacteriostatic activity comparison diagram that adopt Bactericidal test to record.
Fig. 3-1 and Fig. 3-2 is respectively daptomycin standard substance (A) and mutagenesis obtained strains Ge-27(B) fermentation broth sample HPLC comparison diagram.
Embodiment
The bacterial strain Ge-27 of high expression level daptomycin, Classification And Nomenclature: Streptomyces roseosporus (Streptomycesroseosporus), preservation date: on November 30th, 2012, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number: CGMCCNO.6899.
The bacterial strain RG-4 of high expression level daptomycin, Classification And Nomenclature: Streptomyces roseosporus (Streptomycesroseosporus), preservation date: on December 18th, 2012, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number: CGMCCNO.7014.
The bacterial strain Ri-5 of high expression level daptomycin, Classification And Nomenclature: Streptomyces roseosporus (Streptomycesroseosporus), preservation date: on December 18th, 2012, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number: CGMCCNO.7015.
By embodiment, the present invention is further described by reference to the accompanying drawings below.
The screening method of the bacterial strain of high expression level daptomycin of the present invention, first, screening has the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance; Then, from the Streptomyces roseosporus bacterial strain with Rifampin and/or gentamicin resistance, screen the bacterial strain of high expression level daptomycin.
In the present embodiment, the starting strain that screening has the Streptomyces roseosporus bacterial strain of Rifampin and/or gentamicin resistance selects Streptomyces roseosporus ATCC31586, buy in American Type Culture Collecti (ATCC), be numbered StreptomycesroseosporusATCC31586, this bacterial strain is the original strain producing daptomycin.
Prepare Streptomyces roseosporus spore suspension: in ripe Spore cultivation ware (90mm) of Streptomyces roseosporus, add 5mL physiological saline, scrape spore, (concentration is generally 10 to gained spore liquid
9-10
10individual/mL) with after normal saline dilution 100 times, pour into and be equipped with in the sterilizing triangular flask of granulated glass sphere, in 30 DEG C, 200rpm vibrates 30min, make spore disperse activation, filter through sterilized non-fat cotton, obtained monospore suspension, with blood counting chamber counting, spore concentration is made to be 10
5-10
6individual/mL.
One, the mensuration of Rifampin and gentamicin minimal inhibitory concentration
Prepare Gause I solid medium, wherein composition proportion is in table 1.Get Rifampin respectively and gentamicin standard substance add Gause I substratum, be down flat plate, make the Concentration of Rifampicin gradient plate of 0.4 μ g/mL, 0.6 μ g/mL, 0.8 μ g/mL, 1.0 μ g/mL, 1.2 μ g/mL, 1.4 μ g/mL, 1.6 μ g/mL and 1.8 μ g/mL, and 0.4 μ g/mL, 0.5 μ g/mL, 0.6 μ g/mL, 0.7 μ g/mL, 0.8 μ g/mL, 0.9 μ g/mL, 1.0 μ g/mL and 1.1 μ g/mL gentamicin concentration gradient plate, the flat board of not added with antibiotic is set simultaneously in contrast.To be coated on prepared flat board after Streptomyces roseosporus spore suspension dilution 10 ~ 100 times, after cultivating 5d, observe, and calculate lethality rate.The tolerance of Streptomyces roseosporus to Rifampin and gentamicin measures with reference to table 2.
The formula of table 1 Gause I solid medium
| Zulkovsky starch | 20g/L |
| KNO 3 | 1g/L |
| Nacl | 0.5g/L |
| K 2HPO 4·3H 2O | 0.5g/L |
| MgSO 4·7H 2O | 0.5g/L |
| FeSO 4.7H 2O | 0.1g/L |
| Agar powder | 15g/L |
The tolerance of table 2 Streptomyces roseosporus to Rifampin and gentamicin measures
| Rifampin (μ g/mL) | Lethality rate (%) | Gentamicin (μ g/mL) | Lethality rate (%) |
| 0.4 | 43 | 0.4 | 51 |
| 0.6 | 57 | 0.5 | 64 |
| 0.8 | 70 | 0.6 | 73 |
| 1.0 | 84 | 0.7 | 85 |
| 1.2 | 93 | 0.8 | 92 |
| 1.4 | 100 | 0.9 | 98 |
| 1.6 | 100 | 1.0 | 100 |
| 1.8 | 100 | 1.1 | 100 |
Known according to table 2: Rifampin and the MIC value of gentamicin to starting strain are respectively 1.4 μ g/mL, 1.0 μ g/mL.
Two, Rifampin and gentamicin are to the compound minimal inhibitory concentration of Streptomyces roseosporus
Prepare Gause I solid medium, add Rifampin and gentamicin to substratum simultaneously, be down flat plate, make the gradient plate of two kinds of independent minimal inhibitory concentrations of antibiosis: 0.1 × MIC, 0.2 × MIC, 0.3 × MIC, 0.4 × MIC, 0.5 × MIC, 0.6 × MIC, 0.8 × MIC and 1.0 × MI.To be coated on prepared flat board after Streptomyces roseosporus spore suspension dilution 10 ~ 100 times, after cultivating 5d, observe, and calculate lethality rate.Streptomyces roseosporus measures with reference to table 3 tolerance of Rifampin and gentamicin simultaneously.
Table 3 Streptomyces roseosporus measures the tolerance of Rifampin and gentamicin simultaneously
| Rifampin & gentamicin (μ g/mL) | Lethality rate (%) |
| 0.1×MIC | 49 |
| 0.2×MIC | 61 |
| 0.3×MIC | 70 |
| 0.4×MIC | 83 |
| 0.5×MIC | 95 |
| 0.6×MIC | 100 |
| 0.8×MIC | 100 |
| 1.0×MIC | 100 |
Known according to table 3: the minimal inhibitory concentration of two kinds of antibiotic combinations is 0.6 × MIC.
Three, the best ultraviolet mutagenesis time is determined
Get the plate being equipped with 5mL Streptomyces roseosporus spore suspension, through uv irradiating, (power is 30W respectively, distance is 30cm) after 20s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 70s, with the spore suspension without uv irradiating as a control group, be coated with non-resistant flat board and (prepare Gause I solid medium, be down flat plate), add up lethality rate and positive mutation rate after cultivating 5d in juxtaposition 30 DEG C of camera bellows, to determine best mutation time.Fig. 1 is the determination result of ultraviolet mutagenesis time.
As shown in Figure 1, when mutation time is 45s, lethality rate is 78%, and positive mutation rate is the highest, therefore this experiment adopts 45s to be the ultraviolet mutagenesis time.
Four, ultraviolet mutagenesis combination antibiotic resistance screening
Described mutagenic treatment is ultraviolet mutagenesis, and uv irradiating power is 30W, and distance is 30cm, and irradiation time is 20s ~ 70s
Get the plate being equipped with 5mL Streptomyces roseosporus spore suspension, after uv irradiating 45s, with the spore suspension without uv irradiating as a control group, the resistant panel of minimal inhibitory concentration of coating containing Rifampin, gentamicin and the two combination respectively, after cultivating 5d in juxtaposition 30 DEG C of camera bellows, picking colony is comparatively large, the plumpish single bacterium colony of spore; Shake flask fermentation, obtains fermented liquid, is specially: picking individual colonies, to seed culture medium, is forwarded to fermention medium after amplification culture (30 DEG C, 48h), cultivates (30 DEG C, 7d).Wherein seed culture medium is: analysis for soybean powder 0.5%, yeast powder 0.5%, calglucon 1%, KCl0.02%, MgSO
40.02%, defoamer 0.03%, pH7.0; Fermention medium is: analysis for soybean powder 2.2%, Fe (NH
4)
2sO
40.66%, dextrin 4.125%, molasses 0.275%, pH7.0.Then, with HPLC external standard method daptomycin content, wherein, analytical column is anti-phase C18 post; Moving phase is for respectively containing 0.1(v) acetonitrile of % trifluoroacetic acid and water (volume ratio is 45:54), flow velocity 1mL/min; Column temperature adopts 30 DEG C; Detector adopts 220nm UV-light to detect.Table 4 is the positive mutating strain of antibiotics resistance screening under ultraviolet mutagenesis condition.
The positive mutating strain of antibiotics resistance screening under table 4 ultraviolet mutagenesis condition
As shown in table 4, adopt ultraviolet mutagenesis combination antibiotic resistance screening to add the sudden change probability of bacterial strain, the bacterial strain of the high expression level daptomycin obtained produces the output of daptomycin all at more than 59.86mg/L, and wherein Ge-27 fermentation level amplification is maximum, amplification 42.2%, output reaches 79.44mg/L.
Five, the Bactericidal test of fermented liquid
Use subtilis (BS168) as indicator, drawing 0.6mL bacteria concentration is 10
8the subtilis bacteria suspension of individual/mL, on flat board, leaves standstill with after spreader coating evenly, makes it naturally dry up; With tweezers, aseptic Oxford cup is put into culture dish gently; Draw the fermented liquid after the sterile filtration of certain volume in the cup of Oxford; Carefully culture dish is moved in incubator, cultivate 16h for 37 DEG C; Observe fungistatic effect.Fig. 2 is the original strain (starting strain) and Ge-27 fermented liquid bacteriostatic activity comparison diagram that adopt Bactericidal test to record.As shown in Figure 2, the fungistatic effect of Ge-27 fermented liquid is remarkable, compared with original strain, has had obvious lifting.
Six, HPLC compares
With foregoing HPLC external standard method daptomycin standard substance (A) and mutagenesis obtained strains Ge-27(B) the daptomycin content of fermentation broth sample.Fig. 3-1 and Fig. 3-2 is respectively daptomycin standard substance (A) and mutagenesis obtained strains Ge-27(B) fermentation broth sample HPLC comparison diagram.X-coordinate is appearance time/min, and ordinate zou is relative peak area.Visible Ge-27 goes out peak more early, and output is higher.
Bacterial strain Ge-27, RG-4, Ri-5 of the high expression level daptomycin that the present invention obtains also can be used as starting strain, utilize method of the present invention screening to obtain output further higher, namely higher than the mutant strain of about 79.44mg/L.
Although with reference to above embodiments illustrating aforementioned embodiments of the present invention, the present invention has been not limited to these embodiments and can various different form have implemented.It will be understood by those skilled in the art that except illustrating in specification sheets, under the condition not changing technical spirit of the present invention or essential characteristic, the present invention embodied in other can realize technique effect of the present invention.Therefore, these embodiments are interpreted as illustrative in every respect, should not consider in a restricted way.
Claims (3)
1. a bacterial strain for high expression level daptomycin, its Classification And Nomenclature is Streptomyces roseosporus (Streptomycesroseosporus), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNO.6899.
2. the bacterial strain of high expression level daptomycin as claimed in claim 1 is producing the application in daptomycin.
3. the bacterial strain of high expression level daptomycin as claimed in claim 2 is producing the application in daptomycin, it is characterized in that: the output that described bacterial strain produces daptomycin reaches 79.44mg/L.
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| CN101892287B (en) * | 2009-05-19 | 2015-06-17 | 上海医药工业研究院 | Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains |
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| CN102586147A (en) * | 2012-02-17 | 2012-07-18 | 厦门大学 | Method for screening streptomyces roseosporus capable of producing Daptomycin at high yield |
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