CN101748178B - Process for producing aromatic polyene antibiotic through fermentation - Google Patents
Process for producing aromatic polyene antibiotic through fermentation Download PDFInfo
- Publication number
- CN101748178B CN101748178B CN 200810204283 CN200810204283A CN101748178B CN 101748178 B CN101748178 B CN 101748178B CN 200810204283 CN200810204283 CN 200810204283 CN 200810204283 A CN200810204283 A CN 200810204283A CN 101748178 B CN101748178 B CN 101748178B
- Authority
- CN
- China
- Prior art keywords
- antibiotic
- aromatic polyene
- fermentation
- polyene antibiotic
- engineering bacteria
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a process for producing aromatic polyene antibiotic through fermentation. The method comprises the following steps: in the initial process of growing engineering bacteria, cultivating the engineering bacteria under the condition from neutrality to alkalescence so as to promote the synthesis of an aromatic side chain precursor of the antibiotic, namely p-aminobenzoic acid; and continuously fermenting under the condition from alkalescence to neutrality, and converting the p-aminobenzoic acid into the aromatic polyene antibiotic. The method can effectively improve the yield of the aromatic polyene antibiotic and reduce byproducts.
Description
Technical field
The present invention relates to the fermentation field, relate more specifically to a kind of fermentation method for producing of aromatic polyene antibiotic.
Background technology
Aromatic polyene antibiotic is a class microbiotic, and it is mainly by interacting with the sterol of eukaryotic cell membrane, causes that small molecules loses and cause necrocytosis thereby form passage.
Aromatic polyene macrolide antifungal antibiotic such as amphotericin are restricted in application, and its principal element is that its toxicity is too strong, especially renal toxicity.
Find that in the pharmacological research of aromatic polyene macrolide antifungal antibiotic such as amphotericin the C-16 position of chemosynthesis is that the amphotericin derivative of methyl chains reduces greatly than the toxicity of amphotericin (its C-16 position is carboxylic side-chain).
People such as Liang Rongfang have found a kind ofly new can produce antibiotic streptomycete bacterial strain in research farming anti-5192 produces the process of bacterium protoplast fusion breedings, i.e. streptomycete FR-008.Show through chemical structure analysis, the secondary metabolite FR-008 of this streptomycete bacterial strain, with the chemical structure of candicidin (Candicidin) be identical (Liang Rongfang, week opens. the researchs that farming anti-5102 produces the bacterium protoplast fusion breedings.The biotechnology journal, 1987,3,130-136; Application number is the Chinese patent application " seven alkene macrolide polyketone antibiotic FR-008 mixtures " of CN200310108746.6).
Compare with amphotericin, the aromatic nucleus side chain, 38 membered macrolides and side chain and glycosyl are all very similar, therefore except many for meta-bolites FR-008 or candicidin, after the outer carboxyl of FR-008 or candicidin ring was taken off, the toxicity of its polyketone antibiotic of deriving also reduced greatly.
People such as the new academician of the Deng Zi of Shanghai Communications University adopt the combination biosynthetic means, have synthesized a series of microbiotic FR-008 or candicidin genetically engineered derivative compound.Interrupting mutant strain CS101, ketose aminotransferase gene fscMII comprising glycosyltransferase gene fscMI interrupts mutant strain CS102 and FscP gene fscP gene and interrupts mutant strain CS103 genetically engineered derivative CS101, the CS102 and the CS103 that obtain of fermentation culture respectively.These compounds have stronger anti-mycotic activity, and wherein some toxicity of compound reduces greatly than FR-008 or candicidin, have higher clinical development prospect.Be that the Chinese patent application " seven alkene macrolide polyketone antibiotic FR-008 mixtures " of CN200310108746.6, the Chinese patent application that application number is CN200310108745.1 " desugar and deamination sugar FR-008 derive polyketone antibiotic " and application number are the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0 referring to application number.These compounds can be used to prevent prostatauxe and kill mosquitoes larva and are used for preventing that trichomonas vaginitis from infecting and candidal vaginitis (claiming colpitis mycotica again).But these seven alkene macrolide polyketone antibiotics are antibacterium also.
Yet the output of producing these aromatic polyene antibiotics by genetically engineered is very low, usually only about the 10mg/L fermented liquid, thereby has restricted the process of clinical trial and the industrialization of these aromatic polyene antibiotics.
In sum, this area presses for the method that exploitation is a kind of efficiently, pass through the fermentative production aromatic polyene antibiotic.
Summary of the invention
Purpose of the present invention just provide a kind of efficiently, the method by the fermentative production aromatic polyene antibiotic.
In a first aspect of the present invention, a kind of method of fermentative production aromatic polyene antibiotic is provided, described method comprises:
(1) engineering bacteria that will produce aromatic polyene antibiotic is inoculated in fermention medium, and carries out fermentation culture 12-60 hour under pH7.6-8.5, thereby makes described engineering bacterium fermentation produce para-amino benzoic acid;
(2) regulate pH, and under pH6.4-7.4, continue the described engineering bacteria of fermentation culture, thereby make described engineering bacteria that para-amino benzoic acid is converted into aromatic polyene antibiotic; With
(3) from fermented liquid, isolate described aromatic polyene antibiotic.
In another preference, pH maintains 7.8-8.2 in described step (1); And/or
In described step (2), pH maintains 6.5-7.2.
In another preference, in step (1), and under pH7.6-8.5, carried out fermentation culture 18-48 hour, more preferably cultivated 20-36 hour.
In another preference, in step (2), also comprise in fermentation system and add para-amino benzoic acid.
In another preference, the engineering bacteria of described production aromatic polyene antibiotic is selected from down group: streptomycete FR-008, glycosyltransferase gene fscM I interrupt mutant strain CS101, ketose aminotransferase gene fscMII and interrupt mutant strain CS102 or FscP gene fscP interruption mutant strain CS103.
In another preference, described aromatic polyene antibiotic comprises the aromatic polyene macrolide antibiotic; More preferably described aromatic polyene antibiotic comprises seven alkene macrolide polyketone antibiotics.
In another preference, described aromatic polyene antibiotic is selected from down group: microbiotic FR-008, candicidin, glycosyltransferase gene fscM I interrupt mutant strain CS101 and cultivate the de-glycosylation compound CS101, the ketose aminotransferase gene fscMII that obtain and interrupt mutant strain CS102 and cultivate the deamination sugar compounds CS102 that obtains or FscP gene fscP and interrupt mutant strain CS103 and cultivate the FR-008 decarboxylation that the obtains polyketone antibiotic CS 103 that derives.
In another preference, in step (1), also comprise the concentration of measuring aromatic polyene antibiotic described in the fermented liquid, thereby judge whether described engineering bacteria begins to produce aromatic polyene antibiotic.
In another preference, the addition altogether of para-amino benzoic acid is the 0.002-0.2g/L fermented liquid, more preferably is 0.01-0.1g/L.
In another preference, fermentation total time≤160 of step (1) and (2) hour, more preferably≤120 hour.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1 has shown under the situation of not controlling the pH value, the fermenting process curve of CS103 bacterial strain.
Fig. 2 showed pH value control at 6.9 o'clock, the fermenting process curve of CS103 bacterial strain.
Fig. 3 has shown when controlling the pH value stage by stage, the fermenting process curve of CS103 bacterial strain.
Fig. 4 has shown and has controlled the pH value stage by stage, and added under the synthetic precursor microbiotic situation fermenting process curve of CS103 bacterial strain at the fermentation initial stage.
Fig. 5 has shown the method for conventional separation and purification aromatic polyene antibiotic.
Embodiment
The inventor is through extensive and deep research, be surprised to find that a kind of fermentation process of effective raising aromatic polyene antibiotic or derivatives thereof output, namely by in the different steps of fermenting process, controlling the method for the different pH of fermented liquid, make growth, metabolism and the production of engineering bacteria be in desirable state, thereby improved the fermentation yield of aromatic polyene antibiotic or derivatives thereof greatly.
Particularly, in the initial stage of growth of engineering bacteria, under the culture condition of pH7.6-8.5, the culturing engineering bacterium, thus promote microbiotic aromatic side chain precursor---synthesizing of para-amino benzoic acid; When engineering bacteria begins or be about to begin synthetic aroma family polyene antibiotic, rapidly pH is transferred to 6.4-7.4 and under pH, continue fermentation, para-amino benzoic acid is converted into aromatic polyene antibiotic, thereby has improved the output of aromatic polyene antibiotic extremely effectively, and reduced by product.
In a preference of the present invention, also comprise step: add the precursor substance para-amino benzoic acid at the aromatic polyene antibiotic synthesis phase.
Engineering bacteria
As used herein, " fermentation " namely refer to cultivate the described recombinant bacterial strain that carries expression vector, make it produce aromatic polyene antibiotic and derivative thereof.More particularly, " fermentation " is a kind of fairly large production process, is the 2-10000 liter as the fermentation scale, and more preferably fermentation scale is the 5-5000 liter, can be 50 liters, 100 liters, 1000 liters or higher as the fermentation scale.
Can be used for engineering bacteria of the present invention and be not particularly limited, can be that this area is for the production of the engineering bacteria of aromatic polyene antibiotic.These engineering bacterias can be that screening obtains, also can be through genetic engineering modified.Usually, these engineering bacterias need precursor substance para-amino benzoic acid or similar substance in synthetic aroma family polyene antibiotic.
But the preferred engineering bacteria of a class is the engineering bacteria of fermentative production aromatic polyene macrolide antibiotic (more preferably seven alkene macrolide polyketone antibiotics).Representational engineering bacteria comprises (but being not limited to): streptomycete FR-008 (application number is the Chinese patent application " seven alkene macrolide polyketone antibiotic FR-008 mixtures " of CN200310108746.6), glycosyltransferase gene fscM I interrupts mutant strain CS101 (application number is the Chinese patent application " desugar and deamination sugar FR-008 derive polyketone antibiotic " of CN200310108745.1), ketose aminotransferase gene fscMII interrupts mutant strain CS102 (application number is the Chinese patent application " desugar and deamination sugar FR-008 derive polyketone antibiotic " of CN200310108745.1), or FscP gene fscP interrupts mutant strain CS103 (seeing that application number is the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0) or its derivative strain.
Be applicable to that engineering bacteria of the present invention is generally streptomycete, but also can be other bacterial strains, for example intestinal bacteria, actinomycetes, fungi etc.
Aromatic polyene antibiotic
The aromatic polyene antibiotic that available the inventive method is produced is not particularly limited, and can be that any precursor substance para-amino benzoic acid or similar substance of needing in building-up process is as the aromatic polyene antibiotic of aromatic side chain.Preferred aromatic polyene antibiotic is the aromatic polyene macrolide antibiotic, more preferably is seven alkene macrolide polyketone antibiotics (referring to CN200310108746.6, CN200310108745.1 and CN200710037060.0).
Representational aromatic polyene antibiotic comprises (but being not limited to):
1) microbiotic FR-008 or candicidin and derivative thereof;
2) glycosyltransferase gene fscM I interrupts the Compound C S101 that mutant strain CS101 cultivates the desugar base that obtains;
3) ketose aminotransferase gene fscMII interrupts the Compound C S102 that mutant strain CS102 cultivates the deamination sugar that obtains;
4) FscP gene fscP interrupts mutant strain CS103 and cultivates the toxicity that obtains and reduce the FR-008 decarboxylation polyketone antibiotic CS103 that derives;
5) microbiotic FR-008 or candicidin; With
6) other passes through the combination FR-008 that obtains of biosynthesis technology or the genetically engineered derivative of candicidin.
Substratum and fermentation condition
Being applicable to that the substratum of the inventive method and culture condition (except the pH value and add the para-amino benzoic acid) are not particularly limited, can be conventional substratum and the culture condition that is used for described engineering bacteria in this area.Certainly, according to different fermentation scale and needs, those skilled in the art can rule of thumb or test conditions such as air flow, stirring velocity are adjusted.
Compare with existing fermentation condition, the key distinction of the present invention is to control stage by stage pH, thereby promotes the synthetic of microbiotic aromatic side chain precursor and microbiotic itself.
In order to determine the time of regulation and control pH, can be during the fermentation, the concentration of aromatic polyene antibiotic described in the results of regular determination fermented liquid, thus judge whether described engineering bacteria begins to produce aromatic polyene antibiotic.In case engineering bacteria begins or is about to begin produce aromatic polyene antibiotic, then can regulate pH to 6.4-7.4.
Certainly, under the situation that factors such as fermentation condition and inoculum size are determined, in case determined that by measuring engineering bacteria begins to produce the time of aromatic polyene antibiotic, then can measure the concentration of aromatic polyene antibiotic described in the fermented liquid.
In the present invention, the corresponding fermentation starting stage of step (1) is generally 12-60 hour, preferably is 18-48 hour, more preferably is 20-36 hour.
After fermentation culture finishes, can be from the described aromatic polyene antibiotic of ordinary method isolated or purified from fermented liquid and derivative thereof.Isolated or purified aromatic polyene antibiotic and derivative thereof can adopt various separating and purifying technology well known to those skilled in the art from cultured products, and comprising (but being not limited to): solvent extraction adds the precipitator method.A kind of initial gross separation purge process of routine as shown in Figure 5.
Major advantage of the present invention is:
(1) according to aromatics polyenoid polyketone antibiotic structure and synthetic characteristic, by controlling the synthetic of pH regulator microbiotic aromatic side chain precursor and microbiotic itself stage by stage, improve pH at the synthetic initial stage and promote microbiotic aromatic side chain precursor---synthesizing of para-amino benzoic acid, and then improve the antibiotic fermentation level.
Compared with former zymotechnique, it is about 200% that the inventive method can make aromatic polyene antibiotic output significantly improve, and unit thalline microbiotic output obviously improves, and it is about 280% that the productive rate of unit volume fermented liquid improves,
(2) reduce by product, be beneficial to the separation and purification work in downstream.
(3) fermenting process is easy to control, is convenient to scale operation, thereby has fabulous industry using value.
(4) fermentation period obviously shortens, and production cost significantly reduces.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise defined or explanation, same meanings of being familiar with of all specialties used herein and scientific words and those skilled in the art.Any method similar or impartial to described content and material all can be applicable in the inventive method in addition.
Embodiment 1
In order to determine the time of regulation and control pH, can be during the fermentation, the concentration of aromatic polyene antibiotic described in regular (as per 5 minutes or per 10 minutes) or the The real time measure fermented liquid, thus judge whether described engineering bacteria begins to produce aromatic polyene antibiotic.In case engineering bacteria begins or is about to begin produce aromatic polyene antibiotic, then can regulate pH to 6.4-7.4.
Be example with the FR-008 decarboxylation polyketone antibiotic CS103 that derives, the fermentation initial stage is detected by the following method: high performance liquid chromatograph (Agenlent 1100), chromatographic column is SB-C8 (4.6mm*250mm), moving phase is 20mM, pH4.6 ammonium acetate buffer-acetonitrile (60: 40), flow velocity is 1mL/min, column temperature is 25 ℃, detects wavelength 380nm, obtains regression equation under the above-mentioned chromatographic condition: y=5.5015x+87.599 (R
2=0.9987).When the CS103 fermentation level reaches 10-30mg/L, in the time of best 20mg/L, namely fermented 24 hours, can regulate pH to 6.4-7.4.
Embodiment 1 (Comparative Examples)
The derive fermentation of polyketone antibiotic CS103 of FR-008 decarboxylation
300 μ l FscP gene fscP are interrupted mutant strain CS103 (application number is the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0) spore suspension to be seeded to the 250mL that contains the 50mL seed culture medium and to shake in the bottle, inoculation is placed under 28 ℃, cultivate about 30h on the 200rpm/min shaking table, receive the 3.7L fermentor tank by inoculum size 10%; 450rpm, 200L/h air flow, 28 ℃ of cultivations.(fermentation process is the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0 with application number)
Whole fermentation process is not controlled the pH value, the initial pH6.9 of substratum, and at 0-12h, pH is elevated to 7.5 gradually.The result as shown in Figure 1, thalline raises in order to adapt to the synthetic of the synthetic precursor PABA of fermentation initial stage microbiotic and self to regulate pH; PH descends gradually after the 12h, and this moment, microbiotic began to synthesize, and up to 96h some rise slightly, thalline begins self-dissolving, and the thalline maximum amount reaches 6.5g/L; At this moment, put bottle and detect the derive fermentation level of polyketone antibiotic CS103 of FR-008 decarboxylation and reach about maximum 46 μ g/mL, no longer continue synthetic.
The derive fermentation of polyketone antibiotic CS103 of FR-008 decarboxylation
With embodiment 1, difference is: pH6.9 is kept with hydrochloric acid and sodium hydroxide in the inoculation back, cultivates 120 to fermentation ends.
The result as shown in Figure 2.When fermenting process was kept pH6.9, because fermenting process pH is more stable, the thalli growth ability also was improved, and the final cell dry weight reaches 9.4g/L.Substrate consumption speed is accelerated, and glucose consumes 10g/L at 48-72h, and 120h almost exhausts, and does not control the batch fermentation of pH among the embodiment 1, just consumes about 10g/L up to 120h.The consumption of amino nitrogen is also obviously accelerated.As seen bacterial metabolism speed is accelerated, and this also can significant reaction come out from dissolved oxygen and RQ value.The dissolved oxygen Schwellenwert is down to about 30% by original 50%, and the RQ maximum value brings up to 0.18 by original 0.12.Putting bottle and detect the derive fermentation level of polyketone antibiotic CS103 of FR-008 decarboxylation and reach maximum at 120h, is about 88.2 μ g/mL, has improved 90% than embodiment 1.
Embodiment 3
The derive fermentation of polyketone antibiotic CS103 of FR-008 decarboxylation
With embodiment 1, difference is: pH8.0 is kept with hydrochloric acid and sodium hydroxide in the inoculation back.Behind the 24h, rapidly pH is transferred to 6.9, and be maintained until fermentation ends.
The result as shown in Figure 3.Take to control stage by stage after the pH strategy, cultivate 120h, putting bottle detects the derive fermentation level of polyketone antibiotic CS103 of FR-008 decarboxylation and further brings up to 111 μ g/mL, do not improve 141% than not controlling the pH batch fermentation among the embodiment 1, improved 26% than control pH6.9 batch fermentation among the embodiment 2 yet.
In addition, the highest dry cell weight DCW
MaxBe 9.5g/L, do not have considerable change with control pH6.9 batch fermentation.The raising of this explanation microbiotic output is not because cell concentration increases influence.
Embodiment 4
The derive fermentation of polyketone antibiotic CS103 of FR-008 decarboxylation
In embodiment 1, difference is: (a) pH8.0 is kept with hydrochloric acid and sodium hydroxide in the inoculation back.Behind the 24h, rapidly pH is transferred to 6.9, and be maintained until fermentation ends; (b) with the para-amino benzoic acid of 0.02g/L respectively at 0h, 12h, 24h and 48h add fermentor tank to four times.
The result as shown in Figure 4.Add after the precursor, CS103 output is further brought up to nearly 140 μ g/mL, improves 26% than embodiment 3.
In addition, the highest dry cell weight DCW
MaxBe 9.4g/L, do not have notable difference with a few job fermentations in front.This explanation " is controlled the pH strategy " among the embodiment 3 stage by stage though pass through to improve the activity of para-amino benzoic acid synthetic enzyme, has promoted branched acid to be converted into para-amino benzoic acid, and the scale of construction increases before making, and does not reach the best preceding scale of construction; And because the thalli growth environmental limit can't further improve the pH at fermentation initial stage; Therefore add the accumulation that para-amino benzoic acid can further promote CS103 by one or many (preferably in batches repeatedly) on this basis, reached effect preferably.And after adding outer source precursor, the time that the biosynthesizing of CS103 begins advances to 6h.Because generated time shifts to an earlier date, bacterial metabolism is more vigorous, and dissolved oxygen further reduces, and the RQ maximum reaches 0.56.At this moment, the consumption of glucose and amino nitrogen is accelerated, and particularly glucose exhausts at 96h.
Four groups of embodiment contrast and experiment of table 1.
The derive fermentation of polyketone antibiotic CS101 of FR-008 desugar
300 μ l glycosyltransferase gene fscM I are interrupted mutant strain CS101 (application number is the Chinese patent application " desugar and deamination sugar FR-008 derive polyketone antibiotic " of CN200310108745.1) spore suspension to be seeded to the 250mL that contains the 50mL seed culture medium and to shake in the bottle, inoculation is placed under 28 ℃, cultivate about 30h on the 200rpm/min shaking table, receive the 3.7L fermentor tank by inoculum size 10%; 450rpm, 200L/h air flow, 28 ℃ of cultivations.(fermentation process is the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0 with application number)
Whole fermentation process is not controlled the pH value, and the initial pH6.7 of substratum is cultured to 96h and puts bottle and detect the derive fermentation level of polyketone antibiotic CS101 of FR-008 desugar and reach about maximum 79.3 μ g/mL, no longer continues synthetic.
Embodiment 6
The derive fermentation of polyketone antibiotic CS101 of FR-008 desugar
In embodiment 5, difference is: (a) pH7.7 is kept with hydrochloric acid and sodium hydroxide in the inoculation back.Behind the 24h, rapidly pH is transferred to 6.6, and be maintained until fermentation ends; (b) with the para-amino benzoic acid of 0.02g/L respectively at 0h, 12h, 24h and 48h add fermentor tank to four times.Be cultured to 96h, CS101 output is further brought up to nearly 236.8 μ g/mL.
Embodiment 7
The fermentation of microbiotic FR-008
300 μ l streptomycete FR-008 (application number is the Chinese patent application " seven alkene macrolide polyketone antibiotic FR-008 mixtures " of CN200310108746.6) spore suspension is seeded to the 250mL that contains the 50mL seed culture medium and shakes in the bottle, inoculation is placed under 28 ℃, cultivate about 30h on the 200rpm/min shaking table, receive the 3.7L fermentor tank by inoculum size 10%; 450rpm, 200L/h air flow, 28 ℃ of cultivations.(fermentation process is the Chinese patent application " decarboxylation FR-008 derive polyketone antibiotic and application thereof " of CN200710037060.0 with application number)
Whole fermentation process is not controlled the pH value, and the initial pH6.9 of substratum is cultured to 96h and puts bottle fermentation level of detect antibiotics FR-008 and reach about maximum 150.9 μ g/mL, no longer continues synthetic.
The fermentation of microbiotic FR-008
In embodiment 7, difference is: (a) pH8.4 is kept with hydrochloric acid and sodium hydroxide in the inoculation back.Behind the 24h, rapidly pH is transferred to 7.2, and be maintained until fermentation ends; (b) with the para-amino benzoic acid of 0.02g/L respectively at 0h, 12h, 24h and 48h add fermentor tank to four times.Be cultured to 96h, microbiotic FR-008 output is further brought up to nearly 421.3 μ g/mL.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the method for a fermentative production aromatic polyene antibiotic is characterized in that, described method comprises:
(1) engineering bacteria that will produce aromatic polyene antibiotic is inoculated in fermention medium, and carries out fermentation culture 12-60 hour under pH7.6-8.5, thereby makes described engineering bacterium fermentation produce para-amino benzoic acid;
Wherein, the engineering bacteria of described production aromatic polyene antibiotic is that to interrupt mutant strain CS103 and described aromatic polyene antibiotic be the FR-008 decarboxylation polyketone antibiotic CS103 that derives to FscP gene fscP; Or the engineering bacteria of described production aromatic polyene antibiotic is that to interrupt mutant strain CS101 and described aromatic polyene antibiotic be the FR-008 desugar polyketone antibiotic CS101 that derives to glycosyltransferase gene fscMI; Or the engineering bacteria of described production aromatic polyene antibiotic is that streptomycete FR-008 and described aromatic polyene antibiotic are microbiotic FR-008;
(2) regulate pH, and under pH6.4-7.4, continue the described engineering bacteria of fermentation culture, thereby make described engineering bacteria that para-amino benzoic acid is converted into aromatic polyene antibiotic; With
(3) from fermented liquid, isolate described aromatic polyene antibiotic.
2. the method for claim 1 is characterized in that, pH maintains 7.8-8.2 in described step (1); And/or
In described step (2), pH maintains 6.5-7.2.
3. the method for claim 1 is characterized in that, in step (1), and carries out under pH7.6-8.5 fermentation culture 18-48 hour.
4. the method for claim 1 is characterized in that, in step (1), and carries out under pH7.6-8.5 fermentation culture 20-36 hour.
5. the method for claim 1 is characterized in that, also comprises in fermentation system in step (2) and adds para-amino benzoic acid.
6. as claim 1 or 5 described methods, it is characterized in that, in step (1), also comprise the concentration of measuring aromatic polyene antibiotic described in the fermented liquid, thereby judge whether described engineering bacteria begins to produce aromatic polyene antibiotic.
7. method as claimed in claim 5 is characterized in that, the addition altogether of para-amino benzoic acid is the 0.002-0.2g/L fermented liquid.
8. method as claimed in claim 7 is characterized in that, the addition altogether of para-amino benzoic acid is the 0.01-0.1g/L fermented liquid.
9. as claim 1 or 5 described methods, it is characterized in that fermentation total time≤160 of step (1) and (2) hour.
10. method as claimed in claim 9 is characterized in that, fermentation total time≤120 of step (1) and (2) hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810204283 CN101748178B (en) | 2008-12-10 | 2008-12-10 | Process for producing aromatic polyene antibiotic through fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810204283 CN101748178B (en) | 2008-12-10 | 2008-12-10 | Process for producing aromatic polyene antibiotic through fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101748178A CN101748178A (en) | 2010-06-23 |
| CN101748178B true CN101748178B (en) | 2013-07-10 |
Family
ID=42475791
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810204283 Expired - Fee Related CN101748178B (en) | 2008-12-10 | 2008-12-10 | Process for producing aromatic polyene antibiotic through fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101748178B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544437A (en) * | 2003-11-20 | 2004-11-10 | 上海交通大学 | Desugar and deaminosugar FR-008 derived polyketone antibiotics |
| WO2007024997A2 (en) * | 2005-08-22 | 2007-03-01 | Fermalogic, Inc. | Methods of increasing production of secondary metabolites |
| CN101024665A (en) * | 2007-02-01 | 2007-08-29 | 华东理工大学 | Decarboxylated FR-008 derivative polyketone antibiotic and use thereof |
-
2008
- 2008-12-10 CN CN 200810204283 patent/CN101748178B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544437A (en) * | 2003-11-20 | 2004-11-10 | 上海交通大学 | Desugar and deaminosugar FR-008 derived polyketone antibiotics |
| WO2007024997A2 (en) * | 2005-08-22 | 2007-03-01 | Fermalogic, Inc. | Methods of increasing production of secondary metabolites |
| CN101024665A (en) * | 2007-02-01 | 2007-08-29 | 华东理工大学 | Decarboxylated FR-008 derivative polyketone antibiotic and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| Biology》.2003,第10卷1065-1076. * |
| Shi Chen et al..Organizational and Mutational Analysis of a Complete FR-008/Candicidin Gene Cluster Encoding a Structurally Related Polyene Complex.《Chemistry & Biology》.2003,第10卷1065-1076. |
| Shi Chen et al..Organizational and Mutational Analysis of a Complete FR-008/Candicidin Gene Cluster Encoding a Structurally Related Polyene Complex.《Chemistry & * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101748178A (en) | 2010-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rymowicz et al. | High-yield production of erythritol from raw glycerol in fed-batch cultures of Yarrowia lipolytica | |
| Novak et al. | Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process | |
| Takagi et al. | Continuous 2-Keto-L-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia | |
| CN103403155B (en) | New saccharomyces cerevisiae strains | |
| CN101831414B (en) | Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase | |
| CN102586216A (en) | Method for producing sodium alginate lyase by utilizing vibrio vulnificus | |
| KR20110042058A (en) | Methods and processes for producing organic acids | |
| Fu et al. | High-efficiency l-lactic acid production by Rhizopus oryzae using a novel modified one-step fermentation strategy | |
| Zhu et al. | A three-stage culture process for improved exopolysaccharide production by Tremella fuciformis | |
| CN107586810A (en) | A kind of biofermentation production technology of hyaluronic acid | |
| Guo et al. | Efficient production of 2, 3-butanediol from cheese whey powder (CWP) solution by Klebsiella pneumoniae through integrating pulsed fed-batch fermentation with a two-stage pH control strategy | |
| Xu et al. | Production of mycophenolic acid by Penicillium brevicompactum immobilized in a rotating fibrous-bed bioreactor | |
| CN105483064A (en) | Method for fermentation production of bacitracin | |
| CN101613712A (en) | Improve the method for Avrmectin and/or ivermectin output and produce bacterial strain | |
| CA2719581C (en) | Improved fermentation process for higher yield coefficient of lipase-inhibitor with respect to consumed fatty acid | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN101671706B (en) | Carbohydrate supplementing method in fermentation process of mycophenolic acid | |
| Bai et al. | Ammonium lactate production by Lactobacillus lactis BME5-18M in pH-controlled fed-batch fermentations | |
| Yu et al. | Enhanced production of L (+)-lactic acid by floc-form culture of Rhizopus oryzae | |
| CN102021214A (en) | Oxygen consumption rate-based vitamin B12 fermentation production control process | |
| CN101748178B (en) | Process for producing aromatic polyene antibiotic through fermentation | |
| Mostafa | Production of acetic acid and glycerol from salted and dried whey in a membrane cell recycle bioreactor | |
| Mulchandani et al. | Production of acetone-butanol-ethanol by Clostridium acetobutylicum using a spin filter perfusion bioreactor | |
| Liu et al. | Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source | |
| CN110964754B (en) | Method for reducing proportion of succinic acid fermentation by-products of actinobacillus succinogenes |
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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130710 Termination date: 20131210 |