CN112680387A - Streptomyces rubicerae strain for producing actinomycin D through fermentation and application thereof - Google Patents
Streptomyces rubicerae strain for producing actinomycin D through fermentation and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microorganisms, and particularly relates to a streptomyces rubiginosis mutant strain for producing actinomycin D by fermentation, and further discloses an application of the streptomyces rubiginis mutant strain for producing actinomycin D by fermentation. The Streptomyces rubiginosis FIM-Z19-12(Streptomyces rubiginosolvelus) capable of producing actinomycin D at high yield is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, the strain can efficiently ferment actinomycin D, in a fermentation experiment, the titer of the actinomycin D produced by fermenting the Streptomyces rubiginosis FIM-Z19-12 is as high as 1383mg/L, the yield of the actinomycin D is greatly improved, and the actinomycin D can be applied to industrial fermentation production.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a streptomyces rubiginosus strain capable of producing actinomycin D through fermentation, and further discloses an application of the streptomyces rubiginicus strain in producing actinomycin D through fermentation.
Background
Actinomycin D (actinomycin D, AMD, dactinomycin) was originally isolated from actinomycetes s.melanochromogenes No.1779 or s.parvulus, and belongs to a member of the actinomycin family, and such compounds are produced by various microorganisms of the genus streptomyces. Actinomycin D is a pigment peptide lactone antibiotic, is one of classical antitumor drugs, has good antitumor efficacy, and can obtain good effect when being combined with other antitumor drugs for treating tumors by matching with radiation. The anticancer effect of actinomycin D is probably closely related to the chromophore structure thereof, the cyclic peptide part can play the role of a carrier, and the unique chemical structure of the cyclic peptide, the antitumor mechanism for inhibiting RNA synthesis and the characteristic of difficult cross drug resistance with other chemotherapeutic drugs are the inexhaustible advantages of the chemotherapeutic drugs. Therefore, in recent years, research on actinomycin D has been actively carried out, new targets and new mechanisms of antitumor action are continuously discovered, and research on producing bacteria from different sources of actinomycin D, structural modification and analog screening with low toxicity and high antitumor activity are also new concerns of developers.
Currently, the commonly used strain breeding techniques (strain improvement) can be divided into two major categories, random breeding (random screening) and rational breeding (rationalized selection) according to the principle. Although genetic engineering, metabolic engineering and systematic biological research consisting of various omics based on molecular biology provide wide prospects for directionally constructing functional cell strains, it is extremely difficult to truly realize the goal of modification by culturing the functional cells. Due to the complexity and multinodality of the metabolic network in the microorganism, the metabolic flux of the strain after gene manipulation is not shifted toward the expected design direction. Especially in a complex system for generating secondary metabolites such as antibiotics, random breeding technology is mainly adopted to obtain excellent industrial production strains. For a long time, traditional mutation breeding means such as ultraviolet, microwave and chemical mutagens are mainly adopted in the random breeding technology for improving the Streptomyces rubiginosus strain, and a certain effect is achieved, however, when the mutagens are used for treating the same strain for a long time, due to the non-tropism of mutation and the accumulation of mutation, the high yield of the strain is obtained, meanwhile, the life capacity, the sporulation amount and the like of the strain are obviously weakened, and multiple times of mutagenesis often cause higher negative mutation rate and saturated resistance, so that the space for improving the strain is insufficient.
The normal pressure room temperature plasma breeding technology (ARTP) is a microorganism genome rapid mutation technology, the generated plasma is rich in various chemical active particles, and has multiple effects of generating genetic substance damage to strain cells, causing cell membrane permeability and protein structure change and the like, and inducing the cells to start an SOS repair mechanism, and generating various mismatching sites in the repair process. Therefore, how to screen and obtain the strain which is suitable for industrial production and can produce actinomycin D with high yield has positive significance for the industrial production and application of the actinomycin D.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a streptomyces rubiginosis mutant strain for producing actinomycin D by fermentation, so as to solve the problem that the fermentation strain of actinomycin D in the prior art is not ideal in performance;
the second technical problem to be solved by the invention is to provide the application of the streptomyces rubiginosus mutant strain in fermentation production of actinomycin D.
In order to solve the technical problems, the Streptomyces rubicerasus strain is classified and named as Streptomyces rubiginosolveusFIM-Z19-12, is preserved in Guangdong province microorganism strain preservation center, addresses 5 th experimental building of Michelia furiosa No. 100 college in Fujiu province in Guangzhou city, Guangdong province, has the preservation number of GDMCC No.61323, and has the preservation date of 2020, 12 months and 2 days.
The invention also discloses application of the streptomyces erythreus strain in fermentation production of actinomycin D.
The invention also discloses a method for producing actinomycin D by fermentation, which comprises the step of inoculating the streptomyces erythreus strain into a suitable fermentation culture medium for fermentation culture.
Specifically, the fermentation medium comprises the following components in percentage by mass: 2-3% of soluble starch, 3-4% of soybean meal, 0.1-1% of peptone, 0.5-1.5% of corn flour, 0.01-0.1% of dipotassium hydrogen phosphate and 7.0-7.2% of pH value adjustment.
Preferably, the fermentation medium comprises the following components in percentage by mass: 2.5 percent of soluble starch, 3.5 percent of soybean meal, 0.5 percent of peptone, 1.0 percent of corn flour, 0.05 percent of dipotassium hydrogen phosphate and the balance of distilled water, and adjusting the pH value to 7.0-7.2.
Specifically, the conditions of the fermentation culture comprise: the rotation speed is controlled at 150-300rpm, and fermentation culture is carried out at 28-32 ℃ for 3-5 d.
Specifically, the method for producing actinomycin D by fermentation further comprises the step of inoculating the streptomyces erythreus strain into a seed culture medium for seed liquid culture;
the seed culture medium comprises the following components in percentage by mass: 2-3% of soluble starch, 0.1-1% of glucose, 0.5-1.5% of soybean flour, 0.1-1% of peptone and 0.1-0.5% of yeast extract, and adjusting the pH value to 7.0-7.2.
Preferably, the seed culture medium comprises the following components in percentage by mass: 2.5 percent of soluble starch, 0.5 percent of glucose, 1.0 percent of soybean meal, 0.5 percent of peptone and 0.3 percent of yeast extract, and the pH value is adjusted to 7.0-7.2.
Specifically, the seed liquid culture conditions comprise: the seed liquid culture is carried out for 40-60h at 28-32 ℃ by controlling the rotation speed of 180-300 rpm.
Specifically, the method for producing actinomycin D by fermentation further comprises the step of inoculating the streptomyces erythraea strain into a slant culture medium for activation;
the slant culture medium comprises the following components in percentage by mass: 0.5-1.5% of soluble starch, 0.5-1.5% of glucose, 0.01-0.1% of magnesium sulfate, 0.05-0.15% of potassium nitrate, 0.01-0.1% of dipotassium phosphate and 2.0% of agar, and adjusting the pH value to 7.0-7.2.
Preferably, the slant culture medium comprises the following components by mass: 1.0 percent of soluble starch, 1.0 percent of glucose, 0.05 percent of magnesium sulfate, 0.1 percent of potassium nitrate, 0.05 percent of dipotassium phosphate and 2.0 percent of agar, and adjusting the pH value to 7.0-7.2.
Specifically, the conditions of the slant culture medium activation step include: culturing at 25-30 deg.C for 8-12 days.
The Streptomyces rubiginosis FIM-Z19-12(Streptomyces rubiginosolvelus) capable of producing actinomycin D at high yield is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, the strain can efficiently ferment actinomycin D, in a fermentation experiment, the titer of the actinomycin D produced by fermenting the Streptomyces rubiginosis FIM-Z19-12 is as high as 1383mg/L, the yield of the actinomycin D is greatly improved, and the actinomycin D can be applied to industrial fermentation production; the screened strain FIM-Z19-12 has good stability, the titer of actinomycin D produced by the strain FIM-Z19-12 after five generations of continuous passage is basically stable, the same higher level is maintained, and the strain FIM-Z19-12 can be used as a production strain for further research and development.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 is a graph of ARTP efflux time versus lethality in mutagenesis experiments;
FIG. 2 is a phylogenetic tree of the strain FIM-Z19-12 according to the present invention.
Detailed Description
In the following examples of the present invention, the culture medium includes:
the separation plate culture medium, the slant culture medium and the plate culture medium comprise the following components in percentage by mass: 1.0% of soluble starch, 1.0% of glucose, 0.05% of magnesium sulfate, 0.1% of potassium nitrate, 0.05% of dipotassium hydrogen phosphate, 2.0% of agar and the balance of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing for 30min by high-pressure steam at 121 ℃;
the seed culture medium comprises the following components in percentage by mass: 2.5% of soluble starch, 0.5% of glucose, 1.0% of soybean meal, 0.5% of peptone, 0.3% of yeast extract and the balance of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing for 30min by high-pressure steam at 121 ℃;
the fermentation medium comprises the following components in percentage by mass: 2.5% of soluble starch, 3.5% of soybean meal, 0.5% of peptone, 1.0% of corn flour, 0.05% of dipotassium hydrogen phosphate and the balance of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing for 30min by high-pressure steam at 121 ℃.
In the following examples of the present invention, the content of actinomycin D is detected by HPLC, and the specific detection method refers to the method described in Chinese patent CN 105254712B.
Example 1 isolation of the fermentation Strain FIM-Z19
Collecting soil in Taishan underfoot vegetable garden of Jinan, Shandong, specifically, removing floating soil with a surface layer of about 10cm by using a sampling shovel, and collecting 10-25 g of soil sample at a position of 10 cm; weighing 2g of soil sample, adding 10mL of sterile normal saline, oscillating, standing for 30min, collecting supernatant as stock solution, and performing gradient dilution with sterile normal saline with dilution degree of 10-2、10-3、10-4And 10-5A suspension of (a).
0.1mL of the stock solution and the selected suspension are respectively taken and coated on a separate agar medium plate prepared by adding 50mg/L of potassium dichromate water, 3 parallel plates are repeated on each sample, then the coated plate is cultured at 28 ℃, and the appearance, the size, the color, the edge shape, the surface dry and wet state and other morphological characteristics of a bacterial colony are observed.
Culturing for 8-12 days under aseptic condition, picking out representative single colony such as round colony, central bulge, surface wrinkle, white or yellowish colony, streaking, inoculating on slant of agar separating culture medium, culturing at 28 deg.C, separating and purifying to obtain strain, and naming the obtained strain as strain FIM-Z19, and storing at-80 deg.C with 20% glycerol.
EXAMPLE 2 obtaining of mutagenized Strain FIM-Z19-12
Taking the screened strain FIM-Z19, transferring the strain to a slant culture medium, and culturing for 8-12 days in a constant temperature incubator at 28 ℃; washing off spores on the slant culture medium with normal saline, scattering glass beads, filtering with warp cloth, and making into 106Spore suspension per mL.
10 μ L of the spore suspension prepared above was pipetted into a circular iron plate with a diameter of 1cm, placed in a normal pressure room temperature plasma mutagenesis system with helium as working gas, power supply of 110W, and working gas flow of 10L/min, and a treatment distance of 2mm, and treated for 5s, 10s, 15s, 30s, 45s, 60s, 75s, and 90s, respectively, and the treated spore suspension was subjected to gradient dilution and plating to prepare a lethality curve (as shown in fig. 1). As can be seen from the figure, there is a clear dose-effect relationship between the mutagenic treatment dose and the lethality of the strain FIM-Z19, which gradually increases with the treatment time.
Selecting three irradiation times with different lethal doses of 10s, 30s and 60s according to the lethal rate curve, carrying out plasma mutagenesis on spore suspensions of the strain FIM-Z19, mixing the spore suspensions with the three different treatment times in a test tube filled with normal saline, and uniformly mixing to obtain mutagenized spore suspensions for later use.
Taking the spore suspension, and respectively coating the spore suspension in a streptomycin-containing plate culture medium, wherein the streptomycin concentration (mu g/mL) in the plate culture medium is controlled to be 5mg/L, 10mg/L, 25mg/L, 50mg/L, 100mg/L and 200mg/L respectively; after culturing at 30 ℃ for 8-10 days, the growth of colonies on different plates was observed, and the results are shown in Table 1 below. As can be seen, the minimal concentration of streptomycin in the plate medium without colonies is the Minimal Inhibitory Concentration (MIC) against streptomycin, which is determined from the results in Table 1 as the 50mg/L MIC against streptomycin, which is the lethal resistance mutation marker;
TABLE 1 Streptomycin lethal concentration assay for spores of FIM-Z19 Strain
| Streptomycin (mg/L) | 5 | 10 | 25 | 50 | 100 | 200 |
| Growth of colonies | ++ | ++ | + | - | - | - |
Injection: + growth with colonies, -growth with no colonies
Performing gradient dilution on the obtained mutagenic spore suspension, and respectively controlling the dilution degree to be 10-1、10-2、10-3、10-4、10-5、10-6 Choose 10-4、10-5、10-6The spore suspensions of the three dilutions were spread on resistant plates containing 50mg/L streptomycin, respectively, and incubated at 28 ℃ for 8-12 days in the dark.
Inoculating the single colony grown on each prepared resistant plate to a slant culture medium for 10 days, inoculating the single colony to a seed culture medium in an inoculation amount of 1%, and culturing at 30 ℃ and 230r/min for 45-50h to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium with an inoculation amount of 10%, and culturing at 30 deg.C and 230r/min for 3-6d to obtain a fermentation liquid.
Taking a proper amount of the obtained fermentation liquor, and adding absolute ethyl alcohol with the volume being two times that of the fermentation liquor, wherein the volume ratio of the fermentation liquor to the absolute ethyl alcohol is 1: 2, fully oscillating, standing, centrifuging at 4000r/min for 10min, and measuring the yield of actinomycin D by using high performance liquid chromatography after the supernatant passes through an organic filter membrane, and determining that the product in the fermentation liquor of the embodiment has a correct structure.
By this method, the strain having the highest actinomycin D production was selected, and for convenience of description, the selected strain was named strain FIM-Z19-12, and strain FIM-Z19-12 was preserved in glycerol.
Example 3 identification of the mutagenized Strain FIM-Z19-12
Physiological and biochemical characteristic identification
Streaking the obtained strain FIM-Z19-12 on a separation culture medium plate, inserting a cover glass, culturing at 30 ℃ for 7-20d, and observing morphological characteristics and hyphae of a single colony by using an optical microscope, a transmission electron microscope and a scanning electron microscope.
The main morphological and physiological and biochemical characteristics of the obtained strain FIM-Z19 are as follows: the flat colony is round, the center is raised, the surface is wrinkled, aerial hyphae are white or yellowish, the hyphae in the substrate are light yellow, and soluble yellow or red brown pigment is produced; the method comprises the steps of gelatin liquefaction, peptonization after milk coagulation, starch hydrolysis, good growth on cellulose, nitrate reduction, utilization of glucose, fructose, rhamnose, arabinose and mannitol, and no utilization of sucrose, raffinose and inositol. The strain is a high oxygen consumption bacterium, the optimal growth temperature is 28-32 ℃, and the optimal growth pH is 7.0-8.0; when the rotating speed of the shaking table is 230-280r/min and the culture lasts for 3-5D, the yield of the actinomycin D is the highest, and the dissolved oxygen has a remarkable effect on the generation of the actinomycin D in the fermentation process.
Molecular biological identification
Sequencing the 16SrDNA sequence of the mutagenic strain FIM-Z19-12, comparing the 16SrDNA sequence of the strain with the existing sequence in a GenBank database, and performing homology analysis; selecting a corresponding model strain 16SrDNA gene sequence on an LPSN (http:// www.bacterio.cict.fr) website, comparing the systematic evolution analysis by using CLUSTAL-X software, performing the systematic evolution analysis on the generated comparison file by using a MEGA software adjacency method, wherein the topological analysis is the result of 1000 repeated sampling, and the evolutionary tree of the strain FIM-Z19-12 is shown in figure 2. Sequence analysis of 16S rDNA showed that the strain FIM-Z19-12 has 99.25% sequence homology with S.rubiginosolveus.
By combining the above morphological, physiological and biochemical characteristics and molecular biological identification, the strain FIM-Z19-12 can be determined to belong to the genus Streptomyces rubiginosus (Streptomyces rubiginosolvelus), which is classified and named as Streptomyces rubiginosolvelus FIM-Z19-12, and is deposited in Guangdong province microorganism culture collection center, and is addressed to Xiuxiaozuo No. 100 Dazhonglu experimental building 5 building in Guangdong province, the collection number is GDMCC No.61323, and the storage date is 12 months and 2 days in 2020.
EXAMPLE 4 fermentative production of actinomycin D by the mutagenized Strain FIM-Z19-12
Activation of Strain FIM-Z19-12: transferring the strain FIM-Z19-12 preserved by adopting glycerol to a slant culture medium, and culturing for 8-12d in a constant temperature incubator, wherein the culture temperature is 28 ℃.
Preparing a seed solution: inoculating the activated single colony of the strain FIM-Z19-12 into seed culture medium (60 mL seed culture medium in 500mL triangular flask), and culturing at 30 deg.C and 250r/min for 40-60h to obtain seed solution.
Fermentation culture: the seed solution prepared above was inoculated into a fermentation medium (60 mL of the fermentation medium in a 500mL Erlenmeyer flask) at an inoculum size of 5% (v/v), and the resulting fermentation broth was subjected to fermentation culture at 30 ℃ for 4 days at 250r/min, followed by detection.
The detection result shows that the yield of actinomycin D produced by shake flask fermentation of the three batches of strain FIM-Z19-12 is 1362 mu g/mL, 1308 mu g/mL and 1350mg/L respectively.
Example 5 genetic stability verification of the mutagenized Strain FIM-Z19-12
The screened high-yield actinomycin D strain FIM-Z19-12 is subjected to 500mL shake flask continuous culture for 3-5 generations to detect the genetic stability, and the strain passage fermentation experiment result is as follows:
the high-yield resistant strain FIM-Z19-12 was passaged 6 times in succession: f1, F2, F3, F4, F5 and F6, the fermentation titer is measured after shake flask fermentation, the relative titer is 100% by taking a primary strain (F0) with good growth as a control, and the results are shown in the following Table 2.
TABLE 2 Effect of passages on actinomycin D production by the strain FIM-Z19-12
| Strain algebra | F1 | F2 | F3 | F4 | F5 | F6 |
| Relative potency (%) | 100 | 100.2 | 101.3 | 99.8 | 98.5 | 94.3 |
As can be seen from the data in Table 2, the five-passage fermentation level of the strain FIM-Z19-12 screened by the invention has no obvious influence, and the titer of actinomycin D is basically stable and can be maintained at the same higher level; therefore, the strain FIM-Z19-12 has better genetic stability, and the actinomycin D level produced by the target strain, namely the strain FIM-Z19-12, is finally maintained to be more than 1308 mg/L. Therefore, the mutagenized strain Streptomyces rubigenosolveus FIM-Z19-12(Streptomyces rubigenosolvolus) can be used as a production strain for further research and development.
Example 6 differences in fermentation Performance between the mutagenized strains
In previous studies, the applicant conducted mutagenic screening of Streptomyces rubicerasus FIM-N31 suitable for fermentation of actinomycin D based on microwave mutagenesis in combination with resistance mutation method, in which the relative fermentation titer of the mutagenized strain Str186 was optimized.
The present example is based on the mutant strain Str186 screened in advance and the mutant strain Streptomyces rubiginosus FIM-Z19-12 screened in the present invention, the fermentation effect was verified according to the fermentation method in the foregoing example 4, and the results of testing the fermentation titer (μ g/mL) of actinomycin D in 3 batches of fermentation broth are shown in Table 3 below, which proves that the mutant strain of the present invention has better fermentation capability and can ferment actinomycin D with high yield.
Table 3 differences in fermentation performance of different mutagenized strains (n ═ 3)
| Batches of | 1 | 2 | 3 | Average (μ g/mL) |
| Str186 titer | 547 | 498 | 532 | 525.7±25.1 |
| FIM-Z19-12 potency | 1358 | 1325 | 1343 | 1342.0±16.5 |
EXAMPLE 7 production of actinomycin D by fermentation of the mutagenized Strain FIM-Z19-12 in-tank
And (3) seed culture in a shaking flask: inoculating the strain FIM-Z19-12 lawn into seed culture medium (250 mL seed culture medium in 1000mL triangular flask), and culturing at 30 deg.C and 200r/min for 48h to obtain shake flask seed solution.
Seed tank seed culture: inoculating the shake flask seed solution into seed culture medium (70L seed culture medium in 100L tank) at 0.5%, and culturing at 30 deg.C, 0.05MPa, air flow 1:1vvm, and stirring speed of 200r/min for 48 hr to obtain seed solution in seed tank.
Fermentation culture in a fermentation tank: the prepared seed solution was inoculated into a fermentation medium (1 ton of a tank-packed 700L fermentation medium) at an inoculation amount of 5% (v/v), and the mixture was cultured at a culture temperature of 30 ℃, a tank pressure of 0.05MPa, and an air flow rate of 1: 0.8-1.8vvm, the stirring speed is 150-.
The detection result shows that the yield of actinomycin D produced by three batches of fermentation is 1369 mug/mL, 1383 mug/mL and 1352 mug/mL respectively, and further proves that the screened strain can efficiently ferment actinomycin D.
Therefore, the mutagenic strain obtained by screening based on the normal-pressure room-temperature plasma breeding technology has better fermentation performance, can ferment high-yield actinomycin D, has the fermentation titer as high as 1383mg/L, greatly improves the yield of actinomycin D, and can be applied to industrial fermentation production; and the FIM-Z19-12 strain has good stability, the titer of actinomycin D produced by the strain after five generations of continuous passage is basically stable, the same higher level is maintained, and the strain can be used as a production strain for further research and development.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (9)
1. A Streptomyces rubicerasus strain classified and named as Streptomyces
rubiginosolveluusFIM-Z19-12, which has been deposited with the Guangdong province culture Collection with the deposit number GDMCC No.61323 and the deposit date of 2020, 12 months and 2 days.
2. Use of the strain of Streptomyces rubiginosus of claim 1 for the fermentative production of actinomycin D.
3. A method for producing actinomycin D by fermentation, which comprises inoculating the Streptomyces rubiginosus strain of claim 1 in a suitable fermentation medium and carrying out fermentation culture.
4. The method for the fermentative production of actinomycin D according to claim 3, characterised in that the fermentation medium comprises the following components in mass: 2-3% of soluble starch, 3-4% of soybean meal, 0.1-1% of peptone, 0.5-1.5% of corn flour, 0.01-0.1% of dipotassium hydrogen phosphate and 7.0-7.2% of pH value adjustment.
5. The method for fermentative production of actinomycin D according to claim 3 or 4, characterised in that the conditions of the fermentative culture comprise: the rotation speed is controlled at 150-300rpm, and fermentation culture is carried out at 28-32 ℃ for 3-5 d.
6. The method for the fermentative production of actinomycin D according to any of claims 3 to 5, which further comprises inoculating the strain of Streptomyces rubicundunculatus according to claim 1 in a seed medium for seed culture;
the seed culture medium comprises the following components in percentage by mass: 2-3% of soluble starch, 0.1-1% of glucose, 0.5-1.5% of soybean flour, 0.1-1% of peptone and 0.1-0.5% of yeast extract, and adjusting the pH value to 7.0-7.2.
7. The method of claim 6, wherein the conditions of said seed culture comprise: the seed liquid culture is carried out for 40-60h at 28-32 ℃ by controlling the rotation speed of 180-300 rpm.
8. The method for fermentative production of actinomycin D according to any of claims 3 to 7, further comprising the step of inoculating the strain of Streptomyces rubicens according to claim 1 in a slant medium for activation;
the slant culture medium comprises the following components in percentage by mass: 0.5-1.5% of soluble starch, 0.5-1.5% of glucose, 0.01-0.1% of magnesium sulfate, 0.05-0.15% of potassium nitrate, 0.01-0.1% of dipotassium phosphate and 2.0% of agar, and adjusting the pH value to 7.0-7.2.
9. The method for fermentative production of actinomycin D according to claim 8, characterised in that the conditions of said slant medium activation step comprise: culturing at 25-30 deg.C for 8-12 days.
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