阅读说明：本技术 利用深海链霉菌生产怡莱霉素e的培养基及其制备方法 (Culture medium for producing bleomycin E by using deep sea streptomycete and preparation method thereof ) 是由 安法梁 郑高帆 朱云飞 鞠建华 马俊英 于 2021-01-15 设计创作，主要内容包括：本发明公开了一种利用深海链霉菌生产怡莱霉素E的培养基的制备方法,包括如下步骤：(1)以重量份计,准确称取可溶性淀粉80-120g、酵母浸膏20-30g、玉米浆1.6-2.4g、氯化钠4-6g、硝酸钠6.4-9.6g、硫酸铵6.4-9.6g、磷酸二氢钾0.16-0.24g,以去离子定容至1000mL；(2)以3M氢氧化钠溶液调节pH至7.2-7.4,以重量份计,补加终浓度为6.4-9.6g的碳酸钙；所述深海链霉菌为深海链霉菌SCSIO ZH16△ilaR。本发明还公开了采用上述方法制备获得的培养基以及采用该培养基发酵生产怡莱霉素E的方法,采用该方法可提高怡莱霉素E的产量。(The invention discloses a preparation method of a culture medium for producing bleomycin E by utilizing deep sea streptomycete, which comprises the following steps: (1) accurately weighing 80-120g of soluble starch, 20-30g of yeast extract, 1.6-2.4g of corn steep liquor, 4-6g of sodium chloride, 6.4-9.6g of sodium nitrate, 6.4-9.6g of ammonium sulfate and 0.16-0.24g of monopotassium phosphate in parts by weight, and fixing the volume to 1000mL by deionization; (2) adjusting the pH value to 7.2-7.4 by using 3M sodium hydroxide solution, and supplementing calcium carbonate with the final concentration of 6.4-9.6g in parts by weight; the deep sea streptomycete is deep sea streptomycete SCSIO ZH16 delta ilaaR. The invention also discloses a culture medium prepared by the method and a method for producing the bleomycin E by fermenting the culture medium, and the yield of the bleomycin E can be improved by adopting the method.)

1. A preparation method of a culture medium for producing bleomycin E by using deep sea streptomyces is characterized by comprising the following steps:

firstly, accurately weighing 80-120g of soluble starch, 20-30g of yeast extract, 1.6-2.4g of corn steep liquor, 4-6g of sodium chloride, 6.4-9.6g of sodium nitrate, 6.4-9.6g of ammonium sulfate and 0.16-0.24g of monopotassium phosphate in parts by weight, dissolving the components in deionized water, and fixing the volume to 1000 mL;

secondly, regulating the pH value to 7.2-7.4 by using 3M sodium hydroxide solution, and supplementing calcium carbonate with the final concentration of 6.4-9.6g in parts by weight;

the deep sea streptomycete is deep sea streptomycete SCSIO ZH16 delta ilaaR.

2. The method for preparing a culture medium for producing bleomycin E according to claim 1, comprising the steps of:

firstly, accurately weighing 120g of soluble starch, 20g of yeast extract, 1.6-2.4g of corn steep liquor, 4-6g of sodium chloride, 6.4-9.6g of sodium nitrate, 6.4-9.6g of ammonium sulfate and 0.16-0.24g of monopotassium phosphate in parts by weight, dissolving the components in deionized water, and fixing the volume to 1000 mL;

and step two, regulating the pH to 7.2-7.4 by using 3M sodium hydroxide solution, and supplementing calcium carbonate with the final concentration of 6.4-9.6g in parts by weight.

3. The method for preparing a culture medium for producing bleomycin E according to claim 1, comprising the steps of:

firstly, accurately weighing 120g of soluble starch, 20g of yeast extract, 2.4g of corn steep liquor, 6g of sodium chloride, 9.6g of sodium nitrate, 6.4g of ammonium sulfate and 0.24g of monopotassium phosphate in parts by weight, dissolving the components in deionized water, and fixing the volume to 1000 mL;

and step two, after the pH value is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution, 9.6g of calcium carbonate is added according to parts by weight.

4. A culture medium for producing bleomycin E by using Streptomyces abyssochlii, which is prepared by the preparation method according to any one of claims 1 to 3.

5. A process for producing bleomycin E by fermentative culture of Streptomyces deep-sea using the medium according to claim 4, comprising the steps of:

firstly, unfreezing Streptomyces abyssocensis SCSIO ZH16 delta ilaR spore liquid frozen and stored at the temperature of 20 ℃ below zero, streaking and passaging in a solid culture medium, and growing for 7-14 days to obtain a fresh solid culture medium with abundant spore amount;

step two, digging out the solid culture medium of fresh deep sea streptomycete SCSIO ZH16 delta ilaaR with the size of about 0.5cm²Inoculating the agar block into a seed culture medium, and fermenting and culturing for 60h to obtain a fresh seed solution;

step three, taking the fresh seed liquid, inoculating the fresh seed liquid into the culture medium of claim 4 according to the inoculation amount of 10% by volume ratio, and performing shaking culture on a shaker at 28 ℃ and 200rpm for 6-7 days to obtain a fermentation liquid;

and step four, carrying out suction filtration, leaching and purification on the fermentation liquor to obtain the bleomycin E.

6. The method of claim 5, wherein the solid plating medium comprises per liter: 4g of soluble starch, 10g of malt extract, 4g of yeast extract powder, 5-10g of pure oatmeal and 20g of agar powder, dissolving the raw materials in deionized water, cooking for 20 minutes, subpackaging, sterilizing at 121 ℃ for 20 minutes, and adding the apramycin with the final concentration of 25mg/L before flatting.

7. The method of claim 5, wherein the seed medium comprises per liter: 20g of glucose, 2g of peptone, 2g of yeast extract powder, 5g of soybean powder, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate, 4g of sodium chloride and 2g of calcium carbonate, and dissolving the components in deionized water.

Technical Field

The invention belongs to the technical field of microbial fermentation culture media, particularly relates to the technical field of culture media for culturing deep sea streptomycete, and more particularly relates to a culture medium for producing bleomycin E by utilizing deep sea streptomycete and a preparation method thereof.

Background

The sea is a treasure house of human resources and contains abundant biological resources, the number of known biological species of the sea is about 40 to over ten thousand, and 80 percent of the total number of the biological species of the ball is occupied, wherein many species are not available on the land. The ocean covers 70.8% of the earth's surface area, and 95% of the sea depth exceeds 1000 meters, which is called deep sea. Nowadays, deep sea is recognized as a future important gene resource source due to its complex and unique habitat, and has good application and development prospects. Due to the uniqueness of the environment, deep sea contains abundant extreme microorganisms which can generate biomacromolecules and micromolecules with unique structure and function, and the biomacromolecules and the micromolecules are different from the structure and the function of the terrestrial compounds. With the critical point of multidrug resistance, especially in antibiotic resistance, it is imperative to enter unknown waters to explore new chemical entities. A large number of active substances contained in marine rare microorganisms are under development.

The actinomycete strain SCSIO ZH16 was isolated from a sediment sample collected deep at 3536m (120 ℃ 0.250'E, 20 ℃ 22.971' N) from the south sea. Phylogenetic analysis based on the nearly complete 16S rRNA gene sequence showed that strain SCSIO ZH16 belongs to the genus Streptomyces. The 16S rRNA gene sequence has been deposited in GenBank under accession number KT 9708. The strain is preserved in China general microbiological culture Collection center of China institute of microbiology (Beijing, China), and is named as Streptomyces atratus SCSIO ZH 16. Streptomyces abyssochlaini SCSIO ZH16 has the ability to produce a variety of secondary metabolites, but most biosynthetic genes are "silent". The wild strain can produce a kind of cyclic heptapeptide natural products which are named as 'Yi lai mycin'. Only two amino acids of seven amino acids of the bleomycin are natural and unmodified, and comprise special 3-nitrotyrosine, L-2-amino-4-hexenoic acid and isopentenylated tryptophan structural units. The ilaR gene for coding cytochrome P450 monooxygenase is knocked out by a Jujianhua researcher team of south China sea of the Chinese academy of sciences through genetic engineering modification, the epoxidation reaction of isopentenyl is interrupted, and a genetic engineering modified strain delta ilaR is obtained. The strain can generate bleomycin E with strong anti-tubercle bacillus activity, the activity of the bleomycin E is 30 times of that of a first-line anti-tubercle drug rifampicin, and the bleomycin E is an excellent potential anti-tubercle candidate drug.

However, the relatively low yield of telithromycin E in the initial fermentation process has become a bottleneck limiting the use of telithromycin E for preclinical studies. Therefore, it is imperative to improve the yield of the bleomycin E by improving the fermentation process.

Disclosure of Invention

Aiming at the current situation, the culture medium for producing the bleomycin E by fermentation is deeply researched, the culture medium which is simple in components, convenient to operate and low in cost and does not need special equipment is obtained, and the yield of the bleomycin E can be obviously improved by fermenting the deep sea streptomycete to produce the high-efficiency antituberculosis drug of the bleomycin E by using the culture medium. Therefore, the first object of the present invention is to provide a method for preparing a medium for producing bleomycin E by using Streptomyces deep sea. The second purpose of the invention is to provide a culture medium for producing the bleomycin E by using the deep sea streptomycete. A third object of the present invention is a process for the fermentative production of pleithromycin E using the culture medium described above.

In order to achieve the purpose, the invention adopts the following technical scheme:

as a first aspect of the present invention, a method for preparing a medium for producing bleomycin E by using Streptomyces deep sea comprises the steps of:

firstly, accurately weighing 80-120g of soluble starch, 20-30g of yeast extract, 1.6-2.4g of corn steep liquor, 4-6g of sodium chloride, 6.4-9.6g of sodium nitrate, 6.4-9.6g of ammonium sulfate and 0.16-0.24g of monopotassium phosphate in parts by weight, dissolving the components in deionized water, and fixing the volume to 1000 mL;

and step two, regulating the pH value to 7.2-7.4 by using a 3M sodium hydroxide solution, supplementing calcium carbonate with the final concentration of 6.4-9.6g in parts by weight, subpackaging the mixture into a shake flask, and sterilizing the shake flask for 20 minutes at 121 ℃.

According to the invention, the deep-sea streptomyces is deep-sea streptomyces SCSIO ZH16 delta ilaaR.

Further, in the first step, by weight, 120g of soluble starch, 20g of yeast extract, 1.6-2.4g of corn steep liquor, 4-6g of sodium chloride, 6.4-9.6g of sodium nitrate, 6.4-9.6g of ammonium sulfate and 0.16-0.24g of monopotassium phosphate are accurately weighed, dissolved in deionized water and subjected to constant volume to 1000 mL;

and step two, regulating the pH value to 7.2-7.4 by using a 3M sodium hydroxide solution, supplementing calcium carbonate with the final concentration of 6.4-9.6g in parts by weight, subpackaging the mixture into a shake flask, and sterilizing the shake flask for 20 minutes at 121 ℃.

Preferably, the preparation method of the culture medium comprises the following steps: firstly, accurately weighing 120g of soluble starch, 20g of yeast extract, 2.4g of corn steep liquor, 6g of sodium chloride, 9.6g of sodium nitrate, 6.4g of ammonium sulfate and 0.24g of monopotassium phosphate in parts by weight, dissolving the components in deionized water, and fixing the volume to 1000 mL;

and step two, adjusting the pH to 7.2-7.4 by using a 3M sodium hydroxide solution, and adding 9.6g of calcium carbonate.

As a second aspect of the present invention, a medium for producing bleomycin E by Streptomyces abyssal is prepared by the above-mentioned preparation method.

As a third aspect of the present invention, a method for producing bleomycin E by fermenting and culturing Streptomyces abyssinicus with the culture medium described above comprises the following steps:

thawing Streptomyces abyssocensis spore liquid frozen and stored at the temperature of-20 ℃, streaking and passaging in a solid culture medium, and growing for 7-14 days to obtain a fresh solid culture medium with abundant spore amount;

step two, digging out the solid culture medium of fresh deep sea streptomycete with the size of about 0.5cm²Inoculating the agar block into a seed culture medium, and fermenting and culturing for 60h to obtain a fresh seed solution;

step three, taking the fresh seed liquid, inoculating the fresh seed liquid into the culture medium according to the inoculation amount of 10% (v/v), and performing shaking culture on a shaker at the temperature of 28 ℃ and the speed of 200rpm for 6-7 days to obtain a fermentation liquid;

step four, carrying out suction filtration, leaching and purification on the fermentation liquor to obtain the bleomycin E;

and step five, the thallus growth is characterized by the thallus dry weight. 10mL of the well-mixed fermentation broth was taken, filtered under vacuum, rinsed with 100mL of tap water and drained. The cells and filter paper were dried at 55 ℃ for 24h, then transferred to a desiccator to be cooled and weighed. The mycelium stem is obtained by calculation by a differential subtraction method.

According to the invention, the deep-sea streptomyces is deep-sea streptomyces SCSIO ZH16 delta ilaaR.

According to the invention, the solid plating medium comprises per liter: 4g of soluble starch, 10g of malt extract, 4g of yeast extract powder, 5-10g of pure oatmeal and 20g of agar powder, dissolving the raw materials in deionized water, cooking for 20 minutes, subpackaging, sterilizing at 121 ℃ for 20 minutes, and adding the apramycin with the final concentration of 25mg/L before flatting.

According to the invention, the seed medium comprises per liter: 20g of glucose, 2g of peptone, 2g of yeast extract powder, 5g of soybean powder, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate, 4g of sodium chloride and 2g of calcium carbonate, and dissolving the components in deionized water.

The invention has the beneficial effects that:

1. the components of the culture medium comprise soluble starch, yeast extract, corn steep liquor, sodium chloride, sodium nitrate, ammonium sulfate, monopotassium phosphate and calcium carbonate, the raw materials are easy to obtain, the price is low, and the popularization and the application are facilitated;

2. the culture medium is prepared by dissolving the raw materials in deionized water, and the method is simple, low in difficulty and free of special technical training and special instruments and equipment;

3. the culture medium and the method are adopted to ferment and culture the Streptomyces abyssocensis SCSIO ZH16 delta ilaR, the strain grows rapidly in the culture medium, the flux of a secondary metabolic pathway is improved, the culture medium is used to ferment and culture the Streptomyces abyssocensis SCSIO ZH16 delta ilaR to produce the bleomycin E, the yield of a target product of the bleomycin E can be improved, the difficulty is reduced for the later purification work, and the maximum yield of the bleomycin E can reach 202.96mg/L which is 12.76 times of the original level.

Drawings

FIG. 1 is a graph comparing the results of production of bleomycin E in examples of the present invention with that of a control group.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not specified, in the following examples are generally conducted under conventional conditions, or under conditions provided by the manufacturers.

It should be noted that the components of the culture medium selected for use in the present invention are all commercially available products. Yeast extract was purchased from national drug group chemical reagents, Inc.; soluble starch is purchased from Shanghai Tantake technologies, Inc.; corn steep liquor was purchased from amelaner hao biotechnology limited.

It should be noted that in small scale experiments, to ensure complete sterilization, the dry corn steep liquor powder should be dissolved sufficiently to prevent the recovery of residual bacteria during fermentation.

1. The strains used in the present invention:

streptomyces abyssalis SCSIO ZH16 Δ ilaaR, provided by Jujia researchers team at south China sea of Chinese academy of sciences. The deep sea streptomycete SCSIO ZH16 is separated from sediments in 3536 meters deep in south China sea, and is subjected to genetic engineering modification to obtain a modified strain SCSIO ZH16 delta ilaR.

2. Seed culture medium:

accurately weighing 20g of glucose, 2g of peptone, 2g of yeast extract powder, 5g of soybean powder, 0.5g of magnesium sulfate heptahydrate, 0.5g of monopotassium phosphate and 4g of sodium chloride, dissolving with deionized water, fixing the volume to 1000mL, adjusting the pH to 7.2-7.4 with 3M sodium hydroxide solution, and adding 2g of calcium carbonate.

3. Solid culture medium

Accurately weighing 4g of soluble starch, 10g of malt extract, 4g of yeast extract powder, 5-10g of pure oatmeal and 20g of agar powder, dissolving in deionized water, steaming for 20 minutes, subpackaging, sterilizing at 121 ℃ for 20 minutes, and adding 25mg/L of apramycin before paving.

Example 1

1) Preparation of fermentation Medium

120g of glucose, 20g of soybean meal, 2.4g of corn steep liquor, 6g of sodium chloride, 9.6g of sodium nitrate, 6.4g of ammonium sulfate and 0.24g of monopotassium phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, and 9.6g of calcium carbonate is added after the pH is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

Thawing Streptomyces abyssocensis SCSIO ZH16 delta ilaR spore liquid frozen and stored at the temperature of 20 ℃ below zero, streaking and passaging in a solid culture medium, and growing for 7-14 days to obtain a fresh solid culture medium with abundant spore amount. Digging out the solid culture medium of fresh Streptomyces abyssochlii SCSIO ZH16 delta ilaaR with the size of about 0.5cm²The agar block was inoculated into a 250mL shake flask containing 25mL of seed medium, shake-cultured at 28 ℃ for 60 hours at 200rpm to obtain a fresh seed solution, the novel medium was inoculated at an inoculum size of 10% (v/v), and shake-cultured at 28 ℃ and 200 rpm.

3) Yield detection of bleomycin E

25mL of the fermentation liquor obtained are taken, filtered off under vacuum, rinsed with 100mL of tap water and filtered off. Extracting the supernatant with ethyl acetate for three times, evaporating to dryness at 45 deg.C under reduced pressure, dissolving with methanol, transferring methanol into EP tube, centrifuging at 12000rpm, collecting supernatant into EP tube, ultrasonic dissolving, filtering with 0.22 μm filter membrane, and storing in 4 deg.C refrigerator for HPLC detection. The filtered cells were dried, ground in a mortar, extracted with methanol, and the solvent was evaporated to dryness at 45 ℃ under reduced pressure, then eluted with methanol, transferred to an EP tube, centrifuged at 12000rpm, the supernatant was taken into an EP tube, ultrasonically dissolved, filtered through a 0.22 μm filter, and stored in a refrigerator at 4 ℃ for HPLC detection. In conclusion, the content of the bleomycin E was measured by high performance liquid chromatography, and the actual content of the bleomycin E in the supernatant and the bacterial cells was converted from a calibration curve.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 46.8mg/L and the biomass was found to be 26.6 g/L.

Example 2

1) Preparation of fermentation Medium

120g of glucose, 20g of yeast extract, 2.4g of corn steep liquor, 6g of sodium chloride, 9.6g of sodium nitrate, 6.4g of ammonium sulfate and 0.24g of monopotassium phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, the pH is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution, and 9.6g of calcium carbonate is added. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 121.55mg/L and the biomass was found to be 27.6 g/L.

Example 3

1) Preparation of a fermentation medium:

120g/L of soluble starch, 20g/L of soybean meal, 2.4g/L of corn steep liquor, 6g/L of sodium chloride, 9.6g/L of sodium nitrate, 6.4g/L of ammonium sulfate and 0.24g/L of potassium dihydrogen phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, the pH value is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution, and 9.6g of calcium carbonate is added. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 54.05mg/L and the biomass was found to be 23.5 g/L.

Example 4

1) Preparation of fermentation Medium

120g of soluble starch, 20g of yeast extract, 2.4g of corn steep liquor, 6g of sodium chloride, 9.6g of sodium nitrate, 6.4g of ammonium sulfate and 0.24g of monopotassium phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, and 9.6g of calcium carbonate is added after the pH is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 202.96mg/L and the biomass was found to be 25.7 g/L.

Example 5

1) Preparation of fermentation Medium

120g of soluble starch, 20g of yeast extract, 1.6g of corn steep liquor, 4g of sodium chloride, 6.4g of sodium nitrate, 9.6g of ammonium sulfate and 0.24g of monopotassium phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, and 9.6g of calcium carbonate is added after the pH is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 199.77mg/L and the biomass was found to be 26.6 g/L.

Example 6

1) Preparation of fermentation Medium

120g of soluble starch, 20g of yeast extract, 2.4g of corn steep liquor, 4g of sodium chloride, 9.6g of sodium nitrate, 9.6g of ammonium sulfate and 0.16g of monopotassium phosphate are accurately weighed, dissolved in deionized water, and the volume is determined to be 1000mL, the pH is adjusted to 7.2-7.4 by using 3M sodium hydroxide solution, and then 6.4g of calcium carbonate is added. Then, the mixture was dispensed into a shake flask and sterilized at 121 ℃ for 20 minutes.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was found to be 133.93mg/L and the biomass was found to be 28.6 g/L.

Comparative example 1 initial fermentation Process

1) Preparation of fermentation Medium

Accurately weighing 3g of soluble starch, 3g of peptone, 1g of soybean meal, 3g of glycerol and 6g of sea salt, dissolving the soluble starch, the peptone, the soybean meal, the glycerol and the sea salt in deionized water, fixing the volume to 1000mL, adjusting the pH value to 7.2-7.4 by using a 3M sodium hydroxide solution, and adding 0.4g of calcium carbonate.

2) Fermentation process

The same procedure as in example 1 was used.

3) Yield detection of bleomycin E

The yield of bleomycin E was measured as in example 1.

Finally, the yield of the target product, i.e., the bleomycin E, was 15.9mg/L and the biomass was 13.3 g/L.

The results of examples 1-6 and comparative example 1 are shown in FIG. 1.

The results show that, for the screened preferred culture medium (example 1), carbon-nitrogen source optimization (examples 3 and 2) is respectively carried out, the optimal culture medium formula (example 4) of the invention is obtained, so that the yield of the leiomycinE reaches 202.96mg/L, and the yield of the leiomycinE in examples 5 and 6 also reaches 199.77mg/L and 133.93mg/L respectively, compared with a control group (example of the control group), the yield of the leiomycinE is obviously improved by fermenting and culturing the deep sea streptomyces SCSIO ZH16 Δ ilaR by using the culture medium of the invention.

The foregoing is merely an example of the embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.