阅读说明：本技术 一种生产去甲四环素的方法 (Method for producing nortetracycline ) 是由 王雪峰 韩冰 闫彩洁 郑万静 王绘砖 徐珍 袁昉 于 2020-05-28 设计创作，主要内容包括：本发明属于微生物发酵技术领域,具体涉及一种生产去甲四环素的方法,将去甲金霉素生产菌株接种斜面培养基,25-30℃,20％-60％湿度条件下培养10-15天；成熟后接种至种瓶培养基,25-30℃,20％-60％湿度条件下220rpm震荡培养36-50h；种液成熟后,按照5％-10％接种量接种到发酵培养基中,相同条件下震荡培养5-8天,发酵结束,得到去甲四环素发酵液；本发明提高了去甲四环素的发酵单位,达到了工业化生产水平,对米诺环素、替加环素等高附加值产品生产过程中简化合成步骤,减少危险因素,降低生产成本具有重要的意义和应用价值。(The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing nortetracycline, which comprises the steps of inoculating a noraureomycin production strain into a slant culture medium, and culturing for 10-15 days at 25-30 ℃ under the condition of 20-60% humidity; inoculating to a seed bottle culture medium after maturation, and performing shake culture at 220rpm under the conditions of 25-30 ℃ and 20-60% humidity for 36-50 h; after the liquid is mature, inoculating the liquid into a fermentation medium according to the inoculation amount of 5% -10%, performing shake culture for 5-8 days under the same condition, and finishing fermentation to obtain a nortetracycline fermentation liquid; the invention improves the fermentation unit of the nortetracycline, achieves the industrial production level, and has important significance and application value for simplifying the synthesis steps, reducing the risk factors and reducing the production cost in the production process of high value-added products such as minocycline, tigecycline and the like.)

1. A method of producing nortetracycline, comprising the steps of:

(1) slant culture: inoculating the demethylated aureomycin production strain into a slant culture medium, and culturing for 10-15 days at 25-30 ℃ under the humidity condition of 20% -60%;

(2) culturing in a seed bottle: inoculating the mature slant culture to a seed bottle culture medium, and performing shake culture at the temperature of 25-30 ℃ and the humidity of 20% -60% at 220rpm for 36-50 h;

(3) fermentation culture: after the liquid is mature, inoculating the liquid into a fermentation medium according to the inoculation amount of 5% -10%, performing shake culture for 5-8 days under the same condition, and finishing fermentation to obtain the nortetracycline fermentation liquid.

2. A method of producing nortetracycline according to claim 1, wherein the slant medium comprises the following components and amounts thereof: 1-2% of flour, 0.05-0.15% of peptone, 0.01-0.1% of potassium dihydrogen phosphate, 0-0.005% of magnesium sulfate and 2.0-2.5% of agar.

3. A method of producing nortetracycline according to claim 1, wherein the seed bottle medium comprises the following components and amounts: 1.0-3.0% of glucose, 1.0-3.0% of starch, 1.0-3.0% of high-temperature soybean powder, 0.5-1.5% of calcium carbonate, 0.1-0.5% of yeast powder, 0.5-3.0% of peptone, 0.1-0.5% of ammonium sulfate, 0-0.5% of sodium bromide, 0.01-0.05% of potassium dihydrogen phosphate, 0-0.5% of corn steep liquor, 0.5-2% of soybean oil (V/V) and natural pH.

4. A method of producing nortetracycline according to claim 1, wherein the fermentation medium comprises the following components and amounts: 6-10% of starch, 2-4% of dextrin, 2-4% of high-temperature soybean powder, 0.5-3% of soybean protein powder, 0.5-1.5% of calcium carbonate, 1.0-3.0% of peptone, 0.1-0.5% of ammonium sulfate, 0.5-1.5% of L-lysine, 0.1-0.5% of sodium bromide, 0.005-0.05% of potassium dihydrogen phosphate, 0.3-1.5% of corn steep liquor, 0.5-3% of soybean oil (V/V), 0.01-0.05% of defoaming agent, 0.05-0.1% of alpha-amylase (W/W of the amount of starch) and 0.001-0.002% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid or nitric acid.

5. A method of producing nortetracycline according to claim 1, wherein the fermentation medium comprises the following components and amounts: 9% of starch, 2% of dextrin, 3% of high-temperature soybean powder, 1% of soybean protein powder, 1% of calcium carbonate, 1% of peptone, 0.1% of ammonium sulfate, 0.8% of L-lysine, 0.3% of sodium bromide, 0.005% of potassium dihydrogen phosphate, 0.3% of corn steep liquor, 1% of soybean oil (V/V), 0.01% of defoaming agent, 0.05% of alpha-amylase (W/W of the amount of starch) and 0.0015% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid.

6. A method of producing nortetracycline according to claim 1, wherein the fermentation medium comprises the following components and amounts: 10% of starch, 2% of dextrin, 3.5% of high-temperature soybean powder, 2% of soybean protein powder, 1% of calcium carbonate, 1% of peptone, 0.1% of ammonium sulfate, 1.0% of L-lysine, 0.3% of sodium bromide, 0.005% of potassium dihydrogen phosphate, 0.3% of corn steep liquor, 2% of soybean oil (V/V), 0.01% of antifoaming agent, 0.05% of alpha-amylase (W/W of the amount of starch) and 0.0015% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid.

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing nortetracycline.

Background

Tetracyclines antibiotics (Tetracyclines) are a class of broad spectrum antibiotics with a tetracene (Naphthacene) core structure (figure 1), including Oxytetracycline (OTC), chlortetracycline (CTC), Tetracycline (TC), norchlortetracycline (DMCTC), nortetracycline (DMTC), doxycycline, minocycline, tigecycline, and the like (Nguyen F, StarostaAL, Arenz S, SohmenD, donhofer a, Wilson DN (2014) Tetracyclines antibiotics and restristanse mechanisms, biological 395(5): 559-75). Among these antibiotics, OTC, CTC, TC, DMCTC and DMTC are natural products, OTC is produced by Streptomyces rimosus, and CTC, TC, DMCTC and DMTC are produced by Streptomyces aureus or mutants thereof.

With the emergence of drug-resistant strains and the urgent need of broad-spectrum antibiotics, researchers developed and synthesized more efficient tetracyclic antibiotics such as doxycycline, minocycline, tigecycline, etc. through chemical modification on the basis of these natural products, such as: doxycycline can be synthesized by taking terramycin as a raw material, and minocycline, tigecycline and the like can be synthesized by taking demeclocycline as a raw material. It is worth noting that in 2018 there were 3 tetracycline antibiotics that were FDA approved: eravacyline, Sarecocyline and Omadacyline. Therefore, the method has important significance for improving the productivity of the biosynthesis products.

In the actual production, nortetracycline (figure 2) is synthesized by taking noraureomycin as a raw material, and then minocycline is synthesized by taking nortetracycline as a raw material, so that the minocycline can be further synthesized into tigecycline with higher value. The process of synthesizing the nortetracycline by taking the noraureomycin as the raw material needs high-pressure hydrogenation dechlorination reaction catalyzed by metal palladium (Pd), and the reaction is dangerous and high in cost, so that if the nortetracycline can be produced by a fermentation method, the steps of synthesizing minocycline, tigecycline and the like can be simplified, the dangerous factors are reduced, and the production cost is reduced.

In the current research, OTC, CTC, TC, and DMCTC are synthesized by fermentation, and are all produced industrially, while the research on nortetracycline (DMTC) is rarely reported. CN201910693880.8 obtains an engineering strain DMT1 for producing the nortetracycline by knocking out the ctcK gene of the coding methyltransferase in the aureofaciens strain S.aureofaciens CGMCC4.1043, and further improves the yield of the nortetracycline by expressing strategies such as activating gene ctcB in a synthetic cluster, resistance gene ctcR, synthetic gene ctcQ and the like on the basis; CN201911007748.3 obtains a method for producing nortetracycline by fermentation of streptomyces aureofaciens by adjusting the culture medium composition of a fermentation tank, adjusting a feed supplement formula and matching with the process control of the fermentation process.

In the fermentation process of the noraureomycin, the nortetracycline is one of impurities, and the yield is low. The structural difference between the nortetracycline and the noraureomycin is that the 7-position lacks a chlorine atom, so the synthesis process is supposed to be firstly to synthesize the nortetracycline, and then to synthesize the noraureomycin by halogenating and chloridizing the 7-position. In the fermentation process of the demethyltetracycline, if the fermentation main product can be converted from the demethyltetracycline to the demethyltetracycline by changing the process conditions, the method is used for producing the demethyltetracycline, and has important application value.

In view of the above, the present invention develops a method and a culture medium for producing nortetracycline by the noraureomycin producing strain s.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for producing nortetracycline, which adopts a noraureomycin production strain streptomyces aureofaciens to produce the nortetracycline by changing a culture medium and fermentation conditions.

The invention adopts the specific technical scheme that: (1) slant culture: inoculating the demethylated aureomycin production strain into a slant culture medium, and culturing for 10-15 days at 25-30 ℃ under the humidity condition of 20% -60%;

(2) culturing in a seed bottle: inoculating the mature slant culture to a seed bottle culture medium, and performing shake culture at the temperature of 25-30 ℃ and the humidity of 20% -60% at 220rpm for 36-50 h;

(3) fermentation culture: after the liquid is mature, inoculating the liquid into a fermentation medium according to the inoculation amount of 5% -10%, performing shake culture for 5-8 days under the same condition, and finishing fermentation to obtain the nortetracycline fermentation liquid.

Preferably, the components and contents of the slant culture medium are as follows: 1-2% of flour, 0.05-0.15% of peptone, 0.01-0.1% of potassium dihydrogen phosphate, 0-0.005% of magnesium sulfate and 2.0-2.5% of agar.

Preferably, the seed bottle culture medium comprises the following components in percentage by weight: 1.0-3.0% of glucose, 1.0-3.0% of starch, 1.0-3.0% of high-temperature soybean powder, 0.5-1.5% of calcium carbonate, 0.1-0.5% of yeast powder, 0.5-3.0% of peptone, 0.1-0.5% of ammonium sulfate, 0-0.5% of sodium bromide, 0.01-0.05% of potassium dihydrogen phosphate, 0-0.5% of corn steep liquor, 0.5-2% of soybean oil (V/V) and natural pH.

Preferably, the fermentation medium comprises the following components in percentage by weight: 6-10% of starch, 2-4% of dextrin, 2-4% of high-temperature soybean powder, 0.5-3% of soybean protein powder, 0.5-1.5% of calcium carbonate, 1.0-3.0% of peptone, 0.1-0.5% of ammonium sulfate, 0.5-1.5% of L-lysine, 0.1-0.5% of sodium bromide, 0.005-0.05% of potassium dihydrogen phosphate, 0.3-1.5% of corn steep liquor, 0.5-3% of soybean oil (V/V), 0.01-0.05% of defoaming agent, 0.05-0.1% of alpha-amylase (W/W of the amount of starch) and 0.001-0.002% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid or nitric acid.

Preferably, the fermentation medium comprises the following components in percentage by weight: 9% of starch, 2% of dextrin, 3% of high-temperature soybean powder, 1% of soybean protein powder, 1% of calcium carbonate, 1% of peptone, 0.1% of ammonium sulfate, 0.8% of L-lysine, 0.3% of sodium bromide, 0.005% of potassium dihydrogen phosphate, 0.3% of corn steep liquor, 1% of soybean oil (V/V), 0.01% of defoaming agent, 0.05% of alpha-amylase (W/W of the amount of starch) and 0.0015% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid.

Preferably, the fermentation medium comprises the following components in percentage by weight: 10% of starch, 2% of dextrin, 3.5% of high-temperature soybean powder, 2% of soybean protein powder, 1% of calcium carbonate, 1% of peptone, 0.1% of ammonium sulfate, 1.0% of L-lysine, 0.3% of sodium bromide, 0.005% of potassium dihydrogen phosphate, 0.3% of corn steep liquor, 2% of soybean oil (V/V), 0.01% of antifoaming agent, 0.05% of alpha-amylase (W/W of the amount of starch) and 0.0015% of 2-mercaptobenzothiazole. Adjusting pH to 5.2-5.5 with sulfuric acid.

The invention has the beneficial effects that: by changing the culture medium and the fermentation conditions, the nortetracycline can be produced by using the noraureomycin production strain streptomyces aureofaciens, and the fermentation unit of the nortetracycline is obviously improved by process optimization, thereby reaching the industrial production level. The nortetracycline is used as a raw material to synthesize minocycline, tigecycline and the like, so that the synthesis steps can be simplified, the risk factors are reduced, the production cost is reduced, and the method has important application value.

Drawings

FIG. 1 is a structural formula of tetracycline antibiotics;

FIG. 2 is a structural formula of nortetracycline.

Detailed Description

The invention will be further illustrated with reference to specific examples: