阅读说明：本技术 一种提高甾药生产菌株活力的甾药前体发酵生产方法 (Steroid precursor fermentation production method for improving activity of steroid production strain ) 是由 系祖斌 卢方欣 潘高峰 贺一君 于 2019-11-27 设计创作，主要内容包括：本发明属于生物工程技术领域,尤其涉及一种提高甾药生产菌株活力的甾药前体发酵生产方法,通过在菌种发酵的过程中培养基中磷酸盐含量进行控制,并在菌体的发酵过程中采用变温培养方式,可以有效解决微生物法甾药前体生产时存在的生产效率低的问题,为甾药前体生产成本的降低提供新方法。(The invention belongs to the technical field of bioengineering, and particularly relates to a steroid precursor fermentation production method for improving the activity of a steroid production strain.)

1. A steroid precursor fermentation production method for improving the activity of a steroid production strain is characterized by comprising the following steps:

(1) weighing a proper amount of rice, putting the rice into a sieve, washing the rice with distilled water, draining, and pouring the rice into a special containing basin;

(2) adding nutrient solution into the placing basin in the step 1, stirring to adjust the pH value, wherein the mass ratio of the nutrient solution to the rice is 1: 3, stirring for 10-20min to obtain a first mixture;

(3) subpackaging the first mixture obtained in the step 2, and respectively filling the first mixture into a plurality of shake flasks;

(4) placing the shake flask filled in the step 3 into a sterilization device for sterilization;

(5) taking out the shake flasks after the sterilization in the step 4, adding a proper amount of spore suspension into each shake flask, and then putting all the shake flasks into a shaking table for cultivation, wherein the cultivation temperature is controlled to be 25-28 ℃, the shaking speed of the shaking table is controlled to be 90-120rpm, and the cultivation time is controlled to be 20-37 h;

(6) taking out the spores cultured in the shake flask in the step 5, performing vacuum drying, and then storing the strains by using a sand-soil tube method, wherein the temperature of the storage environment is controlled at 0-4 ℃;

(7) taking out the strain stored in the step 6, inoculating the strain into a 1000L seed fermentation tank filled with a culture medium according to the inoculation amount of 5-10%, controlling the pH value to be 6.3-6.7, reducing the pH value by adding a proper amount of ammonium sulfate when the pH value is higher than the optimal pH value, increasing the pH value by supplementing ammonia water when the pH value is lower than the optimal pH value, controlling the culture time to be 20-25h and controlling the temperature to be 29-32 ℃;

(8) inoculating the strain in 1000L seed fermentation tank into 8000L seed fermentation tank filled with culture medium according to 20-30% of inoculation amount, controlling pH value at 6.8-7.3, reducing pH value by adding appropriate amount of ammonium sulfate when pH is higher than optimum pH, increasing pH value by adding ammonia water when pH is lower than optimum pH, maintaining temperature at 25 deg.C within 0-45h, then reducing temperature to 20 deg.C at constant speed, maintaining for 175h, and returning to 25 deg.C within 20h for culturing.

2. A process for the fermentative production of a steroid precursor with improved activity of a steroid producing strain according to claim 1, wherein the rice in the step 1 is new rice of high quality.

3. A process for the fermentative production of a steroid precursor according to claim 1, wherein the shake flask used in step 3 is a 250ml shake flask, and wherein the percentage of the volume of the first mixture in each shake flask to the volume of the shake flask is 16-20%.

4. A process for the fermentative production of a steroid precursor for enhancing the activity of a steroid producing strain according to claim 1, wherein the sterilization means in step 4 is irradiation sterilization.

5. A process for the fermentative production of a steroid precursor with improved activity of a steroid producing strain according to claim 1, wherein the formulation of the culture medium in the 1000L fermenter in step 7 is: maltose 130-140g/L, nitrate 2.8-3.2g/L, phosphate 3.1-4.0g/L, etc.

6. A process for the fermentative production of a steroid precursor with improved activity of a steroid producing strain according to claim 1, wherein in step 8, 8000L of the fermentation tank medium is formulated as: maltose 130-140g/L, nitrate 2.8-3.2g/L, phosphate 1.0-1.2g/L, etc.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a steroid precursor fermentation production method for improving the activity of steroid production strains.

Background

Steroids have prominent positions in the chemical industry, and are second only to antibiotics. The steroid drugs play important regulating roles on organisms, including improving protein metabolism, restoring and enhancing physical strength, inducing diuresis and reducing blood pressure and the like; the medicine can be used for treating skin diseases such as rheumatic arthritis and eczema, and endocrine diseases such as prostate and Edison; can be used in the fields of contraception, miscarriage prevention, operation anesthesia and the like. More than 300 steroid drugs are currently produced globally, the main of which are steroid hormone drugs. The global steroid hormone drug sales in 2016 exceed 1000 billion dollars, and are second only to antibiotics in the second major class of chemicals. At present, our country has developed new resources of steroid hormone drugs as one of the recent development directions and key points of the pharmaceutical industry, and the export of hormone bulk drugs and intermediates has become an important variety of bulk drugs in our country going to the world.

Phytosterols are known to be catabolized by many microorganisms into a range of steroid precursors. Of these, 4-androstene-3, 17-dione (AD) and 1, 4-androstene-3, 17-dione (ADD) are the main precursors for the synthesis of steroid drugs such as progestogens, contraceptives and estrogens. It is reported that rapidly growing M.tumefaciens has a strong ability to degrade phytosterol side chains to accumulate AD and ADD. As an alternative to chemical synthesis, bioconversion has become the primary production method in the pharmaceutical industry for the production of steroid precursors. However, both the original strain and the engineering strain have long transformation period (more than or equal to 5 days), and the vitality of the strain is obviously reduced in the middle and later transformation period (after 3 days), and the transformation efficiency is sharply reduced. This is also one of the reasons for the high cost of production of steroid precursors.

Disclosure of Invention

The invention provides a steroid precursor fermentation production method for improving the activity of steroid producing strains and a preparation process thereof, which aim to solve the problems in the background technology.

The invention provides a steroid precursor fermentation production method for improving the activity of steroid production strains, which is characterized by comprising the following steps:

(1) weighing a proper amount of rice, putting the rice into a sieve, washing the rice with distilled water, draining, and pouring the rice into a special containing basin;

(2) adding nutrient solution into the placing basin in the step 1, stirring to adjust the pH value, wherein the mass ratio of the nutrient solution to the rice is 1: 3, stirring for 10-20min to obtain a first mixture;

(3) subpackaging the first mixture obtained in the step 2, and respectively filling the first mixture into a plurality of shake flasks;

(4) placing the shake flask filled in the step 3 into a sterilization device for sterilization;

(5) taking out the shake flasks after the sterilization in the step 4, adding a proper amount of spore suspension into each shake flask, and then putting all the shake flasks into a shaking table for cultivation, wherein the cultivation temperature is controlled to be 25-28 ℃, the shaking speed of the shaking table is controlled to be 90-120rpm, and the cultivation time is controlled to be 20-37 h;

(6) taking out the spores cultured in the shake flask in the step 5, performing vacuum drying, and then storing the strains by using a sand-soil tube method, wherein the temperature of the storage environment is controlled at 0-4 ℃;

(7) taking out the strain stored in the step 6, inoculating the strain into a 1000L seed fermentation tank filled with a culture medium according to the inoculation amount of 5-10%, controlling the pH value to be 6.3-6.7, reducing the pH value by adding a proper amount of ammonium sulfate when the pH value is higher than the optimal pH value, increasing the pH value by supplementing ammonia water when the pH value is lower than the optimal pH value, controlling the culture time to be 20-25h and controlling the temperature to be 29-32 ℃;

(8) inoculating the strain in 1000L seed fermentation tank into 8000L seed fermentation tank filled with culture medium according to 20-30% of inoculation amount, controlling pH value at 6.8-7.3, reducing pH value by adding appropriate amount of ammonium sulfate when pH is higher than optimum pH, increasing pH value by adding ammonia water when pH is lower than optimum pH, maintaining temperature at 25 deg.C within 0-45h, then reducing temperature to 20 deg.C at constant speed, maintaining for 175h, and returning to 25 deg.C within 20h for culturing.

Preferably, the rice in the step 1 is high-quality new rice.

Preferably, the shake flask in step 3 is a 250ml capacity shake flask, wherein the percentage of the volume of the first mixture in each shake flask to the capacity of the shake flask is 16-20%.

Preferably, the sterilization mode in the step 4 is irradiation sterilization.

Preferably, the formula of the 1000L fermentation tank culture medium in the step 7 is as follows: maltose 130-140g/L, nitrate 2.8-3.2g/L, phosphate 3.1-4.0g/L, etc.

Preferably, in the step 8, the formula of the 8000L fermentation tank culture medium is as follows: maltose 130-140g/L, nitrate 2.8-3.2g/L, phosphate 1.0-1.2g/L, etc.

Compared with the prior art, the invention has the beneficial effects that:

(1) by controlling the content of phosphate in the step 7 and the step 8, the concentration of phosphate is properly increased in the growth period of the thalli, and the concentration of phosphate is properly reduced in the production period of the thalli, so that the effects of promoting primary metabolism, inhibiting secondary metabolism, inhibiting the synthesis of certain key enzymes in the synthesis of secondary metabolites, inhibiting the activity of certain key enzymes in the synthesis of secondary metabolites, increasing the energy load of the thalli and simultaneously promoting the primary metabolism are achieved, the activity of the thalli is improved, and the production efficiency of the thalli is improved;

(2) in step 8, the thalli in the production period are cultured in a variable temperature culture mode, so that the environmental temperature can adapt to the requirements of different stages in the production period of the thalli, the activity of the thalli is further improved, and the production efficiency of the thalli is improved.

Detailed Description

The invention is further illustrated by the following examples.