CN114717282A

CN114717282A - Composition and application thereof

Info

Publication number: CN114717282A
Application number: CN202210271267.9A
Authority: CN
Inventors: 佘卢; 夏平宇; 赵邦红; 周良娟
Original assignee: Beijing Strowin Biotechnology Co ltd
Current assignee: Beijing Strowin Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2022-03-18
Publication date: 2022-07-08

Abstract

The application belongs to the technical field of microbial fermentation, and particularly discloses a composition which comprises the following components in parts by weight: 0.5-1.5 parts of defoaming agent, 0.08-0.5 parts of adenosine triphosphate, 10-20 parts of glycerol and 2-10 parts of ammonium sulfate. The application has at least one of the following beneficial effects: according to the composition provided by the application, in the continuous fermentation process, the viscosity of the fermentation liquor can be reduced by feeding the composition, dissolved oxygen is increased, nutrition is supplemented, and the growth environment of thalli is improved, so that the yield of a target product and the utilization rate of equipment are increased.

Description

Composition and application thereof

Technical Field

The application belongs to the technical field of microbial fermentation, and particularly relates to a composition and application thereof in a continuous fermentation process of pichia pastoris engineering bacteria.

Background

The continuous fermentation process is that when the microorganism is cultured to the exponential growth phase, on one hand, a fresh liquid culture medium is continuously and continuously added into a fermentation tank at a certain speed, and after the fresh liquid culture medium is fully stirred and utilized by the produced strains, the fermentation liquid is continuously discharged at the same speed, so that the growth and the metabolism of the microorganism in the fermentation tank are kept in a vigorous and stable state.

In the fermentation process of pichia pastoris, the wet weight in the fermentation liquor is gradually increased (the highest amount can be about 500g/L at the end of fermentation) along with the continuous growth and passage of thalli, the fermentation liquor is more and more viscous, the Dissolved Oxygen (DO) is reduced under the same air condition, and the growth environment of the thalli becomes worse due to the accumulation of various metabolites. In the later stage of fermentation, the amount of target protein secreted by the unit thallus is gradually reduced. A large amount of water is also taken away along with the discharge of air, so that a concentration process is formed, the protein expression of the strain is influenced, the utilization rate of equipment is reduced, and the production cost is increased invisibly.

Disclosure of Invention

In order to enhance the growth and fermentation of the strain in a continuous fermentation process, the present application provides a composition. The composition can promote growth and fermentation of the strain during continuous fermentation.

The application is realized by the following scheme:

the application provides a composition, which comprises the following components in parts by weight: 0.5-1.5 parts of defoaming agent, 0.08-0.5 parts of adenosine triphosphate, 10-20 parts of glycerol and 2-10 parts of ammonium sulfate.

The present application has obtained a composition by long-term exploration. During the continuous fermentation process, the fed-batch addition of the composition can effectively reduce foam, improve dissolved oxygen and store the growth and metabolism of thalli.

The application also provides application of the composition in a continuous fermentation process of pichia pastoris engineering bacteria.

In one embodiment of the present application, the continuous fermentation process of pichia pastoris engineering bacteria comprises the following steps:

step 1: inoculating the pichia pastoris engineering strain seed liquid to a fermentation culture medium for culturing until the wet weight of the strain reaches over 90 g/L. Preferably, the wet weight of the thalli reaches 90-110 g/L;

step 2: adding glucose solution containing PTM1 in a flowing manner, and adjusting the adding speed to control the dissolved oxygen to be more than 20% until the wet weight of the thalli reaches more than 160 g/L. Preferably, the wet weight of the thallus reaches 160-;

and step 3: adding a glucose solution containing PTM1 in a flowing manner, adding methanol in a flowing manner to induce the expression of the foreign protein, and adjusting the feeding rate according to the dissolved oxygen to ensure that the dissolved oxygen is controlled to be more than 20%;

and 4, step 4: continuously fermenting, reducing the fed-batch amount of the glucose solution containing the PTM1, increasing the fed-batch amount of the methanol, and starting to collect the fermentation liquor;

and 5: stopping feeding the glucose solution containing PTM1, and increasing the feeding amount of methanol again until the wet weight of the bacteria reaches above 300g/L, preferably, the wet weight of the bacteria reaches 300-360 g/L;

step 6: feeding the composition of claim 1 while feeding methanol until the foreign protein expression level is not reached.

In one embodiment of the present application, the composition is first formulated as a solution according to: 0.5-1.5 g of defoaming agent, 0.08-0.5 g of adenosine triphosphate, 10-20g of glycerol and 2-10g of ammonium sulfate are added into 1L of water for preparation.

In one embodiment of the present application, in step 6, the composition is supplemented in such a way that:

1-12 h: 19-22L/h/L, dissolved oxygen is controlled to be more than 25%, and the pH value is 5.0-5.5;

13-19 h: 39-42L/h/L, dissolved oxygen is controlled to be more than 20%, and the pH value is 5.0-5.5;

20-65 h: 50-53L/h/L, dissolved oxygen is controlled to be more than 10%, and the pH value is 5.0-5.5;

after 66 h: 60-62L/h/L, dissolved oxygen is controlled to be more than 10%, and the pH value is 5.0-5.5.

In one embodiment of the present application, in step 1, the fermentation medium includes 5% of glucose, 4% of ammonium dihydrogen phosphate, 1% of magnesium sulfate, 1% of potassium sulfate, 0.5% of potassium dihydrogen phosphate, 0.1% of calcium sulfate, and 0.16% of potassium hydroxide.

In a specific embodiment of the present application, the culture time in step 1 is 12-16h, and the pH value is maintained at 4.8-5.0 during the culture process.

In one embodiment of the present application, in the step 2, the concentration of the glucose solution is 20% to 30%, and the concentration of the PTM1 is 10 to 15 mL/L.

In one embodiment of the present application, in the step 2, the glucose solution is added in an amount of 30-40 mL/h/L, for example, 30mL/h/L, 32mL/h/L, 34mL/h/L, 36mL/h/L, 38mL/h/L or 40 mL/h/L.

In one embodiment of the present application, in the step 2, the glucose solution is fed in an amount of 36 mL/h/L.

In a specific embodiment of the present application, the culture time in step 2 is 5-6 h.

In a specific embodiment of the present application, in the step 3, the volume ratio of methanol to glucose solution is 1: (5-10). For example, the volume ratio of methanol to glucose solution is 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, etc.

In one embodiment of the present application, in the step 3, the volume ratio of the methanol to the glucose solution is 1: 8.

In one embodiment of the present application, the glucose is fed in an amount of 8mL/h/L in step 3.

In one embodiment of the present application, the methanol is fed in the step 3 in an amount of 1 mL/h/L.

In a specific embodiment of the present application, the culture time in step 3 is 3.5 to 4.5 hours.

In one embodiment of the present invention, in the step 4, the volume ratio of methanol to the glucose solution is (3-5): 1, for example, the volume ratio of methanol to the glucose solution is 3:1, 4:1 or 5: 1.

In one embodiment of the present application, in the step 4, the volume ratio of the methanol to the glucose solution is 4: 1.

In one embodiment of the present application, the methanol is fed in the amount of 1.0 to 1.5ml/h/L in said step 4. For example, the amount of methanol fed may be 1.0mL/h/L, 1.1mL/h/L, 1.2mL/h/L, 1.3mL/h/L, 1.4mL/h/L, or 1.5mL/h/L, etc.

In one embodiment of the present application, the methanol is fed in the step 5 in an amount of 2.6 to 4.0 ml/h/L.

The compositions provided herein have at least one of the following benefits:

the composition provided by the application can reduce the viscosity of fermentation liquor, improve dissolved oxygen and improve the growth environment of thalli by feeding the composition in a continuous fermentation process, thereby improving the yield of a target product and the utilization rate of equipment.

Drawings

FIG. 1 is a flow chart of fermentation process of Pichia pastoris engineering strain provided in the examples of the present application.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The technical solutions of the present application will be described clearly and completely in conjunction with the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The composition provided by the application comprises the following components in parts by weight: 0.5-1.5 parts of defoaming agent, 0.08-0.5 parts of adenosine triphosphate, 10-20 parts of glycerol and 2-10 parts of ammonium sulfate.

When the composition is used in a strain fermentation process, the composition is prepared into a solution, and the preparation process comprises the following steps: 0.5-1.5 g of defoaming agent, 0.08-0.5 g of adenosine triphosphate, 10-20g of glycerol and 2-10g of ammonium sulfate are added into 1L of tap water, uniformly mixed, prepared and sterilized for 30min at 121 ℃.

The function of the composition is illustrated by taking the growth and fermentation process of pichia pastoris engineering strains as an example.

Fermentation medium: 5% of glucose, 4% of ammonium dihydrogen phosphate, 1% of magnesium sulfate, 1% of potassium sulfate, 0.5% of monopotassium phosphate, 0.1% of calcium sulfate and 0.16% of potassium hydroxide.

PTM 1: 0.6 percent of copper sulfate, 0.009 percent of potassium iodide, 0.3 percent of manganese sulfate, 0.02 percent of sodium molybdate, 0.002 percent of boric acid, 0.05 percent of cobalt chloride, 2 percent of zinc chloride, 6.5 percent of ferrous sulfate, 0.5 percent of sulfuric acid and 0.02 percent of biotin.

Example compositions

The ingredients and amounts of the compositions in each example are provided in table 1.

Ingredients and amounts of compositions in Table 11L of Water

Preparation of composition solution: taking example 4 as an example, the specific process is as follows:

1.0g of defoaming agent (Dow DF103 selected in each example of the application, PPE defoaming agent can be selected) is added into 1L of water, 0.2g of adenosine triphosphate, 15g of glycerol and 5g of ammonium sulfate are uniformly mixed, and the mixture is sterilized at 121 ℃ for 30min for later use after preparation.

The composition solutions in the other examples were prepared in the same manner as in example 4.

Example 7 fermentation of Pichia engineered Strain to produce Phytase

The fermentation process of the pichia pastoris engineering strain for producing phytase is shown in figure 1. This example describes in detail the application of the composition in fermentation process of pichia pastoris engineering strain for producing phytase, taking the composition formed in example 4 as an example, and the specific process is as follows:

firstly, a thallus culture stage: a65-ton fermentation tank is adopted, 3 tons of seed liquid are inoculated into 30 tons of fermentation medium according to the inoculation proportion of 10 percent, the stirring speed is 160rpm, the ventilation amount is 3000-.

② carbon source feeding stage: continuously adding 25% glucose solution (containing 12mL PTM1 per liter), wherein the adding amount is 36mL/h/L, the dissolved oxygen is controlled to be above 20%, the wet weight of the thallus is measured to reach about 170g/L, and the whole process is about 5 h.

③ a glucose-methanol mixed feeding stage: the 25% glucose solution (containing 12mL of PTM1 per liter) was adjusted to 8mL/h/L while methanol was fed at the same time, the methanol feed rate was 1mL/h/L and the dissolved oxygen was 20% or more, and the culture was continued for 4 hours.

Fourthly, methanol induction stage: the 25% glucose solution (12 mL of PTM1 per liter) was adjusted to a feed rate of 0.4mL/h/L and the methanol feed rate was adjusted to 1.2mL/h/L, and the culture was continued for 2 hours.

A continuous fermentation stage: stopping feeding the glucose solution containing the PTM1, adjusting the feeding amount of methanol to be 3.2mL/h/L, measuring the wet weight of the thalli to reach 300g/L in the period, observing the liquid level of the fermentation liquid in the period, discharging when the liquid level distance of the fermentation liquid is less than 1m from the top of the fermentation liquid, and collecting the fermentation liquid, wherein the discharging amount is 1000L each time.

Sixthly, a feeding stage: the solution of the composition prepared in example 4 was fed-batch, and the fermentation broth was collected continuously by the following procedure:

1-12 h: 19L/h/L, and the dissolved oxygen is controlled to be more than 25 percent;

13-19 h: 39L/h/L, and the dissolved oxygen is controlled to be more than 20 percent;

20-65 h: 53L/h/L, and the dissolved oxygen is controlled to be more than 10 percent;

after 66 h: 62L/h/L, and the dissolved oxygen is controlled to be more than 10 percent until the expression quantity of the foreign protein does not reach the standard (the thalli are aged, and the enzyme production is obviously reduced).

Samples were taken every 12 hours from the start of collection of the fermentation broth to determine the amount of enzyme expressed in the fermentation broth.

Comparative example 1

The difference between the comparative example 1 and the example 7 is that the fermentation stage is continued until the expression level of the foreign protein does not reach the standard without the step (C) in the comparative example 1. The results of phytase production are shown in Table 2.

TABLE 2

As can be seen from table 2, in example 7, the average enzyme activity was 3183176.8U × T, and the average volume was 56.1T; in contrast, in comparative example 1, the average enzyme activity is 2822475.2U × T, and the average volume is 51.9T, it can be seen that, compared with comparative document 1, the enzyme activity of the phytase produced by using the process provided in this embodiment is improved by 12.8%, and the tank-discharging volume is increased by 8.1%.

Example 8 fermentation Process of Pichia engineering Strain for production of glucose oxidase

The fermentation process of the pichia pastoris engineering strain for producing glucose oxidase is shown in figure 1. This example describes in detail the application of the composition in the fermentation process of pichia pastoris engineering strains for producing glucose oxidase, taking the composition formed in example 1 as an example, and the specific process is as follows:

firstly, a thallus culture stage: a65-ton fermentation tank is adopted, 3 tons of seed liquid are inoculated into 30 tons of fermentation medium according to the inoculation proportion of 10 percent, the stirring speed is 180rpm, the ventilation quantity is 3000-.

② carbon source feeding stage: continuously adding 30% glucose solution (containing 12mL PTM1 per liter), wherein the adding amount is 36mL/h/L, the dissolved oxygen is controlled to be above 20%, the wet weight of the thallus is measured to reach about 170g/L, and the whole process is about 6 h.

③ mixing glucose and methanol for feeding: the addition amount of 30% glucose solution (containing 12mL of PTM1 per liter) was adjusted to 5mL/h/L, while methanol was added, the addition amount of methanol was 1mL/h/L, and dissolved oxygen was 20% or more, and the culture was continued for 4 hours.

Fourthly, methanol induction stage: the 30% glucose solution (12 mL PTM1 per liter) was adjusted to 0.3mL/h/L and methanol was adjusted to 1.5mL/h/L for 2 h.

A continuous fermentation stage: stopping feeding the glucose solution containing the PTM1, adjusting the feeding amount of methanol to be 3.6mL/h/L, continuously collecting the fermentation liquor, measuring the wet weight of the thalli to be 340g/L in the period, observing the liquid level of the fermentation liquor in the period, discharging and collecting the fermentation liquor when the liquid level distance of the fermentation liquor is less than 1m from the top of the fermentation tank, and discharging 1200L each time.

Sixthly, a feeding stage: feeding the composition solution prepared in example 1, and collecting the fermentation liquor continuously, wherein the specific process is as follows:

1-12 h: 21L/h/L, and the dissolved oxygen is controlled to be more than 25 percent;

13-19 h: 40L/h/L, and the dissolved oxygen is controlled to be more than 20 percent;

20-65 h: 51L/h/L, and the dissolved oxygen is controlled to be more than 10 percent;

after 66 h: 60L/h/L, and the dissolved oxygen is controlled to be more than 10 percent until the expression quantity of the foreign protein does not reach the standard.

Comparative example 2

Comparative example 2 differs from example 8 in that the fermentation medium without glucose is fed during the feed phase in comparative example 2. The results of glucose oxidase production are shown in table 3.

TABLE 3

As can be seen from table 3, in example 8, the average enzyme activity was 435614.3U × T, and the average volume was 56.3T; in contrast, in comparative example 1, the average enzyme activity is 2822475.2U × T, and the average volume is 51.8T, it can be seen that, compared with comparative document 1, the enzyme activity of the phytase produced by using the process provided in this embodiment is improved by 10.9%, and the tank-placing volume is increased by 8.5%.

EXAMPLE 9 fermentation of engineered Pichia Strain for xylanase production

The fermentation process of the pichia pastoris engineering strain for producing xylanase is shown in figure 1. This example describes the application of the composition in fermentation process of pichia pastoris engineering strain for producing xylanase, taking the composition formed in example 6 as an example, and the specific process is as follows:

firstly, a thallus culture stage: 3 tons of seed liquid are inoculated into 30 tons of fermentation medium by adopting a 65 ton fermentation tank according to the inoculation ratio of 10 percent, the stirring speed is 170rpm, the ventilation quantity is 3000-3300, the tank pressure is 0.05MP, the culture is carried out at 30 ℃ and pH5.0, the wet weight of the thalli is measured to reach about 100g/L, and the whole process is about 13 hours.

And a thallus culture stage: continuously adding 20% glucose solution (containing 12mL PTM1 per liter), wherein the adding amount is 36mL/h/L, the dissolved oxygen is controlled to be above 20%, the wet weight of the thallus is measured to reach about 170g/L, and the whole process is about 6 h.

③ mixing glucose and methanol for feeding: the 20% glucose solution (containing 12mL of PTM1 per liter) was adjusted to 10mL/h/L while methanol was fed at the same time, the methanol feed rate was 1mL/h/L and the dissolved oxygen was 20% or more, and the culture was continued for 4 hours.

Fourthly, methanol induction stage: the 20% glucose solution (12 mL of PTM1 per liter) was adjusted to a feed rate of 0.4mL/h/L and the methanol feed rate was adjusted to 1.5mL/h/L, and the culture was continued for 2 hours.

A continuous fermentation stage: stopping feeding the glucose solution containing the PTM1, adjusting the feeding amount of methanol to be 4.0mL/h/L, continuously collecting the fermentation liquor, measuring the wet weight of the thalli to reach 360g/L in the period, observing the liquid level of the fermentation liquor in the period, discharging and collecting the fermentation liquor when the liquid level distance of the fermentation liquor is less than 1m from the top of the fermentation liquor, and discharging 1500L each time.

Sixthly, a feeding stage: the solution of the composition prepared in example 6 was fed-batch, and the fermentation broth was collected continuously by the following procedure:

1-12 h: 22L/h/L, and the dissolved oxygen is controlled to be more than 25 percent;

13-19 h: 42L/h/L, and the dissolved oxygen is controlled to be more than 20 percent;

20-65 h: 50L/h/L, and the dissolved oxygen is controlled to be more than 10 percent;

after 66 h: 61L/h/L, and the dissolved oxygen is controlled to be more than 10 percent until the expression quantity of the foreign protein does not reach the standard.

Comparative example 3

Comparative example 3 differs from example 9 in that the fermentation medium without glucose is fed during the feed phase in comparative example 3. The xylanase production results are shown in Table 4.

TABLE 4

As can be seen from table 4, in example 9, the average enzyme activity was 9481753.1U × T, and the average volume was 55.8T; in contrast, in comparative example 1, the average enzyme activity is 8414465.7U × T, and the average volume is 51.5T, it can be seen that, compared with comparative document 1, the enzyme activity of the phytase produced by using the process provided in this embodiment is improved by 12.7%, and the tank-discharging volume is increased by 8.3%.

In conclusion, the composition provided by the application has the advantages of safe and reasonable formula, low cost, no pollution, convenience in use, simple and feasible process, capability of greatly improving the fermentation productivity and equipment utilization rate of pichia pastoris engineering strains to reduce cost and improve efficiency, convenience in popularization and suitability for large-scale production.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. The composition is characterized by comprising the following components in parts by weight: 0.5-1.5 parts of defoaming agent, 0.08-0.5 parts of adenosine triphosphate, 10-20 parts of glycerol and 2-10 parts of ammonium sulfate.

2. The composition of claim 1 for use in a pichia pastoris engineering bacteria semi-continuous fermentation process.

3. The application of claim 2, wherein the pichia pastoris engineering bacteria semi-continuous fermentation process comprises the following steps:

step 1: inoculating the pichia pastoris engineering strain seed liquid to a fermentation culture medium for culture until the wet weight of the strain reaches more than 90 g/L;

step 2: feeding a glucose solution containing PTM1, and adjusting the feeding rate to control the dissolved oxygen to be more than 20% until the wet weight of the thalli reaches more than 160 g/L;

and 5: stopping feeding the glucose solution containing the PTM1, and increasing the feeding amount of the methanol again until the wet weight of the thalli reaches more than 300 g/L;

4. The method of claim 3, wherein the composition is first formulated as a solution according to: 0.5-1.5 g of defoaming agent, 0.08-0.5 g of adenosine triphosphate, 10-20g of glycerol and 2-10g of ammonium sulfate are added into 1L of water for preparation.

5. The method of claim 3, wherein in step 6, the composition is supplemented by:

1-12 h: 19-22L/h/L, and the dissolved oxygen is controlled to be more than 25%;

13-19 h: 39-42L/h/L, and the dissolved oxygen is controlled to be more than 20%;

20-65 h: 50-53L/h/L, and the dissolved oxygen is controlled to be more than 10%;

after 66 h: 60-62L/h/L, and the dissolved oxygen is controlled to be more than 10%.

6. The method according to claim 3, wherein the concentration of the glucose solution in the step 2 is 20-30%.

7. The method according to claim 3, wherein in the step 3, the volume ratio of the methanol to the glucose solution is 1: (5-10).

8. The method according to claim 3, wherein in the step 4, the volume ratio of the methanol to the glucose solution is (3-5): 1.

9. The method according to claim 3, wherein the methanol is fed in an amount of 1.2 to 1.5ml/h/L in the step 4.

10. The method according to claim 3, wherein the methanol is fed in the step 5 in an amount of 3.2 to 4.0 ml/h/L.