Method for reducing inclusion bodies in protein fermentation production

Abstract

The invention relates to a method for producing foreign proteins by fermenting recombinant Escherichia coli, said fermentation being a fed-batch culture process, said foreign proteins being expressed in the form of inclusion bodies in a reduced proportion compared to a control method without said method, the method comprises the following steps: after the recombinant Escherichia coli grows and induces expression for a period of time, stopping feeding a carbon source in a short time and / or carrying out heat shock treatment on fermentation liquor through instantaneous temperature rise after the recombinant Escherichia coli grows and induces expression for a period of time. The invention also relates to a method for reducing the proportion of inclusion bodies of the foreign protein or increasing the proportion of soluble forms of the foreign protein in a fermentation process of expressing the foreign protein by using recombinant Escherichia coli.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular to a method for producing a foreign protein (particularly L-aspartate-α-decarboxylase) by fermenting recombinant Escherichia coli, wherein the proportion of the foreign protein expressed in the form of inclusion bodies is reduced compared to a control method not using the method. In addition, the present invention also relates to a method for reducing the proportion of inclusion bodies of the foreign protein or increasing the proportion of its soluble form during a fermentation process in which the foreign protein is expressed using recombinant Escherichia coli. Background Art

[0002] β-Alanine, also known as 3-aminopropionic acid, is the only β-amino acid in nature. The physiological function of β-alanine is mainly as an intermediate product of metabolism. It is widely present in the metabolic process of animals, plants and microorganisms, and plays an indispensable role in the growth and development of organisms. It is mainly used to synthesize pantothenic acid and calcium pantothenate, carnosine, sodium pamidronate, balsalazide, etc., and is widely used in medicine, feed, food and other fields. In recent years, there have been more and more reports on β-alanine abroad, most of which are studies on β-alanine derivatives. The role of β-alanine derivatives in medical, environmental and daily chemical products is also being discovered, so the market demand for β-alanine is also increasing. At present, the production of β-alanine at home and abroad is mainly based on chemical synthesis, which causes certain pollution to the environment, and its products contain certain harmful substances. The use of bioenzymatic conversion instead of traditional chemical synthesis to produce bulk chemicals is a hot topic in recent biological research. This method has the advantages of mild production conditions, relatively simple process, and little environmental pollution. There are many studies on bacterial-derived L-aspartate-α-decarboxylase, including those from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis. However, most bacterial L-aspartate-α-decarboxylases are small molecule proteins with unstable enzyme activity. The small molecule target protein of L-aspartate-α-decarboxylase from microorganisms expressed by recombinant Escherichia coli requires subsequent protein self-cleavage to produce active subunits to have catalytic ability. The small molecule protein is easily degraded by proteases during fermentation, which has led to the failure of biosynthesis to be industrialized. This experimental scheme is selected from the L-aspartate-α-decarboxylase gene (TcADC) from the insect Tribolium castaneum. The L-aspartate-α-decarboxylase gene from the insect Tribolium castaneum is easily expressed in the prokaryotic expression system Escherichia coli to form inclusion bodies with misfolded proteins. In response to this phenomenon, the schemes commonly used by researchers include: co-expression of molecular chaperones or fusion tags, replacement of hosts and vectors suitable for protein soluble expression, cooling down during fermentation induction to slow down the protein expression rate, and other operations. These schemes can reduce the generation of inclusion bodies, but require a series of operations in molecular construction, or consume a large amount of cooling water by cooling induction during fermentation, increase energy consumption, and slow down cell growth rate at low temperatures, thereby increasing fermentation costs.

[0003] Common methods and drawbacks of existing technologies for solving inclusion bodies:

[0004] First, lower the culture and induction temperature to slow down the protein expression rate so that the target protein can be correctly folded. Although lowering the temperature can reduce inclusion bodies, the growth rate of E. coli is greatly affected after cooling, and the fermentation cycle needs to be extended. The biomass of the final product will be reduced. In industrial production, cooling requires more cooling water, which increases energy consumption and labor costs. In addition, the growth rate of bacteria is limited, which increases the risk of contamination.

[0005] Second, a soluble chaperone protein and a commonly used fusion tag protein are constructed and then co-expressed with the target protein. However, the expression level of the target protein is partially occupied by the chaperone protein and the commonly used fusion tag protein, so the expression level of the target protein is reduced. In addition, complex genetic manipulation is required, and appropriate chaperones and fusion tags need to be selected. After co-expression, it is difficult to control the balance of the expression levels of the target protein, chaperone, and fusion tag protein, thereby affecting the stability of the fermentation batch, and it is difficult to achieve industrial scale-up.

[0006] Third, use other chassis cells, such as yeast, Corynebacterium glutamicum, animal cells, etc. Although eukaryotic cells such as yeast have relatively complete protein modification systems, the fermentation cycle is very long, the culture medium cost of Corynebacterium glutamicum animal cells is more expensive, and molecular operations are relatively difficult.

[0007] Therefore, the present invention is in urgent need of a method for reducing the proportion of inclusion bodies of the exogenous protein or increasing the proportion of its soluble form during the fermentation process of expressing the exogenous protein using recombinant Escherichia coli and / or a method for producing exogenous protein (especially L-aspartate-α-decarboxylase) with a low inclusion body ratio by fermenting recombinant Escherichia coli. Summary of the invention

[0008] The present invention reduces the generation of inclusion bodies during fermentation and enhances the activity of the strain by controlling and regulating the fermentation process (for example, stopping the carbon source addition for a short time to form a "starvation" state during the culture process and then resuming the carbon source addition and / or raising the culture temperature for a short time, etc.) when fermenting the recombinant Escherichia coli into which the gene encoding the foreign protein is transferred. The present invention does not require modification mutation of the original encoding gene, reduces the workload of molecular gene operation, does not require too low a temperature during induction, reduces the use of cooling water, reduces energy consumption, and is simple and convenient to operate.

[0009] Specifically, the present invention solves the technical problems existing in the prior art through the following technical solutions:

[0010] 1. A method for producing foreign protein by fermenting recombinant Escherichia coli, wherein the fermentation is a fed-batch culture process, and the proportion of the foreign protein expressed in the form of inclusion bodies is reduced compared with a control method not using the method, the method comprising:

[0011] (a) after the recombinant Escherichia coli grows and is induced to express for a period of time, stopping the addition of the carbon source for 1-10 minutes, and then continuing the addition of the carbon source; and / or

[0012] (b) 1-10 minutes after stopping the addition of the carbon source, the fermentation broth is thermally stimulated by instantaneously increasing the temperature.

[0013] 2. The method according to claim 1, wherein when the OD of the fermentation broth is 600 When the OD value of the fermentation liquid is 18-25, stop adding the carbon source for 1-8 minutes. 600 When the temperature is 20-22, stop adding the carbon source for 2-5 minutes.

[0014] 3. The method according to item 1 or 2, wherein the temperature of the fed-batch culture process is 37° C. during the growth of the recombinant Escherichia coli and is 30° C. during the induced expression of the recombinant Escherichia coli.

[0015] 4. The method according to any one of items 1 to 3, wherein the instantaneous temperature increase is from 30°C to 35-50°C, preferably to 37-46°C, more preferably to 40-44°C.

[0016] 5. The method according to any one of items 1 to 4, wherein the instantaneous temperature increase is maintained for 30-120 seconds, preferably 45-110 seconds, and more preferably 60-100 seconds.

[0017] 6. The method according to any one of items 1 to 5, wherein the carbon source is selected from the group consisting of carbohydrates, lipids, organic acids, alcohols and hydrocarbons, preferably the carbon source is selected from the group consisting of monosaccharides, disaccharides and polysaccharides, more preferably the carbon source is selected from the group consisting of glucose, maltose, molasses and starch.

[0018] 7. The method according to any one of items 1 to 6, wherein the culture conditions of the fed-batch culture process are selected from one or more of the following:

[0019] (i) adjusting the dissolved oxygen level in the fed-batch culture process to 30%-40% by adjusting the stirring speed and / or the tank pressure;

[0020] (ii) controlling the pH of the fed-batch culture process to be between 6.8 and 7.2 by adding ammonia or hydrochloric acid;

[0021] (iii) the flow rate of the carbon source is 4-8 g / L / hour, preferably 5-7 g / L / hour; and / or

[0022] (iv) The fermentation is carried out for a total of 20-48 hours, preferably 22-42 hours, more preferably 24-36 hours.

[0023] 8. The method according to any one of items 1 to 7, wherein the induced expression of the recombinant Escherichia coli is performed by adding an inducer, and preferably the inducer is 0.01-0.5 mmol / L IPTG.

[0024] 9. The method according to any one of items 1 to 8, wherein the exogenous protein is L-aspartate-α-decarboxylase.

[0025] 10. The method according to any one of items 1 to 9, wherein the recombinant Escherichia coli comprises a gene encoding L-aspartate-α-decarboxylase.

[0026] 11. A method according to any one of items 1 to 10, wherein the proportion of the exogenous protein expressed in the form of inclusion bodies is reduced by 10%, 15%, 20%, 25% or 30% compared with the control method in which the carbon source is not stopped and / or heat stimulation is not performed; or the proportion of the exogenous protein expressed in a soluble form is increased by 10%, 15%, 20%, 25% or 30% compared with the control method in which the carbon source is not stopped and / or heat stimulation is not performed.

[0027] 12. A method for reducing the proportion of inclusion bodies of a foreign protein or increasing the proportion of a soluble form thereof or improving the specific enzyme activity of the foreign protein during a fermentation process in which a foreign protein is expressed by recombinant Escherichia coli, comprising: during the fermentation process of the recombinant Escherichia coli:

[0028] (a) after the recombinant Escherichia coli grows and is induced to express for a period of time, stopping the addition of the carbon source for 1-10 minutes, and then continuing the addition of the carbon source; and / or

[0029] (b) 1-10 minutes after stopping the addition of the carbon source, the fermentation broth is thermally stimulated by instantaneously increasing the temperature.

[0030] 13. The method according to claim 12, wherein when the OD of the fermentation broth is 600 When the OD value of the fermentation liquid is 18-25, stop adding the carbon source for 1-8 minutes. 600 When the temperature is 20-22, stop adding the carbon source for 2-5 minutes.

[0031] 14. The method according to item 12 or 13, wherein the temperature of the fed-batch culture process is 37°C during the growth of the recombinant Escherichia coli and is 30°C during the induced expression of the recombinant Escherichia coli.

[0032] 15. The method according to any one of items 12 to 14, wherein the instantaneous temperature increase is from 30°C to 35-50°C, preferably to 37-46°C, more preferably to 40-44°C.

[0033] 16. The method according to any one of items 12 to 15, wherein the instantaneous temperature increase is maintained for 30 to 120 seconds, preferably 45 to 110 seconds, and more preferably 60 to 100 seconds.

[0034] 17. The method according to any one of items 12-16, wherein the carbon source is selected from the group consisting of carbohydrates, lipids, organic acids, alcohols and hydrocarbons, preferably the carbon source is selected from the group consisting of monosaccharides, disaccharides and polysaccharides, more preferably the carbon source is selected from the group consisting of glucose, maltose, molasses and starch.

[0035] 18. The method according to any one of items 12 to 17, wherein the culture conditions of the fed-batch culture process are selected from one or more of the following:

[0036] (i) adjusting the dissolved oxygen level in the fed-batch culture process to 30%-40% by adjusting the stirring speed and / or the tank pressure;

[0037] (ii) controlling the pH of the fed-batch culture process to be between 6.8 and 7.2 by adding ammonia or hydrochloric acid;

[0038] (iii) the flow rate of the carbon source is 4-8 g / L / hour, preferably 5-7 g / L / hour; and / or

[0039] (iv) The fermentation is carried out for a total of 20-48 hours, preferably 22-42 hours, more preferably 24-36 hours.

[0040] 19. The method according to any one of items 12 to 18, wherein the induced expression of the recombinant Escherichia coli is performed by adding an inducer, and preferably the inducer is 0.01-0.5 mmol / L IPTG.

[0041] 20. The method according to any one of items 12 to 19, wherein the exogenous protein is L-aspartate-α-decarboxylase.

[0042] 21. The method according to any one of items 12 to 20, wherein the recombinant Escherichia coli comprises a gene encoding L-aspartate-α-decarboxylase.

[0043] 22. A method according to any one of items 12-21, wherein the proportion of the exogenous protein expressed in the form of inclusion bodies is reduced by 10%, 15%, 20%, 25% or 30% compared with the control method in which the carbon source is not stopped and / or heat stimulation is not performed; or the proportion of the exogenous protein expressed in a soluble form is increased by 10%, 15%, 20%, 25% or 30% compared with the control method in which the carbon source is not stopped and / or heat stimulation is not performed.

[0044] In order to make the technical solution of the present invention clearer, the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1The effect of glucose addition on bacterial viability after a short sugar suspension (0 (control), 1, 2, 3, 4 or 5 minutes) during 1-liter tank fermentation of strain PET32a-TcADC-BL21 (DE3) is shown;

[0046] Figure 2 The effect of heat stimulation at different temperatures (37°C (control), 40°C, 42°C, 44°C, 46°C or 48°C) on bacterial viability during the 1-liter tank fermentation of strain PET32a-TcADC-BL21 (DE3) is shown;

[0047] Figure 3 The effect of different durations of 42°C heat stimulation (30 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds or 180 seconds) on bacterial viability during 1-liter tank fermentation of strain PET32a-TcADC-BL21 (DE3) is shown;

[0048] Figure 4 The electrophoresis diagram obtained by SDS-PAGE electrophoresis of the supernatant obtained after centrifugation of the three groups of fermentation broths subjected to heat stimulation for 30 seconds, 60 seconds or 90 seconds respectively and the control fermentation broth without heat stimulation is shown, and the expression amount of the total target protein, the target protein expressed in the form of inclusion bodies and the soluble target protein can be detected and analyzed by image analysis; and

[0049] Figure 5 The diagram shows (1) whole cell samples of cells after normal fermentation were disrupted, (2) supernatant samples of cells after normal fermentation were disrupted and centrifuged, (3) whole cell samples of cells after being treated with sugar deprivation for 4 minutes and heat stimulation at 42°C for 90 seconds were disrupted, and (4) supernatant samples of cells after being disrupted and centrifuged after being treated with sugar deprivation for 4 minutes and heat stimulation at 42°C for 90 seconds.

[0050] Figure 6 The diagram shows (1) the precipitate (inclusion body) sample after disruption and centrifugation of cells subjected to normal fermentation, (2) the supernatant sample after disruption and centrifugation of cells subjected to normal fermentation, (3) the precipitate (inclusion body) sample after disruption and centrifugation of cells subjected to sugar withdrawal for 4 minutes and heat stimulation at 42°C for 90 seconds, and (4) the supernatant sample after disruption and centrifugation of cells subjected to sugar withdrawal for 4 minutes and heat stimulation at 42°C for 90 seconds. DETAILED DESCRIPTION OF THE INVENTION

[0052] 1. Definition

[0053] In the present application, inclusion bodies refer to high-density, insoluble protein particles wrapped by membranes that are formed when exogenous genes are expressed in prokaryotic cells, especially when they are efficiently expressed in Escherichia coli. It is a high-refractive area when observed under a microscope, which is significantly different from other components in the cytoplasm. The formation of inclusion bodies is relatively complex and is related to the rate of protein production in the cytoplasm. The concentration of newly generated polypeptides is high and there is not enough time for folding, thus forming non-crystalline, amorphous protein aggregates; in addition, the formation of inclusion bodies is also believed to be related to the culture conditions of the host bacteria, such as culture medium composition, temperature, pH value, ionic strength and other factors. Biologically active proteins in cells often exist in the form of soluble or molecular complexes, and functional proteins are always folded into specific three-dimensional structures. The proteins in the inclusion bodies are aggregates in an unfolded state and have no biological activity.

[0054] In this application, heat shock proteins (HSP) refer to a class of heat stress proteins that are widely present in bacteria and mammals. When an organism is exposed to high temperatures, it will be stimulated by heat to synthesize this protein to protect the organism itself. Many heat shock proteins have molecular chaperone activity. According to the size of the protein, heat shock proteins are divided into five categories, namely HSP110, HSP90, HSP70, HSP60 and small molecule heat shock proteins.

[0055] 2. The method for producing exogenous protein by fermentation of the present invention

[0056] In one aspect, the present invention provides a method for producing exogenous protein by fermenting recombinant Escherichia coli, wherein the fermentation is a fed-batch culture process, and the proportion of the exogenous protein expressed in the form of inclusion bodies is reduced compared with a control method that does not adopt the method, and the method comprises: (a) after the recombinant Escherichia coli grows and induces expression for a period of time, stopping the flow of carbon source for 1-10 minutes, and then continuing the flow of carbon source; and / or (b) after stopping the flow of carbon source for 1-10 minutes, thermally stimulating the fermentation liquid by instantaneous temperature increase.

[0057] In one embodiment, after the recombinant E. coli is grown and induced to express, when the OD of the fermentation broth is 600 When the OD of the fermentation broth is 18-25, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued. In one embodiment, after the recombinant Escherichia coli is grown and induced to express, when the OD of the fermentation broth is 18-25, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued. 600 When the OD value of the fermentation broth is 18-22, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued to be added. In one embodiment, after the recombinant Escherichia coli is grown and induced to express, when the OD value of the fermentation broth is 18-22, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued to be added.600 When the carbon source is about 20, the addition of the carbon source is stopped for 2-4 minutes, and then the addition of the carbon source is continued.

[0058] In one embodiment, the temperature during the growth of the recombinant E. coli during the fermentation is about 37° C. In one embodiment, the temperature during the induced expression of the recombinant E. coli during the fermentation is about 30° C.

[0059] In one embodiment, the carbon source added during the fermentation and / or the carbon source added during the fed-batch culture is selected from the group consisting of carbohydrates, lipids, organic acids, alcohols and hydrocarbons. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is selected from the group consisting of monosaccharides, disaccharides and polysaccharides. In one embodiment, the carbon source is selected from the group consisting of glucose, maltose, molasses and starch.

[0060] In one embodiment, the exogenous protein is L-aspartate-α-decarboxylase. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from insects. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from the family of Tenebriophyta. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from the genus Tribolium. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from Tribolium castaneum. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase as shown in SEQ ID NO:2. In one embodiment, the recombinant Escherichia coli comprises a gene encoding an L-aspartate-α-decarboxylase. In one embodiment, the recombinant Escherichia coli comprises a gene encoding an L-aspartate-α-decarboxylase derived from Tribolium castaneum. In one embodiment, the recombinant E. coli comprises a gene encoding L-aspartate-α-decarboxylase as shown in SEQ ID NO: 2. In one embodiment, the recombinant E. coli comprises a L-aspartate-α-decarboxylase gene as shown in SEQ ID NO: 1.

[0061] In one embodiment, the culture conditions of the fermentation process are selected from one or more of the following:

[0062] (i) adjusting the stirring speed and / or the tank pressure to maintain the dissolved oxygen level at 20%-50%;

[0063] (ii) controlling the pH of the fed-batch culture process to be between 6.5 and 7.5 by feeding ammonia or hydrochloric acid;

[0064] (iii) the flow rate of the carbon source is 4-8 g / L / hour; and / or

[0065] (iv) The fermentation is carried out for a total of 20-48 hours.

[0066] In one embodiment, the culture conditions of the fermentation process are selected from one or more of the following:

[0067] (i) maintaining a dissolved oxygen level of 30% to 40% by adjusting the stirring speed and / or tank pressure;

[0068] (ii) controlling the pH of the fed-batch culture process to be between 6.8 and 7.2 by adding ammonia or hydrochloric acid;

[0069] (iii) the carbon source has a flow rate of 5-7 g / L / hour; and / or

[0070] (iv) The fermentation is carried out for a total of 22-42 hours, preferably 24-36 hours.

[0071] In one embodiment, the induced expression of the recombinant Escherichia coli is performed by adding an inducer. In one embodiment, the inducer is 0.01-0.5 mmol / L IPTG. In one embodiment, the inducer is 0.1 mmol / L IPTG. In one embodiment, the induced expression of the recombinant Escherichia coli is at an OD of 0.1% to 0.5% of the fermentation broth. 600 In one embodiment, the induced expression of the recombinant Escherichia coli is carried out when the OD of the fermentation broth is 28-32. 600 It is carried out at 30 hours.

[0072] In one embodiment, after the recombinant E. coli is grown and induced to express, when the OD of the fermentation broth is 600 When the OD value of the fermentation liquid is 18-22, the carbon source is stopped for 1-10 minutes, and then the carbon source is continued. In one embodiment, after the recombinant E. coli is grown and induced to express, when the OD value of the fermentation liquid is 18-22, the carbon source is stopped for 1-10 minutes, and then the carbon source is continued. 600 When the OD value of the fermentation liquid is 18-22, the carbon source is stopped for 1-5 minutes, and then the carbon source is continued. 600 When the carbon source is about 20, the addition of the carbon source is stopped for 2-4 minutes, and then the addition of the carbon source is continued.

[0073] In one embodiment, the instantaneous temperature rise is from 30°C to 35-50°C. In one embodiment, the instantaneous temperature rise is from 30°C to 37-46°C. In one embodiment, the instantaneous temperature rise is from 30°C to 40-44°C. In one embodiment, the instantaneous temperature rise is from 30°C to 42°C. In one embodiment, the instantaneous temperature rise is maintained for 30-120 seconds. In one embodiment, the instantaneous temperature rise is maintained for 45-110 seconds. In one embodiment, the instantaneous temperature rise is maintained for 60-100 seconds. In one embodiment, the instantaneous temperature rise is maintained for 90 seconds.

[0074] In one embodiment, the proportion of the exogenous protein expressed in the form of inclusion bodies is reduced by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% compared with a control method in which the carbon source is not stopped and / or heat stimulation is not performed; or the proportion of the exogenous protein expressed in a soluble form is increased by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% compared with a control method in which the carbon source is not stopped and / or heat stimulation is not performed.

[0075] 2. The method of reducing the proportion of inclusion bodies of exogenous proteins produced by fermentation or increasing the proportion of soluble forms of exogenous proteins or improving the specific enzyme activity of the exogenous proteins according to the present invention

[0076] In one aspect, the present invention provides a method for reducing the proportion of inclusion bodies of exogenous proteins, increasing the proportion of soluble forms, or improving the specific enzyme activity of exogenous proteins during fermentation using recombinant Escherichia coli to express exogenous proteins, which comprises: (a) after the recombinant Escherichia coli grows and induces expression for a period of time, stopping the addition of carbon source for 1-10 minutes, and then continuing the addition of carbon source; and / or (b) after stopping the addition of carbon source for 1-10 minutes, thermally stimulating the fermentation liquid by instantaneous temperature increase.

[0077] In one embodiment, the fermentation process is a fed-batch process.

[0078] In one embodiment, the temperature during the growth of the recombinant E. coli during the fermentation is about 37° C. In one embodiment, the temperature during the induced expression of the recombinant E. coli during the fermentation is about 30° C.

[0079] In one embodiment, the carbon source added during the fermentation and / or the carbon source added during the fed-batch culture is selected from the group consisting of carbohydrates, lipids, organic acids, alcohols and hydrocarbons. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is selected from the group consisting of monosaccharides, disaccharides and polysaccharides. In one embodiment, the carbon source is selected from the group consisting of glucose, maltose, molasses and starch.

[0080] In one embodiment, the exogenous protein is L-aspartate-α-decarboxylase. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from insects. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from the family of Tenebriophyta. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from the genus Tribolium. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase derived from Tribolium castaneum. In one embodiment, the exogenous protein is an L-aspartate-α-decarboxylase as shown in SEQ ID NO:2. In one embodiment, the recombinant Escherichia coli comprises a gene encoding an L-aspartate-α-decarboxylase. In one embodiment, the recombinant Escherichia coli comprises a gene encoding an L-aspartate-α-decarboxylase derived from Tribolium castaneum. In one embodiment, the recombinant E. coli comprises a gene encoding L-aspartate-α-decarboxylase as shown in SEQ ID NO: 2. In one embodiment, the recombinant E. coli comprises a L-aspartate-α-decarboxylase gene as shown in SEQ ID NO: 1.

[0081] In one embodiment, the culture conditions of the fermentation process are selected from one or more of the following:

[0082] (i) adjusting the stirring speed and / or the tank pressure to maintain the dissolved oxygen level at 20%-50%;

[0083] (ii) controlling the pH of the fed-batch culture process to be between 6.5 and 7.5 by feeding ammonia or hydrochloric acid;

[0084] (iii) the flow rate of the carbon source is 4-8 g / L / hour; and / or

[0085] (iv) The fermentation is carried out for a total of 20-48 hours.

[0086] In one embodiment, the culture conditions of the fermentation process are selected from one or more of the following:

[0087] (i) maintaining a dissolved oxygen level of 30% to 40% by adjusting the stirring speed and / or tank pressure;

[0088] (ii) controlling the pH of the fed-batch culture process to be between 6.8 and 7.2 by adding ammonia or hydrochloric acid;

[0089] (iii) the carbon source has a flow rate of 5-7 g / L / hour; and / or

[0090] (iv) The fermentation is carried out for a total of 22-42 hours, preferably 24-36 hours.

[0091] In one embodiment, the induced expression of the recombinant Escherichia coli is performed by adding an inducer. In one embodiment, the inducer is 0.01-0.5 mmol / L IPTG. In one embodiment, the inducer is 0.1 mmol / L IPTG. In one embodiment, the induced expression of the recombinant Escherichia coli is at an OD of 0.1% to 0.5% of the fermentation broth. 600 In one embodiment, the induced expression of the recombinant Escherichia coli is carried out when the OD of the fermentation broth is 28-32. 600 It is carried out at 30 hours.

[0092] In one embodiment, after the recombinant E. coli is grown and induced to express, when the OD of the fermentation broth is 600 When the OD of the fermentation broth is 18-25, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued. In one embodiment, after the recombinant Escherichia coli is grown and induced to express, when the OD of the fermentation broth is 18-25, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued. 600 When the OD value of the fermentation broth is 18-22, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued to be added. In one embodiment, after the recombinant Escherichia coli is grown and induced to express, when the OD value of the fermentation broth is 18-22, the carbon source is stopped for 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 minutes, and then the carbon source is continued to be added. 600 When the carbon source is about 20, the addition of the carbon source is stopped for 2-4 minutes, and then the addition of the carbon source is continued.

[0093] In one embodiment, the instantaneous temperature rise is from 30°C to 35-50°C. In one embodiment, the instantaneous temperature rise is from 30°C to 37-46°C. In one embodiment, the instantaneous temperature rise is from 30°C to 40-44°C. In one embodiment, the instantaneous temperature rise is from 30°C to 42°C. In one embodiment, the instantaneous temperature rise is maintained for 30-120 seconds. In one embodiment, the instantaneous temperature rise is maintained for 45-110 seconds. In one embodiment, the instantaneous temperature rise is maintained for 60-100 seconds. In one embodiment, the instantaneous temperature rise is maintained for 90 seconds.

[0094] In one embodiment, the proportion of the exogenous protein expressed in the form of inclusion bodies is reduced by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% compared with a control method in which the carbon source is not stopped and / or heat stimulation is not performed; or the proportion of the exogenous protein expressed in a soluble form is increased by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% compared with a control method in which the carbon source is not stopped and / or heat stimulation is not performed.

[0095] 3. Advantages and beneficial effects of the present invention

[0096] Compared with the methods of the prior art, the method of the present invention has at least the following advantages or beneficial effects:

[0097] (1) During fermentation, the fermentation process is controlled and regulated (sugar supplementation is stopped for a short period of time during culture to form a "starvation" state), and then glucose is resumed after sugar supplementation is stopped for a period of time to enhance the vitality of the strain, so that the strain has stronger vitality to adapt to high temperature heat stimulation without losing growth vitality.

[0098] (2) Briefly raising the temperature induces the strain to produce heat shock stress proteins. The produced heat shock proteins can assist the correct folding of the target protein and thus reduce the formation of inclusion bodies.

[0099] (3) The methods of reducing inclusion bodies of the present invention are all based on the regulation of the fermentation process and do not require the addition of additional reagents or complex molecular operations.

[0100] (4) The present invention does not require modification or mutation of the original gene, which can greatly reduce the workload of molecular gene manipulation. In addition, the induction does not require a very low temperature, which can reduce the use of cooling water and energy consumption. It is a simple and convenient method for effectively reducing inclusion bodies. DETAILED DESCRIPTION

[0101] The present invention will be further described below in conjunction with specific embodiments, but these specific embodiments cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and the changed and modified embodiments still fall within the scope of protection of the present invention.

[0102] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0103] Bacterial strains and plasmids

[0104] The Escherichia coli host bacteria used in this application is commercially available BL21 (DE) 3, and is stored in liquid nitrogen in the laboratory of Moga (Shanghai) Biotechnology Co., Ltd. The expression vector is commercially available PET32a (ampicillin resistance), and the L-aspartate-α-decarboxylase gene (TcADC) sequence derived from the insect Tribolium castaneum is provided by Moga (Shanghai) Biotechnology Co., Ltd. (see CN115261364B). Nanjing GenScript was commissioned to perform full gene synthesis to construct the expression vector PET32a-TcADC. After obtaining the plasmid, Escherichia coli BL21 (DE) 3 was simultaneously transformed to obtain the strain PET32a-TcADC-BL21 (DE3), which was stored in liquid nitrogen.

[0105] Preparation of host bacteria BL21(DE3) competent cells

[0106] (1) Streak the host bacteria on an agar plate and culture at 37°C for 16 to 20 hours.

[0107] (2) Take a single colony from the plate and transfer it to a 250-ml Erlenmeyer flask containing 50 ml of LB medium. Incubate at 37°C with shaking at 220 rpm overnight.

[0108] (3) Add 1 ml of overnight culture medium to 50 ml of LB medium and culture at 37°C with a shaker at 220 rpm for 3 hours.

[0109] (4) The culture medium was placed into a pre-cooled 50 ml sterile centrifuge tube, incubated on ice for 5 to 10 minutes, and centrifuged at 4,000 rpm for 7 minutes at 4°C. The supernatant was discarded.

[0110] (5) Precipitation with 10 ml of ice-cold CaCl 2 The solution was resuspended, centrifuged at 4,000 rpm at 4°C for 5 minutes, and the supernatant was discarded.

[0111] (6) Add 30 ml of ice-cold 0.1 mol / L CaCl 2 The suspended cells were placed on ice for 30 min, centrifuged at 4000 rpm for 5 min at 4°C, and the supernatant was discarded;

[0112] (7) Use 3 ml of ice-cold 0.1 mol / L CaCl 2 The cells were suspended, 1.0 ml of pre-cooled 80% glycerol solution was added, and the suspension was dispensed into pre-cooled 1.5 ml sterile eppendorf tubes, 200 μl per tube, and frozen at below -60°C as competent cells.

[0113] Preparation of bacterial strain PET32a-pCDFDuet-ADC-BL21(DE3)

[0114] Take plasmids PET32a-ADC (ampicillin resistance) and pCDFDuet-ADC (streptomycin resistance) and competent cells BL21(DE)3 from liquid nitrogen, quickly put them in an ice bath, and prepare for the transformation steps:

[0115] (1) Aseptically take 1 μl of plasmid and slowly add it to a centrifuge tube containing 100 μl of competent cells. Immediately place on ice for 30 minutes. Be sure to handle it with care and do not shake it during the operation. Competent cells are relatively fragile and can easily affect the transformation efficiency.

[0116] (2) After 30 minutes of ice bath, take out and place in a 42°C circulating water bath for heat shock for 90 seconds, take out of the ice bath for 2 minutes, add 400 μl of LB liquid medium without resistance to the centrifuge tube under sterile conditions, and shake on a shaker for recovery culture at 37°C, 160 rpm, for 60 minutes;

[0117] (3) After the recovery, 50 μl of the culture medium was spread on a plate of LB solid medium with ampicillin resistance and streptomycin resistance under sterile conditions and cultured in an incubator at 37°C overnight;

[0118] (4) A single colony was picked and transferred to 10 mL LB liquid culture medium with ampicillin resistance and streptomycin resistance. After culturing at 37°C and 180 rpm for 6 hours, 15% glycerol was added to preserve the seed, and the engineered strain PET32a-pCDFDuet-ADC-BL21 (DE3) was obtained, which was stored in liquid nitrogen.

[0119] Preparation of engineered strain PET32a-TcADC-BL21(DE3)

[0120] Take the plasmid PET32a-TcADC (ampicillin resistance) and competent cells BL21(DE)3 from liquid nitrogen, quickly put them in an ice bath, and prepare for the transformation steps:

[0121] (1) Aseptically take 1 μl of plasmid and slowly add it to a centrifuge tube containing 100 μl of competent cells. Immediately place on ice for 30 minutes. Be sure to handle it with care and do not shake it during the operation. Competent cells are relatively fragile and can easily affect the transformation efficiency.

[0122] (2) After 30 minutes of ice bath, take out and place in a 42°C circulating water bath for heat shock for 90 seconds, take out of the ice bath for 2 minutes, add 400 μl of LB liquid medium without resistance to the centrifuge tube under sterile conditions, and shake on a shaker for recovery culture at 37°C, 160 rpm, for 60 minutes;

[0123] (3) After the recovery, 50 μl of the culture medium was spread on a plate of LB solid medium with ampicillin resistance and streptomycin resistance under sterile conditions and cultured in an incubator at 37°C overnight;

[0124] (4) A single colony was picked and transferred to 10 mL LB and added with ampicillin resistance liquid culture medium. After culturing at 37° C. and 180 rpm for 6 hours, 15% glycerol was added to preserve the seed, and the engineered strain PET32a-TcADC-BL21 (DE3) was obtained, which was stored in liquid nitrogen.

[0125] Shake flask seed preparation

[0126] (1) Rejuvenation, purification and preservation of strains

[0127] Take 100 μL of the initial bacterial solution (glycerol tube) and inoculate it into 10 ml (30 ml test tube) LB medium (containing antibiotics). Incubate overnight at 37°C and 200 rpm in a shaking incubator for 17 hours. Measure the OD value of the culture solution. 600 The OD value of the culture medium was greater than 2.0, and there was no foreign bacteria under microscopic examination. The culture medium was streaked with an inoculation loop on LB solid medium (containing antibiotics) at 37°C for 24 hours. A single colony was picked and inoculated into 10 ml (30 ml test tube) of LB medium (containing antibiotics). The culture medium was shaken at 37°C and 200 rpm overnight for 17 hours, and the OD value of the culture medium was measured. 600 If the value is greater than 2.0 and there are no foreign bacteria under microscopic examination, the strain can be preserved in freezing tubes (several glycerol tubes can be preserved) to obtain purified strains, which are stored at -80°C. Glycerol tubes will be used as the starting strains for subsequent fermentations.

[0128] (2) Primary LB shake flask seed preparation

[0129] Inoculate 100 μL of the glycerol bacteria frozen in liquid nitrogen into 50 ml (250 ml baffled shake flask) of LB medium (containing antibiotics), and culture overnight at 30°C and 200 rpm for 17 hours. Measure the OD value of the culture solution. 600 About 3.0, no foreign bacteria were found under microscopic examination, and the sample was kept for transfer to shake flask organic nitrogen TB fermentation medium.

[0130] (3) Secondary TB shake flask seed preparation

[0131] Take 2 ml of the first-level seed solution and inoculate it into 200 ml (500 ml of liquid in a baffled shake flask) of the second-level organic nitrogen TB shake flask medium (containing antibiotics), and culture it in a shaker at 37°C and 200 rpm for about 6 hours. Measure the OD of the culture solution. 600 About 5.0, no foreign bacteria found under microscopic examination, keep a sample for preparation of seeds for canning.

[0132] LB primary seed medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract powder.

[0133] TB shake flask fermentation medium: 24 g / L yeast extract powder, 12 g / L peptone, 4 g / L glycerol.

[0134] TB fermenter medium: 24 g / L yeast extract powder, 12 g / L peptone, 10 g / L glucose, magnesium chloride hexahydrate: 0.5 g / L, manganese sulfate monohydrate: 0.05 g / L.

[0135] Example 1. Test on the activity of fermentation broth enhanced by short-term sugar suspension during fermentation of strain PET32a-TcADC-BL21 (DE3) in 1-liter parallel fermenter

[0136] The prepared secondary seeds were placed into a 1-liter parallel fermentation tank (basic liquid volume: 0.4 liter) under flame protection, and at the same time, a fermentation liquid was added to make the final concentration of 0.1 g / L ampicillin, and various metal ion solutions (magnesium chloride hexahydrate, manganese sulfate monohydrate) were added to the inoculation port. The fermentation tank temperature was controlled at 37°C, the stirring speed was controlled at 300 rpm, the tank pressure was 0.03 MPa, ammonia water was added to control the pH to 7.0, 10 g / L glucose was added, the dissolved oxygen and stirring were linked to be controlled at 30%-40%, the maximum stirring speed was 800 rpm, and the ammonia water was automatically controlled to pH 7.0.

[0137] -Dissolved oxygen control: Control the stirring speed and tank pressure to keep DO at 30%-40%. The stirring speed must be increased and decreased slowly.

[0138] -Feed control: constant flow of glucose to keep residual sugar below 1g / L, control specific growth rate, growth stage, glucose flow acceleration is 5-7g / L / H, temperature control: 37℃ during fermentation growth stage, temperature drops to 30℃ after induction

[0139] -pH control: constant 7.0

[0140] -Induction mechanism: When the fermentation broth OD 600 When the temperature reached 30, 0.1mmol / L IPTG was added for induction at 30℃. The total fermentation time was 24 hours and the tank was collected.

[0141] - Test on the activity of fermentation liquid by briefly stopping sugar during fermentation to rejuvenate and enhance the fermentation liquid:

[0142] When the fermentation liquid OD 600 When the temperature reaches 20, stop adding glucose. After stopping sugar, the dissolved oxygen in the fermentation liquid rises rapidly, and the pH also rises accordingly. Control the stirring speed and tank pressure to control the dissolved oxygen at 30%-40%. Investigate the pH rise data after different sugar-stopping times. After reaching the set sugar-stopping time, add 5g / L / H of glucose immediately. At this time, the pH and dissolved oxygen drop rapidly, and the activity of the fermentation liquid is improved. The short-term sugar-deficiency "starvation" improves the tolerance and stress resistance of the strain, produces heat shock proteins for subsequent heat stimulation, and does not cause the cells to lose activity after high temperature. The following are the experimental data of different sugar-stopping times (final fermentation liquid OD600 and pH recovery value). Figure 1 As shown in Table 1.

[0143] Experimental data analysis:

[0144] The experiment was tested using six 1L parallel fermentation tanks, with sugar withdrawal times of 0 (control), 1, 2, 3, 4, and 5 minutes, respectively, and fermentation ended after 24 hours.

[0145] Table 1. Comparison of different sugar withdrawal time plans

[0146]

[0147] Result analysis:

[0148] After 0, 1, 2, 3, 4 or 5 minutes of sugar withdrawal, the corresponding pH value recovered and increased in sequence, and the corresponding final fermentation liquid OD 600 The highest OD value was 116 after 4 minutes of glucose withdrawal, while the blank control did not stop glucose withdrawal. 600 OD is 93, stop sugar for 1-3 minutes 600 The results are 93, 98 and 106 respectively, indicating that the activity of the fermentation bacteria was improved after different sugar withdrawal times, and 4 minutes had the best effect. After 5 minutes, the long-term lack of sugar may have changed the metabolic pathway of the cells, and the activity of the fermentation bacteria was reduced. In short, OD 600 When the temperature reaches 20, stop adding glucose for 4 minutes. After stopping the addition of glucose, the dissolved oxygen in the fermentation liquid rises rapidly, and the pH also rises accordingly. Control the stirring speed and tank pressure to control the dissolved oxygen at 30%-40%. The activity of the bacterial solution is greatly improved by immediately adding 5g / L / H of glucose for 4 minutes after stopping the addition of glucose. The subsequent experimental plans all adopt the plan of stopping the addition of glucose for 4 minutes.

[0149] Example 2. Experiments on different thermal stimulation temperatures in 1-liter parallel fermentation tanks of strain PET32a-TcADC-BL21 (DE3)

[0150] The prepared secondary seeds were placed into a 1-liter parallel fermentation tank (basic liquid volume: 0.4 liter) under flame protection, and at the same time, a fermentation liquid was added to make the final concentration of 0.1 g / L ampicillin, and various metal ion solutions (magnesium chloride hexahydrate, manganese sulfate monohydrate) were added to the inoculation port. The fermentation tank temperature was controlled at 37°C, the stirring speed was controlled at 300 rpm, the tank pressure was 0.03 MPa, ammonia water was added to control the pH to 7.0, 10 g / L glucose was added, the dissolved oxygen and stirring were linked to be controlled at 30%-40%, the maximum stirring speed was 800 rpm, and the ammonia water was automatically controlled to pH 7.0.

[0151] -Dissolved oxygen control: Control the stirring speed and tank pressure to keep DO at 30%-40%. The stirring speed must be increased and decreased slowly.

[0152] -Feed control: constant flow of glucose to keep residual sugar below 1g / L, control specific growth rate, growth stage, glucose flow acceleration is 5-7g / L / H, temperature control: 37℃ during fermentation growth stage, temperature drops to 30℃ after induction

[0153] -pH control: constant 7.0

[0154] -Induction mechanism: When the fermentation broth OD 600 When the temperature reached 30, 0.1mmol / L IPTG was added for induction at 30℃. The total fermentation time was 24 hours and the tank was collected.

[0155] - Experiments on stopping sugar for 4 minutes during fermentation to strengthen the fermentation liquid and different thermal stimulation temperatures:

[0156] When the fermentation liquid OD 600 When it reaches 20, stop adding glucose for 4 minutes. After stopping sugar, the dissolved oxygen in the fermentation liquid rises rapidly, and the pH also rises accordingly. Control the stirring speed and tank pressure to control the dissolved oxygen at 30%-40%. Investigate the pH rise data after different sugar stopping times. After 4 minutes, immediately add 5g / L / H of glucose. At this time, the pH and dissolved oxygen drop rapidly, and the activity of the fermentation bacteria is improved. Immediately implement heating measures for the fermentation liquid, and heat it to 40℃, 42℃, 44℃, 46℃, 48℃ and 37℃ blank control culture without heating. After the temperature rises to the set value, keep it for 90 seconds and then quickly cool it to 37℃ for continued cultivation. After 24 hours of fermentation, the tank is collected. After heating, the host cells will produce corresponding heat shock proteins to reduce protein misfolding in response to heat stimulation, which will also have a certain effect on the growth of cells. The fermentation end OD of 40℃, 42℃, 44℃, 46℃, 48℃ and 37℃ blank control culture without heating 600 like Figure 2 And as shown in Table 2.

[0157] Table 2. Comparison of different thermal stimulation temperatures

[0158]

[0159] Experimental data analysis:

[0160] The experiment was tested using six 1L parallel fermentation tanks, with thermal stimulation temperatures of 37°C (control), 40°C, 42°C, 44°C, 46°C, and 48°C, respectively. The thermal stimulation time was 90 seconds, and the fermentation ended after 24 hours.

[0161] Data analysis shows that thermal stimulation temperatures of 44°C, 46°C, and 48°C have a significant inhibitory effect on cell growth, while thermal stimulation at 42°C for 90 seconds has little effect on growth. This implementation case proves that 42°C is the preferred thermal stimulation temperature, and subsequent experiments will continue to investigate the thermal stimulation temperature of 42°C at different retention times.

[0162] Example 3. Experiments on thermal stimulation of strain PET32a-TcADC-BL21 (DE3) in 1-liter parallel fermentation tank at 42°C with different retention times

[0163] The prepared secondary seeds were placed into a 1-liter parallel fermentation tank (basic liquid volume: 0.4 liter) under flame protection, and at the same time, a fermentation liquid was added to make the final concentration of 0.1 g / L ampicillin, and various metal ion solutions (magnesium chloride hexahydrate, manganese sulfate monohydrate) were added to the inoculation port. The fermentation tank temperature was controlled at 37°C, the stirring speed was controlled at 300 rpm, the tank pressure was 0.03 MPa, ammonia water was added to control the pH to 7.0, 10 g / L glucose was added, the dissolved oxygen and stirring were linked to be controlled at 30%-40%, the maximum stirring speed was 800 rpm, and the ammonia water was automatically controlled to pH 7.0.

[0164] -Dissolved oxygen control: Control the stirring speed and tank pressure to keep DO at 30%-40%. The stirring speed must be increased and decreased slowly.

[0165] -Feed control: constant flow of glucose to keep residual sugar below 1g / L, control specific growth rate, growth stage, glucose flow acceleration is 5-7g / L / H, temperature control: 37℃ during fermentation growth stage, temperature drops to 30℃ after induction

[0166] -pH control: constant 7.0

[0167] -Induction mechanism: When the fermentation broth OD 600 When the temperature reached 30, 0.1mmol / L IPTG was added for induction at 30℃. The total fermentation time was 24 hours and the tank was collected.

[0168] During the fermentation process in a 1-liter parallel fermenter, the experiment of stopping sugar for 4 minutes and adding 42°C heat stimulation with different retention times:

[0169] When the fermentation liquid OD 600 When the temperature reached 20, the addition of glucose was stopped for 4 minutes. After the sugar was stopped, the dissolved oxygen in the fermentation liquid rose rapidly, and the pH also rose accordingly. The stirring speed and tank pressure were controlled to control the dissolved oxygen at 30%-40%. The pH rise data after different sugar withdrawal times were examined. After 4 minutes, 5g / L / H of glucose was immediately added. At this time, the pH and dissolved oxygen dropped rapidly, and the activity of the fermentation liquid was improved. The fermentation liquid was immediately heated to 42°C and retained for 30 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds, and 180 seconds for six groups of experiments. After the retention time ended, the temperature was quickly lowered to 37°C for normal culture. After 24 hours of fermentation, samples were taken to detect OD 600 , data such as Figure 3 And as shown in Table 3.

[0170] Table 3. Comparison of retention time of different thermal stimulations

[0171]

[0172] Experimental data analysis:

[0173] -The experiment was tested using six 1L parallel fermenters, with a thermal stimulation temperature of 42°C and retention times of 30 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds, and 180 seconds.

[0174] -Through data analysis, the final OD values ​​of the three groups of fermentation broths were 42°C for thermal stimulation and 120, 150 and 180 seconds for retention time. 600 The results are relatively low, which may be due to the fact that the heat stimulation time is too long, causing cell damage and affecting cell viability. However, the other three groups of heat stimulation at 42℃ for 30 seconds, 60 seconds, and 90 seconds have little effect on growth. The final fermentation liquid OD 600 The values ​​are relatively close.

[0175] -In order to verify the selection of appropriate heat stimulation retention time among heat stimulation of 30 seconds, 60 seconds, and 90 seconds, the fermentation broth of three groups of heat stimulation for 30 seconds, 60 seconds, and 90 seconds was centrifuged and the cells were broken, and the supernatant was taken for SDS-PAGE to detect and analyze the expression of soluble target protein. Figure 4 shown.

[0176] -SDS-PAGE analysis: The soluble expression of the target protein in the three samples with heat stimulation for 30 seconds, 60 seconds, and 90 seconds was higher than that in the control sample (without heat stimulation treatment). Among the three samples with heat stimulation for 30 seconds, 60 seconds, and 90 seconds, the soluble protein expression at 30 seconds was the lowest, and the soluble protein expression at 60 seconds and 90 seconds was basically the same. Considering the adequacy of the retention time of heat stimulation, 90 seconds is preferred.

[0177] -The experimental data of this implementation case proves that the best effect is achieved when the thermal stimulation temperature is 42°C and the retention time is 90 seconds.

[0178] Comparative Example 4. Normal fermentation in 1-liter parallel fermentation tank of strain PET32a-TcADC-BL21 (DE3) (control experiment)

[0179] The prepared secondary seeds were placed into a 1-liter parallel fermentation tank (basic liquid volume: 0.4 liter) under flame protection, and at the same time, a fermentation liquid was added to make the final concentration of 0.1 g / L ampicillin, and various metal ion solutions (magnesium chloride hexahydrate, manganese sulfate monohydrate) were added to the inoculation port. The fermentation tank temperature was controlled at 37°C, the stirring speed was controlled at 300 rpm, the tank pressure was 0.03 MPa, ammonia water was added to control the pH to 7.0, 10 g / L glucose was added, the dissolved oxygen and stirring were linked to be controlled at 30%-40%, the maximum stirring speed was 800 rpm, and the ammonia water was automatically controlled to pH 7.0.

[0180] -Dissolved oxygen control: Control the stirring speed and tank pressure to keep DO at 30%-40%. The stirring speed must be increased and decreased slowly.

[0181] -Feed control: constant flow of glucose to keep residual sugar below 1g / L, control specific growth rate, growth stage, glucose flow acceleration is 5-7g / L / H, temperature control: 37℃ during fermentation growth stage, temperature drops to 30℃ after induction

[0182] -pH control: constant 7.0

[0183] -Induction mechanism: When the fermentation broth OD 600 When the temperature reached 30, 0.1mmol / L IPTG was added for induction at 30℃. The total fermentation time was 24 hours and the tank was collected.

[0184] -Note: Sugar was not stopped during the entire fermentation process, and the fermentation liquid was not heated to stimulate the fermentation liquid, as a control experiment for normal fermentation.

[0185] Example 5. Inclusion body content and specific enzyme activity determination

[0186] 40 ml of the fermentation broth of the optimization scheme of the present invention (heat stimulation at 42°C for 90 seconds after sugar withdrawal for 4 minutes) and the control fermentation broth of Comparative Example 4 were taken at 7000 rpm, centrifuged for 10 minutes, and the supernatant was removed to collect wet cells. 1 g of the centrifuged wet cells was added with 10 g of deionized water and mixed. After ultrasonic disruption for 20 minutes, the whole cell solution and the supernatant of the centrifuge were taken for SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis are shown in FIG. Figure 5 As shown; take the supernatant and the precipitate after crushing, dilute them ten times with deionized water, and perform SDS-PAGE electrophoresis on the dilution. The results of SDS-PAGE electrophoresis are shown as follows Figure 6 shown.

[0187] Depend on Figure 5-6 The results show that, compared with the control experiment, although the total amount of protein in the whole cell remains basically unchanged, the amount of protein expressed in the form of inclusion bodies is significantly reduced, and the proportion of soluble protein is greatly increased in the optimized scheme of the present invention (heat stimulation at 42°C for 90 seconds after sugar withdrawal for 4 minutes).

[0188] The total protein expression (g) was determined by image analysis, and the enzyme activity was determined by the following method:

[0189] Take 100 μL of the supernatant in a 1.5 mL centrifuge tube, add sodium L-aspartate at a final concentration of 100 mmol / L and pyridoxal phosphate (PLP) at a final concentration of 1 mmol / L, and react at 37°C, pH 6.5 for 30 minutes to detect the enzyme activity. Enzyme activity is defined as: the amount of enzyme required to convert sodium L-aspartate to 1 mmol of β-alanine per hour at 37°C, pH 6.5 is one enzyme activity unit U.

[0190] The specific enzyme activity was determined based on the total protein expression (g) and enzyme activity (U). The specific enzyme activity is defined as the enzyme activity per gram of protein, in U / g. The results of the specific enzyme activity determination are shown in Table 4 below.

[0191] Table 4. Enzyme activity test results of different samples:

[0192] serial number Sample plan Specific enzyme activity (U / g) 1 Comparative Example 4, control 143 2 Example 3: Heat shock at 42°C for 30 seconds after 4 minutes of sugar withdrawal 189 3 Example 3: After 4 minutes of sugar withdrawal, heat shock at 42°C for 60 seconds 243 4 Example 3: After 4 minutes of sugar withdrawal, heat shock at 42°C for 90 seconds 255

[0193] Experimental data analysis:

[0194] Compared with the control experiment of Comparative Example 4, the experimental scheme of the present invention (heat stimulation at 42°C for different times after 4 minutes of sugar withdrawal) reduced the protein expressed in the form of inclusion bodies and increased the proportion of soluble protein, thereby significantly improving the specific enzyme activity. The specific enzyme activity of the scheme with heat stimulation for 90 seconds reached 255 U / g, which was 78.3% higher than the specific enzyme activity (143 U / g) of the control experiment of Comparative Example 4 (see Table 4).

[0195] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the specific implementation of the present invention is described above, it should be understood by those skilled in the art that this is only an example, and the protection scope of the present invention is defined by the attached claims. Those skilled in the art may make various changes or modifications to the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A method for producing foreign protein by fermenting recombinant Escherichia coli, wherein the fermentation is a fed-batch culture process, and the proportion of the foreign protein expressed in the form of inclusion bodies is reduced compared with a control method not using the method, the method comprising: (a) after the recombinant Escherichia coli grows and is induced to express for a period of time, stopping the feeding of the carbon source for 1-10 minutes, and then continuing the feeding of the carbon source; and / or (b) 1-10 minutes after stopping the addition of the carbon source, the fermentation broth is thermally stimulated by instantaneously increasing the temperature.

2. The method according to claim 1, wherein the OD of the fermentation broth is 600 When the OD value of the fermentation liquid is 18-25, stop adding the carbon source for 1-8 minutes. 600 When the temperature is 20-22, stop adding the carbon source for 2-5 minutes.

3. The method according to claim 1 or 2, wherein the temperature of the fed-batch culture process is 37°C during the growth of the recombinant Escherichia coli, and the temperature is 30°C during the induced expression of the recombinant Escherichia coli.

4. The method according to any one of claims 1 to 3, wherein the instantaneous temperature increase is from 30°C to 35-50°C, preferably to 37-46°C, more preferably to 40-44°C.

5. The method according to any one of claims 1 to 4, wherein the instantaneous temperature increase is maintained for 30 to 120 seconds, preferably 45 to 110 seconds, more preferably 60 to 100 seconds.

6. The method according to any one of claims 1-5, wherein the carbon source is selected from the group consisting of carbohydrates, lipids, organic acids, alcohols and hydrocarbons, preferably the carbon source is selected from the group consisting of monosaccharides, disaccharides and polysaccharides, more preferably the carbon source is selected from the group consisting of glucose, maltose, molasses and starch.

7. The method according to any one of claims 1 to 6, wherein the culture conditions of the fed-batch culture process are selected from one or more of the following: (i) adjusting the dissolved oxygen level in the fed-batch culture process to 30%-40% by adjusting the stirring speed and / or the tank pressure; (ii) controlling the pH of the fed-batch culture process to be between 6.8 and 7.2 by adding ammonia or hydrochloric acid; (iii) the flow rate of the carbon source is 4-8 g / L / hour, preferably 5-7 g / L / hour; and / or (iv) The fermentation is carried out for a total of 20-48 hours, preferably 22-42 hours, more preferably 24-36 hours.

8. The method according to any one of claims 1 to 7, wherein the induced expression of the recombinant Escherichia coli is performed by adding an inducer, preferably the inducer is 0.01-0.5 mmol / L IPTG.

9. The method according to any one of claims 1 to 8, wherein the exogenous protein is L-aspartate-α-decarboxylase.

10. The method according to any one of claims 1 to 9, wherein the recombinant Escherichia coli comprises a gene encoding L-aspartate-α-decarboxylase.

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