A method for producing L-threonine by cyclic fermentation

Abstract

The invention provides a method for producing L-threonine by cyclic fermentation. By coupling the dissolved oxygen feedback feeding strategy and the cell cyclic fermentation strategy, on the one hand, the sugar feeding rate can be regulated according to the level of dissolved oxygen, thereby relieving the substrate inhibition caused by high-concentration glucose; on the other hand, in the cell cyclic fermentation process, as the supernatant is continuously discharged, not only the accumulation of the product L-threonine and the by-products acetic acid, glutamic acid, etc. in the reaction system is reduced, but also the inhibition of the products and by-products can be appropriately relieved; at the same time, in the cyclic fermentation process, the cells are fully utilized, the activity of the cells is high, and the cell density and production efficiency in the fermenter are improved; in addition, the method realizes the continuous separation and discharge of the clear liquid, saves time and investment in separation equipment for subsequent extraction processes, directly reduces the production cost of threonine, and can effectively improve the market competitiveness of domestic threonine.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a method for producing L-threonine through cyclic fermentation. Background Art

[0002] L-threonine is the second limiting amino acid after L-lysine in pig feed, and the third limiting amino acid after L-methionine and L-lysine in poultry feed. It can improve the body's immune function and fat metabolism, promote growth, improve meat quality, promote small intestinal mucus protein secretion, maintain intestinal health, etc., and has important physiological functions. In addition, L-threonine is also widely used in many fields such as food, medicine and cosmetics.

[0003] At present, L-threonine is mainly produced in industry through Escherichia coli fermentation. Although the level of L-threonine produced by Escherichia coli used in industrial fermentation is very high, there is still a certain gap with the theoretical sugar-acid conversion rate (81%). Therefore, it is of great significance to explore how to further improve the ability of Escherichia coli to produce L-threonine and improve its sugar-acid conversion rate.

[0004] As an L-threonine producing bacterium, Escherichia coli easily produces metabolic byproducts such as acetic acid and glutamic acid during fermentation and cultivation. The accumulation of byproducts not only causes a waste of carbon sources, but also seriously inhibits bacterial growth and the synthesis of L-threonine to a certain extent. Among them, the inhibitory effect of acetic acid is the most obvious. When acetic acid accumulates to a certain concentration, it not only has an adverse effect on cell metabolism, but also seriously affects the expression of exogenous genes, thereby greatly reducing the synthesis of L-threonine. Based on this, exploring the appropriate fermentation medium formula and optimizing the fermentation process are one of the effective methods to improve the fermentation capacity of Escherichia coli engineered bacteria. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing L-threonine by cyclic fermentation.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A method for producing L-threonine by cyclic fermentation, using Escherichia coli engineered bacteria A.BU14 to produce L-threonine by microbial fermentation, the specific steps are as follows:

[0008] (1) Activate the E. coli engineered bacteria A.BU14 by two generations of slant culture;

[0009] (2) inoculating the activated strain into a seed tank for seed culture to obtain a seed solution;

[0010] (3) Biomass OD during seed culture600 When the pH value is 15-25, the seed solution is inoculated into the fermentation tank, and the cyclic fermentation is controlled based on the yield of L-threonine in the fermentation tank to obtain L-threonine.

[0011] The above-mentioned Escherichia coli engineered bacteria A.BU14 is the Escherichia coli engineered bacteria A.BU14 in ZL202410552098.5.

[0012] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the specific method of activation culture in step (1) is: taking the Escherichia coli engineered bacteria A.BU14 and streaking it on the activated slant, culturing it for 12 hours and then transferring it to the second generation slant and continuing to culture it for 12 hours; the specific components of the activated slant culture medium used are glucose 1 g / L, peptone 4 g / L, sodium chloride 1 g / L, yeast extract powder 5 g / L, KH2PO42 g / L, MgSO4·7H2O 0.5 g / L, agar powder 25 g / L, and the rest is water, pH = 6.8-7.0, and the activation culture temperature is 32-37°C.

[0013] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the specific method of seed culture in step (2) is: the activated strain is transferred to 5L seed culture medium for culture, the pH is maintained at 6.8-7.0, the temperature is constant at 37°C, and the dissolved oxygen is 30%-50%; the specific components of the seed culture medium used are glucose 25 g / L, yeast powder 5 g / L, peptone 4 g / L, citric acid 2 g / L, (NH4)2SO43 g / L, MgSO4·7H2O 0.8 g / L, KH2PO42 g / L, FeSO4·7H2O 20 mg / L, MnSO410 mg / L, VB1, VB3, VB5, VB 12 2 mg / L each, VH 1 mg / L, and the rest is water.

[0014] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the specific method of fermentation culture in step (3) is as follows: when the seed cell mass (OD 600 ) reaches 15-25, and the seed liquid is inoculated into a 30L fermentation tank for fermentation culture; the specific components of the fermentation medium used are yeast powder 4g / L, peptone 3g / L, corn steep liquor 20ml / L, ammonium sulfate 3g / L, methionine 0.1g / L, KH2PO4·3H2O 4g / L, glutamic acid 2g / L, MgSO4·7H2O 1.5g / L, FeSO4·7H2O 20mg / L, MnSO410mg / L, VB1, VB3, VB5 each 2mg / L, and the rest is water.

[0015] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the fermentation culture conditions in step (3) are: temperature of 37°C, pH of 6.8-7.0, pH maintained by adding ammonia water by a peristaltic pump, dissolved oxygen of 30%-50%, tank pressure of 0.03-0.05 MPa, ventilation volume of 0.3-0.5 vvm; glucose concentration in the tank of 0-0.5 g / L (maintained by adding 80% glucose solution, and 1-3 ml of fermentation liquid was sampled every 2 h for testing during the process).

[0016] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the specific steps of cyclic fermentation in step (3) are:

[0017] 1) First, couple the 30L fermenter with the ceramic membrane, and then ferment and produce L-threonine in the 30L fermenter. When the L-threonine yield is ≥90g / L, open the valve to start the fermentation liquid to flow out (simultaneously feed an equal volume of glucose solution), separate through the ceramic membrane, extract and recover L-threonine directly from the supernatant, and return the concentrate to the fermenter;

[0018] 2) When the L-threonine production in the fermentation liquid drops to ≤70g / L, close the valve and allow the fermentation tank to continue normal fermentation until the fermentation liquid flows out again when the production reaches 90g / L. Repeat the fermentation cycle to maintain the L-threonine production in the fermentation liquid at 70-90g / L.

[0019] 3) After 50 h of co-fermentation, the L-threonine production showed a pulse curve change.

[0020] The above-mentioned method for producing L-threonine by cyclic fermentation couples the dissolved oxygen feedback feeding strategy and the cell cycle fermentation strategy. On the one hand, the sugar feeding rate is regulated according to the level of dissolved oxygen, thereby relieving the substrate inhibition caused by high concentration of glucose; on the other hand, with the continuous discharge of the clear liquid during the cell cycle, the concentrations of L-threonine, acetic acid and glutamate in the reaction system are reduced, thereby relieving the inhibition of products and by-products.

[0021] The dissolved oxygen feedback feeding strategy means that the level of dissolved oxygen is inversely proportional to the feeding rate. The dissolved oxygen value detected by the dissolved oxygen electrode is used as a signal. By adjusting the sugar feeding rate to control the dissolved oxygen level of 30-50%, the glucose concentration and specific growth rate are controlled below the critical value that causes acetic acid accumulation, thereby reducing acetic acid secretion. During the fermentation process, the cyclic fermentation is started when the L-threonine production reaches a certain value (≥90g / L), and the cyclic fermentation is stopped when it drops to a certain value (≤70g / L).

[0022] Preferably, in the above-mentioned method for producing L-threonine by cyclic fermentation, the cyclic fermentation time during the fermentation process is 16-20 hours, 20-24 hours or 24-28 hours.

[0023] Beneficial effects:

[0024] The above-mentioned method for producing L-threonine by cyclic fermentation, by coupling the dissolved oxygen feedback feeding strategy and the cell cyclic fermentation strategy, can, on the one hand, adjust the sugar feeding rate according to the level of dissolved oxygen, thereby relieving the substrate inhibition caused by high concentration of glucose; on the other hand, during the cell cyclic fermentation process, as the supernatant is continuously discharged, not only the accumulation of the product L-threonine and the by-products acetic acid, glutamic acid, etc. in the reaction system is reduced, but the inhibition of the products and by-products can be appropriately relieved; at the same time, during the cyclic fermentation process, the cells are fully utilized, the cell activity is high, and the cell density and production efficiency in the fermenter are improved; in addition, the method realizes the continuous separation and discharge of the clear liquid, saves time and investment in separation equipment for the subsequent extraction process, directly reduces the production cost of threonine, and can effectively improve the market competitiveness of domestic threonine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the cell recycling fermentation bioreactor.

[0026] Figure 2 This is a curve chart showing the changes in L-threonine production during the fermentation process. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0028] The production starting bacteria used in the following examples is strain A.BU14 in Example No. ZL202410552098.5.

[0029] Example 1

[0030] A method for producing L-threonine by cyclic fermentation, using a pulsed cyclic fermentation method, the specific steps are as follows:

[0031] (1) Activation of strains: Take strain A.BU14 and streak it on the activation slant. After culturing for 12 h, transfer it to the second generation slant and continue culturing for 12 h. The components of the activation slant culture medium are glucose 1 g / L, peptone 4 g / L, sodium chloride 1 g / L, yeast extract powder 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, agar powder 25 g / L, and the rest is water. The pH value is 6.8-7.0. The activation culture temperature is 32-37°C.

[0032] (2) Seed culture: The activated strain was transferred to 5L seed culture medium for culture, with the pH maintained at 6.8-7.0, the temperature constant at 37°C, and the dissolved oxygen at 30%-50%; the specific components of the seed culture medium included 25 g / L glucose, 5 g / L yeast powder, 4 g / L peptone, 2 g / L citric acid, 3 g / L (NH4)2SO4, 0.8 g / L MgSO4·7H2O, 2 g / L KH2PO4, 20 mg / L FeSO4·7H2O, 10 mg / L MnSO4, VB1, VB3, VB5, VB 12 2 mg / L each, VH 1 mg / L, and the rest is water.

[0033] (3) Fermentation culture: The amount of seed bacteria (OD 600 ) reaches about 20, and the seed liquid is inoculated into a 30L fermentation tank for fermentation culture. The specific components of the fermentation medium include yeast powder 4g / L, peptone 3g / L, corn steep liquor 20ml / L, ammonium sulfate 3g / L, methionine 0.1g / L, KH2PO4·3H2O 4g / L, glutamic acid 2g / L, MgSO4·7H2O 1.5g / L, FeSO4·7H2O20mg / L, MnSO410mg / L, VB1, VB3, VB5 2mg / L each, the rest is water; the fermentation temperature is 37℃, pH is 7.0, pH is maintained by adding ammonia water through a peristaltic pump, and the cycle fermentation is started when the L-threonine production is fermented to 90g / L, the fermentation cycle is 50h, the dissolved oxygen is 30%-50%, the tank pressure is 0.03-0.05MPa, the ventilation volume is 0.3-0.5vvm, and the glucose concentration in the tank is maintained at 0-0.5g / L by adding 80% glucose solution. During the process, 1-3 ml of the fermentation liquid is sampled every 2 hours for testing.

[0034] Example 2

[0035] A method for producing L-threonine by pulsed cyclic fermentation, wherein the specific steps are as described in Example 1, except that the fermentation culture process in step (3) is as follows: Figure 1 As shown, firstly, the 30L fermentation tank is coupled with the ceramic membrane, and then the L-threonine is produced by normal fermentation in the 30L fermentation tank. When the L-threonine production is ≥90g / L, the valve at the bottom of the fermentation tank is opened to start the outflow of the fermentation liquid (at the same time, an equal volume of glucose solution is added), and the supernatant is directly extracted and recovered for L-threonine, and the concentrate is returned to the fermentation tank; when the L-threonine production in the fermentation liquid drops to ≤70g / L, the valve is closed, and the fermentation tank continues to ferment normally until the fermentation liquid flows out again when it reaches 90g / L. The fermentation cycle is repeated for 20-24h, so that the L-threonine production in the fermentation liquid is maintained at 70-90g / L. Finally, the fermentation is carried out for 50h. Figure 2 As shown in the figure, the L-threonine production showed a pulse curve change, and the sugar-acid conversion rate increased from 59.1% to 63.3%, an increase of 7.1% compared with before optimization.

[0036] Example 3

[0037] A method for producing L-threonine by pulsed cyclic fermentation, referring to Example 2, the numerical optimization of starting cyclic fermentation when the L-threonine yield is ≥90 g / L and stopping cyclic fermentation when the L-threonine yield is ≤70 g / L during the fermentation process is shown in Table 1. The cyclic fermentation time selected in this embodiment is 20-24 h.

[0038] Table 1 Results of starting or stopping cycle fermentation with different L-threonine yields

[0039] Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 L-threonine production in open cycle fermentation (g) 85 85 85 90 90 90 95 95 95 L-threonine production in stopped cycle fermentation (g) 65 70 75 65 70 75 65 70 75 <![CDATA[Bacterial biomass (OD 600 )]]> 127.3 130.3 132.4 131.6 133.5 135.8 131.6 132.9 136.1 Sugar-acid conversion rate (%) 62.3 62.7 62.4 62.8 63.3 63.0 62.7 62.9 62.5

[0040] As can be seen from Table 1, when the L-threonine production reaches 90 g / L, the cyclic fermentation is started and the cyclic fermentation is stopped when it drops to 70 g / L, the conversion rate of L-threonine reaches a maximum value of 63.3%, and the bacterial biomass is also relatively high. Therefore, the subsequent examples are carried out on the basis of the best optimized values ​​of Example 3.

[0041] Example 4

[0042] A method for producing L-threonine by pulsed cyclic fermentation, referring to Example 2, the cyclic fermentation durations in the three fermentation processes are 16-20 hours, 20-24 hours, and 24-28 hours respectively. The fermentation results are shown in Table 2.

[0043] Table 2 Effects of different cycle fermentation durations on fermentation results

[0044] The first group: 16-20h The second group 20-24h Group 3 24-28h <![CDATA[Cell biomass (OD 600 )]]> 127.3 133.5 136.8 Sugar-acid conversion rate (%) 62.8 63.3 63.0

[0045] It can be seen from Table 2 that when the cycle fermentation time is 20-24h, the sugar-acid conversion rate of L-threonine is the highest.

[0046] In summary, the best process method verified by experiments is: start the circulating fermentation when the L-threonine production reaches 90g / L and stop the circulating fermentation when it drops to 70g / L. The circulating fermentation time is 20-24h. Finally, after fermentation process optimization and a total fermentation time of 50h, the L-threonine production showed a pulse curve change, and the sugar-acid conversion rate increased from 59.1% to 63.3%, an increase of 7.1%.

[0047] It can be seen that the method of the present invention achieves an improvement in the conversion rate of L-threonine and improves the fermentation index of the existing strain.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention, which are all considered to be within the protection scope of the present invention.

Claims

1. A method for producing L-threonine by cyclic fermentation, characterized in that: The L-threonine is produced by microbial fermentation using Escherichia coli engineered bacteria A.BU14. The specific steps are as follows: (1) Activate the E. coli engineered bacteria A.BU14 by two generations of slant culture; (2) inoculating the activated strain into a seed tank for seed culture to obtain a seed solution; (3) Biomass OD during seed culture 600 When the temperature is 15-25, the seed liquid is inoculated into the fermentation tank, and the cyclic fermentation is controlled based on the L-threonine production in the fermentation tank. The sugar supplement rate is adjusted to control the dissolved oxygen level of 30-50%. When the L-threonine production during the fermentation process is ≥90g / L, the cyclic fermentation is started, and when it drops to ≤70g / L, the cyclic fermentation is stopped to obtain L-threonine.

2. The method for producing L-threonine by cyclic fermentation according to claim 1, characterized in that: The specific method of activation culture in step (1) is as follows: take the Escherichia coli engineered bacteria A.BU14 and inoculate it on the activation slant, culture it for 12 hours, and then transfer it to the second generation slant and continue to culture it for 12 hours; the specific components of the activated slant culture medium used are glucose 1 g / L, peptone 4 g / L, sodium chloride 1 g / L, yeast extract powder 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, agar powder 25 g / L, and the rest is water, pH=6.8-7.0, and the activation culture temperature is 32-37°C.

3. The method for producing L-threonine by cyclic fermentation according to claim 1, characterized in that: The specific method of seed culture in step (2) is as follows: the activated strain is transferred to 5L seed culture medium for culture, the pH is maintained at 6.8-7.0, the temperature is constant at 37°C, and the dissolved oxygen is 30%-50%; the specific components of the seed culture medium used are glucose 25 g / L, yeast powder 5 g / L, peptone 4 g / L, citric acid 2 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4 10 mg / L, VB1, VB3, VB5, VB 12 2 mg / L each, VH 1 mg / L, and the rest is water.

4. The method for producing L-threonine by cyclic fermentation according to claim 1, characterized in that: The specific method of fermentation culture in step (3) is as follows: 600 When the pH value reaches 15-25, the seed liquid is inoculated into a 30L fermentation tank for fermentation culture; the specific components of the fermentation medium used are yeast powder 4g / L, peptone 3g / L, corn steep liquor 20ml / L, ammonium sulfate 3g / L, methionine 0.1g / L, KH2PO4·3H2O 4g / L, glutamic acid 2g / L, MgSO4·7H2O 1.5g / L, FeSO4·7H2O20mg / L, MnSO4 10mg / L, VB1, VB3, VB5 each 2mg / L, and the rest is water.

5. The method for producing L-threonine by cyclic fermentation according to claim 1, characterized in that: The fermentation culture conditions in step (3) are as follows: temperature of 37°C, pH of 6.8-7.0, pH maintained by adding ammonia water via a peristaltic pump, dissolved oxygen of 30%-50%, tank pressure of 0.03-0.05 MPa, ventilation volume of 0.3-0.5 vvm; and glucose concentration in the tank of 0-0.5 g / L.

6. The method for producing L-threonine by cyclic fermentation according to claim 1, characterized in that: The specific steps of the cyclic fermentation in step (3) are: 1) First, couple the 30L fermentation tank with the ceramic membrane, and then ferment and produce L-threonine in the 30L fermentation tank. When the L-threonine yield is ≥90g / L, open the valve to start the outflow of the fermentation liquid, separate through the ceramic membrane, extract and recover L-threonine directly from the supernatant, and return the concentrate to the fermentation tank; 2) When the L-threonine production in the fermentation liquid drops to ≤70g / L, close the valve and allow the fermentation tank to continue normal fermentation until the fermentation liquid flows out again when the production reaches 90g / L. Repeat the fermentation cycle to maintain the L-threonine production in the fermentation liquid at 70-90g / L. 3) Fermentation for 50h.

7. The method for producing L-threonine by cyclic fermentation according to claim 1 or 6, characterized in that: During the fermentation process, the cyclic fermentation time is 16-20h, 20-24h or 24-28h.

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