Method for increasing fermentation yield of L-serine by regulating ORP (Oxidation Reduction Potential)

Abstract

The invention discloses a method for increasing the fermentation yield of L-serine by regulating ORP (Oxidation Reduction Potential), and belongs to the technical field of fermentation. According to the method disclosed by the invention, the ORP control in the fermentation process is optimized by controlling the carbon source supplement in stages, the fermentation yield of the L-serine can be effectively improved, and compared with the fermentation effect of regulating and controlling the ORP through the ventilatory capacity, the yield of the L-serine is improved by more than 20%, so that the method is beneficial to improving the comprehensive benefits of industrialization.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for increasing the yield of L-serine fermentation by regulating ORP, belonging to the technical field of fermentation. BACKGROUND

[0002] L-serine is a natural amino acid, which is an important component of protein synthesis in organisms and participates in the growth, repair and metabolism of cells. At present, L-serine has wide application prospects in the fields of amino acid drugs, food processing and cosmetic industry. There are technologies for fermenting and producing L-serine by modifying wild-type Escherichia coli. At present, the yield can be improved by methods such as selecting excellent strains, improving fermentation conditions such as temperature and pH, and controlling microbial metabolic activities to control metabolic products.

[0003] Oxidation-reduction potential (ORP) not only directly reflects the oxidation-reduction ability of the environment where the microorganism is located, but also indirectly represents the metabolic activity of the microorganism. And ORP has the universality and sensitivity in various states similar to pH, temperature and other fermentation parameters, which can overcome the limitations of dissolved oxygen electrode application in micro-aerobic and anaerobic fermentation, and can monitor the growth state of microorganisms in real time. Therefore, online monitoring and controlling ORP can effectively improve the fermentation results. However, there are few methods for improving product yield by controlling ORP in aerobic fermentation. In the production fermentation process, ORP also changes with the growth of bacterial cells. At present, the optimal oxidation-reduction potential in the production of L-serine fermentation process is not known, which limits the growth space of L-serine titer and conversion rate. Therefore, the present application develops a method for regulating L-serine yield by regulating ORP through feeding. SUMMARY

[0004] The present application provides a method for increasing the yield of L-serine fermentation by dynamically regulating ORP, using L-serine-producing Escherichia coli as the fermentation strain, and controlling the carbon source feeding parameters in stages; the stage is to start feeding at the 4th hour of fermentation, and the feeding speed is controlled according to Y=aX+b until the current ORP reaches the lowest ORP range, and then the feeding speed at that time is constant; wherein Y represents the feeding amount per unit time per unit volume of fermentation broth, and X represents the feeding time; the lowest ORP range includes-120 mV ~-130 mV, -130 mV ~-140 mV, -140 mV ~-150 mV.

[0005] In one embodiment, the a is 1.5-1.6, and the b is 7-8.

[0006] In an embodiment, the method is first fermented at 35-40℃ for 3 h, and then fed according to Y=aX+b from the 4th to the 13th hour, until the current ORP reaches the range of-120 to-130 mV, and then fed at the feeding rate at that time until the end of fermentation.

[0007] In an embodiment, the method is first fermented at 35-40℃ for 3 h, and then fed according to Y=aX+b from the 4th to the 15th hour, until the current ORP reaches the range of-130 to-140 mV, and then fed at the feeding rate at that time until the end of fermentation.

[0008] In an embodiment, the method is first fermented at 35-40℃ for 3 h, and then fed according to Y=aX+b from the 4th to the 17th hour, until the current ORP reaches the range of-140 to-150 mV, and then fed at the feeding rate at that time until the end of fermentation.

[0009] In an embodiment, the minimum ORP range includes-120 to-130 mV, -130 to-140 mV, -140 to-150 mV; preferably-130 to-140 mV.

[0010] In an embodiment, the carbon source includes but is not limited to glucose.

[0011] In an embodiment, the method is fermentation using E. coli.

[0012] In an embodiment, the E. coli includes but is not limited to ALE8-8 (DE3 strain) in CN107406864B, SER_151, SER_160, SER_166, SER_164, SER_162 involved in CN117625508A.

[0013] The method of the present application does not have a special limitation on the fermentation volume, and can be various fermentation volumes used in current conventional production or research.

[0014] The present application also provides a method for fermentatively producing L-serine, which is fermenting E. coli as a fermenting microorganism under the following conditions: (1) fermenting at 35-40℃ within 3 hours after the start of fermentation; (2) feeding at the 4th hour of fermentation; the feeding is according to Y=aX+b, wherein Y represents the feeding amount of the fermentation broth per unit volume per unit time (g / (L·h)), X represents the feeding time (h); when the current ORP is in the required range, constant-speed feeding is started, the speed of the constant-speed feeding is the current value when the minimum ORP range is reached, the a is 1.5-1.6, and the b is 7-8.

[0015] In an embodiment, the carbon source includes, but is not limited to, glucose.

[0016] In an embodiment, fermentation is completed in 3 hours, and feeding of glucose is started in the 4th hour.

[0017] In an embodiment, when the titer of L-serine reaches 90 g / L, the feeding of glucose is stopped, and other fermentation conditions are maintained until the residual glucose is consumed and the dissolved oxygen returns to above 60%, and then the fermentation is stopped.

[0018] The application also provides use of the method in preparation of a product containing L-serine.

[0019] Advantages: (1) The application sets different ORP control ranges between batches, forms an ORP control gradient between batches, and in the case of ensuring production, combines the product titer and conversion rate under the ORP condition to determine the optimal ORP value in the fermentation process, improve the comprehensive benefits of industrialization, and provide a reference for other optimization of the technology.

[0020] (2) The application realizes precise regulation of ORP by feeding carbon sources, which can effectively improve the fermentation yield of L-serine, and the yield of L-serine is increased by more than 20% compared with other ways of regulating ORP (such as air flow regulation). DETAILED DESCRIPTION

[0021] In the embodiments of the application, the strain used is ALE8-8 (DE3) in patent CN107406864B.

[0022] The formula of the seed medium and the fermentation medium is: glucose 11 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, yeast extract powder 1 g / L, ammonium sulfate 5 g / L, glycine 1 g / L, and defoaming agent 0.5 mL / L. The fermentation feeding medium is: glucose 480 g / kg.

[0023] The determination method of L-serine is as follows: 0.2 g of L-serine fermentation liquor is taken in a 50 mL centrifuge tube, 35 g of aqueous phosphoric acid is added, and shaken uniformly, 0.2 ml of the diluent is taken therefrom, 0.6 ml of o-phthaldehyde derivatization reagent is added, and mixed uniformly, and liquid phase detection is performed within two minutes, the liquid phase conditions are: Agilent C18, mobile phase A:B = 9:1, column temperature 40℃, and detection wavelength: 338 nm. Among them, the mobile phase A: 50 mmol / L sodium acetate: triethylamine: tetrahydrofuran = 950:0.14:5 (V / V); the mobile phase B: 160 mmol / L sodium acetate: methanol: acetonitrile = 200:400:400 (V / V).

[0024] L-serine conversion rate (%) = L-serine yield ÷ consumed pure glucose mass; L-serine yield = L-serine titer in fermentation broth x fermentation broth volume.

[0025] Example 1: The seed solution was prepared as follows: the frozen L-serine-producing E. coli engineering bacteria ALE8-8 (DE3) glycerol bacteria were inoculated into the seed culture medium at a inoculation amount of 0.2%, and cultured at 37°C and 260 rpm for 6-7 h to prepare the seed solution; The seed solution was inoculated into the fermentation medium at a volume ratio of 20% for culture, and the total fermentation system was 2.5 L. The culture temperature was 37°C, the pH was 7.0±0.02, the air volume was 2.5 L / min, the tank pressure was 0.03 MPa, the rotation speed was 300-1200 rpm, and the dissolved oxygen was 15%. The rotation speed was adjusted according to the dissolved oxygen range.

[0026] At the 4th hour of fermentation, the glucose was supplemented according to Y=1.58X+7.6, wherein Y represents the unit of time and unit of volume of the feeding amount (g / (L·h)), and X represents the feeding time (h). During the fermentation process, the ORP was displayed and monitored through the pH / ORP electrode. At the 11th hour of fermentation (7th hour of feeding), the current ORP reached the range of-120 mV to-130 mV, and the feeding speed (18.66 g / (L·h)) at this time point was used for constant-speed feeding. When the titer reached 90 g / L, the sugar feeding was stopped, and the other fermentation conditions were continuously maintained. When the residual sugar was consumed and the dissolved oxygen rose to more than 60%, the fermentation was stopped, and at this time, the total fermentation time was 75 h. The ORP during the fermentation process was directly displayed through the pH / ORP electrode. The L-serine content during the fermentation process was detected by high-performance liquid chromatography, and the conversion rate of glucose converted into L-serine was calculated.

[0027] Example 2: The seed solution prepared in Example 1 was used for fermentation culture. The fermentation medium and culture conditions were the same as in Example 1, except that when the fermentation was 15 h (11 h of feeding), the current ORP reached the range of-130 mV to-140 mV, and the feeding speed (24.98 g / (L·h)) at this time point was used for constant-speed feeding. When the titer reached 90 g / L, the sugar feeding was stopped, and the other fermentation conditions were continuously maintained. When the residual sugar was consumed and the dissolved oxygen rose to more than 60%, the fermentation was stopped, and at this time, the total fermentation time was 72 h. The ORP during the fermentation process was directly displayed through the pH / ORP electrode. The L-serine content during the fermentation process was detected by high-performance liquid chromatography, and the conversion rate of glucose converted into L-serine was calculated.

[0028] Example 3: The seed liquid prepared in Example 1 was used for fermentation culture. The fermentation medium and culture conditions were the same as in Example 1, except that the current ORP reached the range of -140 mV to -150 mV at 17 h (13 h of feeding) of fermentation, and the feeding speed (28.14 g / (L·h)) at this time point was used for constant feeding. When the titer reached 90 g / L, the sugar feeding was stopped, and other fermentation conditions were continued to be maintained. When the residual sugar was consumed and the dissolved oxygen rose to more than 60%, the fermentation was stopped, and the total fermentation time was 78 h. The ORP during the fermentation process was directly displayed through the pH / ORP electrode. The L-serine content during the fermentation process was detected by high performance liquid chromatography, and the conversion rate of glucose to L-serine was calculated.

[0029] Example 4: The seed liquid prepared in Example 1 was used for fermentation culture. The fermentation medium and culture conditions were the same as in Example 1, except that the initial fermentation volume was 8 L, and the feeding was performed according to Y=1.6X+8.0 starting at 4 h of fermentation; wherein Y represents the feeding amount per unit time per unit volume (g / (L·h)), and X represents the feeding time (h). The ORP during the fermentation process was displayed and monitored through the pH / ORP electrode. The current ORP reached the range of -130 mV to -140 mV at 15 h (11 h of feeding time) of fermentation, and the sugar feeding speed at this time point, 25.60 g / (L·h), was used for constant feeding. When the titer reached 90 g / L, the sugar feeding was stopped, and other fermentation conditions were continued to be maintained. When the residual sugar was consumed and the dissolved oxygen rose to more than 60%, the fermentation was stopped, and the total fermentation time was 72 h. The L-serine content during the fermentation process was detected by high performance liquid chromatography, and the conversion rate of glucose to L-serine was calculated.

[0030] The results (Table 1) showed that in this process, the L-serine titer and conversion rate were the highest in the range of -130 mV to -140 mV of ORP, and -130 mV to -140 mV of ORP was determined as the optimal ORP range during the fermentation process.

[0031] Comparative Example 1: The specific method was the same as in Example 2, except that the ORP control method was changed from sugar feeding regulation to air volume regulation. After 15 h (11 h of feeding time) of fermentation, when the ORP reached the range of -130 mV to -140 mV, the sugar feeding formula continued to run, and the ORP was maintained in the range by adjusting the air volume or speed.

[0032] Table 1 lists the cycle, titer, and conversion rate of Examples 1-4 and the comparative example.

[0033] Table 1 Cycle, titer, and conversion rate of examples and comparative examples

[0034] Note: "Sugar stopping time" refers to the corresponding fermentation time when sugar feeding is stopped.

[0035] The results show that: although the ORP in the comparative examples can also be controlled by air volume or rotation speed, the sugar stopping time, tank release titer, and tank release conversion rate are lower than the feeding control scheme, and the optimal method for regulating ORP in the process is feeding regulation.

[0036] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for dynamically regulating ORP to increase L-serine fermentation yield, characterized in that, Using L-serine-producing *Escherichia coli* as the fermentation strain, carbon source feeding parameters were controlled in stages. The staged feeding began at the 4th hour of fermentation, with the feeding rate controlled according to Y=1.58X+7.6 until the current ORP reached the minimum ORP range, at which point the feeding rate was kept constant. Here, Y represents the amount of feed per unit volume per unit time, and X represents the feeding duration. The minimum ORP range included -120 mV to -130 mV, -130 mV to -140 mV, and -140 mV to -150 mV.

2. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at a rate of Y=1.58X+7.6 from the 4th to the 13th hour until the current ORP reaches -120 mV to -130 mV, and then feeding at a constant rate until fermentation is complete.

3. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at a rate of Y=1.58X+7.6 from the 4th to the 15th hour until the current ORP reaches -130 mV to -140 mV, and then feeding at a constant rate until fermentation is complete.

4. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at a rate of Y=1.58X+7.6 from the 4th to the 17th hour until the current ORP reaches a range of -140 mV to -150 mV, and then feeding at a constant rate until fermentation is complete.

5. The method according to any one of claims 1 to 4, characterized in that, The carbon source includes, but is not limited to, glucose.

6. The method according to any one of claims 1 to 5, characterized in that, The L-serine-producing Escherichia coli include Escherichia coli ALE8-8, SER_151, SER_160, SER_166, SER_164, or SER_162.

7. A method for producing L-serine by fermentation, characterized in that, Fermentation was carried out using Escherichia coli as the fermenting microorganism under the following conditions: (1) Ferment at 35~40℃ for the first 3 hours of fermentation; (2) Feeding is carried out from the 4th to the 17th hour of fermentation. The feeding rate is based on Y=1.58X+7.6; where Y represents the amount of feed per unit volume of fermentation liquid per unit time, and X represents the feeding time (h); when the current ORP reaches the control range, the feeding rate at that moment is kept constant until the end of fermentation.

8. The method according to claim 7, characterized in that, After fermentation for 3 hours, glucose is added as feed.

9. The method according to claim 7 or 8, characterized in that, The Escherichia coli includes, but is not limited to, Escherichia coli ALE8-8(DE3).

10. The use of the method according to any one of claims 1 to 9 in the preparation of products containing L-serine.

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