Method for improving fermentation yield of tryptophan synthetase

Abstract

The invention discloses a method for improving the fermentation yield of tryptophan synthetase, and belongs to the technical field of fermentation engineering. According to the method disclosed by the invention, by optimizing linear material supplementing parameters and combining linear material supplementing and constant-speed material supplementing, the fermentation cost and the process difficulty are reduced, and the yield of product synthesis is improved. The method provided by the invention can reduce the manual monitoring of process parameters, save the labor cost and improve the automation level, and is suitable for industrial production of tryptophan synthetase.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for improving the fermentation yield of tryptophan synthetase, and belongs to the technical field of fermentation engineering. BACKGROUND

[0002] Cystine is used as a raw material for producing detoxicants and expectorants in medicine; as a dairy additive and a bread quickening agent in food industry, which can produce various special flavors after heating with sugar in food and prevent oil oxidation, and has a wide range of uses in food processing; and as a raw material for producing cold perm in cosmetic industry. Tryptophan synthetase is a key enzyme for synthesizing cystine, and improving the fermentation yield of tryptophan synthetase is an effective way to improve the yield of cystine.

[0003] At present, there is no efficient and convenient method for improving the yield of tryptophan synthetase for industrial production. The present application is based on the fermentation characteristics of Escherichia coli engineering bacteria capable of producing tryptophan synthetase, and the glucose content in the fermentation broth is monitored during the cultivation process to control the glucose concentration in the fermentation system to be maintained at 0.2-0.7 g / L, the feeding speed is calculated, and the feeding equation is fed back to reduce the frequency of human intervention during the fermentation feeding process. SUMMARY

[0004] The present application provides a method for preparing tryptophan synthetase and / or improving the fermentation yield of tryptophan synthetase, which comprises fermenting Escherichia coli in a culture medium, starting feeding when the dissolved oxygen rises to 60% during fermentation, linear feeding first, and then adding an inducer and reducing the temperature after the OD 600 value reaches a certain value, and constant-speed feeding.

[0005] In one embodiment, the linear feeding is performed according to the relationship Y=aX+b, wherein Y represents the feeding amount of the fermentation broth per unit volume per unit time (g / (L·h)), and X represents the feeding time.

[0006] After the OD 600 value is greater than or equal to 40, an inducer is added and the temperature is reduced, and constant-speed feeding is started.

[0007] In one embodiment, a is 0.5-0.6, preferably 0.53-0.58, and b is 9-12; wherein the unit of a is g / (L·h²), and the unit of b is g / (L·h).

[0008] In one embodiment, the linear feeding is performed according to the relationship Y=0.55X+9.

[0009] In one embodiment, the constant-speed feeding is to feed glucose at a flow rate of 9-12 g / (L·h).

[0010] In one embodiment, the method involves fermentation using Escherichia coli.

[0011] In one embodiment, the Escherichia coli includes, but is not limited to, ALE8-8 (DE3 strain) disclosed in patent CN107406864B, and strains SER151, SER_160, SER_166, SER_162, and SER_164 disclosed in CN117625508A.

[0012] In one embodiment, the fermentation medium contains 8-12 g / L glucose, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 0.5-1.5 g / L ammonium sulfate, 1-3 g / L yeast extract, 0.05-0.15 g / L ferrous sulfate heptahydrate, and 0.5-1.5 g / L citric acid.

[0013] In one embodiment, the supplement is glucose.

[0014] In one embodiment, the concentration of the glucose solution used for feeding is 300-800 g / kg, for example 300 g / kg, 350 g / kg, 400 g / kg, 450 g / kg, 460 g / kg, 470 g / kg, 480 g / kg, 490 g / kg, or 500 g / kg, 550 g / kg, 600 g / kg, 650 g / kg, 700 g / kg, 750 g / kg, or 800 g / kg.

[0015] The method described in this invention does not have any particular limitation on the fermentation volume and can be any fermentation volume used in current conventional production or research.

[0016] In one embodiment, the fermentation volume is 1.5~2.5 L, the aeration rate during fermentation is 2~5 L / min, and the rotation speed is 300-1200 rpm. The dissolved oxygen is controlled at 20%~40% by adjusting the aeration or rotation speed.

[0017] In one embodiment, the temperature is controlled at 35℃~38℃ during linear feeding; the temperature is controlled at 25℃~30℃, dissolved oxygen at 20%~40%, and the pH of the fermentation broth is controlled at 6.5~7.5 during constant-rate feeding.

[0018] In one embodiment, the constant rate feeding means controlling the glucose feeding rate to be 9 ± 0.2 g / (L·h), i.e. 0.88~0.92 g / (L·h).

[0019] In one embodiment, the dissolved oxygen is controlled by adjusting the aeration rate or the stirring speed.

[0020] In one embodiment, the pH is controlled by adding ammonia.

[0021] In one embodiment, the fermentation cycle is 20-30 hours, and optionally, the fermentation cycle is 22 hours, 24 hours, 26 hours, 28 hours or 30 hours.

[0022] Beneficial effects: (1) This invention proposes a feeding method that is more in line with the fermentation characteristics, with the wet weight of the fermentation broth reaching 160-180 g / L (based on wet cell weight), and the yield of cystine, a product synthesized by 4 h catalysis, is increased to over 90%, taking into account both biomass and enzyme activity, and achieving excellent catalytic effect.

[0023] (2) This invention combines linear feeding with constant-rate feeding, which reduces fermentation costs and process difficulty, and increases the synthesis of the target product, making it suitable for industrial production of tryptophan synthase. Detailed Implementation

[0024] Seed culture medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L. Sterilize at 121℃ for 20 min, cool before use.

[0025] Fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium sulfate 1 g / L, yeast extract 2 g / L, ferrous sulfate heptahydrate 0.1 g / L, citric acid 1 g / L. Sterilize at 121℃ for 30 min, cool before use. Feeding medium: glucose aqueous solution with a concentration of 480 g / kg.

[0026] Method for determining the catalytic activity of tryptophan synthase per unit volume of fermentation broth: The catalytic activity of tryptophan synthase per unit volume of fermentation broth was calculated based on the yield of L-serine to cysteine. The specific determination method is as follows: (1) Take 50 mL of fermentation broth, centrifuge at 12000 rpm for 5 min to prepare wet cells for later use; (2) Weigh 100 g of 8% serine solution (8 g of pure serine) into a 250 mL Erlenmeyer flask with baffle. (3) Weigh 1.5 times the molar amount of pure serine in NaHS into the solution; (4) Add all the wet bacterial cells obtained in step (1) to the solution; (5) Add 0.05% of the wet bacterial mass of pyridoxal phosphate (if the amount added is less than 0.01 g, weigh 0.01 g). (6) The reaction system was reacted at 37℃ and 220rpm for 6 h; (7) During the reaction, samples were taken every 2 hours, and the contents of the substrate serine and the product cystine were determined by HPLC and the yield was calculated. HPLC conditions: Agilent C18, column temperature 35℃, detection wavelength 192 nm, mobile phase 5 mmol / L sodium dodecyl sulfate: acetonitrile = 80:20.

[0027] Yield calculation: Yield (%) = (molar amount of cystine after enzymatic reaction × 2) ÷ molar amount of serine before enzymatic reaction × 100%.

[0028] Example 1 (1) Preparation of strains: The strain ALE8-8(DE3) disclosed in CN107406864B was inoculated into seed culture medium at an inoculum rate of 0.15% and cultured at 37°C and 220 rpm for 6.5 h in a shaker to obtain OD. 600 Seed liquid with a concentration of ≥ 3.

[0029] (2) Fermentation: The seed liquid prepared in step (1) was inoculated into the cooled fermentation medium at a volume ratio of 2%-5%, and fermented by stirring and ventilation. The culture temperature was 37℃, the induction temperature was 28℃, the pH was controlled at 6.8-7.2, the dissolved oxygen was controlled at 20%~30%, and the rotation speed was adjusted according to the dissolved oxygen range. Feeding begins when dissolved oxygen levels rise to 60%. For example, with a fermentation volume of 2 L, the feeding method is as follows: ① Linear feeding stage: The feeding start time is taken as feeding time 0. Starting from feeding time 0, the feeding equation is set according to the form Y=aX+b, where a=0.55, which represents the slope of the increase in feeding amount; b=9, which represents the feeding rate at the beginning of feeding; Y represents the feeding amount per unit volume of fermentation broth per unit time (g / (L·h)); and X represents the feeding time (h). ② Constant-rate feeding stage: waiting for the bacterial cell OD 600 After reaching ≥40℃, the temperature was lowered to the induction temperature of 28℃, and then IPTG with a final concentration of 0.5 mM was added. The feeding rate was set to a constant rate of 9 g / (L·h). After a cumulative fermentation time of 28 h, the fermentation was completed.

[0030] Example 2: Fermentation culture was carried out according to the method in Example 1. The difference was that the values ​​of a and b were different, with a being 0.53 and b being 10.

[0031] Example 3: Fermentation culture was carried out according to the method in Example 1. The difference lies in the values ​​of a and b, where a is 0.57 and b is 12.

[0032] Comparative Example 1: Fermentation culture was carried out according to the method in Example 1. The difference was that the value of a was different, and a was 0.4.

[0033] Comparative Example 2: Fermentation culture was carried out according to the method in Example 1. The difference was that the value of a was different, and a was 0.7.

[0034] Comparative Example 3: Fermentation was carried out according to the method in Example 1. The difference was that the constant feeding rate after induction was adjusted to 15 g / (L·h).

[0035] Table 1 shows the wet weight and process yield after fermentation for Examples 1-3 and Comparative Examples 1-3. Fermentation using the methods of Examples 1-3 can result in a fermentation broth with high overall catalytic capacity. Although the method of Comparative Example 3 can achieve a high biomass, the rapid cell growth prevents tryptophan synthase from properly folding, thus limiting the enzyme activity of the fermentation broth.

[0036] Table 1 Wet weight and conversion rate of the examples and comparative examples

[0037] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing tryptophan synthase and / or increasing the fermentation yield of tryptophan synthase, wherein the method involves fermenting *Escherichia coli* in a culture medium, and starting feed feeding when the dissolved oxygen level rises to 60%; the feed feeding is initially linear, and fermentation continues until the OD level reaches 60%. 600 Once a certain value is reached, an inducing agent is added and the temperature is lowered to switch to constant-rate feeding.

2. The method according to claim 1, characterized in that, Fermentation is carried out using Escherichia coli, including but not limited to ALE8-8 (DE3 strain) in CN107406864B, and SER151, SER_160, SER_166, SER_162, and SER_164 involved in CN117625508A.

3. The method according to claim 1 or 2, characterized in that, The culture medium used for fermentation contains 8-12 g / L glucose, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 0.5-1.5 g / L ammonium sulfate, 1-3 g / L yeast extract, 0.05-0.15 g / L ferrous sulfate heptahydrate, and 0.5-1.5 g / L citric acid.

4. The method according to any one of claims 1 to 3, characterized in that, The linear feeding is performed according to the relationship Y=aX+b, where Y represents the amount of feed per unit volume of fermentation broth per unit time (g / (L·h)) and X represents the feeding time; wherein a is 0.5~0.6, preferably 0.53~0.58, b is 9~12, and the constant rate feeding is to feed glucose at a flow rate of 9~12 g / (L·h).

5. The method according to any one of claims 1 to 4, characterized in that, Fermentation to OD 600 After reaching ≥40, an inducing agent is added and constant-rate feeding begins; the inducing agent includes, but is not limited to, IPTG, with a concentration of 0.1 mM to 1 mM.

6. The method according to any one of claims 1 to 5, characterized in that, The supplement is glucose.

7. The method according to any one of claims 1 to 6, characterized in that, During linear feeding, the temperature is controlled at 35℃~38℃, and during constant-rate feeding, the temperature is controlled at 25℃~30℃, dissolved oxygen at 20%~40%, and the pH of the fermentation broth is controlled at 6.5~7.

5.

8. The method according to any one of claims 5 to 7, characterized in that, The constant rate feeding refers to controlling the glucose feeding rate to be 9 ± 0.2 g / (L·h).

9. The method according to any one of claims 1 to 8, characterized in that, The fermentation cycle is 20-30 hours.

10. Use of the method according to any one of claims 1 to 9 in the preparation of tryptophan synthase or products containing tryptophan synthase.

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