Fermentation method for efficiently producing fucose by using genetically engineered bacteria

Abstract

The invention provides a fermentation method for efficiently producing fucose by using genetically engineered bacteria, the preservation number of the genetically engineered bacteria is CCTCC NO: M 20252837, and the fermentation method comprises the following steps: S1, preparing a seed culture medium, a fermentation culture medium and a fed-batch culture medium; wherein the fed-batch culture medium contains glucose and glycerol in a mass ratio of (1-2): 1; s2, inoculating the genetically engineered bacteria into a seed culture medium, and culturing to obtain a seed solution; s3, inoculating the seed solution into a fermentation culture medium, and controlling the temperature to be 35-37 DEG C, the pH to be 7.0 and the dissolved oxygen to be greater than or equal to 30%; when dissolved oxygen is increased to 60% or above, culture conditions are controlled in four stages for culture; and when the OD600 value is reduced, putting into a tank for centrifugation, and collecting supernate to finish fermentation. The fucose is produced by a fermentation process designed for specific engineering bacteria, the product yield is high, the purity is high, and the method is suitable for industrial production and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a fermentation method for the efficient production of fucose using genetically engineered bacteria. Background Technology

[0002] Fucose is a naturally occurring six-carbon deoxymonosaccharide widely distributed in marine algae (such as brown algae), higher plants, mammalian cell surface glycoproteins / glycolipids, and breast milk. It is named after its initial extraction from the cell walls of brown algae. Its unique chemical structure (the C6 hydroxyl group is replaced by hydrogen) endows it with special physiological activities, and it is currently widely used in food, medicine, cosmetics, and animal feed. However, traditional production methods are inefficient and costly, limiting its large-scale industrial application. In recent years, with in-depth research into the physiological functions of L-fucose, its application value in areas such as gut health, lowering blood lipids, anti-cancer effects, and skin moisturizing has been gradually discovered. Developing efficient and low-cost production technologies has become a key need in this field.

[0003] The production methods of fucose have been continuously iterating with technological advancements. The core objectives are to achieve high purity, low cost, and large-scale production, while also considering the sustainability of raw materials and the environmental friendliness of the process. Currently, commercially available or research-stage production methods are mainly divided into three categories: natural extraction, microbial fermentation, and chemical synthesis. These three methods differ significantly in terms of raw materials, processes, product purity, and application scenarios. The mainstream methods currently are extraction from brown algae and microbial fermentation. Natural extraction methods suffer from seasonality affecting raw material availability, low efficiency, high production costs, and the generation of large amounts of waste acid that pollute the environment. Chemical synthesis methods involve complex reaction steps and low yields, resulting in high costs, numerous byproducts, and difficulties in purification, while also having a significant environmental impact. In contrast, bio-fermentation offers controllable production conditions, stable yields, high production efficiency, easy industrial scale-up, high product purity, and is environmentally friendly.

[0004] Therefore, it is essential to develop high-yield fucose strains and provide supporting fermentation processes to further achieve high purity, low cost, and large-scale production, while also taking into account the sustainability of raw materials and the environmental friendliness of the process. Summary of the Invention

[0005] In view of this, the present invention provides accession number CCTCC NO: M 20252837, accession date: December 10, 2025, depositary institution: China Center for Type Culture Collection (CCTCC), classification and naming: Escherichia coli The Fuc-WT22 genetically engineered bacteria fermentation method is used to efficiently obtain fucose.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A fermentation method for efficiently producing fucose using genetically engineered bacteria, wherein the genetically engineered bacteria has the accession number CCTCC NO: M 20252837, and the fermentation method includes the following steps: S1. Prepare seed culture medium, fermentation culture medium, and fed culture medium; wherein, the fed culture medium contains glucose and glycerol in a mass ratio of (1~2):1; S2. Inoculate the genetically engineered bacteria into the seed culture medium and culture them to obtain the seed liquid; S3. Inoculate the seed culture into the fermentation medium, controlling the temperature at 35-37℃, pH 7.0, and dissolved oxygen ≥30%; once the dissolved oxygen rises to above 60%, continue culturing under controlled conditions in four stages; until OD... 600 When the value decreases, centrifuge the mixture in the lower tank, collect the supernatant, and the fermentation is complete; The first stage includes: rotation speed of 200-400 rpm, temperature of 35-37℃, feeding rate of 2.5-3.5 g / L, dissolved oxygen content of 45-55%, and incubation time of 8-9 hours. Second stage: Rotation speed 500~600rpm, temperature 23~25℃, feeding rate 4.0~5.0g / L, dissolved oxygen 30~35%, incubation time 4~6h; The third stage: rotation speed 500~700rpm, temperature 28~30℃, feeding rate 8.0~9.0g / L, dissolved oxygen 30~35%, and incubation time 30~35h; Fourth stage: Rotation speed 500~700rpm, temperature 28~30℃, feeding rate 1.5~2.5g / L, dissolved oxygen 30~35%, incubation time 20~24h; During the second stage, lactose was added at a rate of 1-3 g / L to induce lactation for 4-6 hours.

[0007] Furthermore, the seed culture medium in step S1 consists of: 5 g / L yeast extract, 10 g / L soybean peptone, and 10 g / L NaCl. Fermentation medium composition: yeast extract powder (5-10) g / L, diammonium hydrogen phosphate (3-4) g / L, anhydrous potassium dihydrogen phosphate (10-13.5) g / L, magnesium sulfate heptahydrate (1-1.4) g / L, trace element solution 1 ml / L, calcium chloride (1-2) g / L, vitamin B (10-13) mg / L, citric acid (1-2) g / L, glucose (5-10) g / L, glycerol (10-15) g / L, ammonium sulfate (5-8) g / L, PPE defoamer; The composition of the supplemental culture medium is: glucose 250~500g / L, magnesium sulfate 20g / L, glycerol 250g / L, and trace element solution 1ml / L. The micronutrient solutions in the fermentation medium and fed-batch medium consist of: NaCl 5 g / L, MnCl2·4H2O 4 g / L, ZnSO4·7H2O 1 g / L, sodium molybdate dihydrate 0.5 g / L, FeCl3·6H2O 4.75 g / L, boric acid 0.575 g / L, CuSO4·5H2O 0.4 g / L, and concentrated sulfuric acid 2.038 ml / L.

[0008] In some specific embodiments, the preferred composition of the supplemental culture medium is: 250 g / L glucose, 20 g / L magnesium sulfate, 250 g / L glycerol, and 1 ml / L trace element solution. Furthermore, the seed solution is obtained in step S2 as follows: The genetically engineered bacteria with accession number CCTCC NO: M 20252837 were inoculated into seed culture medium at an inoculum rate of 0.3–0.6% (w / v) and cultured at 37°C with shaking until OD reached. 600 When the pH reaches 0.8-1.2, the primary seed culture medium is obtained; then, the primary seed culture medium is inoculated into the seed culture medium at an inoculation rate of 0.08-0.12% (v / v), and cultured at a constant temperature of 37℃ with shaking until the OD reaches 0.8-1.2. 600 When the value reaches 0.8-1.2, the seed solution is obtained.

[0009] In some specific embodiments, preferably, the inoculum amount of genetically engineered bacteria is 0.5% (w / v) when preparing the primary seed culture medium; and the inoculum amount of the primary seed culture medium is 0.1% (v / v) when preparing the seed solution.

[0010] Furthermore, in step S3, the seed culture inoculation amount is 1~5% (v / v).

[0011] In some specific embodiments, preferably, the first stage of step S3 is characterized by: rotation speed of 300 rpm, temperature of 37°C, feeding rate of 3.0 g / L, dissolved oxygen content of 50%, and incubation time of 8 h. Second stage: Rotation speed 550 rpm, temperature 25℃, feeding rate 4.5 g / L, dissolved oxygen 30%, incubation time 5 h; Third stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 8.5 g / L, dissolved oxygen 30%, incubation time 32 h; Fourth stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 2.0 g / L, dissolved oxygen 30%, incubation time 22 h.

[0012] In some specific embodiments, preferably, lactose is added at a feeding rate of 2 g / L for 5 hours during the second stage.

[0013] Furthermore, the centrifugation conditions in step S3 are: rotation speed 6000~8000 rpm, time 25~35 min.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The genetically engineered bacterial substrate strain used in this invention is Escherichia coli W3110, which is a food safety production strain. The substrate used for fermentation is lactose and glucose. During the fermentation process, lactose is added at a feeding rate of 1-3 g / L. In the subsequent fucose synthesis reaction, 2'-fucosylated lactose can be synthesized from lactose and then decomposed into fucose and lactose, realizing substrate recycling. This solves the problem of high concentration of lactose inhibiting cell growth, thereby achieving high-density cell fermentation, high-yield fucose production, and reducing production costs.

[0015] (2) In the fucose fermentation production process, by optimizing the culture medium composition, the carbon source is glucose and glycerol co-fermentation, and by adjusting the ratio and feeding rate of the feeding carbon source (glucose and glycerol), high-density fermentation (5L fermenter fermentation OD) is achieved. 600 The highest value is 210, the yield is 80g / L, and the purity is as high as 93.5%.

[0016] (3) The fermentation process adopts staged precise control, which significantly prolongs the growth cycle of bacteria, thereby increasing the accumulation of products by 237% (from 25.4 g / L to 85.8 g / L). Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the metabolic pathway of the genetically engineered bacteria in Example 1 of the present invention.

[0018] Figure 2 This is a graph showing the HPLC detection results of the fermentation products in Example 1 of the present invention.

[0019] Figure 3 This is a TLC detection result of the fermentation product in Example 1 of the present invention.

[0020] Figure 4 This is a growth curve diagram of the strains under five feeding schemes in Example 1 of the present invention. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0022] Example 1 This embodiment provides a fermentation method for the efficient production of fucose using genetically engineered bacteria. The genetically engineered bacteria have the preservation number CCTCC NO: M 20252837 (see the metabolic pathway diagram). Figure 1 The specific fermentation process is as follows: 1. Culture medium preparation Seed culture medium composition: 5 g / L yeast extract, 10 g / L soybean peptone, 10 g / L NaCl.

[0023] Fermentation medium composition: yeast extract powder FM885 5g / L, diammonium hydrogen phosphate 4g / L, anhydrous potassium dihydrogen phosphate 13.5g / L, magnesium sulfate heptahydrate 1.4g / L, trace element solution 1ml / L, calcium chloride 2g / L, vitamin B 12.2mg / L, citric acid 1g / L, glucose 5g / L, glycerol 10g / L, ammonium sulfate 5g / L, PPE defoamer.

[0024] The supplemental culture medium consists of: 250 g / L glucose, 20 g / L magnesium sulfate, 250 g / L glycerol, and 1 ml / L trace element solution.

[0025] The micronutrient solution in the fermentation culture and fed culture medium consisted of the following components: NaCl 5 g / L, MnCl2·4H2O 4 g / L, ZnSO4·7H2O 1 g / L, sodium molybdate dihydrate 0.5 g / L, FeCl3·6H2O 4.75 g / L, boric acid 0.575 g / L, CuSO4·5H2O 0.4 g / L, and concentrated sulfuric acid 2.038 ml / L.

[0026] 2. Seed liquid preparation First, sterilize the seed culture medium in a shake flask and cool it to room temperature. Then, inoculate the strain into the seed culture medium at an inoculation rate of 0.5% (w / v) in a clean bench and place it in a shaker at a constant temperature of 37°C for OD culture. 600 Once the pH reaches 1.0, the primary seed culture medium is obtained. The seed culture medium is then sterilized in a shake flask and cooled to room temperature. In a clean bench, the primary seed culture medium is inoculated into the seed culture medium at an inoculation rate of 0.1% (v / v), and the flask is placed in a shaker and incubated at 37°C with constant temperature shaking until the OD reaches 1.0. 600 When the value reaches 1.0, the seed solution is obtained.

[0027] 3. Fermentation process In a 5L fermenter, fermentation medium was added according to the fermentation medium formula and sterilized at 121℃ for 20 minutes. Seed culture was inoculated into the fermenter at a rate of 3% (v / v). The temperature was controlled at 37℃, pH 7.0, and dissolved oxygen ≥30%. Once the dissolved oxygen rapidly rose above 60%, the fermentation process was divided into four stages: logarithmic growth, protein expression, product synthesis, and substrate sugar consumption. By controlling the dissolved oxygen, rotation speed, feed rate, and temperature at different stages, high-yield fucose fermentation could be successfully achieved. The specific parameters for the four stages are as follows: Phase 1: Rotation speed 300 rpm, temperature 37℃, feeding rate 3.0 g / L, dissolved oxygen 50%, incubation time 8 h; Second stage: Rotation speed 550 rpm, temperature 25℃, feeding rate 4.5 g / L, dissolved oxygen 30%, incubation time 5 h; Third stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 8.5 g / L, dissolved oxygen 30%, incubation time 32 h; Fourth stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 2.0 g / L, dissolved oxygen 30%, incubation time 22 h.

[0028] During the second stage, lactose was added at a rate of 1 g / L to induce lactation for 5 hours.

[0029] OD was measured after the above fermentation stages were completed. 600 Value, when OD 600 When the flow rate drops, centrifuge in the tank (7000 rpm, 30 min), collect the supernatant, and fermentation is complete. To understand the product acquisition and test the supernatant, the test results are shown below. Figure 2 , 3 .

[0030] Furthermore, during the fermentation process, this application also investigated the following conditions while keeping other conditions unchanged: (1) Regarding the mass ratio of glucose to glycerol in the fed culture medium; (2) Four-stage control of dissolved oxygen, rotation speed, feeding speed, and temperature parameters; (3) Amount of lactose added for induction.

[0031] The specific research results are shown in Tables 1-3.

[0032] Table 1. Fermentation results of different glucose-glycerol ratios in fed culture medium.

[0033] The fermentation volume in Table 1 varies depending on the carbon source because the OD is continuously adjusted during fermentation. 600Measurements were performed to determine the optimal fermentation volume for different carbon sources (see specific growth curves). Figure 4 As shown in Table 1, when the mass ratio of glucose to glycerol is (1~2):1, all indicators are relatively high, with the optimal mass ratio being 1:1. However, using glucose alone or mixing glucose and glycerol will severely affect the fermentation results if the glucose content is too low.

[0034] Table 2. Fermentation results under different parameter control at four stages of the fermentation process.

[0035] As shown in Table 2, dissolved oxygen, rotation speed, feeding rate, and temperature have a significant impact on the fermentation results during the four stages of fermentation: logarithmic growth, protein expression, product synthesis, and substrate sugar consumption. Stage-specific control is necessary to successfully achieve high-yield fucose fermentation.

[0036] Table 3. Fermentation results with different lactose induction amounts

[0037] Table 3 shows that the amount of lactose added has a significant impact on the fermentation results, reaching a maximum OD value of [missing information]. 600 The values ​​and corresponding fermentation times vary. A addition rate of 1-3 g / L yields suitable results, with an optimal overall result achieved at a rate of 2 g / L; however, excessively high lactose levels can inhibit the final product formation.

[0038] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation method for efficiently producing fucose using genetically engineered bacteria, characterized in that, The genetically engineered bacteria has the preservation number CCTCC NO: M 20252837, and the fermentation method includes the following steps: S1. Prepare seed culture medium, fermentation culture medium, and fed culture medium; wherein, the fed culture medium contains glucose and glycerol in a mass ratio of (1~2):1; S2. Inoculate the genetically engineered bacteria into the seed culture medium and culture them to obtain the seed liquid; S3. Inoculate the seed culture into the fermentation medium, controlling the temperature at 35-37℃, pH 7.0, and dissolved oxygen ≥30%; once the dissolved oxygen rises to above 60%, continue culturing under controlled conditions in four stages; until OD... 600 When the value decreases, centrifuge the mixture in the lower tank, collect the supernatant, and the fermentation is complete; The first stage includes: rotation speed of 200-400 rpm, temperature of 35-37℃, feeding rate of 2.5-3.5 g / L, dissolved oxygen content of 45-55%, and incubation time of 8-9 hours. Second stage: Rotation speed 500~600rpm, temperature 23~25℃, feeding rate 4.0~5.0g / L, dissolved oxygen 30~35%, incubation time 4~6h; The third stage: rotation speed 500~700rpm, temperature 28~30℃, feeding rate 8.0~9.0g / L, dissolved oxygen 30~35%, and incubation time 30~35h; Fourth stage: Rotation speed 500~700rpm, temperature 28~30℃, feeding rate 1.5~2.5g / L, dissolved oxygen 30~35%, incubation time 20~24h; During the second stage, lactose was added at a rate of 1-3 g / L to induce lactation for 4-6 hours.

2. The fermentation method according to claim 1, characterized in that, The seed culture medium in step S1 consists of: 5 g / L yeast extract, 10 g / L soybean peptone, and 10 g / L NaCl. Fermentation medium composition: yeast extract powder (5-10) g / L, diammonium hydrogen phosphate (3-4) g / L, anhydrous potassium dihydrogen phosphate (10-13.5) g / L, magnesium sulfate heptahydrate (1-1.4) g / L, trace element solution 1 ml / L, calcium chloride (1-2) g / L, vitamin B (10-13) mg / L, citric acid (1-2) g / L, glucose (5-10) g / L, glycerol (10-15) g / L, ammonium sulfate (5-8) g / L, PPE defoamer; The composition of the supplemental culture medium is: glucose 250~500g / L, magnesium sulfate 20g / L, glycerol 250g / L, and trace element solution 1ml / L. The micronutrient solutions in the fermentation medium and fed-batch medium consist of: NaCl 5 g / L, MnCl2·4H2O 4 g / L, ZnSO4·7H2O 1 g / L, sodium molybdate dihydrate 0.5 g / L, FeCl3·6H2O 4.75 g / L, boric acid 0.575 g / L, CuSO4·5H2O 0.4 g / L, and concentrated sulfuric acid 2.038 ml / L.

3. The fermentation method according to claim 2, characterized in that, The supplemental culture medium consists of: 250 g / L glucose, 20 g / L magnesium sulfate, 250 g / L glycerol, and 1 ml / L trace element solution.

4. The fermentation method according to claim 1, characterized in that, The seed solution was obtained in step S2 as follows: The genetically engineered bacteria with accession number CCTCC NO: M 20252837 were inoculated into seed culture medium at an inoculum rate of 0.3–0.6% (w / v) and cultured at 37°C with shaking until OD reached. 600 When the pH reaches 0.8-1.2, the primary seed culture medium is obtained; then, the primary seed culture medium is inoculated into the seed culture medium at an inoculation rate of 0.08-0.12% (v / v), and cultured at a constant temperature of 37℃ with shaking until the OD reaches 0.8-1.

2. 600 When the value reaches 0.8-1.2, the seed solution is obtained.

5. The fermentation method according to claim 1, characterized in that, When preparing the primary seed culture medium, the inoculum amount of genetically engineered bacteria is 0.5% (w / v); when preparing the seed solution, the inoculum amount of the primary seed culture medium is 0.1% (v / v).

6. The fermentation method according to claim 1, characterized in that, In step S3, the seed culture inoculation amount is 1~5% (v / v).

7. The fermentation method according to claim 1, characterized in that, Step S3, first stage: rotation speed 300 rpm, temperature 37℃, feeding rate 3.0 g / L, dissolved oxygen 50%, incubation time 8 h; Second stage: Rotation speed 550 rpm, temperature 25℃, feeding rate 4.5 g / L, dissolved oxygen 30%, incubation time 5 h; Third stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 8.5 g / L, dissolved oxygen 30%, incubation time 32 h; Fourth stage: Rotation speed 600 rpm, temperature 30℃, feeding rate 2.0 g / L, dissolved oxygen 30%, incubation time 22 h.

8. The fermentation method according to claim 7, characterized in that, During the second stage, lactose was added at a feeding rate of 2g / L for 5 hours to induce induction.

9. The fermentation method according to claim 1, characterized in that, Centrifugation conditions in step S3: rotation speed 6000~8000 rpm, time 25~35 min.

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