Fermentation conversion method of salidroside

Abstract

This invention discloses a fermentation conversion method for rhodioloside. The method includes: fermenting and culturing recombinant *Escherichia coli*, collecting the bacterial cells by centrifugation after fermentation, and adding the bacterial cells to a reaction solution using glucose and tyrosol as substrates for the biosynthesis of rhodioloside. This invention accumulates glucosidase during the fermentation stage and constructs an enzyme-catalyzed reaction system to convert glucose and tyrosol into rhodioloside. Through continuous optimization of the fermentation and conversion stage feed formulations, reaction conditions, and control strategies, the production of rhodioloside using this method can achieve high yields in a short time, with a maximum rhodioloside content of up to 9206 mg / L in the conversion reaction system and a tyrosol conversion rate exceeding 80%. This method for preparing rhodioloside is simple, easy to implement, low-cost, and energy-efficient, and can be used for large-scale industrial production of rhodioloside, offering significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and more specifically, to a fermentation conversion method for rhodioloside. Background Technology

[0002] Rhodioloside is a phenolic compound widely found in plants of the Rhodiola genus. It is one of the main active monomers in Rhodiola genus and has pharmacological activities such as anti-radiation, anti-oxidation, immune enhancement, and promotion of cancer cell apoptosis. It is widely used in the pharmaceutical, cosmetic, and food industries.

[0003] With in-depth research into the mechanism of action and the clarification of pharmacological effects of rhodioloside, its demand has further increased. Traditional natural plant sources cannot meet market demand due to limitations in growth environment, content, and separation and extraction processes. Chemical synthesis methods, on the other hand, have harsh reaction conditions, are prone to environmental pollution, and have high hazardous waste treatment costs, which are not conducive to industrial production.

[0004] The method of preparing rhodioloside through fermentation using genetically modified microorganisms has mild reaction conditions, low cost, and a wide range of raw material sources, making it a major trend in the industrial production of rhodioloside. However, currently available journal and patent literature mostly uses recombinant Saccharomyces cerevisiae, Monascus purpureus, and other fungi for deep fermentation, which has problems such as long fermentation cycles and low biosynthetic yields. Summary of the Invention

[0005] The purpose of this invention is to provide a fermentation and conversion method for rhodioloside.

[0006] To achieve the objective of this invention, a fermentation conversion method for rhodioloside is provided, comprising the following steps:

[0007] A. Fermentation culture of recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs, centrifugation to collect the bacterial cells after fermentation;

[0008] B. Add the bacterial cells obtained in step A to a reaction solution with glucose and tyrosol as substrates to carry out the biosynthesis of rhodioloside.

[0009] For recombinant Escherichia coli, please refer to paragraphs 119-147 of the instruction manual in US20190264221A1.

[0010] The aforementioned method, step A includes the following sub-steps:

[0011] A1. Preparation of recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs seed culture;

[0012] A2. Fermentation culture is carried out using a supplementary method.

[0013] In step A2, when the fermentation broth OD 600nm Value reaches 2-5 (preferred OD) 600nm When the value reaches 3), start continuously feeding the culture medium until the fermentation broth OD 600nm Fermentation should be stopped when the value reaches 40 and the bacterial cell content detected by centrifugation reaches 10-12% (volume percentage).

[0014] Seed culture medium used: 10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0;

[0015] Fermentation medium used: glycerol 5g / L, yeast extract 5g / L, soybean peptone 5g / L, dipotassium hydrogen phosphate 18g / L, potassium dihydrogen phosphate 6.8g / L, sodium sulfate 0.7g / L, magnesium sulfate 0.5g / L, ammonium chloride 3.2g / L, calcium chloride 0.01g / L.

[0016] The supplemental culture medium used consisted of 10 g / L yeast extract, 10 g / L soybean peptone, and 500 g / L glycerol.

[0017] In step A2, the preferred flow rate of the feeding medium is 20 mL / L·h.

[0018] Further, step A1 includes: inoculating recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs with an inoculum age of 7 hours into the seed culture medium and culturing at 37±1℃ until OD 600nm A value of 0.5-1.0 indicates that it is seed liquid.

[0019] Further, step A2 includes: inoculating the seed culture obtained in step A1 into the fermentation medium at a volume ratio of 0.02%-0.04% (preferably 0.03%), and fermenting at 35-38°C and a pressure of 0.05±0.01MPa; and waiting for the fermentation broth to OD 600nm When the value reaches 8-10, lower the temperature to 23-26℃ and continue culturing.

[0020] The initial dissolved oxygen level is 100%, and during fermentation, the dissolved oxygen level is controlled above 20%. Aeration is controlled at 1 ± 0.3 VVM, and the rotation speed is controlled at 150-450 rpm. Throughout the fermentation process, the pH of the fermentation system is controlled at 6.8-7.0. Preferably, ammonia is used to adjust the pH of the system.

[0021] In the aforementioned method, the reaction solution in step B is: tyrosol 0.7 g / L, glucose 30 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 2.7 g / L, calcium chloride 0.1 g / L, prepared with water.

[0022] Step B includes: taking 108 -10 9 1.5 kg of CFU / g bacterial cells were added to a reaction vessel containing a reaction solution. The reaction vessel had a liquid volume of 30 L / 50 L. The biosynthesis of rhodioloside was carried out at 35-38℃ and a pressure of 0.05±0.01 MPa.

[0023] The initial dissolved oxygen in the reaction vessel is 100%, and the dissolved oxygen is controlled to be no less than 60% during the reaction. The pH value of the system is controlled at 7.2-7.5 throughout the biosynthesis process. Preferably, sodium hydroxide is used to adjust the pH value of the system.

[0024] Furthermore, step B also includes the step of adding glucose solution and tyrosol solution to the reaction solution during the biosynthesis of rhodioloside.

[0025] Preferably, glucose solution is added starting when the glucose concentration in the reaction solution is reduced to 2%, and the glucose concentration in the reaction solution is controlled at 2-3%.

[0026] More preferably, the flow rate of the glucose solution is equivalent to adding 3-6g of glucose per liter of reaction solution per hour.

[0027] Preferably, the tyrosol solution is added starting when the concentration of tyrosol in the reaction solution is consumed to 0.03%, and the concentration of tyrosol in the reaction solution is controlled at 2-5 mmol / L.

[0028] More preferably, the flow rate of the tyrosol solution is equivalent to adding 200-280 mg of tyrosol per liter of reaction solution per hour.

[0029] In step B, the synthesis reaction is stopped when the rhodioloside content increases to less than 100 μg / mL.

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] This invention accumulates glucosidase during fermentation and constructs an enzyme-catalyzed reaction system to convert glucose and tyrosol into rhodioloside. Through continuous optimization of the fermentation and conversion stage feed formulations, reaction conditions, and control strategies, the method of this invention can achieve high rhodioloside yields in a short time, with a maximum rhodioloside content of up to 9206 mg / L in the conversion reaction system and a tyrosol conversion rate exceeding 80%. This method for preparing rhodioloside is simple, easy to implement, low-cost, and energy-efficient, making it suitable for large-scale industrial production of rhodioloside and offering significant economic and social benefits.

[0032] (I) This process replaces glucose, the common carbon source for Escherichia coli, with glycerol and optimizes the ratio of various materials in the fermentation broth accordingly, thereby obtaining the optimal combination to reduce the growth rate and prevent enzymes from forming inclusion bodies. At the same time, it reduces the accumulation of metabolites such as acetic acid and glutamate, and prevents the generation of glucosidase from being hindered by feedback inhibition.

[0033] (II) A gradient cooling control strategy was adopted during the fermentation process. Extensive experiments were conducted to verify the cooling time and range in different culture media, ultimately determining the temperature control conditions in this invention. A suitable temperature can effectively regulate the growth rate, achieving a high cell count while preventing inclusion body formation. Furthermore, a suitable temperature allows for better peptide chain folding, thereby enhancing glucosidase activity.

[0034] (III) This invention also synergistically optimizes the reaction concentrations of glucose and tyrosol and the amount of centrifuged bacterial cells used in the reaction stage. Extensive exploratory experiments were conducted on the centrifuged bacterial cells, glucose concentration, and tyrosol concentration under industrial production equipment conditions to maintain the reaction rate at the optimal level and increase the yield of rhodioloside. Too low a substrate concentration during the catalytic reaction will reduce the reaction rate, while too high a substrate concentration will cause *E. coli* to enter a vigorous metabolic state, producing a large number of byproducts that affect the synthesis rate of rhodioloside. Too little bacterial cell usage will also reduce conversion efficiency, while too much will lead to an excessively high growth and metabolic rate, disrupting the stability of the enzyme-catalyzed reaction system conditions such as dissolved oxygen content and temperature uniformity. Therefore, it is necessary to strictly control the feeding rate during the reaction stage according to the optimal conditions obtained through extensive experiments to increase the yield of rhodioloside. Detailed Implementation

[0035] This invention provides a fermentation conversion method for rhodioloside, the specific method comprising:

[0036] (1) Add the seed liquid of the fermentation strain to the fermentation medium; the initial dissolved oxygen in the fermenter is 100%, and the dissolved oxygen is controlled to be above 20% during the fermentation process. Ammonia water is used to adjust the pH of the fermentation liquid throughout the process.

[0037] (2) When OD 600nm Value reaches 2-5 (preferred OD) 600nm When the value is 3), continuously add a mixed solution of carbon and nitrogen sources (feed culture medium) and continue culturing;

[0038] (3) When the fermentation broth OD 600nm When the value reaches 8-10, lower the culture temperature (reduce the temperature to 23-26℃);

[0039] (4) Ferment for 15-17 hours, when OD 600nm Fermentation should be stopped when the value reaches 40 and the bacterial cell content detected by centrifugation reaches 10-12%.

[0040] In step (1), the seed liquid of the fermentation strain is added to the fermentation medium at a volume ratio of 0.02%-0.04% (preferably 0.03%) to the fermentation medium.

[0041] The fermentation strain used was the recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs disclosed in US20190264221A1, which acquires the ability to biosynthesize rhodioloside by overexpressing exogenous 4-hydroxyphenylacetaldehyde reductase gene and glucosidase gene.

[0042] During the fermentation process described above, the initial temperature is controlled at 35-38℃ (preferably 36-38℃), and after cooling, it is controlled at 23-26℃ (preferably 24-26℃). The pressure is 0.05±0.01MPa, and ammonia water is added throughout the fermentation process to adjust the pH value to not be lower than 6.8.

[0043] In the above method, the fermentation medium in step (1) is formulated as follows: 5 g / L glycerol, 5 g / L yeast powder, 5 g / L soybean peptone, 18 g / L dipotassium hydrogen phosphate, 6.8 g / L potassium dihydrogen phosphate, 0.7 g / L sodium sulfate, 0.5 g / L magnesium sulfate, 3.2 g / L ammonium chloride, and 0.01 g / L calcium chloride, and is prepared with water.

[0044] In the above method, step (2) involves adding a mixed carbon and nitrogen source solution by adding 10-15g of glycerol, 0.4-0.6g of yeast powder, and 0.4-0.6g of soybean peptone per liter of fermentation broth per hour, calculated by pure substance mass. The mixed solution used for feeding is prepared separately, and the glycerol concentration can be 40-70%.

[0045] Furthermore, the present invention provides a method for synthesizing rhodioloside from tyrosol and glucose by constructing a glucosidase-catalyzed reaction system using the above-mentioned fermentation products, comprising the following steps:

[0046] (5) Add the bacterial cells obtained by centrifuging the fermentation broth to the reaction solution; the initial dissolved oxygen in the reaction tank is 100%, and the dissolved oxygen is controlled to be no less than 60% during the reaction process. Sodium hydroxide is used to adjust the pH of the fermentation broth throughout the process.

[0047] (6) When the glucose concentration in the reaction solution is consumed to 2%, glucose solution is started to be added continuously.

[0048] (7) When the concentration of tyrosol in the reaction solution is consumed to 0.03%, start adding tyrosol solution. Control the concentration of tyrosol in the reaction solution to 2-5 mmol / L according to the actual conversion.

[0049] (8) Stop the reaction when the content of rhodioloside increases to less than 100 μg / mL.

[0050] During the reaction process described above, the temperature is controlled at 35-38℃ (preferably 36-38℃), the pressure is 0.05±0.01MPa, and sodium hydroxide is added throughout the reaction to adjust the pH value to be no lower than 7.2.

[0051] In the above method, the reaction solution in step (5) is formulated as follows: tyrosol 0.7 g / L, glucose 30 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 2.7 g / L, calcium chloride 0.1 g / L, and water.

[0052] In the above method, step (6) involves adding 3-6g of glucose per liter of fermentation broth per hour, calculated based on the mass of glucose solids. The glucose solution used for feeding is prepared separately, with a concentration of 40-70%.

[0053] In the above method, step (7) involves adding tyrosol by adding 200-280 mg of tyrosol per liter of fermentation broth per hour, calculated based on the solid mass of tyrosol. The tyrosol solution used for the feed is prepared separately, with a concentration not exceeding 5%.

[0054] The recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs used in this invention were provided by the Whitehead Institute for Biomedical Research, Cambridge, MA.

[0055] The reagents used in the following examples are as follows:

[0056] Seed culture medium: 10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0057] Fermentation medium: glycerol 5g / L, yeast powder 5g / L, soybean peptone 5g / L, dipotassium hydrogen phosphate 18g / L, potassium dihydrogen phosphate 6.8g / L, sodium sulfate 0.7g / L, magnesium sulfate 0.5g / L, ammonium chloride 3.2g / L, calcium chloride 0.01g / L.

[0058] Reaction solution: Tyrosol 0.7 g / L, glucose 30 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 2.7 g / L, calcium chloride 0.1 g / L

[0059] Feeding medium: yeast extract 10g / L, soybean peptone 10g / L, glycerol 500g / L.

[0060] Prepare a separate 600 g / L glucose solution and a 300 g / L sodium hydroxide solution, sterilize them separately, and prepare for feed addition. Filter and sterilize a 42 g / L tyrosol solution for use. Additionally, prepare a 25%-28% ammonia solution for feed addition.

[0061] Example 1: Fermentation and transformation of rhodioloside (50L small-scale experiment)

[0062] Prepare 3L of seed culture medium in a 5L seed tank, sterilize at 121℃ for 25 min, cool to 37℃, and inoculate recombinant Escherichia coli cultured in shake-flask seed culture medium at an inoculum rate of 0.3% (v / v) at a seed age of 7 hours. Then incubate at 37±1℃ for 7 hours until OD reaches 0.5%. 600nm A value of 0.6 indicates that it is seed liquid.

[0063] Prepare 30L of fermentation medium in a 50L fermenter, sterilize at 121℃ for 25min, cool to 37℃, adjust pH to 6.8 with ammonia, and inoculate the cultured seed liquid at an inoculation rate of 0.03% (v / v). During fermentation, control the temperature at 37±1℃ and the pressure at 0.05±0.01MPa. Control the aeration and rotation speed according to the dissolved oxygen status to ensure that the dissolved oxygen is above 20% (assuming the initial dissolved oxygen in the fermenter is 100%). Control the aeration at 1±0.3VVM and the rotation speed at 150-450rpm. Continuously add ammonia to maintain the pH at no less than 6.8.

[0064] Monitoring fermentation broth OD 600nm Value, when OD 600nm When the OD value reaches 3, the fed culture medium is started at a fixed rate, specifically 20 ml of mixed solution per liter of fermentation broth per hour, i.e., 20 mL / L·h. When the OD value of the fermentation broth reaches 3... 600nm When the OD value reaches 10, lower the culture temperature to 22℃ and continue culturing. 600nm When the pH reaches 40 and the cell count exceeds 10% (by volume), stop fermentation. Quickly centrifuge the fermentation broth, remove the supernatant, and collect the cells.

[0065] Prepare 30L of reaction solution in a 50L reaction vessel, sterilize at 121℃ for 25 minutes, cool to 37℃, and adjust the pH to 7.2 with sodium hydroxide solution.

[0066] All the centrifuged bacterial cells were added to the reaction solution. During the reaction, the temperature was controlled at 37±1℃ and the pressure at 0.07±0.01MPa. The aeration and rotation speed were controlled according to the dissolved oxygen status to ensure that the dissolved oxygen was above 60% (assuming the initial dissolved oxygen in the reaction tank was 100%). The aeration was controlled at 1±0.3VVM and the rotation speed was controlled at 200-600rpm. Sodium hydroxide was added throughout the process to keep the pH not lower than 7.2.

[0067] Monitor the glucose concentration. When the concentration falls below 2%, begin adding glucose solution in a feedstock at a rate of 5 g glucose per liter of reaction solution per hour (calculated by the mass of glucose solids), i.e., 5 g / L·h. Two hours after the start of the reaction, begin adding tyrosol solution in a feedstock at a rate of 220 mg tyrosol per liter of reaction solution per hour (calculated by the mass of tyrosol solids), i.e., 220 mg / L·h. After the feedstock begins, maintain the glucose concentration in the reaction solution at 2% and the tyrosol concentration at 5 mM.

[0068] The reaction was stopped when the rhodioloside content increased to less than 100 mg / L·h, and the final rhodioloside content was determined to be 9206 mg / L.

[0069] Example 2: Fermentation and transformation of rhodioloside (5000L pilot-scale experiment)

[0070] Prepare 300L of seed culture medium in a 500L seed tank, sterilize at 121℃ for 25 min, cool to 37℃, inoculate with recombinant Escherichia coli cultured in shake flask seed culture medium at an inoculation rate of 0.3% (volume ratio) for 7 hours, and then incubate at 37±1℃ for 7 hours.

[0071] Prepare 3000L of fermentation medium in a 5000L fermenter, sterilize at 121℃ for 25min, cool to 37℃, adjust pH to 6.8 with ammonia, and inoculate the cultured seed liquid at an inoculation rate of 0.03% (v / v). During fermentation, control the temperature at 37±1℃ and the pressure at 0.05±0.01MPa. Control the aeration and rotation speed according to the dissolved oxygen status to ensure that the dissolved oxygen is above 20% (assuming the initial dissolved oxygen level of the fermenter is 100%). Control the aeration at 0.5-1VVM and the rotation speed at 50-150rpm. Continuously add ammonia to maintain the pH at no less than 6.8.

[0072] Monitoring fermentation broth OD 600nm Value, when OD 600nm When the OD value reaches 3, the fed culture medium is started at a fixed rate, specifically 200 ml of mixed solution per liter of fermentation broth per hour, i.e., 20 mL / L·h. When the OD value of the fermentation broth reaches 3... 600nm When the OD value reaches 10, lower the culture temperature to 22℃ and continue culturing. 600nm When the pH reaches 40 and the cell count exceeds 10% (by volume), stop fermentation. Quickly centrifuge the fermentation broth, remove the supernatant, and collect the cells.

[0073] Prepare 3000L of reaction solution in a 5000L reaction vessel, sterilize at 121℃ for 25 minutes, cool to 37℃, and adjust the pH to 7.2 with sodium hydroxide solution.

[0074] All the centrifuged bacterial cells were added to the reaction solution. During the reaction, the temperature was controlled at 37±1℃ and the pressure at 0.07±0.01MPa. The ventilation and rotation speed were controlled according to the dissolved oxygen status to ensure that the dissolved oxygen was above 60% (assuming the initial dissolved oxygen in the reaction tank was 100%). The ventilation was controlled at 1±0.3VVM and the rotation speed was controlled at 50-150rpm. Sodium hydroxide was added throughout the process to keep the pH not lower than 7.2.

[0075] Monitor the glucose concentration. When the concentration falls below 2%, begin adding glucose solution in a feedstock at a rate of 5 g glucose per liter of reaction solution per hour (calculated by the mass of glucose solids), i.e., 5 g / L·h. Two hours after the start of the reaction, begin adding tyrosol solution in a feedstock at a rate of 220 mg tyrosol per liter of reaction solution per hour (calculated by the mass of tyrosol solids), i.e., 220 mg / L·h. After the feedstock begins, maintain the glucose concentration in the reaction solution at 2% and the tyrosol concentration at 5 mM.

[0076] The reaction was stopped when the rhodioloside content increased to less than 100 mg / L·h, and the final rhodioloside content was determined to be 9097 mg / L.

[0077] Example 3: Control Experiment (50L Small-Scale Experiment)

[0078] In the preliminary experiment, it was verified that the same carbon source was used in the fermentation stage and the reaction conversion stage, that is, glucose was used as the main carbon source in the fermentation stage.

[0079] The amount of glucose added was adjusted based on the LB medium formula, and the inorganic salt ratio was optimized in conjunction with the formula to form a fermentation medium.

[0080] Fermentation medium (carbon source: glucose) formula: glucose 20g / L, soybean peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, dipotassium hydrogen phosphate 18g / L, potassium dihydrogen phosphate 6.8g / L, sodium sulfate 0.35g / L, magnesium sulfate 0.6g / L, calcium chloride 0.01g / L.

[0081] Feeding medium 1: yeast extract 10 g / L, soybean peptone 10 g / L;

[0082] Feeding medium 2: glucose 500g / L.

[0083] The fermentation and conversion were carried out in the same manner as in Example 1, as follows:

[0084] Prepare 3L of seed culture medium in a 5L seed tank, sterilize at 121℃ for 25 min, cool to 37℃, and inoculate recombinant Escherichia coli cultured in shake-flask seed culture medium at an inoculum rate of 0.3% (v / v) at a seed age of 7 hours. Then incubate at 37±1℃ for 7 hours until OD reaches 0.5%. 600nm A value of 0.6 indicates that it is seed liquid.

[0085] Prepare 30L of fermentation medium in a 50L fermenter, sterilize at 121℃ for 25min, cool to 37℃, adjust pH to 6.8 with ammonia, and inoculate the cultured seed liquid at an inoculation rate of 0.03% (v / v). During fermentation, control the temperature at 37±1℃ and the pressure at 0.05±0.01MPa. Control the aeration and rotation speed according to the dissolved oxygen status to ensure that the dissolved oxygen is above 20% (assuming the initial dissolved oxygen in the fermenter is 100%). Control the aeration at 1±0.3VVM and the rotation speed at 150-450rpm. Continuously add ammonia to maintain the pH at no less than 6.8.

[0086] Monitoring fermentation broth OD 600nm Value, when OD 600nm When the OD value reaches 3, feed medium 1 and feed medium 2 are added at a fixed rate. Specifically, 20 ml of feed medium 1 mixed solution is added per liter of fermentation broth per hour, i.e., 20 mL / L·h; 8 ml of glucose solution from feed medium 2 is added per liter of fermentation broth per hour (calculated based on glucose solid mass, 4 g glucose is added per liter of reaction solution per hour), i.e., 8 mL / L·h. When the OD value of the fermentation broth reaches 3... 600nm When the OD value reaches 10, lower the culture temperature to 22℃ and continue culturing. 600nm When the pH reaches 40 and the cell count exceeds 10% (by volume), stop fermentation. Quickly centrifuge the fermentation broth, remove the supernatant, and collect the cells.

[0087] Prepare 30L of reaction solution in a 50L reaction vessel, sterilize at 121℃ for 25 minutes, cool to 37℃, and adjust the pH to 7.2 with sodium hydroxide solution.

[0088] All the centrifuged bacterial cells were added to the reaction solution. During the reaction, the temperature was controlled at 37±1℃ and the pressure at 0.07±0.01MPa. The aeration and rotation speed were controlled according to the dissolved oxygen status to ensure that the dissolved oxygen was above 60% (assuming the initial dissolved oxygen in the reaction tank was 100%). The aeration was controlled at 1±0.3VVM and the rotation speed was controlled at 200-600rpm. Sodium hydroxide was added throughout the process to keep the pH not lower than 7.2.

[0089] Monitor the glucose concentration. When the concentration falls below 2%, begin adding glucose solution at a rate of 5 g glucose per liter of reaction solution per hour (calculated by the mass of glucose solids), i.e., 5 g / L·h. Two hours after the start of the reaction, begin adding tyrosol solution at a rate of 130 mg tyrosol per liter of reaction solution per hour (calculated by the mass of tyrosol solids), i.e., 130 mg / L·h (due to poor enzyme activity and slow substrate consumption, reduce the feed rate). After feed begins, maintain the glucose concentration in the reaction solution at 2% and the tyrosol concentration at 5 mM.

[0090] The reaction was stopped when the rhodioloside content increased to less than 100 mg / L·h. The final rhodioloside content was measured to be 4816 mg / L, with a conversion rate of approximately 50%.

[0091] The above comparative experiments demonstrate that optimizing the production process according to this method can significantly increase the yield of rhodioloside and the conversion rate of tyrosol. In a 50L fermentation conversion experiment, the yield increased by nearly 90%, and the tyrosol conversion rate increased by approximately 60%.

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A fermentation and transformation method for rhodioloside, characterized in that, Includes the following steps: A. Fermentation culture of recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs, centrifugation to collect the bacterial cells after fermentation; B. Add the bacterial cells obtained in step A to a reaction solution with glucose and tyrosol as substrates to carry out the biosynthesis of rhodioloside. Step A includes the following sub-steps: A1. Preparation of recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs seed culture; A2. Fermentation culture using a fed-batch method; In step A2, when the fermentation broth OD 600nm When the OD value reaches 2-5, start continuously feeding the culture medium until the fermentation broth OD value reaches 2-5. 600nm Fermentation should be stopped when the value reaches 40 and the bacterial cell content detected by centrifugation reaches 10-12%. Seed culture medium used: 10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0; Fermentation medium used: glycerol 5g / L, yeast powder 5g / L, soybean peptone 5g / L, dipotassium hydrogen phosphate 18g / L, potassium dihydrogen phosphate 6.8g / L, sodium sulfate 0.7g / L, magnesium sulfate 0.5g / L, ammonium chloride 3.2g / L, calcium chloride 0.01g / L; The supplemental culture medium used was: yeast extract 10 g / L, soybean peptone 10 g / L, and glycerol 500 g / L. The reaction solution described in step B is: tyrosol 0.7 g / L, glucose 30 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 2.7 g / L, calcium chloride 0.1 g / L, prepared with water; Step B includes: taking 10 8 -10 9 1.5 kg of CFU / g bacterial cells were added to a reaction vessel containing a reaction solution. The reaction vessel had a liquid volume of 30 L / 50 L. The biosynthesis of rhodioloside was carried out at 35-38℃ and a pressure of 0.05±0.01 MPa. The initial dissolved oxygen in the reaction vessel is 100%, and the dissolved oxygen is controlled to be no less than 60% during the reaction. The pH of the system is controlled to be 7.2-7.5 throughout the entire biosynthesis process. Step B also includes the step of adding glucose solution and tyrosol solution to the reaction solution during the biosynthesis of rhodioloside; In step B, glucose solution is added continuously once the glucose concentration in the reaction solution is reduced to 2%, and the glucose concentration in the reaction solution is controlled at 2-3%. In step B, tyrosol solution is added starting when the concentration of tyrosol in the reaction solution is consumed to 0.03%, and the concentration of tyrosol in the reaction solution is controlled at 2-5 mmol / L.

2. The method according to claim 1, characterized in that, In step A2, the feed rate of the culture medium is 20 mL / L·h.

3. The method according to claim 2, characterized in that, Step A1 includes: inoculating recombinant Escherichia coli BL21(DE3)E.coli-(pHhis8-4)-UGTs with an inoculum age of 7 hours into the seed culture medium and culturing at 37±1℃ until OD2000. 600nm A value of 0.5-1 indicates that it is seed liquid; Step A2 includes: inoculating the seed culture obtained in step A1 into the fermentation medium at a volume ratio of 0.02%-0.04%, and fermenting at 35-38℃ and a pressure of 0.05±0.01MPa; waiting for the fermentation broth to OD 600nm When the value reaches 8-10, lower the temperature to 23-26℃ and continue culturing; The initial dissolved oxygen level was 100%, and the dissolved oxygen level was controlled above 20% during fermentation. The aeration rate was controlled at 1 ± 0.3 VVM, and the rotation speed was controlled at 150-450 rpm. Throughout the fermentation process, the pH value of the fermentation system was controlled at 6.8-7.

0.

4. The method according to claim 1, characterized in that, In step B, the flow rate of the glucose solution is equivalent to adding 3-6g of glucose per liter of reaction solution per hour.

5. The method according to claim 1, characterized in that, In step B, the flow rate of the tyrosol solution is equivalent to adding 200-280 mg of tyrosol per liter of reaction solution per hour.

6. The method according to any one of claims 1-5, characterized in that, In step B, the synthesis reaction is stopped when the rhodioloside content increases to less than 100 μg / mL.