A fermentation medium and its application in a fermentation process for improving the production of 7-dhc in saccharomyces cerevisiae

Abstract

This invention discloses a fermentation medium and its application in the fermentation process for increasing 7-DHC production in *Saccharomyces cerevisiae*. By optimizing key nutrients and precisely controlling fermentation conditions, the production efficiency of 7-DHC is significantly improved while reducing costs. The medium is carefully formulated with proportions of glucose, corn steep liquor, amino acids (such as L-leucine and L-histidine), vitamins, and metal ions to ensure an optimal growth environment for *Saccharomyces cerevisiae*. During fermentation, dissolved oxygen levels are maintained by adjusting parameters such as temperature, pH, fermentation speed, and aeration rate. A high-concentration glucose solution is added at appropriate residual sugar concentrations, and the ethanol concentration is strictly controlled to avoid its inhibitory effect on cells. Furthermore, using molasses as an alternative carbon source is not only economical but also ensures efficient 7-DHC synthesis. This enables a transition from small-scale trials to large-scale production, promoting its widespread application in the pharmaceutical and health product fields.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically designing a fermentation culture medium and its application in the fermentation process for increasing the yield of 7-DHC in Saccharomyces cerevisiae. Background Technology

[0002] 7-Dehydrocholesterol (7-DHC) is a sterol and a key precursor in the synthesis of vitamin D3 (VD3). It is also an intermediate in the synthesis of some steroidal drugs and has important pharmaceutical and clinical applications. Currently, VD3 is mainly produced through chemical synthesis and extraction methods. Extraction methods involve extracting from specific mosses, resulting in low yields and high costs. Chemical synthesis methods primarily use cholesterol derived from lanolin or pine wood as starting materials, first synthesizing 7-DHC, and then using photocatalysis and thermal conversion to synthesize VD3. Chemical synthesis methods suffer from problems such as numerous byproducts, difficulty in controlling conditions, and environmental pollution. Regardless of the method, 7-DHC is the precursor for VD3 synthesis, while naturally sourced phytosterols are limited in quantity and expensive, failing to meet the production needs of industrial-scale synthesis.

[0003] Compared with chemical synthesis, microbial synthesis of 7-DHC is a promising alternative for solving the 7-DHC source problem due to its advantages of being greener, more economical, environmentally friendly, and sustainable. Current research mainly focuses on strain screening and molecular modification. Saccharomyces cerevisiae has recognized advantages such as a clear genetic background, generally accepted safety (GRAS), short growth cycle, ease of genetic manipulation, and low culture cost, making it a foundational organism for the biosynthesis of many terpenoids and steroids. Patent CN 202310661079.1 achieved a 7-DHC yield of 370.68 mg / L at the shake-flask level by optimizing the ferrous ion concentration in the culture medium; Patent CN 202310058515.6 further increased the yield by adding n-dodecane to the fermentation medium to enhance the strain's ability to excrete 7-DHC.

[0004] However, the yield of 7-DHC obtained by fermentation of Saccharomyces cerevisiae is currently low, and the culture medium still requires a large amount of expensive organic nitrogen source to meet the growth requirements of Saccharomyces cerevisiae, which directly leads to the inability to achieve large-scale production of this process. At the same time, there are a large number of 7-DHC structural analogs in the by-products, which greatly affects the extraction and purification of downstream products.

[0005] Therefore, developing an efficient fermentation medium and optimizing the corresponding fermentation process to increase 7-DHC yield, reduce costs and decrease byproduct generation is of great significance for promoting the green biomanufacturing of 7-DHC and even VD3. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to improve the production capacity of 7-DHC by Saccharomyces cerevisiae and reduce product manufacturing costs through the optimization of culture medium design and precise control of fermentation process, thereby promoting the large-scale industrial application of 7-DHC.

[0007] This invention discloses a fermentation culture medium, the components and concentrations of which include: L-tryptophan: 0.5-2.5 g / L; L-leucine: 0.2-1.2 g / L; L-sodium glutamate: 0.2-1.0 g / L; uracil: 0.2-1.0 g / L; ferrous sulfate: 0.02-0.1 g / L; biotin: 30-70 mg / L; vitamin B1: 100-200 mg / L; L-histidine: 0.1-0.6 g / L; and at least one carbon source and at least one nitrogen source.

[0008] For the technical solution described above, a further preferred embodiment is that the concentrations of each component in the fermentation culture medium are: L-tryptophan 1.5-2.0 g / L, L-leucine 0.5-0.8 g / L, L-sodium glutamate 0.5-0.8 g / L, uracil 0.5-0.8 g / L, ferrous sulfate 0.06-0.08 g / L, biotin 50-60 mg / L, vitamin B1 150-180 mg / L, and L-histidine 0.3-0.4 g / L.

[0009] For the technical solution described above, more preferably, the carbon source is selected from glucose, sucrose, molasses and mixtures thereof, with a concentration of 10-100 g / L.

[0010] For the technical solution described above, a further preferred embodiment is that the glucose concentration is 20-70 g / L and the molasses concentration is 0-50 g / L.

[0011] For the technical solution described above, more preferably, the nitrogen source can be an organic nitrogen source or an inorganic nitrogen source, with a concentration of 10-80 g / L.

[0012] In a further preferred embodiment of the above-described technical solution, the nitrogen source is corn steep liquor with a concentration of 25-60 g / L.

[0013] For the technical solution described above, a further preferred range of the fermentation culture medium is shown in the following table:

[0014]

[0015]

[0016] Another aspect of the present invention is the application of the fermentation medium in the fermentation process for increasing the yield of 7-DHC in Saccharomyces cerevisiae, comprising the following steps: controlling dissolved oxygen and sugar concentration during fermentation, controlling the ethanol concentration to 0-15 g / L, and collecting the fermentation broth at the end of fermentation.

[0017] In a further preferred embodiment of the above-described technical solution, the ethanol concentration is controlled to be 0-10 g / L.

[0018] In a further preferred embodiment of the above-described technical solution, the ethanol concentration is controlled to be 0-5.0 g / L.

[0019] For the technical solution described above, a further preferred embodiment is that the dissolved oxygen control method is to alternately adjust the rotation speed and ventilation rate to control the dissolved oxygen to be higher than 20%.

[0020] For the technical solution described above, a further preferred embodiment is that the dissolved oxygen control method is to alternately adjust the rotation speed and ventilation rate to control the dissolved oxygen to be higher than 40%.

[0021] For the technical solution described above, a further preferred embodiment includes: adjusting the bacterial cell concentration OD... 600 Seed culture of 5-8 is inoculated into fermentation medium. At 25-40℃ (preferably 30-35℃) and pH 5.0-6.0 (5.0-5.8; most preferably 5.0-5.5), the aeration rate and rotation speed are alternately adjusted to control dissolved oxygen above 20%. When the residual sugar drops to 0.5-2.0 g / L, 400-800 g / L glucose solution is added, and the ethanol concentration is controlled at 0-15 g / L. The fermentation broth is collected at the end of fermentation.

[0022] For the technical solution described above, a further preferred embodiment is that the rotational speed is 150-800 rpm and the air volume is 0.5-2.0 vvm.

[0023] For the technical solution described above, more preferably, under shake flask fermentation culture conditions, the rotation speed is 150-800 rpm and the aeration rate is 0.5-2.0 vvm; more preferably, the rotation speed is 200-600 rpm and the aeration rate is 1.0-1.8 vvm; most preferably, the rotation speed is 300-500 rpm and the aeration rate is 1.2-1.5 vvm.

[0024] For the technical solution described above, more preferably, under 1-60L fermentation culture conditions, the rotation speed is 200-800rpm, more preferably 300-600rpm; the ventilation volume is 0.5-2.0vvm, more preferably 1.0-1.8vvm.

[0025] For the technical solution described above, a further preferred embodiment is that the sugar concentration is controlled by adding 300-1000 g / L glucose solution when the residual sugar concentration drops to 0.5-2.0 g / L. More preferably, the residual sugar concentration drops to 0.5-1.5 g / L; most preferably, the residual sugar concentration drops to 0.5-1.0 g / L.

[0026] For the technical solution described above, the preferred glucose concentration is 500-700 g / L;

[0027] For the technical solution described above, under shake-flask fermentation culture conditions, the ethanol concentration is more preferably 0-5.0 g / L; most preferably 0-2.0 g / L.

[0028] For the technical solution described above, under fermentation culture conditions of 1-10L, the ethanol concentration is more preferably 0-15g / L; most preferably 0-10g / L.

[0029] For the technical solution described above, under fermentation culture conditions of 10-60L, the ethanol concentration is more preferably 0-10g / L; further preferably 0-5g / L; and most preferably 0-2.0g / L.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. By optimizing the culture medium formulation and fermentation conditions, the yield of 7-DHC was significantly increased, reaching the high efficiency level required for industrial production.

[0032] 2. The addition of specific trace element combinations effectively promoted the synthesis of 7-DHC, while reducing the formation of the byproduct yeast sterol and improving product purity.

[0033] 3. Molasses was successfully used as an economical and effective carbon source alternative, reducing production costs without affecting 7-DHC production.

[0034] 4. The effects of different types of organic nitrogen sources (such as corn steep liquor) on cell growth and 7-DHC production were verified, providing a more flexible raw material selection scheme.

[0035] 5. Precise control of ethanol concentration during the feeding process ensures optimal 7-DHC synthesis efficiency and avoids inhibition caused by excessive ethanol concentration.

[0036] 6. The technology has been successfully scaled up from small-scale trials to pilot-scale trials and then to large-scale production, proving its feasibility and stability in industrial applications. Attached Figure Description

[0037] Figure 1 Optimize the amount of trace elements added when using peptone to add organic nitrogen to shake flasks.

[0038] Figure 2 Comparison of the effects of adding trace elements to different nitrogen sources on a 5L fermenter.

[0039] Figure 3 Optimize the amount of trace elements added when using peptone for organic nitrogen in a 5L fermenter.

[0040] Figure 4 Optimize the amount of corn syrup added to a 5L fermenter. Detailed Implementation

[0041] To better understand the technical solution of the present invention, detailed descriptions are provided below through specific embodiments. These embodiments are intended to explain the specific applications and technical effects of the present invention, but do not limit the scope of protection of the present invention. All materials, equipment, and methods involved in all embodiments are conventional choices in the art, unless otherwise stated. The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0042] The components of the seed culture medium in the following examples are as follows:

[0043] Glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L. Accurately weigh the ingredients according to the above formula, dissolve in deionized water and bring to a final volume, then sterilize at 121°C for 20 minutes.

[0044] The shake flask culture process in the following examples is as follows:

[0045] Transfer 0.2 mL of bacterial culture from the glycerol tube to a 50 mL Erlenmeyer flask containing 6 mL of shake-flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the primary seed culture. Take 1 mL of the primary seed culture and transfer it to a 500 mL Erlenmeyer flask containing 100 mL of shake-flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the secondary seed culture. The resulting secondary seed culture has a cell concentration of 5-8, which is sufficient for the scale-up fermentation culture in subsequent examples.

[0046] The strain used in the following examples is Saccharomyces cerevisiae SC007X, and its detailed construction process has been described in Example 3 of paragraph

[0073] of patent CN115044603B.

[0047] The following examples illustrate the methods for 7-DHC extraction and detection after fermentation:

[0048] Take 5 mL of fermentation broth, centrifuge at 8000 rpm for 10 min, discard the supernatant to obtain bacterial cells. Add 5 mL of 1.5 M potassium hydroxide-methanol solution and 3 g of 2 mm diameter glass beads to the bacterial cells, vortex for 10 min, then sonicate for 10 min, and vortex again for 10 min to fully disrupt the cell walls. Add another 10 mL of 1.5 M potassium hydroxide-methanol solution to the bacterial cells, heat in an 80 °C water bath for 1 h, then rapidly cool in an ice bath for 3 min. Add 15 mL of n-hexane and 15 mL of pure water, vortex for 20 min, and allow to separate into layers. After separation, take 1 mL of the upper layer solution into a 2 mL brown centrifuge tube, dry it with nitrogen gas, and then add 1 mL of methanol to dissolve it completely. Take 500 μL and use liquid chromatography to detect the yield of 7-DHC and yeast sterols.

[0049] The following examples mainly focus on optimizing the fermentation medium components:

[0050] 1. Optimize the shake-flask fermentation medium, which typically includes the following components: glucose 25-50 g / L, sucrose 0-25 g / L, peptone 40-60 g / L, and dipotassium hydrogen phosphate 0.6-1.8 g / L. Accurately weigh the ingredients according to the above formula, dissolve them in deionized water and bring the volume to a final depth. Adjust the pH to 5.0-6.0 with sodium hydroxide and sterilize at 121°C for 20 min.

[0051] 2. Optimize the pre-fermentation medium, which typically includes the following components: glucose 25-50 g / L, sucrose 0-25 g / L, peptone 30-50 g / L, and dipotassium hydrogen phosphate 0.6-1.8 g / L. Accurately weigh the components according to the above formula, dissolve them in deionized water, and bring the volume to a final depth. Sterilize at 121°C for 20 minutes.

[0052] Comparison table of culture medium components

[0053] Components Optimize the pre-shake flask fermentation medium Optimize the fermentation medium on the pre-tank glucose 25-50g / L 25-50g / L sucrose 0-25g / L 0-25g / L peptone 40-60g / L 30-50g / L dipotassium hydrogen phosphate 0.6-1.8g / L 0.6-1.8g / L

[0054] Example 1: Culture on a shake flask

[0055] (1) Accurately weigh 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. Dissolve the remainder in water and bring the volume to a final volume. Sterilize at 121°C for 20 min to obtain the seed culture medium. Accurately weigh 33 g / L glucose, 34 g / L molasses, 50 g / L corn steep liquor, 0.5 g / L L-glutamate, 0.3 g / L L-histidine, 0.5 g / L L-leucine, 1.5 g / L L-tryptophan, 0.5 g / L uracil, 0.06 g / L ferrous sulfate, 50 mg / L biotin, 150 mg / L vitamin B1, and 1.2 g / L dipotassium hydrogen phosphate. Dissolve in water and bring the volume to a final volume. Sterilize at 121°C for 20 min to obtain the shake flask fermentation medium.

[0056] (2) 0.2 mL of bacterial culture from the glycerol tube was transferred to a 50 mL Erlenmeyer flask containing 6 mL of shake-flask seed culture medium. The mixture was incubated at 30°C and 220 rpm for 24 h to obtain the primary seed culture. 1 mL of the primary seed culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of shake-flask seed culture medium. The mixture was incubated at 30°C and 220 rpm for 24 h to obtain the secondary seed culture. The secondary seed culture was inoculated at 6% (v / v) into a 500 mL Erlenmeyer flask containing 80 mL of fermentation medium. The mixture was incubated at 30°C and 220 rpm for 96 h, and the fermentation broth was collected. Extraction and analysis showed that the highest yield of 7-DHC reached 454 mg / L.

[0057] Under shake-flask conditions, the yield of 7-DHC reached 454 mg / L by optimizing the fermentation medium composition. This result indicates that *Saccharomyces cerevisiae* SC007X can efficiently synthesize 7-DHC under appropriate nutritional conditions and culture parameters. This yield represents a significant improvement compared to conventional laboratory-scale operations, demonstrating the importance of specific medium formulations for the synthesis of the target product.

[0058] Example 2: Cultivation in a 5L fermenter

[0059] (1) Accurately weigh 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. Dissolve the remainder in deionized water and bring the volume to a final volume. Sterilize at 121°C for 20 min to obtain shake flask seed culture medium. Accurately weigh 33 g / L glucose, 34 g / L molasses, 50 g / L corn steep liquor, 0.5 g / L L-glutamate, 0.3 g / L L-histidine, 0.5 g / L L-leucine, 1.5 g / L L-tryptophan, 0.5 g / L uracil, 0.06 g / L ferrous sulfate, 50 mg / L biotin, 150 mg / L vitamin B1, and 1.2 g / L dipotassium hydrogen phosphate. Dissolve the remainder in water and bring the volume to a final volume. Sterilize at 121°C for 20 min to obtain tank fermentation culture medium.

[0060] (2) Take 1.0 mL of bacterial culture from the glycerol tube and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of shake flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the primary seed culture. Take 1 mL of the primary seed culture and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of shake flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the secondary seed culture. Inoculate the secondary seed culture at a ratio of 10% (v / v) into a 5 L tank containing 3 L of tank fermentation medium. Control the fermentation temperature at 30°C, use ammonia to control the pH at 5.5, rotate the turbine at 300–800 rpm, and aerate at 1.0–2.0 vvm. Alternately adjust the turbine and aeration to keep the dissolved oxygen above 20%. When the residual sugar drops below 1.0 g / L, start adding 600 g / L glucose solution and control the ethanol concentration at 0–5.0 g / L. Collect the fermentation broth after culturing for 96 h. Extraction and testing revealed that the highest yield of 7-DHC reached 2400 mg / L.

[0061] In the above experiments, many parameters were set as range values ​​because these parameters are not constant during fermentation but are dynamically adjusted according to the actual situation. Specifically:

[0062] Rotation speed (300–800 rpm) and aeration rate (1.0–2.0 vvm): These two parameters need to be adjusted in real time according to changes in dissolved oxygen levels. This dynamic adjustment strategy can effectively cope with changes in oxygen demand during fermentation and maintain the optimal cell growth environment.

[0063] Ethanol concentration (0-5.0 g / L): This parameter is closely related to the rate of glucose replenishment and the rate of ethanol consumption. During fermentation, ethanol production can be controlled by monitoring the residual sugar concentration and adding glucose solution. Simultaneously, cells continuously consume the ethanol already produced, therefore the ethanol concentration will fluctuate within the range of 0-5.0 g / L. Maintaining an ethanol concentration within this range helps balance the accumulation and consumption of metabolites.

[0064] By scaling up the fermentation process to a 5L fermenter and further optimizing fermentation conditions, including pH control, dissolved oxygen levels, and feeding strategies, the yield of 7-DHC was significantly increased to 2400 mg / L. This result not only demonstrates the successful scale-up from small-scale to pilot-scale production but also verifies the crucial role of precise control of fermentation parameters in improving biosynthetic efficiency. Compared to shake-flask experiments, the yield of 7-DHC in the fermenter increased sixfold, indicating significant potential for large-scale production.

[0065] Example 3: Shake flask fermentation effect before optimization

[0066] (1) Accurately weigh 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone, dissolve them in water and bring the volume to 1 L. Sterilize at 121 °C for 20 min to obtain the shake flask seed culture medium. Accurately weigh 25 g / L glucose, 25 g / L sucrose, 50 g / L peptone, and 0.6 g / L dipotassium hydrogen phosphate, dissolve them in water and bring the volume to 1 L. Sterilize at 121 °C for 20 min to obtain the pre-optimized shake flask fermentation culture medium.

[0067] (2) 0.2 mL of bacterial culture from the glycerol tube was transferred to a 50 mL Erlenmeyer flask containing 6 mL of shake-flask seed culture medium. The mixture was incubated at 30°C and 220 rpm for 24 h to obtain the primary seed culture. 1 mL of the primary seed culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of shake-flask seed culture medium. The mixture was incubated at 30°C and 220 rpm for 24 h to obtain the secondary seed culture. The secondary seed culture was inoculated at 6% into a 500 mL Erlenmeyer flask containing 80 mL of pre-optimized shake-flask fermentation medium. After 120 h of shake-flask incubation, the fermentation broth was collected. Extraction and analysis showed that the 7-DHC yield reached 206 mg / L.

[0068] Under unoptimized conditions, the yield of 7-DHC was only 206 mg / L. Comparing this to subsequent optimized experiments (such as Example 1), the impact of the initial formulation and process parameters on the product synthesis is clearly evident. This baseline data provides a benchmark for evaluating the effectiveness of subsequent optimization measures.

[0069] Example 4: Fermentation effect of a 5L fermenter before optimization

[0070] (1) Accurately weigh 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. Dissolve the remainder in deionized water and bring the volume to 1 L. Sterilize at 121 °C for 20 min to obtain shake flask seed culture medium. Accurately weigh 25 g / L glucose, 25 g / L sucrose, 50 g / L peptone, and 0.6 g / L dipotassium hydrogen phosphate. Dissolve the remainder in water and bring the volume to 1 L. Sterilize at 121 °C for 20 min to obtain optimized pre-fermentation medium.

[0071] (2) Take 1.0 mL of bacterial culture from the glycerol tube and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of shake flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the primary seed culture. Take 1 mL of the primary seed culture and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of shake flask seed culture medium. Incubate at 30°C and 220 rpm for 24 h to obtain the secondary seed culture. Inoculate the secondary seed culture at a ratio of 10% (v / v) into a 5 L tank containing 3 L of optimized pre-fermentation medium. Control the fermentation temperature at 30°C, use ammonia to control the pH at 5.0-6.0, the rotation speed at 300-800 rpm, and the aeration rate at 1.0-2.0 vvm. Alternately adjust the rotation speed and aeration rate to control dissolved oxygen above 20%. When the residual sugar drops to 1.0 g / L, start adding 600 g / L glucose solution and control the ethanol concentration at 0-10.0 g / L. Collect the fermentation broth after 120 h of incubation. After extraction and testing, the yield of 7-DHC reached 805 mg / L.

[0072] Under unoptimized 5L fermenter conditions, the yield of 7-DHC reached 805 mg / L. Compared to Example 2, despite using the same equipment, the unoptimized conditions resulted in a significant difference in yield. This highlights the importance of optimizing fermentation conditions for improving the synthesis of the target product.

[0073] Example 5: When peptone is used as the organic nitrogen source on the shake flask, trace elements are added.

[0074] (1) Prepare the optimized shake-flask fermentation medium according to step (1) in Example 3. Initially add trace element combination 1. The addition amounts of each component in the combination are as follows: L-glutamate sodium 0.2 g / L, L-histidine 0.1 g / L, L-leucine 0.2 g / L, L-tryptophan 0.5 g / L, uracil 0.2 g / L, ferrous sulfate 0.02 g / L, biotin 30 mg / L, vitamin B1 100 mg / L; other operations are the same as in step (1) in Example 3. Seed liquid preparation and fermentation culture are carried out according to step (2) in Example 3. After culturing for 120 h, the fermentation broth is collected, and the 7-DHC yield is detected. The experimental results are as follows. Figure 1 As shown.

[0075] (2) Prepare the optimized shake-flask fermentation medium according to step (1) in Example 3. Initially add trace element combination 2. The addition amounts of each component in the combination are as follows: L-glutamate sodium 1.0 g / L, L-histidine 0.5 g / L, L-leucine 1.0 g / L, L-tryptophan 2.5 g / L, uracil 1.0 g / L, ferrous sulfate 0.1 g / L, biotin 70 mg / L, and vitamin B1 200 mg / L. Other operations are the same as in step (1) of Example 3. Seed culture preparation and fermentation culture are carried out according to step (2) in Example 3. After culturing for 120 h, the fermentation broth is collected, and the 7-DHC yield is detected. The experimental results are as follows. Figure 1 As shown.

[0076] (3) Prepare the optimized shake-flask fermentation medium according to step (1) in Example 3. Initially add trace element combination 3. The addition amounts of each component in the combination are as follows: L-glutamate sodium 1.2 g / L, L-histidine 0.6 g / L, L-leucine 1.2 g / L, L-tryptophan 3.0 g / L, uracil 1.2 g / L, ferrous sulfate 0.12 g / L, biotin 90 mg / L, and vitamin B12 50 mg / L; other operations are the same as in step (1) of Example 3. Seed liquid preparation and fermentation culture are carried out according to step (2) in Example 3. After culturing for 120 h, the fermentation broth is collected, and the 7-DHC yield is detected. The experimental results are as follows. Figure 1 As shown.

[0077] (4) Prepare the optimized shake-flask fermentation medium according to step (1) in Example 3. Initially add trace element combination 4. The addition amounts of each component in the combination are as follows: L-glutamate sodium 0.5 g / L, L-histidine 0.3 g / L, L-leucine 0.5 g / L, L-tryptophan 1.5 g / L, uracil 0.5 g / L, ferrous sulfate 0.06 g / L, biotin 50 mg / L, vitamin B1 150 mg / L; other operations are the same as in step (1) in Example 3. Seed liquid preparation and fermentation culture are carried out according to step (2) in Example 3. After culturing for 120 h, the fermentation broth is collected, and the 7-DHC yield is detected. The experimental results are as follows. Figure 1 As shown.

[0078] When using peptone as the organic nitrogen source in a shake flask, a trace element supplement is added (see table).

[0079]

[0080]

[0081] Figure 1 The results showed that adding a certain concentration range of trace element combinations to the pre-optimized shake-flask fermentation medium increased the yield of 7-DHC to varying degrees. Simultaneously, the yield of the byproduct yeast sterol was significantly reduced. Specifically, with trace element combination 4, the 7-DHC yield increased from 206 mg / L to a maximum of 356 mg / L, a 73% increase compared to before addition, while the byproduct yeast sterol decreased by 50%. This demonstrates the regulatory effect of trace elements on metabolic pathways and their important role in increasing the yield of the target product and reducing byproducts. Since yeast sterol and 7-DHC have very similar chemical structures, the reduction of byproducts is more conducive to separation and extraction. However, more trace element combinations are not necessarily better; excessive addition can also inhibit 7-DHC synthesis, proving that appropriate adjustment of trace element concentration is crucial for improving 7-DHC yield.

[0082] Example 6: Molasses was used to replace glucose or sucrose in the shake flask.

[0083] The optimized shake-flask fermentation medium was prepared according to step (1) in Example 3. Different amounts of molasses were used to replace glucose and sucrose, and other operations were the same as in step (1) of Example 3. After culturing for 120 h, the fermentation broth was collected, and the 7-DHC yield was detected. The experimental results are shown in the table below.

[0084] Molasses is used instead of glucose or sucrose on the shake flask.

[0085]

[0086] The results in the table show that the optimal 7-DHC yield was achieved with glucose at 33 g / L and molasses at 34 g / L, reaching 454 mg / L, demonstrating the best effect of this combination. This indicates that molasses, as a more economical carbon source alternative, can reduce costs without affecting or even increasing 7-DHC yield, thereby increasing the economic efficiency of the production process.

[0087] Example 7

[0088] Comparison of the effects of adding trace elements when using peptone as the organic nitrogen source in a 5L fermenter

[0089] Following step (1) in Example 4, the optimized pre-fermentation medium was prepared, with the initial addition of a micronutrient combination. The amounts of each component in the combination were as follows: L-glutamate 0.5 g / L, L-histidine 0.3 g / L, L-leucine 0.5 g / L, L-tryptophan 1.5 g / L, uracil 0.5 g / L, ferrous sulfate 0.06 g / L, biotin 50 mg / L, and vitamin B1 150 mg / L. Other operations were the same as in 1.2. After culturing for 120 h, the fermentation broth was collected, and the 7-DHC yield was detected. The experimental results are shown in the table.

[0090] From the table and Figure 2 The results showed that the production of 7-DHC in a 5L fermenter increased to 1200 mg / L after the addition of the trace element combination, which was 50% higher than before the addition. At the same time, the production of the byproduct yeast sterol was significantly reduced, decreasing by 50% compared to before the addition. This further proves that the addition of the trace element combination is beneficial to the synthesis of 7-DHC.

[0091] Comparison of the effects of adding trace elements when using corn steep liquor as the organic nitrogen source in a 5L fermentation tank

[0092] (1) Prepare the optimized pre-fermentation medium according to step (1) in Example 4, replacing 50 g / L of peptone with 50 g / L of corn steep liquor. Other operations are the same as in Example 1. After culturing for 96 h, collect the fermentation broth and detect the 7-DHC yield. The experimental results are shown in the table.

[0093] The results in the table show that when the peptone in the pre-optimized fermentation medium was replaced with corn steep liquor, cell growth was inhibited without the addition of trace elements, and the OD500 concentration at 40 hours was significantly lower. 600 It has almost stagnated, with both biomass and 7-DHC production being very low.

[0094] (2) Based on the above culture medium, add trace element combination 1. The addition amounts of each component in the combination are as follows: L-glutamate sodium 0.2 g / L, L-histidine 0.1 g / L, L-leucine 0.2 g / L, L-tryptophan 0.5 g / L, uracil 0.2 g / L, ferrous sulfate 0.02 g / L, biotin 30 mg / L, and vitamin B1 100 mg / L. Other operations are the same as in Example 1. After culturing for 96 h, collect the fermentation broth and detect the 7-DHC yield. The experimental results are as follows. Figure 3 As shown.

[0095] (3) Based on the above culture medium, add trace element combination 2. The addition amounts of each component in the combination are as follows: L-glutamate sodium 1.0 g / L, L-histidine 0.5 g / L, L-leucine 1.0 g / L, L-tryptophan 2.5 g / L, uracil 1.0 g / L, ferrous sulfate 0.1 g / L, biotin 70 mg / L, and vitamin B1 200 mg / L. Other operations are the same as in Example 1. After culturing for 96 h, collect the fermentation broth and detect the 7-DHC yield. The experimental results are as follows. Figure 3 As shown.

[0096] (4) Based on the above culture medium, add trace element combination 3. The addition amounts of each component in the combination are as follows: L-sodium glutamate 1.2 g / L, L-histidine 0.6 g / L, L-leucine 1.2 g / L, L-tryptophan 3.0 g / L, uracil 1.2 g / L, ferrous sulfate 0.12 g / L, biotin 90 mg / L, and vitamin B12 50 mg / L. Other operations are the same as in Example 1. After culturing for 96 h, collect the fermentation broth and detect the 7-DHC yield. The experimental results are as follows. Figure 3 As shown.

[0097] (5) Based on the above culture medium, add trace element combination 4. The addition amounts of each component in the combination are as follows: L-sodium glutamate 0.5 g / L, L-histidine 0.3 g / L, L-leucine 0.5 g / L, L-tryptophan 1.5 g / L, uracil 0.5 g / L, ferrous sulfate 0.06 g / L, biotin 50 mg / L, and vitamin B1 150 mg / L. Other operations are the same as in Example 1. After culturing for 96 h, collect the fermentation broth and detect the 7-DHC yield. The experimental results are as follows. Figure 3 As shown.

[0098] Comparison of the effects of adding trace elements to different nitrogen sources in a 5L fermenter

[0099] Formula 1 Formula 2 Formula 3 Formula 4 Types of organic nitrogen sources peptone Corn syrup peptone Corn syrup Do trace elements need to be added? yes yes no no <![CDATA[40h bacterial concentration OD 600 > 60 70 50 28 Culture cycle (h) 120 96 120 40 7-DHC production (mg / L) 1350 230 800 129

[0100] From the table above and Figure 3 The results showed that cell growth returned to normal under the condition of adding a combination of trace elements, indicating that when using corn steep liquor as the organic nitrogen source, a certain concentration range of trace element combination must be added to achieve a cell growth level comparable to that when using peptone as the organic nitrogen source. Furthermore, 7-DHC production increased with bacterial concentration and OD... 600 With the increase of [specific element], the 7-DHC yield increased from 800 mg / L to a maximum of 1350 mg / L, and the fermentation cycle was shortened to 96 h. This result once again demonstrates the importance of trace elements in promoting 7-DHC synthesis, especially in larger-scale fermentation processes.

[0101] Example 8: Optimization of corn steep liquor addition in a 5L fermenter

[0102] The fermentation medium was prepared according to Example 1, with corn steep liquor added at concentrations of 30, 40, 50, and 60 g / L. Other procedures were the same as in Example 1. After 96 hours of cultivation, the fermentation broth was collected, and the 7-DHC yield was measured. The experimental results are as follows: Figure 4 As shown.

[0103] As shown in the figure, when the corn steep liquor addition was 40 g / L in a 5L fermenter, the 7-DHC yield reached 2400 mg / L. This is one of the higher yields among all the examples. These results demonstrate the direct impact of the type and amount of organic nitrogen source on 7-DHC synthesis and provide guidance for industrial applications.

[0104] Example 9: Optimization of culture conditions in a 5L tank

[0105] When using peptone as an organic nitrogen source, the ethanol concentration is controlled during the feeding process.

[0106] The fermentation medium for tank fermentation was prepared according to Example 7, and other operations were the same as in Example 4. The ethanol concentration was controlled at 0-5.0 g / L, 5.0-10.0 g / L, and 10.0-15.0 g / L during the feeding process, respectively. The experimental results are shown in the table.

[0107] The effect of ethanol concentration control during feeding when peptone is the organic nitrogen source.

[0108]

[0109]

[0110] The results in the table show that when peptone was used as the organic nitrogen in the 5L fermenter, the 7-DHC yield was 1100 mg / L when the ethanol concentration was controlled at 10.0-15.0 g / L during the feeding process. The yields were similar when the ethanol concentration was controlled at 0-5.0 and 5.0-10.0 g / L, at 1500 mg / L and 1450 mg / L respectively. This indicates that the ethanol concentration needs to be controlled at 0-10.0 g / L during the feeding process to be more conducive to the synthesis of 7-DHC. This suggests that maintaining a low ethanol concentration is necessary for the efficient synthesis of 7-DHC; excessively high ethanol concentrations may inhibit 7-DHC formation.

[0111] When using corn steep liquor as the organic nitrogen source, control of ethanol concentration during feeding is crucial.

[0112] The fermentation medium for tank fermentation was prepared according to Example 8, and other operations were the same as in Example 1. The ethanol concentration was controlled at 0-5.0 g / L and 5.0-10.0 g / L during the feeding process, respectively. The experimental results are shown in the table.

[0113] The Influence of Ethanol Concentration on Feeding Process When Corn Stew is Used as an Organic Nitrogen Source (Table)

[0114] Types of organic nitrogen sources Corn syrup Corn syrup Ethanol concentration during feeding 0-5.0g / L 5.0-10.0g / L 7-DHC production (mg / L) 2400 1540

[0115] The results in the table show that when corn steep liquor was used as the organic nitrogen source in the 5L fermenter, the 7-DHC yields were 2400 mg / L and 1540 mg / L when the ethanol concentration was controlled at 0-5.0 and 5.0-10.0 g / L during the feeding process, respectively. This indicates that the ethanol concentration needs to be controlled within the range of 0-5.0 g / L during the feeding process, which is more conducive to the synthesis of 7-DHC and yields significantly higher yields than under high ethanol concentration conditions. The control range of ethanol concentration during the feeding process needs to be more precise when using corn steep liquor as the organic nitrogen source; this further confirms the influence of ethanol concentration on the synthesis of 7-DHC under different organic nitrogen source conditions.

[0116] Example 10: Fermentation effect when scaled up to a 50L fermenter

[0117] The secondary seed culture was inoculated at a ratio of 10% (v / v) into a 50L tank containing 30L of fermentation medium. The fermentation temperature was controlled at 30℃, the tank pressure at 0.5MPa, and the pH was maintained at 5.0-6.0 using ammonia. The fermentation speed was 300-500 rpm, and the aeration rate was 1.0-2.0 vvm. The speed and aeration rate were alternately adjusted to maintain dissolved oxygen above 20%. When the residual sugar dropped to 1.0 g / L, a 600 g / L glucose solution was added, and the ethanol concentration was controlled at 0-5.0 g / L. After culturing for 88 hours, the fermentation broth was collected. Extraction and testing confirmed that the fermentation process was successfully scaled up to a 50L fermenter, with a maximum 7-DHC yield of 2780 mg / L. This result not only validates the process from small-scale to pilot-scale and then to industrial-scale production, but also demonstrates the good scalability and potential commercial value of the technology of this invention.

[0118] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A fermentation medium, characterized in that: Its components and concentrations include: L-tryptophan: 0.5-2.5 g / L; L-leucine: 0.2-1.2 g / L; L-monosodium glutamate: 0.2-1.0 g / L; uracil: 0.2-1.0 g / L; ferrous sulfate: 0.02-0.1 g / L; biotin: 30-70 mg / L; vitamin B1: 100-200 mg / L; L-histidine: 0.1-0.6 g / L; and at least one carbon source and at least one nitrogen source.

2. The fermentation medium of claim 1, characterized in that: The concentrations of each component are as follows: L-tryptophan 1.5-2.5 g / L, L-leucine 0.5-1.0 g / L, L-sodium glutamate 0.5-1.0 g / L, uracil 0.5-1.0 g / L, ferrous sulfate 0.06-0.1 g / L, biotin 50-70 mg / L, vitamin B1 50-200 mg / L, and L-histidine 0.3-0.5 g / L.

3. The fermentation medium of claim 1, wherein: The concentrations of each component are as follows: L-tryptophan 1.5-2.0 g / L, L-leucine 0.5-0.8 g / L, L-sodium glutamate 0.5-0.8 g / L, uracil 0.5-0.8 g / L, ferrous sulfate 0.06-0.08 g / L, biotin 50-60 mg / L, vitamin B1 50-180 mg / L, and L-histidine 0.3-0.4 g / L.

4. The fermentation medium according to claim 1, characterized in that: The carbon source is selected from glucose, sucrose, molasses and mixtures thereof, with a concentration of 10-100 g / L.

5. The fermentation medium according to claim 4, characterized in that: The glucose concentration is 20-70 g / L; the molasses concentration is 0-50 g / L.

6. The fermentation medium according to claim 1, characterized in that: The nitrogen source can be an organic nitrogen source or an inorganic nitrogen source, with a concentration of 10-80 g / L.

7. The fermentation medium according to claim 6, characterized in that: The nitrogen source is corn steep liquor at a concentration of 25-60 g / L.

8. The application of the fermentation medium as described in claim 1 in the fermentation process for increasing the yield of 7-DHC in Saccharomyces cerevisiae, characterized in that: The process includes the following steps: controlling dissolved oxygen and sugar concentration during fermentation, controlling ethanol concentration at 0-15 g / L, and collecting the fermentation broth at the end of fermentation.

9. The application according to claim 8, characterized in that: Control the ethanol concentration to 0-10 g / L.

10. The application according to claim 8, characterized in that: Control the ethanol concentration to 0-5.0 g / L.

11. The application according to claim 8, characterized in that: The dissolved oxygen control method is as follows: the rotation speed and ventilation rate are alternately adjusted to control the dissolved oxygen to be above 20%, preferably above 40%.

12. The application according to claim 11, characterized in that: The specified rotational speed is 150-800 rpm, and the air volume is 0.5-2.0 vvm.

13. The application according to claim 8, characterized in that: The method for controlling sugar concentration is as follows: when the residual sugar drops to 0.5-2.0 g / L, start adding 300-1000 g / L glucose solution.

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