Mixed fermentation method for producing salidroside from escherichia coli and reducing metabolic accumulation of by-product acetic acid
By using a three-stage fermentation method and the auxiliary strain Bacillus megaterium, the problem of acetic acid accumulation in Escherichia coli fermentation was solved, thereby increasing the yield of rhodioloside and fermentation efficiency.
Patent Information
- Application Number
- CN202511096044.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-10-28
AI Technical Summary
During E. coli fermentation, acetic acid accumulation has a toxic effect on the bacteria, leading to growth inhibition and reduced production of the target metabolite rhodioloside. Therefore, effectively reducing acetic acid accumulation has become a key issue.
A three-stage fermentation method was adopted. First, the glycosyltransferase mutant SiUGT1R47K was used for the first stage of fermentation. Then, when acetic acid accumulated to a certain level, Bacillus megaterium was inoculated for the second stage of fermentation. The third stage of fermentation was carried out by controlling the fermentation conditions and IPTG induction, and glucose was added to maintain an appropriate concentration.
It significantly reduces acetic acid accumulation, increases rhodioloside yield by 118-153%, shortens fermentation time by 21-37%, reduces alkali usage, and improves the microbial growth environment.
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Figure CN120843631A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a mixed fermentation method for producing rhodioloside from Escherichia coli and reducing the accumulation of the byproduct acetic acid metabolism. Background Technology
[0002] Salidroside is a naturally occurring glycoside found in plants of the Rhodiola genus. It is formed by the glycosylation reaction of p-hydroxyphenylethanol (tyrosol) and UDP-glucose, and its chemical name is p-hydroxyphenylethyl-β-D-glucosinolate. Salidroside is a naturally occurring phenylpropionate compound found in various medicinal plants, including Rhodiola rosea, Ligustrum lucidum, and Aconitum tanguticum. Its molecular formula is C2. 17 H 20 O7, with a relative molecular mass of 300 and a melting point of 158-160℃, is extremely soluble in water, readily soluble in ethanol, and slightly soluble in ether and acetone, appearing as colorless, transparent, needle-like crystals. Rhodioloside was first discovered in Rhodiola rosea and has since been found in other plants of the Ligustrum genus. Rhodioloside is the most important medicinal component of Rhodiola rosea plants, possessing various effects including anti-fatigue, anti-hypoxia, anti-aging, anti-cancer, anti-tumor, anti-hypothyroidism, anti-inflammation, protection of cardiovascular system activity, protection of central nervous system activity, and cognitive enhancement. It has wide applications in food, health products, daily chemicals, and medicine.
[0003] The traditional method for producing rhodioloside is plant extraction, using Rhodiola rosea as the primary plant. Rhodiola rosea typically grows at altitudes of 3500-5000 meters. my country has over 70 species of Rhodiola rosea, distributed in the high-altitude mountainous regions of Northeast, North, Northwest, and Southwest China. These plants are difficult to cultivate artificially, making them quite precious. In recent years, they have been designated as endangered species by the state, and harvesting is prohibited. Therefore, the high cost, high pollution, and low yield of this plant extraction method have become limiting factors for its utilization.
[0004] Bio-fermentation is currently the best choice for source substitution, as its low cost, high efficiency, and low pollution make it more sustainable and meet market demands. However, during microbial fermentation (especially E. coli fermentation), when the microorganisms utilize glucose as a carbon source, the byproduct acetic acid is also produced and accumulated throughout the entire growth cycle. Excessive accumulation of acetic acid can be toxic to the microorganisms and can also have an irreversible inhibitory effect on their metabolism. Reports show that acetic acid concentrations of 5–10 g / L can observably inhibit the lag phase, maximum specific growth rate, cell concentration, and final protein yield. When the acetic acid concentration is greater than 10 or 20 g / L, cell growth will stop, and when the acetic acid concentration in the fermentation system is greater than 12 g / L, the expression of exogenous proteins is completely inhibited. Therefore, reducing the production and accumulation of acetic acid is a crucial aspect of rhodioloside microbial fermentation. Summary of the Invention
[0005] In view of this, the present invention aims to propose a mixed fermentation method for Escherichia coli to produce rhodioloside and reduce the accumulation of the byproduct acetic acid metabolism, so as to solve the technical problem of acetic acid accumulation.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: Furthermore, the mixed fermentation method for producing the metabolite rhodioloside includes the following steps: The Escherichia coli strain was inoculated into a fermentation medium sterilized by high temperature steam for the first stage of fermentation; when the acetic acid in the fermentation system reached a certain level, the seed liquid of auxiliary fermentation bacteria was inoculated for the second stage of fermentation; when the OD of the bacterial cells in the fermentation system reached a certain value, IPTG was added to induce the third stage of fermentation, with the glucose content in the fermentation system as the control index, and specific culture medium was added for feeding operations.
[0007] This invention provides a mixed fermentation method for producing rhodioloside from Escherichia coli and reducing the accumulation of the byproduct acetic acid metabolism. The steps of the method are as follows: Step 1: The glycosyltransferase mutant SiUGT1 R47K The genetically engineered bacteria are inoculated into a fermenter for the first stage of fermentation; Step 2: When the acetic acid content of the byproduct in the fermentation system reaches 1.5-2.0 g / L, inoculate with Bacillus megaterium (BME). Bacillus megaterium The second stage of fermentation will then begin; Step 3: After the OD of the bacterial cells in the system reaches 25-30, add an inducer to carry out the third stage of fermentation, maintain the glucose content at 3-5 g / L, and obtain rhodioloside.
[0008] Further specifying, when the bacterial cell OD reaches 3 in step 1, the glycosyltransferase mutant SiUGT1... R47K The genetically engineered bacteria were inoculated into a 1000 L fermenter with a loading coefficient of 0.6 at a 5% inoculum. The pH was 7.0±0.15, the fermentation temperature was 36-38℃, the initial aeration ratio was 0.53-0.67 V / Vm, and the initial rotation speed was 200 r / min. The dissolved oxygen during fermentation was controlled between 30-35% by increasing the aeration ratio and rotation speed.
[0009] Further specifying, the glycosyltransferase mutant SiUGT1 R47K Methods for obtaining genetically engineered bacteria: Introducing bacteria containing the phenylpyruvate decarboxylase gene... ARO10 Ethanol dehydrogenase gene ADH6 phosphoglucosuricase gene pgm and UDP-glucose pyrophosphorylase gene galUOverexpression plasmids and genes containing mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. aroG fbr Cyclohexadiene dehydrogenase gene tyrC and mutant glycosyltransferase gene SiUGT1 R47K The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered bacteria.
[0010] To further specify, the OD of Bacillus megaterium in step 2 is 5-8.
[0011] Further specified, in step 2 the inoculum size is 4%, the initial pH is 6.9±0.1, the fermentation temperature is 36-38℃, the initial aeration ratio is 0.53-0.67 V / Vm, and the constant rotation speed is 200 r / min.
[0012] Further specifying, in step 2, when the second-stage fermentation reaches 15-19 hours, IPTG with a final fermentation concentration of 0.5 mM is added to induce expression, and then the third-stage fermentation is carried out.
[0013] Further restrictions were imposed, with the temperature reduced to 29°C, dissolved oxygen controlled at 30%, and IPTG at a final fermentation concentration of 0.5 mM added for induction.
[0014] Further specifying, in step 3, glucose content is maintained using supplemental culture medium.
[0015] Further specified, the feed medium is: 500 g / L glucose, 20 g / L potassium dihydrogen phosphate, 20 g / L dipotassium hydrogen phosphate, 5 g / L magnesium sulfate, and 15 g / L peptone.
[0016] Further specify that fermentation should be completed in 47-55 hours.
[0017] Compared with the prior art, the beneficial effects of the present invention are: Bacillus megaterium ( Bacillus megaterium This method balances the pH of the fermentation system, resulting in minimal pH fluctuations after inoculation. This significantly reduces the total amount of alkali used for pH adjustment, decreasing ion concentration and mitigating the damage caused by high ion concentrations to the bacteria. It effectively reduces the production and accumulation of acetic acid, a metabolic byproduct of E. coli fermentation, relieving the inhibitory effect of acetic acid on the growth and metabolism of the target bacteria, E. coli. It also significantly increases the accumulation of rhodioloside, greatly shortens fermentation time, and ultimately increases the final content of rhodioloside by 118-153%, while significantly reducing fermentation time by 21-37%.
[0018] [Biological Preservation Information]: A type of Bacillus megaterium ( Bacillus megateriumStrain LXB4070 was deposited on March 4, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 21867, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description
[0019] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings: Figure 1 The HPLC chromatogram shows the content of rhodioloside in the fermentation broth. Figure 2 This is a comparison chart of the yield of the fermentation method (Example 3) involved in this invention and the traditional fermentation method (the only difference between this invention and the traditional method is that there is no auxiliary bacteria added, but the other steps are the same as those of this invention); Figure 3 This is a comparison chart of the fermentation time of the fermentation method (Example 3) involved in this invention and a traditional fermentation method (the only difference between this invention and the traditional method is that no auxiliary bacteria are added, but the other steps are the same as those of this invention); Figure 4 The graphs show a comparison of the dynamic changes in cell OD during the fermentation process of Examples 1-3 and Comparative Examples 1-5. Figure 5 This is a comparison chart showing the amount of alkali solution used to adjust pH during the fermentation process of Examples 1-3 and Comparative Examples 1-5; Figure 6 This is a comparison chart showing the real-time changes in the content of acetic acid, a byproduct, in the fermentation broth during the fermentation process of Examples 1-3 and Comparative Examples 1-5. Figure 7 The graph shows a comparison of the yields of rhodiolosides from fermentation in Examples 1-3 and Comparative Examples 1-5. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.
[0021] Overexpression originates from Escherichia coli (E. coli) Escherichia coli ) phosphoglucose mutase gene pgm UDP-glucose pyrophosphorylase gene galU、 Derived from brewer's yeast ( Saccharomycescerevisiae ) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana mutant glycosyltransferase gene AtUGT85A1 A21G Recombinant plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 A21G From Zhou Jingwen's team at Jiangnan University (Engineering) Escherichia coli for Efficient De Novo Synthesis of Salidroside ） .
[0022] Overexpression plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 A21G glycosyltransferase gene AtUGT85A1 A21G Replace with information from Sesame ( Sesamum indicum mutant glycosyltransferase SiUGT1 R47K SEQ ID NO: 1 (Mutant glycosyltransferase SiUGT1) R47K (nucleotide sequence)
[0023] The seed culture medium for Escherichia coli includes: glucose 10-15 g / L, peptone 10-15 g / L, ammonium sulfate 3-5 g / L, potassium dihydrogen phosphate 2-5 g / L, dipotassium hydrogen phosphate 2-5 g / L, magnesium sulfate 1-2 g / L, and trace element stock solution 1-2 mL.
[0024] The auxiliary fermentation medium for the seed culture of the fermentation bacteria was: 15 g / L glycerol, 16 g / L yeast powder, 9 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, 5 g / L citric acid monohydrate, 1 mL / L trace element stock solution, and 0.3‰ defoamer.
[0025] The three-stage fed culture medium consisted of 500 g / L glucose, 20 g / L potassium dihydrogen phosphate, 20 g / L dipotassium hydrogen phosphate, 5 g / L magnesium sulfate, and 15 g / L peptone.
[0026] Example 1. This embodiment provides a mixed fermentation method for Escherichia coli to produce rhodioloside and reduce the accumulation of the byproduct acetic acid metabolism, comprising the following steps: 1. Cultivation of the target strain: The target strain of Escherichia coli was streaked from the frozen glycerol tube onto a solid culture medium and cultured at 35°C for 24 h to obtain the original planar seed. The original slant seed was then inoculated into a 3 L Erlenmeyer flask containing liquid seed medium and cultured at 37°C and a shaking speed of 200 r / min for 15 h before use. 2. Propagation of the target strain: The primary shake flask seed culture from step 1 was inoculated at a rate of 5% into a 50 L fermenter with a loading coefficient of 0.6. The seed tank was sterilized at 121℃ for 30 min beforehand. The initial pH of the culture medium was 6.9, the fermentation temperature was 36℃, the initial aeration ratio was 0.53 V / Vm, and the initial rotation speed was 150 r / min. The dissolved oxygen during fermentation was controlled at 30% by increasing the aeration ratio and rotation speed.
[0027] 3. First-stage fermentation: When the OD of the seed culture cells in step 2 reaches 3, inoculate 5% into a 1000L fermenter with a loading coefficient of 0.6. The fermentation medium is pre-sterilized at 121℃ for 30 min, with an initial pH of 6.85, a fermentation temperature of 36℃, an initial aeration ratio of 0.53 V / Vm, and an initial rotation speed of 200 r / min. Dissolved oxygen is controlled at 30% by increasing aeration and rotation speed. When the first-stage fermentation reaches 7-10 h and the acetic acid content (a byproduct) in the fermentation system reaches 1.5-2.0 g / L, introduce the auxiliary fermentation bacteria *Bacillus megaterium* for the second-stage fermentation. 4. Second stage fermentation: The auxiliary fermentation bacteria (Bacillus megaterium) were inoculated using conventional methods and cultured in pre-sterilized seed culture medium with an initial pH of 6.8, a fermentation temperature of 36℃, an initial aeration ratio of 0.53 V / Vm, and a constant rotation speed of 200 r / min.
[0028] 5. When fermentation has progressed for 7 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.53 g / L, the seed culture of auxiliary fermentation bacteria with an OD of 5 is inoculated at a rate of 4%, and the second stage of fermentation begins.
[0029] 6. Third stage fermentation: When fermentation reaches 15 h and the OD of the system cells (mixed microorganisms) reaches 25, the culture temperature is reduced to 29℃, the dissolved oxygen is controlled at 30%, and IPTG with a final fermentation concentration of 0.5 mM is added to induce expression and carry out the three-stage fermentation.
[0030] 7. Throughout the entire process, the glucose content in the system was monitored. When the glucose content in the fermentation medium was lower than 3 g / L, a feeding operation was performed to control the glucose content between 3-5 g / L. At the same time, the cell OD, the alkaline solution used to adjust the pH, and the content of the by-product acetic acid were monitored at regular intervals.
[0031] 8. When the fermentation time reached 47 h, the fermentation was stopped. At this time, the content of rhodioloside in the fermentation broth was measured to be 35.2 g / L.
[0032] Example 2. This embodiment provides a mixed fermentation method for Escherichia coli to produce rhodioloside and reduce the accumulation of the byproduct acetic acid metabolism, comprising the following steps: 1. Same as step 1 of Example 1; 2. The seed culture from step 1 was inoculated into a 50 L fermenter with a loading coefficient of 0.6 at a 5% inoculation rate. The seed tank was sterilized at 121℃ for 30 min beforehand. The initial pH of the culture medium was 7.0, the fermentation temperature was 37℃, the initial aeration ratio was 0.6 V / Vm, and the initial rotation speed was 150 r / min. The dissolved oxygen during fermentation was controlled at 30% by increasing the aeration ratio and rotation speed.
[0033] 3. When the OD of the bacterial cells in the seed culture of step 2 reaches 3, inoculate 5% into a 1000 L fermenter with a loading coefficient of 0.6. The fermentation medium is sterilized at 121℃ for 30 min in advance. The initial pH of the medium is 7.0, the fermentation temperature is 37℃, the initial aeration ratio is 0.6 V / Vm, and the initial rotation speed is 200 r / min. By increasing the aeration rate and rotation speed, the dissolved oxygen during fermentation is controlled at 35%.
[0034] 4. The auxiliary fermentation bacteria were inoculated using conventional methods and cultured in pre-sterilized seed culture medium with an initial pH of 6.9, a fermentation temperature of 37℃, an initial aeration ratio of 0.6 V / Vm, and a constant rotation speed of 200 r / min.
[0035] 5. When fermentation has progressed to 8.5 hours and the content of acetic acid, a byproduct, in the fermentation broth is measured to be 1.68 g / L, an auxiliary fermentation bacteria seed liquid with an OD of 7 is inoculated at a rate of 5%, and the second stage of fermentation begins.
[0036] 6. When fermentation reaches 17 h and the cell OD of the system reaches 27.3, the culture temperature is reduced to 30℃, the dissolved oxygen is controlled at 35%, and IPTG with a final fermentation concentration of 0.5 mM is added to induce expression, and the three-stage fermentation is carried out.
[0037] 7. Throughout the entire process, the glucose content in the system was monitored. When the glucose content in the fermentation medium was lower than 3 g / L, a feeding operation was performed to control the glucose content between 3-5 g / L. At the same time, the cell OD, the alkaline solution used to adjust the pH, and the content of the by-product acetic acid were monitored at regular intervals.
[0038] 8. When the fermentation time reached 51 h, the fermentation was stopped. At this time, the content of rhodioloside in the fermentation broth was measured to be 36.8 g / L.
[0039] Example 3. This embodiment provides a mixed fermentation method for Escherichia coli to produce rhodioloside and reduce the accumulation of the byproduct acetic acid metabolism, comprising the following steps: 1. Same as step 1 of Example 1; 2. The seed culture from step 1 was inoculated into a 50L fermenter with a loading coefficient of 0.6 at a 5% inoculation rate. The seed tank was sterilized at 121℃ for 30 min beforehand. The initial pH of the culture medium was 7.1, the fermentation temperature was 38℃, the initial aeration ratio was 0.67 V / Vm, and the initial rotation speed was 150 r / min. The dissolved oxygen during fermentation was controlled to be between 35% by increasing the aeration ratio and rotation speed.
[0040] 3. When the OD of the bacterial cells in the seed culture of step 2 reaches 4, inoculate 5% into a 1000 L fermenter with a loading coefficient of 0.6. The fermentation medium is sterilized at 121℃ for 30 min in advance. The initial pH of the medium is 7.15, the fermentation temperature is 38℃, the initial aeration ratio is 0.67 V / Vm, and the initial rotation speed is 200 r / min. By increasing the aeration rate and rotation speed, the dissolved oxygen during fermentation is controlled to be between 35%.
[0041] 4. The auxiliary fermentation bacteria were inoculated using conventional methods and cultured in pre-sterilized seed culture medium with an initial pH of 7.0, a fermentation temperature of 38℃, an initial aeration ratio of 0.67 V / Vm, and a constant rotation speed of 200 r / min.
[0042] 5. When fermentation has progressed for 10 hours and the content of acetic acid, a byproduct, in the fermentation broth is 2.0 g / L, the seed culture of auxiliary fermentation bacteria with an OD of 8 is inoculated at a rate of 6%, and the second stage of fermentation begins.
[0043] 6. When fermentation reaches 19 h and the cell OD of the system reaches 30, the culture temperature is reduced to 31℃, the dissolved oxygen is controlled at 35%, and IPTG with a final fermentation concentration of 0.5 mM is added to induce expression, and the three-stage fermentation is carried out.
[0044] 7. Throughout the entire process, the glucose content in the system was monitored. When the glucose content in the fermentation medium was lower than 3 g / L, a feeding operation was performed to control the glucose content between 3-5 g / L. At the same time, the cell OD, the alkaline solution used to adjust the pH, and the content of the by-product acetic acid were monitored at regular intervals.
[0045] 8. When the fermentation time reaches 55 h, stop the fermentation. At this time, the content of rhodioloside in the fermentation broth is measured to be 38 g / L.
[0046] To more clearly illustrate the advantages and innovations of this invention, a comparative fermentation experiment was conducted, as follows: Comparative Example 1. 1. Same as Example 1; 2. Same as Example 1; 3. Same as Example 1; 4. Use water instead of auxiliary fermentation bacteria and sterilize it in advance.
[0047] 5. When fermentation has been going on for 7 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.53 g / L, inoculate with 4% of the water that was sterilized in step 4, and enter the second stage of fermentation.
[0048] 6. Same as Example 1; 7. Same as Example 1; 8. When the fermentation time reached 47 h, the fermentation was stopped. At this time, the content of rhodioloside in the fermentation broth was measured to be 11.2 g / L.
[0049] Comparative Example 2. 1. Same as Example 2; 2. Same as Example 2; 3. Same as Example 2; 4. Use water instead of auxiliary fermentation bacteria and sterilize it in advance.
[0050] 5. When fermentation has progressed to 8.5 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.66 g / L, inoculate with water that was sterilized in step 4 in advance, with an inoculation volume of 5%, and enter the second stage of fermentation.
[0051] 6. Same as Example 2; 7. Same as Example 2; 8. When the fermentation time reached 51 h, the fermentation was stopped. At this time, the content of rhodioloside in the fermentation broth was measured to be 12.1 g / L.
[0052] Comparative Example 3. 1. Same as Example 3; 2. Same as Example 3; 3. Same as Example 3; 4. Use water instead of auxiliary fermentation bacteria and sterilize it in advance.
[0053] 5. When fermentation has been going on for 10 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.95 g / L, water that was sterilized in step 4 is inoculated at a rate of 6%, and the second stage of fermentation begins.
[0054] 6. Same as Example 3; 7. Same as Example 3; 8. When the fermentation time reaches 55 h, the fermentation is stopped. At this time, the content of rhodioloside in the fermentation broth is measured to be 12.9 g / L.
[0055] Comparative Example 4. 1. Same as Example 3; 2. Same as Example 3; 3. Same as Example 3; 4. Use water instead of auxiliary fermentation bacteria and sterilize it in advance.
[0056] 5. When fermentation has been going on for 10 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.95 g / L, water that was sterilized in step 4 is inoculated at a rate of 6%, and the second stage of fermentation begins.
[0057] 6. Same as Example 3; 7. Same as Example 3; 8. When the fermentation time reaches 70 h, the fermentation is stopped. At this time, the content of rhodioloside in the fermentation broth is measured to be 16.1 g / L.
[0058] Comparative Example 5. 1. Same as Example 3; 2. Same as Example 3; 3. Same as Example 3; 4. Use water instead of auxiliary fermentation bacteria and sterilize it in advance.
[0059] 5. When fermentation has been going on for 10 hours and the content of acetic acid, a byproduct, in the fermentation broth is 1.95 g / L, water that was sterilized in step 4 is inoculated at a rate of 6%, and the second stage of fermentation begins.
[0060] 6. Same as Example 3; 7. Same as Example 3; 8. When the fermentation time reaches 75 hours, fermentation is stopped. At this point, the rhodioloside content in the fermentation broth reaches 15.2 g / L. The specific embodiments of the present invention disclosed above are only for illustrating the invention. The specific embodiments do not describe all details exhaustively, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.
[0061] Depend on Figure 4 It can be seen that the OD changes in the examples follow a normal trend throughout the fermentation cycle, while the OD changes in the comparative examples are combined with... Figure 6 The acetic acid content changed gradually after 10-15 hours of fermentation. In the comparative experiment, the acetic acid content began to increase, and the corresponding OD liquid began to be affected. When the fermentation time was about 40 hours, the acetic acid had accumulated to more than 10 g / L, which led to a significant inhibition of cell growth. Compared with the example, the OD was significantly lower, showing that the present invention has a significant beneficial effect on normal cell growth.
[0062] The amount of alkali solution used in Examples 1-3 was only 15, 14.3 and 16 L, respectively, while the amounts used in Comparative Examples 1-5 were 16, 18, 22, 25, 24 and 27 L, respectively. It can be seen that the amount of alkali solution used in the Examples was significantly lower than that in the Comparative Examples, showing an advantage.
[0063] Depend on Figure 6 In conjunction with the operation of the embodiments, after about 10 hours, when the auxiliary fermentation bacteria Bacillus megaterium was introduced into the embodiments, the accumulation of acetic acid began to tend to a balanced state, with the acetic acid content being less than 1 g / L. In contrast, after 10 hours, due to the different operation compared with the embodiments, the accumulation of acetic acid in Comparative Examples 1-5 gradually increased, and the final accumulation was all higher than 15 g / L, with Comparative Example 5 reaching a maximum of 23 g / L, which has been verified to exceed the specific tolerance limit.
[0064] Depend on Figure 7 As can be seen, the final fermentation rhodioloside content in the examples reached 35.2, 36.8 and 38 g / L, respectively, while the content in the comparative examples was between 11.2 and 16.1 g / L, demonstrating the advantage of this invention in increasing the rhodioloside content.
Claims
1. A mixed fermentation method for producing rhodioloside from Escherichia coli and reducing the accumulation of the byproduct acetic acid, characterized in that, The steps of the method are as follows: Step 1: The glycosyltransferase mutant SiUGT1 R47K The genetically engineered bacteria are inoculated into a fermenter for the first stage of fermentation; Step 2: When the acetic acid content of the byproduct in the fermentation system reaches 1.5-2.0 g / L, inoculate with Bacillus megaterium (BME). Bacillus megaterium The second stage of fermentation will then begin; Step 3: After the OD of the bacterial cells in the system reaches 25-30, add an inducer to carry out the third stage of fermentation, maintain the glucose content at 3-5 g / L, and obtain rhodioloside.
2. The mixed fermentation according to claim 1, characterized in that, When the bacterial cell OD reaches 3 in step 1, the glycosyltransferase mutant SiUGT1... R47K The genetically engineered bacteria were inoculated into a 1000L fermenter with a loading coefficient of 0.6 at a 5% inoculum. The pH was 7.0±0.15, the fermentation temperature was 36-38℃, the initial aeration ratio was 0.53-0.67 V / Vm, and the initial rotation speed was 200 r / min. The dissolved oxygen during fermentation was controlled between 30-35% by increasing the aeration ratio and rotation speed.
3. The mixed fermentation according to claim 2, characterized in that, glycosyltransferase mutant SiUGT1 R47K Methods for obtaining genetically engineered bacteria: Introducing bacteria containing the phenylpyruvate decarboxylase gene... ARO10 Ethanol dehydrogenase gene ADH6 phosphoglucosuricase gene pgm and UDP-glucose pyrophosphorylase gene galU Overexpression plasmids and genes containing mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. aroG fbr Cyclohexadiene dehydrogenase gene tyrC and mutant glycosyltransferase gene SiUGT1 R47K The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered bacteria.
4. The mixed fermentation according to claim 1, characterized in that, In step 2, the OD of Bacillus megaterium is 5-8.
5. The mixed fermentation according to claim 1, characterized in that, In step 2, the inoculum size was 4%, the initial pH was 6.9±0.1, the fermentation temperature was 36-38℃, the initial aeration ratio was 0.53-0.67 V / Vm, and the constant rotation speed was 200 r / min.
6. The mixed fermentation according to claim 1, characterized in that, In step 2, when the second-stage fermentation reaches 15-19 h, IPTG with a final fermentation concentration of 0.5 mM is added to induce expression, and then the third-stage fermentation is carried out.
7. The mixed fermentation according to claim 1, characterized in that, The temperature was lowered to 29℃, dissolved oxygen was controlled at 30%, and IPTG with a final fermentation concentration of 0.5 mM was added for induction.
8. The mixed fermentation according to claim 1, characterized in that, In step 3, glucose levels are maintained using supplemental culture medium.
9. The mixed fermentation method according to claim 8, characterized in that, The supplemental culture medium consisted of 500 g / L glucose, 20 g / L potassium dihydrogen phosphate, 20 g / L dipotassium hydrogen phosphate, 5 g / L magnesium sulfate, and 15 g / L peptone.
10. The mixed fermentation according to claim 1, characterized in that, Fermentation is complete in 47-55 hours.