Method for screening chiral inositol producing strain by utilizing culture medium containing inositol and glutathione
By adding inositol and glutathione to the culture medium, combined with gradient dilution, selection culture and fermentation induction, recombinant E. coli with high chiral inositol conversion rate was screened, and the screening efficiency and strain stability in the prior art were solved. The problem of sexual insemination has achieved efficient and stable production of hypersonic inositol.
Patent Information
- Application Number
- CN202411367770.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2024-12-27
AI Technical Summary
The existing screening methods are difficult to efficiently and stably produce recombinant E. coli with high chiral inositol conversion rate, and the screening efficiency is low, and the resulting strain is insufficient.
The method of screening chiral inositol production bacteria using inositol and glutathione-containing culture medium is used to screen for recombinant with high chiral inositol conversion through gradient dilution, selection culture, colony selection, liquid culture and fermentation induction. E. coli, and the best bacterial combination was selected through HPLC detection.
The screening success rate was significantly improved, and the obtained strains had higher chiral inositol conversion rate, the optimal conversion rate could reach 10%, and were easy to operate and low cost, which was suitable for large-scale industrial production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for screening chiral inositol-producing bacteria by using a culture medium containing inositol and glutathione. Background Art
[0002] Recombinant Escherichia coli is often used to produce a variety of valuable compounds, including chiro-inositol. However, existing screening methods still face challenges in efficiently and stably producing recombinant Escherichia coli with ideal chiro-inositol conversion rates. Existing screening culture media often fail to fully protect the redox environment inside the bacteria, resulting in low screening efficiency and a low chance of screening strains with high chiro-inositol conversion rates. Summary of the invention
[0003] The technical problem to be solved by the present invention is to provide a method for screening chiral inositol-producing bacteria using a culture medium containing inositol and glutathione, which overcomes the defects in the prior art. By improving the culture medium formula and culture conditions, recombinant Escherichia coli with a high chiral inositol conversion rate can be screened out in a targeted and efficient manner, and the obtained strain has strong stability and can be applied to the production of chiral inositol.
[0004] In order to solve the above technical problems, the technical solution of the present invention is:
[0005] The method for screening chiral inositol producing bacteria using a culture medium containing inositol and glutathione comprises the following steps:
[0006] a. Recombinant Escherichia coli containing inositol dehydrogenase and recombinant Escherichia coli containing inositol monoketone isomerase were diluted with sterile water to obtain dilutions;
[0007] b. The dilution in step a was inoculated onto a selective medium, wherein the selective medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L glutathione, and the rest was deionized water, with a natural pH value; and cultured at 37 ° C for 12 to 16 h;
[0008] c. In step b, the colonies cultured on the culture medium with uniform single color, raised colonies and a colony diameter of 1 to 2 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; cultured at a constant temperature of 37°C for 12 to 16 hours;
[0009] d. A portion of the colony obtained in step c was used for preservation, and a portion was inoculated into a shake flask containing 50 ml of liquid culture medium, wherein the liquid culture medium was formulated as 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.6 to 2.0;
[0010] e. The single colony cultured in the shake flask in step d was inoculated into 1L TB medium at an inoculum size of 1% for fermentation culture, and the culture was cultured at 37°C and 220rpm until the OD600 value of the bacterial solution was 1.7, and then the inducer IPTG was added at a final concentration of 1.0mM. The fermentation was completed after constant temperature induction at 37°C for 5 hours; the fermentation broth was centrifuged to collect the bacteria, and then the collected recombinant Escherichia coli containing inositol dehydrogenase (5g) and the recombinant Escherichia coli containing inositol monoketone isomerase (10g) were randomly paired and combined in a weight ratio of 1:2, 15g of bacteria were weighed, and the bacteria were resuspended in 45ml of physiological saline to obtain a resuspended solution, and then the resuspended solution was added to 1L of substrate, and the reaction was carried out at 37°C for 24h to obtain a conversion solution; the content of chiral inositol in the conversion solution was detected by HPLC, and the best bacterial combination was selected.
[0011] Preferably, the gradient dilution in step a is from 10 -1 to 10 -8 .
[0012] Preferably, the step b is carried out by constant temperature culture at 37° C. for 14 hours.
[0013] Preferably, in step c, the colonies are white in morphology, have raised colonies, and have a colony diameter of 1 to 2 mm; and are cultured at a constant temperature of 37° C. for 14 hours.
[0014] Preferably, in step d, the colonies are white in morphology, have raised colonies, and have a colony diameter of 1 to 2 mm; and the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 1.8.
[0015] Preferably, the substrate in step e is prepared by 50 g / L inositol, 50 mM phosphate buffer with a pH of 7, and the rest is purified water, and the pH value of the substrate is 7.
[0016] Preferably, the components of the TB culture medium in step e are 12 g / L peptone, 24 g / L yeast powder, 4 ml / L glycerol, and the pH value is natural.
[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0018] The invention adds inositol and glutathione into the selection culture medium to carry out selection culture and screening, thereby improving the success rate of screening.
[0019] The recombinant Escherichia coli screened by the present invention has a higher chiro-inositol conversion rate, and the optimal conversion rate can reach 10%, which is significantly improved compared with the prior art;
[0020] The screening method of the present invention is simple to operate and has low cost, and the screened strains are suitable for large-scale industrial production;
[0021] In conclusion, the method of the present invention is simple and can efficiently screen out recombinant Escherichia coli with a high chiral inositol conversion rate, thereby obtaining a strain with high conversion rate and strong stability. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 HPLC chart of the first group of products of Example 1 of the present invention;
[0023] Figure 2 This is the HPLC chart of D-chiro-inositol standard. DETAILED DESCRIPTION
[0024] The technical solution of the present invention is further described below in conjunction with embodiments:
[0025] Embodiment 1:
[0026] a. Recombinant E. coli containing inositol dehydrogenase and recombinant E. coli containing inositol monoketone isomerase were diluted with sterile water to obtain 10 -1 to 10 -8 diluent.
[0027] b. Add the result in step a to 10 -1 to 10 -8 The dilutions were inoculated on the selection medium, the formula of which was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L glutathione, and the rest was deionized water, with a natural pH value; and cultured at a constant temperature of 37°C for 14 hours.
[0028] c. After the culture in step b, the colonies on the culture medium with white color, raised colonies and a colony diameter of 1 to 2 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; the culture is kept at 37° C. for 14 hours; a portion of the colonies with white color, raised colonies and a colony diameter of 1 to 2 mm is selected for preservation and the recombinant Escherichia coli containing inositol dehydrogenase is named A101, A102, A103, A104, and A105, and the recombinant Escherichia coli containing inositol monoketone isomerase is named B101, B102, B103, B104, and B105, and the other portion is transferred to a liquid culture medium for culture.
[0029] d. The strains obtained in step c were inoculated into 50 ml of liquid culture medium shake flasks, the liquid culture medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, the rest was deionized water, pH value natural; constant temperature culture at 37 ° C, 240 rpm speed until the bacterial solution OD600 value of 1.8.
[0030] e. The products of the shake flask culture in step d were inoculated into 1L TB medium at an inoculum size of 1%, the components of the TB medium being 12 g / L peptone, 24 g / L yeast powder, 4 ml / L glycerol, and natural pH; the culture was carried out at a constant temperature of 37°C and 220 rpm until the OD600 value of the bacterial solution was 1.7, and then the inducer IPTG was added at a final concentration of 1.0 mM, and the fermentation was terminated by constant temperature induction at 37°C for 5 hours. Then the fermentation liquid was centrifuged, the cells were collected, and then A101 cells and B101 cells were mixed according to a weight ratio of 1:2 to form group 1, A102 cells and B102 cells were mixed to form group 2..., and so on, the weight of the mixture was 15g, and then the cells were resuspended with 45ml of physiological saline to obtain a resuspension, and then the resuspension was added to 1L of substrate (made of 50g / L inositol, 50mM phosphate buffer with a pH of 7, and the rest was pure water, and the pH value of the substrate was 7), and the reaction was carried out at a constant temperature of 37°C for 24h to obtain a conversion liquid. The content of chiro-inositol in the conversion liquid was detected by HPLC, and the weights of chiro-inositol were 5.0g, 4.8g, 4.2g, 4.6g, and 5.0g, respectively, and the best cell combinations were selected as 1, 2, and 5.
[0031] Embodiment 2:
[0032] a. Recombinant E. coli containing inositol dehydrogenase and recombinant E. coli containing inositol monoketone isomerase were diluted with sterile water to obtain 10 -1 to 10 -8 diluent.
[0033] b. Add the result in step a to 10 -1 to 10 -8 The dilutions were inoculated on the selection medium, the formula of which was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L glutathione, and the rest was deionized water, with a natural pH value; and cultured at a constant temperature of 37°C for 12 hours.
[0034] c. After the culture in step b, the colonies on the culture medium with white color, raised colonies and a colony diameter of 1 to 2 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; the culture is kept at 37° C. for 12 hours; a part of the colonies with white color, raised colonies and a colony diameter of 1 to 2 mm is selected for preservation and the recombinant Escherichia coli containing inositol dehydrogenase is named A201, A202, A203, A204, and A205, and the recombinant Escherichia coli containing inositol monoketone isomerase is named B201, B202, B203, B204, and B205; and the other part is transferred to a liquid culture medium for culture.
[0035] d. Inoculate each strain obtained in step c into a shake flask containing 50 ml of liquid culture medium, the liquid culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest is deionized water, the pH value is natural. Cultivate at 37°C and 220 rpm until the OD600 value of the bacterial liquid is 1.6.
[0036] e. Each strain cultured in step d was inoculated into 1L TB medium at an inoculum size of 1%, wherein the components of the TB medium were 12 g / L peptone, 24 g / L yeast powder, 4 ml / L glycerol, and natural pH; the culture was cultured at 37°C and 220 rpm until the OD600 value of the bacterial solution was 1.7, and then the inducer IPTG was added at a final concentration of 1.0 mM, and the fermentation was terminated by constant temperature induction at 37°C for 5 hours. Then the fermentation liquid was centrifuged, the cells were collected, and then A201 cells and B201 cells were mixed according to a weight ratio of 1:2 to form group 1, A202 cells and B202 cells were mixed to form group 2..., and so on, the weight of the mixture was 15g, and then the cells were resuspended with 45ml of physiological saline to obtain a resuspension, and then the resuspension was added to 1L of substrate (made of 50g / L inositol, 50mM phosphate buffer with a pH of 7, and the rest was pure water, and the pH value of the substrate was 7), and the reaction was carried out at 37°C for 24h to obtain a conversion liquid. The content of chiro-inositol in the conversion liquid was detected by HPLC, and chiro-inositol 3.9g, 3.8g, 3.2g, 4.6g, and 3.0g were obtained, and the best cell combination was selected as 4.
[0037] Embodiment 3:
[0038] a. Recombinant E. coli containing inositol dehydrogenase and recombinant E. coli containing inositol monoketone isomerase were diluted with sterile water to obtain 10 -1 to 10 -8 diluent.
[0039] b. Add the result in step a to 10 -1 to 10 -8 The dilutions were inoculated on the selection medium, the formula of which was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L glutathione, and the rest was deionized water, with a natural pH value; and cultured at a constant temperature of 37°C for 16 hours.
[0040] c. After the culture in step b, the colonies on the culture medium with white color, raised colonies and a colony diameter of 1 to 2 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; the culture is kept at 37° C. for 16 hours; a portion of the colonies with white color, raised colonies and a colony diameter of 1 to 2 mm is selected for preservation and the recombinant Escherichia coli containing inositol dehydrogenase is named A301, A302, A303, A304, and A305, and the recombinant Escherichia coli containing inositol monoketone isomerase is named B301, B302, B303, B304, and B305; and the other portion is transferred to a liquid culture medium for culture.
[0041] d. The strains obtained in step c were inoculated into shake flasks containing 50 ml of liquid culture medium, the liquid culture medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37°C and 260 rpm until the OD600 value of the bacterial solution reached 2.0.
[0042] e. Each strain cultured in step d was inoculated into 1L TB medium at an inoculum size of 1%, wherein the components of the TB medium were 12 g / L peptone, 24 g / L yeast powder, 4 ml / L glycerol, and natural pH; the culture was cultured at 37°C and 220 rpm until the OD600 value of the bacterial solution was 1.7, and then the inducer IPTG was added at a final concentration of 1.0 mM, and the fermentation was terminated by constant temperature induction at 37°C for 5 hours. Then the fermentation liquid was centrifuged, the cells were collected, and then A201 cells were mixed with B201 cells according to a weight ratio of 1:2 to form group 1, A202 cells were mixed with B202 cells to form group 2, and so on, the weight of the mixture was 15g, and then the cells were resuspended with 45ml of physiological saline to obtain a resuspension, and then the resuspension was added to 1L of substrate (made of 50g / L inositol, 50mM phosphate buffer with a pH of 7, and the rest was pure water, and the pH value of the substrate was 7), and the reaction was carried out at a constant temperature of 37°C for 24h to obtain a conversion liquid. The content of chiro-inositol in the conversion liquid was detected by HPLC, and chiro-inositol 4.0g, 3.8g, 3.5g, 3.6g, and 4.0g were obtained, and the best cell combinations were selected as 1 and 5.
[0043] The fermentation results and conversion rate determination results of the strains of Examples 1-3 are shown in Table 1:
[0044] Table 1 Example 1-3 strain fermentation results and conversion rate determination results
[0045]
[0046] From the above results, it can be seen that the strains in Groups 1, 2, and 5 in Example 1 and Group 4 in Example 2 have higher yields.
[0047] Example 4: Stability test
[0048] The results of the genetic stability test of the bacteria in groups 1, 2, and 5 in Example 1 and group 4 in Example 2 showed that the yield of the bacteria in groups 1 and 2 in Example 1 remained unchanged after 50 generations, which proved that the bacteria can be used in industrial production.
[0049] Comparative Example 1:
[0050] No glutathione was added to the culture medium, and the culture and screening were performed according to the experimental conditions of Examples 1-3, respectively, to obtain a total of 15 groups of strain combinations, of which the first 1-5 groups were the results of culture and screening according to the experimental conditions of Example 1, groups 6-10 were the results of culture and screening according to the experimental conditions of Example 2, and groups 11-15 were the results of culture and screening according to the experimental conditions of Example 3. The specific data are shown in Table 2.
[0051] Table 2 Comparative Example 1 strain fermentation results and conversion rate determination results
[0052]
[0053] Result analysis:
[0054] Through the analysis of the conversion rates of the examples and the comparative examples, it can be seen that the strain combinations obtained by the method of the present invention have a conversion rate greater than or equal to 92% for 5 of the 15 combinations, while only 2 of the 15 combinations in the comparative example have a conversion rate greater than or equal to 92%; thus, it can be seen that adding glutathione to the culture medium can effectively increase the probability of screening high-expressing strains.
[0055] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.
Claims
1. A method for screening chiral inositol producing bacteria using a culture medium containing inositol and glutathione, characterized in that: The following steps are involved: a. Recombinant Escherichia coli containing inositol dehydrogenase and recombinant Escherichia coli containing inositol monoketone isomerase were diluted with sterile water to obtain dilutions; b. The dilution in step a was inoculated onto a selective medium, wherein the selective medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L glutathione, and the rest was deionized water, with a natural pH value; and cultured at 37 ° C for 12 to 16 h; c. In step b, the colonies cultured on the culture medium with uniform single color, raised colonies and a colony diameter of 1 to 2 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; cultured at a constant temperature of 37°C for 12 to 16 hours; d. A portion of the colony obtained in step c was used for preservation, and a portion was inoculated into a shake flask containing 50 ml of liquid culture medium, wherein the liquid culture medium was formulated as 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.6 to 2.0; e. Inoculate the single colony cultured in the shake flask in step d into 1L TB medium at an inoculum size of 1% for fermentation culture, and culture at 37°C and 220rpm until the OD600 value of the bacterial solution is 1.7, add the inducer IPTG at a final concentration of 1.0mM, and induce at 37°C for 5 hours until the fermentation is completed; centrifuge the fermentation broth to collect the bacteria, and then randomly pair the collected recombinant Escherichia coli containing inositol dehydrogenase and the recombinant Escherichia coli containing inositol monoketone isomerase in a weight ratio of 1:2, weigh 15g of bacteria, and then resuspend the bacteria with 45ml of physiological saline to obtain a resuspended solution, and then add the resuspended solution to 1L of substrate, and react at 37°C for 24h to obtain a conversion solution; detect the content of chiral inositol in the conversion solution by HPLC, and select the best bacterial combination.
2. The method for screening chiral inositol production bacteria by utilizing a culture medium containing inositol and glutathione as claimed in claim 1, wherein: The gradient dilution in step a is from 10 -1 to 10 -8 .
3. The method for screening chiral inositol production bacteria by utilizing a culture medium containing inositol and glutathione as claimed in claim 1, wherein: In step b, the mixture is cultured at a constant temperature of 37° C. for 14 hours.
4. The method for utilizing a medium containing inositol and glutathione to screen chiral inositol production bacteria as claimed in claim 1, wherein: In the step c, the colonies are white in morphology, convex in colonies, and have a colony diameter of 1 to 2 mm; and the colonies are cultured at a constant temperature of 37° C. for 14 hours.
5. The method for utilizing a culture medium containing inositol and glutathione to screen chiral inositol producing bacteria as claimed in claim 1, wherein: In the step d, the colonies are white in morphology, convex in colonies, and have a colony diameter of 1 to 2 mm; the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 1.
8.
6. The method for screening chiral inositol producing bacteria by utilizing a culture medium containing inositol and glutathione as claimed in claim 1, wherein: In the step e, the substrate is prepared by 50 g / L inositol, 50 mM phosphate buffer with a pH of 7, and the rest is purified water, and the pH value of the substrate is 7.
7. The method for screening chiral inositol producing bacteria by utilizing a culture medium containing inositol and glutathione as claimed in claim 1, wherein: The components of the TB culture medium in step e are 12 g / L peptone, 24 g / L yeast powder, 4 ml / L glycerol, and the pH value is natural.