Culture medium for promoting growth of genetically modified bacillus subtilis and preparation method thereof

Abstract

The invention provides a culture medium for promoting growth of genetically modified bacillus subtilis and a preparation method thereof. The culture medium for promoting growth of genetically modified bacillus subtilis is prepared from the following raw materials: pure fermentation liquor, anhydrous dextrose, peptone, yeast extract, monopotassium phosphate, calcium carbonate and distilled water. The culture medium for promoting the growth of the genetically modified bacillus subtilis can relieve the metabolic pressure caused by the gene modification of the bacillus subtilis, improve the growth rate and metabolic activity of cells, ensure the high-throughput expression of riboflavin, realize the efficient and stable culture of strains, lay a foundation for industrial production, and have wide application prospects. The problem of growth inhibition of the bacillus subtilis after gene modification is solved.

Description

Technical Field

[0001] The invention relates to the field of biological preservation fluid, and in particular to a culture medium for promoting the growth of genetically modified Bacillus subtilis and a preparation method thereof. Background Art

[0002] With the rapid development of biotechnology, the application of genetic engineering technology in the field of microorganisms is becoming more and more extensive. As an important industrial microorganism, Bacillus subtilis has many advantages such as clear genetic background, easy cultivation, and high safety, and has become one of the hot research objects. The precise editing of its genes through gene scissors technology aims to achieve efficient synthesis of specific metabolites, such as high-throughput expression of riboflavin, which is of great significance for industrial production and biotechnology research. However, genetically modified strains often have problems such as changes in growth characteristics, which requires in-depth research and optimization of their culture conditions to meet the needs of industrial production.

[0003] In Bacillus subtilis, by designing crRNA for specific genes (such as ribG, ribA, ribH / purR, etc.), these genes can be accurately knocked out using CRISPR-Cas9 technology. For example, the knockout of genes such as ribG, ribA, and ribH may affect the key steps in the riboflavin biosynthesis pathway, thereby changing the synthesis flux of riboflavin. These genes are involved in the synthesis or conversion process of different precursor substances in the riboflavin synthesis pathway. After knocking them out, the original metabolic flow may be blocked or changed, so that more metabolic intermediates flow to the synthesis direction of riboflavin, thereby achieving high-throughput expression of riboflavin. As a regulatory gene, the knockout of the purR gene may relieve the inhibitory effect on genes related to riboflavin synthesis, and also help to increase the yield of riboflavin. In order to further optimize the effect after genetic modification, RBS (ribosome binding site) library screening technology is often combined with gene scissors technology. RBS is a specific region on mRNA that binds to the ribosome. Its sequence and structure will affect the binding efficiency of the ribosome to the mRNA, thereby regulating the translation initiation rate of the gene. By constructing a library containing different RBS sequences and combining it with a knockout Bacillus subtilis strain for screening, the RBS sequence that is most suitable for the efficient expression of the target gene (such as riboflavin synthesis-related genes) can be found. This method can achieve fine regulation of the riboflavin synthesis pathway by adjusting the translation efficiency of the gene without changing the gene coding sequence, further improving the yield and production efficiency of riboflavin.

[0004] Although high-throughput expression of riboflavin can be achieved by knocking out genes and screening RBS libraries in Bacillus subtilis using gene scissors technology, some new problems have also been brought about, the most prominent of which is that the growth of the modified strains is inhibited. Gene knockout may cause changes in metabolic pathways in cells, and some originally balanced metabolic flows are broken. For example, after knocking out genes such as ribG, ribA, and ribH / purR, intermediate metabolites in the riboflavin synthesis pathway may accumulate, which will not only have toxic effects on cells, but may also affect the normal operation of other related metabolic pathways, thereby inhibiting cell growth and reproduction. In order to achieve high-throughput synthesis of riboflavin, cells may need to consume more energy and precursors, which may lead to an imbalance in energy metabolism and material metabolism in cells. For example, excessive metabolic flows directed to riboflavin synthesis may reduce the energy and material supply required for cell growth and maintenance of basic physiological functions, thereby limiting cell growth. Operations such as gene knockout and RBS library screening may affect the overall regulatory network of gene expression in cells. The expression of some genes related to cell growth and metabolism may be indirectly affected, resulting in abnormal expression levels, which in turn affects the normal physiological function and growth status of cells.

[0005] In view of the problems existing in the prior art, the present application aims to find suitable culture conditions that can provide a suitable growth environment for the genetically modified Bacillus subtilis, alleviate the metabolic pressure caused by genetic modification, and increase the growth rate and metabolic activity of the cells, thereby ensuring high-throughput expression of riboflavin while achieving efficient and stable culture of the strain, laying the foundation for industrial production and solving the problem of growth inhibition of Bacillus subtilis after genetic modification. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a culture medium for promoting the growth of genetically modified Bacillus subtilis and a preparation method thereof, so as to solve the problem of growth inhibition of genetically modified Bacillus subtilis.

[0007] The technical solution of the present invention:

[0008] The invention provides a culture medium for promoting the growth of genetically modified Bacillus subtilis. The raw materials for preparing the culture medium for promoting the growth of genetically modified Bacillus subtilis include: pure fermentation liquid, anhydrous glucose, peptone, yeast extract, potassium dihydrogen phosphate, calcium carbonate and distilled water.

[0009] Furthermore, the raw materials for preparing the culture medium for promoting the growth of genetically modified Bacillus subtilis include, by weight: 8-20 parts of pure fermentation broth, 3-5 parts of anhydrous glucose, 0.5-1 parts of peptone, 0.3-0.8 parts of yeast extract, 0.3-0.5 parts of potassium dihydrogen phosphate, 0.2-0.4 parts of calcium carbonate, and 1000-1500 parts of distilled water.

[0010] Furthermore, the raw materials of the pure fermentation liquid are malt, brewer's yeast and distilled water.

[0011] The method for preparing the pure fermentation broth comprises the following steps:

[0012] S1: Add malt and appropriate amount of distilled water to a beaker, heat to 60-70°C, start saccharification, obtain wort, then transfer the wort into a flask, boil and sterilize, and then cool it down to the fermentation temperature in a water bath;

[0013] S2: Add brewer's yeast into a flask filled with distilled water, let it stand for 20-45 minutes, shake the flask to make it uniform, and let it stand for another 20-45 minutes to prepare yeast solution;

[0014] S3: adding the wort prepared in step S1 to the yeast solution prepared in step S2, and then passing sterile air into the mixture of the wort and the yeast solution for aeration and oxygenation for 12-24 hours;

[0015] S4: After the aeration and oxygenation in step S3 is stopped, the mixture of the wort and the yeast solution is allowed to stand for 24-38 hours, and then the fermented mixture is sterilized at high temperature;

[0016] S5: Filter the sterilized mixed solution to remove solids and obtain pure fermentation liquid.

[0017] Furthermore, the mass ratio of malt to water is 1:2-5; the sugar content of the wort obtained by saccharification in step S1 is 8-10 degrees, and the fermentation temperature is 27-30°C.

[0018] Furthermore, the mass ratio of brewer's yeast to distilled water in step S2 is 1:10-15.

[0019] Furthermore, the wort temperature in step S3 is 3-5° C. higher than the yeast solution.

[0020] Furthermore, the brewer's yeast is a mixture of one or more of Angel CN36, Fermentis S-04, and Fermentis WB-06.

[0021] The present invention also provides a method for preparing a culture medium for promoting the growth of genetically modified Bacillus subtilis, comprising the following steps:

[0022] Step 1: Add distilled water into a beaker, then add anhydrous glucose, peptone, yeast extract, potassium dihydrogen phosphate and calcium carbonate, mix well, and prepare a bacterial culture medium;

[0023] Step 2: adding pure fermentation broth to the bacterial culture medium obtained in step 1, and then adjusting the pH value to 7.0-7.6 to obtain a final mixed solution;

[0024] Step 3: The final mixed solution obtained in step 2 is sterilized and then packaged to obtain the culture medium for promoting the growth of the genetically modified Bacillus subtilis.

[0025] Furthermore, in step 2, the volume ratio of the bacterial culture medium to the pure fermentation liquid is 5-10:1.

[0026] Beneficial effects:

[0027] The invention provides a culture medium for promoting the growth of genetically modified Bacillus subtilis, which can alleviate the metabolic pressure of Bacillus subtilis caused by genetic modification, improve the growth rate and metabolic activity of cells, and realize efficient and stable culture of strains while ensuring high-throughput expression of riboflavin, thereby laying a foundation for industrial production and solving the problem of growth inhibition of Bacillus subtilis after genetic modification. DETAILED DESCRIPTION

[0028] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following embodiments are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Other combinations and various modifications within the concept of the present invention may be performed without departing from the spirit or scope of the present invention.

[0029] Unless otherwise specified, the chemical reagents used in the present invention are all commercially available analytically pure.

[0030] The yeast extract was purchased from Wuhan Jiyesheng Chemical Co., Ltd., with a CAS number of 8013-01-2. The peptone was purchased from Guangdong Huankai Microbiological Technology Co., Ltd., with a CAS number of 050240.

[0031] Preparation of pure fermentation broth A:

[0032] S1: Add 500g malt and 1L distilled water into a beaker, heat to 65℃, start saccharification, and obtain wort at 8-10℃. Then transfer 1L wort into a flask and boil for 15 minutes to sterilize, then cool it down to 28℃ in a water bath.

[0033] S2: 10 g of brewer's yeast (Fumandis WB-06) was added to a flask containing 100 ml of distilled water. After standing for 30 minutes, the flask was shaken to make it uniform, and then stood for another 30 minutes to prepare a yeast solution;

[0034] S3: adding 1000 ml of the wort prepared in step S1 to the yeast solution prepared in step S2, wherein the wort temperature is 3°C higher than the yeast solution, and then passing sterile air into the mixture of the wort and the yeast solution for aeration and oxygenation for 16 hours;

[0035] S4: After stopping aeration and oxygenation in step S3, the mixture of wort and yeast solution is left to stand for 32 hours, and then the fermented mixture is sterilized at 100°C;

[0036] S5: Filter the sterilized mixed solution to remove solids to obtain pure fermentation solution A.

[0037] Preparation of pure fermentation broth B:

[0038] The difference between this preparation and the preparation of pure fermentation broth A is that 10 g of brewer's yeast in step S2 is replaced by 10 g of brewer's yeast 7 g.

[0039] Preparation of pure fermentation broth C:

[0040] The difference between this preparation and the preparation of pure fermentation broth A is that 10 g of brewer's yeast in step S2 is replaced by 10 g of brewer's yeast 6 g.

[0041] Preparation of culture medium to promote the growth of genetically modified Bacillus subtilis:

[0042] Step 1: Add distilled water into a beaker, then add anhydrous glucose, peptone, yeast extract, potassium dihydrogen phosphate and calcium carbonate, mix well, and prepare a Bacillus subtilis culture medium;

[0043] Step 2: adding pure fermentation liquid to the Bacillus subtilis culture medium obtained in step 1, then adjusting the pH value to 7.2, and finally fixing the volume to obtain a final mixed solution;

[0044] Step 3: The final mixed solution obtained in step 2 is sterilized and then packaged to obtain the culture medium for promoting the growth of the genetically modified Bacillus subtilis.

[0045] Embodiment 1:

[0046] Step 1: Add 1L of distilled water to a beaker, then add 3.5g of anhydrous glucose, 0.83g of peptone, 0.5g of yeast extract, 0.35g of potassium dihydrogen phosphate and 0.25g of calcium carbonate, mix well, and prepare a bacterial culture medium;

[0047] Step 2: Add 200 ml of pure fermentation liquid A to 1 L of bacterial culture medium obtained in step 1, and then adjust the pH value to 7.2 to obtain a final mixed solution;

[0048] Step 3: The final mixed solution obtained in step 2 is sterilized and then packaged to obtain the culture medium for promoting the growth of the genetically modified Bacillus subtilis.

[0049] Embodiment 2:

[0050] The difference between this preparation and Example 1 is that 200 ml of pure fermentation liquid A in step 2 is replaced by 200 ml of pure fermentation liquid B.

[0051] Embodiment 3:

[0052] The difference between this preparation and Example 1 is that 200 ml of pure fermentation liquid A in step 2 is replaced by 100 ml of pure fermentation liquid A.

[0053] Embodiment 4:

[0054] The difference between this preparation and Example 1 is that 200 ml of pure fermentation liquid A in step 2 is replaced by 200 ml of pure fermentation liquid C.

[0055] Embodiment 5:

[0056] The difference between this preparation and Example 1 is that 200 ml of pure fermentation liquid A in step 2 is replaced by 50 ml of pure fermentation liquid A.

[0057] Comparative Example 1:

[0058] The difference between this preparation and Example 1 is that no pure fermentation broth is added in step 2.

[0059] Comparative Example 2:

[0060] 1L of distilled water was added to a beaker, followed by 20g of anhydrous glucose, 15g of peptone, 0.5g of yeast extract, 0.35g of potassium dihydrogen phosphate and 0.25g of calcium carbonate, mixed evenly, the pH value was adjusted to 7.2, and a culture medium for promoting the growth of genetically modified Bacillus subtilis was prepared after high-temperature sterilization.

[0061] The culture medium for promoting the growth of genetically modified Bacillus subtilis prepared in the above examples and comparative examples was subjected to the following experiments to perform performance tests:

[0062] 1. Single colonies of genetically modified Bacillus subtilis and non-genetically modified Bacillus subtilis were picked with a sterile inoculation loop and inoculated into sterile test tubes containing 5 mL of the culture medium of the embodiment and the comparative example. The seed liquid was inoculated into 250 ml conical flasks containing 100 ml of the sterilized culture medium of the embodiment and the comparative example at an inoculum amount of 1-2% (v / v). The conical flasks were placed in a 37°C constant temperature shaker and cultured at a rotation speed of 200 rpm.

[0063] 2. Growth Curve Determination

[0064] During the culture process, 1 ml of bacterial solution was taken out from the conical flasks of the experimental group and the control group every 2 hours, and the taken bacterial solution was placed in a sterile centrifuge tube and immediately placed on ice to prevent the bacteria from continuing to grow. The concentration of the bacterial solution was determined by measuring the optical density (OD600) of the bacterial solution at a wavelength of 600nm using a spectrophotometer, and a blank culture medium was used as a control. The growth curve drawn based on the OD600 value was used to compare the growth rate (maximum specific growth rate μmax) of the genetically modified Bacillus subtilis in the example and comparative culture medium.

[0065] 3. Metabolic Activity Assay

[0066] The oxygen consumption rate of the bacterial solution was measured using a dissolved oxygen electrode. The dissolved oxygen electrode was inserted into a sealed container containing the bacterial solution to record the changes in dissolved oxygen concentration at different time points. The oxygen consumption rate was calculated based on the dissolved oxygen concentration change curve to evaluate the metabolic activity of the bacteria and compare the differences in metabolic activity of genetically modified Bacillus subtilis in the two culture media.

[0067] 4. Determination of Riboflavin Expression

[0068] Sample treatment After 48 hours of culture, 10 ml of bacterial solution was taken out from the conical flasks of the examples and comparative examples, and the bacterial solution was centrifuged (4000 rpm, 10 minutes) to collect the supernatant. For intracellular riboflavin, the bacterial precipitate needs to be resuspended with an appropriate buffer, ultrasonically disrupted or chemically broken, and then centrifuged to obtain the supernatant. Riboflavin content determination High performance liquid chromatography (HPLC) was used to determine the content of riboflavin.

[0069] Table 1: Test results

[0070] Inspection items Growth rate (μ) h-1 Oxygen consumption rate μmol O / (min·L) Riboflavin content mg / L Example 1 1.65 26.5 4.8 Example 2 1.63 26.0 4.5 Example 3 1.61 25.8 4.2 Example 4 1.23 20.2 3.0 Example 5 1.26 20.5 3.2 Comparative Example 1 0.89 12.6 1.8 Comparative Example 2 0.76 11.5 1.2

[0071] From the above data, it can be seen that the culture medium for promoting the growth of genetically modified Bacillus subtilis prepared in the present invention can alleviate the metabolic pressure of Bacillus subtilis caused by genetic modification, can increase the growth rate and metabolic activity of cells, express riboflavin with high throughput, and culture the strain efficiently and stably. Specifically, it can be seen from the comparison between Example 4 and Example 1 that when preparing pure fermentation broth, an unreasonable mass ratio of brewer's yeast to distilled water will cause the prepared pure fermentation broth to affect the growth rate and metabolic activity of the culture medium for Bacillus subtilis cells, as well as the amount of riboflavin expressed; Example 5 can be seen from the comparison between Example 1 that when preparing pure fermentation broth, an unreasonable mass ratio of bacterial culture medium to brewer's yeast will cause the prepared pure fermentation broth to affect the growth rate and metabolic activity of the culture medium for Bacillus subtilis cells, as well as the amount of riboflavin expressed; Comparative Example 1 can be seen from the comparison between Example 1 that not adding pure fermentation broth will cause the prepared culture medium for promoting the growth of genetically modified Bacillus subtilis to fail to achieve the expected effect on the growth rate and metabolic activity of Bacillus subtilis; From the comparison between Comparative Example 2 and Example 1, it can be seen that the culture medium for promoting the growth of genetically modified Bacillus subtilis prepared with unreasonable component content will fail to achieve the expected effect on the growth rate and metabolic activity of Bacillus subtilis.

[0072] The present invention can also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A culture medium for promoting the growth of genetically modified Bacillus subtilis, characterized in that: The raw materials for preparing the culture medium for promoting the growth of genetically modified Bacillus subtilis include: pure fermentation liquid, anhydrous glucose, peptone, yeast extract, potassium dihydrogen phosphate, calcium carbonate and distilled water.

2. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 1, characterized in that: The raw materials for preparing the culture medium for promoting the growth of genetically modified Bacillus subtilis include, by weight: 8-20 parts of pure fermentation liquid, 3-5 parts of anhydrous glucose, 0.5-1 part of peptone, 0.3-0.8 part of yeast extract, 0.3-0.5 part of potassium dihydrogen phosphate, 0.2-0.4 part of calcium carbonate, and 1000-1500 parts of distilled water.

3. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 1, characterized in that: The raw materials of the pure fermentation liquid are malt, brewer's yeast and distilled water.

4. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 3, characterized in that: The method for preparing the pure fermentation broth comprises the following steps: S1: Add malt and appropriate amount of distilled water to a beaker, heat to 60-70°C, start saccharification, obtain wort, then transfer the wort into a flask, boil and sterilize, and then cool it down to the fermentation temperature in a water bath; S2: Add brewer's yeast into a flask filled with distilled water, let it stand for 20-45 minutes, shake the flask to make it uniform, and let it stand for another 20-45 minutes to prepare yeast solution; S3: adding the wort prepared in step S1 to the yeast solution prepared in step S2, and then passing sterile air into the mixture of the wort and the yeast solution for aeration and oxygenation for 12-24 hours; S4: After the aeration and oxygenation in step S3 is stopped, the mixture of the wort and the yeast solution is allowed to stand for 24-38 hours, and then the fermented mixture is sterilized at high temperature; S5: Filter the sterilized mixed solution to remove solids and obtain pure fermentation liquid.

5. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 4, characterized in that: The mass ratio of malt to water is 1:2-5; the sugar content of the wort obtained by saccharification in step S1 is 8-10 degrees, and the fermentation temperature is 27-30°C.

6. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 4, characterized in that: The mass ratio of brewer's yeast to distilled water in step S2 is 1:10-15.

7. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 4, characterized in that: The wort temperature in step S3 is 3-5° C. higher than the yeast solution.

8. The culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 4, characterized in that: The brewer's yeast is a mixture of one or more of Angel CN36, Fermentis S-04, and Fermentis WB-06.

9. The method for preparing a culture medium for promoting the growth of genetically modified Bacillus subtilis according to any one of claims 1 to 8, characterized in that: The following steps are involved: Step 1: Add distilled water into a beaker, then add anhydrous glucose, peptone, yeast extract, potassium dihydrogen phosphate and calcium carbonate, mix well, and prepare a bacterial culture medium; Step 2: adding pure fermentation broth to the bacterial culture medium obtained in step 1, and then adjusting the pH value to 7.0-7.6 to obtain a final mixed solution; Step 3: The final mixed solution obtained in step 2 is sterilized and then packaged to obtain the culture medium for promoting the growth of the genetically modified Bacillus subtilis.

10. The method for preparing a culture medium for promoting the growth of genetically modified Bacillus subtilis according to claim 9, characterized in that: In the step 2, the volume ratio of the bacterial culture medium to the pure fermentation liquid is 5-10:1.