Method for improving lincomycin yield by optimizing fermentation culture medium for lincomycin genetically engineered bacteria

Abstract

According to the invention, the lincomycin fermentation culture medium is optimized through a Plackett-Burman design, a steepest climbing experiment and a Box-Behnken experiment, so that the lincomycin fermentation culture medium has higher matching property with genetically engineered bacteria. In particular, the present invention relates to the addition of one or more of inorganic salts, small molecular mercaptans and amino acids to a fermentation medium. More specifically, the additive substances comprise cobalt chloride, sodium sulfate and methionine. According to the method, the yield of lincomycin is effectively increased by adding inorganic salt and amino acid into the fermentation culture medium.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and more specifically, relates to a method for optimizing a fermentation medium for lincomycin genetically engineered bacteria to increase lincomycin yield. Background Art

[0002] Lincomycin is a lincosamide antibiotic with broad-spectrum anti-Gram-positive activity and is widely used in clinical medicine. In recent years, the demand for lincomycin and its derivatives has been increasing year by year, necessitating increased production of lincomycin.

[0003] Currently, lincomycin is primarily produced through fermentation of Streptomyces lincomycin. A common and effective approach involves obtaining high-yield strains and optimizing the fermentation process. However, since different strains have their own optimal fermentation processes, existing fermentation processes often do not work well with genetically engineered high-yield strains, preventing them from fully realizing their high-yield potential. This presents an urgent problem to be addressed. Summary of the Invention

[0004] The present invention provides a method for optimizing the fermentation medium of lincomycin genetically engineered bacteria to increase lincomycin yield. The method increases lincomycin yield by adding one or more of inorganic salts, small molecule thiols, and amino acids to the fermentation medium.

[0005] In a first aspect, the present invention provides a method for increasing lincomycin production during the production of lincomycin using Streptomyces lincomycin, comprising adding one or more of an inorganic salt, a small molecule thiol, and an amino acid to the fermentation medium. The inorganic salts include cobalt chloride, magnesium chloride, ferrous sulfate, sodium sulfate, and the like; the small molecule thiol includes ethyl mercaptan, inositol, and the like; the amino acids include proline, alanine, methionine, and valine; the inorganic salts are preferably cobalt chloride and sodium sulfate; the small molecule thiol is preferably inositol; and the amino acids are preferably proline, methionine, and valine.

[0006] In a preferred example: the dosage of the inorganic salt may be further preferably: 1-10 mg / L of cobalt chloride, 0.15-1.5 g / L of sodium sulfate; the dosage of the small molecule thiol may be further preferably: 0.2-0.8 g / L of inositol; the dosage of the amino acid may be further preferably: 0.02-0.2 g / L of proline, 0.02-0.2 g / L of methionine, and 0.02-0.2 g / L of valine.

[0007] In another preferred embodiment, the dosage of the inorganic salt may be further preferably: 3-6 mg / L of cobalt chloride, 0.8-1.6 g / L of sodium sulfate; and the dosage of the amino acid may be further preferably: 0.1-0.3 g / L of methionine.

[0008] In another preferred embodiment, the dosage of the inorganic salt may be further preferably: 3.6 mg / L of cobalt chloride and 1.23 g / L of sodium sulfate; and the dosage of the amino acid may be further preferably: 0.26 g / L of methionine.

[0009] Therefore, in one aspect, the present invention provides a method for increasing lincomycin production by adding one or more of inorganic salts, small molecule thiols and amino acids to a fermentation medium.

[0010] In another aspect, the inorganic salts and amino acids of the present invention are used as additives to fermentation media, or are used in combination with other inorganic salts and amino acids.

[0011] In another aspect, the fermentation medium of the present invention comprises a lincomycin-producing strain, such as Streptomyces lincomycin, preferably the genetically engineered lincomycin strain L-427Δ2790. L-427Δ2790 is derived from the industrial lincomycin strain L-427 maintained in our laboratory by genetically engineering the deletion of the SLCG_2790 gene. Among them, L-247 is a high-yielding strain isolated from soil in our laboratory through multiple rounds of mutagenesis and screening, resulting in potential industrial applications and maintained in our laboratory.

[0012] The beneficial effects of the present invention are embodied in:

[0013] The present invention establishes an optimization method for the fermentation culture medium adapted to lincomycin genetically engineered bacteria, solves the problem of low compatibility between existing fermentation processes and genetically engineered bacteria, and improves lincomycin yield by optimizing the fermentation culture conditions of lincomycin genetically engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A Pareto chart showing the effects of various factors provided in the implementation case of the present invention;

[0015] Figure 2 A normal plot of the effects of various factors provided in the implementation case of the present invention;

[0016] Figure 3 This is a yield graph of a ramp test provided in an implementation case of the present invention;

[0017] Figure 4 Response surface diagram of the interaction between cobalt chloride and sodium sulfate in an embodiment of the present invention;

[0018] Figure 5Response surface diagram of the interaction between cobalt chloride and methionine in an embodiment of the present invention;

[0019] Figure 6 Response surface diagram of the interaction between sodium sulfate and methionine in an embodiment of the present invention;

[0020] Figure 7 This is a graph showing the yield before and after optimization of the shake flask fermentation medium in an embodiment of the present invention;

[0021] Figure 8 This is a graph showing the yield before and after optimization of the horizontal fermentation medium in a 5L fermenter in an embodiment of the present invention. Specific implementation plan

[0022] The present invention will be further described below with reference to the accompanying drawings in the embodiment of the present invention:

[0023] Specific technologies or conditions not specified in the following implementation cases are all conventional technologies or conditions, or technologies or conditions described in the literature in this field.

[0024] 1. The culture medium formula involved in the embodiment of the present invention:

[0025] Slant culture medium: 20 g / L glucose, 5 g / L soybean meal, 1 g / L potassium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium hydrogen phosphate, 0.5 g / L sodium chloride, 0.01 g / L ferrous sulfate heptahydrate, 20 g / L agar, balance water. Sterilize at 121°C for 20 min.

[0026] Seed culture medium: 20 g / L corn starch, 15 g / L soybean meal, 32 g / L corn steep liquor, 15 g / L glucose, 1.5 g / L ammonium sulfate, 4 g / L calcium carbonate, balance water; after constant volume, adjust the pH to 7.2-7.3 with 6 mol / L sodium hydroxide solution. Sterilize at 121°C for 20 min.

[0027] Fermentation medium: corn starch 40 g / L, glucose 10 g / L, soybean meal 28 g / L, corn steep liquor 12 g / L, sodium chloride 5.2 g / L, sodium nitrate 6.2 g / L, potassium dihydrogen phosphate 0.26 g / L, ammonium nitrate 2.1 g / L, ammonium sulfate 2.1 g / L, calcium carbonate 7 g / L, balance water; after constant volume, adjust the pH to 7.2-7.3 with 6 mol / L sodium hydroxide solution. Sterilize at 121°C for 20 min.

[0028] 2. Cultivation method in the embodiment of the present invention:

[0029] Slant culture: Take out the bacteria stored in the glycerol tube from the -80℃ freezer, aspirate 50 μl and spread it on a plate, and culture it in a 30℃ incubator for 5-7 days.

[0030] Seed bottle culture: Take the grown plate out of the 30℃ constant temperature incubator and dig a 1cm block with an inoculation shovel. 2 The size of the microorganisms was inoculated into the seed culture medium and cultured at 30°C and 220 rpm for 48 h.

[0031] 500 mL shake flask fermentation culture: aspirate the seed solution in a clean bench, inoculate 10% of the inoculum into the fermentation medium, and culture at 30°C and 220 rpm for 7 days.

[0032] Fermentation in a 5L bioreactor: The fermentation medium volume in the 5L bioreactor is 3L, with 1g / L of defoamer added. The fermentation seed is cultured using the same method as shake flask fermentation and flame inoculated with an inoculum volume of 300mL. The reactor agitator speed is set at 100-600rpm, ventilation at 1vvm, culture temperature at 30°C, and tank pressure at 0.04mPa. During fermentation, the dissolved oxygen level is maintained at 30-60%, and ammonia is added to maintain a pH of no less than 6.6.

[0033] III. Description of reagents involved in the embodiments of the present invention:

[0034] The reagents involved in the embodiments of the present invention, including molecular biology reagents, chemical reagents and antibiotics, are all commercial products and can be purchased.

[0035] The following is a detailed description of an implementation case of the present invention. This embodiment is implemented based on the technical solution of the present invention. The following is a detailed implementation method and specific operation process.

[0036] Example 1

[0037] Plackett-Burman experiment

[0038] To investigate the combined effects of inorganic salts, small thiols, and amino acids on the growth of genetically engineered lincomycin strains and lincomycin synthesis, and to identify the optimal additive combination, the present invention conducted experiments on the combined addition and concentration optimization of multiple ingredients. Six ingredients—cobalt chloride, sodium sulfate, inositol, proline, methionine, and valine—were selected for a Plackett-Burman (PB) experiment to investigate the primary factors influencing lincomycin synthesis.

[0039] The factors represented by each variable and their levels are shown in Table 1, and the response value is the yield of lincomycin A. The PB experimental design table is shown in Table 2, and the PB experimental results are shown in Table 3.

[0040] Table 1 Plackett-Burman experimental factor table

[0041] variable factor High level (g / L) Low level (g / L) A Cobalt chloride 0.001 0.01 B sodium sulfate 0.15 1.5 C Inositol 0.2 0.8 D Proline 0.02 0.2 E Methionine 0.02 0.2 F Valine 0.02 0.2

[0042] Table 2Plackett-Burman experimental design table

[0043] RUN A B C D E F LinA (mg / L) 1 1 1 -1 -1 -1 1 1041 2 1 -1 1 1 1 -1 1311 3 -1 1 -1 1 1 -1 2144 4 1 -1 -1 -1 -1 -1 1034 5 1 -1 -1 -1 1 -1 1486 6 -1 1 1 1 -1 -1 1081 7 -1 -1 -1 1 -1 1 752 8 1 1 1 -1 -1 -1 1070 9 -1 1 1 -1 1 1 2088 10 1 1 -1 1 1 1 1818 11 -1 -1 1 -1 1 1 1570 12 1 -1 1 1 -1 1 673

[0044] Table 3 Analysis of variance of the Plackett-Burman experimental model

[0045]

[0046] The regression analysis was conducted with lincomycin A production as the dependent variable. The results are shown in Table 3. Figure 1 、 Figure 2 As shown, using lincomycin A production as an indicator, three significant factors were screened: cobalt chloride (A), sodium sulfate (B), and methionine (E), all with P values ​​< 0.05. Sodium sulfate (B) and methionine (E) had P values ​​< 0.01, indicating they were extremely significant factors. A model P value < 0.05 indicates the model is significant and statistically significant.

[0047] Example 2

[0048] Steepest climb test

[0049] To further optimize the added ingredients, cobalt chloride (A), sodium sulfate (B), and methionine (E) were selected for a steepest ramp experiment. Based on the estimated coefficient model obtained from the PB experiment, these were sequentially increased or decreased. Sodium sulfate (B) and methionine (E) had positive effects, while cobalt chloride (A) had a negative effect. The design and results of the steepest ramp experiment are shown in Table 4.

[0050] Table 4 Design and results of the steepest climbing experiment

[0051] Serial number Cobalt chloride (g / L) Sodium sulfate (g / L) Methionine (g / L) LinA (mg / L) 1 0.006 0.4 0 1487.1 2 0.005 0.8 0.1 1939.2 3 0.004 1.2 0.2 2187.2 4 0.003 1.6 0.3 1931.3 5 0.002 2.0 0.4 1623.2 6 0.001 2.4 0.5 1356.5

[0052] The results of the steepest climbing test are as follows Figure 3 As shown in the figure, when the cobalt chloride (A) concentration is 0.004 g / L, the sodium sulfate (B) concentration is 1.2 g / L, and the methionine (E) concentration is 0.2 g / L, the yield reaches its maximum, which is the maximum response value region. Therefore, the subsequent response surface experiment was designed with the levels of each factor in sequence number 3 as the center value.

[0053] Experimental Example 3

[0054] Box-Behnken experiment

[0055] Based on the above experimental results, cobalt chloride (A), sodium sulfate (B), and methionine (E) were selected for the Box-Behnken experiment. The factors and levels of the BB experiment are shown in Table 5, and the design and results of the BB experiment are shown in Table 6.

[0056] Table 5 Factors and levels of Box-Behnken experiment

[0057] variable factor Low level (-1) Medium level (0) High level (1) A Cobalt chloride 0.003 0.004 0.005 B sodium sulfate 0.8 1.2 1.6 E Methionine 0.1 0.2 0.3

[0058] Table 6 Box-Behnken experimental design and results

[0059]

[0060]

[0061] The data in Table 6 were subjected to multiple regression fitting by software analysis, and the quadratic polynomial linear regression equation for lincomycin A production was obtained as follows:

[0062] Y=2173.97–171.78A+133B+309.79E–2.01AB–83.57AE–19.2BE–224A 2 -248.32

[0063] B 2 –274.74E 2

[0064] Wherein Y is the yield of lincomycin A, A is cobalt chloride, B is sodium sulfate, and E is methionine.

[0065] The experimental results were subjected to regression variance analysis and credibility analysis, and the results are shown in Tables 7 and 8.

[0066] Table 7 Regression analysis of variance

[0067]

[0068] Table 8 Model credibility analysis

[0069] Std.Dev. 18.88 <![CDATA[R 2 ]]> 0.9988 Mean 1822.41 <![CDATA[Adjusted R 2 ]]> 0.9972 CV% 1.04 <![CDATA[Predicted R 2 ]]> 0.9908 Adeq Precision 72.6243

[0070] According to the results in Tables 7 and 8, the P value of this model is <0.0001, and the model is extremely significant; the lack-of-fit value is 0.4950, and the model error is not significant; the correlation coefficient of this model is 0.9988, and the fit is high; the Adeq Precision (signal-to-noise ratio) is 72.6243>4, and the credibility is high.

[0071] Represent the prediction model as a 3D response surface plot, such as Figure 4 、 Figure 5 、 Figure 6 The model was used to predict the maximum lincomycin production, which was 2277.8 mg / L when the cobalt chloride content was 0.0032 g / L, the sodium sulfate content was 1.22 g / L, and the methionine content was 0.24 g / L.

[0072] Based on the original culture medium, the fermentation medium of lincomycin genetically engineered bacteria was optimized through methods such as Plackett-Burman experiment, steepest climbing test, and response surface experimental design. The optimized fermentation medium achieved a yield of 2217.27 mg / L in shake flasks, which was 38.1% higher than the original culture medium for lincomycin production (1604.88 mg / L). Figure 7 At the 5L fermentation tank level, the optimized fermentation medium produced lincomycin at 4598.12 mg / L, which was 26.3% higher than the original medium fermentation production of lincomycin (3640.6 mg / L). Figure 8 It can be seen that the addition of the inorganic salt and amino acid combination of the present invention can significantly increase the fermentation yield of lincomycin.

[0073] The above is a detailed introduction to one embodiment of the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the technical solutions and core ideas of the present invention. Those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for optimizing the fermentation medium of lincomycin genetically engineered bacteria to increase the production of lincomycin, characterized in that One or more combinations of inorganic salts, small molecule thiols and amino acids are added to the lincomycin fermentation medium.

2. The method of the preceding claim, wherein the types of inorganic salts include cobalt chloride, magnesium chloride, ferrous sulfate, sodium sulfate, etc.; wherein the types of small molecule thiols include ethanethiol, inositol, etc.; wherein the types of amino acids include proline, alanine, methionine, valine, etc.; wherein the types of inorganic salts can be further preferably cobalt chloride and sodium sulfate; wherein the types of small molecule thiols can be further preferably inositol; wherein the types of amino acids can be further preferably proline, methionine and valine.

3. The method of the preceding claim, wherein the amount of the inorganic salt can be further preferably: cobalt chloride 1-10 mg / L, sodium sulfate 0.15-1.5 g / L; wherein the amount of the small molecule thiol can be further preferably: inositol 0.2-0.8 g / L; wherein the amount of the amino acid can be further preferably: proline 0.02-0.2 g / L, methionine 0.02-0.2 g / L, valine 0.02-0.2 g / L.

4. The method of the preceding claim, wherein the dosage of the inorganic salt may further preferably be: 3-6 mg / L of cobalt chloride, 0.8-1.6 g / L of sodium sulfate; wherein the dosage of the amino acid may further preferably be: 0.1-0.3 g / L of methionine.

5. The method of the preceding claim, wherein the dosage of the inorganic salt may further preferably be: 3.6 mg / L of cobalt chloride, 1.23 g / L of sodium sulfate; wherein the dosage of the amino acid may further preferably be: 0.26 g / L of methionine.

6. The process of the preceding claims, wherein the inorganic salts and amino acids are used as additions to the fermentation medium or in combination with other inorganic salts and amino acids.

7. The method of the preceding claim, wherein the fermentation medium comprises a lincomycin producing strain, such as Streptomyces lincomycin, preferably the lincomycin genetically engineered strain L-427Δ2790.

8. The method of the preceding claim, a kit for optimizing the fermentation medium of lincomycin genetically engineered bacteria to increase the production of lincomycin, characterized in that: The kit comprises the additive combination described in any one of claims 1 to 5.

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