Fermentation method for improving yield of polymyxin B

Abstract

The invention discloses a fermentation method for increasing the yield of polymyxin B. The fermentation method comprises the following steps that 1, bacillus polymyxa is inoculated into an initial culture medium containing Mg < 2 + > and Mn < 2 + > for fermentation, the concentration of Mg < 2 + > in the culture medium is 0.5-10 mM, and the concentration of Mn < 2 + > in the culture medium is 5-100 mu M; and step 2, sampling the fermentation liquor at set intervals, measuring the concentration of Mg < 2 + > / Mn < 2 + >, and supplementing a concentrated solution containing Mg < 2 + > and / or Mn < 2 + > into the fermentation liquor when the concentration of Mg < 2 + > is lower than a first threshold value and / or the concentration of Mn < 2 + > is lower than a second threshold value, so that the concentration of Mg < 2 + > in the fermentation liquor is maintained at 0.5-10mM, and the concentration of Mn < 2 + > is maintained at 5-100mu M. In the fermentation process of paenibacillus polymyxa, the concentration, the proportion and the adding time sequence of Mg < 2 + > and Mn < 2 + > ions in the culture medium are accurately controlled and optimized, the maximum catalytic efficiency of an NRPS enzyme system is met, and therefore synthesis of polymyxin B is efficiently driven.

Description

Technical Field

[0001] This invention belongs to the technical field of microbial fermentation engineering, specifically relating to a fermentation method for increasing polymyxin B production. Background Technology

[0002] Polymyxins are a class of basic cyclic polypeptide antibiotics produced by *Bacillus polymyxa*, composed of various amino acids and fatty acids. Different strains produce polymyxins A, B, C, D, and E with different chemical structures. Clinically available products are mainly polymyxin B and polymyxin E sulfates and mesylates. Polymyxin B has superior antibacterial activity compared to polymyxin E. Polymyxins have strong bactericidal activity against Gram-negative bacteria. Due to their significant nephrotoxicity, ototoxicity, and neuromuscular blocking effects, their clinical application is primarily topical or localized. They are particularly effective in preventing infections caused by *Pseudomonas aeruginosa* after burns and surgery, as well as drug-resistant *Shigella* and diseases caused by other Gram-negative bacteria, exhibiting high safety. Furthermore, they serve as a last resort for treating infections caused by multidrug-resistant Gram-negative bacteria, such as meningitis and dysentery, without inducing drug resistance. Global sales of polymyxin B topical medications remain stable at around $400 million annually. With the establishment of rational dosing regimens and further research on toxicity, the clinical application of polymyxin B will become more widespread.

[0003] Currently, polymyxin B is produced entirely through microbial fermentation. There are relatively few domestic fermentation manufacturers, and the overall fermentation technology and level are low, with fermentation potency generally below 8000 u / mL. The main reason is that polymyxin B is produced by *Bacillus polymyxa*, which has a rapid metabolism, short culture cycle, and makes precise control of the fermentation process difficult. Therefore, it is necessary to explore new fermentation formulations and processes. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a fermentation method for increasing polymyxin B yield. This method is based on the Mg2+ production process in the NRPS enzymatic reaction. 2+ / Mn 2+ A deep understanding of the mechanism is needed to address the role of Mg in the culture medium during the fermentation process of Bacillus polymyxa. 2+ and Mn 2+ The concentration, ratio, and timing of ion addition are precisely controlled and optimized to maximize the catalytic efficiency of the NRPS enzyme system, thereby efficiently driving the synthesis of polymyxin B.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A fermentation method for increasing polymyxin B yield includes the following steps: Step 1: Inoculate Bacillus polymyxa into a solution containing Mg 2+ and Mn 2+ Fermentation was carried out in the initial culture medium, wherein the culture medium contained Mg 2+ Concentrations of 0.5–10 mM, Mn 2+ Concentrations range from 5 to 100 μM; Step 2: Take samples of the fermentation broth at regular intervals and determine the Mg content. 2+ / Mn 2+ The concentration of Mg 2+ Concentration below the first threshold and / or Mn 2+ When the concentration is below the second threshold, add Mg-containing substances to the fermentation broth. 2+ and / or Mn 2+ A concentrated solution to reduce Mg in the fermentation broth. 2+ The concentration was maintained at 0.5–10 mM, Mn 2+ The concentration was maintained at 5–100 μM.

[0006] Furthermore, the polymyxin Bacillus was fermented at 25-35°C and pH 6.8-7.4.

[0007] Furthermore, Mg in the culture medium 2+ With Mn 2+ The molar concentration ratio is 70-120:1.

[0008] Furthermore, Mg in the culture medium 2+ Concentration of 2–5 mM, supplemented with Mg 2+ After concentrating the liquid, the Mg in the fermentation broth is reduced. 2+ The concentration was maintained at 2–5 mM.

[0009] Furthermore, Mn in the culture medium 2+ Concentrations of 2–80 μM, supplemented with Mn 2+ After concentration, the Mn² concentration in the fermentation broth is maintained at 2–80 μM.

[0010] Furthermore, the first threshold is 2.5mM.

[0011] Furthermore, the second threshold is 20 μM.

[0012] Furthermore, magnesium ions come from magnesium sulfate or magnesium chloride, and manganese ions come from magnesium sulfate or manganese chloride.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) Increased yield; in the catalytic cycle of nonribosomal peptide synthases (NRPS), the A domain is responsible for recognizing specific amino acids (such as D-Phe, Leu, Thr in polymyxin B) and catalyzing their formation of aminoacyl-AMP. Mg2+ By neutralizing the negative charge of the ATP phosphate group through coordination, it stabilizes the leaving group of pyrophosphate (PPi) and induces the active conformational closure of the A domain. Studies have found that in some NRPS systems, Mn... 2+ (even low concentrations of Ca) 2+ It can not only replace Mg 2+ This can even improve the selectivity of the A domain for specific substrate amino acids. When Mg 2+ When Mn is scarce or in a state of imbalance of metal ion homeostasis, 2+ It can occupy metal-binding sites and maintain the basic activity of the A-domain. Therefore, this invention optimizes Mg... 2+ / Mn 2+ The optimal catalytic state of the NRPS adenylation domain was ensured, significantly improving the activation efficiency and utilization of amino acid precursors, thereby increasing the final yield of polymyxin B by more than 20% compared to conventional culture media. 2) Improved production efficiency: This invention improves the utilization efficiency of ATP, reduces energy waste, accelerates the assembly rate of polypeptide chains, and may shorten the fermentation cycle.

[0014] 3) Stable process; the feeding strategy avoids the synthesis bottleneck caused by premature depletion of ion concentration, making the fermentation process more stable and controllable; 4) Cost-effectiveness: Without significantly increasing production costs, a substantial increase in output was achieved through precise control rather than simply increasing the amount of raw materials, which has extremely high industrial application value. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0017] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0018] This invention provides a fermentation method for increasing polymyxin B production. Each 1L of basal fermentation medium contains: 80–90 g wheat flour; 1–2 g amylase; 4–6 g glucose; 4–6 g low-temperature soybean flour; 2–4 g corn steep liquor; 4–6 g ammonium sulfate; 0.2–0.3 g potassium dihydrogen phosphate; 3–5 g calcium carbonate; an initial magnesium ion concentration controlled between 0.5 mM and 10 mM; an initial manganese ion concentration controlled between 5 μM and 100 μM; 0.5 g L-threonine; 0.5 g L-leucine; 0.5 g L-phenylalanine; and water; pH 6.8–7.4; sterilized at 115 °C for 20 min; and then fermented at 25–35 °C.

[0019] In the above-mentioned culture medium, magnesium ions are obtained by adding MgSO4·H2O or MgCl2 to the culture medium, preferably with the initial concentration controlled at 2 mM to 5 mM.

[0020] In the above-mentioned culture medium, manganese ions are obtained by adding MnSO4·H2O or MnCl2 to the culture medium, preferably with the initial concentration controlled between 20 μM and 80 μM.

[0021] After preparing the culture medium, the inoculum size was 5% (v / v), the temperature was 30 °C, the pressure was 0.05 MPa, the aeration was 1 vvm, the initial stirring was 200 rpm, and the dissolved oxygen was ≥30%. During fermentation, samples were aseptically taken every 3-5 hours, and Mg was determined offline using ICP-OES. 2+ / Mn 2+ When Mg 2+ Concentration below the first threshold and / or Mn 2+ When the concentration is below the second threshold, add Mg-containing substances to the fermentation broth. 2+ and / or Mn 2+ A concentrated solution to reduce Mg in the fermentation broth. 2+ The concentration is maintained between 0.5 mM and 10 mM, preferably between 2-5 mM; Mn 2+ The concentration is maintained between 5 μM and 100 μM, preferably between 20 and 80 μM.

[0022] Preferably, the first threshold is 2.5 mM and the second threshold is 20 μM.

[0023] To better illustrate the effect of Mg 2+ and Mn 2+ Precise control and optimization of the concentration, ratio, and timing of ion addition can maximize the catalytic efficiency of the NRPS enzyme system, thereby efficiently driving the synthesis of polymyxin B. The following section discusses different concentrations of Mg... 2+ Mn2+ The following are examples of regulation to illustrate this.

[0024] Mg 2+ Different concentrations Use 3.0 L of basal culture medium in a 5 L fermentation tank. Each L of culture medium contains 80–90 g wheat flour; 1–2 g amylase; 4–6 g glucose; 4–6 g low-temperature soybean flour; 2–4 g corn steep liquor; 4–6 g ammonium sulfate; 0.2–0.3 g potassium dihydrogen phosphate; 3–5 g calcium carbonate; and 0, 0.493, 0.740, 0.986, and 1.232 g of MgSO4·7H2O respectively. 2+ 0, 2, 3, 4, 5 mM); manganese sulfate 8.54 mg; L-threonine 0.5 g; L-leucine 0.5 g; L-phenylalanine 0.5 g; 0.1% defoamer and water; pH 7.0±0.1, sterilized at 115 ℃ for 20 min.

[0025] Among them, Mg 2+ The concentrations were 0, 2, 3, 4, and 5 mM, respectively, for Mn. 2+ The concentration was fixed at 50 μM. The bacteria were inoculated with *Bacillus polymyxin B* strain and fermented at 30°C and 200 rpm for 96 hours. The potency of polymyxin B in the final fermentation broth was determined by HPLC, and the results are shown in Table 1. Table 1 indicates that within the range of 0-5 mM, 4 mM Mg... 2+ At the optimal concentration, its potency was 7300 U / mL higher than that of the 20 mM control group (16500 U / mL) (an increase of 44.2%). When the concentration was increased to 5 mM, the potency began to decline.

[0026] Table 1 Optimal Mg 2+ Determination of initial concentration (n=3)

[0027] Mn 2+ Synergistic effect verification The 5 L container contains 3.0 L of liquid, and the culture medium (per 1 L of water) is the same as in Example 1, with 0.986 g of MgSO4·7H2O fixed (Mg 2+ 4 mM), examining Mn 2+ Concentrations were determined, and the results are shown in Table 2. Table 2 indicates that within the 0-80 μM range, 50 μM Mn... 2+ At the optimal concentration, it exhibits the best synergistic effect. When the concentration increases to 80 μM, the potency shows a plateau or a slight downward trend.

[0028] Table 2 Mn 2+ Synergistic effect verification (n=3)

[0029] Comparison of Concentration Maintenance and Feeding Effect A comparison was made between batch fermentation and dynamic feeding systems. The batch fermentation system used optimal initial conditions (Mg... 2+ :4 mM, Mn 2+ Fermentation was carried out at 50 μM, with no additional metal ions or other nutrients added throughout the fermentation process. The initial conditions of the dynamic feeding group were consistent with the batch fermentation group. During fermentation, ICP-OES monitoring revealed that the ion concentration was below the trigger threshold. Therefore, Mg was added at 30 h and 54 h of fermentation. 2+ Mn 2+ The concentrated salt solution maintained the concentrations of both ions within a set range, and the results are shown in Table 3. Table 3 shows that the fermentation cycle of the dynamic feeding group was reduced by 9.4%, the polymyxin production rate increased by 27.7%, the polymyxin production capacity per cell increased by 15.20%, and the final potency increased by 15.4%. This demonstrates that the dynamic feeding strategy brought comprehensive performance improvements to the fermentation production of polymyxin B.

[0030] Table 3. Comparison of overall performance between dynamic feeding process and batch fermentation (n=3)

[0031] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the content of this specification should be included within the protection scope of the present invention.

Claims

1. A fermentation method for increasing polymyxin B yield, characterized in that, Includes the following steps: Step 1: Inoculate Bacillus polymyxa into a solution containing Mg 2+ and Mn 2+ Fermentation was carried out in the initial culture medium, wherein the culture medium contained Mg 2+ Concentrations of 0.5–10 mM, Mn 2+ Concentrations range from 5 to 100 μM; Step 2: Take samples of the fermentation broth at regular intervals and determine the Mg content. 2+ / Mn 2+ The concentration of Mg 2+ Concentration below the first threshold and / or Mn 2+ When the concentration is below the second threshold, add Mg-containing substances to the fermentation broth. 2+ and / or Mn 2+ A concentrated solution to reduce Mg in the fermentation broth. 2+ The concentration was maintained at 0.5–10 mM, Mn 2+ The concentration was maintained at 5–100 μM.

2. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, Fermentation of Bacillus polymyxa was carried out at 25-35℃ and pH 6.8-7.

4.

3. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, Mg in culture medium 2+ With Mn 2+ The molar concentration ratio is 70-120:

1.

4. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, Mg in culture medium 2+ Concentration of 2–5 mM, supplemented with Mg 2+ After concentrating the liquid, the Mg in the fermentation broth is reduced. 2+ The concentration was maintained at 2–5 mM.

5. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, Mn in culture medium 2+ Concentrations of 2–80 μM, supplemented with Mn 2+ After concentration, the Mn² concentration in the fermentation broth is maintained at 2–80 μM.

6. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, The first threshold is 2.5mM.

7. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, The second threshold is 20 μM.

8. The fermentation method for increasing polymyxin B yield according to claim 1, characterized in that, Magnesium ions come from magnesium sulfate or magnesium chloride, while manganese ions come from magnesium sulfate or manganese chloride.

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