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CN-122012657-A - Fermentation method for improving yield of polymyxin B

CN122012657ACN 122012657 ACN122012657 ACN 122012657ACN-122012657-A

Abstract

The invention discloses a fermentation method for improving polymyxin B yield, which comprises the following steps of 1, inoculating polymyxin bacillus into an initial culture medium containing Mg 2+ and Mn 2+ for fermentation, wherein the concentration of Mg 2+ in the culture medium is 0.5-10mM, the concentration of Mn 2+ in the culture medium is 5-100 mu M, 2, sampling fermentation liquid at intervals, measuring the concentration of Mg 2+ /Mn 2+ in the fermentation liquid, and supplementing a concentrated solution containing Mg 2+ and/or Mn 2+ into the fermentation liquid 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 as to maintain the concentration of Mg 2+ in the fermentation liquid at 0.5-10mM and the concentration of Mn 2+ at 5-100 mu M. In the fermentation process of the Paenibacillus polymyxa, the concentration, the proportion and the addition time sequence of Mg 2+ and Mn 2+ ions in a culture medium are precisely controlled and optimized, so that the maximum catalytic efficiency of an NRPS enzyme system is met, and the synthesis of polymyxin B is efficiently driven.

Inventors

  • DU QIANQIAN
  • HAN BING
  • LI MENG

Assignees

  • 河北圣雪大成制药有限责任公司

Dates

Publication Date
20260512
Application Date
20260329

Claims (8)

  1. 1. A fermentation process for increasing polymyxin B production comprising the steps of: Step 1, inoculating bacillus polymyxa into an initial culture medium containing Mg 2+ and Mn 2+ for fermentation, wherein the concentration of Mg 2+ in the culture medium is 0.5-10mM, and the concentration of Mn 2+ in the culture medium is 5-100 mu M; And 2, sampling the fermentation liquor at intervals, measuring the concentration of Mg 2+ /Mn 2+ in the fermentation liquor, 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 as to maintain the concentration of Mg 2+ in the fermentation liquor at 0.5-10mM and the concentration of Mn 2+ at 5-100 mu M.
  2. 2. The fermentation method for improving yield of polymyxin B according to claim 1, wherein the fermentation is carried out by placing the Bacillus polymyxin under conditions of 25-35 ℃ and pH 6.8-7.4.
  3. 3. The fermentation process for increasing yield of polymyxin B according to claim 1, wherein the molar concentration ratio of Mg 2+ to Mn 2+ in the medium is 70-120:1.
  4. 4. The fermentation process for increasing polymyxin B production according to claim 1, wherein the concentration of Mg 2+ in the medium is 2-5 mM and the concentration of Mg 2+ in the fermentation broth is maintained at 2-5 mM after the Mg 2+ concentrate is added.
  5. 5. The fermentation process for increasing polymyxin B production according to claim 1, wherein the concentration of Mn 2+ in the medium is 2-80. Mu.M, and the concentration of Mn2 in the fermentation broth is maintained at 2-80. Mu.M after the Mn 2+ concentrate is added.
  6. 6. The fermentation process for increasing yield of polymyxin B according to claim 1, wherein the first threshold is 2.5mM.
  7. 7. The fermentation process for increasing yield of polymyxin B according to claim 1, wherein the second threshold is 20 μm.
  8. 8. The fermentation process for increasing yield of polymyxin B according to claim 1, wherein the magnesium ion is derived from magnesium sulfate or magnesium chloride and the manganese ion is derived from magnesium sulfate or manganese chloride.

Description

Fermentation method for improving yield of polymyxin B Technical Field The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a fermentation method for improving yield of polymyxin B. Background Polymyxin is a generic term for a family of basic cyclic polypeptides consisting of a variety of amino acids and fatty acids produced by paenibacillus polymyxa. Polymyxin A, B, C, D, E, which is different in chemical structure due to different strains, is produced. The products on the market in clinical application are mainly sulfate and mesylate of polymyxin B and polymyxin E. Wherein the antibacterial effect of polymyxin B is superior to that of polymyxin E. Polymyxin has strong bactericidal effect on gram-negative bacteria, and clinical application of the medicine mainly adopts external or local medicine due to great nephrotoxicity, ototoxicity and neuromuscular blocking effect. Especially, the medicine has good effect on preventing scald, post-surgical infection of pseudomonas aeruginosa, drug-resistant dysentery bacteria and diseases caused by other gram-negative bacteria, and has high safety in use. At the same time, the composition is used as the final choice for treating multi-drug resistant gram-negative bacteria infection, is used for treating meningitis, dysentery and the like caused by gram-negative bacteria, and does not generate drug resistance. The global sales of polymyxin B for external use is stabilized around 4 billion dollars each year, and clinical application of polymyxin B will be more widespread with establishment of reasonable dosing regimens and further study of toxicity. At present, the production of polymyxin B adopts a microbial fermentation method. The domestic fermentation manufacturers are few, the fermentation process and the fermentation level are low, and the fermentation titer is basically lower than 8000 u/mL. The main reason is that the polymyxin B producing strain is bacillus polymyxa, and the strain is fast in metabolism, short in culture period and difficult to accurately regulate and control in fermentation process. It is therefore necessary to explore new fermentation formulas and processes. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a fermentation method for improving the yield of polymyxin B, which is based on the deep understanding of the mechanism of Mg 2+/Mn2+ in the NRPS enzymatic reaction, and accurately controls and optimizes the concentration, proportion and addition time sequence of Mg 2+ and Mn 2+ ions in a culture medium in the fermentation process of the polymyxin B, thereby meeting the maximum catalytic efficiency of an NRPS enzyme system and efficiently driving the synthesis of the polymyxin B. In order to solve the technical problems, the invention adopts the following technical scheme: A fermentation process for increasing yield of polymyxin B comprising the steps of: Step 1, inoculating bacillus polymyxa into an initial culture medium containing Mg 2+ and Mn 2+ for fermentation, wherein the concentration of Mg 2+ in the culture medium is 0.5-10mM, and the concentration of Mn 2+ in the culture medium is 5-100 mu M; And 2, sampling the fermentation liquor at intervals, measuring the concentration of Mg 2+/Mn2+ in the fermentation liquor, 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 as to maintain the concentration of Mg 2+ in the fermentation liquor at 0.5-10mM and the concentration of Mn 2+ at 5-100 mu M. Further, the Bacillus polymyxa is subjected to fermentation at 25-35 ℃ and pH 6.8-7.4. Further, the molar concentration ratio of Mg 2+ to Mn 2+ in the culture medium is 70-120:1. Further, the concentration of Mg 2+ in the culture medium is 2-5 mM, and the concentration of Mg 2+ in the fermentation broth is maintained at 2-5 mM after the concentrated solution of Mg 2+ is added. Further, the concentration of Mn 2+ in the culture medium is 2-80 mu M, and the concentration of Mn2 in the fermentation broth is maintained at 2-80 mu M after the Mn 2+ concentrate is added. Further, the first threshold is 2.5mM. Further, the second threshold is 20. Mu.M. Further, the magnesium ion is derived from magnesium sulfate or magnesium chloride, and the manganese ion is derived from magnesium sulfate or manganese chloride. Compared with the prior art, the invention has the beneficial effects that: 1) The A domain is responsible for recognizing and catalyzing the formation of aminoacyl-AMP of specific amino acids (e.g., D-Phe, leu, thr in polymyxin B, etc.) in the catalytic cycle of non-ribosomal peptide synthetases (NRPS) to increase yield. Mg 2+ neutralizes the negative charge of the ATP phosphate group by complexation, stabilizes the leaving group of pyrophosphate (PPi), and