Method for regulating and controlling components of polymyxin B homolog

A technology of polymyxin and bacterial strains, which is applied in the field of regulating homologue components of polymyxin B, and can solve the problems that the regulation mechanism of polymyxin B components is not very clear and lacks effective methods

Active Publication Date: 2020-04-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many literatures have reported the mechanism of non-ribosomal peptide synthetase, and optimized the regulation of the components of other lipopeptide substances through medium optimization. At the same time, the gene cluster and synthesis mechanism of polymyxin B have also been studied,

Method used

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  • Method for regulating and controlling components of polymyxin B homolog
  • Method for regulating and controlling components of polymyxin B homolog
  • Method for regulating and controlling components of polymyxin B homolog

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1L seed medium contains the following ingredients: 3-7g glucose, 25-35g starch, 0.5-3g yeast powder, 2-6g peptone, NaCl 0.3-1g, KH 2 PO 4 0.8-2g, MgSO 4 ·7H 2 O 0.1-1.2g, distilled water dilute to 1L, natural pH.

Embodiment 2

[0047] 1L fermentation medium contains the following components: glucose 50-80g, yeast powder 0.1-2.0g, beef extract 1-3g, peptone 6-12g, NaCl 3-8g, KH 2 PO 4 0.4-1.4g, KCl 0.2-0.6g, MgSO 4 ·7H 2 O 0.4-1.2g, FeSO 4 ·7H 2 O0.01-0.05mg, MnSO 4 ·7H 2 O 3-9mg, distilled water to 1L, natural pH.

Embodiment 3

[0048] Example 3 (control)

[0049] (1) Seed culture: Culture the Bacillus polymyxa CJX518 (Paenibacillus polymyxa) with the deposit number CGMCC 7096 in the seed medium of Example 1 to obtain first-level seeds; cultivate the first-level seeds in the seed medium, Get secondary seeds;

[0050] (2) Production of polymyxin B: According to the initial OD of the secondary seeds obtained in step (1) 600 The inoculum amount of 0.2 was inserted into 200 mL of the fermentation medium of Example 2, and fermentation was carried out in a shake flask for 72 hours at a rotation speed of 200 rpm and a temperature of 30°C.

[0051] The content of polymyxin B1 is 41%, the content of B2 is 23%, the content of B3 is 17%, and the content of B1-1 is 19%.

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Abstract

The invention discloses a method for regulating and controlling the components of a polymyxin B homolog. The method comprises the following steps: (1) constructing pMAD-DeltaA and pMAD-DeltaC; (2) transforming pMAD-DeltaA into CJX518 strains, conducting screening to obtain successfully-transformed strains, performing culturing and subculturing to obtain plasmid-lost strains, verifying the plasmid-lost strains as structural domain-replaced strains 518-A through PCR sequencing, transforming pMAD-DeltaC into the CJX518 strains, screening the pMAD-DeltaC transformed strains 518-A, carrying out culturing and subculturing to obtain plasmid-lost strains, and verifying the plasmid-lost strain as structural domain-replaced strains 518-AC through PCR sequencing; (3) carrying out seed culture to obtain secondary seeds; and (4) producing polymyxin B by inoculating the secondary seeds into a fermentation medium, and carrying out fermenting for 72 hours. According to the method disclosed by the invention, the components of the polymyxin B homolog are regulated and controlled, a ratio of B1 to B2 is increased, and a ratio of B3 to B1-1 is reduced.

Description

Technical field [0001] The invention belongs to the technical field of biology and new medicine, and relates to a method for regulating the homologous components of polymyxin B. Background technique [0002] Polymyxin B has strong inhibitory activity against super bacteria and is currently the last line of defense for humans to use antibiotics. It has nephrotoxicity and neurotoxicity during use. Polymyxin B is a complex mixture containing multiple homologues produced by Bacillus polymyxa catalyzed by non-ribosomal peptide synthetase. Clinical and commercial polymyxin B preparations usually include B1, B2, B3, B1 -1 Four homologues, the main components of which are B1 and B2. Different single components have different activities, and the combined effects are different, and the clinical effects caused are also different. Many literatures reported on the mechanism of non-ribosomal peptide synthetase, the components of other lipopeptides were optimized and regulated by the medium, ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12P21/04C07K7/62C12R1/01
CPCC12N15/66C12P21/02C07K7/62
Inventor 程景胜袁野元英进
Owner TIANJIN UNIV
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