Key exchange domain for controlling lipid chain length change of lipopeptide, mutants and application of mutants

A technology for exchanging structures and domains, applied in the biological field, to achieve the effects of reducing toxic side effects, promoting clinical drug production, and increasing activity

Active Publication Date: 2021-03-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, so far, there has been no report on the co-crystal structure of the Cs domain and its fatty acyl substrate, so it is difficult to control the substrate specificity of the Cs domain through rational modification, and further directional modification of lipopeptides

Method used

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  • Key exchange domain for controlling lipid chain length change of lipopeptide, mutants and application of mutants
  • Key exchange domain for controlling lipid chain length change of lipopeptide, mutants and application of mutants
  • Key exchange domain for controlling lipid chain length change of lipopeptide, mutants and application of mutants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The Cs-AL regions of rzmA and holA were exchanged to obtain mutants rzmACsholA and holACsrzmA, and the catalytic activity of the mutants was verified from in vivo and in vitro levels. That is, the ability of the full-length rzmA / holA gene cluster and the corresponding mutants to catalyze the formation of the final product rzmA / holA and its derivatives shall prevail (the structure is as follows figure 1 , shown in a and b), the quantification adopts absolute quantification, after separating and purifying the derivative and wild-type lipopeptide product, it is used as a standard to quantify the mutant, and the quantitative instrument is liquid chromatography-mass spectrometry (LC-MS).

[0027] The specific implementation steps are as follows:

[0028] (1) According to antismash ( https: / / antismash.secondarymetabolites.org / #! / start ) to predict the Cs-AL region of rzmA and holA, and determine its amino acid sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0029] ...

Embodiment 2

[0036] Further, the glbA gene cluster derived from DSM7029 was manipulated, and the Cs-AL region of glbA was replaced by the Cs-AL of holA from the heterologous strain DS19002, and the Cs-AL of glpA from the homologous strain DSM7029. The obtained mutants were glbACsholA and glbACsglpA, and the catalytic activity of the mutants was verified from in vivo and in vitro levels. That is, the ability of the full-length glbA gene cluster and corresponding mutants to catalyze the formation of the final product glbA and its derivatives shall prevail (the structure is as follows figure 2 , shown in a and b), the quantification adopts absolute quantification, after separating and purifying derivatives and wild-type lipopeptide products, it is used as a standard to quantify the mutants, and the quantitative instrument is liquid chromatography-mass spectrometry (LC-MS).

[0037] The Red / ET recombination system and operation in DSM7029 involved in Example 2 were all used in literature 3 (W...

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Abstract

The invention discloses a key exchange domain for controlling lipid chain length change of lipopeptide. The key exchange domain is a Cs-AL domain after exchange of a Cs-AL domain of RhizomideA and a Cs-AL domain of Holrhizin, or a Cs-AL domain after replacement of a Cs-AL domain of glbA by the Cs-AL domain of the Holrhizin, or a Cs-AL domain after replacement of the Cs-AL domain of the glbA by a Cs-AL domain of glpA. The invention also discloses mutants obtained by exchanging the key exchange domain and application of the mutants. Experiments prove that for the RhizomideA, the mutant rzmACsholA can directionally obtain a derivative with C8 lipid chain length; for the Holrhizin, the mutant holACsrzmA can directionally obtain a derivative with C2 lipid chain length; and the two mutants glbACsholA and glbACsglpA of the glbA can obtain derivatives with C8 and C10 lipid chain lengths respectively. It is indicated that the key exchange domain has huge application value in guiding modification of lipopeptide which has a medicinal prospect and even is already a patent medicine.

Description

technical field [0001] The invention relates to a key structural domain for controlling the change of lipopeptide lipid chain length and its mutant and application, belonging to the field of biotechnology. Background technique [0002] Non-ribosomal peptides are an important class of microbial natural products, which have a wide range of biological activities, such as antibacterial, antitumor, immunosuppressive, etc. The gene clusters of non-ribosomal peptides mainly synthesize peptide chains of different lengths through an iterative module structure (module), and the modules can be further subdivided into domains, mainly including: condensation domain (C domain), Adenylation domain (Adenylation domain: A domain), thiolation domain (Thiolation domain: T domain). These three domains constitute a basic module, and multiple modules constitute a huge non-ribosomal peptisome, in which the initial thiolation domain is converted into The activated form with the long arm of phosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P21/00C12R1/19
CPCC12N9/93C12P21/00C12Y603/02
Inventor 卞小莹钟林张娜陈汉娜武大雷张友明
Owner SHANDONG UNIV
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