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A key exchange domain that controls changes in the lipid chain length of lipopeptides and its mutants and applications

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

Active Publication Date: 2022-07-22
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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  • A key exchange domain that controls changes in the lipid chain length of lipopeptides and its mutants and applications
  • A key exchange domain that controls changes in the lipid chain length of lipopeptides and its mutants and applications
  • A key exchange domain that controls changes in the lipid chain length of lipopeptides and its mutants and applications

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Experimental program
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Effect test

Embodiment 1

[0026] The Cs-AL regions of rzmA and holA were exchanged to obtain mutants rzmACsholA and holACsrzmA, and the catalytic activities of the mutants were verified from in vitro and in vivo 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 shown in figure 1 , a, b), absolute quantification was used for quantification. After separation and purification of derivatives and wild-type lipopeptide products, they were used as standards to quantify mutants. The quantitative instrument was liquid chromatography-mass spectrometry (LC-MS).

[0027] The specific implementation steps are as follows:

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

[0029] (2) Usin...

Embodiment 2

[0036] Further, the glbA gene cluster derived from DSM7029 was manipulated to replace the Cs-AL region of glbA with Cs-AL from holA of heterologous bacteria DS19002 and Cs-AL of glpA from homologous bacteria DSM7029. The obtained mutants were glbACsholA and glbACsglpA respectively, and the catalytic activities of the mutants were verified from in vitro and in vivo levels. That is, the ability of the full-length glbA gene cluster and the corresponding mutants to catalyze the formation of the final product glbA and its derivatives shall prevail (structures such as figure 2 , shown in a, b), absolute quantification was used for quantification. After separating and purifying the derivatives and wild-type lipopeptide products, the mutants were quantified by using them as standards. The quantitative instrument was liquid chromatography-mass spectrometry (LC-MS).

[0037] The Red / ET recombination system and operation in DSM7029 involved in the embodiment 2 all adopt document 3 (Wang...

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Abstract

The invention discloses a key exchange domain for controlling the change of the lipid chain length of lipopeptides, wherein the key exchange domain refers to the exchange of the Cs-AL domain of rhizopeptide with the Cs-AL domain of rhizopeptide The latter Cs-AL structural domain, or the Cs-AL structural domain after the Cs-AL structural domain of the chrysanthemum peptide is replaced by the Cs-AL structural domain of the rhizopeptide, or the Cs-AL structural domain of the chrysanthemum peptide The Cs-AL domain after being replaced by the Cs-AL domain of calicheamicin. The invention also discloses mutants obtained by exchanging the key exchange domains and their applications. Experiments show that for rhizopamide, the rzmACsholA mutant can directionally obtain derivatives with C8 lipid chain length, and for rhizopeptides , the holACsrzmA mutant can be directed to obtain derivatives of C2 lipid chain length, while the two mutants of synobactin, glbACsholA and glbACsglpA, can obtain derivatives of C8 and C10 lipid chain lengths, respectively. It is predicted that the present invention has great application value in guiding the transformation of lipopeptides with medicinal prospects and even drugs.

Description

technical field [0001] The invention relates to a key structural domain for controlling the change of the lipid chain length of a lipopeptide, a mutant thereof and an application thereof, belonging to the field of biotechnology. Background technique [0002] Non-ribosomal peptides are an important class of microbial natural products with 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 (domains), mainly including: condensation domain (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 peptidase, in which the initial thiolated domain is activated by pho...

Claims

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Application Information

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Patent Type & Authority Patents(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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