Artificial non-ribosomal peptide synthetases

A technology of variants and structural domains, applied in the direction of enzymes, ligases, drug combinations, etc., can solve problems such as module and/or structural domain compatibility uncertainty

Pending Publication Date: 2018-03-27
JOHANN WOLFGANG GOETHE UNIV FRANKFURT AM MAIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A problem with such swaps or combinations is often uncertainty about the compatibility of modules and / or domains with each other

Method used

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  • Artificial non-ribosomal peptide synthetases
  • Artificial non-ribosomal peptide synthetases
  • Artificial non-ribosomal peptide synthetases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Comparing the present invention (EU concept) with German Patent Application No. 1999151196

[0067] In order to compare the present invention with state-of-the-art methods, the following experiments were performed. In this experiment, we attempted to exchange several domains (yellow) in the Ambactin-producing NRPS AmbS to generate new Ambactin derivatives ( Figure 5 ): on the one hand according to the EU conception ( Figure 5 D), on the other hand according to the prior art method suggested by Marahiel et al. (WO200130985) ( Figure 5 C). Only the present invention yields the desired cyclic peptide. Another recombinant NRPS ( Figure 5 C) shows no generation of any new derivatives.

Embodiment 2

[0068] Example 2: De novo assembly of the NRPS biosynthetic cluster Plu3263 (GxpS) responsible for GameX peptide synthesis

[0069] In support of the accuracy of the present invention, we reassembled the NRPS to generate the GameX peptide from known NRPS building blocks. As predicted, the artificial NRPS was able to produce the desired peptide ( Image 6 ).

Embodiment 3

[0070] Example 3. De novo assembly of the NRPS biosynthetic cluster XtpS responsible for Xenotetrapeptide synthesis and generation of threonine-containing derivatives

[0071] In order to support the accuracy of the present invention and its application in the construction of new artificial NRPS to generate new peptides (from the newly constructed XtpS Figure 7 , artificial NRPS Figure 8 ).

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Abstract

The present invention concerns a novel method for the modification and / or custom-made design of artificial non-ribosomal peptide synthetases (NRPSs) from naturally available NRPSs. The artificial NRPSs are of predetermined length and amino acid composition and sequence. Via fusion of well-defined NRPS units (so-called "exchange units") in a certain manner, using a specific sequence motif in the linker areas it is possible to construct artificial and / or modified NRPS assembly lines, which have the ability of synthesizing peptides of a desired structure.

Description

Background of the invention [0001] Nonribosomal peptide synthases (NRPS) and polyketide synthases (PKS) are multifunctional enzyme complexes that contain a modular architecture (Marahiel 1997). Due to its drug-related properties, including antimicrobial (eg, teixobactin), antineoplastic (eg, bleomycin), antifungal (fengycin), and immunosuppressive (cyclosporine) activities , many natural products synthesized by these enzymes are of pharmaceutical and / or biotechnological interest (Ling et al. 2015, Ishizuka et al. 1967, Loeffler et al. 1986, Emmel et al. 1989). Although peptides generated from NRPS display a wide range of biological activities and enormous structural diversity (e.g., non-proteinogenic amino acids, N-methylation, epimerization, heterocycles), they share a common mode of synthesis, The so-called "multiple-carrierthiotemplate mechanism". [0002] The structure of NRPS is an obligatory module ( figure 1 ). A module is defined as the catalytic unit that incorpor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P21/02C12N15/62
CPCC12P21/02C12N15/1093C40B40/06C40B40/08C12N9/93C12Y603/02A61P31/04A61P31/10A61P35/00A61P37/06
Inventor C-P.尼泽特H.B.博德K.博茨许于克F.弗莱施哈克
Owner JOHANN WOLFGANG GOETHE UNIV FRANKFURT AM MAIN
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