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Novel non-ribosome polypeptide synthetase resolution expression method

An expression method and a sub-unit technology, applied in the field of non-ribosomal polypeptide synthetase split expression technology, can solve difficult problems and achieve the effect of increasing production

Pending Publication Date: 2022-06-24
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is increasingly difficult to obtain novel non-ribosomal peptides with practical value using traditional screening techniques

Method used

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  • Novel non-ribosome polypeptide synthetase resolution expression method
  • Novel non-ribosome polypeptide synthetase resolution expression method
  • Novel non-ribosome polypeptide synthetase resolution expression method

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the determination of NRPS splitting unit

[0040] The auxotrophic Saccharomyces cerevisiae BJ5464-NpgA was used as the chassis cell, and the beauvericin NRPS BbBEAS (the amino acid sequence shown in SEQ ID NO. ) as an example (Xu YQ, Orozco R, Wijeratne EMK, Gunatilaka AAL, Stock SP, Molnar I. 2008. Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulencefactor of the entomopathogenic fungus Beauveria bassiana. Chem Biol15(9):898-907.) , BbBEAS was split and expressed in two sections with the structural domain as the node, and the production of beauvericin in the transformants was detected by LC-MS, and the efficient splitting unit was screened.

[0041] experimental method

[0042] (1) Construction of heterologous expression cassettes for BbBeas and its split fragments

[0043] According to the sequence information of BbBeas and its structural domain, its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in...

Embodiment 2

[0054] Example 2. Determination of NRPS splitting site

[0055] Using auxotrophic Saccharomyces cerevisiae BJ5464-NpgA as chassis cells to split BbBEAS into C at different split sites 1 -A 1 -T 1 and T 1 -C 2 -A 2 -T 2a -T 2b -C 3 Two splitting units are used to detect the production of beauvericin in transformants by LC-MS, and screen for efficient splitting sites.

[0056] experimental method:

[0057] (1)C 1 -A 1 -T 1 and T 1 -C 2 -A 2 -T 2a -T 2b -C3 Construction of heterologous expression cassettes.

[0058] According to the sequence information of BbBeas and its domains (see Sequence Listing), specific primers (see Table 1) were designed, and corresponding start codons, stop codons and vector homologous fragments were added to the ends of the primers. According to the instructions of the seamless cloning kit, these fragments are connected with the expression vector, and finally the expression vector containing the heterologous expression cassette of BbB...

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Abstract

The invention relates to the technical field of biology, and discloses a non-ribosome polypeptide synthetase resolution expression method, which is characterized in that according to the prediction of an NRPS structural domain and a three-dimensional structure, NRPS is subjected to resolution expression at a special resolution site by taking Tn-1-Cn-An-Tn as a unit, so that the expression of an NRPS gene is converted into multi-gene co-expression by taking a resolution unit as an independent gene, and the non-ribosome polypeptide synthetase is obtained. The corresponding non-ribosome polypeptide product is efficiently obtained. The method not only can be used for heterologous expression of the giant NRPS in fungi and increase the yield of the non-ribosome polypeptide, but also provides a theoretical basis and way for synthesizing novel non-ribosome polypeptide by performing inter-module combination through a combinatorial biosynthesis method. The method has relatively high practical application value in the aspects of excavation of natural non-ribosome polypeptides by utilizing synthetic biology, yield improvement, creation of non-natural non-ribosome polypeptides and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a novel non-ribosomal polypeptide synthase splitting and expression technology. Background technique [0002] Non-ribosomal polypeptide natural products are an important source of modern drug creation, such as antifungal drug echinocandin, immunosuppressive drug cyclosporine, etc. However, it is increasingly difficult to obtain novel non-ribosomal polypeptides with practical value using traditional screening techniques. With the rapid development of synthetic biology technologies such as high-throughput genome sequencing technology, gene synthesis, and multi-gene assembly, it has provided a way to mine and produce valuable non-ribosomal polypeptides through heterologous biosynthesis technology. [0003] The core backbone of nonribosomal peptide compounds is assembled by modular nonribosomal peptide synthetase (NRPS). Generally, NRPS is composed of multiple modules, and each module co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/81C12N1/19C12R1/865
CPCC12N9/93C12N15/81C12Y602/01
Inventor 徐玉泉岳群尹淼淼张礼文
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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