CN-121975831-A - Discontinuous pairing homology arm mediated Red/ET homologous recombination method and application thereof

CN121975831ACN 121975831 ACN121975831 ACN 121975831ACN-121975831-A

Abstract

The invention belongs to the technical fields of genetic engineering and synthetic biology, and particularly relates to a discontinuous pairing homologous arm mediated Red/ET homologous recombination method and application thereof. The method of the invention is characterized in that a discontinuous pairing homologous arm mediated homologous recombination engineering technology is utilized, and the method is essentially different from the traditional Red/ET type recombinase mediated continuous homologous arm homologous recombination. Experiments prove that the method can be applied to modularized gene cluster modification of non-ribosomal polypeptide synthetase (NRPS), the modification efficiency of the NRPS is remarkably improved, the modification success rate of natural products synthesized by the NRPS is greatly improved, and key technical support is provided for fully releasing the application potential of the active natural products. Therefore, the method not only innovates and effectively supplements the existing Red/ET editing method, but also is an ideal expansion of the current genetic engineering technology system, and has good practicability and wide application value.

Inventors

ZHONG LIN
BIAN XIAOYING
TU QIANG
ZHANG YOUMING
WANG XINGYAN
SHI XINGXING

Assignees

湖南文理学院

Dates

Publication Date: 20260505
Application Date: 20260120

Claims (10)

1. A non-continuous paired homology arm mediated Red/ET homology recombination method, characterized in that the homology arms used are non-continuous paired homology arms comprising a plurality of continuously paired regions, wherein the length of a single continuously paired region is no more than 50bp, compared to Red/ET class recombinase mediated homology recombination methods.
2. The method of non-contiguously paired homology-arm mediated Red/ET homology recombination according to claim 1, wherein the non-contiguously paired homology arms comprise a homology pairing region and a non-homology pairing region.
3. The discontinuous paired homology arm mediated Red/ET homology recombination method according to claim 2, wherein the discontinuous paired homology arms comprise n homology paired regions and n-1 non-homology paired regions, and the homology paired regions and the non-homology paired regions are arranged in interval, wherein n is an integer not less than 2.
4. The method of discontinuous paired homology-arm mediated Red/ET homologous recombination according to claim 2, wherein the length of the DNA homologous sequence in the homologous pairing zone is no more than 50bp and the length of the DNA non-homologous sequence in the non-homologous pairing zone is greater than 1bp.
5. The method of non-continuous paired homology arm mediated Red/ET homologous recombination according to any of claims 2-4, wherein the homology arms comprise an upstream homology arm and a downstream homology arm.
6. The method of discontinuous paired homology arm mediated Red/ET homology recombination according to claim 5, wherein at least one of the upstream homology arm and the downstream homology arm is a discontinuous paired homology arm.
7. The use of the discontinuous paired homology arm mediated Red/ET homologous recombination method according to any one of claims 1-6 in the modification of a non-ribosomal polypeptide synthase modular gene cluster.
8. The use of claim 7, wherein the non-ribosomal polypeptide synthase modular gene cluster engineering comprises one or more of non-ribosomal polypeptide synthase modular gene cluster internal engineering, inter-gene cluster engineering, and gene cluster rational design.
9. A recombinant vector for modifying a non-ribosomal polypeptide synthetase modularized gene cluster is characterized by comprising a basic vector and a DNA fragment of the non-ribosomal polypeptide synthetase modularized gene cluster with an upstream homology arm and a downstream homology arm, wherein the basic vector carries the non-ribosomal polypeptide synthetase gene cluster, the DNA fragment of the non-ribosomal polypeptide synthetase modularized gene cluster with the upstream homology arm and the downstream homology arm is subjected to homologous recombination with the basic vector, at least one of the upstream homology arm and the downstream homology arm is a non-continuous pairing homology arm, and a plurality of continuous pairing regions are contained in the non-continuous pairing homology arm, wherein the length of a single continuous pairing region is not more than 50bp.
10. The engineering bacterium for modifying the non-ribosomal polypeptide synthetase modularized gene cluster is characterized by comprising a basic bacterium and the recombinant vector of claim 9, wherein the basic bacterium is a host bacterium with Red/ET recombinant enzymes.

Description

Discontinuous pairing homology arm mediated Red/ET homologous recombination method and application thereof Technical Field The invention belongs to the technical fields of genetic engineering and synthetic biology, and particularly relates to a discontinuous pairing homologous arm mediated Red/ET homologous recombination method and application thereof. Background Natural Products (NPs) are an important source of clinical drugs and are also key active ingredients in agricultural biocontrol agents. Non-ribosomal polypeptides (Non-Ribosomal Peptide, NRP) are an important proportion of natural product drugs, including antibiotics such as penicillin, vancomycin, polymyxin, daptomycin, and the like, as well as the immunosuppressant cyclosporin and the anticancer drug bleomycin. NRPs natural products are synthesized by multi-module enzyme catalysis called non-ribosomal polypeptide synthases (NRPSs), but the natural products with complex and various structures face a plurality of challenges through total chemical synthesis, so that the pharmaceutical properties of the natural products are improved through semisynthesis, and the method is still a mainstream method in the current medicine research and development field. Compared with the traditional total chemical synthesis or semisynthesis method, the combined biosynthesis technology of natural products provides a faster and more economical way to exploit its pharmaceutical potential. In the preparation of natural products using combinatorial biosynthesis techniques, while researchers have invested a great deal of effort in trying to find universal fusion sites for reprogramming of NRPS, strategies that rely on traditional trial-and-error approaches remain significantly limited. The method generally needs to carry out protein crystallography analysis, manual screening of potential fusion sites and subsequent demonstration test and application. However, due to the influence of NRPS evolutionary diversity and structural complexity, such methods have the problems of low efficiency and poor generality, and it is difficult to meet the wider demands of NRPS engineering. Therefore, the current art still cannot realize the "plug and play" replacement of NRPS modules or domains in a true sense, and lacks a systematic, efficient and high-throughput method that can comprehensively modify NRPS gene clusters. Disclosure of Invention The invention aims to provide a discontinuous pairing homologous arm mediated Red/ET homologous recombination method (PARTIALLY PAIRED Homologous Arms (ppHA) -MEDIATED ET Recombineering) and application thereof, wherein the method can remarkably improve the transformation efficiency of NRPS, and further greatly improve the transformation success rate of natural products synthesized by the NRPS. The invention provides a non-continuous pairing homologous arm mediated Red/ET homologous recombination method, which is characterized in that the used homologous arm is a non-continuous pairing homologous arm, the non-continuous pairing homologous arm comprises a plurality of continuous pairing regions, and the length of a single continuous pairing region is not more than 50bp. Preferably, the non-contiguous pairing homology arms include a homologous pairing region and a non-homologous pairing region. Preferably, the non-continuous pairing homology arms comprise n homologous pairing regions and n-1 non-homologous pairing regions, the homologous pairing regions and the non-homologous pairing regions are arranged at intervals, and n is an integer more than or equal to 2. Preferably, the length of the DNA homologous sequence in the homologous pairing zone is not more than 50bp, and the length of the DNA non-homologous sequence in the non-homologous pairing zone is more than 1bp. Preferably, the homology arm includes an upstream homology arm and a downstream homology arm. Preferably, at least one of the upstream homology arm and the downstream homology arm is a non-contiguous pairing homology arm. The invention also provides application of the discontinuous pairing homology arm mediated Red/ET homologous recombination method in non-ribosome polypeptide synthetase modularized gene cluster modification. Preferably, the non-ribosomal polypeptide synthase modular gene cluster modification comprises one or more of a non-ribosomal polypeptide synthase modular gene cluster internal modification, an inter-gene cluster modification, and a gene cluster rational design. The invention also provides a recombinant vector for modifying the non-ribosomal polypeptide synthetase modularized gene cluster, which comprises a basic vector and a DNA fragment of the non-ribosomal polypeptide synthetase modularized gene cluster with an upstream homology arm and a downstream homology arm, wherein the basic vector is provided with the non-ribosomal polypeptide synthetase gene cluster, the DNA fragment of the non-ribosomal polypeptide synthetase modularized gene cluster with the upstream ho