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CN-122012630-A - Recombinant enzyme combination, application and homologous recombination method suitable for bacteroides

CN122012630ACN 122012630 ACN122012630 ACN 122012630ACN-122012630-A

Abstract

The invention discloses a recombinase combination, application and homologous recombination method suitable for bacteroides, belonging to the technical field of microbial genetic engineering. The invention discloses a method for constructing and screening a combination of a single-chain annealing protein and a single-chain binding protein for the first time. The single-chain annealing protein and the single-chain binding protein are constructed in a shuttle expression vector of escherichia coli-bacteroides fragilis to realize the co-expression of the single-chain annealing protein and the single-chain binding protein in the bacteroides fragilis, so that the homologous recombination capacity of the bacteroides fragilis is obviously improved. By the method, the homologous recombination efficiency of the genome editing of the bacteroides fragilis is greatly improved, the absolute genome editing efficiency is improved to 0.052% from the level which can not be detected originally, and the research and application progress of genome engineering of the strain are obviously promoted.

Inventors

  • ZHENG WENTAO
  • WANG XUE
  • SONG JINGWEI
  • TU QIANG
  • ZHANG YOUMING

Assignees

  • 山东大学
  • 山东大学苏州研究院

Dates

Publication Date
20260512
Application Date
20260228

Claims (10)

  1. 1. A combination of recombinases suitable for use in bacteroides comprising a single-chain annealing protein and a single-chain binding protein of bacteroides.
  2. 2. The combination of recombinases as claimed in claim 1, wherein the single stranded annealed protein comprises any one of BaSSAP 9 -BaSSAP 22 , preferably any one of BaSSAP 2 、BaSSAP 6 and BaSSAP 9 , further preferably BaSSAP 9 .
  3. 3. The combination of claim 1, wherein the single-chain binding protein comprises any one of BaSSB 1 -BaSSB 74 , preferably BaSSB 39 .
  4. 4. The combination of recombinases of claim 1, wherein the combination of recombinases is a combination of BaSSAP 9 and BaSSB 39 ; Preferably, the nucleotide sequence of BaSSAP 9 is shown as SEQ ID NO.1, and the nucleotide sequence of BaSSB 39 is shown as SEQ ID NO. 2.
  5. 5. Use of a combination of recombinases as claimed in any one of claims 1 to 4 in homologous recombination of bacteroides sp; preferably, the bacteroides is bacteroides fragilis.
  6. 6. A homologous recombination method suitable for bacteroides, comprising using the recombinase combination of any one of claims 1-4, thereby improving homologous recombination efficiency.
  7. 7. The method of homologous recombination according to claim 6, wherein the method comprises designing a homology arm, constructing the recombinase combination on an E.coli-bacteroides shuttle vector, and electrically transforming bacteroides to obtain a corresponding transformant; preferably, the bacteroides is bacteroides fragilis.
  8. 8. The homologous recombination method according to claim 7, wherein the E.coli-bacteroides shuttle vector is pNBU.about. intN.about.2-ermF plasmid, and the nucleotide sequence of the pNBU.about.2-intN.about.2-ermF plasmid is shown in SEQ ID NO. 3.
  9. 9. The method for homologous recombination according to claim 8, wherein the recombinant enzyme is constructed on the E.coli-Bacteroides shuttle vector by constructing BaSSAP and BaSSB downstream of the P BfP1E6 promoter respectively to coexpress them and then placing them on the E.coli-Bacteroides shuttle vector to obtain pNBU2-intN2-intN 2-ermF-P BfP1E6 -BaSSAP-BaSSB.
  10. 10. Use of the recombinase combination according to any one of claims 1-4 or the homologous recombination method according to any one of claims 6-9 for gene editing of bacteroides, further comprising bacteroides fragilis, preferably bacteroides fragilis.

Description

Recombinant enzyme combination, application and homologous recombination method suitable for bacteroides Technical Field The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a recombinase combination, application and homologous recombination method suitable for bacteroides. Background The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. With the deep research of microbiome, intestinal symbiotic bacteria, in particular Bacteroides (bacterioides), have become the most potential candidate flora in the development of 'next generation probiotics' due to the core roles of the intestinal symbiotic bacteria in host metabolism, immune regulation and micro-ecological homeostasis. However, the transformation of such symbiotic bacteria from basic research into safe, controllable engineered strains faces a fundamental challenge of lacking efficient, precise and species-adapted genetic manipulation tools. This tool bottleneck severely constrains the targeted enhancement of its probiotic function, the rational knockdown of potential pathogenicity, and the systematic construction of complex synthetic biological pathways. Genetic manipulation of bacteroides is predicated on its unique physiological niches and genotypes. Taking the model strain Bacteroides fragilis (Bacteroides fragilis) of this genus as an example, it exhibits a typical "symbiotic-pathogenic" two-way image. In intestinal canal steady state, it can play the role of immunoregulation by synthesizing capsular polysaccharide A and other substances, however, once translocated to the external environment of intestinal canal, the complete oxidative stress resistance system and the drug resistance gene library mediated by movable genetic elements can be quickly converted into pathogenic potential. The two sides of this biological property makes it desirable not only for the efficiency of the editing tool, but also for it to be highly specific and controllable when genetically modified, to avoid introducing new biosafety risks due to off-target or unintended modifications. Currently, the genetic manipulation system applied to bacteroides depends mainly on its endogenous homologous recombination system or site-specific integrase. These methods have the common problems of long requirement for homology arms, long operation period, and most of the methods are limited to single-site editing. Although CRISPR-Cas systems have been introduced to improve targeting, in Bacteroides, the editing efficiency is still not ideal, and there is a risk of accumulation of off-target effects, which is more debilitating in complex operations such as polygenic collaborative editing or large fragment DNA integration. Therefore, the development of a platform technology which does not depend on a long homologous arm, is not limited by specific enzyme cutting sites and can synchronously realize the precise editing of multiple gene sites is a key for releasing the application potential of the synthetic biology of the bacteroides. Homologous recombination mediated "recombination engineering" (Recombineering) technology provides an ideal path for solving the above-described bottlenecks. The core of the technology is to use a phage-derived homologous recombinase system, such as Red alpha/Red beta or RecE/RecT, to greatly improve the recombination efficiency mediated by the short homology arms (40-50 bp) in cells. The mechanism of action is divided into two steps, first, an exonuclease (e.g., red. Alpha. Or RecE) processes a double-stranded DNA donor to produce single-stranded DNA with a 3' overhang, and then, a single-stranded annealing protein (SSAP, e.g., red. Beta. Or RecT) binds to and protects the single strand and facilitates its pairing and recombination with chromosomal target sequences. Studies have shown that the introduction of single-stranded binding proteins (SSB) during recombination can act synergistically with SSAP to further stabilize single-stranded DNA intermediates against degradation by host nucleases, thereby hopefully increasing recombination efficiency by several orders of magnitude. Notably, the efficiency of recombinant engineering is highly dependent on the suitability of SSAP and SSB proteins for host cells. In the prior art, research has been carried out to successfully establish efficient recombination systems in E.coli, lactic acid bacteria and even bifidobacteria by screening and optimizing specific protein combinations. However, these successful protocols derived from other bacteria cannot be directly transplanted into bacteroides. The bacteroides have unique and complex cell envelope structures, DNA repair mechanisms with obvious differences and endogenous nucl