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A group of homologous recombinases, expression vector and applications thereof

A technology of homologous recombination enzyme and Burkholderia, applied in the field of microbial genetic engineering, to achieve the effect of increasing growth rate and increasing biomass

Active Publication Date: 2018-02-16
DEZHOU MICROP BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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After searching, there are no reports about the homologous recombinase and its expression vector that can efficiently mediate the recombination in Burkholderia, the recombination system in Burkholderia and its construction and application

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  • A group of homologous recombinases, expression vector and applications thereof
  • A group of homologous recombinases, expression vector and applications thereof
  • A group of homologous recombinases, expression vector and applications thereof

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preparation example Construction

[0050] Preparation of electroporated cells for recombination:

[0051] Escherichia coli competent cells were prepared according to the procedures reported in the previous literature. Preparation method of Burkholderia competent cells: Pick a single colony from the plate with a yellow tip and inoculate it into a 2.0ml centrifuge tube filled with 1.6ml of liquid CYMG medium with suitable resistance, culture at 30°C, 950rpm for 24h , to about OD600 5.0; transfer 40μl to a new 2.0ml centrifuge tube containing 1.6ml liquid CYMG medium, culture at 30°C, 950rpm for 12-14h, so that OD600 reaches about 1.8; when recombining, you need to add Corresponding inducer, 30°C, 950rpm, induced for 90min, induced the expression of the recombinant enzyme; centrifuged at 9300rpm for 1min, collected the bacteria, and discarded the supernatant. Add 1ml ddH 2 O resuspend the cells, centrifuge again at 9700rpm for 1min, discard the supernatant, and add 1 ml ddH again 2 O resuspend the cells, centri...

Embodiment 1

[0086] Embodiment 1: the screening of Burkholderia homologous recombinase, the construction of expression vector and the optimization of working condition (see list for the primer sequence that embodiment relates to)

[0087] (1) The Burkholderia homologous recombinase Redβ / α-7029 and the construction of the recombinase expression vector were obtained through bioinformatics and transcriptomics analysis and screening.

[0088] It was previously reported that the Red / ET recombination technology in E. coli cannot be used in non-naturally transformed Burkholderia. The present invention proves that the Red / ET recombination technology in E. coli cannot efficiently regulate DSM 7029 and other Homologous recombination in naturally transformed Burkholderia. Genomic deletion and integration in Burkholderia remain a challenge, implying that host specificity is an essential requirement for exonuclease-recombinases. The present invention analyzes DSM 7029 and other putative DNA recombinan...

Embodiment 2

[0103] Example 2: The application of Burkholderia homologous recombination enzyme in obtaining the compound glidopeptin.

[0104] The constructed genetic manipulation system can be used to explore new secondary metabolites. The present invention predicts the presence of several PKSs and NRPSs-like natural product biosynthesis gene clusters on the DSM 7029 genome based on bioinformatics analysis: cluser 6A (3068287-311501 ), cluster 7 (3161734-3234609) and cluster 11 (3950755-4033151). Transcriptomics analysis showed that the core gene expression of clusterer 6A (3068287-3115010) was very low under different conditions. By comparing the LC-MS analysis results of DSM7029 wild type and DSM 7029 and cluster 6A knockout strain fermentation broth, no corresponding gene expression was found. secondary metabolites, so we infer that these two gene clusters are silenced in wild-type DSM7029. In the research of the present invention, RedG-Redα / Redβ7029 was inserted into the constitutive...

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Abstract

The present invention discloses a group of homologous recombinases capable of efficiently mediating the in vivo recombination of Burkholderia, wherein the homologous recombinase comprises a protein Red[alpha]7029 having the function of exonuclease, and a protein Red[beta]7029 having the function of single-stranded annealed protein. The invention further discloses an expression vector pBBR1-Rha-BA7029-Km containing the homologous recombinase, applications of the combination of the homologous recombinase and an E. Coli-derived double-stranded DNA exonuclease inhibitory protein Red[gamma] in improvement of the in vivo recombination efficiency of Burkholderia, and applications of the homologous recombinase in the obtaining of a compound glidopeptin or rhizopeptin. According to the present invention, the multiple genes can be iteratively deleted so as to simplify the genome of Burkholderia, or the functional promoter is inserted to activate the unknown organism in an in-situ manner to synthesize the gene cluster, and the like, such that the important significance is provided for the bio-exploration and the functional genomics study of Burkholderia.

Description

technical field [0001] The present invention relates to a group of Gram-negative bacteria homologous recombinases and their expression vectors and applications, in particular to a group of Burkholderia homologous recombinases and their expression vectors, the recombination system in Burkholderia and its construction and application The invention belongs to the field of microbial genetic engineering. Background technique [0002] Bacteria are an important source of drugs and pesticides. In recent years, bacterial genome sequencing projects have revealed a large number of undiscovered natural product synthesis pathways, and these undiscovered biosynthetic pathways can provide new sample libraries for drug screening. There are two approaches to the discovery of these compounds, in situ activation and heterologous expression. Simple gene manipulation methods such as gene deletion and insertion on natural product genomes are the main strategies for in situ activation. However, ...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/74C12P1/04
CPCC07K14/245C12N9/22C12N15/74C12P1/04
Inventor 卞小莹王雪陈汉娜周海波张友明
Owner DEZHOU MICROP BIO TECH CO LTD
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