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A kind of genetically engineered rhodococcus and its construction method and application

A technology of genetic engineering and rhodococcus, applied in the field of genetic engineering, can solve problems such as low efficiency of homologous recombination and difficulty in gene manipulation of rhodococcus, achieve good application prospects, avoid plasmid stability, and improve catalytic stability

Active Publication Date: 2021-11-23
北京衍微科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rhodococcus gene manipulation is difficult and homologous recombination efficiency is low, which increases the difficulty of the application and development of CRISPR / Cas9 in Rhodococcus. At present, the application of CRISPR / Cas9 gene knockout in Rhodococcus has not been reported

Method used

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  • A kind of genetically engineered rhodococcus and its construction method and application
  • A kind of genetically engineered rhodococcus and its construction method and application
  • A kind of genetically engineered rhodococcus and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of plasmid vectors pRCTc and pBNVCm with different resistance genes and different replicons

[0043] The CRISPR / Cas9 gene editing method established in the present invention needs to use different plasmid vectors to express different elements, so it is necessary to modify the existing plasmids to meet the experimental requirements. Plasmids pRCTc and pBNVCm are Escherichia coli-Rhodococcus shuttle plasmids, based on plasmid pNV18.1 (Chiba, et al., Jpn J Infect Dis 2007, 60(1), 45-47.), by replacing the resistance gene and Rhodococcus replicon engineered.

[0044] The plasmid pRCTc carries the tetracycline resistance gene, and the replicon is pRC4. The construction process is as follows: using the genome of Rhodococcus R. ruber TH3 (WO2009117843-A1) as a template, using primers fusion-Tc-F and fusion-Tc-R, PCR Amplify the tetracycline resistance gene; use the plasmid pNV18.1 as a template, use primers pNV-F and pNV-R, and amplify the pNV18.1 backb...

Embodiment 2

[0061] Example 2 Construction of Rhodococcus Overexpressing Recombinase Che9c60&61 and Analysis of Its Impact on Knockout Efficiency

[0062] This embodiment constructs the plasmid pRCTc-Pa2-Che9c60&61 carrying the recombinase (as attached figure 1 shown), transformed into Rhodococcus R.ruber TH to obtain R.ruber TH (Che9c60&61) overexpressing the recombinase. Taking amidase gene knockout as an example to evaluate the effect of recombinase on knockout efficiency, transform the homologous arm of amidase gene with chloramphenicol resistance gene into R.ruber TH (Che9c60&61) and R.ruber TH, and perform statistical transformation Number of children and knockout efficiency.

[0063]The construction method of the plasmid pRCTc-Pa2-Che9c60&61 is as follows: the recombinase Che9c60&61 was synthesized by Huada Qinglan Biotechnology (Wuxi) Co., Ltd., and the two segments contained XbaI and KpnI restriction sites, and the Che9c60&61 gene fragment was digested with XbaI and KpnI; Rhodoc...

Embodiment 3

[0078] Example 3 Establishment of a CRISPR / Cas9-based gene editing method for Rhodococcus to achieve seamless knockout of amidase

[0079] CRISPR / Cas9 requires two important elements to function, nuclease Cas9 and guide RNA (sgRNA). This embodiment needs to construct their expression vector pNV-Pa2-Cas9 (see attached figure 2 shown) and pBNVCm-BbsI-sgRNA (as attached image 3 shown); at the same time, in order to achieve gene knockout, it is also necessary to provide the homology arms needed to repair DNA double-strand breaks. The process of gene editing of Rhodococcus using CRISPR / Cas9 is attached Figure 5 shown, the specific details are described below.

[0080] The construction method of pNV-Pa2-Cas9 is as follows: use the plasmid pCAS9-mCherry (Addgene #80975) as a template, use primers XbaI-Cas9-F, BamHI-Cas9-R to amplify the Cas9 gene, and use XbaI and BamHI to digest the Cas9 gene and plasmid pNV-Pa2 (CN109182241A), after purification and recovery, T4 DNA ligase wa...

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Abstract

The invention discloses a genetically engineered rhodococcus in the technical field of genetic engineering, a construction method and application thereof. Among them, the method for constructing genetically engineered Rhodococcus is a gene editing method based on CRISPR / Cas9, which has high gene knockout efficiency, can realize traceless knockout, and superimposed knockout of genes, providing an efficient operation for the genetic modification of Rhodococcus tool. Taking amidase gene knockout as an example, the knockout efficiency can reach 75%. At the same time, the rhodococcus with low by-product synthesis and high catalytic stability constructed by the above-mentioned editing method can be applied to catalyze the hydration of acrylonitrile to produce acrylamide or the preparation of ammonium acrylate. It has good catalytic stability and high efficiency, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered rhodococcus and its construction method and application. Background technique [0002] Rhodococcus is a Gram-positive actinomycete with rich metabolic enzymes and good tolerance to organic solvents, so it is widely used in biocatalysis, bioremediation and biosynthesis. For example, free cells of Rhodococcus ruber and Rhodococcus purpura can highly express nitrile hydratase, have good tolerance to polar solvents acrylonitrile and acrylamide, and have been successfully used in the production of chemicals such as acrylamide and nicotinamide. Production (CN101892228A; CN104762338A); Rhodococcus erythropolis has a desulfurase system, which can be applied to biological desulfurization and plays an important role in the field of environmental protection (CN107557106A); Rhodococcus turbide can degrade and utilize lignin to produce oil, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/02C12R1/01
CPCC12N9/88C12P13/02C12Y402/01084
Inventor 于慧敏梁有向焦松唐玲珺
Owner 北京衍微科技有限公司
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