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Double knockout recombinant rhodococcus as well as construction method and application thereof

A rhodococcus and double-gene technology, applied in the field of microbial genetic engineering in the Ming Dynasty

Active Publication Date: 2016-03-23
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the impact of factors such as cost, raw material price, nitrilase activity and catalytic process, the biological method of acrylic acid (ammonium) produced by nitrilase catalysis has not been widely used.

Method used

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  • Double knockout recombinant rhodococcus as well as construction method and application thereof
  • Double knockout recombinant rhodococcus as well as construction method and application thereof
  • Double knockout recombinant rhodococcus as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Cloning of embodiment 1 nitrilase gene and construction of high expression shuttle plasmid pNV-Nit

[0085] Using the total DNA of Rhodococcus R. rhodochrousATCC33278 as a template, the nitrilase gene was amplified by polymerase chain reaction (PCR). The upstream primer is: 5'-TGCACTGCAGATGGTCGAATACACAAAACACATTCA-3' (SEQ ID NO: 7), the downstream primer is: 5'-CCCAAGCTTTCAGATGGAGGCTGTCGCC-3' (SEQ ID NO: 8), and the introduced enzyme cutting site is PstI / HindIII. After the primers were designed, they were synthesized by Beijing Saibaisheng Bioengineering Co., Ltd., and dissolved in sterile water to a concentration of 50 μmol / L. The polymerase used in PCR, the corresponding amplification buffer, and four kinds of deoxynucleotide solutions were purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[0086] The PCR reaction system is:

[0087]

[0088] The reaction conditions are: 94°C, 5min; 94°C, 1min, 50°C, 1.5min, 72°C, 1.5min, 30 cycles; finally 72°C, 10min. ...

Embodiment 2

[0090] Embodiment 2 Recombinant rhodococcus construction of amidase gene-nitrile hydratase gene double knockout

[0091] According to the nitrile hydratase gene sequence of Rhodococcus TH, primers were firstly designed to amplify the upstream fragment A of the nitrile hydratase gene. The upstream and downstream primers used were: 5'-GGAATTCCACCCTGCCGCCGTTGGACGACCAC-3' (SEQ ID NO: 1), 5'-GCTCTAGATCCTTTCATCGGAGCTGGGCTCAAA-3' (SEQ ID NO: 2). The enzyme cutting sites introduced are EcoRI / XbaI. The primers were synthesized by Beijing Saibaisheng Bioengineering Co., Ltd. and dissolved in sterile water to a concentration of 50 μmol / L. The polymerase used in PCR, the corresponding amplification buffer, and four kinds of deoxynucleotide solutions were purchased from Treasure Bioengineering (Dalian) Co., Ltd. The PCR reaction system is the same as in Example 1.

[0092] Similarly, according to the nitrile hydratase gene sequence of Rhodococcus TH, primers were first designed to ampli...

Embodiment 3

[0097] Example 3 Construction of Nitrilase High Expression Recombinant Rhodococcus

[0098] The nitrilase high-expression shuttle plasmid pNV-Nit constructed in Example 1 was transformed by electroporation into the competent cells of the double-gene knockout host strain TH (dAm-dNH) constructed in Example 2 to obtain recombinant Rhodococcus THdAdN (Nit).

[0099] Similarly, the nitrilase high-expression shuttle plasmid pNV-Nit constructed in Example 1 is transformed by electroporation into the competent cells of the single-gene knockout host strain TH (dAm) (see Chinese invention patent ZL200880000969.1), which can be A comparative recombinant Rhodococcus THdA(Nit) was obtained.

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Abstract

The invention discloses a construction method of double knockout recombinant rhodococcus. The construction method comprises the following steps: (1) primers are designed according to nitrile hydratase gene sequence of the rhodococcus for respective amplification of an upstream sequence and a downstream sequence of a nitrile hydratase gene, and an upstream fragment and a downstream fragment of the nitrile hydratase gene are obtained; (2) the upstream fragment and the downstream fragment of the nitrile hydratase gene are inserted into a suicide plasmid, and a recombinant suicide plasmid is obtained, wherein the suicide plasmid carries a kanamycin resistant gene and a levansucrase gene; (3) competent cells of recombinant rhodococcus of an amidase gene are knocked out through transformation of the recombinant suicide plasmid, and the double knockout recombinant rhodococcus is obtained: first screening is performed through kanamycin resistance, and a first recombinant colony is obtained and subjected to second screening through a sucrose plate. The invention further discloses recombinant rhodococcus with high nitrilase expression, a construction method of the recombinant rhodococcus and a method for preparing acrylamide.

Description

technical field [0001] The present invention relates to the field of microbial genetic engineering. Specifically, the present invention relates to double-gene knockout recombinant Rhodococcus and a construction method thereof. More specifically, the present invention relates to a construction method for double-gene knockout Rhodococcus, a double-gene knockout rhodococcus Gene knockout recombinant rhodococcus, a method for constructing recombinant rhodococcus with high nitrilase expression, a recombinant rhodococcus with high nitrilase expression, the use of recombinant rhodococcus with high nitrilase expression in the preparation of carboxylic acid compounds, and A method for preparing ammonium acrylate. Background technique [0002] Rhodococcus is an aerobic, Gram-positive actinomycete with a relatively fast growth rate. Due to their strong ability to synthesize enzymes and metabolites, as well as good tolerance to organic solvents, Rhodococcus is widely used in the biolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74C12N1/21C12P13/02C12R1/01
CPCC12N9/80C12N9/88C12P13/02C12Y305/01004C12Y402/01084
Inventor 于慧敏孙继哲陈杰罗晖沈忠耀
Owner TSINGHUA UNIV
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