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Genetically engineered bacterium producing L-isoleucine, construction method and application thereof

A technology of genetically engineered bacteria and isoleucine, which is applied in the field of genetically engineered bacteria producing L-isoleucine and its construction, can solve problems such as insufficient capacity, and achieve the effects of reducing production costs and widening industrial application prospects.

Active Publication Date: 2019-03-29
WUHAN GRAND HOYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a genetic engineering bacterium producing L-isoleucine and its build method and application

Method used

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  • Genetically engineered bacterium producing L-isoleucine, construction method and application thereof
  • Genetically engineered bacterium producing L-isoleucine, construction method and application thereof
  • Genetically engineered bacterium producing L-isoleucine, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of Δddh-pk18mobsacB recombinant plasmid

[0047] This example constructs the Δddh-pk18mobsacB recombinant plasmid, which contains the upstream and downstream fragments of the ddh gene encoding diaminopimelate dehydrogenase, and the transposable gene mob and sucrose-sensitive gene that act on homologous recombination in Corynebacterium glutamicum The gene sacB.

[0048] The plasmid pk18mobsacB was digested with EcoRI / SalI double enzymes (the plasmid pk18mobsacB was purchased from Wuhan Miaoling Biotechnology Co., Ltd.), and the reaction system was: plasmid 1 μg, 10×buffer 5 μl, EcoRI 1 μl, SalI 1 μl, and water to 50 μl. Enzyme digestion at 37°C for 5h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification kit to recover 5.6kb nucleotide fragments;

[0049] Compared with the standard strain C. glutamicum ATCC 13032, the glycerol-preserved strain C. glutamicum H5ΔargGΔalaT::ilvC (self-constructed strain, see patent CN 10670...

Embodiment 2

[0061] Example 2 Construction of recombinant strain C. glutamicum H5ΔargGΔalaT::ilvCΔddh

[0062] Transfer the correctly sequenced transformant in Example 1 to 5ml of LB medium, shake and culture at 37°C and 200rpm for 16 hours, take 5ml of the bacterial liquid, centrifuge at 10,000rpm for 2 minutes, discard the supernatant; use plasmid mini-extraction reagent for wet bacteria cassette to extract the plasmid. Electrotransfer the plasmid into the starting strain C. glutamicum H5ΔargGΔalaT::ilvC, spread the transformation product on LBHIS solid medium containing kanamycin sulfate, culture at 30°C for 48 hours until the transformant grows, select the transformant and transfer 3ml LB culture medium, shake culture at 30°C, 200rpm for 24h, spread the bacterial solution on a plate containing 10% sucrose, culture at 30°C for 48h until a single colony grows, pick a single colony and inoculate it on a plate containing kanamycin sulfate and On a plate containing 10% sucrose, select a si...

Embodiment 3

[0065] Example 3 Construction of lysC-Δddh-pk18mobsacB recombinant plasmid

[0066] The lysC-Δddh-pk18mobsacB recombinant plasmid was constructed, which contained the pgro promoter encoding the chaperone protein groES gene in the genome of the strain C. glutamicumH5ΔargGΔalaT::ilvC, the lysC gene encoding the aspartokinase, and the lysC gene from Escherichia coli- The rrnB terminator of the Corynebacterium glutamicum shuttle vector, the upstream and downstream fragments of the ddh gene encoding diaminopimelate dehydrogenase, and the transposable genes mob and sucrose that can effectively act on homologous recombination in Corynebacterium glutamicum Sensitive gene sacB, specific steps are as follows.

[0067] The vector Δddh-pk18mobsacB was digested with EcoR V, and the reaction system was: 1 μg of plasmid, 5 μl of 10×buffer, 1 μl of EcoR V, and 50 μl of water. Enzyme digestion at 37°C for 5h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification kit ...

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Abstract

The present invention discloses a genetically engineered bacterium of corynebacterium glutamicum producing L-isoleucine. ddh gene encoding diaminopimelate dehydrogenase is knocked out in a genome of astarting strain. A nucleotide sequence of the ddh gene is shown as SEQ ID No:2; a lysC operon encoding an aspartokinase is inserted, the lysC operon contains a lysC mutant gene, a 5'-end promoter anda 3' end termination thereof, and a nucleotide sequence of the lysC mutant gene is shown as SEQ ID No:1; and besides, the starting strain is C. glutamicum H5 delta argG delta alaT::ilvC with a preservation number of CCTCC NO:M2016609. The present invention also discloses a preparation method and an application of the genetically engineered bacterium. The production of the L-isoleucine by the genetically engineered bacterium is increased by 5%, synthesis of a by-product lysine of a metabolic branch is reduced by 45.24%, production cost is lowered, and the genetically engineered bacterium has abroad industrial application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered bacterium producing L-isoleucine and its construction method and application. Background technique [0002] L-isoleucine (L-isoleucine, ILE), chemical name "β-methyl-α-aminovaleric acid", molecular formula: C 6 h 13 NO 2 , relative molecular mass: 131.17, is one of the eight essential amino acids, and is collectively referred to as "branched chain amino acids" (BCAAs) together with L-valine and L-leucine. L-isoleucine is a raw material for human hormones, protein synthesis and energy generation, which can promote protein synthesis and inhibit its decomposition, and also plays a vital role in the interaction of transmembrane domains of phospholipid bilayer membrane proteins. Therefore, L-isoleucine is widely used in industries such as food and medicine, and has great commercial value. [0003] The traditional production methods of L-isole...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/77C12P13/06C12R1/15
CPCC12N9/1217C12N15/77C12P13/06C12Y207/02004
Inventor 苏海霞朱程军邢盼盼王炯李敬皮莉左江黄治华鲁凡
Owner WUHAN GRAND HOYO
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