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Genetically engineered bacterium synthesizing L-asparaginate and construction method and application thereof

A technology of genetically engineered bacteria and asparagine, which is applied in the field of genetic engineering and fermentation engineering, can solve the problems of difficult wastewater treatment, heavy pollution, and low product yield, and achieve the effects of reducing consumption, increasing yield, and increasing accumulation

Inactive Publication Date: 2019-09-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production of L-asparagine is mainly made from L-aspartic acid through esterification and ammonolysis, but the yield of the process product is low, and a large amount of organic solvent is involved, which causes great pollution and difficult treatment of waste water.

Method used

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  • Genetically engineered bacterium synthesizing L-asparaginate and construction method and application thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0041] This example illustrates the construction of target plasmids pTarget T2-1, pTarget T2-2, pTarget T2-3, pTarget T2-4, pTarget T2-5, pTargetT2-6 containing gene targeting sequences and homologous recombination fragments using overlapping PCR technology method.

[0042] 1. Using the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11 as primers and plasmid pTarget F as a template, linear fragment 1 was obtained by PCR amplification;

[0043] 2. Using the nucleotide sequence shown in SEQ ID NO:10 and SEQ ID NO:12 as a primer, repeat step 1 to obtain linear fragment 2; use the nucleotide sequence shown in SEQ ID NO:10 and SEQ ID NO:13 Sequence is primer, repeat step 1 to obtain linear fragment 3; With the nucleotide sequence shown in SEQ ID NO:10 and SEQ ID NO:14 as primer, repeat step 1 to obtain linear fragment 4; With SEQ ID NO:10 and SEQ ID NO:14 The nucleotide sequence shown in ID NO:15 is a primer, and step 1 is repeated to obtain a linear fragment 5; the nu...

Embodiment 2

[0053] This example illustrates the method of using arabinose to induce the preparation of a competent protein containing cas9, and the specific steps include:

[0054] 1. Introduce the pCas plasmid into the Escherichia coli recombinant bacteria to be gene-edited. This biological material is disclosed in the patent literature of the Chinese patent (application number 201811346790.3, application date 2018.11.13), and its preservation number is CCTCC NO: M2018521. Positive recombinants were screened out on the LB plate of mycin;

[0055] 2. Induce the positive recombinants in the above steps with 30 mM arabinose, shake the bacteria for 3-4 hours in an environment of 30° C., the OD value is about 0.4-0.6, and prepare as competent.

Embodiment 3

[0057] This example illustrates the use of CRISPR / Cas9 technology to knock out the parental Escherichia coli fumarase encoding gene fumABC, DNA transcription binding regulator encoding gene arcA, glucose transport-related gene ptsG, citrate synthase encoding gene gltA and argininosuccinic acid synthesis The enzyme encodes the process of the gene argG.

[0058] 1. Introduce the pTarget T2-1 plasmid into competent cells containing cas9 protein, and select positive recombinants with LB plates added with spectinomycin and kanamycin.

[0059] 2. Identify the above-mentioned positive recombinants by PCR, and screen out the target strains.

[0060] 3. Transfer the strain 1 screened out in step 2 into the LB culture medium containing kanamycin induced by IPTG, and then transfer to the LB medium plate containing kanamycin and simultaneously containing kanamycin Mycin and spectinomycin on the LB medium plate, screen out the recombinant strain 1 that has degraded the pTarget T2-1 plasmi...

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Abstract

The invention discloses a genetically engineered bacterium synthesizing L-asparaginate. The genetically engineered bacterium is classified and named Escherichia coliDeltafumABCDeltaarcADeltaptsGDeltagltADeltaargG / pAA, and the preservation number of the genetically engineered bacterium is CCTCC NO:M2019170. The construction process of the genetically engineered bacterium comprises the steps that aspawn running strain fumarase encoding gene, a DNA transcription binding regulatory factor encoding gene, a glucose transport related gene, a citrate synthase encoding gene and an argininosuccinate synthase encoding gene are knocked out; an aspartase encoding gene and an asparagine synthetase A encoding gene are cloned to an expression plasmid, the recombinant plasmid is transformed into recombinant escherichia coli after gene knockout, and a target strain is obtained. The genetically engineered bacterium is synthesized into the L-asparaginate through fermentation, achieves a route for biosynthesizing and preparing the L-asparaginate by completely using glucose as a raw material, is efficient, green and environmentally friendly, and has economy.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and specifically relates to a genetic engineering bacterium for preparing L-asparagine by using glucose biosynthesis, a construction method and application thereof. Background technique [0002] L-Asparagine is a hydroxyl amide compound of L-Aspartic acid, which is widely used in food, medicine, chemical synthesis and microbial cultivation and other fields. In medicine, it is mainly used as one of the twenty kinds of amino acid infusions, and has the effect of treating skin diseases; in food, it can be used as a food additive for refreshing drinks; in the chemical industry, it is mainly used as a stabilizer for iron salts and ceramic surfaces In the environmental protection industry, it is an important raw material for water treatment membranes; at the same time, it is also an important additive for microbial culture and animal cell culture. Has a good ma...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/90C12P13/20C12R1/19
CPCC12N9/88C12N9/1025C12N9/93C12N15/902C07K14/245C12P13/20C12Y402/01002C12Y203/03003C12Y403/02001C12Y403/01001C12Y603/05004
Inventor 马江锋储乐乐方艳信丰学董维亮章文明陈可泉姜岷欧阳平凯
Owner NANJING UNIV OF TECH
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