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Strain for maleic acid whole-cell catalysis synthesis of L-aspartic acid and method

A technology of aspartic acid and maleic acid is applied in the fields of genetic engineering and fermentation engineering to achieve the effects of low price, significant economic and social benefits, and abundant resources

Inactive Publication Date: 2019-07-12
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, L-aspartic acid is mainly synthesized from fumaric acid by enzymatic method, and there are few studies on maleic acid as raw material.

Method used

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  • Strain for maleic acid whole-cell catalysis synthesis of L-aspartic acid and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example illustrates the method of constructing target plasmids pTarget T2-1, pTarget T2-2, pTarget T2-3, pTarget T2-4 containing gene targeting sequences and homologous recombination fragments by overlapping PCR technology.

[0043] 1. Using the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 as primers and plasmid pTarget F as a template, linear fragment 1 was obtained by PCR amplification;

[0044] 2. Using the nucleotide sequence shown in SEQ ID NO:8 and SEQ ID NO:10 as a primer, repeat step 1 to obtain linear fragment 2; use the nucleotide sequence shown in SEQ ID NO:8 and SEQ ID NO:11 The sequence is a primer, and step 1 is repeated to obtain linear fragment 3; the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:12 are used as primers, and step 1 is repeated to obtain linear fragment 4

[0045] 3. The linear fragment 1 was digested with SpeI and ligated, and the ligated product was transformed into a competent medium, and the LB plate coate...

Embodiment 2

[0054] This example illustrates the method of using arabinose to induce the preparation of a competent protein containing cas9, and the specific steps include:

[0055] 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;

[0056] 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

[0058] This example illustrates the process of knocking out the fumarase-encoding gene fumABC, the citrate synthase-encoding gene gltA, and the argininosuccinate synthase-encoding gene argG of the parental Escherichia coli using CRISPR / Cas9 technology.

[0059] 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;

[0060] 2. Identify the above-mentioned positive recombinants by PCR, and screen out the target strains;

[0061] 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 plasmid.

[0062] 4. Inducing the recombinant strains screened in step 3 with arabinose to prepare competen...

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Abstract

The invention discloses a strain for maleic acid whole-cell catalysis synthesis of L-aspartic acid and a method. The strain is named as Escherichia coli delta fumABC delta gltA delta argG / pMA, the preservation number of the strain is CCTCC NO:M 2019171, and the method comprises the construction processes that knocking out fumarase encoding gene, a citrate synthase encoding gene and an argininosuccinate synthase encoding gene in an original strain, meanwhile, aspartase encoding gene mutation N217K-T233R-V367G and maleic cis-trans isomerase encoding gene mutation G8A-G179A are cloned to expression plasmids to form recombinant plasmids, the recombinant plasmids are converted into the strain with knockout genes, and the target strain is obtained. According to the method for genetically engineered bacterium whole-cell catalysis synthesis of L-aspartic acid, the path for catalysis synthesis of L-aspartic acid by adopting maleic acid as raw materials is achieved, the production cost is lowered, efficient catalysis is achieved, and economy is achieved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and specifically relates to a bacterial strain and a method for catalytically synthesizing L-aspartic acid by utilizing maleic acid whole cells. Background technique [0002] L-Aspartic acid is widely used in medicine, food and chemical industry. In medicine, it is the main component of amino acid preparations; in chemical industry, it can be used as a raw material for the manufacture of synthetic resins, and is widely used in the synthesis of polyaspartic acid, an environmentally friendly material; especially in the food industry, L-aspartic acid is a It is a good nutritional supplement and is also the main raw material for the production of sugar substitute aspartame. Has a good market prospect. [0003] At present, L-aspartic acid mainly uses fumaric acid as raw material, and is synthesized by biological enzymatic method. There are relatively few stud...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/54C12N15/52C12N15/60C12N15/61C12P13/20C12R1/19
CPCC12N9/1025C12N9/93C12N9/88C12N9/90C12N15/70C12N15/902C12P13/20C12Y603/04005C12Y403/01001C12Y502/01001
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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