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Genetically engineered bacterium for producing L-aspartic acid through fermentation and construction method and application thereof

A technology of genetically engineered bacteria and aspartic acid, which is applied to the field of genetically engineered bacteria that ferment L-aspartic acid and its construction, can solve the problem of high cost of raw materials, low product concentration, and low economical implementation of industrialization. problems, to achieve the effect of improving yield and concentration, green process route, significant economic and social benefits

Inactive Publication Date: 2019-02-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The domestic full biosynthesis of the above-mentioned products is still in the laboratory research stage, and the economical implementation of its industrialization is low. The main problems causing this situation include: 1) high cost of raw materials; 2) low conversion rate of raw materials
[0004] A strain that can use glucose to ferment L-aspartic acid under anaerobic conditions has been obtained earlier, but the concentration of the product is not high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] This example illustrates the method of constructing the target plasmid pTarget T2 containing gene targeting sequence and homologous recombination fragment by overlapping PCR technology.

[0030] 1. Using the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 as primers and plasmid pTarget F as a template, linear fragment 1 was obtained by PCR amplification;

[0031] 2. The linear fragment 1 was digested with SpeI and ligated, and the ligated product was transformed into a competent medium, and the positive recombinant was screened out on the LB plate coated with spectinomycin;

[0032] 3. The positive recombinants were inoculated in LB liquid culture and multiplied with spectinomycin, and then the plasmid was extracted to obtain the pTarget T1 plasmid;

[0033] 4. Using the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 as primers, and the E. coli genome as a template, linear fragment 2 was obtained by PCR amplification; using SEQ ID NO: 6 and SEQ ...

Embodiment 2

[0038] This example illustrates the method of using arabinose to induce the preparation of a competent protein containing cas9, and the specific steps include:

[0039] 1. Introduce the pCas plasmid into the Escherichia coli recombinant bacteria to be gene-edited. The preservation number is CGMCC NO: 2301, and use the LB plate of kanamycin to screen out positive recombinants;

[0040] 2. Induce the positive recombinants in the above steps with 30mM arabinose, and shake the bacteria for 3-4 hours at 30°C to prepare competent cells with an OD value of about 0.4-0.6.

Embodiment 3

[0042] This example illustrates the use of CRISPR / Cas9 technology to knock out parental Escherichia coli aceBAC repressor gene iCR the process of.

[0043] 1. Introduce the pTarget T2 plasmid into competent cells containing cas9 protein, and select positive recombinants with LB plates added with spectinomycin and kanamycin;

[0044] 2. Identify the above-mentioned positive recombinants by PCR, and screen out the target genetically engineered strains.

[0045]3. After the positive recombinants screened in step 2 were transferred to the LB medium containing kanamycin induced by IPTG and cultured, they were transferred to the LB medium plate containing kanamycin and simultaneously containing kanamycin. Namycin and spectinomycin LB medium plate, screened out recombinant strains that have degraded the pTarget T2 plasmid.

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Abstract

The invention discloses a genetically engineered bacterium for producing L-aspartic acid through fermentation and a construction method and application thereof. The bacterial strain is classified andnamed ad Escherichia coli deltaiclR, and its preservation number is CCTCC NO: M2018521. The construction process mainly includes the steps that a repressor gene iclR of an aceBAC operon in a recombinant bacterium with the preservation number CCTCC NO: 2301 is knocked out so as to open glyoxylic acid pathway for sharing the flux, acetic acid accumulated in the fermentation process is fully utilizedand is used as a raw material, fumaric acid is obtained by fermentation, the utilization efficiency of a carbon source is improved, and thus the yield of L-aspartic acid is further improved. The invention also discloses a culture medium of the L-aspartic acid by the genetically engineered bacterium through fermentation and culture conditions. The route of adopting only glucose as the raw materialfor fermentation to prepare the L-aspartic acid is realized, the yield of L-aspartic acid is improved, and the genetically engineered bacterium is friendly to the environment and economical.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and in particular relates to a genetic engineering bacterium fermenting to produce L-aspartic acid and its construction method and application. 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 is mainly synthesized from fumaric acid by biological enzyme method, while fumaric acid is mainly prepared by chemical meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/31C12P13/20C12R1/19
CPCC12N15/70C12N15/902C12P13/20C07K14/245
Inventor 马江锋储乐乐董维亮信丰学章文明方艳姜岷
Owner NANJING UNIV OF TECH
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