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Genetic engineering strain capable of high yielding L-cysteine and construction and application of genetic engineering strain

A technology of genetically engineered bacteria and cysteine, applied to genetically engineered bacteria with high L-cysteine ​​yield and its construction and application fields, can solve problems such as toxicity

Active Publication Date: 2021-05-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high concentrations of L-cysteine ​​also exhibit certain toxicity to cells

Method used

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  • Genetic engineering strain capable of high yielding L-cysteine and construction and application of genetic engineering strain
  • Genetic engineering strain capable of high yielding L-cysteine and construction and application of genetic engineering strain
  • Genetic engineering strain capable of high yielding L-cysteine and construction and application of genetic engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049]Example 1: Determination of L-cysteine ​​content

[0050]The detection method is as follows:

[0051]Acid nuttexone formulated: It is weighed with 250 mg of nitrialketone to add 6 ml of acetic acid and 4 ml of hydrochloric acid, formulated into an acid nut trisotane solution.

[0052]Sample treatment: dilute the sample concentration between 0.1 to 1 g / L;

[0053]Reaction conditions: 500 μl of samples, 500 μl of acetic acid and 500 μL of acidic ninol mix, boiling water bath for 10 min;

[0054]Detection conditions: The reaction sample was determined at OD560 nm at OD560 nm.

Embodiment 2

[0055]Example 2: Constructing effective strain E.COLI W3110 EYC (TRC-CYSM) and shake flask fermentation

[0056]In Escherichia coli W3110 EYC (CCTCC NO: M 20191026) gene editing technology as the original strain using the CRISPR-Cas9 mediated (Yu Jiang et al.2015Multigene Editing in theEscherichia coli Genome via the CRISPR-Cas9 System.Applied EnvironmentalMicrobiology.81: 2506-2514) The original promoter of Cysm is replaced with the genome to enhance the expression strength of Cysm in the genome by the TRC promoter (Nucleotide sequence as shown in SEQ ID NO.1).

[0057](1) Construction of PTarget-Cysm plasmid: Template with PTARGET F plasmid (AddGene Plasmid # 62226), PT-CYSM-F / PT-CYSM-R is amplified, and the PCR product is incubated at 37 ° C at 37 ° C for digest. 3H, then transformed to E.COLI DH5α, spectacular enzyme flat screen, sequencing to obtain the correct PTARGET-CYSM plasmid for subsequent connection Donordna.

[0058](2) Constructing a PTD-Cysm containing Donor plasmid: Templa...

Embodiment 3

[0066]Example 3: Constructing effective strain E.COLI W3110EYC (TRC-CYSM NRDH) and shake flask fermentation

[0067](1) Constructing a PTARGET-NRDH plasmid: PTARGET F plasmid (AddGene Plasmid # 62226), PT-NRDH-F / PT-NRDH-R is amplified, and the PCR product is incubated at 37 ° C at 37 ° C for dpn I. 3H, then converted to E.COLI DH5α, spectacular enzyme flat screen, sequencing to obtain the correct PTARGET-NRDH plasmid for subsequent connection Donordna.

[0068](2) Constructing a PTD-NRDH plasmid: Template, PTD-NRDH-UP-F, PTD-NRDH-UP-R, PTD-NRDH-UP-R, PTD-NRDH-UP-F, PTD-NRDH-UP-F, PTD-NRDH-UP-F, PTD-NRDH-UP-F. The primer, the construction steps were in Example 2 (2) to obtain a PTD-NRDH plasmid.

[0069](3) Import a PCAS plasmid (AddGene Plasmid # 62225) into the E.COLI W3110EY (TRC-CYSM) sensing state obtained in Example 2, E.COLI W31110EY (TRC-CYSM) sensing state preparation method as in Example 2 (3) ).

[0070](4) Constructing the positive colonies of E Coli W3110 EC (TRC-Cysm NRDH), the m...

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Abstract

The invention relates to a genetic engineering strain capable of high yielding L-cysteine, a construction method for the genetic engineering strain and application of the genetic engineering strain in microbial-fermentation preparation of the L-cysteine. According to the method, the Escherichia coli genetic engineering strain capable of high yielding the L-cysteine is obtained through (1) strengthening the efficiency of conversion of the L-cysteine from S-Sulfocysteine in an Escherichia coli L-cysteine synthesis way and (2) strengthening the utilization ratio of thiosulfate radicals in the Escherichia coli L-cysteine synthesis way, and the yield of the L-cysteine is increased to 4.88g / L from 3.90g / L.

Description

[0001](1) Technical field[0002]The present invention relates to a genetic engineering of high-yield L-cysteine ​​and a construction method thereof, as well as its application of L-cysteine ​​in microbial fermentation.[0003](2) Background[0004]Cysteine ​​(Cysteine, Symbol Cys or C) has two homomers: L-cysteine ​​and D-cysteine. Most of the biological body is L-cysteine, which is one of the main presence forms of S element in the organism, which is a metabolic source of a large sulfur-containing compound. L-cysteine ​​is a semi-essential amino acid having a molecular formula HSCH2CH (NH2) COOH, which is usually involved in certain enzymatic reactions as a nucleophilic reactor due to a mercapto-side chain. L-cysteine ​​sulfhydryl can easily oxidize cystine, and they play an important role in maintaining protein structures. In addition, L-cysteine ​​also has a variety of physiological effects in biosurbs, such as anti-oxidation, participating antibiotics. Tolerance, also has important r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/113C12N15/54C12N15/31C12P13/12C12R1/19
CPCC12N9/1085C12N9/13C07K14/245C12P13/12C12Y205/01047C12Y208/01001
Inventor 柳志强黄子瀚张博陈开杨辉郑裕国
Owner ZHEJIANG UNIV OF TECH
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