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A kind of genetic engineering bacterium producing L-cysteine, construction method and application

A technology of genetically engineered bacteria and cysteine ​​is applied in the application field of genetically engineered bacteria in the production of L-cysteine ​​by microbial fermentation, which can solve the problem of the lack of in-depth and meticulous research on the production of L-cysteine ​​by fermentation, etc. question

Active Publication Date: 2022-04-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the fermentative production of L-cysteine ​​has not been thoroughly studied in the past.

Method used

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  • A kind of genetic engineering bacterium producing L-cysteine, construction method and application
  • A kind of genetic engineering bacterium producing L-cysteine, construction method and application
  • A kind of genetic engineering bacterium producing L-cysteine, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the mensuration of L-cysteine ​​content

[0062] The detection method is as follows:

[0063] Preparation of acidic ninhydrin: Weigh 250 mg of ninhydrin and add 6 mL of acetic acid and 4 mL of hydrochloric acid to prepare an acidic ninhydrin solution.

[0064] Sample treatment: dilute the sample concentration to between 0.1 and 1g / L;

[0065] Reaction conditions: Mix 500 μL sample, 500 μL acetic acid and 500 μL acidic ninhydrin respectively, and bathe in boiling water for 10 minutes;

[0066] Detection conditions: put the reaction sample at OD 560 Measure its OD at nm 560 value.

Embodiment 2

[0067] Embodiment 2: Construction effective bacterial strain E.coli W3110EY (Trc-pgk) and shaking flask fermentation

[0068]Using Escherichia coli W3110EY as the starting strain, using CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al.2015 Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514), using The trc promoter derived from pTrc99A (nucleotide sequence shown in SEQ ID No.1) replaces the original promoter of pgk in the genome to enhance the expression intensity of pgk.

[0069] (1) Construction of pTarget-pgk plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pT-pgk-F / pT-pgk-R as primers for PCR amplification, and the PCR product is digested by Dpn I at 37°C 3h, then transformed into E.coli DH5α, screened with spectaclease plate, and sequenced to verify that the correct pTarget-pgk plasmid was obtained, which was used for subsequent connection of DonorDNA.

[0070] (2...

Embodiment 3

[0078] Example 3: Construction and Shake Flask Fermentation of E coli W3110 EY (trc-pgk△cycA) Knockout Serine Translocation Gene

[0079] (1) Construction of pTarget-cycA plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pT-cycA-F / pT-cycA-R as primers for PCR amplification, and the PCR product is digested by Dpn I at 37°C 3h, then transformed into E.coli DH5α, screened with spectacle enzyme plate, and sequenced to verify that the correct pTarget-cycA plasmid was obtained, which was used for subsequent connection of DonorDNA.

[0080] (2) Construction of pTD-cycA plasmid: with the E.coli W3110 genome as a template, pTD-cycA-up-F, pTD-cycA-up-R, pTD-cycA-down-F and pTD-cycA-down-R are The primers and construction steps were the same as in Example 2 (2) to obtain the pTD-cycA plasmid.

[0081] (3) Introduce the pCas plasmid (Addgene Plasmid #62225) into the competent E.coli W3110EY (trc-pgk) obtained in Example 2, and the preparation method of the compete...

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Abstract

The invention discloses a genetic engineering bacterium of L-cysteine ​​and a construction method thereof, and an application of the genetic engineering bacterium in preparation of L-cysteine ​​by microbial fermentation. The present invention strengthens the serine module of Escherichia coli L-cysteine ​​synthesis pathway by (1) enhancing the ability of Escherichia coli to utilize serine; Influence, (3) increase the expression of sulfur module synthesis gene (4) heterologously express the serC gene of Corynebacterium glutamicum on the plasmid, obtain the Escherichia coli genetically engineered strain of L-cysteine ​​high production, L-cysteine ​​output from 2.7 g / L increased to 3.83g / L.

Description

(1) Technical field [0001] The invention relates to a genetically engineered bacterium producing L-cysteine ​​and a construction method thereof, as well as the application of the genetically engineered bacterium in preparing L-cysteine ​​by microbial fermentation. (2) Background technology [0002] As an important sulfur-containing amino acid, L-cysteine ​​plays an important physiological role in organisms; it is not only a precursor substance for the synthesis of certain amino acids; Folding, assembly, stability, etc. play an important role. The use of L-cysteine ​​is mainly concentrated in 4 industries: food industry - used as a bread improver; cosmetics industry - used for beauty water, perm liquid, skin care cream for sun protection; pharmaceutical industry - Used as a expectorant to treat bronchitis; feed industry - used as a feed additive. According to statistics, the annual demand for L-cysteine ​​is about 5,000 tons, and the demand continues to rise. [0003] At p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/90C12P13/12C12R1/19
CPCC07K14/245C12N15/902C12P13/12
Inventor 柳志强陈勇贞张博郑裕国
Owner ZHEJIANG UNIV OF TECH
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