Genetically engineered bacterium for high yield of L-cysteine, construction method and application

A technology of genetically engineered bacteria and cysteine, applied in the field of genetically engineered bacteria, construction and application of high-yield L-cysteine, can solve the problems of L-cysteine ​​fermentation production without in-depth and meticulous research , to achieve the effect of enhancing the utilization capacity, weakening the translocation and weakening the impact

Active Publication Date: 2020-04-17
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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  • Genetically engineered bacterium for high yield of L-cysteine, construction method and application
  • Genetically engineered bacterium for high yield of L-cysteine, construction method and application
  • Genetically engineered bacterium for high yield of L-cysteine, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: the mensuration of L-cysteine ​​content

[0067] The detection method is as follows:

[0068] 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.

[0069] Sample treatment: dilute the sample concentration to between 0.1 and 1g / L;

[0070] Reaction conditions: Mix 500 μL sample, 500 μL acetic acid and 500 μL acidic ninhydrin respectively, and bathe in boiling water for 10 minutes;

[0071] Detection conditions: put the reaction sample at OD 560 Measure its OD at nm 560 value.

Embodiment 2

[0072] Embodiment 2: Construction effective bacterial strain E.coli W3110EY (Trc-pgk) and shaking flask fermentation

[0073] Using Escherichia coli W3110EY as the starting strain, use CRISPR-Cas9-mediated gene editing technology (Yu Jiang et al.2015Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514), using source In the trc promoter of pTrc99A (the nucleotide sequence is shown in SEQ ID No.1), the original promoter of pgk was replaced in the genome to enhance the expression intensity of pgk.

[0074] (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.

[...

Embodiment 3

[0083] Example 3: Construction and Shake Flask Fermentation of E coli W3110 EY (trc-pgk△cycA) Knockout Serine Translocation Gene

[0084] (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.

[0085] (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 Primer, construction step is the same as embodiment 2

[0086] (2) to obtain pTD-cycA plasmid.

[0087] (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 competent E....

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Abstract

The invention discloses a genetically engineered bacterium of L-cysteine, a construction method of the genetically engineered bacterium and application of the genetically engineered bacterium in preparation of L-cysteine through microbial fermentation. The construction method comprises the following steps: (1) strengthening a serine module of an escherichia coli L-cysteine synthetic route, so thatthe serine utilization capability of escherichia coli is enhanced; (2) weakening the transfer of serine, so that the influence of byproducts on the L-cysteine synthesis is weakened; (3) increasing the expression of a sulfur module synthesis gene; and (4) heterologously expressing serC gene of Cornebacterium glutarum on a plasmid to obtain an escherichia coli genetically engineered bacterial strain with high L-cysteine yield, wherein the L-cysteine yield is increased from 2.7 g/L to 3.83 g/L.

Description

[0001] (1) Technical field [0002] The invention relates to a genetically engineered bacterium capable of high-yielding L-cysteine ​​and its construction method, as well as the application of the genetically engineered bacterium in preparing L-cysteine ​​by microbial fermentation. [0003] (2) Background technology [0004] 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...

Claims

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Application Information

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Patent Type & Authority Applications(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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