Method for constructing high-yield glutathione recombinant strain and application

A technology of glutathione and recombinant strains, applied in the field of bioengineering, can solve the problems of easy oxidation and high cysteine ​​price, and achieve the effect of cost saving

Pending Publication Date: 2021-02-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, as another essential precursor of GSH, cysteine ​​is relatively expensive and easily oxidized when added as a substrate

Method used

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  • Method for constructing high-yield glutathione recombinant strain and application
  • Method for constructing high-yield glutathione recombinant strain and application
  • Method for constructing high-yield glutathione recombinant strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The construction of embodiment 1 recombinant plasmid

[0022] In this example, plasmids for knockout and knockin and expression of related genes in Corynebacterium glutamicum were respectively constructed, and the plasmid map is as follows figure 1 with figure 2 shown.

[0023] 1.1 Helper plasmid pEC-XK99E-P tuf -ATcysE and pEC-XK99E-P tuf -build of cysE-cysK

[0024] Plasmid pEC-XK99E is linearized with primer pair pEC-XK99E1-For(SEQ ID No.1) / pEC-XK99E1-Rev(SEQ ID No.2), the fragment contains kanamycin resistance gene, E. coli replicon , Corynebacterium glutamicum replicon and rrnBT1 terminator; Primer pair Ptuf-ATcysE-For (SEQ ID No.3) / Ptuf-ATcysE-Rev (SEQ ID No.4) from pGH-Ptuf-ATcysE plasmid (Jierui Biology Synthesis, amplified Ptuf-ATcysE fragment in SEQ ID No.5). The two fragments were circularized by Gibson recombination to construct plasmid pEC-XK99E-Ptuf-ATcysE.

[0025] Utilize the primer pair pEC-XK99E2-For(SEQ ID No.6) / pEC-XK99E2-Rev(SEQ ID No.7) to ...

Embodiment 2

[0038] Example 2 Constructing GSH high-yield recombinant strains CgGsh-13032-1 and CgGsh-13032-2 with Corynebacterium glutamicum ATCC13032 as the starting bacterium

[0039] The wild-type Corynebacterium glutamicum ATCC13032 was used as the starting bacterium, and the plasmid pXMJ19-Ptac-gshF was electrotransduced, and the bacterial solution was coated with chloramphenicol-containing BHIS plate for culture, and the correct transformant was obtained after screening, which was named CgGsh-13032-1.

[0040] Using wild-type Corynebacterium glutamicum ATCC13032 as the starting bacterium, the plasmid pK19mobsacB-ΔaecD was knocked out by electroporation to construct a cysteine ​​desulfhydrylase-deleted strain of Corynebacterium glutamicum. The specific operation process is as follows:

[0041] (2) Extract the plasmid pK19mobsacB-ΔaecD from Escherichia coli DH5α, and wash it with pH 8.0 water for later use

[0042] (2) Electroporation of the plasmid pK19mobsacB-ΔaecD to wild-type Cor...

Embodiment 3

[0048] Example 3 Taking Corynebacterium glutamicum ATCC13032 as the starting strain cysteine ​​synthesis enhanced strain to synthesize cysteine

[0049] The medium formula used in this embodiment is as follows:

[0050] Pick the activated single colony and put it in 1-100mL seed culture medium, cultivate it at 25-37℃, 50-500rpm for 5-30h, press

[0051] 0.1-30% inoculum amount was transferred to 1-500mL seed medium, cultivated at 25-37℃, 50-500rpm for 5-30h, and then pressed

[0052]The initial OD600 is 0.1-3 and transferred to 1-10L / 3-50L fermenter; the pH is adjusted to 5.0-8.0 by adding ammonia water, and the

[0053] Control the dissolved oxygen to 10-50% by controlling the speed and aeration rate; add 0-100mM glycine in the early logarithmic period,

[0054] Add 0-100mM glycine and 0-20g / L sodium thiosulfate.

[0055] Seed medium: 1~20g / L glucose, 0.5~2g / L yeast extract, 1~5g / L peptone, 1~3g / L

[0056] (NH4)2SO4, 0.1~1g / L K2HPO4, 0.1~1g / L KH2PO4.

[0057] Fermentatio...

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Abstract

The invention discloses a method for constructing a high-yield glutathione recombinant strain and application, and belongs to the field of bioengineering. The method for constructing the high-yield glutathione recombinant strain is characterized in that corynebacterium glutamicum for synthesizing a large amount of glutamic acid is used as an original strain, and a gene gshF from streptococcus agalactiae or genes gshA and gshB from escherichia coli are introduced to endow glutathione with the synthesis capability; further genome is subjected to deletion of genes aceD, mcbR and sdaA; a truncatedgene serA, a gene pgk, genes cysE and cysK derived from the escherichia coli and a gene cysE derived from arabidopsis thaliana are expressed at the same time, and a cysteine pathway is strengthened;and as the corynebacterium glutamicum can synthesize a large amount of the glutamic acid and the cysteine pathway is strengthened, the glutathione can be highly produced only by adding glycine in a fermentation process of obtained engineering bacteria.

Description

technical field [0001] A method and application for constructing a high-yield glutathione recombinant strain belongs to the field of bioengineering, and specifically relates to a high-yield glutathione method. [0002] technical background [0003] So far, glutathione (GSH) is mainly synthesized by microbial fermentation. In view of the low production of GSH in wild bacteria, genetic engineering and metabolic engineering are widely used to enhance the synthesis of GSH in fermentation hosts such as Saccharomyces cerevisiae, Pichia pastoris, Candida utilis and Escherichia coli. However, three precursor amino acids need to be added to achieve the purpose of high GSH production. Since Corynebacterium glutamicum can synthesize glutamate in a large amount by itself, using it as a host can avoid the addition of glutamate, a precursor for GSH synthesis. In addition, as another essential precursor of GSH, cysteine ​​is relatively expensive and easily oxidized when added as a substra...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N15/31C12P21/02
CPCC12N15/77C07K14/315C07K14/245C12P21/02C07K5/0215
Inventor 徐志南刘伟黄磊连佳长蔡谨
Owner ZHEJIANG UNIV
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