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Preparation method of genetically engineered bacterium for synthesizing glutathione and product thereof

A technology of genetically engineered bacteria and glutathione, applied in the field of bioengineering, can solve the difficulties in the purification process of fermentation and synthesis of glutathione, unfavorable synthesis of reduced glutathione, low conversion efficiency of exogenous cysteine, etc. problem, to achieve the effect of enhancing the activity of intracellular glutathione synthase, improving the utilization efficiency of raw materials, and reducing production costs

Active Publication Date: 2014-09-24
唐星
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0020] The above-mentioned literature reports are mainly aimed at the high expression of the standard glutathione biosynthetic pathway in different host cells, thereby increasing the activity of intracellular glutathione synthase, thereby increasing the level of glutathione synthesis in cells; but always It does not solve the problem of fast and efficient coupling of the two-step catalytic reaction of GSH I and GSH II, nor does it solve the problem of low conversion efficiency of adding exogenous cysteine
Not only that, Escherichia coli itself produces pyrogens during the growth process, which brings difficulties to the fermentation and synthesis of glutathione and the subsequent purification process, increasing production costs
As a protein expression system, Pichia pastoris has the advantages of high-density fermentation and high yield of target products, but it has a strong preference for aerobic growth, which is not conducive to the synthesis of reduced glutathione

Method used

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  • Preparation method of genetically engineered bacterium for synthesizing glutathione and product thereof
  • Preparation method of genetically engineered bacterium for synthesizing glutathione and product thereof
  • Preparation method of genetically engineered bacterium for synthesizing glutathione and product thereof

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preparation example Construction

[0057] The invention provides a method for preparing a genetically engineered bacterium for synthesizing glutathione, comprising:

[0058] i) transfer the gene encoding γ-glutamylcysteine ​​synthetase-connecting peptide-glutathione synthetase fusion protein into the host bacterial cell, and make it highly expressed;

[0059] ii) transfer the gene encoding γ-glutamate kinase-connecting peptide-glutathione synthetase fusion protein into the host bacterial cell, and make it highly expressed;

[0060] iii) knocking out the gamma-glutamyl transpeptidase gene of the host bacterial cell;

[0061] iv) knocking out the inositol-3-phosphate synthase gene of the host bacterial cell;

[0062] After said i), ii), iii), and iv) are completed in any order, the genetically engineered bacteria for synthesizing glutathione can be obtained;

[0063] The connecting peptide in said i) and ii) is composed of 6-34 amino acid residues, and its general formula is: G-X-G-Y-G-Z, wherein G is glycine, ...

Embodiment 1

[0076] The extraction of embodiment 1 yeast genomic DNA

[0077] Use YPD medium (2% Peptone, 1% Yeast Extract, 2% Glucose, solid medium with 1.5% agar) to cultivate Saccharomyces cerevisiae or Pichia pastoris, using yeast genomic DNA from Beijing Kangwei Century Biotechnology Co., Ltd. The extraction kit (Cat.No.CW0569) extracts its genomic DNA, and the specific operation steps are as follows:

[0078] 1. Take 1-5ml of yeast culture, centrifuge at 12000rpm for 1 minute, collect the bacterial pellet, and discard the supernatant.

[0079] 2. Removal of yeast cell walls: Add 600 μl Lyticase Working Buffer (check the addition of β-mercaptoethanol before use to make the final concentration 0.1%) to the cells, add 5 μl Lyticase (10 U / μl), and mix well. 30°C for 30 minutes. Centrifuge at 4000rpm for 10 minutes, discard the supernatant, and collect the precipitate.

[0080] 3. Add 200 μl of Buffer GTL to the pellet, add 40 mg of glass beads (Glass Beads), vortex for 5 minutes, cent...

Embodiment 2G

[0090] Construction of embodiment 2 GSH I-GSH II homologous integration expression vector

[0091] 1. Cloning of homologous integration fragment δDNA fragment

[0092] 4 primers were designed and synthesized according to the δDNA sequence of Saccharomyces cerevisiae S288C (GenBank registration number U20865) (see SEQ ID NO.15-18 for the nucleotide sequence of the primers):

[0093] TY_R1:5′-AATGCGGTACCGCGGCCGCGAGCTGAGAAATTTGTGGGT-3′

[0094] TY_R2:5′-TGTAATAGGGATATCCACCTAAGTCTCGAGTCTAGAACTAGTGGATCCCC-3′

[0095] TY_R3:5′-ACTTAGGTGGATATCCCCTATTACA-3′

[0096] TY_R4: 5'-AAGCTGGAGCTCGCGGCCGCCCGCGGCCGCTATAATGTTG-3';

[0097] Using yeast genomic DNA as a template, two sets of primers TY_R1 / TY_R2 and TY_R3 / TY_R4 were used for PCR amplification (95°C, 5min; 95°C, 45S, 50°C, 50S, 72°C, 30S, 30cycles; 72°C, 10min ; 4°C, 10min), to obtain DNA fragments with lengths of 315bp and 199bp respectively, and to use the multifunctional DNA purification and recovery kit (Beijing Baitaike Bio...

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Abstract

The invention discloses a preparation method of a genetically engineered bacterium for synthesizing glutathione. The preparation method comprises the following steps: (i) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase fusion protein into a host bacterium cell, and highly expressing the gene; (ii) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase into the host bacterium cell, and highly expressing the gene; (iii) knocking off the gamma-glutamyltranspeptidase of the host bacterium cell; (iv) knocking off an inositol-3-phosphatep synthase gene of the host bacterium cell; and completing the steps (i), (ii), (iii) and (iv) in any sequence, thereby obtaining the genetically engineered bacterium for synthesizing glutathione. The invention further relates to the prepared genetically engineered bacterium. The genetically engineered bacterium for synthesizing glutathione, which is disclosed by the invention, is not only high in efficiency in synthesizing glutathione, but also can greatly improve the conversion efficiency of exogenous cysteine, and can be applied to glutathione production.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for preparing genetically engineered bacteria for synthesizing glutathione and its products. Background technique [0002] Glutathione (GSH), namely γ-L-glutamyl-L-cysteinyl-glycine, is a compound composed of L-glutamic acid, L-cysteine ​​and glycine, widely Biologically active tripeptide compounds present in living organisms (Meister A, Anderson ME. Glutathione. Annu Rev Biochem, 1983, 52:711-760). In all organisms, glutathione exists in two forms, namely reduced glutathione (GSH) and oxidized glutathione (GSSG), among which the reduced glutathione that plays a major role in the body In the majority (Akerboom TP, Bilizer MM, Sies H. The relationship of biliary GSSG efflux and intracellular GSSG content in perfused rat liver. J Biol Chem, 1982, 257:4248–4252). Glutathione has the functions of anti-oxidation, scavenging free radicals, and inhibiting HIV in the body (Carmel...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12R1/865
Inventor 王伟唐星唐亮王玮玮杨燕周文龙朱义波
Owner 唐星
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