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Glutathione produced bacterial strain and construction method thereof

A technique for producing glutathione and bacterial strains, which is applied in the field of genetic engineering, can solve problems such as the inability to purposefully regulate cell expression, achieve high cell density, biotransformation rate, high bioaccumulation, and reduce synthetic production costs Effect

Inactive Publication Date: 2008-07-16
南京华锦生物制品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the general method of genetic engineering or metabolic engineering is to add foreign genes or the host's own genes, which generally change the original regulatory promoter or still use the original gene promoter but change the copy number, because GSH I and GSH II have not been studied in depth The proportional relationship of gene content cannot purposely regulate the expression of GSHI and GSHII in cells, and the result may not necessarily achieve the purpose of efficient and coordinated expression of GSHI and GSH II [Yaohui. Wei Dongzhi, Zhou Yuxun et al. (2002 ).Journal of East China University of Science and Technology, 28:144-148]

Method used

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  • Glutathione produced bacterial strain and construction method thereof
  • Glutathione produced bacterial strain and construction method thereof
  • Glutathione produced bacterial strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of recombinant GSH-related synthetase expression pAOG1G2 plasmid (scheme 1)

[0046] 1. Extraction of Saccharomyces cerevisiae genomic DNA

[0047] Saccharomyces cerevisiae genomic DNA was extracted according to the "Experimental Guide to Yeast Genetics Methods". Inoculate Saccharomyces cerevisiae S288c in 4ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose), at 28-30°C for 16-18 hours. Take the culture solution and centrifuge the sedimented cells (3000rpm, 10min), then wash with cold, equal volume of sterile distilled water and centrifuge again, transfer the cell pellet to a 1.5ml centrifuge tube, in 200μl lysis buffer (2% (v / v) Triton X-100, 1% (v / v) SDS, 100mM NaCl, 10mM Tris base (pH8.0), 1mM EDTA (pH8.0)), then add about 300μl acid-washed glass beads and 200μl 1:1 phenol: chloroform mixture, vortexed for 1-2min, added 200μl of TE buffer (10mM Tris base (pH8.0), 1mM EDTA (pH8.0)), mixed by inversion 6-10 times. The samples were cen...

Embodiment 2

[0062] Embodiment 2: Construction of CYS3, CYS4 gene expression vector

[0063] 1. Extraction of the genome of the host strain Pichia pastoris GS115

[0064] The method is the same as the extraction of Saccharomyces cerevisiae genomic DNA in Example 1.

[0065] 2. Cloning of G418 resistance gene and CBS gene

[0066] The upstream and downstream primers were designed according to the plasmid pPIC3.5K, the upstream primer (5'sense primer) pkan-1TGAGGGAGCCACGGTTGATGAGAGC, the downstream primer (3'anti-sense primer) pkan-2GGGGTGTTATGAGCCATATTCAACG.

[0067] Using the plasmid pPIC3.5K as a template and the above primer pair as primers, Pyrobest polymerase was used to amplify the Kan fragment derived from the transposon Tn903 G418 resistance gene of Escherichia coli.

[0068] The upstream and downstream primers were designed according to the CBS gene of the GS115 strain genome (GenBank No.AF367364). The upstream primer (5′sense primer) pCBS-1 AGAAATACATACTATGTCAGACAACAACC, and the...

Embodiment 3

[0083] Example 3: Construction of recombinant cells expressing exogenous cystathionine synthase and cystathionine-γ-lyase genes

[0084] 1. Electroporation of Pichia pastoris cell preparation

[0085] Inoculate GS115 strain in 2ml YPD medium, culture at 30°C for 12-20 hours, inoculate in 100ml YPD medium at a ratio of 1:100, and culture at 30°C until OD 600 =1.3~1.5, 4°C, centrifuge at 1500g for 10min, suspend and wash the precipitate twice with pre-cooled sterile water, centrifuge at 1500g at 4°C for 10min, then suspend and wash the precipitate twice with pre-cooled 1mol / L D-sorbitol, After centrifugation at 1500 g for 10 min at 4°C, the obtained GS115 was resuspended in 0.3 ml of pre-cooled 1 mol / LD-sorbitol, and placed on ice until use.

[0086]2. Construction of recombinant cells expressing exogenous cystathionine synthase and cystathionine-γ-lyase genes

[0087] Use the PCR product gel recovery kit to extract about 10ug of the integrated expression vector PCR product of...

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Abstract

The invention discloses a glutathione producing strain, in which a GSH related synthetic enzyme Gamma-L-glutamyl-L-cysteine synthetic enzyme gene GSH I, a glutathione synthetic enzyme gene GSH II, and a cysteine related synthetic enzyme cystathionine synthetic enzyme gene CYS3 and a cystathionine-Gamma-lyase CYS4 gene are introduced. The glutathione producing strain of the invention has the advantages of high density and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a glutathione-producing bacterial strain and a construction method thereof. Background technique [0002] Glutathione (GSH) is an important non-protein sulfhydryl compound in organisms. Due to its many important physiological functions, it is not only widely used in the fields of medicine and cosmetics, but also widely used in various food processing. Fields, such as flavored food, dairy products, oral health products, etc. [Sies H. (1999) Free Rad Biol Med.27, 916-921; Zheng Yunlang (1995) Biological Bulletin 30, 22-24]. The industrial production of glutathione is mainly chemical synthesis, enzymatic and biological fermentation. Optical activity and environmental problems in chemical methods. There are two main ways to biosynthesize GSH: enzymatic conversion and fermentation. The common feature of the two is that there must first be a microorganism with good performance, an...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/52C12N15/60C12N15/81C07K5/083C12R1/84
Inventor 许善峰金大勇蔡宇杰
Owner 南京华锦生物制品有限公司
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