Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine and glycine that has a wide range of applications in the pharmaceutical, food and cosmetic industries.
At present, traditional industrial production methods mainly rely on yeast fermentation process to synthesize, but this process has some limitations, GSH will accumulate in yeast cells, and the extraction efficiency is low, limiting its high yield.
Therefore, a new biotechnological method is needed to improve the production efficiency and reduce the cost of glutathione.
Previous studies have shown that in the amino acid fermentation process of E. coli, the product is usually secreted from the cell, so as to obtain a high yield, which has certain advantages over yeast.
Shingo Kobayashi et al. Engineering Escherichia coli for efficient GSH production in Metabolic Engineering.
The authors used Escherichia coli as chasse to produce glutathione. Through metabolic engineering and genetic modification, the biosynthetic pathway of GSH in Escherichia coli was enhanced to improve its yield and secretion efficiency.
Glutathione production was improved by modifying Escherichia coli
Strategy 1
Overexpression of genes encoding key enzymes in GSH biosynthesis, such as the gshA gene encoding cysteine-glutamate ligase and the gshB gene encoding GSH synthase.
Strategy 2
Reduced glutathione degradation.
ggt gene encoding gamma-glutamyltransferase and pepT gene encoding tripeptide peptidase were deleted.
Strategy 3
Promote the oxidation of GSH.
The gor gene encoding glutathione reductase is deleted, thus promoting GSH secretion.
Strategy 4
Inhibits cell reuptake of glutathione.
YliABCD is an ABC transporter involved in the reabsorption of secreted GSH. The authors deleted the yliABCD gene to reduce the reuptake of GSH by cells and promote its accumulation in the medium.
Strategy 5
Enhanced cysteine production.
Cysteine is an important precursor of glutathione synthesis, and some studies have shown that CysE is a key enzyme to increase cysteine production. Through overexpression of cysE gene, ompF promoter is directly inserted into the upstream of cysE coding sequence, increasing the conversion rate from serine to O-acetylserine.
Strategy 6
Partially blocks the glycolysis pathway and increases the carbon flux from 3-phosphoglyceric acid to serine.
The authors deleted the gpmA gene encoding phosphoglycerate mutase, partially blocking the glycolytic pathway and shifting more carbon flow from 3-phosphoglycerate to serine, thereby indirectly promoting GSH production.
Through the above series of strategies, the strain that can produce 19.6g/L glutathione was finally obtained.
The subsequent authors explored the effect of continuous feeding of glycine on GSH production, and when the strain was cultured without glycine supplementation, its growth was significantly inhibited and only a small amount of GSH was produced, suggesting that glycine supplementation is essential for glutathione production.
Then, by analyzing the metabolic flow and metabolomics results, we found that the conversion of O-acetylserine to cysteine is the rate-limiting step for GSH production, and by using sodium sulfite as the sulfur source, this limitation is largely overcome, and finally the GSH production of the strain is increased to 22.0g/L, which is the highest yield reported in the literature so far.
In conclusion, the authors’ research provides a new biotechnological method for the industrial-scale production of glutathione.
GSH Bio-Tech
As a company focusing on the production of high-quality API raw materials, Anhui GSH Bio-technology Co., Ltd. is committed to providing excellent products and services to the global health industry through innovative technology. Our product line covers glutathione, NMN, Citicoline Sodium and adenosine methionine, which are widely used in medicine, food, health care products and other fields.
GSH Bio-Tech main products are L-glutathione reduced (GSH), β-Nicotinamide Mononucleotide (NMN), Citicoline Sodium (CDPC) and other raw materials. The acid market share ranks among the top three in China, and the products are exported to Europe, America, South Korea, India and other parts of the world.
