Production of glutathione by engineered strain of Yarrowia lipolytica using alternative substrates from by products.

The tripeptide glutathione (Gamma-L-Glutamyl-L-Cysteinylglycine) is the most abundant low-molecular-weight intracellular thiol. It plays important roles in cellular redox balance, nutrient metabolism and regulation of cellular mechanism. Glutathione deficiency contributes to oxidative stress, which considered as key factor in aging and pathogensis of many diseases including kwashiorkor, seizure, Alzheimer’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, HIV, AIDS, cancer, heart attack, stroke and diebetes. Glutathione, that could exist as a reduced (GSH) and oxidized (GSSG) state is sytnsised in a two step process. Firstly, glutamate and cysteine are ligated by an gamma-glutamylcysteine synthase (gamma-GCS, EC 6.3.2.2) encoded by GSH1 gene to form a L-gamma-glutamyl-L-cysteine. In a second step a L-gamma-glutamylcysteine-glycine gamma ligase (GS or GSH synthetase, EC 6.3.2.3) encoded by gene GSH2 link a glycin residue on the dipeptide. .
In the present research, the yeast Y. lipolytica, considered as an emerging industrial chassis was used as a cell factory to produce glutathione from raw material. First, we overexpress GSH1 and GSH2 genes under the control of pTEF, a strong constitutive promoter. This yielded to a significant increase (69%) of glutathione production. In a second step, we genetically adapt the producing strain to different cheap raw sugar-sources, including glycerol, inulin, lactose and starch. Among them, inulin revealed the most suited for GSH synthesis. The production titre was increased by 61 % as compared to glucose media used as a reference.
In this study, utilization of promoter to boost the expression of the two enzymes (L-cysteine gamma-ligase and glutathione synthase) which regulate the production of GSH in Y. lipolytica is implemented. The transformation of these two cassettes including promoter-GCL-ter and promoter-GS-ter into Y. lipolytica genome for stable intrgration and secrection, expectedly. The newly constructed strain for glutathionge overproduction shows lightly different comparing to control. Following the successfulness of GSH-overexpressed genes, this strain was used as initial host for further modification of exploiting other cheap raw sugar-source usability in by-products such as glycerol, starch and inulin. Among those strains we constructed, inulin consumption strain shows its ability to produce higher amount of glutathione comparing to others.