Abstract :
[en] Tripeptide glutathione (GSH), the most abundant non-protein thiol compounds in cells, plays an important role in many cellular mechanisms. GSH is also a biotechnology-oriented molecule with applications in medicine, food, and cosmetics. The GSH precursor, namely gamma-glutamylcysteine (γ-GC), has also shown its therapeutic potential. Y. lipolytica is a non-conventional yeast and is of interest as a cell factory for pharmaceuticals, food- and feed-related applications.
In this thesis, we firstly improve the glutathione production in Y. lipolytica by overexpression of the endogenous biosynthetic genes GSH1 and GSH2, encoding γ-glutamylcysteine ligase (GCL) and glutathione synthetase (GS) enzymes, respectively. The strain was further engineered for the ability to metabolize carbon sources from organic wastes including glycerol, lactose, starch, and inulin. The glutathione overproducing strain with inulin-adapted metabolism showed the highest GSH productivity. During bioreactor culture, maximal productivity and yield from inulin were 510 μmol/gDCW and 626 mg/L, respectively.
One of the main bottlenecks in glutathione overproduction is the feedback inhibition of the GCL enzyme by glutathione. Unfortunately, multicopy integration of the GSH1 gene did not overcome this bottleneck. As a second strategy to increase thiol accumulation, we focused on glutathione precursor γ-GC, a dipeptide that can substitute for GSH in some cell mechanisms. We firstly disrupted the GSH2 gene before overexpressing GSH1 in multicopy. The constructed strains showed an increase in intracellular thiol production as demonstrated by means of NMR and LC-QTOF/MS as well as thiol measurement in the cell extract.
