Three-pathway Combinatorial Expression For Glutathione Biosynthesis In Saccharomyces Cerevisiae

Glutathione (GSH) is a biological active tripeptide widely distributed in living organisms. The free sulfhydryl moiety of cysteine residue contributes to a wide variety of biological activities, such as anti-oxidization, detoxification and immune regulation, etc. GSH has been widely used in pharmaceutical, food and cosmetic industries.GSH is primarily synthesized intracellularly through two consecutive reactions catalyzed by y-glutamylcysteine synthetase (Gsh1 in eukaryotes and GshA in prokaryotes, encoded by gshl and gshA, respectively) and glutathione synthetase (Gsh2 in eukaryotes and GshB in prokaryotes, encoded by gsh2 and gshB, respectively); A novel bifunctional enzyme GshF (encoded by gshF) found in some Gram-positive pathogenic bacteria is able to perform complete synthesis of GSH; In other studies, compensatory pathway for GSH synthesis was characterized in yeast and Escherichia coli that lack Gshl and GshA. y-glutamyl kinase (GK) encoded by pro1 in eukaryotes or proB in prokaryotes, the first enzyme in proline biosynthetic pathway, could to some extent achieve the function of Gsh1. As research in biosynthesis and metabolism of GSH went further and more detailed, using a high-GSH-accumulated strain constructed by genetic engineering for GSH production via fermentation is the most effective means for a high GSH yield on an industrial scale. Many efforts have been focused on single overexpression of Gshl/Gsh2 or GshF in various microorganisms. In the present study, a new method of three-pathway combination was developed to improve synthetic capacity of GSH in engineered Saccharomyces cerevisiae for GSH production on an industrial scale. The main results are presented as below:1. Alternative pathway for GSH biosynthesis by y-glutamyl kinaseWith the replacement of gshl gene of genomic DNA of wild type S. cerevisiae W303-lb by recombinant DNA fragment of y-glutamyl kinase, the resulting strains were compared for their H2O2 tolerances. Overexpression of S. cerevisiae Pro1 in Gsh1 deleted strain could give a higher H2O2 tolerances. Recombinant strain W303-lb/P with overexpression of y-glutamyl kinase-linker-glutathione synthetase fusion protein (Prol-GshB) provided a slightly higher GSH production than strain W303-1b in shake flask cultures. From these data, it appears that overexpression of Prol could devote to more y-glutamyl phosphate involved in an alternative pathway for a small amount of GSH formation.2. Three biosynthetic pathways combined to increase GSH productionThe artificially recombinant enzyme glutathione synthetase-linker-y-glutamylcysteine synthetase fusion protein (Gsh2-Gshl) was overexpressed in S. cerevisiae W303-lb, and the resulting strain W303-lb/G together with W3O3-lb/P and W303-1b/F (constructed previously in our lab) represent different pathway strains. Three biosynthetic pathways of GSH were combinatorially expressed in S. cerevisiae W303-lb, and two-pathway strains W303-lb/FG, W303-lb/FP and three-pathway strain W303-1b/FGP were further constructed. The highest intracellular yield of GSH achieved by modulation of single GSH biosynthetic pathway was 187 mg/L produced by strain W303-1b/F and the GSH concentration was 2.45 mg/L/OD. Introduction of a second pathway could enhance the synthetic capacity of GSH. Among all the engineered strains, the strain W303-lb/FGP with three biosynthetic pathways presented the highest yield of GSH of 217 mg/L and the GSH concentration was 2.78 mg/L/OD. The result of shake flask cultures with addition of amino acid precursors further proved the advantages of three-pathway combinatorial method which presents higher GSH yield and utilization efficiency of amino acid precursors than that of a single pathway. After addition of amino acid precursors, GSH production of W303-lb/FGP was improved by 61.37%, while that of W303-lb/F was 43.17%.3. Scale-up cultivation of engineered strain harbouring three biosynthetic pathwaysTo exploit the GSH productive capacity of engineered strain W303-lb/FGP harbouring three pathways in a larger scale, batch cultivation for GSH production was carried out in a 10 L fermentor. The result showed that the GSH productive capacity had no obvious change after scale-up fermentation, suggesting that the pathway genes were stably integrated into the genome of S. cerevisiae. The fermentation time was shortened obviously by controlling the fermentation parameters. After fermentation for 24 h, the engineered strain reached a GSH yield of 273 mg/L, and the GSH concentration was 2.7 mg/L/OD. Glucose feeding could improve both GSH yield and Biomass.In all, a three-pathway combinatorial method of GSH biosynthesis presented higher GSH biosynthetic capacity and could break the bottleneck of single pathway modulation. This strategy could be used for optimizing engineered strain and would be an important development on way to more effective industrial production of GSH using S. cerevisiae.