1. Improvement of GSH production by metabolic engineering the sulfate assimilation pathway of S.cerevisiae Kiyotaka Y. Hara & Kentaro Kiriyama & Akiko Inagaki & Hideki Nakayama & Akihiko Kondo

2. Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries.Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. Glutathione

3. pathway In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production.

4. Materials and methods S. cerevisiae YPH499 1Sulfate assimilation 2Glutathione synthesis 3Combinatorial engineered

5. over-expressed each gene of 1.Sulfate assimilation The related genes for sulfate assimilation metabolism (APA1, MET3, MET14, and MET16) were amplified by PCR from S. cerevisiae genomic DNA. construct pAUR-APA1, pAUR-MET3 pAUR-MET14 and pAUR- MET16, respectively.

6. over-expressed each gene (APA1, MET3,MET14, and MET16) involved in sulfate assimilation metabolism

7. 2. Glutathione synthesis The fundamental δ-integration vector: pδAUR, was constructed as follows: the DNA fragment encoding a large portion of the promoter-deficient AUR1-C marker gene was amplified from pAUR123 by PCR. The amplified fragment was digested with XhoI and inserted into the XhoI site of the plasmid pδseq

8. The expression plasmid for the GCS: the DNA fragment conjugating the S. cerevisiae phosphoglycerate kinase (PGK) promoter gene, S. cerevisiae γ-GC synthetase gene, and S. cerevisiae PGK terminator gene was obtained from pGK402- GCS (Yoshida et al. 2011) by digestion with XhoI and NotI. The digested fragment was inserted into the SalI / NotI site of the plasmid pδAUR to construct the plasmid pδAUR-GCS. pδAUR-GCS and pδAUR-GS, were digested with AscI and transformed into S. cerevisiae YPH499 using a lithium acetate method.

9. The calculated copy numbers of GCS/GS genes Cocktail 1, and Cocktail 2 strains were 1/1, 3/2 and 7/14, respectively.

10. 3.Combinatorial engineered To construct combinatorial mutant strains which expressed δ-integrated GCS/GS genes and a single- integrated MET14 gene or MET16 gene, the GCS/GS δ-integrated host strain was transformed by EcoRV- digested pRS405-MET14 or pRS406-MET16, respectively.

11. over-expressed theMET14 andMET16 gene in Cocktail 2

12. discussion

13. Thank you