Download Improvement of GSH production by metabolic engineering the

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
Glutathione
• 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.
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.
Materials and methods
• S. cerevisiae YPH499
1 Sulfate assimilation
2 Glutathione synthesis
3 Combinatorial engineered
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 pAURMET16, respectively.
over-expressed each gene (APA1, MET3,MET14,
and MET16) involved in sulfate assimilation
metabolism
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
• 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 pGK402GCS (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.
• The calculated copy numbers of GCS/GS genes
Cocktail 1, and Cocktail 2 strains were 1/1, 3/2
and 7/14, respectively.
3.Combinatorial engineered
• To construct combinatorial mutant strains which
expressed δ-integrated GCS/GS genes and a singleintegrated MET14 gene or MET16 gene, the GCS/GS
δ-integrated host strain was transformed by EcoRVdigested pRS405-MET14 or pRS406-MET16,
respectively.
over-expressed theMET14 andMET16
gene in Cocktail 2
discussion
Thank you