Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation

• Published in: Applied biochemistry and biotechnology Vol. 180; no. 7; pp. 1446 - 1455
• Main Authors: Jiang, Yu, Tao, Rongsheng, Shen, Zhengquan, Sun, Liangdong, Zhu, Fuyun, Yang, Sheng
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.12.2016
• Subjects: Acetate Kinase - metabolism; Adenosine Triphosphate - biosynthesis; Biochemistry; Biotechnology; Biotechnology - methods; Chemistry; Chemistry and Materials Science; Dipeptides - metabolism; Escherichia coli; Escherichia coli - metabolism; Glutamate-Cysteine Ligase - metabolism; Glutathione - biosynthesis; Glutathione Synthase - metabolism; Mutation - genetics; Streptococcus - enzymology; Streptococcus thermophilus; Acetate kinase; ATP generation; Bifunctional γ-glutamylcysteine synthetase/glutathione synthetase; Glutathione
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-016-2178-5

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l −1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol −1 added cysteine and a productivity of 6.1 ± 0.0 g l −1  h −1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.