title: Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.
abstract: 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-1added cysteine and a productivity of 6.1 ± 0.0 g l-1h-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.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China.
authors.forename: Rongsheng
authors.initials: R
authors.lastname: Tao
authors.affiliation: Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China.
authors.forename: Zhengquan
authors.initials: Z
authors.lastname: Shen
authors.affiliation: Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China.
authors.forename: Liangdong
authors.initials: L
authors.lastname: Sun
authors.affiliation: Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China.
authors.forename: Fuyun
authors.initials: F
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. syang@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China. syang@sibs.ac.cn.|Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China. syang@sibs.ac.cn.|Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, 200237, China. syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D000084:Acetate Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001709:Biotechnology / Q000379:methods*; D004151:Dipeptides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000096:biosynthesis*; D005981:Glutathione Synthase / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology
chemical_list: D004151:Dipeptides; D000255:Adenosine Triphosphate; D000084:Acetate Kinase; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione; C017341:gamma-glutamylcysteine
keywords: ATP generation; Acetate kinase; Bifunctional γ-glutamylcysteine synthetase/glutathione synthetase; Glutathione