One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5 mM glutathione was produced within 5 h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7 mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione.