Efficient production of l‐glutathione by whole‐cell catalysis with ATP regeneration from adenosine

Abstract l ‐glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole‐cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine‐based adenosine triphosphate (ATP) regeneration system in Escherichia coli . Concretely, to enhance GSH production in E. coli , several genes associated with GSH and l ‐cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP‐binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine‐based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole‐cell catalytic conditions, the engineered strain GSH17‐FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added l ‐Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high‐value metabolites that rely on ATP consumption.