Adenosine triphosphate (ATP) is necessary in the enzymatic production of glutathione (GSH). Our aim was to improve GSH production by increasing the efficiency of ATP regeneration in a coupled system. Previous results Suggested that low GSH production in the Coupled system is due to the irreversible transformation of adenosine (Ado) to hypoxanthine (Hx) via inosine (Ino) pathway in Escherichia coli JM109 (pBV03). In this Study. to block the transformation of Ado into Hx and enhance GSH production. a coupled ATP regeneration system was constructed with adenosine deaminase-deficient recombinant E coli JW1615 (pBV03) and Saccharomyces cerevisiae WSH2. GSH production was improved (2.94-fold of the control), and ATP regeneration reaction was established in the coupled system. The results are applicable to ATP regeneration in other microbial processes. (C) 2009 Elsevier Inc. All rights reserved.