A process of L-theanine production by the recombinant gamma-glutamylmethylamide synthetase (GMAS) coupled with an adenosine triphosphate (ATP) regeneration system that utilized inorganic polyphosphate (polyP) via polyphosphate kinase (PPK) was designed. The gmas gene from Methylovorus mays and ppk gene from Rhodobacter sphaeroides were cloned into pET-21a(+), then introduced into E. coli BL21(DE3) respectively. GMAS and PPK were both overexpressed in soluble forms. L-Theanine was efficiently produced from sodium glutamate, ethylamine hydrochloride and polyP with a very small amount of ATP. After 5 h at 37 degrees C and pH at 7.0, the 93% productivity of L-theanine was achieved at concentration of 200mM sodium glutamate and 200 mM ethylamine hydrochloride with 5 mM ATP. Productivity of L-theanine decreased to 84% when using 400 mM sodium glutamate, and 26% when using 600 mM sodium glutamate. The productivity of L-theanine obtained at a very small amount of ATP and high substrate concentration provides the basis for the large-scale industrial production of L-theanine. (C) 2016 Elsevier Ltd. All rights reserved.