Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by γ-glutamylmethylamide synthetase

L-theanine, an amino acid found in green tea leaves, holds promise for natural flavor enhancement and the food industry due to its unique taste and numerous therapeutic effects. The production of L-theanine through enzymatic synthesis has been actively studied, leveraging advantages such as high purity of the L-form and short reaction times. The ATP-dependent enzyme γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays No. 9 achieves remarkable L-theanine production. In this study, to produce L-theanine with less ATP during the enzymatic synthesis catalyzed by GMAS, the process was coupled with an ATP regeneration system catalyzed by acetate kinase (ACK). During the optimization process, the ACK ATP regeneration could be achieved without the need for heterologous ACK, thereby streamlining cell preparation. Employing ACK ATP regeneration in a whole-cell reaction with recombinant Escherichia coli produced 186 mM L-theanine from 200 mM glutamate and ethylamine, with a productivity of 93 mmol/L/h, using only 30 mM ATP. To our knowledge, this study represents the first attempt to apply and evaluate the endogenous ACK ATP regeneration system for L-theanine production.