Metabolic Engineering of for Fermentative Production of l-Theanine.

-alkylated amino acids are intermediates of natural biological pathways and can be found incorporated in peptides or have physiological roles in their free form. The -ethylated amino acid l-theanine shows taste-enhancing properties and health benefits. It naturally occurs in green tea as major free amino acid. Isolation of l-theanine from shows low efficiency, and chemical synthesis results in a racemic mixture. Therefore, biochemical approaches for the production of l-theanine gain increasing interest. Here, we describe metabolic engineering of KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from for l-theanine production and heterologous enzymes from for growth with xylose. l-Theanine (15.4 mM) accumulated in shake flasks with minimal medium containing monoethylamine and glucose, 15.2 mM with glycerol and 7 mM with xylose. Fed-batch bioreactor cultures yielded l-theanine titers of 10 g L with glucose plus xylose, 17.2 g L with glycerol, 4 g L with xylose, and 21 g L with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using and the first compatible with the use of various alternative carbon sources.