Metabolic Engineering of <i>Pseudomonas putida</i> for Fermentative Production of <scp>l</scp>-Theanine

Abstract

<i>N</i>-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 <i>N</i>-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 <i>Camilla sinensis</i> 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 <i>Pseudomonas putida</i> KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from <i>Methylorubrum extorquens</i> for l-theanine production and heterologous enzymes from <i>Caulobacter crescentus</i> 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^-1 with glucose plus xylose, 17.2 g L^-1 with glycerol, 4 g L^-1 with xylose, and 21 g L^-1 with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using <i>P. putida</i> and the first compatible with the use of various alternative carbon sources.