Efficient fermentative production of l-theanine by Corynebacterium glutamicum

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. l-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in l-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of l-theanine in Corynebacterium glutamicum, an industrially safe host. For l-theanine synthesis, we used gamma-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of l-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an l-glutamate overproducing strain, to produce l-theanine upon ethylamine addition to the hosts. Second, the l-glutamate exporter in host cells was disrupted, which markedly increased the l-theanine titer in GDK-9 cells and almost eliminated the accumulation of l-glutamate in the culture medium. Third, a chromosomally gmas(Mm)-integrated l-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived l-serine/l-alanine decarboxylases; however, these enzymes showed no l-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced similar to 42 g/L l-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of l-theanine achieved using ethylamine supplementation.