Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum.

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.