High-level bioproduction of L-theanine by systematically engineered Escherichia coli without ethylamine feeding

Engineered autonomous ethylamine biosynthesis via dual-pathway strategy, eliminating the need for exogenous ethylamine. • Achieved record-breaking L-theanine titer of 118.2 g/L in fed-batch fermentation, the highest reported production to date. • Elucidated the ethylamine preference mechanism of L-theanine synthetase Mm GMAS, providing insights for enzyme engineering. • Developed systematic metabolic engineering combining precursor enhancement, pathway knockout, and adaptive evolution. L-Theanine, the most abundant free amino acid in tea, is a non-proteinogenic amino acid widely used in food and cosmetics, with documented health benefits and therapeutic potential. To circumvent the reliance on hazardous exogenous ethylamine in L-theanine production, we engineered an integrated and efficient strategy for high-level L-theanine production in Escherichia coli via dual-pathway ethylamine biosynthesis. First, by enhancing the expression and elucidating the ethylamine preference mechanism of the L-theanine synthetase Mm GMAS, the obtained strain THE-3 produced 15.8 g/L L-theanine. Building on this, the knock-out of L-theanine degradation pathway combined with bolstered precursors supply further increased the titer to 16.8 g/L and 22.8 g/L in strain THE-6 and THE-9, respectively. Then, developing the ethylamine intracellular biosynthesis via Cs AlaDC-mediated decarboxylation pathway in THE-13 resulted in the production of 19.5 g/L L-theanine and 5.4 g/L L-alanine without exogenous ethylamine. Subsequently, by synergizing the Pp SpuC-mediated transamination pathway with L-alanine as the amino donor, and subjecting this strain to evolution for acetaldehyde resistance, the final engineered strain, THE-17, achieved a record-breaking titer of 118.2 g/L in fed-batch fermentation, with a yield of 0.389 g/g glucose and a productivity of 2.46 g/L/h. This study establishes the highest reported L-theanine production to date and provides a transformative approach for industrial L-theanine production, shifting the paradigm from reliance on exogenous ethylamine addition to autonomous ethylamine supply.