Systematic Metabolic Engineering and Model‐Guided Optimization for High‐Level Production of L‐Theanine from Xylose in <i>Escherichia coli</i>

Lignocellulosic biomass represents a promising sustainable feedstock for biomanufacturing, yet the efficient conversion of its dominant pentose, D-xylose, into high-value α-ketoglutarate derivatives like L-theanine remains challenging due to the inherent carbon loss and low yield of conventional metabolic pathways. To overcome this limitation, a novel microbial platform was developed by reconstituting the carbon-conserving Weimberg pathway in E. coli, enabling the direct and de novo biosynthesis of L-theanine from xylose in just 7 enzymatic steps. Through comprehensive metabolic engineering, including the blocking of competitive pathways, enhancing the precursor supply, and fine-tuning cofactor balance, the overproducing strain TH 4-4 achieved a titer of 9.94 g/L and a yield of 0.33 g/g. Furthermore, flux balance analysis of enzyme-constrained metabolic network model was used to quantitatively assess metabolic trade-offs, and a two-stage microaerobic-aerobic cultivation strategy was implemented, resulting in the highest titer of 14.31 g/L and a yield of 0.48 g/g, representing a 2811.4-fold increase compared to the original strain. Finally, a fed-batch fermentation of the engineered strain achieved a titer of 95.42 g/L, a yield of 0.55 g/g xylose, and a productivity of 1.33 g/L/h. This work pioneers the high-level production of L-theanine from xylose and provides a transformative framework for the sustainable valorization of lignocellulosic sugars into valuable TCA cycle derivatives.