ABSTRACT 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.
Han et al. (2026) studied this question.