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

## TL;DR

Scientists engineered E. coli to efficiently produce L-theanine from xylose, a sugar from plant biomass, using a new metabolic pathway and optimization techniques.

## Contribution

The study introduces the shortest synthetic pathway for L-theanine production from xylose in E. coli, achieving record-high yields.

## Key findings

- The engineered strain achieved a titer of 95.42 g/L of L-theanine with a yield of 0.55 g/g xylose.
- A two-stage cultivation strategy increased titer to 14.31 g/L and yield to 0.48 g/g.
- The new pathway represents a 2811.4-fold improvement over the original strain.

## 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.

This study is pioneering in constructing the shortest known synthetic pathway for L‐theanine production from xylose within E coli. Through comprehensive metabolic engineering strategies, our engineered strain achieved the highest reported L‐theanine titer from xylose, with a titer of 95.42 g/L, and a yield of 0.55 g/g. This work establishes a scalable and economically attractive platform for the sustainable production of L‐theanine as well as other TCA‐derived biochemicals.