Metabolic Construction of a Glucose-Based Biosynthetic Pathway in <i>Escherichia coli</i> toward Sustainable de Novo Xylitol Production

Xylitol is industrially synthesized by d-xylose, which is more expensive than glucose. However, de novo xylitol biosynthesis from glucose remains challenging in <i>Escherichia coli</i>. To address these challenges, this study first developed a biosynthetic pathway from glucose via an arabitol intermediate with arabitol phosphate dehydrogenase (APDH). Chassis optimization through knocking out key genes (<i>rpiA/B</i>, <i>pgi</i>, <i>tktA</i>, <i>tktB</i>, <i>xylB</i>) enhanced flux and increased arabitol yield to 1.61 g/L. Subsequently, the arabitol-to-xylitol route was established using arabitol dehydrogenase (ArDH2) and xylitol dehydrogenase (XDH1) enzymes, showing a 52.1% conversion rate in vitro. The heterologous AXA module (APDH, ArDH2, XDH1) was multicopy integrated into the optimized chassis W3P-5ΔB. The strain W3P4A produced 5.04 g/L arabitol and 1.89 g/L xylitol in shake-flasks. Fed-batch fermentation with a plasmid-carrying strain achieved 5.11 g/L xylitol and 18.5% (g/g) yield from glucose. This work establishes a new platform for xylitol bioproduction from glucose and provides a foundational framework for industrial-scale implementation.