De Novo Biosynthesis of <scp>d</scp>-Allitol from Sucrose via Engineered Modular Metabolic Pathways

d-Allitol is a rare sugar alcohol that has garnered interest owing to its potential applications in the food, pharmaceutical, and cosmetics industries. Conventional d-allitol production mostly relies on enzymatic processes or whole-cell catalysis, which typically require exogenous cofactor supplementation and multiple substrate inputs, limiting economic viability. This study offers a novel approach for the green synthesis of d-allitol using sucrose as a substrate. First, we engineered a phosphoenolpyruvate-phosphotransferase system (PTS)-independent sucrose utilization pathway. Nevertheless, d-fructose from sucrose hydrolysis was diverted to cell growth. Subsequently, to redirect flux, we enhanced d-glucose metabolism by overexpressing <i>glk</i>, <i>pgi</i>, and <i>zwf</i> and deleted <i>mak</i> to block d-fructose consumption. Introduction of d-allitol biosynthetic genes enabled the accumulation of d-allitol from sucrose. Finally, further optimization of intracellular byproducts and cofactors increased the yield to 10.11 g/L (40.4%). This approach not only provides a new strategy for sucrose utilization but also offers a potential pathway for d-allitol biosynthesis.