Coexpression of <scp>D</scp>-Allulose 3-Epimerase and <scp>L</scp>-Rhamnose Isomerase in <i>Bacillus subtilis</i> through a Dual Promoter Enables High-Level Biosynthesis of <scp>D</scp>-Allose from <scp>D</scp>-Fructose in One Pot

D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of <i>Blautia produca</i> D-allulose 3-epimerase (Bp-DAE) and <i>Bacillus subtilis</i> L-rhamnose isomerase (BsL-RI) within a single cell using <i>B. subtilis</i> WB800N as the host. Using this system, D-allose was synthesized via a simple, cost-effective, one-pot enzymatic process, employing whole cells as catalysts and D-fructose as the substrate. The system exhibited optimal activity at 65 °C, pH 8.5, with 1 mM Mn²⁺ and 20 g/L of whole-cell dry weight. Initial production reached 12.5 g/L D-allose with a 12.5% yield from 100 g/L D-fructose. Optimization of dual promoter combinations enhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields of 15.00, 14.55, and 14.40%, respectively. This D-allose production biocatalyst offers a scalable and economically viable platform for the industrial production of rare sugar.