Coexpression of D‑Allulose 3‑Epimerase\nand L‑Rhamnose Isomerase in Bacillus\nsubtilis through a Dual Promoter Enables High-Level\nBiosynthesis of D‑Allose from D‑Fructose\nin One Pot

D-Allose, a rare sugar, has gained significant\nattention\nnot only as a low-calorie sweetener but also for its anticancer, antitumor,\nanti-inflammatory, antioxidant, and other pharmaceutical properties.\nDespite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low\nconversion rates, and the high cost of substrates. Here, we explored\nthe food-grade coexpression of Blautia produca D-allulose 3-epimerase (Bp-DAE) and Bacillus\nsubtilis L-rhamnose isomerase (BsL-RI) within\na single cell using B. subtilis WB800N\nas the host. Using this system, D-allose was synthesized\nvia a simple, cost-effective, one-pot enzymatic process, employing\nwhole cells as catalysts and D-fructose as the substrate.\nThe system exhibited optimal activity at 65 °C, pH 8.5, with\n1 mM Mn<sup>2+</sup> and 20 g/L of whole-cell dry weight. Initial\nproduction reached 12.5 g/L D-allose with a 12.5% yield from\n100 g/L D-fructose. Optimization of dual promoter combinations\nenhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields\nof 15.00, 14.55, and 14.40%, respectively. This D-allose\nproduction biocatalyst offers a scalable and economically viable platform\nfor the industrial production of rare sugar.