L-Xylulose is a potentially valuable rare sugar used as starting material for antiviral and anticancer drug development in pharmaceutical industries. In this study, cofactor engineering was applied to improve the efficiency of L-xylulose production from xylitol. A water-forming NAD⁺ regeneration enzyme (NADH oxidase) from Streptococcus mutans ATCC 25175 was introduced into E. coli with xylitol-4-dehydrogenase (XDH) of Pantoea ananatis resulting in recombinant cells harboring the vector pETDuet-xdh-SmNox. The co-expression system exhibited optimal activity at a temperature of 37 °C and pH 8.5, and the addition of Mg²⁺ enhanced the catalytic activity by 1.19 fold. Co-expression of NADH oxidase with XDH enzyme resulted in increased L-xylulose concentration and productivity from xylitol as well as the intracellular NAD⁺ concentration. In a 1 L bioconversion system the final concentration and productivity of L-xylulose from 50 g/L of xylitol reached 48.45 g/L, and 2.42 g/L.h respectively. Overall, this study is a suitable approach for large-scale production of L-xylulose from xylitol.

L-木酮糖是一种具有潜在价值的稀有糖，用作制药行业抗病毒和抗癌药物开发的起始原料。在这项研究中，辅因子工程被应用于提高从木糖醇生产L-木酮糖的效率。将来自变形链球菌ATCC 25175 的成水 NAD ⁺再生酶（NADH 氧化酶）与Pantoea ananatis 的木糖醇-4-脱氢酶 (XDH) 一起引入大肠杆菌，从而产生含有载体 pETDuet -xdh-SmNox 的重组细胞。共表达系统在 37 °C 和 pH 8.5 的温度下表现出最佳活性，添加 Mg ²⁺催化活性提高了 1.19 倍。NADH 氧化酶与 XDH 酶的共表达导致来自木糖醇的L-木酮糖浓度和生产力以及细胞内 NAD ⁺浓度的增加。在 1 L 生物转化系统中，来自 50 g/L 木糖醇的L-木酮糖的最终浓度和生产率分别达到 48.45 g/L 和 2.42 g/Lh。总的来说，这项研究是从木糖醇大规模生产L-木酮糖的合适方法。