D-阿洛酮糖作为一种重要的稀有糖,以其低能量甜味特性、脂质代谢调节功能以及血糖平衡调节能力而著称。该研究在枯草芽孢杆菌中,通过异源表达醛醇氧化酶(alditol oxidase,AldO)、L-墨角藻糖-1-磷酸醛缩酶(L-fuculose-1-phosphate aldolase,FucA)和果糖-1-磷酸酶(fructose-1-phosphatase,YqaB),成功构建了一条从甘油到D-阿洛酮糖的生物合成途径。为了进一步提升生物合成效率,对FucA的启动子进行精细优化,并对全细胞反应中双细胞的比例进行调整。此外,为简化双细胞转化系统,构建了单细胞转化系统。在此基础上,还考察了全细胞的反应条件,包括温度、pH、全细胞浓度以及甘油浓度,以确定最优的反应参数。经过一系列优化实验,最终确定全细胞反应条件:温度为30 ℃,pH值为6.5,全细胞质量浓度为150 g/L,甘油质量浓度为50 g/L。在优化条件下,通过甘油补料策略,提高了D-阿洛酮糖的产量,使其最高产量达到5.25 g/L,与对照组相比产量提高了26.5%。该研究展示了一种从甘油出发合成D-阿洛酮糖的食品级方法。
D-allulose, an important rare sugar, serves as a low-calorie sweetener with the ability to regulate lipid metabolism and blood glucose balance.In Bacillus subtilis, a biosynthetic pathway from glycerol to D-allulose has been successfully constructed through heterologous expression of alditol oxidase (AldO), L-fuculose-1-phosphate aldolase (FucA), and fructose-1-phosphatase (YqaB).After optimizing the promoter of FucA, the ratio of mixed whole-cell was optimized.Then, to simplify the mixed whole-cell conversion system, a whole-cell conversion system using a single strain was constructed.Furthermore, the whole-cell conversion conditions, including temperature, pH, whole-cell concentration, and glycerol concentration, were investigated.The optimal conditions for whole-cell conversion were 30 ℃, pH 6.5, whole-cell concentration 150 g/L, and glycerol concentration 50 g/L.Under optimized conditions, the production of D-allulose was achieved through glycerol feeding strategy, with the highest yield reaching 5.25 g/L, representing a 26.5% increase compared to the control group.This study presents a food-grade method for synthesizing D-allulose from glycerol.
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