Multienzyme Cascade Synthesis of Rare Sugars From Glycerol in Bacillus subtilis

BACKGROUND: Rare sugars are valuable and unique monosaccharides extensively utilized in the food, cosmetics, and pharmaceutical industries. Considering the high purification costs and the complex processes of enzymatic synthesis, whole-cell conversion has emerged as a significantly important alternative. The Escherichia coli strain was initially used in whole-cell synthesis of rare sugars. However, its pathogenic nature poses limitations to its widespread applications. Consequently, there is an urgent need to explore biologically safe strains for the efficient production of rare sugars. RESULTS: In this study, the generally regarded as safe (GRAS) strain Bacillus subtilis was employed as the chassis cells to produce rare sugars via whole-cell conversion. Three genes encoding alditol oxidase (AldO), L-rhamnulose-1-phosphate aldolase (RhaD), and fructose-1-phosphatase (YqaB) involved in rare sugars biosynthesis were heterogeneously expressed in B. subtilis to convert the only substrate glycerol into rare sugars. To enhance the expression levels of the relevant enzymes in B. subtilis, different promoters for aldO, rhaD, and yqaB were investigated and optimized in this system. Under the optimized reaction conditions, the maximum total production titer was 16.96 g/L of D-allulose and D-sorbose with a conversion yield of 33.9% from glycerol. Furthermore, the engineered strain produced 26.68 g/L of D-allulose and D-sorbose through fed-batch for the whole-cell conversion, representing the highest titer from glycerol reported to date. CONCLUSION: This study demonstrated an efficient and cost-effective method for the synthesis of rare sugars, providing a food-grade platform with the potential to meet the growing demand for rare sugars in industries.