Development of an in vitro multi-enzyme system for efficient one-pot biosynthesis of sorbitol from fructose-6-phosphate.

Fructose-6-phosphate is a key ketose monophosphate intermediate that plays a central role in the metabolic pathway of glycolysis. Sorbitol is a naturally occurring sugar alcohol with diverse biological properties, which is widely applied in the food, cosmetic, and pharmaceutical industries due to its favorable physicochemical and physiological characteristics. Although a biosynthetic pathway from starch to fructose-6-phosphate has been well established in metabolic engineering studies, the downstream enzymatic route leading to the biosynthesis of sorbitol from this intermediate has not yet been elucidated in the literature. In this study, we performed the design and implementation of an artificial multi-enzymatic cascade system for the biosynthesis of sorbitol from fructose-6-phosphate. Firstly, candidate enzymes sorbitol-6-phosphate dehydrogenase (S6PDH) and sorbitol-6-phosphate dephosphatase (S6PDP) were selected with an initial activity of 147 U/mg and 1.7 U/mg, respectively. Secondly, molecular modifications were then carried out to improve the thermostability of EcS6PDH, EcS6PDH-M4 displays markedly improved thermostability, with a significant extension of half-life at 40 °C from less than 1 min to 375 min and an elevated T by 9.1 °C. Although multiple rounds of protein engineering were undertaken to improve the catalytic activity of EcS6PDP, no variant exhibiting substantially enhanced catalytic efficiency was identified. Finally, a one-pot biosynthetic system was established by integrating EcS6PDH-M4, EcS6PDP, and glucose dehydrogenase. Following systematic optimization of this multi-enzyme cascade system, 82.6 mM of sorbitol was efficiently produced from 200 mM fructose-6-phosphate. Our work provides a foundational framework for the further enzymatic synthesis of sorbitol directly from starch, offering a promising route for sustainable sugar alcohol production.