Gram-scale one-pot enzymatic synthesis of CDP-ribitol by a designed bifunctional fusion enzyme

D-Ribitol-5-phosphate (Rbo5P) plays vital roles in bacterial and mammalian development. In mammals, Rbo5P is an essential component of the O-mannosyl glycan on α-dystroglycan, and its defective biosynthesis causes a group of dystroglycanopathies. Supplementation with cytidine diphosphate ribitol (CDP-ribitol, CDP-Rbo) and its analogues has shown therapeutic potential for certain dystroglycanopathies. However, facile and scalable strategies to prepare CDP-Rbo remain unavailable. Here, we report a practical one-pot enzymatic strategy for gram-scale CDP-Rbo production. Starting from inexpensive ribitol, Rbo5P is first generated by L-ribulokinase (AraB) and then converted to CDP-Rbo by CDP-ribitol pyrophosphorylase (TarI) in 90% yield. To streamline the process, AraB and TarI were fused into a single bifunctional biocatalyst, AraB-TarI, which converts ribitol directly into CDP-Rbo on a gram-scale in a one-step reaction. Furthermore, a nucleotide recycling strategy was developed to lower the cost and increase the atom economy from 47% to 75%. This work provides a green and scalable route to CDP-Rbo and a reliable material supply for developing therapeutics against dystroglycanopathies.