Continuous‐Flow Biocatalysis of UDP‐Glucuronic Acid: Engineering the Rate‐Limiting Enzyme and Optimizing Multienzyme Immobilization

UDP‐GlcA, the activated form of glucuronic acid, is essential for glycosylation and polysaccharide biosynthesis. Despite advances in UDP‐GlcA synthesis, economical and scalable production methods remain limited. Here, we developed a multienzyme immobilization system for the continuous‐flow synthesis of UDP‐GlcA. We first constructed a UDP‐glucose synthesis module that maintained stable operation for over 200 h with a space‐time yield of 3.9 g·L −1 ·h −1 . To address the bottleneck of the rate‐limiting enzyme UDP‐glucose dehydrogenase (TuaD), we obtained the mutant TuaD M8, using a combined strategy of protein repair one‐stop shop engineering and rational reversion of synergistic deleterious mutations, achieving significantly enhanced activity and stability. Integration of both modules yielded a UDP‐GlcA flow synthesis system with a space‐time yield of 1.3 g·L −1 ·h −1 and 220 h continuous operation, demonstrating strong industrial potential. This strategy provides an efficient, sustainable, and cost‐effective approach for large‐scale UDP‐GlcA production and establishes a framework for biosynthesizing other high‐value nucleotide sugars via immobilized enzyme systems.