Construction of Bienzyme Self-Assembling Clusters Based on SpyCatcher/SpyTag for the Efficient Biosynthesis of Homogeneous Dextran.

The development of spatially organized enzyme systems with enhanced substrate channeling is critical for biocatalytic applications in food and agricultural biotechnology. Here, we constructed bienzyme self-assembling clusters (BESCs) using SpyCatcher/SpyTag technology to integrate prokaryotic dextransucrase (YG) and eukaryotic dextranase (padex). Through the modular engineering of eight enzyme chimeras, six covalently linked BESCs were generated, with YGST-SCpadex exhibiting superior performance. This optimized assembly reduced lag time by 59% and produced low-molecular-weight dextran (3500 Da) with enhanced uniformity (D̵ = 1.28), features desirable for food-grade applications. Mechanistic analysis confirmed that precise spatial organization enabled efficient intramolecular substrate channeling, improving cascade catalysis while maintaining operational stability. This modular strategy provides a versatile framework for engineering tailored enzyme complexes, offering transformative potential for sustainable biomanufacturing of polysaccharide-based ingredients in the food and agriculture sectors.