A Sequence-to-Scaffold Platform Enables Programmable Carrier-Supported Biocatalysis for Nucleoside Analog Synthesis

Abstract Industrial biocatalysis increasingly requires strategies that convert enzyme discovery into robust catalyst systems for process implementation. Here, we report Spatially Programmable Assembly of Computationally Mined Enzymes (SPACE), a sequence-to-scaffold workflow coupling multiparametric enzyme mining with programmable immobilized assembly. Using nucleoside phosphorylase biocatalysis as a model, SPACE prioritizes enzyme candidates through multiparametric, language-model-assisted mining and organizes them on porous agarose-hydroxypropyl methylcellulose (agarose-HPMC) microsphere carriers through site-specific bioorthogonal scaffold assembly. Scaffold valency tuned enzyme density, balancing loading, catalytic accessibility, and operational stability in single-enzyme catalysts, whereas orthogonal Spy/Snoop assembly adjusted local stoichiometry to coordinate dual-enzyme cascade flux. These architectures improved single-enzyme 5-fluorouridine production and dual-enzyme 2-fluoroadenosine synthesis and extended performance gains across 15 additional nucleoside analog reactions relative to free-enzyme and whole-cell controls. These results establish spatially programmable enzyme immobilization as a platform strategy for transforming computationally mined enzymes into process-oriented biocatalysts.