Optimized 3′-Phosphoadenosine-5′-phosphosulfate Biosynthesis via Yeast-Powered ATP Regeneration and Biotin–Streptavidin Enzyme Immobilization

3'-Phosphoadenosine-5'-phosphosulfate (PAPS), a universal sulfate donor for sulfation reactions, is indispensable for synthesizing bioactive molecules including therapeutic glycosaminoglycans and sulfolipids; however, its enzymatic production on an industrial scale is constrained by ATP overconsumption and the limited free enzyme reusability. We report an integrated biocatalytic platform combining ATP regeneration with affinity immobilization to enable sustainable PAPS biosynthesis. A polyphosphate kinase-driven ATP regeneration system achieved 86% PAPS conversion efficiency by regenerating ADP using low-cost polyphosphate. Biotin-streptavidin affinity immobilization enhanced operational stability, retaining >50% activity over six reuse cycles with a cumulative PAPS titer of 12.02 g/L. Coupling adenosine-converting <i>Saccharomyces cerevisiae</i> whole-cell catalysts with this system decreased substrate costs by 80.7% and delivered 96% molar PAPS yield from adenosine. This work provides a sustainable platform for industrial PAPS biosynthesis to promote sulfated biomolecule production, including glycosaminoglycans and other therapeutics.