Abstract

Here, we describe a stress-tolerant, recyclable, and renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on the Bacillus subtilis spore surface. TIED enables high loading density in the range of 10⁶ to 10⁷ recombinant enzymes per spore, robust catalytic activity of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Furthermore, we demonstrate TIED enzymes to be not only recyclable but also fully renewable after the loss of activity through induction of germination and sporulation, enabling perpetual regeneration of these immobilized biocatalysts.