Stress-tolerant, recyclable, and autonomously renewable biocatalyst platform enabled by engineered bacterial spores

Here, we describe a stress-tolerant, recyclable, and autonomously 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 B. subtilis spore surface. TIED enables a high loading density in the range of 106-107 recombinant enzymes per spore, robust catalytic activities of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Further, we demonstrate TIED-enzymes to be not only recyclable, but fully renewable after loss of activity through induction of germination and sporulation, enabling perpetual reuse of these immobilized biocatalysts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/484680v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@6f50d0org.highwire.dtl.DTLVardef@22a9c3org.highwire.dtl.DTLVardef@1b74e9corg.highwire.dtl.DTLVardef@18a11c8_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic illustration of the T7 RNA polymerase-enabled high-density protein display (TIED) on bacterial spores and its unique features as a biocatalyst platform.