Abstract

Bacillus subtilis (B. subtilis) spores present unique opportunities for practical application due to their genetic tractability, generally regarded as safe (GRAS) status, and extreme stress tolerance. By engineering the surface of B. subtilis spores, researchers have been able to display active enzymes, antigens, and protein-protein interaction motifs for myriad applications. However, traditional techniques resulted in relatively low protein expression (102-104 proteins/spore), limiting utility. This defense will begin by describing an application of a high-density spore display strategy developed in the lab called TIED to arrive at stimuli-responsive biomaterials. B. subtilis spores displaying a high density of pH-responsive protein-protein interaction motifs, referred to simply as “A”, displayed pH-responsive erosion stability by remaining stable at low pH, when A-A interactions are occurring, and dissociation at high pH when A is known to disassociate. The effect was more pronounced with higher protein display, highlighting the utility of TIED high density display. Next, an improvement to TIED itself will be discussed. TIED requires genetically engineering each new B. subtilis strain from scratch to display a new protein of interest. This is an arduous multi-step process that requires empirical optimization of each new construct. This defense will describe a new strategy utilizing the SpyTag/SpyCatcher and SnoopTag/SnoopCatcher protein-peptide pairs. When mixed together, SpyTag and SpyCatcher rapidly and irreversibly form a covalent isopeptide bond in what can be considered a biomolecular “click” chemistry reaction. TIED-Click spores displaying a high density of SpyCatcher and/or SnoopCatcher were created, tested, and were utilized to display three enzymes – galactose oxidase, APEX2, and carbonic anhydrase. TIED-Click showed improved stability over the previously used high-density display strategy, could be reused for catalysis, were successfully used in facile tandem catalysis, could be used to display an enzyme previously unknown to B. subtilis spore display, and could immobilize enzymes directly from E. coli lysates. In each case, display of a new enzyme or multiple enzymes required only the expression and purification of the target enzyme in E. coli.