UC Irvine

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. Chapter 2 begins by leveraging 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 disassociating at high pH when A is known to disassociate. Compared to wild-type spores and spores displaying a low-density of A on the spore surface, stability was significantly improved. This highlights the utility of TIED high-density spore display in achieving more pronounced engineered behaviors, while also showing the limitations of weak non-covalent protein-protein interaction motifs to achieve biomaterials - the mechanical properties remained unchanged with different pH’s despite the extent of A-A interactions changing. Chapter 3 represents an improvement to TIED itself. 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. Chapter 3 describes 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 displaying APEX2 showed improved stability over the previously used high-density display strategy, and TIED-Click displaying galactose oxidase were reused daily for catalysis for over one week with no detectable loss in activity. TIED-Click spores co-displaying galactose oxidase and APEX2 were successfully used in facile tandem catalysis, and showed improved activity over a mixture of spores individually displaying each enzyme. To demonstrate that the strategy can enable facile display of new enzymes, the system was used to display carbonic anhydrase, an enzyme previously unknown to B. subtilis spore display. In each case, display of a new enzyme or multiple enzymes required only the expression and purification of the target enzyme in E. coli. As a further proof-of-concept, direct enzyme immobilization from E. coli lysate expressing galactose oxidase was achieved, although the method was not generalizable to other enzymes, and required significant optimization.