A Computational Framework for Domain Insertion into Type IV Pili for Bacterial Display and Living Material Assembly

Bacterial surface structures have enabled display systems with broad impact across biotechnology, but their narrow host range limits their deployment into diverse species. Conversely, Type IV Pili (TFP) are ubiquitous, structurally conserved appendages found across bacteria, but have been minimally explored for display. Here, we describe a computational framework for predicting viable insertion sites in major pilins for stable TFP-mediated display, which we apply to the major pilin PilA1 of the cyanobacterium Synechocystis sp. PCC 6803 to enable covalent binding to living materials. By analyzing an Alphafold3-generated PilA1 monomer alongside known multimeric TFP multimers, our pipeline identifies non-interfacial, solvent-accessible, and flexible sites for optimal PilA1 display. We probe these sites with both full-length and truncated SpyCatcher003 at two different expression levels. We show that cells expressing these PilA1-SpyCatcher003 fusions maintain up to 8-fold higher levels of cell suspension than previous C-terminal PilA1 display platforms, suggesting improved TFP assembly despite more than a two-fold increase in cargo size. Additionally, we validate SpyCatcher003 reactivity across the engineered strains, enabling covalent attachment of SpyTag003-containing proteins on the Synechocystis surface. Lastly, we utilize this covalent patterning to achieve a four-fold increase in Synechocystis loading into a living material without compromising its viscoelastic or mechanical properties. Taken together, this work provides a predictive framework for TFP engineering, and opens the door towards programmable surface display across the breadth of bacterial species.