A modular biodevice assembly tool kit for combinatorial screening and benchmarking multiple cell surface display platforms in Escherichia coli.

Abstract

Bacterial cell surface display is a versatile platform technology for synthetic biology applications. A successful display platform often requires ad hoc testing of anchor scaffold types and orientations, as well as target protein types and orientations. To facilitate screening for the optimal display platform, this study designed and constructed a modular, combinatorial Escherichia coli surface display toolkit. The modular toolkit not only screens for the optimal combination of diverse anchor proteins to display various bioparts but also benchmarks the performance of multiple surface display platforms in E. coli simultaneously. The design of the modular toolkit was based on five representative types of anchor scaffolds encompassing autotransporters, outer membrane proteins, curli-associated proteins, and fimbriae-associated proteins. Both amino- and carboxy-terminal display was examined across 20 designs of full or truncated scaffolds, each combined with multiple signal peptides, promoters, and terminators. The combinatorial toolkit was validated and optimized using three display reporters with different sizes and features—sfGFP (fluorescence), agarase (enzymatic activity), and NanoLuc (luminescence). The optimal display combinations were primarily dependent on the orientation of anchor scaffolds, which varied with reporter properties. Strong promoters, such as T7, differentially affect surface display activity on substrate-dependent (agarase, NanoLuc) and substrate-independent (sfGFP) proteins, highlighting expression tuning as a critical parameter for surface display optimization. Using this modular kit, two previously uncharacterized anchor scaffolds (YdhQ and YcgV) were identified as functional for surface display. Finally, the applicability of the toolkit across multiple E. coli genetic backgrounds was confirmed, supporting its utility for diverse biotechnological applications.