A Modular System for the Rapid Comparison of Different Membrane Anchors for Surface Display on Escherichia coli

Comparison of different membrane anchor motifs for the surface display of a protein of interest (passenger) is crucial for achieving the best possible performance. However, generating genetic fusions of the passenger to various membrane anchors is time‐consuming. We herein employ a recently developed modular display system, in which the membrane anchor and the passenger are expressed separately and assembled in situ via SpyCatcher and SpyTag interaction, to readily combine a model passenger cytochrome P450 BM3 (BM3) with four different membrane anchors (Lpp‐OmpA, PgsA, INP and AIDA‐I). This approach has the significant advantage that passengers and membrane anchors can be freely combined in a modular fashion without the need to generate direct genetic fusion constructs in each case. We demonstrate that the membrane anchors impact not only cell growth and membrane integrity, but also the BM3 surface display capacity and whole‐cell biocatalytic activity. The previously used Lpp‐OmpA as well as PgsA were found to be efficient for the display of BM3 via SpyCatcher/SpyTag interaction. Our strategy can be transferred to other user‐defined anchor and passenger combinations and could thus be used for acceleration and improvement of various applications involving cell surface display.