<i>Staphylococcus aureus </i>Sortase A (SaSrtA), a calcium-dependent transpeptidase, is a powerful enzymatic tool for covalent site-specific ligation in protein engineering. SaSrtA catalyzes the highly efficient post-translational fusion of two recombinantly tagged proteins, one bearing an LPXTG sequence and the other an N-terminal polyglycine tag. Previous efforts have improved the LPETG-coupling activity of SaSrtA in binding threonine of LPXTG to the N-terminal glycine of (G)<sub>n</sub>, though this design constrains the protein ligation to the polyglycine substrate at the N-terminus. To overcome this, we engineered SaSrtA to improve substrate recognition to a primary amine in a pilin box sequence, a 13-amino acid substrate that mimics the sequence used in sortase-catalyzed pilin assembly in vivo. We modified the yeast surface display to dual display SaSrtA library and LPETG substrate on Aga1 and Aga2 yeast wall proteins. We then screened SaSrtA mutants with broadened substrate specificity toward the pilin box substrate, using fluorescence-activated cell sorting (FACS). These mutants not only demonstrate markedly higher ligation of Pilin Box substrate over the previously engineered SaSrtA mutant but also show over a 15-fold increase in yeast surface expression levels. Many mutations occupy key catalytic regions in the SaSrtA structure like the ß3-ß4 loop, ß6-ß7 loop, and ß7-ß8 loop. This sortase-mediated ligation allows greater liberties in recombinant protein fusion design in comparison to the traditional polyglycine tag. Further, understanding the role of SaSrtA mutants in substrate recognition will facilitate engineering and design of sortase variants with improved enzyme activity and substrate specificity, and broaden the sortase-mediated ligation toolbox.

Engineering Sortase A substrate specificity toward a pilin substrate using directed evolution and yeast surface display

ACS Spring 2022

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