First characterization of a class F sortase and establishment of a microreactor-based assay for its directed evolution

Abstract

Sortases are a family of enzymes responsible for the covalent anchoring of proteins to the cell wall of Gram-positive bacteria via a transpeptidation reaction. These cysteine transpeptidases specifically recognize and cleave a five amino acid long sorting motif on the target proteins and then catalyzed the formation of a new peptide bond between the C-terminus of the cleaved sorting motif and the free amino group of a cell wall component. The transpeptidation activity of the well-characterized class A sortase from Staphylococcus aureus (SaSrtA) and evolved variants thereof continues to see increasing use in a wide range of biotechnological applications (Sortagging). Due to low activity, sortases from classes other than class A are not currently used for this purpose and, with the exception of SaSrtA, laboratory evolution of other sortases has not been performed. In the first part of this work, we report on the exploration of the natural diversity of sortases and de-scribe the in-depth characterization of a sortase enzyme that belongs to the not-yet-investigated class F, sortase F from Propionibacterium acnes (PaSrtF). We showed that PaSrtF exhibits similar behaviour to the wild type SaSrtA in terms of catalytic activity and sequence specificity and demonstrated its usefulness for protein engineering applications. In the second part of the work, the development of a novel assay for the screening of sortase variants with improved properties is described. Hydrogel bead-based microreactors, suitable for high-throughput screening using a large particle flow cytometer, were prepared and evaluated for their capability to act as individual evolutionary units that link sortases activity with a fluorescent readout. The microreactor-based assay, developed and optimized with the wild type SaSrtA, was successfully validated with the newly characterized PaSrtF and can, therefore, be used for its directed evolution.