First Characterization of a Class F Sortase and Establishment of a Microreactor-Based Assay for its Directed Evolution Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Salvatore Di Girolamo aus Italien 2020 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel auf Antrag von Prof. Dr. Florian Seebeck Prof. Dr. Georg Lipps Prof. Dr. Michael Nash Basel, 21 Mai 2019 Prof. Dr. Martin Spiess 1 2 Table of contents ABSTRACT ........................................................................................................................................... 5 LIST OF ABBREVIATIONS ............................................................................................................ 6 1 INTRODUCTION ........................................................................................................................ 8 1.1 Biology and function of sortases ............................................................................................................ 9 1.2 Classification of sortases ..................................................................................................................... 10 1.3 Sortases structure and mechanism of action....................................................................................... 13 1.4 In vitro activity and specificity of sortases .......................................................................................... 17 1.5 Sortases as targets for anti-infective agents development .................................................................. 16 1.6 Sortases as protein engineering tools .................................................................................................. 17 1.7 Optimizing SaSrtA performance ........................................................................................................ 20 1.8 Directed evolution of sortases within hydrogel microcompartments ................................................. 22 2 AIM OF THE WORK ............................................................................................................... 24 3 RESULTS AND DISCUSSION ............................................................................................... 26 3.1 Identification and characterization of novel sortase enzymes ............................................................ 27 3.1.1 Sampling sortase diversity ...................................................................................................................... 27 3.1.2 Recombinant expression and purification............................................................................................... 28 3.1.3 Activity and specificity analysis of BaSrtB, BaSrtD and CpSrtD .......................................................... 30 3.1.4 Structure determination of sortase D2 from C. perfringens .................................................................... 33 3.1.5 Characterization of sortase F from Propionibacterium acnes ................................................................ 34 3.1.6 Analysis of sortase F length-variants ...................................................................................................... 40 3.1.7 Construction and analysis of a chimeric sortase ..................................................................................... 42 3.2 Establishment of a microreactor-based assay for directed evolution of sortases with low activity ... 46 3.2.1 Alginate bead-based compartments as miniature sized culture vessels .................................................. 47 3.2.2 Analysis of clones grown in alginate bead-based microcompartments .................................................. 51 3.2.3 Sortase-mediated conjugation within microreactors ............................................................................... 54 3.2.3.1 Conjugation and product capture strategy based on AlgQ2 ........................................................... 56 3.2.3.1.1 Sortase-mediated conjugation within alginate beads ................................................................ 59 3.2.3.1.2 Analysis of the fusion protein Gly6-AlgQ2-eSaSrtA ............................................................... 63 3.2.3.2 Conjugation strategy based on N-terminally oligo-glycine-modified sortases .............................. 66 3.2.4 Layer-by-layer coating to control capsule permeability ......................................................................... 69 3.2.5 Encapsulated cell lysis optimization ....................................................................................................... 71 3.2.6 Unreacted fluorescent substrate peptide washing optimization .............................................................. 73 3.2.7 Fluorescence normalization with Propidium Iodide ............................................................................... 75 3.2.8 Genotype recovery from individual microreactors ................................................................................. 76 3.2.9 Validation of the microreactor-based assay ............................................................................................ 77 3.2.9.1 Validation of the assay with Gly6-AlgQ2-wtSaSrtA........................................................................ 77 3 3.2.9.2 Validation of the assay with G-SaSrtA .......................................................................................... 83 3.2.10 Microreactor-based assay with sortase B from B. anthracis .............................................................. 86 3.2.11 Microreactor-based assay with PaSrtF ............................................................................................... 90 3.3 Directed evolution of PaSrtF ............................................................................................................... 92 3.3.1 Library construction and analysis ........................................................................................................... 92 3.3.2 Library encapsulation ............................................................................................................................. 94 3.3.3 Preliminary library screening ................................................................................................................. 95 4 CONCLUSIONS AND OUTLOOK ....................................................................................... 97 5 EXPERIMENTAL PROCEDURES ................................................................................... 101 5.1 General information .......................................................................................................................... 102 5.2 PCR oligonucleotide primers, vectors and strains ............................................................................ 103 5.3 Molecular biology .............................................................................................................................. 107 5.3.1 Gene synthesis and cloning .................................................................................................................. 107 5.3.2 Site-directed mutagenesis ..................................................................................................................... 111 5.4 Biochemistry ...................................................................................................................................... 111 5.4.1 Protein expression and purification ...................................................................................................... 111 5.4.2 Sortase activity assays .......................................................................................................................... 115 5.4.3 Structure determination of sortase D from C. perfringens.................................................................... 116 5.5 Microreactors preparation, processing and analysis ........................................................................ 117 5.6 PaSrtF library preparation and screening ........................................................................................ 122 6 BIBLIOGRAPHY .................................................................................................................... 126 ACKNOWLEDGMENTS.............................................................................................................. 139 4 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 recog- nize 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 Staphy- lococcus 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,