Engineering microcin J25 for new biological activities and therapeutic applications Hans Gerstmans 1,2 , Cato Pluym 1 , Jeroen Lammertyn 2 , Joleen Masschelein 1 1 Laboratory for Biomolecular Discovery and Engineering, KU Leuven - VIB, Belgium, 2 MeBioS-Biosensors group,KU Leuven, Belgium Due to their ribosomal origin, unique structural features and pharmacological potential, lasso peptides, such as microcin J25 (MccJ25) represent promising starting points for the development of new macrocyclic peptide drugs. The relaxed substrate specificity of the MccJ25 biosynthetic enzymes, together with the increasing availability of methods to efficiently introduce multiple site-directed mutations, offers tremendous opportunities for generating large libraries of novel-to-nature lasso peptide variants that can be screened for improved pharmaceutical properties or new biological activities 1 . So far, efforts to generate MccJ25 derivatives have been limited to a range of single and carefully selected multiple amino acid substitutions. Using this method, it was discovered which residues are important regarding export, antimicrobial activity etc 2-4 . However, introducing indels or multiple site-specific amino acids changes at the same time has not been explored. Here, we aim to accelerate the development of lasso peptides as clinical drug candidates by using a combination of synthetic biology and a high-throughput, microfluidics-based screening approach. In the first step, we set up a heterologous expression platform in E. coli . We tested different promoters, configurations of the biosynthetic enzymes, and growth media. MccJ25 production was visualised in an antimicrobial activity assay and quantified via UHPLC-ESI-QTOF-MS analysis. The most productive configuration and growth medium were used in further experiments. The second step was to get around the specificity of the MccJ25 exporter, mcjD . This limits the engineering potential of MccJ25, as literature has shown that certain MccJ25 variants are not exported out of the cell. To alleviate this problem, we have characterized two different inducible phage-based lysis systems via colony counting and the release of GFP 5, 6 . Both systems displayed differences regarding the speed of lysis and the amount of lysis. We demonstrated the applicability of this system by showing the antibacterial activity of an MccJ25 variant that is not exported. With the heterologous expression platform and the inducible phage-based lysis system tested, we set up an initial proof-of-concept experiment. We made a library where each possible residue of the loop of MccJ25 was mutagenized. With this, we were able to expand the antibacterial spectrum of MccJ25. References 1. Cheng C, Hua Z-C. 2020. Lasso Peptides: Heterologous Production and Potential Medical Application. Front Bioeng Biotechnol. 2. Ritter SC, Yang ML, Kaznessis YN, Hackel BJ. 2018. Multispecies activity screening of microcin J25 mutants yields antimicrobials with increased specificity toward pathogenic Salmonella species relative to human commensal Escherichia coli. Biotechnol Bioeng 115:2394–2404. 3. Pan SJ, Link AJ. 2011. Sequence Diversity in the Lasso Peptide Framework: Discovery of Functional Microcin J25 Variants with Multiple Amino Acid Substitutions. J Am Chem Soc 133:5016–5023. 4. Ducasse R, Yan K-P, Goulard C, Blond A, Li Y, Lescop E, Guittet E, Rebuffat S, Zirah S. 2012. Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25. ChemBioChem 13:371–380. 5. Cárcel-Márquez J, Flores A, Martín-Cabello G, Santero E, Camacho EM. 2019. Development of an inducible lytic system for functional metagenomic screening. Sci Rep 9:3887. 6. Alex Wong CF, van Vliet L, Bhujbal SV, Guo C, Sletmoen M, Stokke BT, Hollfelder F, Lale R. 2021. A Titratable Cell Lysis-on- Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution. ACS Synth Biol 10:1882–1894.
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