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Heterologous Expression and Characterization of the Antibacterial Lasso Peptide LP2006

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Heterologous Expression and Characterization of the Antibacterial Lasso Peptide LP2006 Heterologous expression and characterization of the antibacterial lasso peptide LP2006 by Gaelen Moore A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Gaelen Moore, 2019 Heterologous expression and characterization of the antibacterial lasso peptide LP2006 Gaelen Moore Master of Science Department of Biochemistry University of Toronto 2019 Abstract The lasso peptides are a class of ribosomally synthesized peptide natural products with diverse bioactivities and structures resembling a lasso. Although the targets of several antibacterial lasso peptides have been investigated to date, the majority remain uncharacterized. Among those that have been characterized, the antibacterial lasso peptides have diverse targets and unique mechanisms of action. One antibacterial lasso peptide with a unique structure, LP2006, is the only member of the class IV peptides. Currently the target and mechanism of action of LP2006 remains unknown. The aim of this study is to develop a system for the heterologous expression of LP2006 to allow for the study of its target and mode of action. I demonstrate that LP2006 can be heterologously expressed using Streptomyces coelicolor M1146, and that purified LP2006 does not appear to activate the cell wall stress response gene liaI . ii Acknowledgments The past two years of my Master’s research project would not have been possible without the support of many people. Firstly, I would like to thank Dr. Justin Nodwell for providing me with the opportunity to work in his laboratory. Despite his busy schedule, he always manages to make plenty of time to meet with his students and never fails to inspire his students. I would also like to thank my committee members, Dr. Karen Maxwell and Dr. Alex Ensminger for their input and thoroughness over the course of my project. I am deeply grateful to Dr. Sheila Pimental-Elardo for her guidance and support in conducting this project. I have thoroughly enjoyed our discussions about marine natural products in addition to our non-scientific discussions. Sheila's kindness and empathy have had a very positive influence on the lab. I am very thankful to have had the opportunity to meet and work with all of the Nodwell Lab members. I am appreciative of not only their scientific suggestions, but also their friendship, which has made my time in the lab highly enjoyable. I would also like to thank my undergraduate thesis project mentor, Sohee Yun, for her guidance and enthusiasm. She inspired me to pursue research and she was always a positive presence during my time as her trainee. Finally, I would like to thank my parents for their steadfast support over the years. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 RiPPs ....................................................................................................................................1 1.1.1 Thiopeptides .............................................................................................................4 1.1.1.1 Biosynthesis of Thiopeptides ....................................................................4 1.1.1.2 Antibiotic Targets of Thiopeptides ............................................................5 1.1.2 Lanthipeptides ..........................................................................................................6 1.1.2.1 Biosynthesis of lanthipeptides ...................................................................9 1.1.2.2 Activity of lanthipeptides ........................................................................11 1.1.3 Lasso peptides ........................................................................................................12 1.1.3.1 Classification of lasso peptides ...............................................................13 1.1.3.2 Biosynthesis of lasso peptides .................................................................17 1.1.3.3 Activity of lasso peptides ........................................................................20 1.1.3.4 Discovery and Heterologous expression of lasso peptides ......................24 1.2 Aim of this work ................................................................................................................26 Chapter 2 Materials and Methods ..................................................................................................27 Materials and Methods ..............................................................................................................27 2.1 General experimental procedures ......................................................................................27 2.1.1 Materials ................................................................................................................27 2.1.2 Strains and plasmids used ......................................................................................27 2.1.3 Primers Used ..........................................................................................................28 iv 2.1.4 Culture conditions ..................................................................................................29 2.1.5 Heterologous expression of LP2006 ......................................................................29 2.2 Isolation and purification of bioactive metabolites ............................................................30 2.2.1 Metabolite extraction .............................................................................................30 2.2.2 Flash chromatography purification ........................................................................30 2.2.3 High-performance liquid chromatography purification .........................................31 2.2.4 Liquid chromatography mass spectrometry analysis .............................................31 2.3 Susceptibility testing ..........................................................................................................32 2.3.1 Disk diffusion assays .............................................................................................32 2.3.2 Broth microtiter dilution assay...............................................................................32 2.4 Target identification ...........................................................................................................32 2.4.1 LacZ reporter assay ................................................................................................32 Results and discussion ..............................................................................................................33 3.1 Nocardiopsis sp. HB141 extract testing.............................................................................33 3.1.1 Extracts of Nocardiopsis sp. HB141 have antibacterial activity ...........................33 3.1.2 Nocardiopsis sp. HB141 is a producer of an antibacterial lasso peptide, LP2006 ...................................................................................................................34 3.1.3 Purification of LP2006 ...........................................................................................35 3.2 Heterologous expression of LP2006 ..................................................................................38 3.2.1 Heterologous expression in Escherichia coli .........................................................38 3.2.2 Heterologous expression in Streptomyces coelicolor M1146 ................................43 3.3 Bioactivity of LP2006 ........................................................................................................46 Conclusions and future directions .............................................................................................48 References ......................................................................................................................................50 Appendix 1 Screen for novel bioactive natural products from marine bacteria ...........................62 Appendix 1 ................................................................................................................................62 v 5.1 Introduction ........................................................................................................................62 5.2 Methods..............................................................................................................................62 5.2.1 Bioactivity screen...................................................................................................62 5.2.1.1 Collection and Isolation of maritime strains ...........................................62 5.2.1.2 Culture conditions ...................................................................................63
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