BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS IN MUSHROOMS By Robert Michael Sgambelluri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry & Molecular Biology – Doctor of Philosophy 2017 ABSTRACT BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS IN MUSHROOMS By Robert Michael Sgambelluri Cyclic peptide compounds possess properties that make them attractive candidates in the development of new drugs and therapeutics. Mushrooms in the genera Amanita and Galerina produce cyclic peptides using a biosynthetic pathway that is combinatorial by nature, and involves an unidentified, core set of tailoring enzymes that synthesize cyclic peptides from precursor peptides encoded in the genome. The products of this pathway are collectively referred to as cycloamanides, and include amatoxins, phallotoxins, peptides with immunosuppressant activities, and many other uncharacterized compounds. This work aims to describe cycloamanide biosynthesis and its capacity for cyclic peptide production, and to harness the pathway as a means to design and synthesize bioactive peptides and novel compounds. The genomes of Amanita bisporigera and A. phalloides were sequenced and genes encoding cycloamanides were identified. Based on the number of genes identified and their sequences, the two species are shown to have a combined capacity to synthesize at least 51 unique cycloamanides. Using these genomic data to predict the structures of uncharacterized cycloamanides, two new cyclic peptides, CylE and CylF, were identified in A. phalloides by mass spectrometry. Two species of Lepiota mushrooms, previously not known to produce cycloamanides, were also analyzed and shown to contain amatoxins, the toxic cycloamanides responsible for fatal mushroom poisonings. The mushroom Galerina marginata, which also produces amatoxins, was used as a model orgasnism for studying cycloamanide biosynthesis due to its culturability. Three enzymes involved in the biosynthesis of cycloamanides were identified in gene knockout studies: a predicted flavin-containing monooxygenase (FMO), P450 monooxygenase, and prolyl oligopeptidase (POP). The gene encoding a specific predicted prolyl oligopeptidase (POPB) was cloned and expressed in Saccharomyces cerevisiae for further characterization, and in vitro studies revealed that the enzyme is bifunctional, catalyzing both a hydrolysis reaction and the key cyclization step in cycloamanide biosynthesis. The utility of POPB as a general catalyst for peptide cyclization was explored by defining its subtrate preferences and limitations. POPB was shown to be highly versatile, catalyzing cyclization of diverse peptide sequences ranging from 8-16 residues in length and sequences containing modified amino acids in addition to the proteinogenic twenty. A method for the use of POPB for the production of combinatorial cyclic peptide libraries is also presented. A total of 100 cyclic peptides, including both novel compounds and bioactive cycloamanides, were produced in these studies and demonstrate the applications of POPB in biotechnology. Copyright by ROBERT MICHAEL SGAMBELLURI 2017 TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix KEY TO ABBREVIATIONS .......................................................................................... xiii CHAPTER 1 INTRODUCTION ........................................................................................1 1.1 Cyclic Peptides ................................................................................................2 1.2 Cycloamanides ................................................................................................5 1.3 Ribosomal Biosynthesis of Cycloamanides ....................................................8 WORKS CITED ...................................................................................................11 CHAPTER 2 DETECTION AND PROFILING OF AMATOXINS IN LEPIOTA MUSHROOMS ..................................................................................................................16 2.1 Abstract .........................................................................................................17 2.2 Introduction ...................................................................................................18 2.3 Methods .........................................................................................................21 2.3.1 Mushroom Collection and Identification .........................................21 2.3.2 Toxin Extraction and LCMS ............................................................22 2.4 Results ...........................................................................................................23 2.4.1 Toxins in Amanita and Galerina Mushrooms .................................23 2.4.2 Toxins in Lepiota Mushrooms .........................................................24 2.5 Discussion .....................................................................................................27 APPENDIX ..........................................................................................................28 WORKS CITED ...................................................................................................31 CHAPTER 3 GENOMIC CAPACITY FOR CYCLOAMANIDE BIOSYNTHESIS IN AMANITA MUSHROOMS .........................................................................................34 3.1 Abstract .........................................................................................................35 3.2 Introduction ...................................................................................................36 3.3 Methods .........................................................................................................37 3.3.1 Genomics and Transcriptomics........................................................37 3.3.2 LC/MS/MS of Predicted Cycloamanides.........................................37 3.4 Results ...........................................................................................................39 3.4.1 MSDIN Genes in Amanita bisporigera and A. phalloides ..............39 3.4.2 New Cycloamanides in Amanita phalloides ....................................39 3.5 Discussion .....................................................................................................43 APPENDIX ..........................................................................................................45 WORKS CITED ...................................................................................................49 v CHAPTER 4 CHARACTERIZATION OF AMANITIN BIOSYNTHESIS IN GALERINA MARGINATA ..............................................................................................52 4.1 Abstract .........................................................................................................53 4.2 Introduction ...................................................................................................54 4.3 Methods .........................................................................................................57 4.3.1 Galerina Growth and Toxin Analysis ..............................................57 4.3.2 Galerina Transformation and Gene Knockouts ...............................58 4.3.3 Purification of an Amanitin Intermediate and NMR .......................58 4.3.4 Analysis of Gene Expression by RT-PCR .......................................59 4.4 Results ...........................................................................................................60 4.4.1 Time Course of Amanitin Production ..............................................60 4.4.2 Genes Involved in Amanitin Biosynthesis .......................................61 4.4.3 Role of a P450 Monooxygenase in Amanitin Biosynthesis.............64 4.4.4 Regulation of Biosynthetic Genes ...................................................66 4.5 Discussion .....................................................................................................68 APPENDIX ..........................................................................................................70 WORKS CITED ...................................................................................................78 CHAPTER 5 BIOCHEMICAL CHARACTERIZATION OF PROLYL OLIGOPEPTIDASE B AS A PEPTIDE MACROCYCLASE .........................................81 5.1 Abstract .........................................................................................................82 5.2 Introduction ...................................................................................................83 5.3 Methods .........................................................................................................85 5.3.1 Protein Expression and Purification.................................................85 5.3.2 Enzyme Assays ................................................................................86 5.3.3 Product Purification and NMR Spectroscopy ..................................86 5.4 Results ...........................................................................................................88 5.4.1 Preparation of Recombinant GmPOPB ...........................................88 5.4.2 GmPOPB Catalyzes Peptide Macrocyclization ...............................89