Biosynthetic Chemistry of Marine Actinomycete Bacteria - A PhD Dissertation by Charles Bradford Larson, University of California San Diego

UNIVERSITY OF CALIFORNIA SAN DIEGO Blurring the Lines Between Natural and Synthetic: The Biosynthetic Chemistry of Marine Actinomycete Bacteria A dissertation submitted in partial satisfaction of the Requirements for the Degree Doctor of Philosophy in Biomedical Sciences by Charles Bradford Larson Committee in charge: Professor Bradley S. Moore, Chair Professor Victor Nizet, Co-Chair Professor Michael Burkart Professor Tracy Handel Professor Susan Taylor 2019 The dissertation of Charles Bradford Larson is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California San Diego 2019 iii Dedication In loving memory of William Larson iv Table of Contents Signature Page. iv Table of Contents. v List of Figures.

viii List of Tables. xiv Abstract of the Dissertation. xvi Chapter 1: Introduction to the Dissertation.1 Natural Products as Traditional Medicines………….2 Microbial Natural Products………………………………………………….3 Technological Advancements in Analytical Chemistry During the Golden Age………………………………………………………………………….4 Marine Natural Products………………………………………………………….5 Actinomycetes Contribution to the Natural Products Pharmacopeia…….6 Natural Products Biosynthesis: Connecting Genes to Molecules………….7 DNA Sequencing and Natural Products Biosynthesis……………………….8 High Throughput and Whole Genome Sequencing Reveals Huge Biosynthetic Potential………………………………………………………………… 18 1.9 Heterologous Expression of Natural Products……………………………….10 Opportunities in Modern Natural Products Discovery and Overview of Dissertation Chapters.11 References for Chapter 1. 24 Chapter 2: Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols……………………….1 Introduction to Chapter 2.2 Chapter 2 Introduction References.3 Reprint of “: Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols”.

39 Chapter 3: PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete……….1 Introduction to Chapter 3.2 Chapter 3 Introduction References.3 Reprint of “PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete”.67 Chapter 4: Marine Actinobacteria Salinispora pacifica Biosynthetic Gene Cluster Produces Phosphonate Degradation Products of Glyphosate………………….1 Phosphorus in Nature and the Marine Phosphorus Cycle.2 Anthropogenic Phosphonate Contamination in the Environment.1 Identification of Salinispora PepM Sequences.2 Heterologous Expression of S. pacifica Phosphonate BGC.3 Purification of Compounds 1-3.4 Cloning and Expression of Phosphonate N-acetyltransferases.5 Distribution of PepM (SppJ) Homologs in the Marine Environment.2 Analysis of the distribution of PepM homologs.3 Cloning and Plasmid Assembly.5 Purification of Phosphonates Produced in Heterologous Host.6 NMR and MS/MS Analysis.7 Cloning and Purification of GNATs from Phosphonate Cluster.6 References for Chapter 4.130 vii List of Figures Figure 1.1: Bioactive chemicals from traditional plant medicines.2: Drugs from the Golden Age of natural products discovery.3: Marine Natural Products.1: Molecular network of all generated extracts.2: Effects of genus and species on molecular diversity-network analysis.3: Effects of additional attributes on molecular diversity-network analysis.4: Time-dependent changes in natural product distribution in Salinispora arenicola CNH-877, grown in ISP2.1: Molecular Network of All Streptomyces Extracts Color Coded By Culture Medium.2: Molecular Network of All Acquired Extracts Color Coded By Genus.3: Molecular Network of All Salinispora Extracts Color Coded By Species.4: Illustration of the major drivers of chemical speciation within the Salinispora data.5: Molecular Network of All Generated Extracts Color Coded by Extraction Solvent.6: Molecular Network of Salinispora Extracts from Different Media Phases.7: Comparison of MS/MS spectra of arenicolide A (upper spectrum, m/z: 827.489,) and a novel, formally hydrated arenicolide analogue (lower spectrum, m/z: 845.501) from the ethyl acetate extract of Salinispora pacifica CNT-138.8: Molecular Network of All Acquired Extracts Color Coded By Strain Location.9: Molecular Network of Salinispora arenicola CNH877 strains grown in four different liquid media and extracted at three different time points.1: A selection of anthracyclines approved for treatment of cancer.2: Capture vector assembly.3: Gene map of biosynthetic gene clusters associated with the biosynthesis of cosmomycins C and D.4: Cosmomycin cluster identified by molecular networking.1: PCR screening of multiple yeast colonies.73 viii Supplementary Figure S3.2: PCR screening of individual yeast colonies.3: Sequencing of integrated cosmomycin pathway.4 PCR amplification of integrated cosmomycin pathway.5: MS2 fragmentation analysis of cosmomycin C (1).6: MS2 fragmentation analysis of cosmomycin D (2).7: MS1 spectrum of cosmomycin analog 3.8: MS2 fragmentation analysis of cosmomycin analog 3.9: MS1 spectrum of cosmomycin analog A.10: MS2 fragmentation analysis of cosmomycin analog A.11: MS2 fragmentation analysis of cosmomycin analog B.12: 1H NMR spectrum of cosmomycin C (1).13: 1H NMR spectrum of A.14: Overlaid 1H NMR spectrum of 1 and A.15: HSQC spectrum of 1.16: HSQC NMR spectrum of A.17: COSY NMR spectrum of 1.18: Molecular network of cosmomycin native producers and heterologous host. CNT-302 molecular network.1: Depiction of a Salinispora colony, the S. pacifica BGC and its small molecule products.2: Biosynthetic scheme for P-C bond installation.3: Depiction of phosphonate BGCs.4: Crude preparation of heterologous host fermentation.5: 1H NMR spectra of both heterologous host and S.

coelicolor M1152 extracts purified by chromatography.6: Broad band decoupling of the NMR spectrum of the enriched fraction of heterologous host extracts reveals the influence of a phosphorus nucleus on three peaks.7: 1H/31P two-dimensional NMR spectrum (HMBC) of enriched preparation of heterologous host extracts.8: 1H NMR analysis of HILIC purified products of fermentation.9: MS/MS analysis of HILIC purified metabolites.10: 31P NMR analysis of heterologous host extracts spiked with synthetic phosphonate standards.11: MS analysis of GNAT enzyme assays with AMPA substrate.12: MS analysis of GNAT enzyme assays with 2-AEP substrate.13: Sequence Similarity Network of S. pacifica PepM homologs found in marine genomes and metagenomes.14: Putative biosynthetic pathway for the production of na-AMPA.1: Gel electrophoresis analysis of phosphonate BGC amplicons prior to assembly.2: Gel digest of assembled S. pacifica BGC in the pCAP03 plasmid.3: HRMS data for m/z=110, including molecular formula calculation.4: HRMS data for m/z=125, including molecular formula calculation.5: HRMS data for m/z=152, including molecular formula calculation.6: 1H NMR analysis of spiking experiments with naAMPA, AMPA, and 2-HEP synthetic standards.7: NMR Analysis of Crude Extract.8: Protein gel (10% acrylamide) of HisTrap purified GNAT enzymes.9: Amplification of small regions of the S. pacifica phosphonate BGC from genomic DNA isolated from the heterologous host.143 x List of Tables Supplementary Table S2.1: Overview of the bacterial strains used in this study.2: Overview of the compounds detected in this study.3: Information about detected staurosporine analogues in the ISP2 time course network.1: 1H NMR chemical shift assignments of compounds 1 and 4 and the related cosmomycin analogs obelmycin C4 and A447C/D.2: Comparison of Streptomyces sp.1: Genes in the Salinispora pacifica phosphonate BGC and their closest homolog determined by NCBI BLAST.2: Multiple Sequence Alignment of S.

pacifica phosphonate BGC GNATs and B. pacifica PepM homologs in marine genomes and metagenomes in the MAR DB database.4: Color coded legend for sequence similarity network.5: Primers used for the amplification of the S. pacifica phosphonate BGC and specific GNATs from the cluster.228 xi Acknowledgements Many thanks to my mentor Brad Moore, who offered me the opportunity to perform my doctoral studies in his laboratory and provided the support and mentorship necessary to complete my studies. His kind but rigorous and thorough instruction and mentorship has shaped me into a competent natural products chemist.

I would also like to thank my Co-Advisor, Victor Nizet, who offered to liaison with the BMS department which allowed me to work at the beautiful laboratory at the Scripps Institute of Oceanography I have called home for the last several years. Additionally, I would like to thank the wonderful natural products community at SIO. Members of the Jensen, Gerwick, Hughes, Fenical, and Allen laboratories provided an environment that encouraged curiosity, collaboration, and fun. Many thanks to the post-doctoral researchers who aided my progress through the years, including Dr.

Yuta Kudo, Dr. Shaun McKinnie, Dr. Hanna Luhavaya, Dr. Jonathan Chekan, Dr.

Ellis O’Neill, Dr. Peter Jordan, and Dr. I would also like to thank my family for their loving encouragement and support throughout my journey in science. Chapter 2, in full, is a reprint of materials as it appears in “Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols” in Journal of Natural Products, 2016, Max Crusman, Ellis C.

da Silva, Paul R. The dissertation author was a secondary investigator and author of this paper. Chapter 3, in full, is a reprint of materials as it appears in “PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin xii Biosynthetic Gene Cluster in an Ocean Streptomycete” in Journal of Natural Products, 2017, Charles Bradford Larson, Max Crusman, Bradley S. The dissertation author was the primary investigator and author of this paper.

xiii Vita 2012-2019 Ph., University of California, San Diego 2006-2010 B., University of Michigan Publications 2019 O'Neill E. Targeted antibiotic discovery through biosynthesis-associated resistance determinants: target directed genome mining. Crit Rev Microbiol. PCR-independent Method of Transformation Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete.

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols. 2016 Wang M, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono CA, Luzzatto-Knaan T, Porto C, Bouslimani A, Melnik AV, Meehan MJ, Liu WT, Crüsemann M, Boudreau PD, Esquenazi E, Sandoval-Calderón M, Kersten RD, Pace LA, Quinn RA, Duncan KR, Hsu CC, Floros DJ, Gavilan RG, Kleigrewe K, Northen T, Dutton RJ, Parrot D, Carlson EE, Aigle B, Michelsen CF, Jelsbak L, Sohlenkamp C, Pevzner P, Edlund A, McLean J, Piel J, Murphy BT, Gerwick L, Liaw CC, Yang YL, Humpf HU, Maansson M, Keyzers RA, Sims AC, Johnson AR, Sidebottom AM, Sedio BE, Klitgaard A, Larson CB, P CAB, Torres-Mendoza D, Gonzalez DJ, Silva DB, Marques LM, Demarque DP, Pociute E, O'Neill EC, Briand E, Helfrich EJN, Granatosky EA, Glukhov E, Ryffel F, Houson H, Mohimani H, Kharbush JJ, Zeng Y, Vorholt JA, Kurita KL, Charusanti P, McPhail KL, Nielsen KF, Vuong L, Elfeki M, Traxler MF, Engene N, Koyama N, Vining OB, Baric R, Silva RR, Mascuch SJ, Tomasi S, Jenkins S, Macherla V, Hoffman T, Agarwal V, Williams PG, Dai J, Neupane R, Gurr J, Rodríguez AMC, Lamsa A, Zhang C, Dorrestein K, Duggan BM, Almaliti J, Allard PM, Phapale P, Nothias LF, Alexandrov T, Litaudon M, Wolfender JL, Kyle JE, Metz TO, Peryea T, Nguyen DT, VanLeer D, Shinn P, Jadhav A, Müller R, Waters KM, Shi W, Liu X, Zhang L, Knight R, Jensen PR, Palsson BO, Pogliano K, Linington RG, Gutiérrez M, Lopes NP, Gerwick WH, Moore BS, Dorrestein xiv PC, Bandeira N. Sharing and Community Curation of Mass Spectrometry Data with Global Natural Products Social Molecular Networking Nature Biotech. Characterization of Recombinant B.

abortus Strain RB51SOD towards Understanding the Uncorrelated Innate and Adaptive Immune Responses Induced by RB51SOD Compared to Its Parent Vaccine Strain RB51 (* co-first authors) Front. VIOLIN: Vaccine Investigation and Online Information Network Nucleic Acids Res. xv Abstract of the Dissertation Blurring the Lines Between Natural and Synthetic: The Biosynthetic Chemistry of Marine Actinomycete Bacteria by Charles Bradford Larson Doctor of Philosophy in Biomedical Sciences University of California San Diego, 2019 Professor Bradley S. Moore, Chair Professor Victor Nizet, Co-Chair Nature is the source of an incredible diversity of complex chemistry.

The wide variety of chemical scaffolds produced as secondary metabolites have been the source xvi of many useful medicinal therapies for the treatment of human disease, in addition to providing useful tools for probing biology. The biosynthetic machinery that produces these molecules is encoded in the genomes of organisms, and recent technological advancements in bioinformatics, DNA sequencing, and gene cloning and capture techniques offer researchers new opportunities for natural products discovery. When combined with modern analytical chemistry techniques, these methods are a powerful engine for compound discovery. The marine environment represents a unique well of biodiversity to investigate with this new methodology, with a unique evolutionary history ancient than terrestrial habitats.

Chapter 2 of this dissertation describes the in-depth analysis of a collection of 146 marine actinomycetes by mass spectrometry and molecular networking to investigate their biosynthetic capacity. These bacteria were cultured in a variety of media, and their extracts were used to generate approximately 1.8 million mass spectra.