Metagenomics-Based Tryptophan Dimer Natural Product Discovery and Development Pipeline Fang-Yuan Chang
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Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2015 Metagenomics-Based Tryptophan Dimer Natural Product Discovery and Development Pipeline Fang-Yuan Chang Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Chang, Fang-Yuan, "Metagenomics-Based Tryptophan Dimer Natural Product Discovery and Development Pipeline" (2015). Student Theses and Dissertations. Paper 276. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. METAGENOMICS-BASED TRYPTOPHAN DIMER NATURAL PRODUCT DISCOVERY AND DEVELOPMENT PIPELINE A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Fang-Yuan Chang June 2015 © Copyright by Fang-Yuan Chang 2015 METAGENOMICS-BASED TRYPTOPHAN DIMER NATURAL PRODUCT DISCOVERY AND DEVELOPMENT PIPELINE Fang-Yuan Chang, Ph.D. The Rockefeller University 2015 Most microbial natural product discovery programs rely on the growth of bacteria in the laboratory, yet it is now well established that the vast majority of bacteria in the environment have not been cultured, particularly from the diverse soil microbiota. By extracting DNA directly from soil samples to construct large archived environmental DNA (eDNA) libraries, thousands of genomes from both cultured and as yet uncultured bacteria can be simultaneously screened for gene clusters encoding natural products of interest. Several natural products with pharmaceutically relevant biological activity arise from the dimerization of tryptophans, such as staurosporine, rebeccamycin, and violacein. To discover novel tryptophan dimers (TDs), we have designed a metagenomics-based TD natural product discovery and development pipeline that consists of seven steps: 1) soil eDNA extraction; 2) eDNA library construction; 3) homology-based screening; 4) bioinformatics analysis; 5) heterologous expression; 6) characterization of compounds and their biosynthesis; 7) target identification. Using a degenerate primer set that targets the CPA synthase gene, one of the conserved genes of tryptophan dimer biosynthesis, we screened the equivalent of ~1 million (over 1 tera base pairs) bacterial genomes from the eDNA libraries, resulting in the discovery of 14 unprecedented TD gene clusters, almost tripling the number of TD gene clusters that have previously been characterized. Using heterologous expression strategies that involve 1) shuttling of pathways into diverse bacterial hosts, 2) overexpression of positive transcriptional regulator, 3) synthetic refactoring of complete pathways, and 4) co-expression of deficient biosynthetic genes, we successfully expressed nine of the 14 gene clusters. This led to the functional characterization of three novel TD families (i.e. indolotryptoline, carboxy-indolocarbazole, and bisindolylmaleimide), consisting of 15 novel natural products (e.g. BE-54017s, borregomycins, erdasporines) with therapeutically relevant bioactivities (e.g. antitumor, antibacterial). Linking biologically active natural products to their cellular targets remains a challenging and critical process in the development of therapeutic agents and small-molecule probes, especially for cytotoxic agents that might serve as anticancer agents. Using multidrug resistance-suppressed (MDR-sup) fission yeast resistant mutant screening, the molecular target of the indolotryptoline family of TD was identified and validated to be the proteolipid subunits of vacuolar H+-ATPase (V-ATPase) at a putative binding site that is distinct from the previously described V-ATPase inhibitors. Together, we demonstrate the utility of this pipeline in the isolation, characterization, and development of novel natural products from the soil bacterial metagenome. Acknowledgments I would especially like to thank the following people for their support: Jeffrey Craig, Paula Calle, Melinda Ternei, and Shigehiro Kawashima as collaborators for my thesis research; Dr. Sean Brady as my thesis advisor; Dr. Tarun Kapoor and Dr. Howard Hang as my thesis committee members; Dr. Jef Boeke as my thesis external committee member, the rest of the members of the Brady laboratory including Hala Iqbal, Louis Cohen, Alex Milshteyn, Hahk-Soo Kang, Zachary Charlop-Powers, Kipchirchir Bitok, John Biggins, Jeremy Owen, Boojala Reddy, Debjani Chakraborty, Jonette Suiter, Jeffrey Kim, Ryan King, Jacob Banik, Zhiyang Feng, John Bauer, and Michael Clarke-Pearson; my Rockefeller classmates including James Letts, Frej Tulin, Jeff Liesch, Maria Maldonado, Mingzi Zhang, Teresa Davoli, and Jabez Bak; members of the Kapoor laboratory including Sarah Wacker, Lei Tan, Anupam Patgiri, Tommaso Cupido, Corynn Kasap, and Lynn Bidermann; members of the Nurse laboratory, including Atanas Kaykov and Jun Funabiki; and my family, James, Anna, and Sha-mei Chang. iii Table of Contents Table of Contents ............................................................................................................. iv List of Figures .................................................................................................................. vii List of Tables .................................................................................................................... xi List of Abbreviations ...................................................................................................... xii Chapter 1: Introduction and background ...................................................................... 1 1.1 Bacterial natural products and their applications ....................................... 1 1.2 Limitations of traditional natural product discovery .................................. 3 1.3 Metagenomics-based natural product discovery ......................................... 4 1.4 Phenotype- versus homology-based screening ............................................. 6 1.5 Tryptophan dimer natural products ........................................................... 10 1.6 Tryptophan dimer discovery and development pipeline ........................... 11 Chapter 2: Screening for tryptophan dimer biosynthetic gene clusters .................... 14 2.1 Divergent biosynthesis of TDs ...................................................................... 14 2.2 Target homology region for TD screening .................................................. 16 2.3 Construction of eDNA libraries ................................................................... 19 2.4 Screening for eDNA-derived TD gene clusters ........................................... 23 2.5 Bioinformatics analysis of TD gene clusters ............................................... 25 Chapter 3: Characterization of novel tryptophan dimer classes ................................ 31 3.1 Heterologous expression strategies .............................................................. 31 3.2 Group E TD class: indolotryptoline – BE-54017 ....................................... 37 3.3 Group E TD class: indolotryptoline – borregomycins ............................... 47 3.4 Group F TD class: carboxy-indolocarbazole .............................................. 59 iv 3.5 Group B TD class: bisindolylmaleimide ..................................................... 66 3.6 Expansion of bacterial TD biosynthetic scheme ........................................ 74 Chapter 4: Investigation of tryptophan dimer’s mode of action ................................ 78 4.1 Resistant mutant screening .......................................................................... 78 4.2 MDR-sup fission yeast as model organism ................................................. 79 4.3 Indolotryptoline as subject for mode of action study ................................ 80 4.4 Mutations conferring indolotryptoline resistance ...................................... 81 4.5 V-ATPase proteolipid subunit as putative target of indolotryptoline ...... 85 4.6 Putative indolotryptoline binding site in V-ATPase .................................. 88 4.7 Summary of MDR-sup S. pombe resistant mutant screening ................... 91 Chapter 5: Discussions ................................................................................................... 93 5.1 Conclusions and future directions ............................................................... 93 5.2 CPA-guided analysis of metagenomic TD biodiversity ............................. 96 5.3 Violacein reporter-based screening of TD gene clusters ........................... 98 5.4 Synthetic biology in heterologous natural product expression ............... 102 5.5 Metagenomic toolbox for natural product analog biosynthesis ............. 107 Chapter 6: Materials and methods .............................................................................. 110 Appendix ........................................................................................................................ 132 Appendix 1: Tryptophan dimer gene cluster annotation .............................. 132 Appendix 2: Compound spectral data summary ........................................... 140 Appendix 3: 1-D NMR spectra ........................................................................ 148 Appendix 4: KinaseProfiler results ................................................................