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Investigating Biosynthetic Steps of an Angucycline Antifungal Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2016 Investigating Biosynthetic Steps of an Angucycline Antifungal S. Gabrielle Gladstone Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biological Engineering Commons Recommended Citation Gladstone, S. Gabrielle, "Investigating Biosynthetic Steps of an Angucycline Antifungal" (2016). All Graduate Theses and Dissertations. 4952. https://digitalcommons.usu.edu/etd/4952 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. INVESTIGATING BIOSYNTHETIC STEPS OF AN ANGUCYCLINE ANTIFUNGAL by S. Gabrielle Gladstone A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biological Engineering Approved: Jixun Zhan Ron Sims Major Professor Committee Member Anhong Zhou Mark R. McLellan Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2016 ii Copyright © S. Gabrielle Gladstone 2016 All Rights Reserved iii ABSTRACT Investigating Biosynthetic Steps of an Angucycline Antifungal by S. Gabrielle Gladstone, Master of Science Utah State University, 2016 Major Professor: Jixun Zhan Department: Biological Engineering From the bacterium Streptomyces sp. SCC-2136 (ATCC 55186), two angucycline nat- ural products are produced, designated Sch 47554 and Sch 47555. These compounds are produced through a type II polyketide biosynthetic pathway. The early biosynthetic steps to these molecules were confirmed. These include the minimal polyketide synthase (PKS), the C-9 ketoreductase, the first-ring aromatase, the subsequent ring cyclase, and two oxy- genases. Also confirmed were the biosynthetic genes responsible for production of the first amicetose moiety, as well as the glycosyltransferase that creates a C -glycosidic bond be- tween the angucyclic scaffold and the amicetose moiety. In confirming these pathways, two new natural products were produced: GG31, an amitosylated rabelomycin, and GG53, ra- belomycin hydroxylated at C-12b. Future work will be to understand the late biosynthetic steps and generate new angucyclines through combinatorial biosynthesis. (64 pages) iv PUBLIC ABSTRACT Investigating Biosynthetic Steps of an Angucycline Antifungal S. Gabrielle Gladstone The species of bacterium Streptomyces sp. SCC-2136 which has the American Type Culture Collection index 55186 naturally produces two chemical compounds, labeled Sch 47554 and Sch 47555. These compounds were previously reported to posess antifungal activity. This thesis sets out to confirm the mechanism by which the bacterium produces these compounds; specifically which genes are responsible for producing the enzymes that make and shape the molecules. The genes and enzymes that were characterized are the minimal polyketide synthase, the ketoreductase that specifically acts on the ninth carbon, the first-ring aromatase, the subsequent ring cyclase, and two oxygenases. Also elucidated were the genes responsible for production of the first sugar (called amicetose) in the chain of two sugars attached to carbon 9 as well as the glycosyltransferase enzyme that attaches this sugar to the rest of the molecule. In confirming these pathways, two new compounds were produced: GG31, which has a base structure of the antibiotic rabelomycin but is attached at carbon 9 to a molecule of the deoxysugar amicetose; and GG53 which has the base structure of rabelomycin but with a hydroxyl group at carbon 12b. Future work will be to understand the later biosynthetic steps and generate new related molecules through combinatorial biosynthesis. v Maria Montessori once said, "The preparations for life are indirect". This is certainly true. This thesis is dedicated to my parents, Melissa and Russell Gladstone; to Andrew Tori Hamblin; to Kandy Napan-Hsieh; and to Aerin Richardson. Wasn't this a wild ride. vi ACKNOWLEDGMENTS I must thank Dr. Jixun Zhan for his guidance and patience while I learned about molecular biology and combinatorial biosynthesis. I also wish to thank the members of my Masters committee, Drs. Anhong Zhou and Ron Sims for their attention and help these last two semesters. Paul Veridian, the graduate coordinator in BioEngineering, was invaluable in the management of all the paperwork, deadlines, and requirements. I could not have finished this without you. Additionally, I am eternally grateful for all of the people who were friendly and en- couraging over the last four years. These include my labmates: Dr. Zeng Jia, Dr. Kandy Napan, Dr. Siyuan Wang, Dr. Jiachen Zi, Dr. Lei Shao, Dr. Tong Zhou, Dr. Bingji Ma, Dr. Shuwei Zhang, Dr. Dayu Yu, Dr. Jianfeng Mei, Dr. Anfeng Xiao, Dr. Riming Yan, Dr. Huiwen Yan, Lei Sun, and Fuchao Xu. For their help outside the lab, I wish to thank the Painter family, Kat Combs, Jessica Bills, Steven Collins, Stetson Barnhouse, Kassie and Rick Cressal, Jesse Walker, the Smurf family, the USU library staff, and anyone who gave me a reassuring smile. And most importantly, I thank my parents, my grandparents, and my partner Andrew Hamblin: we made it. S. G. Gladstone Logan, UT 2016 vii CONTENTS Page ABSTRACT :::::::::::::::::::::::::::::::::::::::::::::::::::::: iii PUBLIC ABSTRACT ::::::::::::::::::::::::::::::::::::::::::::::: iv ACKNOWLEDGMENTS :::::::::::::::::::::::::::::::::::::::::::: vi LIST OF TABLES ::::::::::::::::::::::::::::::::::::::::::::::::: viii LIST OF FIGURES :::::::::::::::::::::::::::::::::::::::::::::::: ix 1 Introduction ::::::::::::::::::::::::::::::::::::::::::::::::::: 1 2 Literature Review ::::::::::::::::::::::::::::::::::::::::::::::: 4 2.1 Type I and Type III PKS ............................ 4 2.2 Type II PKS ................................... 6 2.3 Angucycline Compounds ............................. 9 2.3.1 Angular Hydroxyls ............................ 10 2.3.2 Glycosylation ............................... 11 3 Experimental Methods :::::::::::::::::::::::::::::::::::::::::::: 16 3.1 Extraction of Genomic DNA .......................... 16 3.2 Polymerase Chain Reaction and Amplification of Genes . 16 3.3 Creating Plasmids for Heterologous Expression . 17 3.4 Extraction of Compounds ............................ 18 4 Results ::::::::::::::::::::::::::::::::::::::::::::::::::::::: 19 4.1 Rabelomycin Pathway Elucidation ....................... 19 4.1.1 Minimal PKS ............................... 19 4.1.2 Ketoreductase .............................. 21 4.1.3 Aromatases and Cyclases ........................ 22 4.2 Glycosylation ................................... 23 4.3 Angular Hydroxyls ................................ 24 5 Conclusions :::::::::::::::::::::::::::::::::::::::::::::::::::: 31 6 Future Work ::::::::::::::::::::::::::::::::::::::::::::::::::: 34 6.1 Glycodiversification ................................ 34 6.2 Co-expression of Two Plasmids to Assist with Combinatorial Biosynthesis . 35 APPENDICES :::::::::::::::::::::::::::::::::::::::::::::::::::: 43 A Compounds .................................... 44 B Nuclear Magnetic Resonance Spectra for Structure Confirmation of GG31 . 47 viii LIST OF TABLES Table Page 3.1 Sequences of primers used for plasmid construction . 17 3.2 General PCR mixture .............................. 18 4.1 Nuclear magnetic resonace spectroscopy (NMR) data for GG31 . 25 4.2 Plasmid construction ............................... 26 5.1 Gene functions .................................. 32 A.1 Numbered compounds .............................. 45 ix LIST OF FIGURES Figure Page 1.1 Structures of 1, 2, 3, 4 ............................. 2 2.1 The proposed organization of the herboxidiene biosynthetic gene cluster . 5 2.2 Polyketide chains of certain lengths spontaneously form predictable structures 7 2.3 Biosynthesis of oxytetracycline and reported shunt products . 8 2.4 Oxidoreductase UrdM catalyzes a 2-step reaction . 13 2.5 Catalysis of 12 and 12b oxidation ........................ 14 2.6 Examples of glycosylated natural products ................... 14 2.7 Deoxysugars that can be synthesized from common intermediates . 15 2.8 The four urdamycin glycosyltransferases .................... 15 4.1 Putative gene cluster of ATCC 55186 ...................... 20 4.2 The vector used to build all of the expression plasmids . 20 4.3 HPLC traces of expressed biosynthetic products . 27 4.4 Structures of urdamycin A and simocyclinone D8 . 28 4.5 Structure elucidation of GG31 .......................... 28 4.6 Comparison of retention times of 18 to 16 ................... 29 4.7 Proposed pathway to 1 and 2 .......................... 30 6.1 Putative route from glucose-6-phosphate to the three sugars . 34 CHAPTER 1 Introduction There is a joke about the history of medicine that appeared in the now-discontinued Western Journal of Medicine. It is presented as a table: The History of Medicine 2001 BC Here, eat this root. 1000 AD That root is heathen. Here, say this prayer. 1850 AD That prayer is superstition. Here, drink this potion. 1920 AD That potion is snake oil. Here, swallow this pill. 1945 AD That pill is ineffective. Here, take this penicillin. 1955 AD Oops, bug mutated. Here, take this tetracycline. 1960-1999 AD 39 more \oops"...Here, take this more powerful antibiotic. 2000 AD The bugs have won! Here, eat this root. Natural products, a term
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