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BY XIAOMIN YU DISSERTATION Submitted in Partial Fulfillment of The MOLECULAR CHARACTERIZATION OF PHOSPHONATE BIOSYNTHESIS IN NATURE BY XIAOMIN YU DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor William W. Metcalf, Chair Associate Professor Rachel J. Whitaker Professor John E. Cronan Professor Gary J. Olsen ABSTRACT Phosphonates, compounds characterized by direct C-P bonds, comprise a structurally diverse class of natural products demonstrating an impressive range of biological activities. Although the first biologically produced phosphonate was described more than 50 years ago, the range and diversity of phosphonate production in nature is still not well understood. The biosynthetic pathways of almost all known phosphonates share the same initial step, in which phosphoenolpyruvate (PEP) is isomerized to phosphonopyruvate (PnPy) by PEP mutase (PepM). By using the pepM gene as a molecular marker for phosphonate biosynthetic capacity, I showed that phosphonate biosynthesis is both common and diverse across a wide range of environments, with pepM homologs detected in ~5% of sequenced microbial genomes and 7% of genome equivalents in metagenomic datasets. In addition, PEP mutase sequence conservation was found to be strongly correlated with conservation of other nearby genes, suggesting the diversity of phosphonate biosynthetic pathways could be inferred by examining PEP mutase diversity. By extrapolation, hundreds of unique phosphonate biosynthetic pathways were predicted to exist in nature. As part of a large screening program to uncover new phosphonate-containing natural products, two related phosphonate producers were identified by screening for the pepM gene: Glycomyces sp. NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338. These two actinomycetes produced high amounts of novel phosphonate-containing compounds, which were determined to be 2-hydroxyethylphosphonate (2-HEP) containing polysaccharides (also called phosphonoglycans). The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation and ultrafiltration. Sugar component analyses indicated the presence of various O-methylated galactoses in both phosphonoglycans; the O-methyl groups were shown to derive from S-adenosylmethionine. Partial acid hydrolysis of the purified phosphonoglycans from Glycomyces yielded 2-HEP in ester linkage to the O-5 or O-6 position of a hexose (presumably galactose) and a 2-HEP mono(2,3-dihydroxypropyl) ester. Partial acid ii hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3- dihydroxypropyl) ester. Examination of the genome sequences of the two strains revealed similar pepM-containing gene clusters that are likely to be required for phosphonoglycan synthesis. Like other classes of natural products, most of the phosphonate biosynthetic pathways remain silent under standard laboratory culture conditions. To elicit cryptic phosphonate gene clusters from actinomycetes, two strategies were employed: co-culturing with other microorganisms and selecting for antibiotic-resistant mutants. Although none of the methods successfully turned on phosphonate production, potential avenues for further exploration in terms of inducing the production of unknown phosphonates and increasing the yields of known phosphonates remain possible. To explore the possibility of using the industrial workhorse Corynebacterium glutamicum as a heterologous host to produce phosphonates, a synthetic biology approach was described. A synthetic pathway for the synthesis of 2-HEP was assembled and reconstituted in C. glutamicum, which afforded the production of 2-HEP. However, further modifications are required to improve the stability of the construct and optimize the product yield. iii ToTo my my family iv ACKNOWLEDGEMENTS There are many people without whom I could not have finished this thesis. First and foremost, I would like to give my sincere gratitude to my mentor and thesis advisor, Dr. Bill Metcalf, for his excellent guidance and training. The research presented in this work would not have been possible without his constant support and encouragement throughout my graduate studies. My thanks also go to my thesis committee Dr. John Cronan, Dr. Gary Olsen and Dr. Rachel Whitaker for providing great advice and challenging me to think more critically about my project. I would like to thank Dr. Neil Price, Jun Kai Zhang and Dr. James Doroghazi, who have contributed much of the work presented here. I acknowledge Dr. Feng Lin, Dr. Lingyang Zhu and Dr. Xudong Guan for their assistance with NMR. I would also like to acknowledge Dr. Alvaro Hernandez and Dr. Laura Guest for assistance with DNA sequencing. I am grateful to all members of the Metcalf lab and the Mining Microbial Genome theme of IGB, past and present, for creating a collaborative and enjoyable research environment. I am very fortunate to have them as my colleagues. In particular, I would like to thank Jun Kai Zhang, Dr. Kou-San Ju, Dr. Svetlana Borisova, Dr. James Doroghazi, Dr. Bradley Evans, Dr. Sarath Janga, Joel Cioni, Dr. Jiangta Gao, Courtney Evans, Dr. Benjamin Griffin, Dr. Benjamin Circello, Dr. Juan Velasquez, Dr. Gargi Kulkari, Dr. Annette Erb and Nannan Jiang. I am greatly indebted to their support, encouragement and advice throughout this project. I thank undergraduate students Amla Sampat and Joleen Su for assistance with strain isolation and screening. Thanks are also extended to all the friends who made my past seven years enjoyable outside of the lab, especially Dr. Yuanyuan Liu, Dr. Xuan Zhuang, Dr. Yiran Dong and Dr. Jisen Zhang. Finally, and most importantly, I would like to thank my parents for their endless love, support and understanding. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ....................................................................................................... 1 1.1 The Biological Importance of Phosphorus ................................................................................. 1 1.2 Chemical Properties and Natural Occurrence of Phosphonates ............................................... 1 1.3 Phosphonates as Structural Components of Macromolecules .................................................. 2 1.4 Phosphonates as Antimetabolites .............................................................................................. 6 1.5 Phosphonates as Bioavailable P ............................................................................................... 8 1.6 Biosynthesis of Phosphonates ................................................................................................. 10 1.6.1 Biosynthesis of 2-AEP ...................................................................................................... 10 1.6.2 Biosynthesis of phosphinothricin ....................................................................................... 12 1.6.3 Biosynthesis of fosfomycin ................................................................................................ 16 1.6.4 Biosynthesis of dehydrophos ............................................................................................ 18 1.6.5 Biosynthesis of rhizocticins and plumbemycins ................................................................ 20 1.6.6 Biosynthesis of FR-900098 ............................................................................................... 22 1.6.7 Biosynthesis of methylphosphonate ................................................................................. 23 1.7 Outline of Work Presented in the Thesis ................................................................................. 24 1.8 References ............................................................................................................................... 26 CHAPTER 2: DIVERSITY AND ABUNDANCE OF PHOSPHONATE BIOSYNTHETIC GENES IN NATURE ...................................................................................................................... 37 2.1 Introduction .............................................................................................................................. 37 2.2 Materials and Methods ............................................................................................................. 39 2.3 Results ..................................................................................................................................... 52 2.4 Discussion ................................................................................................................................ 77 2.5 Acknowledgements .................................................................................................................. 77 2.6 References ............................................................................................................................... 78 CHAPTER 3: PURIFICATION AND CHARACTERIZATION OF PHOSPHONOGLYCANS FROM GLYCOMYCES SP. NRRL B-16210 AND STACKEBRANDTIA NASSAUENSIS NRRL B-16338 ............................................................................................................................... 82 3.1 Introduction .............................................................................................................................. 82 3.2 Materials and Methods ............................................................................................................. 82 3.3 Results ....................................................................................................................................
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