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Ether Bridge Formation and Chemical Diversification in Loline Alkaloid Biosynthesis

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Ether Bridge Formation and Chemical Diversification in Loline Alkaloid Biosynthesis University of Kentucky UKnowledge Theses and Dissertations--Plant Pathology Plant Pathology 2014 Ether Bridge Formation and Chemical Diversification in Loline Alkaloid Biosynthesis Juan Pan University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Pan, Juan, "Ether Bridge Formation and Chemical Diversification in Loline Alkaloid Biosynthesis" (2014). Theses and Dissertations--Plant Pathology. 14. https://uknowledge.uky.edu/plantpath_etds/14 This Doctoral Dissertation is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant Pathology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Juan Pan, Student Dr. Christopher L. Schardl, Major Professor Dr. Lisa J. Vaillancourt, Director of Graduate Studies ETHER BRIDGE FORMATION AND CHEMICAL DIVERSIFICATION IN LOLINE ALKALOID BIOSYNTHESIS DISSERTATION A dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By Juan Pan Lexington, Kentucky Director: Dr. Christopher L. Schardl, Professor Plant Pathology Lexington, Kentucky 2014 Copyright © Juan Pan 2014 ABSTRACT OF DISSERTATION ETHER BRIDGE FORMATION AND CHEMICAL DIVERSIFICATION IN LOLINE ALKALOID BIOSYNTHESIS Loline alkaloids, found in many grass-Epichloë symbiota, are toxic or feeding deterrent to invertebrates. The loline alkaloids all share a saturated pyrrolizidine ring with a 1-amine group and an ether bridge linking C2 and C7. The steps in biosynthesis of loline alkaloids are catalyzed by enzymes encoded by a gene cluster, designated LOL, in the Epichloë genome. This dissertation addresses the enzymatic, genetic and evolutionary basis for diversification of these alkaloids, focusing on ether bridge formation and the subsequent modifications of the 1-amine to form different loline alkaloids. Through gene complementation of a natural lolO mutant and comparison of LOL clusters in strains with different loline alkaloid profiles, I found that lolO, predicted to encode a 2-oxoglutarate-dependent nonheme iron (2OG/Fe) dioxygenase, is required in formation of the ether bridge. Through application of isotopically labeled compound to Epichloë uncinata culture, I established that exo-1-acetamidopyrrolizidine (AcAP) and N- acetylnorloline (NANL) are true pathway intermediates. Application of AcAP to yeast expressing lolO resulted in production of NANL, establishing that LolO is sufficient to catalyze this unusual oxygenation reaction. After ether formation, modifications on the 1-amino group give loline, N- methylloline (NML), N-formylloline (NFL) and N-acetylloline (NAL). A double knockout of lolN, predicted to encode an acetamidase, and lolM, predicted to encode a methyltransferase, produced only NANL. Complementation of the double knockout with wild-type lolN and lolM restored the loline alkaloid profile. These results indicate that LolN is involved in deacetylating NANL to produce norloline, which is then modified to form the other lolines. Crude protein extract of a yeast transformant expressing LolM converted norloline to loline and NML, and loline to NML, supporting the hypothesis that LolM functions as a methyltransferase in the loline-alkaloid biosynthesis pathway. The alkaloid NAL was observed in some but not all plants symbiotic with Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline (NAL), indicating that a plant acetyltransferase converts loline to NAL. I further analyzed the basis for loline alkaloid diversity by comparing the LOL clusters in the Epichloë and Atkinsonella species with different loline alkaloid profiles, and found that LOL clusters changed position, orientation and gene content over their evolutionary history. Frequent, independent losses of some or all late pathway genes, lolO, lolN, lolM and lolP, resulted in diverse loline alkaloid profiles. In addition, phylogenetic analyses demonstrated transspecies polymorphism of the LOL clusters. Based on my findings, I established that in Epichloë and Atkinsonella species the ether bridge is formed on acetamidopyrrolizidine. My study of the loline alkaloid profile of Adenocarpus decorticans (Fabaceae) suggests that these plants probably use a similar strategy at least with respect to ether-bridge formation. Further diversification steps of loline alkaloids in grass-Clavicipitaceae symbiota are carried out by enzymes of both Epichloë species and the host plant. Finally, I present evidence that LOL clusters have evolved by balancing selection for chemical diversity. Keywords: loline alkaloids, Epichloë, ether bridge, biosynthesis, chemical diversification _________ Juan Pan ______________ Student’s Signature _________6/25/2014______________ Date ETHER BRIDGE FORMATION AND CHEMICAL DIVERSIFICATION IN LOLINE ALKALOID BIOSYNTHESIS By Juan Pan Dr. Christopher L. Schardl _____________________________ Director of Dissertation Dr. Lisa J. Vaillancourt _____________________________ Director of Graduate Studies 6/25/2014 _____________________________ Date ACKNOWLEDGEMENTS The last four and half years in the University of Kentucky has been both short and long, and there are days of joy and days of frustration, but I am only sure that it has been rewarding. I’d like to take the chance to acknowledge my advisor Dr. Christopher L. Schardl, who has always been patient, supportive, enthusiastic, and inspiring. He showed me a way I would like myself to work with my students if one day I am to take the route of being a faculty; his way of treating a student with such respect as he does with a decent scientist, being patient with even the seemingly silly question or easiest concepts and to lead the discussion to a deeper understanding, and to infect the student simply with his passion for his work. I also greatly appreciate Dr. Robert B. Grossman for his kind advice and help regarding the chemical side of the work I have been involved in, and Minakashi Bhardwaj in his lab for synthesizing the compounds, helping with isolations, explaining to me the chemical concepts, and the moral support as a great friend. I would also like to thank the other committee members, Dr. Mark L. Farman, Dr. Aardra Kachroo, and Dr. Steve Van Lanen for the advice and support. Also I’d like to thank my outside examiner Dr. Robert B. Houtz for his insights and help with my work. I would also like to extend my deep appreciation to Dr. Patrick J. Calie, who kindly allowed me to freely invade his bench when he was busy working in Eastern Kentucky University, and always generously shared his wisdom and kindness as a committed scientist and a great friend. I would also like to express my gratitude to Dr. Carolyn A. iii Young for generously sharing her insights and resources regarding my research, and her encouragements and friendship. I would like to thank past and present CLS lab members and lab manager for their help and support, especially Dr. Padmaja Nagabhyru, who showed me the basics of loline work and has been supportive like an elder sister. I’m thankful to John May at the ERTL of the University of Kentucky for GC-MS analysis and discussion of my work; Dr. Neil Fannin for advice on purification of loline alkaloids with TLC; Dr. Judit Pogany, Jannine Baker, Ching-kai Chuang, and Kai Xu in the laboratory of Dr. Peter Nagy, and Hexiang Luan and Wendy Havens in the laboratory of Dr. Aardra Kachroo for help with protein expression and extraction. I am also grateful for the help I got from undergraduate students, Trevor Kellen, Haley Herndon, and Julien Nolleau. I would also like to thank all the members of the Department of Plant Pathology for their support and encouragement. I would like to share this with my dear friends I’ve made in the past several years: a friend
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