intermediate steps of loline alkaloid biosynthesis

132 Pages·2016·1.7 MB·English

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University of Kentucky Doctoral Dissertations Graduate School
2011
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Jerome Ralph Faulkner
University of Kentucky, [email protected]
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Faulkner, Jerome Ralph, "INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS" (2011). University
of Kentucky Doctoral Dissertations. 209.
https://uknowledge.uky.edu/gradschool_diss/209
This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been
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ABSTRACT OF DISSERTATION

JEROME RALPH FAULKNER

THE GRADUATE SCHOOL
UNIVERSITY OF KENTUCKY
2011
INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS

DISSERTATION

A dissertation in partial fulfillment of the requirements for the degree of Doctor of
Philosophy in the College of Agriculture at the University of Kentucky

By
Jerome Ralph Faulkner

Lexington, Kentucky
Director: Dr. Christopher L. Schardl, Professor Plant Pathology
Lexington, Kentucky
2011

Copyright © Jerome Ralph Faulkner 2011
ABSTRACT OF DISSERTATION

INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS

Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that
inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids
include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1-
aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually
methylated and formylated or acetylated on the 1-amine. In previously published studies
lolines were shown to be derived from the amino acids L-proline and L-homoserine. In
addition the gene cluster involved in loline-alkaloid biosynthesis has also been
characterized. In this dissertation a survey of plant-endophyte symbioses revealed a
phenotype with only N-acetylnorloline. This phenotype provided insights into loline
alkaloid production. This dissertation focuses on determining the steps to loline
biosynthesis after the amino acid precursors. The study involves feeding isotopically
labeled potential precursors to loline-alkaloid-producing cultures of Neotyphodium
uncinatum, as well as RNA interference (RNAi) of N. uncinatum genes for steps in the
pathway. Synthesized deuterated compounds were fed to loline-alkaloid-producing
cultures of N. uncinatum to test their possible roles as precursors or intermediates in the
loline-alkaloid pathway. N-Formylloline was extracted from the cultures and assayed by
GCMS for incorporation of the deuterium label. The results indicated that N-(3-amino,
3-carboxy)propylproline and exo-1-aminopyrrolizidine are intermediates in the loline-
alkaloid biosynthetic pathway. Plasmids were also designed for expression of double-
stranded RNA homologous to loline-alkaloid biosynthesis genes, and introduced by
transformation into N. uncinatum. This RNAi strategy resulted in fungal transformants
altered in loline-alkaloid profiles. The RNAi results indicated that N-acetyl-1-
aminopyrrolizidine is the intermediate before oxygen bridge formation. Based on the
results of this study and the likely roles of the loline-alkaloid biosynthesis genes inferred
from signature sequences of their predicted protein products, I propose a pathway of bond
formation steps in loline-alkaloid biosynthesis.

KEYWORDS: Loline, endophyte, Epichloë, biosynthesis, pyrrolizidine
Jerome Ralph Faulkner
___________________________________
Student’s Signature
August 16, 2011
___________________________________
Date
INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS

By

Jerome Ralph Faulkner

Christopher L. Schardl
_______________________________
Director of Dissertation

Lisa Vaillancourt
_______________________________
Director of Graduate Studies

August 16, 2011
_______________________________
Date
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Name Date
ABSTRACT OF DISSERTATION

JEROME RALPH FAULKNER

THE GRADUATE SCHOOL

UNIVERSITY OF KENTUCKY

2011
ACKNOWLEDGEMENTS

I would first like to thank my Lord and Savior Jesus Christ because without His
grace I would not be the person that was able to do this work. I would like to sincerely
thank my major professor and supervisor Christopher Schardl. Thank you for your
guidance before, during and after this work. I am positive that your helpful suggestions,
assistance and knowledge are the sole reasons that I was able to accomplish this feat.
Your kind leadership, encouraging words, and keen research mind has always impressed
me and I hope that I take some of those traits along with me in my personal and
professional life.
I would like to thank the members of my graduate committee Lisa Vaillancourt,
Pradeep Kachroo and Robert Grossman for their assistance in this process and for helping
to keep me on the right track to reach this final goal. I would also like to thank George
Wagner for being my outside examiner and remaining keeping this commitment.
I would like to express my gratitude and thanks to my family. Thank you Carol,
Cameron and Jordan for being there for me and giving me the support I needed. I would
like to thank my mom and sister for helping me throughout my college years. I am not
good at expressing my feelings but to all of my family thank you for believing and
supporting me.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ................................................................................... iii
LIST OF TABLES ................................................................................................. vi
LIST OF FIGURES .............................................................................................. vii
LIST OF FILES ..................................................................................................... ix

CHAPTER 1: INTRODUCTION
Endophytes of Grasses .................................................................................1
Alkaloids of grass-endophyte symbiosis .....................................................2
Loline alkaloid history .................................................................................3
Loline alkaloid effects..................................................................................6
Loline production in culture.........................................................................7
Genetic analysis of LOL cluster ...................................................................9

CHAPTER 2: LOLINE COMPOSITION IN SELECTED ENDOPHYTES
INTRODUCTION .................................................................................................15
MATERIALS AND METHODS ...........................................................................17
Plant Material .............................................................................................17
Tissue print immunoblot assay ..................................................................18
Loline alkaloid analysis .............................................................................18
Gas chromatography mass spectrometry ...................................................19
Isolation of endophytes ..............................................................................20
Fungal DNA extraction ..............................................................................20
PCR primers and conditions ......................................................................21
PCR product sequencing ............................................................................21
Sequence analysis ......................................................................................22
RESULTS ..............................................................................................................23
Tissue print immunoblot (TPIB) results ....................................................23
Loline alkaloids in plant material ..............................................................23
LOL cluster genes PCR results ..................................................................24
Phylogenetic analysis of lolC.....................................................................25
DISCUSSION ........................................................................................................26

CHAPTER 3: INCORPORATION OF POTENTIAL BIOSYNTHETIC
INTERMEDIATES IN LOLINE ALKALOID-PRODUCING CULTURES
INTRODUCTION .................................................................................................43
MATERIALS AND METHODS ...........................................................................47
Deuterated compounds...............................................................................47
Fungal culture conditions and application of labeled compounds .............47
Loline alkaloid extraction ..........................................................................48
GCMS analysis of lolines ..........................................................................50
RESULTS ..............................................................................................................50
Feeding results ...........................................................................................50
DISCUSSION ........................................................................................................53

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