Year of Publication
Doctor of Philosophy (PhD)
Dr. Christopher L. Schardl
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.
Faulkner, Jerome Ralph, "INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS" (2011). University of Kentucky Doctoral Dissertations. 209.