Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecti cidal alkaloids produced in symbioses of certain Epichloe; (anamorph-Neotyp hodium) species (fungal endophytes) with grasses, particularly of the gener a Lolium and Festuca. Prior to the present study, it was unknown whether lo lines were of plant or fungal origin. Neotyphodium uncinatum, the common en dophyte of meadow fescue (Lolium pratense = Festuca pratensis) produced lol ine, N-acetylnorloline, and N-formylloline when grown in the defined minima l media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in comple x medium cultures. GC-MS and C-13 NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Loline s accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 1 5-30 mM asparagine, ornithine or urea. Kinetics of loline production and fu ngal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationar y phase, as is common for secondary metabolism in other microbes. (C) 2001 Elsevier Science Ltd. All rights reserved.