Experimental Constraints in the Study of the Biosynthesis of Indole Alkaloids in Fungi

Abstract

The disproportionate difficulty in obtaining compelling experimental evidence from 4C-radiolabelling that the indole moiety of the otherwise isoprenoid penitrem A is biosynthesized by Penicillium crustosum directly from tryptophan has been explored. [benzene ring-' 4C]Tryptophan added to the broth beneath the mycelial mat of stationary liquid cultures labelled penitrem A with 1.4% incorporation, only threefold more than that determined for [methylene-14C]tryptophan or [U-14C]tyrosine, incorporation of which could only have been indirect. In contrast, the substituted tryptophan-histidine diketopiperazine roquefortine, biosynthesized concurrently with penitrems by this organism, was labelled with compelling efficiency (23.4% incorporation of [benzene ring-' 4C]tryptophan). In submerged culture, Claviceps paspali concurrently biosynthesized an analogous pair of metabolites, 3-hydroxy-3-methylbutenyl paspalinine and lysergic acid a-hydroxyethylamide. This feature enabled experimental demonstratation of [benzene ring-' 4C]tryptophan incorporation to an extent more consistent with direct contribution of the indole moiety of the indole-diterpenoid paspalinine derivative. The same precursor applied to the sporing surface of P. crustosum stationary cultures also provided stronger evidence for a direct biosynthetic role in the formation of penitrem A. In the absence of competition from any other indolic secondary metabolite, a submerged culture of Penicilliumpaxilfi incorporated 5 % of the [benzene ring-14C]tryptophan given during growth into the indole-diterpenoid paxilline. A double-labelling time-course experiment indicated temporal separation of steps in the biosynthesis of roquefortine. The inadequacy of classical precursor techniques for studying biosynthesis of indole-diterpenoids in P. crustosum is discussed. The more homogeneous submerged culture fermentation system is preferred for experimentation.

The role of tryptophan as a biosynthetic precursor of indole-diterpenoid fungal metabolites: Continuing a debate

“…That the direct source of the indole is tryptophan (6) was inferred from several biosynthetic studies on fungi producing penitrem A (2) (de Jesus et al, 1883;Mantle, 1985, 1989), paxilline (11) (Laws and Mantle, 1989), and pennigritrem (12) (Penn et al, 1992), during which microgram amounts of tryptophan (6), 14 C-labelled uniformly to high specific activity only in the aromatic ring, were given to fermentations during the early part of the idiophase. This probe was available economically from Amersham International at that time only as residue from a custom synthesis; commissioning of another custom synthesis for extended studies would have been beyond university resources.…”

Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels

“…To these groups can be added the more recently discovered terpendoles (Huang et al 1995;Tomoda et al 1995;Gatenby et al 1999), shearinines (Belofsky et al 1995), sulpinines (Laakso et al 1992), nodulisporic acid (Ondeyka et al 1997) and thiersinines (Li et al 2002). All these compounds have a cyclic diterpene skeleton derived from four isoprene units, and an indole moiety derived from tryptophan or a tryptophan precursor (Acklin et al 1977;de Jesus et al 1983;Laws and Mantle 1989), but very little is known about the pathways for their biosynthesis. Biosynthetic schemes have been proposed on the basis of chemical identification of likely intermediates from the organism of interest and related filamentous fungi (Mantle and Weedon 1994;Munday-Finch et al 1996;Gatenby et al 1999), but until recently none of the proposed steps had been validated by biochemical or genetic studies.…”

Biosynthesis of Sespendole

“…5. For most indoloditerpenes, such as penitrem A [4,5], 3-hydroxy-3-methylbutenyl paspalinine [8] and paxilline [8], tryptophan is used as a precursor for the indole moiety, while indole-3-glycerol phosphate, a precursor of tryptophan biosynthesis, is directly incorporated into nodulisporic acid A [6]. In studies of sespendole, anthranilic acid was found to be incorporated into the molecule but tryptophan was not.…”