title: The production and further metabolism of cyanide by the snow mould basidiomycete.
Creators: name: Bunch, Alan William
Publisher: name: University of Kent
Year: 2023

description: Growth and cyanide production by the snow mould basidiomycete was observed in plate, still and shake cultures. It was shown that growth in shake cultures was the most suitable for further investigations of growth and cyanide production by this fungus. Measurable growth and cyanide production was obtained in shake cultures with glucose, acetate or succinate as the carbon source. Cyanide production was shown to have many of the usual characteristics of secondary metabolite production. Cyanogenesis was stimulated by including glycine or serine in the growth medium during growth on glucose. Glycine was not used as either a sole carbon or nitrogen source by this fungus. No further stimulation of cyanide production during growth on glucose plus glycine was obtained by supplementing the medium with methionine. However, supplementation of media containing glucose and glycine with N,N-dimethylglycine or betaine caused greater stimulation of cyanide production. Interpretation of this stimulation was complicated by the effects of betaine and N,N-dimethylglycine on growth and cultural appearance. Inorganic phosphate, Fe²⁺ and Zn²⁺, common effectors of secondary metabolism, did not inhibit or stimulate cyanide production by the snow mould basidiomycete at the concentrations tested. During growth of the snow mould in media containing glucose and either [1-⁻¹⁴C] or [2-⁻¹⁴C] glycine, the carbon-1 atom of glycine was converted principally to CO₂ and the carbon-2 atom was largely converted to cyanide. No intermediates of the cyanogenic pathway were isolated, but the possibility that glyoxylic acid or pyruvic acid cyanohydrins are intermediates was shown to be unlikely. No build-up of glycine occurred prior to cyanogenesis by cultures incubated in media containing [U-⁻¹⁴C] glucose. Glycine stimulated cyanide production by cultures of the snow mould basidiomycete grown with acetate, where cyanide production was always associated with growth. Methionine also slightly stimulated cyanide production when added to media containing both acetate and glycine. In contrast to glucose-containing cultures during growth in media containing acetate and either [1-⁻¹⁴C] or [2-⁻¹⁴C] glycine, both carbon atoms of glycine were catabolised to an equal extent to cyanide, but more ¹⁴CO₂ was produced from the [1-⁻¹⁴C] glycine than [2-⁻¹⁴C] glycine. A role for cyanide production by this fungus is proposed. Cyanide was catabolised by the snow mould basidiomycete mainly to CO₂ during growth on either glucose or acetate The maximal rate of CO₂ production from cyanide during growth on glucose occurred at the time when cyanogenesis was maximal. However, CO₂ production from cyanide during growth on acetate was maximal before cyanogenesis reached its maximum rate. The conversion of cyanide to CO₂ by cell-free extracts has also been observed. Incubation of cultures of the snow mould basidiomycete with K¹⁴CN resulted in only low amounts of radioactivity becoming associated with alanine, glutamate, formamide and β-cyanoalanine. The enzymes for alanine biosynthesis from NH₃,HCN and succinic semialdehyde were not shown to be present or detectable throughout growth in glucose as reported by Strobel (1966, 1967). Formamide hydrolyase activity in cell-free extracts was also absent. A pathway for the conversion of cyanide to CO₂ is proposed. The possibility that cyanogenesis by the snow mould basidiomycete could be used as a model system to elucidate the major role of secondary metabolism (which includes the production of antibiotics and other medically or industrially important compounds) is discussed.