6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using [formula omitted] labels detected by nuclear magnetic resonance and [formula omitted] tracers

Abstract

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2- 14C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1- 14C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [ 14CH 3 ]methionine was incorporated only into the OCH 3 groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2- 13C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH 3 groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2- 13C 2H 3]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.