A sterile glucose-mineral salts broth was fortified with equimolar concentrations (10--3 M) of various organic acids and intermediates in the tricarboxylic acid cycle. Appropriate media were neutralized with 2 N NaOH, inoculated with spore suspensions or mycelial pellets of Penicillium rubrum and incubated quiescently for 14 days or with shaking for 5 days. Rubratoxins were recovered from culture filtrates by ether extraction and resolved by thin-layer chromatography. Toxin formation in quiescent cultures was enhanced by malonate but was not markedly affected by ethyl malonate, shikimate, and acetate or by isocitrate or oxaloacetate added in the presence of malonate. Citrate, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, and malonate when present in the medium alone or in conjunction with malonate caused a 15 to 50% reduction in rubratoxin formation. Acetyl-CoA (10--5 M/flask) caused an 80% increase in toxin yield. Rubratoxin formation in shake cultures was not affected by succinate and malonate. All other combinations of intermediates and malonate caused a 10 to 50% reduction in toxin formation. At 10--3 M, citrate enhanced rubratoxin B formation and stimulated rubratoxin A production by as much as 100%. Above 10--3 M, citrate inhibited toxin production. Incorporation of [2-14C]acetate into rubratoxin was enhanced by malonate, fumarate, and malonate. A combination of pyruvate and malonate produced a 40% increase in [2-14C]acetate incorporation into rubratoxin. The highest reduction of labeled acetate incorporation (36%) was caused by succinate or alpha-ketoglutarate combined with malonate.
Rubratoxin were recovered from cultures of Penicillium rubrum after the mold grew in natural substrates, a semi-synthetic medium, and a glucosemineral salts broth. Substrates that contained rubratoxins were extracted with: diethyl ether, ethyl acetate-benzene, ethanol (100%), ethanol-acetone, acetonitrile, or diethyl ether with refluxing at 45 degrees C. Extracts were screened for rubratoxins by thin-layer chromatography. Some extracts were partially purified with a column of silicic acid using acetone as the eluant. Other extracts were purified (primarily removal of pigments) using columns of silica gel plus Celite and gradient solvent elution. Most rubratoxin B (1.9 mg/g or 0.77 mg/ml) was recovered when corn, rice, or glucose-salts broth were extracted successively with diethyl ether, ethyl acetate-benzene, and diethyl ether or when samples were adjusted to pH 1.5 before refluxing with diethyl ether at 45 degrees C for 1--4h. Most rubratoxin A (0.1--0.15 mg/ml or 1.0 mg/g) was obtained from samples of corn extracted twice each with ethyl alcohol, acetone, and ethyl acetate; from glucose-mineral salts broth extracted with diethyl ether; or from yeast extract sucrose broth extracted with diethyl ether and refluxed for 4 h at 45 degrees C. Large amounts of fairly pure rubratoxin A (up to 400 mg) and rubratoxin B (greater than lg) were obtained with a combination of preparative thin-layer and column chromatography.
A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28 degrees C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1(14)C]acetate, [1,5(14)C]citrate, [2(14)C]malonate, [1(14)C]glucose, [U14C]glucose or [1(14)C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1(14)C]acetate and [2(14)C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.
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