Growth and synthesis of rubratoxin by Penicillium rubrum in a chemically defined medium fortified with organic acids and intermediates of the tricarboxylic acid cycle

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 ofPenicillium 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, -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 -ketoglutarate combined with malonate.