The fungus Alternaria tenuis normally produces tenuazonic acid (3-acetyl-5-secbutyltetramic acid). On supplementation of the culture substrate with L-valine and Ileucine, the organism formed two new tetramic acids, 3-acetyl-5-isopropyltetramic acid and 3-acetyl-5-isobutyltetramic acid, respectively. L-Phenylalanine was not utilized by the organism as a tetramic acid precursor.It has been possible in only a very few cases to change qualitatively the production of a microorganism. The best known example is the influence of side-chain precursors on penicillin fermentations. In this paper, we describe the biological formation of two tenuazonic acid analogues obtained by additions of appropriate precursors to growing cultures of Alternaria tenui8. Tenuazonic acid was first isolated by Rosett et al. from A. tenui8 (3). Its structure was established by Stickings (5), who also showed that it is biosynthetically derived from i-isoleucine and acetate (6). As tenuazonic acid and some synthetic congeneric tetramic acids show certain biological activities (1, 4), it is of interest to achieve microbial production of tetramic acids other than tenuazonic acid. The biosynthetic pattern of tenuazonic acid and the stimulation of its production by the addition of I-isoleucine to the fermentation suggested the possibility of obtaining tetramic acids with different side chains at the 5-position by growing the organism in media supplied with various L-amino acids. The production of the 5-isopropyl and the 5-isobutyl derivatives could be demonstrated by using "IC-carboxyl-labeled L-valine and rleucine, respectively, as additives to the culture media. The radioactive tetramic acids were isolated and identified by isotopic dilution with nonlabeled synthetic tetramic acids as carriers. In this way, it was also shown that phenylalanine could not be utilized by the organism as a tetramic acid precursor.Microorganism and culture conditions. A. tenui8 CMI 89343 was used in this study. Stock cultures were maintained on agar slants of malt extract (1.0%), yeast extract (0.4%), and glucose (0.4%), pH 7.3. The fermentation experiments were carried out in 500-ml conical flasks containing 150 ml of modified Czapek-Dox medium of the following composition (g/liter): glucose, 40.0; NaNO3, 1.0; NH4Cl, 0.25; KH2PO4, 1.0; KCl, 0.25; NaCl, 0.25; MgS0O47H20, 0.5; FeSO4*7H20, 0.01; ZnSO4.7H20, 0.01; and yeast extract, 1.0; pH 5.5. The flasks were incubated at 28 C on a rotary shaker operating at 240 rev/min with a 2.5-cm stroke. Amounts of 50 MCi (0.022 mmole) of aqueous solutions of 14C-carboxylabeled i-amino acids (valine, leucine, and phenylalanine) were added to the cultures after 48 hr of incubation. After 24-hr exposure to the labeled compound, the mycelium was filtered off and the tetramic acid was isolated from the filtrate.Isolation and identification of L-valinederived tetramic acid. The culture filtrate was acidified with HCl and extracted with ether.
