Biosynthesis of Monensin

Abstract

The biosynthesis of monensin by Streptomyces cinnamonensis was studied by using '4C-labeled glucose, acetate, propionate, butyrate, and methionine. The results indicated that the antibiotic is synthesized from five acetate, seven propionate, and one butyrate molecules. The o-methyl group of monensin is derived from methionine, whereas the terminal hydroxymethyl group is incorporated from acetate.Monensin, an antibiotic produced by Streptomyces cinnamonensis (ATCC 15413), was first described by investigators from Eli Lilly and Co. (4). Initial fermentation studies were presented by Stark (7). The structure of the major component, factor A (Fig. 1), was determined by X-ray crystallographic analysis of the silver salt by Agtarap et al. (2). In addition to factor A, three additional factors have been recognized (3): in factor B the ethyl group on ring C is replaced by a methyl group, in factor C the methyl at the carboxyl end is replaced by an ethyl group, and in factor D the methyl group on the B ring is replaced by an ethyl group. The structures of factors C and D are tentative proposals. Factors B, C, and D are minor constituents of the fermentation broth. Factor A, hereafter designated monensin, and its sodium salt are only slightly soluble in water but are very soluble in organic solvents.This study is concerned with the biosynthesis of monensin and the incorporation of labeled intermediates into the antibiotic. MATERIALS AND METHODSOrganism and cultural conditions. The ingredients of the medium used during these studies with S. cinnamonensis, ATCC 15413, were (in mg/ml): glucose (16.7), L-tyrosine (3.3), L-valine (6.6), L-lysine (1.0), CaCO, (1.0), FeSO4 7H20 (0.17), K2HPO4 (0.17), KCl (0.05), MgSO4 7H20 (0.67), biotin (0.025), and folic acid (0.025). Cultures were grown at 32 C in shaken (250 rpm) flasks (100 ml of medium per 500 ml wide-mouth flask), or in 1. Isolation of monensin. The whole broth was harvested and the pH was adjusted to 9.0 with NaOH, after which it was extracted twice with one-half volume of chloroform. The extracts were combined and washed through a column containing carbon (Pittsburgh 12 by 40 mesh). The column was washed with excess chloroform, and the combined extracts were evaporated. The residue was dissolved in methanol and chilled. Cold deionized water was added until the monensin crystallized. The monensin was collected by filtration, washed with cold water, recrystallized from petroleum ether, and assayed for radioactivity.Degradation of labeled monensin. The periodate oxidation of monensin is illustrated in Fig. 2. A sample of "4C-labeled monensin (100 mg) was dissolved in t-butanol (4 ml) with stirring. To 'this solution was added 0.2 M aqueous sodium meta-periodate solution (2 ml). The final mixture was allowed to stand overnight. The reaction mixture was distilled into a receiver containing a dimedone solution (100 mg of dimedone in 2 ml of 50% ethanol-water). The distillate was allowed to stand a few minutes and then concentrated under reduced pressure to induce crystallizat...

Selection of a specifically blocked mutant of. Streptomyces cinnamonensis: Isolation and synthesis of 26-deoxymonensin A.

“…In order to further characterize the pathway to the monensins A (1) and B (2) in S. cinnamonensis, we have generated mutants blocked in monensin production, and describe here the isolation of one such mutant that accumulates mainly 26-deoxymonensin A (3); the mutant must carry a lesion in the hydroxylation step occuring at the terminal C-26 position. The structure assigned to 3 has been confirmed by direct comparison with a sample of 26-deoxymonensin A prepared chemically from monensin A.…”

Precursor Supply for Polyketide Biosynthesis: The Role of Crotonyl-CoA Reductase

“…Surprisingly, no detectable levels of CCR activity were observed in cell extracts of S. cinnamonensis C730.1 grown in chemically defined medium containing mainly glucose (12 gÂL), tyrosine (3.3 gÂL), and either valine (6.7 gÂL) or leucine (7.5 gÂL) (Day et al, 1973) as carbon and nitrogen sources (Table 1). Overall monensin titers were considerably lower in these media.…”

“…Construction of S. cinnamonensis L1, L1ÂpHL18, and C730.1ÂpHL18 was reported previously (Liu and Reynolds, 1999). The chemically defined medium for monensin production was the same as that described previously (Day et al, 1973) with the exception that different amino acids (valine, leucine, isoleucine, and lysine) were used.…”

Deregulation of acetohydroxy-acid synthase: Loss of allosteric inhibition conferred by mutations in the catalytic subunit

“…Unlike its homologue, monensin A contains one butyrate unit (Day et al 1973), derived from the valine precursor 2-oxoisovalerate in the course of the production on chemically defined media (Pospíšil et al 1983). In some valine-analog resistant strains with higher metabolite flow through valine biosynthetic pathway, a proportion of monensin A was markedly elevated via increased availability of the butyrate unit (Pospíšil et al 1984).…”