Biosynthetic relationships among the secalonic acids. Isolation of emodin, endocrocin and secalonic acids from Pyrenochaeta terrestris and Aspergillus aculeatus.

Cynodontin, emodin, endocrocin and secalonic acids A, E and G have been isolated from five strains of Pyrenochaeta terrestis. Aspergillus aculeatus produces emodin, endocrocin and secalonic acids B, D and F. No cynodontin was detected. Isolation of emodin in small amounts supports previous evidence that it is an intermediate in secalonic acid biosynthesis. Isolation of cynodontin and endocrocin, which are co-produced with secalonic acids in other organisms, suggests that these compounds are formed by a common branching pathway. A natural isolate of P. terrestris contained variant strains which produced different relative amounts of secalonic acids A, E and G. From the combinations of secalonic acids produced in organisms so far examined it is concluded that precursor tetrahydroxanthone units or formed in pairs differing in stereo-chemistry only at C-5 or at the trans-invariant C-6 : C-10a positions. A possible biosynthetic pathway is discussed.

3-Hydroxyterphenyllin, a new metabolite of Aspergillus candidus. Structure elucidation by H and C nuclear magnetic resonance spectroscopy.

A new compound isolated from cultures of Aspergillus candidus Link is shown to be 3-hydroxyterphenyllin. The structure was assigned by comparing the 1H and 13C nmr spectra of the metabolite and its acetate derivative with those of terphenyllin and terphenyllin triacetate.

Use of carbon-13 in biosynthetic studies: origin of the malonyl coenzyme A incorporated into tetracycline by Streptomyces aureofaciens.

The proton noise decoupled 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride prepared from Streptomyces aureofaciens cultures supplemented with [1-13C]acetate and [2-13C]acetate showed enrichment of nine alternating ring carbons. In addition, a small enrichment of the carboxamide carbon by [1-13C]acetate was observed. The labelling patterns clearly demonstrated the polyketide origin of the tetracyclic nucleus. The 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride derived from [1,2-13C]acetate showed all 18 ring carbons as doublets with coupling constants appropriate for the incorporation of nine intact two-carbon precursors, confirming that head-to-tail condensation of C2 units had occurred. Absence of bond scission within the C2 units and a low level of uncoupled 13C in the carboxamide substituent indicated that when the organism is supplemented with acetate, malonyl coenzyme A used for tetracycline biosynthesis is formed by direct carboxylation of acetyl coenzyme A.

Biosynthesis of bikaverin in Fusarium oxysporum. Use of 13C nuclear magnetic resonance with homonuclear 13C decoupling to locate adjacent 13C labels.

Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.

Proof for the biosynthetic conversion of L-[indole-15N]tryptophan to [10-15N]anthramycin using (13C, 15N) labelling in conjunction with 13C-NMR and mass spectral analysis.

Using 13C-NMR and mass spectral analysis we have demonstrated that the N-10 nitrogen of anthramycin is biosynthetically derived from the indole-nitrogen of tryptophan. Our experimental approach was to bring a 15N atom, which is derived from L-[indole-15N]tryptophan, and a 13C atom which is derived from DL-[1-13C]tyrosine, into adjacent positions of anthramycin. From resonance intensities and 13C-15N spin-spin coupling in the 13C-NMR spectrum of didehydroanhydroanthramycin, a derivative of anthramycin, we could then determine the 13C enrichment at C-11 and the proportion of 13C bonded to 15N at N-10. These results when combined with mass spectral analysis and isotopic dilution measurements proved that the indole nitrogen of tryptophan was completely retained at N-10 of anthramycin.

Biosynthesis of hydrocarbons by algae: Decarboxylation of stearic acid to N-heptadecane inAnacystis nidulans determined by(13)C- and(2)H-labeling and(13)C nuclear magnetic resonance.

The distribution of isotopic labels inn-heptadecane enriched from [1,2-(13)C] and [2-(13)C, 2-(2)H3) acetates byAnacystis nidulans has been determined by(13)C nuclear magnetic resonance ((13)C NMR). Labeling with [1,2-(13)C] acetate is consistent with assembly from(13)C-(13)C units derived from an acetate "starter" group and 8 malonate units, as in fatty acid biosynthesis, followed by production of a methyl group through bond cleavage of the terminal(13)C-(13)C unit. A comparison of the hydrocarbon with palmitic acid (the only fatty acid produced in sufficient amount for NMR analysis) enriched from [2-(13)C,2-(2)H3]acetate by the same culture shows that they have retained the same fraction of(2)H at corresponding sites, and have therefore undergone identical biosynthetic and hydrogen-deuterium exchange processes, as would be expected ifn-heptadecane originates from de novo-synthesized stearic acid.

Further studies on the biosynthesis of granaticin.

Experiments with cerulenin-inhibited cultures of S. violaceoruber showed conversion of dihydrogranaticin (II) into granaticin (I), but not vice versa, confirming an earlier conclusion that II is the biosynthetic precursor of I. Feeding of CH3(13)C18O2Na followed by 13C-NMR analysis of the product by the 18O shift method indicated the expected incorporation of 18O at carbons 1, 11 and 13 of I and showed that the oxygen of the pyran ring originates from C-3 and not from C-15. Analysis of I biosynthesized from 13C2H3COONa by 13C[1H, 2H] triple resonance NMR spectroscopy showed the incorporation of one atom of deuterium each at C-2 and C-4. C-16 carried a maximum of 2, not 3, atoms of deuterium. These results are discussed in terms of biosynthetic mechanisms.

Pathway of ammonium assimilation in Streptomyces venezuelae examined by amino acid analyses and 15N nuclear magnetic resonance spectroscopy.

To obtain information on the route(s) of ammonium assimilation in Streptomyces venezuelae, cell suspensions transferred to fresh medium lacking nitrogen were pulsed with [15N2]ammonium sulphate. Cells and extracellular fluids were examined by nuclear magnetic resonance and amino acid analysis to assess changes in amino acid pools and the disposition of [15N]ammonium. Following addition of [15N]ammonium, glutamate--glutamine pools of low cell density replacement cultures expanded rapidly and became progressively labelled with 15N, whereas the alanine pool size increased much more slowly and became labelled with 15N to a much lesser extent. These results are consistent with the assimilation of ammonium via glutamate dehydrogenase or glutamine synthetase--glutamate synthase rather than alanine dehydrogenase. Under anaerobic conditions, S. venezuelae assimilates ammonium into alanine rather than glutamate--glutamine. Alanine dehydrogenase may thus function as a vehicle to regenerate NAD+ to maintain substrate-level phosphorylation during periods of anaerobiosis.

Biosynthesis of chloramphenicol. Studies on the origin of the dichloroacetyl moiety.

Chloramphenicol produced by cultures of Streptomyces species 3022a supplemented with sodium [1,2-13C]acetate was labelled with 13C exclusively in the dichloromethine (2.6 +/- 0.1%) and carbonyl (0.59 +/- 0.05% carbon atoms. Satellite signals from 13C-13C coupling between covalently bonded 13C-enriched carbon atoms were too intense to be attributed to random combination of labelled atoms at the average enrichments measured, but their intensity relative to those of the signals for uncoupled 13C atoms indicated that most of the precursor had been incorporated after 13C-13C bond fission. Since [2,3-13c]succinic acid enriched only the carbonyl carbon atom of chloramphenicol, these results suggest that neither acetate nor a Krebs cycle intermediate is a direct precursor of the dichloroacetyl group. Cultures supplemented with [2-3h]-or [2h2]-dichloroacetic acid incorporated negligible amounts of isotope into the antibiotic; on this evidence, the free acid is not an intermediate in chloramphenicol biosynthesis and the acylation step may precede chlorination.

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine.

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.

Glycerol 1,2-cyclic phosphate in centric diatoms. Observation by 31P NMR in vivo, isolation, and structural determination.

We have isolated and identified glycerol 1,2-cyclic phosphate as the compound responsible for a unique prominent resonance at 19.1 ppm in the 31P NMR spectrum in vivo of four species of centric diatoms, where it is responsible for 15 to 30% of the total signals from organophosphates. This appears to be the first observation of this compound as a major metabolite. It was not detectable in nine other species of algae, including four species of pennate diatoms.

Biosynthesis of gibberellins in Gibberella fujikuroi. Gibberellin A47.

Two minor radioactive products in cultures of G. fujikuroi strain ACC917 supplemented with labeled gibberellin A4 have been identified as the 2alpha-hydroxy derivative, gibberellin A47, and the hydrate, gibberellin A2. In addition, evidence was obtained by combined gas chromatography--mass spectrometry for the formation of 3-O-acetylgibberellin A1,3-O-acetylgibberellin A3, and gibberellin A20 by the culture. The time course of gibberellin synthesis in defined and complex media, as well as changes in the relative amounts of radioactive gibberellins formed when [4C]gibberellin A4 was added to cultures at different ages, suggest that the composition of the gibberellin mixture produced is determined by the balance between synthesis and decay of key enzymes in the branching pathway.

Chemical conversion of anthramycin 11-methyl ether to didehydroanhydroanthramycin and its utilization in studies of the biosynthesis and mechanism of action of anthramycin.

Reaction of anthramycin 11-methyl ether (AME) with trifluoroacetic acid results in formation of (1,11a)-didehydroanhydroanthramycin (DAA). Anthramycin biosynthetically labelled from DL-[3'RS(3'-3H)]; DL-[3'S(3'-3H)] and DL-[3'R(3'-3H)] tyrosine each lose approximately 50% of their tritium during this conversion to DAA confirming the labelling pattern of 3'-tritiated species of tyrosine in AME. As expected negligible losses of tritium occurred from AME biosynthetically labelled fron L-[2- or 6-3H] or L-[3- or 5-3H]tyrosine. DAA did not form a stable adduct with DNA in accord with the postulated mechanism of action of anthramycin.