Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase.

BACKGROUND: Modular polyketide synthases catalyse the biosynthesis of medically useful natural products by stepwise chain assembly, with each module of enzyme activities catalysing a separate cycle of polyketide chain extension. Domain swapping between polyketide synthases leads to hybrid multienzymes that yield novel polyketides in a more or less predictable way. No experiments have so far been reported which attempt to enlarge a polyketide synthase by interpolating additional modules. RESULTS: We describe here the construction of tetraketide synthases in which an entire extension module from the rapamycin-producing polyketide synthase is covalently spliced between the first two extension modules of the erythromycin-producing polyketide synthase (DEBS). The extended polyketide synthases thus formed are found to catalyse the synthesis of specific tetraketide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzymes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaketide macrolactones. In each case the predicted products are accompanied by significant amounts of unextended products, corresponding to those of the unaltered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. CONCLUSIONS: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.

Circumdatin G, a new alkaloid from the fungus Aspergillus ochraceus.

The crude extract of the broth of Aspergillus ochraceus was found to inhibit the final stage of polyprotein processing during hepatitis C virus replication. Bioassay-guided fractionation led to the isolation of the known compound mellein as the active component of the extract. Also isolated were circumdatin F and a new alkaloid, circumdatin G. The structure of circumdatin G was determined by spectroscopic analysis.

The production of novel sordarin analogues by biotransformation.

The biotransformation of the fungal protein synthesis inhibitor sordarin is reported. Nine taxonomically diverse organisms supported the isolation and identification of twelve modified products. The structural diversity of the biotransformation products observed and their value in supporting further chemistry is discussed.

A rapid and facile method for the dereplication of purified natural products.

A new approach to the use of commercial databases for the dereplication of purified natural products has been developed. This is based on searching a text file that links each structure with its molecular weight and an exact count of the number of methyl, methylene, and methine groups it contains. Analysis of such a text file, constructed from a database containing more than 126,000 natural product structures, revealed that these data, readily measured using MS and NMR spectroscopy, are highly discriminating. The identification of an alkaloid and a sesquiterpene using this new approach is described.

Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay.

We describe the isolation and characterisation of novel non-benzoquinone ansamycin metabolites related to geldanamycin from a culture of Streptomyces sp. S6699. The compounds possess potent inhibitory activity in a cell-based assay measuring inhibition of oncostatin M signalling in a reporter cell line utilising a secreted placental alkaline phosphatase (sPAP) readout. In this paper we report the isolation and structure elucidation of the compounds and describe some of their biological properties.

Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity.

We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase.

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

Characterization of metabolites in intact Streptomyces citricolor culture supernatants using high-resolution nuclear magnetic resonance and directly coupled high-pressure liquid chromatography-nuclear magnetic resonance spectroscopy.

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional 1H NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures of Streptomyces citricolor. By applying 1H NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC-NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the 1H NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as 1H-1H correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on 1H-1H coupling networks and 13C chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment.

Isolation and characterisation of a prenylated p-terphenyl metabolite of Aspergillus candidus possessing potent and selective cytotoxic activity; studies on mechanism of action.

We describe the discovery and properties of a prenylated p-terphenyl metabolite of the fungus Aspergillus candidus. The compound (1) possesses potent cytotoxic activity against a range of tumour and other hyper-proliferative cell lines. Cell cycle analysis shows that in mouse keratinocyte (BALB/MK) cells treated with 1, the cell cycle is arrested in early S phase, indicative of an antimetabolite effect. Furthermore, cellular cytotoxicity can be reversed by addition of exogenous pyrimidine but not purine nucleosides to the cell culture medium. It is therefore likely that compound 1 selectively inhibits pyrimidine biosynthesis, and it is this property which accounts for its potent cytotoxic properties.

Isolation of translactone-containing triterpenes with thrombin inhibitory activities from the leaves of Lantana camara.

Methanolic extracts prepared from the leaves of Lantana camara have been found to inhibit human thrombin. An assay, in which thrombin activity is measured as a function of clot formation from fibrinogen, was used to guide the fractionation and purification of five principal active constituents (1-5), which were all characterized as 5,5-trans-fused cyclic lactone-containing euphane triterpenes.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Novel inhibitors of fungal protein synthesis produced by a strain of Graphium putredinis. Isolation, characterisation and biological properties.

The isolation and structure determination of 6 analogues of the fungal protein synthesis inhibitor GR135402, from Graphium putredinis, is described. The relative potencies of the compounds as protein synthesis inhibitors and as in vitro antifungal agents provide interesting insights into the structure-activity relationships in this series.

Novel metabolites of warfarin produced by Beauveria bassiana and Streptomyces rimosus: a novel application of hplc-nmr.

1. Biotransformation of warfarin by the fungus Beauveria bassiana (ATCC 7159) yielded the first reported phase II warfarin metabolite, 3'4'-dihydroxywarfarin-3'-[4-methoxyglucoside], another previously unreported metabolite, 3',4'-dihydroxywarfarin and also 4'-hydroxywarfarin. 2. Biotransformation of warfarin by Streptomyces rimosus (NRRL 2234) yielded the previously unreported metabolites, 12-hydroxywarfarin, 4'-hydroxy-11-methoxywarfarin, and also 7-hydroxywarfarin and 4'-hydroxywarfarin, which have not been previously reported as biotransformation products from this organism. 3. Hplc-nmr has been used to identify biotransformation products of warfarin by S. rimosus directly from the microbial broth without prior isolation and purification.

Microbial biotransformation of the angiotensin II antagonist GR117289 by Streptomyces rimosus to identify a mammalian metabolite.

Screening a range of microorganisms incubated with the angiotensin II antagonist GR117289 resulted in the use of Streptomyces rimosus to generate five related biotransformation products. These comprised three compounds hydroxylated on the aliphatic side chain, one further oxidized to a ketone, and one hydroxylated on the phenyl ring. These microbial metabolites were used as standards to identify a human metabolite detected in plasma and urine, but present in insufficient quantities for full structural characterisation. This further demonstrates how the use of microbial biotransformation systems at an early stage of drug metabolism studies can act as a valuable tool in facilitating identification of minor human metabolites.

Novel squalestatins produced by biotransformation.

Microorganisms were screened for the ability to modify the squalene synthase inhibitor squalestatin 1. Biotransformation of 1 by two actinomycetes, S15106 and S15138, yielded three products hydroxylated on the 4,6-dimethyl-oct-2-enoyl side chain either at the 6 position (5) or 7 position (4 two diastereoisomers), and lacking the acetyl ester from the C-1 side chain. Many strains were found to hydrolyse the 4,6-dimethyl-oct-2-enoyl or acetyl esters to yield squalestatins 2 or 3. The 3-methyl ester (6) of 1 was obtained using Fusarium sp. F13945. This fungus also produced a farnesoic acid derivative, possibly in response to inhibition of its squalene synthase by 1. The biotransformation products of 1 all retained potent squalene synthase inhibitory activity.

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. V. Minor metabolites.

The isolation and structure determination of 3H and 13C NMR and MS of 24 novel squalestatins from cultures of Phoma sp. C2932 is described.

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. III. Biosynthesis.

The biosynthetic origin of the carbon and oxygen atoms of the novel fungal secondary metabolite 1 was studied. Incorporation studies with single and multiple labelled 13C precursors indicated that the major portion of the molecule was derived from two polyketide chains made up of acetate units. One of the chains had benzoic acid (which can be derived from phenylalanine) as a starter unit. The remaining carbons were derived from a four-carbon unit related to succinate and from methionine. Studies with [1-(13)C,18O2]acetate and 18O2 indicated that five of the oxygens, including both of the heterocyclic oxygens, were derived from atmospheric oxygen. The oxygens at the two ester carbonyls were derived from acetate.

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. II. Structure elucidation.

Three novel fungal metabolites 1-3 isolated from cultures of a Phoma sp. C2932, are potent and selective inhibitors of squalene synthase. Their structures have been determined by a combination of spectroscopic, X-ray crystallographic and chemical methods; these natural products incorporate the highly functionalised bicyclic core, [1S-(1 alpha, 3 alpha, 4 beta, 5 alpha, 6 alpha, 7 beta]-4,6,7-trihydroxy- 2,8-dioxabicyclo-[3.2.1]octane-3,4,5-tricarboxylic acid.