A Diels-Alder probe for discovery of natural products containing furan moieties.

Natural products (NPs) are fantastic sources of inspiration for novel pharmaceuticals, oftentimes showing unique bioactivity against interesting targets. Specifically, NPs containing furan moieties show activity against a variety of diseases including fungal infections, and cancers. However, it is challenging to discover and isolate these small molecules from cell supernatant. The work described herein showcases the development of a molecular probe that can covalently modify furan moieties via a [4 + 2] Diels-Alder cycloaddition, making them easily identifiable on liquid chromatography-mass spectrometry (LC-MS). The molecular probe, which undergoes this reaction with a variety of furans, was designed with both a UV-tag and a mass tag to enable easy identification. The probe has been tested with a variety of purified furans, including natural products, methylenomycin furan (MMF) hormones, and MMF derivatives. Moreover, the molecular probe has been tested in crude supernatants of various Streptomyces strains and enables identification of MMFs.

Signalling and Bioactive Metabolites from Streptomyces sp. RK44.

Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 µM) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08µM) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7-12 and six siderophores 13-18.

Xanthocidin Derivatives from the Endophytic Streptomyces sp. AcE210 Provide Insight into Xanthocidin Biosynthesis.

Xanthocidin and six new derivatives were isolated from the endophytic Streptomyces sp. AcE210. Their planar structures were elucidated by 1D and 2D NMR spectroscopy as well as by HRMS. The absolute configuration of one compound was determined by using vibrational circular dichroism spectroscopy (VCD). The structural similarities of xanthocidin and some of the isolated xanthocidin congeners to the methylenomycins A, B, and C suggested that the biosynthesis of these compounds might follow a similar route. Feeding studies with isotopically labelled [13 C5 ]-l-valine showed that instead of utilizing acetyl-CoA as starter unit, which has been proposed for the methylenomycin biosynthesis, Streptomyces sp. AcE210 employs an isobutyryl-CoA starter unit, resulting in a branched side chain in xanthocidin. Further evidence for a comparable biosynthesis was given by the analysis of the genome sequence of Streptomyces sp. AcE210 that revealed a cluster of homologues to the mmy genes involved in methylenomycin biosynthesis.

Modeling the architecture of the regulatory system controlling methylenomycin production in Streptomyces coelicolor.

BACKGROUND: The antibiotic methylenomycin A is produced naturally by Streptomyces coelicolor A3(2), a model organism for streptomycetes. This compound is of particular interest to synthetic biologists because all of the associated biosynthetic, regulatory and resistance genes are located on a single cluster on the SCP1 plasmid, making the entire module easily transferable between different bacterial strains. Understanding further the regulation and biosynthesis of the methylenomycin producing gene cluster could assist in the identification of motifs that can be exploited in synthetic regulatory systems for the rational engineering of novel natural products and antibiotics. RESULTS: We identify and validate a plausible architecture for the regulatory system controlling methylenomycin production in S. coelicolor using mathematical modeling approaches. Model selection via an approximate Bayesian computation (ABC) approach identifies three candidate model architectures that are most likely to produce the available experimental data, from a set of 48 possible candidates. Subsequent global optimization of the parameters of these model architectures identifies a single model that most accurately reproduces the dynamical response of the system, as captured by time series data on methylenomycin production. Further analyses of variants of this model architecture that capture the effects of gene knockouts also reproduce qualitative experimental results observed in mutant S. coelicolor strains. CONCLUSIONS: The mechanistic mathematical model developed in this study recapitulates current biological knowledge of the regulation and biosynthesis of the methylenomycin producing gene cluster, and can be used in future studies to make testable predictions and formulate experiments to further improve our understanding of this complex regulatory system.

Correlation between carbon flux through the pentose phosphate pathway and production of the antibiotic methylenomycin in Streptomyces coelicolor A3(2).

Radiorespirometry was employed to study carbon metabolism during the growth of Streptomyces coelicolor A3(2) in a minimal medium which permitted the production of methylenomycin as the sole detectable secondary metabolite. A switch in the pattern of carbon metabolism from the Embden-Myerhof-Parnas pathway to the pentose phosphate pathway occurred during the period of slower growth in batch culture which immediately preceded entry into the stationary phase. This coincided with the period of methylenomycin production. It is proposed that the biosynthesis of methylenomycin is supported by the generation of NADPH during the latter part of growth.