Browsing by Author "Ebel, R."
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Item Accramycin A, A New Aromatic Polyketide, from the Soil Bacterium, Streptomyces sp. MA37(molecules, 2019-09-12) Kyeremeh, K.; Maglangit, F.; Fang, Q.; Leman, V.; Soldatou, S.; Ebel, R.; Deng, H.Abstract: Drug-like molecules are known to contain many di erent building blocks with great potential as pharmacophores for drug discovery. The continued search for unique sca olds in our laboratory led to the isolation of a novel Ghanaian soil bacterium, Streptomyces sp. MA37. This strain produces many bioactive molecules, most of which belong to carbazoles, pyrrolizidines, and fluorinated metabolites. Further probing of the metabolites of MA37 has led to the discovery of a new naphthacene-type aromatic natural product, which wehave named accramycinA1. This molecule was isolated using an HPLC-photodiode array (PDA) guided isolation process and MS/MS molecular networking. The structure of 1 was characterized by detailed analysis of LC-MS, UV, 1D, and 2D NMR data. Preliminary studies on the antibacterial properties of 1 using Group B Streptococcus (GBS) produced a minimum inhibitory concentration (MIC) of 27 g/mL. This represents the first report of such bioactivity amongst the naphthacene-type aromatic polyketides, and also suggests the possibility for the further development of potent molecules against GBS based on the accramycin sca old. A putative acc biosynthetic pathway for accramycin, featuring a tridecaketide-specific type II polyketide synthase, was proposed.Item Biosynthesis of Neocarazostatin A Reveals the Sequential Carbazole Prenylation and Hydroxylation in the Tailoring Steps(Chemistry & Biology, 2015-12) Huang, S.; Elsayed, S.S.; Lv, M.; Tabudravu, J.; Rateb, E.M.; Gyampoh, R.; Kyeremeh, K.; Ebel, R.; Jaspars, M.; Deng, Z.; Yu, YI.; Deng, H.Neocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here, we report the discovery and characterization of the biosynthetic pathway of NZS through genome mining and gene inactivation. The in vitro assays characterized two enzymes: NzsA is a P450 hydroxylase and NzsG is a new phytoene-synthase-like prenyltransferase (PTase). This is the first reported native PTase that specifically acts on the carbazole nucleus. Finally, our in vitro reconstituted experiment demonstrated a coupled reaction catalyzed by NzsG and NzsA tailoring the NZS biosynthesis.Item A Co-Culturing Approach Enables Discovery and Biosynthesis of a Bioactive Indole Alkaloid Metabolite(molecules, 2020-01-08) Kyeremeh, K.; Maglangit, F.; Fang, Q.; Sternberg, J.M.; Ebel, R.; Deng, H.Whole-genome sequence data of the genus Streptomyces have shown a far greater chemical diversity of metabolites than what have been discovered under typical laboratory fermentation conditions. In our previous natural product discovery e orts on Streptomyces sp. MA37, a bacterium isolated from the rhizosphere soil sample in Legon, Ghana, we discovered a handful of specialised metabolites from this talented strain. However, analysis of the draft genome of MA37 suggested that most of the encoded biosynthetic gene clusters (BGCs) remained cryptic or silent, and only a small fraction of BGCs for the production of specialised metabolites were expressed when cultured in our laboratory conditions. In order to induce the expression of the seemingly silent BGCs, we have carried out a co-culture experiment by growing the MA37 strain with the Gram-negative bacterium Pseudomonas sp. in a co-culture chamber that allows co-fermentation of two microorganisms with no direct contact but allows exchange of nutrients, metabolites, and other chemical cues. This co-culture approach led to the upregulation of several metabolites that were not previously observed in the monocultures of each strain. Moreover, the co-culture induced the expression of the cryptic indole alkaloid BGC in MA37 and led to the characterization of the known indolocarbazole alkaloid, BE-13793C 1. Neither bacterium produced compound 1 when cultured alone. The structure of 1 was elucidated by Nuclear Magnetic Resonance (NMR), mass spectrometry analyses and comparison of experimental with literature data. A putative biosynthetic pathway of 1 was proposed. Furthermore, BE-13793C 1 showed strong anti-proliferative activity against HT-29 (ATCC HTB-38) cells but no toxic e ect to normal lung (ATCC CCL-171) cells. To the best of our knowledge, this is the first report for the activity of 1 against HT-29. No significant antimicrobial and anti-trypanosomal activities for 1 were observed. This research provides a solid foundation for the fact that a co-culture approach paves the way for increasing the chemical diversity of strain MA37. Further characterization of other upregulated metabolites in this strain is currently ongoing in our laboratory.Item Discovery of a Single Monooxygenase that Catalyzes Carbamate Formation and Ring Contraction in the Biosynthesis of the Legonmycins.(Angewandte Chemie International Edition, 2015-07) Huang, S.; Tabudravu, J.; Elsayed, S.S.; Travert, J.; Peace, D.; Tong, M.H.; Kyeremeh, K.; Kelly, S.M.; Trembleau, L.; Ebel, R.; Jaspars, M.; Yu, Y.; Deng, H.Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD‐dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so‐called legonmycins. By genome‐driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non‐ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time.Item Legonaridin, a new member of linaridin RiPP from a Ghanaian Streptomyces isolate(Organic and Biomolecular Chemistry, 2015-10) Rateb, M.E.; Zhai, Y.; Ehrner, E.; Rath, C.M.; Wang, X.; Tabudravu, J.; Ebel, R.; Bibb, M.; Kyeremeh, K.; Dorrestein, P.C.; Hong, K.; Jaspars, M.; Deng, H.Linaridins are rare linear ribosomally-synthesized and post-translationally modified peptides (RiPPs) and only two, cypemycin and SGR-1832, in this family have been identified so far. Legonaridin 1 has been discovered as a new member of linaridins through chemical isolation, peptidogenomics, comprehensive 1- and 2-D NMR and advanced Marfey's analyses from the soil bacterium Streptomyces sp. CT34, an isolate collected from Legon, Ghana. Bioinformatics analysis of the gene cluster suggested that the biosynthesis of legonaridin 1 is different from those of cypemycin and SGR-1832. Consistent with bioinformatics and peptidogenomics analyses, 1 has a total of nine post-modifications, 8 dehydrobutyrine residues and a N,N-dimethylated N-terminus with a carboxylic acid at the C-terminus. Legonaridin 1 is structurally different from the two known linaridins comprising a new subfamily. This is the first time that NMR spectroscopy is used to establish the 2-D structure of a linaridin RiPP.Item Signalling and Bioactive Metabolites from Streptomyces sp. RK44(Molecules, 2020-01-22) Kyeremeh, K.; Fang, Q.; Maglangit, F.; Wu, L.; Ebel, R.; Andersen, J.H.; Annang, F.; Pérez-Moreno, G.; Reyes, F.; Deng, H.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 M) 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 = 1.08 M) 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.Item Targeted Dereplication of Microbial Natural Products by High-Resolution MS and Predicted LC Retention Time(Journal of Natural Products, 2017-04) Chervin, J.; Stierhof, M.; Tong, M.H.; Peace, D.; Hansen, K.O.; Urgast, D.S.; Andersen, J.H.; Yu, Y.; Ebel, R.; Kyeremeh, K.; Paget, V.; Cimpan, G.; Wyk, A.V.; Deng, H.; Jaspars, M.; Tabudravu, J.N.A new strategy for the identification of known compounds in Streptomyces extracts that can be applied in the discovery of natural products is presented. The strategy incorporates screening a database of 5555 natural products including 5098 structures from Streptomyces sp., using a high-throughput LCMS data processing algorithm that utilizes HRMS data and predicted LC retention times (tR) as filters for rapid identification of known compounds in the natural product extract. The database, named StrepDB, contains for each compound the structure, molecular formula, molecular mass, and predicted LC retention time. All identified compounds are annotated and color coded for easier visualization. It is an indirect approach to quickly assess masses (which are not annotated) that may potentially lead to the discovery of new or novel structures. In addition, a spectral database named MbcDB was generated using the ACD/Spectrus DB Platform. MbcDB contains 665 natural products, each with structure, experimental HRESIMS, MS/MS, UV, and NMR spectra. StrepDB was used to screen a mutant Streptomyces albus extract, which led to the identification and isolation of two new compounds, legonmaleimides A and B, the structures of which were elucidated with the aid of MbcDB and spectroscopic techniques. The structures were confirmed by computer-assisted structure elucidation (CASE) methods using ACD/Structure Elucidator Suite. The developed methodology suggests a pipeline approach to the dereplication of extracts and discovery of novel natural products.