Genomic scanning enabling discovery of a new antibacterial bicyclic carbamate-containing alkaloid.

Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications. Bacterial pyrrolizidine alkaloids (PAs), containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead, have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates, followed by multistep oxidation, catalysed by single Bayer-Villiger (BV) enzymes, to yield PA scaffolds. Although bacterial PAs are rare in natural product inventory, bioinformatics analysis suggested that the biosynthetic gene clusters (BGCs) that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes. However, most of the strains containing PA-like BGCs are not deposited in the public domain, therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites. Here, we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information. Among the strains tested, we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation. Subsequently one-strain-many-compound (OSMAC) method, supported by a combination of HR-MS, NMR, SMART 2.0 technology, and GNPS analysis, allowed identification and characterization of a new [5 + 7] heterobicyclic carbamate, legoncarbamate, together with five known PAs, bohemamine derivatives, from Streptomyces sp. CT37, a Ghanaian soil isolate. The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra. Legoncarbamate displays antibacterial activity against E. coli ATCC 25922 with an MIC value of 3.1 µg/mL. Finally, a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.

Targeted Isolation of Indole Alkaloids from Streptomyces sp. CT37.

Four compounds (1-4) were isolated from the extracts of Streptomyces sp. CT37 using bioassay in conjunction with mass spectrometric molecular networking (MN) driven isolation. Their complete structures were established by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR) data. Legonimide 1 was identified as a new alkaloid containing a rare linear imide motif in its structure, while compounds 2-4 were already known and their structures were elucidated as 1H-indole-3-carbaldehyde, actinopolymorphol B, (2R,3R)-1-phenylbutane-2,3-diol, respectively. The biosynthetic pathways of 1-4 were proposed based on the reported biogenesis of indole alkaloids in literature. Bioactivity tests for 1 and 2 revealed moderate growth inhibition activity against Candida albicans ATCC 10231 with MIC95 values of 21.54 µg/mL and 11.47 µg/mL, respectively.