Abstract
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.
Highlights
Pyrrolizidines are a group of organic compounds that possess a fused bicyclic five-membered rings with a nitrogen atom at the bridgehead position
With recently discovered pyrrolizidine alkaloids (PAs) metabolites and their corresponding biosynthetic gene clusters (BGCs), we compared the biosynthetic enzymes encoded in these BGCs with key legonmycin biosynthetic enzymes
IR spectra were obtained on a Fourier transform infrared (FTIR) spectrometer (2013, PerkinElmer, UK) equipped with an Attenuated Total Reflection (ATR, PerkinElmer, Buckinghamshire, UK) diamond cell for sample loading was used for infrared spectroscopy experiments. 1D and 2D nuclear magnetic resonance spectroscopy (NMR) spectra were acquired on a Bruker AVANCE IIIHD400MHz (AscendTM14.1 T, UK) with Prodigy TCITM cryoprobe at 298 K in CD3OD and DMSO‐d6 (Goss Scientific, Massachusetts, MA, USA)
Summary
Pyrrolizidines are a group of organic compounds that possess a fused bicyclic five-membered rings with a nitrogen atom at the bridgehead position. While pyrrolizidine alkaloids (PAs) have been mainly found as plant metabolites, some of which are part of plant defence mechanism against insect herbivores, less than 30 PAs have been discovered from bacterial origins [1]. Bacterial PAs exhibit a broad range of bioactivities and as such they have attracted consid erable interest from both academic research groups and the pharma ceutical industry [11]. We disclosed the biosynthetic origin of two bacterial PA specialised metabolites (SMs), denoted as legonmycins A and B [4], isolated from a talented Ghanaian isolate, Streptomyces sp. We have demonstrated that legonmycins are biosynthesized from an NRPS assembly line to generate the key intermediates, lego nindolizidines 8 ([5 + 6] heterobicyclic ring system).
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