Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

Abstract

Bottromycin A2 is a structurally unique ribosomally synthesized and post‐translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug‐resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β‐methylated amino acid residues. The N‐terminus of a precursor peptide (BtmD) is converted into bottromycin A2 by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…A further example of an unusual P450-mediated transformation in RiPP biosynthesis has been identied in bottromycin biosynthesis: here, the P450 BtmJ has been implicated in the oxidative decarboxylation of the thiazoline ring to generate a thiazole moiety. [208][209][210][211] Furthermore, it would appear that this P450 is selective for the unnatural D-isomer of the preceding aspartic acid residue, which is believed be highly prone to epimerisation. 208 Cytochrome P450s TbtJ1 and TbtJ2 from thiomuracin biosynthesis have also been re-purposed to perform novel, synthetic reactions: Bowers and co-workers were able to demonstrate that suitably modied forms of these P450s could perform selective cyclopropanation of dehydroalanine residues within complex linear and cyclic RiPP cores.…”

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols

“…6 In a natural product molecular network, these clusters represent molecular families (MFs) putatively synthesized by gene cluster families (GCFs). 7 Molecular networking is a powerful approach that has advanced several natural product-related research projects involving dereplication and quantification, 8 discovery, 9 biosynthesis, 10 and chemical ecology. 11 It has also been integrated as a central component of the Global Natural Products Social (GNPS) molecular networking platform, where dereplication is performed against a large, community-acquired reference library of spectra.…”

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

“…The genes responsible for bottromycin biosynthesis, including that of the precursor peptide ( botA ) and the necessary tailoring enzymes, are localized within a single biosynthetic gene cluster (Figure ). Bottromycin maturation involves multiple methylation and proteo‐ lysis steps, amidine macrocycle and thiazole heterocycle formation and C‐terminal decarboxylation (Figure ).…”

“…The precursor peptide undergoes a series of post‐translational modifications to become the natural product bottromycin A2. The genes within the gene cluster are coloured according to the modifications their gene products are proposed to catalyse . The order of the modifications have been reported and include proteolysis either side of the core peptide (green), heterocyclisation of the C‐terminal cysteine within the core peptide (purple) oxidation of the resultant thiazoline to thiazole (red), Cβ‐methylation (dark blue), O‐methylation (light blue), and macrocyclisation (dark red).…”

“…The order of the modifications have been reported and include proteolysis either side of the core peptide (green), heterocyclisation of the C‐terminal cysteine within the core peptide (purple) oxidation of the resultant thiazoline to thiazole (red), Cβ‐methylation (dark blue), O‐methylation (light blue), and macrocyclisation (dark red). Epimerisation (pink) is likely spontaneous …”