Integrated metabolomics approach facilitates discovery of an unpredicted natural product suite from Streptomyces coelicolor M145

Abstract

Natural products exhibit a broad range of biological properties and have been a crucial source of therapeutic agents and novel scaffolds. Although bacterial secondary metabolomes are widely explored, they remain incompletely cataloged by current isolation and characterization strategies. To identify metabolites residing in unexplored chemical space, we have developed an integrated discovery approach that combines bacterial growth perturbation, accurate mass spectrometry, comparative mass spectra data analysis, and fragmentation spectra clustering for the identification of low-abundant, novel compounds from complex biological matrices. In this investigation, we analyzed the secreted metabolome of the extensively studied Actinomycete, Streptomyces coelicolor M145, and discovered a low-abundant suite of 15 trihydroxamate, amphiphilic siderophores. Compounds in this class have primarily been observed in marine microorganisms making their detection in the soil-dwelling S. coelicolor M145 significant. At least 10 of these ferrioxamine-based molecules are not known to be produced by any organism, and none have previously been detected from S. coelicolor M145. In addition, we confirmed the production of ferrioxamine D1, a relatively hydrophilic family member that has not been shown to be biosynthesized by this organism. The identified molecules are part of only a small list of secondary metabolites that have been discovered since sequencing of S. coelicolor M145 revealed that it possessed numerous putative secondary metabolite-producing gene clusters with no known metabolites. Thus, the identified siderophores represent the unexplored metabolic potential of both well-studied and new organisms that could be uncovered with our sensitive and robust approach.

Figure 1

Integration of several tools yields a powerful metabolite discovery platform. Crude secondary metabolite samples are assessed using accurate mass spectrometry. These data undergo comparative analysis and features found to be in both sample types are eliminated (Stage 1). Ranking of the remaining leads is performed by manual database searching and deprioritization of features with putative identifications (Stage 2). Compounds remaining after Stage 2 are the novel natural product leads. Fragmentation data (MS²) are obtained for the perturbed metabolite sample and evaluated using molecular networking algorithms to highlight structurally related compounds (Stage 3). Fragmentation clusters containing lead molecules are manually assessed to determine if they contain known natural products (Stage 4). Leads found to cluster with knowns, suggesting structural similarity, are prioritized for further characterization.

Figure 2

Analysis of S. coelicolor M145 grown under normal and perturbing conditions yielded many features likely to represent novel molecules. a) Sample comparison resulted in detection of molecules present under both growth conditions with altered production levels (m/z = 614.27, fold change at 37 °C = 12.2). b) Identification of features that were unique to the culture grown at 37 ° (m/z = 754.44) also occurred. c) A prioritized lead, m/z = 754.44, clustered to several other compounds. Manual database searching revealed that one member of this network was the known natural product, ferrioxamine B, which was produced by the organism at both temperatures (m/z = 614.27, part a).

Figure 3

Accurate mass fragmentation (MS₂) data obtained from ferrioxamine B and three representative compounds. a) Structure of ferrioxamine B (1) and three amphiphilic ferrioxamine family members, C13 with saturated acyl tail (2), C12 with mono-unsaturated acyl tail (3), and C14 with mono-hydoxylated acyl tail (4). In total, 15 amphiphilic ferrioxamines were identified. b) Fragmentation spectrum of ferrioxamine B is identical to known data and exhibits characteristic losses of m/z = 17, 118 and 200. Additionally, a species of m/z = 454 is observed, which is indicative of the core siderophore structure. c) The characteristic fragmentation pattern was seen in the unknowns suggesting that they possess the ferrioxamine core. Observation of the fragment at 454 indicates that the lipid tails appended to these molecules must be at the labeled position (part a). d and e) Compounds predicted to possess a mono-hydroxylated acyl tail showed a mixed fragmentation pattern containing species from both the ferrioxamine core and additional fragments due to dehydration (−18) and cleavage of a hydroxamate resulting in loss of the lipid tail (m/z = 572).

Figure 4

A suite of low-abundant tri-hydroxamate siderophores was identified from S. coelicolor M145. a) The relatively hydrophilic ferrioxamine analog, D₁, was in our molecular network. This compound has been observed in other organisms, but not noted in S. coelicolor M145. b) A suite of fifteen amphiphilic ferrioxamines was discovered. The acyl chain conformation is likely to be branched; however, a linear lipid chain is possible. The location of the unsaturation and hydroxylation is postulated based upon acyl chains previously seen in siderophores.