Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora

Abstract

Although microbial genomes harbor an abundance of biosynthetic gene clusters, there remain substantial technological gaps that impair the direct correlation of newly discovered gene clusters and their corresponding secondary metabolite products. As an example of one approach designed to minimize or bridge such gaps, we employed hierarchical clustering analysis and principal component analysis (hcapca, whose sole input is MS data) to prioritize 109 marine Micromonospora strains and ultimately identify novel strain WMMB482 as a candidate for in-depth "metabologenomics" analysis following its prioritization. Highlighting the power of current MS-based technologies, not only did hcapca enable the discovery of one new, nonribosomal peptide bearing an incredible diversity of unique functional groups, but metabolomics for WMMB482 unveiled 16 additional congeners via the application of Global Natural Product Social molecular networking (GNPS), herein named ecteinamines A-Q (1-17). The ecteinamines possess an unprecedented skeleton housing a host of uncommon functionalities including a menaquinone pathway-derived 2-naphthoate moiety, 4-methyloxazoline, the first example of a naturally occurring Ψ[CH₂NH] "reduced amide", a methylsulfinyl moiety, and a d-cysteinyl residue that appears to derive from a unique noncanonical epimerase domain. Extensive in silico analysis of the ecteinamine (ect) biosynthetic gene cluster and stable isotope-feeding experiments helped illuminate the novel enzymology driving ecteinamine assembly as well the role of cluster collaborations or "duets" in producing such structurally complex agents. Finally, ecteinamines were found to bind nickel, cobalt, zinc, and copper, suggesting a possible biological role as broad-spectrum metallophores.

Figure 1.

Chemical structures of ecteinamines A–T (1–20) described in this paper.

Figure 2.

Processing of a 109-strain library using hcapca revealed strain WMMB482 to be chemically divergent from the remainder of the data set. (A) PCA scores plot of all 109 strains. (B) Different nodes were generated after the application of hcapca. (C) Scores plot of the subcluster “rb” containing 9 strains with WMMB482 highlighted in yellow. (D) Loadings plot of strain WMMB482. The PCA loading plot showed a series of metabolites produced by WMMB482, including one unique metabolite, ecteinamine A (t_R = 7.79 min, m/z 698.241).

Figure 3.

Overlaid MS spectra of the experimental (red traces) and simulated (blue traces) IFS. (A) IFS of ecteinamine A {(C₃₁H₄₄N₃O₁₁S₂)⁺, [M + H]⁺} and its isotopologues ([M + 1 + H]⁺, [M + 2 + H]⁺, and [M + 3 + H]⁺) acquired from the Bruker MRMS instrument. (B) Possible ion fragments of ecteinamine A (ESI-MRMS MS² and ESI-MRMS MS³ of ecteinamine A). (C) Overlaid MS spectra of the experimental (red traces) and simulated (blue traces) IFS of a fragment ion (C₃₀H₄₀N₃O₁₀S₁⁺, fragment A of ecteinamine) and its isotopologue {[M + 1 + H]⁺}. (D) Overlaid MS spectra of the experimental (red traces) and simulated (blue traces) IFS of a fragment ion (C₂₄H₃₀N₃O₆S₁⁺, fragment B of ecteinamine A) and its isotopologue {[M + 1 + H]⁺}.

Figure 4.

Structure determination of ecteinamine A (1). (A) ¹H¹H COSY, key HMBC, ROESY, ¹³C–¹³C COSY, and ¹H–¹⁵N HMBC correlations for ecteinamine A (1). (B) X-ray crystal structure of ecteinamine A (1) illustrating its absolute configuration.

Figure 5.

Metabolomics-based ecteinamine analog discovery. (A) Logic and rationale behind new analog identification based on metabolomics data. Detailed analyses of chemical features obtained from metabolomics enabled the structural elucidation of most ecteinamine family members. (B) Close-up view of molecular networking analysis of ecteinamines in the crude extract of strain WMMB482. In general, two compounds are related if they are connected within the network. A total of 18 ecteinamines were observed, 17 of which were characterized. The determined analogs were grouped and colored in accordance with their structures. (C) Molecular networking analysis elucidated common chemical features. (D) List of structural features (gaps and modules) used for structural elucidation. (E) Detailed MS/MS analysis for structural elucidation of cryptic analogs.

Figure 6.

Overview of methyl group incorporation into 1 and 15 based on isotopic enrichment studies.

Figure 7.

Proposed biosynthetic pathway for ecteinamines. A complete listing of clusters/genes and their putative roles/functions is available in the SI, Section 8. (A) Overview of the ecteinamine biosynthetic gene cluster (ect) within the Micromonospora sp. WMMB482 genome; the ect cluster highlighted is one of more than 20 clusters identified within the WMMB482 genome. Tentative functional annotations for ect genes and their relative organization are indicated by color as shown. Genes colored in white are not proposed to be involved directly in biosynthesis. Black bars represent other unrelated BGCs. (B) Proposed biosynthetic pathway to relevant 2-naphthoic acid moieties. (C) Biosynthesis of the ecteinamine NRPS scaffold. A, adenylation domain; ArCP, aryl carrier protein; Cyc, cyclization domain; T, thioesterase. The MTe domain in module 4 is an MT-like domain postulated to catalyze epimerization of the α-carbon in the cysteinyl thioester. Note that ecteinols A–C (panel C, 21–23) serve as key structural elements for 4–13, 16, and 17 (Figure 1).

Figure 8.

Determination of ecteinamines as broad-spectrum metallophores. (A) Mass spectra of metal-chelating 1 complexes. [Co-1 + H]⁺, m/z_calcd 755.1587; [Cu-1 + H]⁺, m/z_calcd 759.1551; [Ni-1 + H]⁺, m/z_calcd 754.1609; [Zn-1 + H]⁺, m/z_calcd 760.1547. (B) Close-up view of the mass spectrum for Zn-1. {[1-⁶⁴Zn + H]⁺, m/z 760.1554, calcd for C₃₁H₄₂N₃O₁₁S₂⁶⁴Zn, 760.1547} with pseudomolecular ion peaks at m/z 760.2, 762.2, and 764.2 showing 1 bound to zinc isotopes with masses of 64, 66, and 68, respectively. The empirical molecular formula was deduced from exact mass. (C) UV/vis spectra of 1, Ni-1, Co-1, Cu-1, and Zn-1 complexes. In addition to the absorption at λ = 366 nm for 1, Ni-1, Co-1, Cu-1, and Zn-1 complexes showed maximum absorptions at 400, 381, 386, and 390 nm, respectively.