15N Stable Isotope Labeling and Comparative Metabolomics Facilitates Genome Mining in Cultured Cyanobacteria

Abstract

As genome mining becomes a more widely used approach to identify bacterial natural products, the challenge of matching biosynthetic gene clusters to their cognate secondary metabolites has become more apparent. Bioinformatic platforms such as AntiSMASH have made great progress in predicting chemical structures from genetic information, however the predicted structures are often incomplete. This complicates identifying the predicted compounds by mass spectrometry. Secondary metabolites produced by cyanobacteria represent a unique opportunity for bridging this gap. Cultured cyanobacteria incorporate inorganic nitrogen provided in chemically defined media into all nitrogen-containing secondary metabolites. Thus, stable isotope labeling with ¹⁵N labeled nitrate and subsequent comparative metabolomics can be used to match biosynthetic gene clusters to their cognate compounds in cell extracts. Analysis of the sequenced genome of Nostoc sp. UIC 10630 identified six biosynthetic gene clusters predicted to encode the production of a secondary metabolite with at least one nitrogen atom. Comparative metabolomic analysis of the ¹⁵N labeled and unlabeled cell extracts revealed four nitrogen containing compounds that contained the same number of nitrogen atoms as were predicted in the biosynthetic gene clusters. Two of the four compounds were new secondary metabolites, and their structures were elucidated by NMR, HRESIMS, and MS/MS.

Figure 1.

Diagram of the genome mining and comparative metabolomic approach used to match BGCs to their respective natural products.

Figure 2.

Mass spectra from the comparative metabolomics method showing the shift in mass due to incorporation of the ¹⁵N label. Mass spectra from unlabeled compounds are shown in black, and mass spectra from labeled compounds are shown in red. Predicted structures generated by AntiSMASH’s adenylation domain prediction algorithms are shown in each section.

Figure 3.

(A) Dereplication of aeruginosin 865 based on HRESIMS and MS/MS. (B) Putative biosynthetic gene cluster of aeruginosin 865 (BGC6) identified by AntiSMASH and the comparative metabolomics method. Domain abbreviations: A, Adenylation domain; C, Condensation domain; KR, Ketoreductase domain; E, Epimerase domain; Choi, 2-Carboxy-6-Hydroxyoctahydroindole.

Figure 4.

(A) Biosynthetic gene cluster of new anabaenopeptin UIC827 (BGC5). (B) Structure elucidation of anabaenopeptin UIC827 based on adenylation domain substrate prediction and MS/MS fragmentation analysis. Domain abbreviations: A, Adenylation domain; C, Condensation domain; E, Epimerase domain; nMT, N-methyltransferase domain; TE, Thioesterase domain..

Figure 5.

(A) Biosynthetic gene cluster of nostopyrrolidonamide (BGC7). (B) Key 2D NMR correlations of nostopyrrolidonamide. Black arrows indicate ¹³C HMBC correlations, and red arrows indicate ¹⁵N HMBC correlations. Domain abbreviations: CAL, Calcium binding domain; C, Condensation domain; A, Adenylation domain; nMT, N-methyltransferase domain; KS, Ketosynthase domain; AT, Acyltransferase domain; TE, Thioesterase domain.