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 15

Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues**

“…[14] Hence,there is an eed for strategies that can facilitate the discovery of the hidden majority of cyanobacterial NPs.S everal stable-isotope-labeled precursor incorporation methods have been applied successfully to this end. [15][16][17] As triking feature of cyanobacterial NPs is the common presence of fatty acid (FA) derived moieties. [12,18] These building blocks can be incorporated into secondary metabolites through the action of pathway-specific fatty acyl-AMP ligases (FAALs) that load the fatty acyl units into their cognate acyl carrier proteins (ACPs).…”

Methylotroph Natural Product Identification by Inverse Stable Isotopic Labeling

“…Natural products are an important source of small molecule therapeutics (Newman and Cragg, 2020), agricultural compounds (Cantrell et al, 2012), and other bioactive metabolites. Precursor feeding studies are commonly used to determine the biosynthetic route to a compound of interest, and can also be used to identify previously undetected compounds produced by an organism (Gross et al, 2007;Kinnel et al, 2017;May et al, 2020;Schaefer et al, 2018). However, this approach can be limited by the synthetic or commercial availability of an isotopically enriched precursor.…”

“…the biosynthetic route to a compound of interest, and can also be used to identify previously undetected compounds produced by an organism. [3][4][5][6] However, this approach can be limited by the synthetic or commercial availability of an isotopically-enriched precursor. Alternatively, in an inverse stable isotopic labeling approach, a fully 13 C-labeled organism is fed a 12 C-biosynthetic precursor to identify natural products that incorporate this precursor.…”

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products

“…20,23 Variation in the loading and off-loading mechanisms of aeruginosin synthetases contributes to the observed structural diversity of the natural product family. [18][19][20]22,23 Aeruginosin BGCs also encode an assortment of tailoring enzymes that promote structural diversification, including halogenation, acylation, and glycosylation. 18,19,22,23 Suomilide is a complex tetrapeptide that contains unusual Mgs and Aeap moieties common in aeruginosins.…”

“…[18][19][20]22,23 Aeruginosin BGCs also encode an assortment of tailoring enzymes that promote structural diversification, including halogenation, acylation, and glycosylation. 18,19,22,23 Suomilide is a complex tetrapeptide that contains unusual Mgs and Aeap moieties common in aeruginosins. 29 Although suomilide lacks Choi and instead contains a densely functionalized tricyclic azabicyclononane (Abn) moiety, 29 these structural features suggest that suomilide is a part of the aeruginosin family.…”