A Maldiisotopic Approach to Discover Natural Products: Cryptomaldamide, a Hybrid Tripeptide from the Marine Cyanobacterium Moorea producens

Abstract

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen 15N-nitrate to marine cyanobacterial cultures followed by single filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster which putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

Heterologous expression of cryptomaldamide in a cyanobacterial host

“…Moreover, the MS/MS fragmentation pattern matched the previously reported fragmentation pattern of cryptomaldamide ( Figure 4B). 16 These data provided preliminary confirmation of heterologous production of cryptomaldamide in Anabaena. (Table S2).…”

“…(Table S2). 16 Additionally, two singlets at δH 8.03 -8.09 and a singlet at δH 7.78 were indicative of the presence of a monosubstituted guanidine group. Taken together, these data confirmed that cryptomaldamide was being produced in the heterologous host Anabaena.…”

“…Many advances have been made for the screening, detection, and identification of cyanobacterial NPs 16,19,20 and their BGCs. 4,5,21 However, none of these filamentous marine cyanobacteria have been genetically tractable to date.…”

Molecular networking as a dereplication strategy for monitoring metabolites of natural product treated cancer cells

“…Conventionally, spectra of a natural product obtained by liquid chromatography–mass spectrometry, nuclear magnetic resonance and infrared spectroscopy are interpreted based on prior knowledge, or at least “postulation” to the chemical structures of the analyte compounds . Recently, a dereplication strategy utilizing Global Natural Product Social (GNPS) molecular networking has been proposed to leverage the structural features of known compounds to decrypt the tandem mass spectra of unknown compounds . Specifically, cosine similarities among each pair of tandem mass spectra of individual specialized metabolites are calculated, of which the compounds with similar chemical structures are prone to cluster together .…”

“…13 Recently, a dereplication strategy utilizing Global Natural Product Social (GNPS) molecular networking has been proposed to leverage the structural features of known compounds to decrypt the tandem mass spectra of unknown compounds. [14][15][16][17] Specifically, cosine similarities among each pair of tandem mass spectra of individual specialized metabolites are calculated, of which the compounds with similar chemical structures are prone to cluster together. 17,18 This method provides an ability to rapidly categorize hundreds or thousands of specialized metabolomes collected from various organisms based on their chemical structures.…”