Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues*

Abstract

In recent years, extensive sequencing and annotation of bacterial genomes has revealed an unexpectedly large number of secondary metabolite biosynthetic gene clusters whose products are yet to be discovered. For example, cyanobacterial genomes contain a variety of gene clusters that likely incorporate fatty acid derived moieties, but for most cases we lack the knowledge and tools to effectively predict or detect the encoded natural products. Here, we exploit the apparent absence of a functional β-oxidation pathway in cyanobacteria to achieve efficient stable-isotope-labeling of their fatty acid derived lipidome. We show that supplementation of cyanobacterial cultures with deuterated fatty acids can be used to easily detect natural product signatures in individual strains. The utility of this strategy is demonstrated in two cultured cyanobacteria by uncovering analogues of the multidrug-resistance reverting hapalosin, and novel, cytotoxic, lactylate-nocuolin A hybrids-the nocuolactylates.

Keywords: cyanobacteria; isotopic labeling; natural products discovery; oxadiazines; oxidation.

Figure 1

Cyanobacteria appear to lack a canonical β‐oxidation pathway. a) Cladogram depicting the approximate maximum likelihood tree (computed with FastTree) of 16S rRNA genes from KEGG‐annotated genomes of cyanobacteria, highlighting the presence or absence of each of the β‐oxidation genes. b) LC‐HRESIMS analysis of [M−H]⁻ ions of phospholipids (phosphatidylglycerol groups PG 32:0 and PG 34:0) in extracts from cultures of E. coli BL21 pET24d and E. coli pET24d‐aas7942, and in Synechocystis sp. PCC 6803, each cultured with or without d₁₁‐C₆ supplementation, showing that no appreciable deuterium label is detected in E. coli strains.

Figure 2

Supplementation of cyanobacteria with d ₁₁‐C₆ leads to labeling of different secondary metabolites. LC‐HRESIMS‐based detection of labeled 1 (a), 2 a/2 b (b) and 3 (c) in d ₁₁‐C₆ supplemented cultures of Nodularia sp. LEGE 06071, Synechocystis salina LEGE 06099 and Fischerella sp. PCC 9431, respectively. Values next to chromatographic peaks of compounds 1, 2 a/2 b and 3 in EICs from d ₁₁‐C₆ supplemented cultures correspond to peak heights (ion counts).

Figure 3

Discovery of hapalosin analogues following supplementation of Fischerella sp. PCC 9431 with d ₁₁‐C₆. a) LC‐HRESIMS features corresponding to incorporation of a d ₁₁ label were detected in extracts of Fischerella sp. PCC 9431 supplemented with d₁₁‐C₆. b) Optimized separation conditions in LC‐HRESIMS analysis reveals eight metabolites associated with the detected features, including compounds 4–9; EIC peaks marked with an asterisk (*) correspond to minor compounds with substantial overlap with isobaric metabolites and for which structure elucidation was not pursued. c) Structure elucidation of hapalosins 5–9 (see Text S1): LC‐HRESIMS/MS spectra ([M+H]⁺ ions) are shown for the new hapalosins and 3, as a reference. Peak letters correspond to the double fragmentations indicated as “key fragments”; m/z values in red indicate diagnostic fragmentations supporting the structural proposals. d) Proposed labeling pattern for compounds 3, 6, 7, 8 and 9, deduced from supplementation studies with the indicated stable‐isotope‐labeled substrates.

Figure 4

Discovery of nocuolactylates A–C (10–12). a) Detection of 10–12 following supplementation of Nodularia sp. LEGE 06071 with d ₁₁‐C₆, extraction and metabolome analysis, as described. Shown are EICs and the respective MS spectra, illustrating the difference between non‐supplemented and supplemented extracts that enabled detection. b) Structure of 10–12. c) NMR‐derived substructures of 10 with key correlations and ¹³C chemical shifts. d) HRESIMS/MS analysis of 10, establishing the connectivity between the NMR‐derived substructures (asterisks denote major fragments present in all nocuolactylates which result from fragmentation of the 1‐derived portion of the nocuolactylates and likely involve subsequent oxadiazine ring opening, rearrangement and concomitant N₂ loss, as previously proposed for 1. ^[40]

Figure 5

Putative biosynthesis of the nocuolactylates. a) Structure of the chlorosphaerolactylates, cyanobacterial secondary metabolites that are structurally related to the nocuolactylates. b) Architecture of the noc loci in the genomes of the cyanobacterial Nodularia sp. LEGE 06071, Nostoc sp. CCAP 1453/38 and Sphaerospermopsis sp. LEGE 00249, from where nocuolactylates, nocuolin A and chlorosphaerolacylates, respectively, were first reported.