Biosynthesis‐Assisted Structural Elucidation of the Bartolosides, Chlorinated Aromatic Glycolipids from Cyanobacteria

Abstract

The isolation of the bartolosides, unprecedented cyanobacterial glycolipids featuring aliphatic chains with chlorine substituents and C-glycosyl moieties, is reported. Their chlorinated dialkylresorcinol (DAR) core presented a major structural-elucidation challenge. To overcome this, we discovered the bartoloside (brt) biosynthetic gene cluster and linked it to the natural products through in vitro characterization of the DAR-forming ketosynthase and aromatase. Bioinformatic analysis also revealed a novel potential halogenase. Knowledge of the bartoloside biosynthesis constrained the DAR core structure by defining key pathway intermediates, ultimately allowing us to determine the full structures of the bartolosides. This work illustrates the power of genomics to enable the use of biosynthetic information for structure elucidation. Keywordsbiosynthesis; cyanobacteria; dialkylresorcinol; glycolipids; structure elucidation As ancient photoautotrophs, cyanobacteria have a differentiated ecophysiology among prokaryotes [1] that is abundantly reflected in their secondary metabolite diversity. [2] We have explored the chemical diversity of an in-house culture collection of cyanobacteria [3] and herein report the discovery of four unusual secondary metabolites and the use of biosynthesis in their structure elucidation.Examination of a fractionated crude organic extract from the filamentous cyanobacterium Nodosilinea sp. LEGE 06102 led to the 1 H NMR spectroscopy guided isolation of bartoloside A (1, 2.0 mg; Figure 1a). Intriguingly, analogous metabolites, namely bartolosides B, C, and D (2, 5.6 mg; 3, 0.7 mg; and 4, 0.3 mg; Figure 1b), were isolated through bioassay-guided fractionation of a crude extract of Synechocystis salina LEGE 06155, a unicellular, free-living cyanobacterium found at the same beach as the bartoloside A producer. [4] These cyanobacterial strains are morphologically and phylogenetically distant (Figure 1; see also the Supporting Information, Figure S1). Together with their geographical co-occurrence, this observation may suggest pathway acquisition by horizontal gene transfer.Initial structural-elucidation efforts focused on the most abundant metabolites (1 and 2). A combination of 1 H, 13 C, HSQC, HMBC and COSY data revealed a dialkylresorcinol (DAR) scaffold in both compounds (Supporting Information, S7-S39). D-Xylose and L-rhamnose were O-linked to the aromatic rings in 1 and 2, respectively (Figure 2a, see also Figure S33). The DAR scaffold in 2 was further decorated with a xylose residue C-linked to the C4 position (Figure 2a). To the best of our knowledge, this is the first report of a cyanobacterial C-glycoside.The two alkyl substituents on the resorcinol ring accounted for 25 and 27 carbon atoms in 1 and 2, respectively. Considering the HR-ESI-MS derived molecular formulae (C 36 H 62 Cl 2 O 6 for 1 and C 44 H 76 Cl 2 O 10 for 2) and NMR-based partial structures, these groups had to be linear. Overlapping 1 H and 13 C resonances for the two chlorinated methine groups and their corre...

Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

“… 23 This recently described dimetal-carboxylate halogenase class has been implicated in the chlorination of fatty acyl-derived moieties of different cyanobacterial natural products, including the columbamides. 21 , 23 − 25 We found two adjacent homologues of cylC ( clyC and clyD ) in a ∼225 kb contig. No additional cylC homologues (or genes homologous to nonheme iron halogenases, which may also act on unactivated carbon centers) 26 , 27 were found in the genome data.…”

The conifer biomarkers dehydroabietic and abietic acids are widespread in Cyanobacteria

“…Cyanobacterial biomass extraction and VLC fractionation, performed as described above, is routinely carried out in our laboratory as part of bioactive secondary metabolite isolation efforts e.g. 55 . We therefore maintain a small library consisting of solvent-free VLC fractions, stored at −80 °C.The 1 H NMR spectra of fractions from our library originating from the strains depicted in Table 1 (with the exception of the unidentified Synechococcales cyanobacterium strain LEGE 10388 and Plectonema cf.…”

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

“…To date, CylC is the only characterized dimetal-carboxylate halogenase (this classification is based on both biochemical evidence and similarity to other diiron-carboxylate proteins) [27]. Homologs of CylC have been found in the BGCs of the columbamides [29], bartolosides [30], microginin [27], puwainaphycins/minutissamides [31], and chlorosphaerolactylates [32], all of which produce halogenated metabolites. CylC-type enzymes bear low sequence homology to dimetal desaturases and N-oxygenases [27], functionalize C-H bonds in aliphatic moieties at either terminal or mid-chain positions, and are likely able to carry out gem-dichlorination (Kleigrewe 2015, Leão 2015.…”

“…Homologs of CylC have been found in the BGCs of the columbamides [29], bartolosides [30], microginin [27], puwainaphycins/minutissamides [31], and chlorosphaerolactylates [32], all of which produce halogenated metabolites. CylC-type enzymes bear low sequence homology to dimetal desaturases and N-oxygenases [27], functionalize C-H bonds in aliphatic moieties at either terminal or mid-chain positions, and are likely able to carry out gem-dichlorination (Kleigrewe 2015, Leão 2015. The reactivity displayed by CylC and its homologs is of interest for biocatalysis, in particular because this type of carbon center activation is often inaccessible to organic synthesis [15,33].…”

“…. The LEGEcc culture collection maintains cultures isolated from diverse freshwater and marine environments, mostly in Portugal, and, for example, contains all known bartoloside-producing strains[30]. Primers were designed based on 54 nucleotide sequences retrieved from the NCBI that were selected to represent the phylogenetic diversity of CylC homologs(Fig.…”