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Cyanobacteria produce a variety of chemically diverse cyclic lipopeptides with potent antifungal activities. These cyclic lipopeptides have an amphipathic structure comprised of a polar peptide cycle and hydrophobic fatty acid side chain. Many have antibiotic activity against a range of human and plant fungal pathogens. This review article aims to summarize the present knowledge on the chemical diversity and cellular effects of cyanobacterial cyclic lipopeptides that display antifungal activity. Cyclic antifungal lipopeptides from cyanobacteria commonly fall into four structural classes; hassallidins, puwainaphycins, laxaphycins, and anabaenolysins. Many of these antifungal cyclic lipopeptides act through cholesterol and ergosterol-dependent disruption of membranes. In many cases, the cyclic lipopeptides also exert cytotoxicity in human cells, and a more extensive examination of their biological activity and structure–activity relationship is warranted. The hassallidin, puwainaphycin, laxaphycin, and anabaenolysin structural classes are unified through shared complex biosynthetic pathways that encode a variety of unusual lipoinitiation mechanisms and branched biosynthesis that promote their chemical diversity. However, the biosynthetic origins of some cyanobacterial cyclic lipopeptides and the mechanisms, which drive their structural diversification in general, remain poorly understood. The strong functional convergence of differently organized chemical structures suggests that the production of lipopeptide confers benefits for their producer. Whether these benefits originate from their antifungal activity or some other physiological function remains to be answered in the future. However, it is clear that cyanobacteria encode a wealth of new cyclic lipopeptides with novel biotechnological and therapeutic applications. publishedVersion

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring β-hydroxyaspartate (β-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of β-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe(3+) to Fe(2+) photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread β-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe(2+) for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, β-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Bacterial lipopeptides, which contain β-amino fatty acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of β-amino fatty acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain fatty acids of similar length but differ in the amino acid composition of the peptide cycle. Fragmentation energies of 10-55eV were used to obtain the amino acid composition of the peptide macrocycle. However, fragmentation energies of 90-130eV were used to obtain an intense fragment specific for the β-amino fatty acid (CnH2n+2N(+)). The method allowed the number of carbons and consequently the length of the β-amino fatty acid to be estimated. We identified 21 puwainaphycin variants differing in fatty acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence (R(2)=0.9995) on the length of the fatty acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides.

Cyanobacterial lipopeptides have antimicrobial and antifungal bioactivities with potential for use in pharmaceutical research. However, due to their hemolytic activity and cytotoxic effects on human cells, they may pose a health issue if produced in substantial amounts in the environment. In bacteria, lipopeptides can be synthesized via several well-evidenced mechanisms. In one of them, fatty acyl-AMP ligase (FAAL) initiates biosynthesis by activation of a fatty acyl residue. We have performed a bioinformatic survey of the cyanobacterial genomic information available in the public databases for the presence of FAAL-containing non-ribosomal peptide synthetase/polyketide synthetase (NRPS/PKS) biosynthetic clusters, as a genetic basis for lipopeptide biosynthesis. We have identified 79 FAAL genes associated with various NRPS/PKS clusters in 16% of 376 cyanobacterial genomic assemblies available, suggesting that FAAL is frequently incorporated in NRPS/PKS biosynthetases. FAAL was present either as a stand-alone protein or fused either to NRPS or PKS. Such clusters were more frequent in derived phylogenetic lineages with larger genome sizes, which is consistent with the general pattern of NRPS/PKS pathways distribution. The putative lipopeptide clusters were more frequently found in genomes of cyanobacteria that live attached to surfaces and are capable of forming microbial biofilms. While lipopeptides are known in other bacterial groups to play a role in biofilm formation, motility, and colony expansion, their functions in cyanobacterial biofilms need to be tested experimentally. According to our data, benthic and terrestrial cyanobacteria should be the focus of a search for novel candidates for lipopeptide drug synthesis and the monitoring of toxic lipopeptide production.