Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 μM for MIN A and 3.2 μM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 μM) and the plant pathogen Alternaria alternata (0.6 μM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 μM) and A. alternata (MIC = 0.1-0.5 μM), but with partial retention of the cytotoxic effect (∼10-20 μM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 μM), and greatly reduced or abolished cytotoxic activity (>20 μM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.

• Publikační typ
• časopisecké články MeSH

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

• Klíčová slova
• biosynthesis, cyanobacteria, fatty acyl-AMP ligase, lipopeptides, nonribosomal peptide synthetase,
• MeSH
• Anabaena genetika   MeSH
• antifungální látky MeSH
• antiinfekční látky MeSH
• bakteriální geny genetika   MeSH
• bakteriální proteiny genetika   MeSH
• cyklické peptidy biosyntéza   chemie   genetika   MeSH
• lipopeptidy biosyntéza   chemie   genetika   farmakologie   MeSH
• multigenová rodina MeSH
• peptidsynthasy genetika   MeSH
• polyketidsynthasy genetika   MeSH
• sinice genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antifungální látky MeSH
• antiinfekční látky MeSH
• bakteriální proteiny MeSH
• cyklické peptidy MeSH
• lipopeptidy MeSH
• non-ribosomal peptide synthase MeSH Prohlížeč
• peptidsynthasy MeSH
• polyketidsynthasy MeSH

Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.

• MeSH
• Anabaena chemie   klasifikace   růst a vývoj   izolace a purifikace   MeSH
• antifungální látky chemie   izolace a purifikace   farmakologie   MeSH
• Aspergillus flavus účinky léků   růst a vývoj   MeSH
• Candida albicans účinky léků   růst a vývoj   MeSH
• cyklické peptidy chemie   izolace a purifikace   farmakologie   MeSH
• druhová specificita MeSH
• fylogeneze MeSH
• glykolipidy chemie   izolace a purifikace   farmakologie   MeSH
• lipopeptidy chemie   izolace a purifikace   farmakologie   MeSH
• molekulární struktura MeSH
• molekulární typizace MeSH
• Nostoc chemie   klasifikace   růst a vývoj   izolace a purifikace   MeSH
• objevování léků * MeSH
• pyrany chemie   izolace a purifikace   farmakologie   MeSH
• sinice chemie   klasifikace   růst a vývoj   izolace a purifikace   MeSH
• sladká voda mikrobiologie   MeSH
• slané vody MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Brazílie MeSH
• Česká republika MeSH
• Finsko MeSH
• Názvy látek
• antifungální látky MeSH
• cyklické peptidy MeSH
• glykolipidy MeSH
• lipopeptidy MeSH
• pyrany MeSH

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

• MeSH
• anotace sekvence MeSH
• bakteriální geny MeSH
• bakteriální proteiny genetika   fyziologie   MeSH
• biosyntetické dráhy MeSH
• ligasy genetika   fyziologie   MeSH
• lipopeptidy biosyntéza   MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina MeSH
• polyketidsynthasy genetika   fyziologie   MeSH
• sinice enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• ligasy MeSH
• lipopeptidy MeSH
• polyketidsynthasy MeSH
• puwainaphycin F MeSH Prohlížeč
• puwainaphycin G MeSH Prohlížeč