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.

Institute of Microbiology AS CR v v i Department of Phototrophic Microorganisms ALGATECH Třeboň Czech Republic

Institute of Microbiology AS CR v v i Department of Phototrophic Microorganisms ALGATECH Třeboň Czech Republic; Biology Centre of AS CR v v i Institute of Hydrobiology České Budějovice Czech Republic; University of South Bohemia Faculty of Science Department of Botany České Budějovice Czech Republic

Institute of Microbiology AS CR v v i Department of Phototrophic Microorganisms ALGATECH Třeboň Czech Republic; Biology Centre of AS CR v v i Institute of Hydrobiology České Budějovice Czech Republic; University of South Bohemia Faculty of Science Department of Molecular Biology and Genetics České Budějovice Czech Republic

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