A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

Abstract

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.

A combined morphological, ultrastructural, molecular, and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin‐producing clade of cyanobacteria

“…The settings used for HRMS followed the protocol described by Mareš et al. (). Detection of CYN in studied strains was based on the fragmentation spectrum analysis and its comparison to CYN standard (ALX‐350‐149‐C025; Enzo Life Sciences, Farmingdale, NY, USA).…”

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria

“…NRPS enzymes incorporate a range of proteinogenic and nonproteinogenic amino acids which may then be further modified by optional domains for the N-methylation [10][11][12], epimerization [12][13][14][15], or heterocylization of amino acids in the peptide backbone [9,[16][17][18]. Chemical diversity of nonribosomal peptides and polyketides can be further increased through the supply of unusual precursors or enzymatic tailoring reactions [11,14,16,[19][20][21][22][23][24][25][26].…”

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria

“…Lipopeptides are commonly produced by cyanobacteria and present a broad range of bioactivity, including different types of cytotoxicity, growth, and enzyme inhibition of various organisms (fungi, bacteria, plants, protozoa, microalgae, and viruses) (9). A large number of lipopeptides have been described from cyanobacteria, such as barbamide (10), jamaicamide (11), curacin A (12), lyngbyatoxin (13) hassallidin (14), and puwainaphycins (15). Many lipopeptides are synthesized by large multifunctional enzymes known as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) (10)(11)(12)(13).…”

“…PKS modules directly synthesize the lipophilic part of the curacin A (12), whereas the activation of the fatty acid chain incorporated in jamaicamide involves an acyl-acyl carrier protein synthetase (JamA) (10). In the hassallidin gene cluster, produced by Anabaena sp., the involvement of an acyl-protein synthetase and ligase (HasG), an acyl carrier protein (HasH), and a 3-oxoacyl (acyl-carrier-protein) reductase (HasL) are predicted to be involved in the fatty acid synthesis and incorporation in this nonribosomal glycolipopeptide (14), whereas the synthesis of the hybrid puwainaphycins in Cylindrospermum alatosporum involves a fatty-acyl ligase (FAAL) (15).…”