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.

Prenylation is a common step in the biosynthesis of many natural products and plays an important role in increasing their structural diversity and enhancing biological activity. Muscoride A is a linear peptide alkaloid that contain two contiguous oxazoles and unusual prenyl groups that protect the amino-and carboxy-termini. Here we identified the 12.7 kb muscoride (mus) biosynthetic gene clusters from Nostoc spp. PCC 7906 and UHCC 0398. The mus biosynthetic gene clusters encode enzymes for the heterocyclization, oxidation, and prenylation of the MusE precursor protein. The mus biosynthetic gene clusters encode two copies of the cyanobactin prenyltransferase, MusF1 and MusF2. The predicted tetrapeptide substrate of MusF1 and MusF2 was synthesized through a novel tandem cyclization route in only eight steps. Biochemical assays demonstrated that MusF1 acts on the carboxy-terminus while MusF2 acts on the amino-terminus of the tetrapeptide substrate. We show that the MusF2 enzyme catalyzes the reverse or forward prenylation of amino-termini from Nostoc spp. PCC 7906 and UHCC 0398, respectively. This finding expands the regiospecific chemical functionality of cyanobactin prenyltransferases and the chemical diversity of the cyanobactin family of natural products to include bis-prenylated polyoxazole linear peptides.

Aromatic prenylation is an important step in the biosynthesis of many natural products and leads to an astonishing diversity of chemical structures. Cyanobactin pathways frequently encode aromatic prenyltransferases that catalyze the prenylation of these macrocyclic and linear peptides. Here we characterized the anacyclamide (acy) biosynthetic gene cluster from Anabaena sp. UHCC-0232. Partial reconstitution of the anacyclamide pathway, heterologous expression and in vitro biochemical characterization of the enzyme demonstrate that the AcyF enzyme encoded in this biosynthetic gene cluster is a Trp N-prenyltransferase. Bioinformatic analysis suggests the monophyletic origin and rapid diversification of the cyanobactin prenyltransferase enzymes and the multiple origins of N-1 Trp prenylation in prenylated natural products. The AcyF enzyme displayed high flexibility towards a range of Trp-containing substrates and represents an interesting new tool for biocatalytic applications.