Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides

Abstract

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.

Shared PKS Module in Biosynthesis of Synergistic Laxaphycins

“…For example, PKS domain skipping occurs in the synthesis of leinamycin (Tang et al, 2006). Alternative starter modules have been found in the synthesis of anabaenopeptins (Rouhiainen et al, 2010) and puwainaphycins and minutissamides (Mareš et al, 2019). Gene clusters have also been shown to share enzymes in producing non-proteinogenic amino acids as in the case of anabaenopeptin and spumigin (Lima et al, 2017) and aeruginosin and spumigin, which results in the side product pseudoaeruginosin (Liu et al, 2015).…”

Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis

“…Indeed, remarkable studies yielding optimized lipopeptide tails of arylomycins via chemical synthesis have successfully broadened the spectrum of antibiotics . The substrate selectivity of the fatty‐acyl activation step of lipoinitiation has already been thoroughly investigated for its substrate selectivity and has inspired engineering approaches . From a biotechnological perspective, previous studies investigating CDA and surfactin biosynthesis have given us mechanistic insights into the contribution of the C1 domains to lipoinitiation; CDA‐C1 (C1 in CDA NRPS) uses epoxides as donor substrates, whereas srfAA‐C1 (C1 in surfactin NRPS srfAA) uses 3‐hydroxy fatty acids as donor substrates .…”

Novel determination on root-knot nematodes; microspace, mineral profile and transduction of metabolic signals in tomato

“…we find the compound minutissamides (C 55 H 91 N 13 O 16 ) (Table 1), a cyclic polypeptide that 3 0 7 possess cytotoxic activity (Mareš et al, 2019). Four A-D minutissamide of the Anabaena 3 0 8 minutissma cyanobacterium (UTEX 1613) have been isolated (Kang et al, 2011).…”