The Maculalactone Biosynthetic Gene Cluster, a Cryptic Furanolide Pathway Revealed in Nodularia sp. NIES-3585

Cyanobacteria have long been known as a prolific source of bioactive natural products (NPs). One such group of NPs are the furanolides, whose first congeners were discovered in the 1980’s and 1990’s. A structural characteristic of furanolides is a γ-butyrolactone moiety with aromatic or aliphatic substituents at the α- and β-positions and an aromatic substituent at its γ-position. Recent elucidation of the genetic basis of furanolide formation enables a genome mining approaches to discover related cryptic furanolide biosynthetic gene clusters (BGCs).

Within this work, a cryptic BGC (15.5 kb) from Nodularia sp. NIES-3585 was cloned using the Direct Pathway Cloning (DiPaC) strategy and heterologously expressed in E. coli BAP1. Isolation and structural elucidation of the major recombinant products led to the discovery of the known furanolide maculalactone B and the new analogue maculalactone N. Both compounds possess potent antibiotic activity against both gram-positive and gram-negative bacteria.

To identify related maculalactone-like structures, Global Natural Product Social Molecular Networking (GNPS) analysis of high-resolution LCMS data was applied. This led to the identification of 20 maculalactone-related molecules. Structural analysis based on manual annotation of MS/MS fragment ions suggest the maculalactones to strictly possess phenolic α- and γ-substituents with various hydroxylation patterns whilst the β-substituent appears highly variable and may contain a phenolic, aliphatic, or indole portion. Additionally, several structural analogues contain a furan-ring hydroxylation.

These results show the flexibility of the maculalactone core-forming enzymes to utilise various substrates resulting in considerable structural diversity and highlight DiPaC as an efficient strategy to discover the encoded products of cyanobacterial cryptic BGCs.