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

Cyanobacteria have long been recognised as a prolific source of bioactive natural products (NPs). Among these are the furanolides, a structurally diverse class of compounds first discovered in the 1980s and 1990s. Furanolides are characterised by a {gamma}-butyrolactone core bearing aromatic or aliphatic substituents at the - and {beta}-positions and an aromatic substituent at the {gamma}-position. Recent advances in understanding the genetic basis of furanolide biosynthesis has enabled genome mining approaches to discover related cryptic furanolide biosynthetic gene clusters (BGCs). In this work, we identified and cloned a cryptic BGC (15.5 kb) from Nodularia sp. NIES-3585 using the Direct Pathway Cloning (DiPaC) strategy and heterologously expressed it in E. coli BAP1. Through isolation and structural elucidation, we characterized the known compound maculalactone B and discovered two novel analogues: maculalactone N, featuring a 4-hydroxybenzyl substituent at the -position, and furanolide I, bearing an aliphatic group derived from 4-methyl-2-oxopentanoic acid at the {beta}-position. Application of Global Natural Product Social Molecular Networking (GNPS) analysis of high-resolution LCMS data enabled the identification of 20 maculalactone-related molecules. Structural analysis based on manual annotation of MS/MS fragment ions suggests the maculalactones consistently possess aromatic substituents at the - and {gamma}-substituents with various hydroxylation patterns, whilst the {beta}-substituent displays remarkable diversity, accommodating aromatic, aliphatic, or indole moieties. Additionally, several analogues are proposed to exhibit hydroxylation of the furanolide ring. These results demonstrate the substrate promiscuity of the maculalactone biosynthetic enzymes and their capacity to generate considerable structural diversity, while highlighting DiPaC as an effective strategy for accessing cyanobacteria NPs from cryptic BGCs.