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

Cyanobacteria have long been recognized 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. Furanolides are characterized by a γ-butyrolactone core bearing aromatic or aliphatic substituents at the α- and β-positions and an aromatic substituent at the γ-position. Recent advances in understanding the genetic basis of furanolide biosynthesis have 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 compounds maculalactone B and deoxyenhygrolide A and discovered the novel analogue maculalactone N, featuring a 4-hydroxyphenyl substituent at the β-position. Application of Global Natural Product Social Molecular Networking (GNPS) analysis of high-resolution LCMS data enabled the identification of 25 maculalactone-related molecules. Further, a MS/MS fragmentation rationale for furanolides was developed and used to probe for maculalactone-like molecules that were too low in abundance for isolation. The fragmentation analysis suggests the β-substituent displays remarkable diversity, accommodating phenolic, aliphatic, or indole moieties. Additionally, structural diversity occurs through various hydroxylations. 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 to access cyanobacterial NPs from cryptic BGCs.