Complete biosynthesis of the fungal alkaloid zinnimidine: biochemical insights of the isoindolinone core formation

Isoindolinone scaffold is widely presented in bioactive natural products and serves as a fundamental structure in pharmaceutical compounds. However, the key step of the isoindolinone core formation is not well-defined. In this study, we elucidated the complete biosynthetic pathway of the phytotoxic isoindolinone alkaloid zinnimidine, which was isolated from the cultures of various phytopathogenic fungi of the genus Alternaria. This was achieved through a systematic approach that integrated heterologous expression, feeding experiments, and enzymatic characterizations. Enzymes ZinADEF were identified as key catalysts involved in the biosynthesis of isoindolinone scaffold, directing the transformation from the 2-methyl benzoic acid-like tetraketide precursor to form the isoindolinone scaffold, while ZinB and ZinE catalyzes methylation and prenylation modification, respectively. Detailed enzymatic studies of the flavin-dependent oxidoreductase ZinD uncovered the key catalytic processes underlying the conversion from 1,2-benzenediol to the isoindolinone core, accompanied by the generation of hydrogen peroxide. Through enzymatic reactions of ZinD with various amino-containing compounds, porritoxin and several novel isoindolinones were synthesized. The analysis utilizing cblaster highlighted the conservation of ZinADEF genes across different fungi, indicating the existence of common biosynthetic pathways and mechanisms among these fungal species. Additionally, our study explained the late-stage biosynthetic branch details of isoindolinone and isobenzofuranone natural products. Our research unveils the biosynthetic mechanism of fungal isoindolinones, providing a theoretical foundation for the biocontrol of phytotoxins such as zinnimidine, and establishes a foundation for the biosynthesis of isoindolinone alkaloid derivatives.