Chemo-enzymatic Total Synthesis and Combinatorial Biosynthesis of Maculalactones

Maculalactones are bioactive γ-butyrolactone natural products with promising antifouling properties, yet their limited availability constrains further development. Here, we report the identification and characterization of the key biosynthetic enzymes MacE and MacF from the maculalactone (mac) biosynthetic gene cluster in Nodularia sp. NIES-3585. In vitro reconstitution confirmed that MacE catalyzes acyloin condensation between phenylpyruvic acids, while MacF mediates subsequent fusion of the MacE product with cinnamoyl-CoA derivatives. Structure-guided engineering improved enzyme solubility, enabling systematic substrate scope expansion. Through a one-pot enzymatic synthesis, we synthesized 13 maculalactone derivatives with yields reaching more than 96%, while a complementary combinatorial biosynthesis approach enabled the incorporation of sterically demanding indole moieties, thereby significantly expanding the maculalactone structural space. Bioactivity screening against A549 lung adenocarcinoma cells revealed moderate cytotoxicity (IC₅₀ = 20.82–56.64 μM), with methylation of phenol groups significantly enhancing activity. Several analogs exhibited notable antibacterial activity against clinically relevant Gram-positive pathogens, including Streptococcus pneumoniae. Our work provides a sustainable platform for producing structurally diverse maculalactones, laying the foundation for future biomedical applications.