Chemo-enzymatic Total Synthesis and Combinatorial Biosynthesis of Maculalactones Enables Discovery of Anticancer Agents

Abstract Maculalactones are bioactive γ-butyrolactone natural products with promising antifouling properties, yet their limited availability constrains further development. Here, we report the characterization of the key biosynthetic enzymes MacE and MacF from the maculalactone (mac) biosynthetic gene cluster in Nodularia sp. NIES-3585. Exploiting the substrate flexibility of these biocatalysts, we developed a one-pot chemo-enzymatic platform that enabled the synthesis of 13 maculalactone derivatives in isolated yields of up to 96%. Furthermore, a plug-and-play combinatorial biosynthesis strategy with predictable product outcomes was established by assembling an artificial biosynthetic gene cluster from three distinct pathways, enabling the biosynthesis of previously inaccessible maculalactone analogs, including indole-containing derivatives, in a heterologous host (E. coli). Biological evaluation reveals that maculalactone derivatives exhibit cancer-type selective cytotoxicity, with several compounds displaying potent cytotoxicity against colorectal cancer cells (HCT116) with the best IC50 values reaching 2.7 μM. In contrast, only moderate or negligible activity was observed against lung and breast cancer cell lines, indicating pronounced selectivity. Initial mechanistic studies show that these compounds induce DNA damage, leading to apoptosis and inhibition of cancer cell proliferation. In addition, several derivatives exhibited antibacterial activity against Gram-positive pathogens, including Streptococcus pneumoniae. Collectively, our work establishes a general and programmable strategy for maculalactone diversification, expands their chemical and biological space, and highlights their potential as a promising scaffold for the development of selective anticancer agents.