Complete Pathway Refactoring and Combinatorial Biosynthesis of Anthracyclines for Modulation of Histone Eviction and DNA Damage Bioactivities.

Anthracyclines including doxorubicin are cornerstone chemotherapeutic agents with high cytotoxic potency, but their use in cancer treatment is limited by irreversible dose-dependent cardiotoxicity. Recent investigations indicate that cardiotoxicity-free anthracyclines may be achieved through uncoupling DNA damage from chromatin damage. However, semi-synthetic derivatization is complicated by anthracycline structural complexity. Here, we report a modular synthetic biology platform for the complete refactoring and combinatorial biosynthesis of anthracyclines in Streptomyces coelicolor M1152ΔmatAB. Using standardized synthetic DNA parts, we refactored three full biosynthetic pathways of nogalamycin (38 genes), doxorubicin (31 genes), and aclacinomycin (31 genes). The pathways were organized into four functional modules (aglycone biosynthesis, TDP-carbohydrate formation, glycosylation, and tailoring reactions) to facilitate efficient reconstruction. The modular design facilitated streamlined combinatorial biosynthesis yielding 16 derivatives, including 13 novel analogs. Bioactivity profiling across human cancer cell lines identified six potent congeners. Their cytotoxicity correlated with in vitro DNA binding affinities. Two hit molecules harbored desired bioactivity profiles promoting histone eviction at a faster rate than doxorubicin, without causing DNA double strand breaks. Our approach highlights the power of synthetic biology in rational natural product engineering and provides a scalable strategy for exploring new and improved anthracycline anti-cancer agents.