Coordinated Overexpression of Streptomyces Antibiotic Regulatory Protein and Two-Component System Regulators Enables Hyperproduction of the Antitumor Polyketide LL-D49194α1

LL-D49194α1 (LLD) is a natural product with promising antitumor activity; however, its extremely low native fermentation yield has severely hindered further development. This study identified and characterized two key Streptomyces antibiotic regulatory protein-family activators (LldRg1, LldRg5) and a two-component system (LldRg3/Rg4) within its biosynthetic gene cluster. Coordinated overexpression of these three regulators dramatically increased LLD titer >100-fold to ∼400 mg/L. This engineering also led to the coupregulation of an MFS transporter and a DNA repair-related self-resistance gene, suggesting a concerted activation of efflux and self-defense mechanisms alongside biosynthetic enhancement. Our work establishes an efficient microbial cell factory for LLD and demonstrates a generalizable strategy for enhancing the production of valuable, highly cytotoxic natural products.