Metabolic Engineering of Doxorubicin Biosynthesis: Advancing P450 Catalysis through Redox Partner Optimization and Structural Analysis of DoxA
Doxorubicin is a widely used chemotherapy agent produced by Streptomyces peucetius ATCC27952. Key to the biosynthesis is the cytochrome P450 monoxygenase DoxA that catalyzes three consecutive late-stage hydroxylations. However, the final conversion from daunorubicin to doxorubicin is rate-limiting and industrial manufacturing occurs through semi-synthesis from daunorubicin. Here we have discovered three factors that limit catalysis by DoxA. We performed comparative transcriptome analysis to identify the natural redox partners, the ferredoxin FDX4 and ferredoxin reductase FDR3, preferred by DoxA. We show that the vicinal oxygen chelate (VOC) family protein DnrV is a drug-binding protein aiding catalysis by binding doxorubicin and preventing product inhibition of DoxA. Ternary structures of DoxA in complex with ligands and density functional theory calculations revealed that 14-hydroxylation is limited by the preferred anti conformation of the daunorubicin substrate. We employed these findings to convert a daunorubicin over-producing industrial strain to produce 300 mg / L doxorubicin. Overall design: RNA-Seq profiling of Streptomyces peucetius ATCC 27952 and their mutant derivatives GOO1 and X121 at Day1-6