Abstract
Doxorubicin, a widely used chemotherapy drug, is produced by Streptomyces peucetius ATCC27952. The biosynthesis relies on the cytochrome P450 monooxygenase DoxA, which catalyzes three consecutive late-stage oxidation steps. However, conversion from daunorubicin to doxorubicin is inefficient, necessitating semi-synthetic industrial manufacturing. Here, we address key limitations in DoxA catalysis. We identify the natural redox partners ferredoxin Fdx4 and ferredoxin reductase FdR3 by transcriptomic analysis. We discovered the vicinal oxygen chelate family protein DnrV to prevent product inhibition by binding doxorubicin. Structural analysis of DoxA and density functional theory (DFT) calculations reveal that inefficient C14 hydroxylation results from the unfavorable anti-conformation of the methyl ketone side chain of daunorubicin. We harness these advances for rational strain engineering, leading to an 180% increase in doxorubicin yields and an improved production profile. This study provides singular insights into enzymatic constraints in anthracycline biosynthesis and facilitates cost-effective manufacturing to meet the growing global demand for doxorubicin.
Data availability
All data supporting the findings of this study are available within the Article and Supplementary Information file. The final genome assembly data were deposited in the National Center for Biotechnology Information (NCBI) database under the accession numbers JBLNLL000000000 for G001 and JBLNLK000000000 for X121. The RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE289319. The atomic coordinates and structure factors generated in this study have been deposited in the Protein Data Bank. The DoxA–DHD, DoxA–DOD, and DoxA–DNR structures are available under accession codes 9SI5, 9S7F and 9O35, respectively. Source data are provided with this paper.
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Acknowledgements
Support of this research from the Novo Nordisk Foundation NNF19OC0057511 (to M.M.-K.), the Academy of Finland Grants 340013 and 354998 (to M.M.-K.), NIH grant R35 GM118101 (to D.H.S.), Hans W. Vahlteich Professorship (to D.H.S.), NSF Grants CHE-2153972 (to K.N.H.), ENG-2321976 and CHE-2348596 (to S.E.N.), and the UM Pharmacological Sciences Training Program (H.B) is gratefully acknowledged. We thank Dr. Tero Kunnari (Heraeus GmbH) for the gift of anthracycline reference samples and Dr. Kristiina Ylihonko (Care4living Oy) for Streptomyces strains, whilst Dr. Dennis Wander is acknowledged for supplying synthetic DOD. The authors would like to thank Patrick Voskamp (Leiden University crystallization facility) and Matthew Bowler and other beamline scientists at ESRF (MASSIF-1 and -3) for their support in crystallography experiments.
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A.K., E.A., K.Y., and S.A.N. contributed equally to this work. M.K. and K.Y. designed and performed the experiments related to microbiology and transcriptomics. A.K. contributed to fermentation and metabolic engineering experiments. H.T. and M.B. contributed to compound purification and structure elucidation. E.L. performed the large-scale fermentations. H.B., J.S., and R.J.H. carried out the computational modelling and DFT/MD analyses. S.A.N., R.Q.K., M.H., and R.C.M.K. performed protein purification, crystallography, and structural analysis. R.W. contributed to strain construction. E.A. and M.L. carried out protein purification and in vivo assays. M.M.-K., J.N., G.P.W., J.J.N., S.E.N., D.H.S., and K.N.H. conceptualized and supervised the project. All authors contributed to the revision and editing of the final written draft.
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Koroleva, A., Artukka, E., Yamada, K. et al. Metabolic engineering of doxorubicin biosynthesis through P450-redox partner optimization and structural analysis of DoxA. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69194-6
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DOI: https://doi.org/10.1038/s41467-026-69194-6
