Metabolic engineering of doxorubicin biosynthesis through P450-redox partner optimization and structural analysis of DoxA
: Koroleva, Arina; Artukka, Erika; Yamada, Keith; Newmister, Sean A.; Harte, Ralph J.; Boesger, Hannah; Londen, Mikael; Sanders, Jacob N.; Tirkkonen, Heli; Kannisto, Matti; Kuin, Rosan C. M.; Hulst, Mandy; Wang, Rongbin; Leskinen, Ester; Barillec, Morgane; Niemi, Jarmo; van Wezel, Gilles P.; Neefjes, Jacques; Nybo, S. Eric; Houk, Kendall N.; Sherman, David H.; Kim, Robbert Q.; Metsä-Ketelä, Mikko
Publisher: Springer Science and Business Media LLC
: 2026
Nature Communications
: 2358
: 17
: 2041-1723
DOI: https://doi.org/10.1038/s41467-026-69194-6
: https://doi.org/10.1038/s41467-026-69194-6
: https://research.utu.fi/converis/portal/detail/Publication/515625069
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.
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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.
