Modular engineering of the doxorubicin biosynthetic gene cluster based on component functional clustering in Streptomyces.

The anticancer drug doxorubicin is synthesized through the synergistic action of the type II polyketide synthases, along with an array of modification enzymes in Streptomyces. The 33 doxorubicin biosynthetic genes are scattered across at least 15 distinct transcriptional units, posing challenges for their regulatory control. In this study, we firstly achieved heterologous production of doxorubicin in a range of Streptomyces hosts by supplementing functional genes essential for biosynthesis of the sugar moiety. Then, employed a modular engineering approach to reconstruct the doxorubicin gene cluster, grouping the genes responsible for each biosynthetic module into 6 well-defined subclusters. Based on these 6 subclusters, we identified that the glycosylation and post-modification subcluster is the module with the greatest capacity to boost doxorubicin production. When introduced into the heterologous host S. albus J1074, the combination of the reconstructed modular gene cluster and the glycosylation and post-modification module resulted in a doxorubicin production rate that was 15 times greater than that of the natural gene cluster. Modular engineering of the doxorubicin gene cluster, which involves clustering based on component functionality, will streamline the regulatory control of the biosynthetic pathway and facilitate its transplantation into desired hosts for production optimization.