Engineering a Streptomyces albidoflavus cell factory for heterologous biosynthesis of tsukubarubicin

Abstract Background Tsukubarubicin is a nogalamycin-type anthracycline antibiotic with potent cytotoxic activity against human breast cancer cells, making it a promising antitumor candidate. However, its native producer produces only trace amounts of tsukubarubicin and is slow-growing and genetically intractable, limiting further pharmacological evaluation and potential application. To overcome this bottleneck, we developed a multi-layered engineering strategy to improve tsukubarubicin production. Results The complete tsukubarubicin biosynthetic gene cluster ( tsu -BGC) was initially isolated from a PAC library, followed by vector backbone replacement and insertion of an integration module using Red/ET recombineering. The engineered tsu -BGC was introduced into the heterologous host Streptomyces albidoflavus Del14 by conjugation, generating the initial strain D1401. To optimize pathway-specific regulation, the role of the regulatory gene tsuR1 was investigated by gene deletion and overexpression using promoters of different strengths. In situ replacement of the native tsuR1 promoter with the P21 promoter subsequently increased tsukubarubicin production from 3.9 to 37.8 mg/L. Electron beam irradiation mutagenesis was subsequently employed to further improve production, and transcriptomic analysis of the resulting high-producing mutant revealed metabolic changes associated with enhanced tsukubarubicin biosynthesis. Guided by these findings, candidate metabolic pathways were rationally engineered, further improving tsukubarubicin production. The final engineered strain D1404-1 produced 153.0 mg/L tsukubarubicin, representing a 39-fold increase over D1401. Conclusions This study establishes an efficient heterologous production platform for tsukubarubicin and substantially alleviates the supply limitation associated with its native producer. The combination of regulatory engineering, mutagenesis, and rational metabolic optimization provides an effective strategy for improving the production of tsukubarubicin, which provides a valuable foundation for pharmacological evaluation. Furthermore, this multi-level engineering paradigm offers a versatile reference for the heterologous overproduction of other actinomycete-derived polyketide natural products.