Construction and systematic engineering of Saccharomyces cerevisiae for efficient de novo biosynthesis of pentostatin

Pentostatin is a potent adenosine deaminase inhibitor, yet its industrial application is hindered by low extraction yields and complex chemical synthesis. Here, we report an efficient de novo biosynthesis platform for pentostatin in Saccharomyces cerevisiae. Starting with the heterologous expression of cns3 from Cordyceps militaris, we optimized the cell factory via promoter engineering, multicopy integration, and AAH1 knockout. This integration strain achieved a maximum pentostatin titer of 16.28 mg/L in shake-flask cultivation, representing a 19.38-fold improvement over our initial production. Separately, to alleviate severe product toxicity, we implemented flux balance analysis (FBA)-guided transporter engineering; the engineered strain expressing the episomal efflux pump Cns4 yielded a titer of 8.27 mg/L while significantly accelerating the production process. Molecular docking revealed a distinct binding cavity where key residues (e.g., Asp296, Ala292) capture pentostatin via specific hydrogen bonds and hydrophobic interactions. Furthermore, transcriptomics demonstrated that Cns4 globally reprograms carbon and energy metabolism to boost precursor supply and cellular robustness. This work integrates structural insights with systems metabolic engineering, providing a generalizable paradigm for biosynthesizing toxic nucleoside natural products.