Abstract
Recombinant endonucleases are essential for biopharmaceutical manufacturing and molecular biology workflows, yet their intracellular expression in Escherichia coli often leads to host cell toxicity due to non-specific DNA and RNA degradation. To address this, we employed the BacSec® system to secrete Serratia marcescens non-specific endonuclease, nucA (DRNase®) and bovine DNase I in E. coli, aiming to mitigate cytotoxicity and streamlined downstream processing. DRNase® was efficiently secreted, enabling simplified purification at the shake flask level and achieving 1 g/L in high-density fermentation, with over 2 g/L in perfusion-based fermentation. The secreted DRNase® was predominantly monomeric, demonstrated higher specific activity than commercial counterparts, and remained stable at room temperature for over a year. Likewise, secreted bovine DNase I retained strong enzymatic activity without degrading mRNA, making it particularly suitable for mRNA vaccine production. These secreted endonucleases support a wide range of industrial applications, including biologics production, gene therapy, mRNA and viral vector-based vaccines, and therapeutic use. Overall, the BacSec® platform, integrated with perfusion fermentation, provides a scalable, tag-free, and cost-effective solution for high-titer production of active endonucleases. Supplementary Information: The online version contains supplementary material available at 10.1186/s13036-025-00590-0.