Continuous secretory production in E. coli enables scalable, high-titer manufacturing of active recombinant endonucleases.

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.