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<p>Recombinant endonucleases are essential for biopharmaceutical manufacturing and molecular biology workflows, yet their intracellular expression in <i>Escherichia coli</i> often leads to host cell toxicity due to non-specific DNA and RNA degradation. To address this, we employed the BacSec<sup>®</sup> system to secrete <i>Serratia marcescens</i> non-specific endonuclease, nucA (DRNase<sup>®</sup>) and bovine DNase I in <i>E. coli</i>, aiming to mitigate cytotoxicity and streamlined downstream processing. DRNase<sup>®</sup> was efficiently secreted, enabling simplified purification at the shake flask level and achieving 1&#xa0;g/L in high-density fermentation, with over 2&#xa0;g/L in perfusion-based fermentation. The secreted DRNase<sup>®</sup> 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<sup>®</sup> platform, integrated with perfusion fermentation, provides a scalable, tag-free, and cost-effective solution for high-titer production of active endonucleases.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月15日

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  • Sudarsana Reddy Lokireddy,
  • Chennakesavulu Thummadhi,
  • Pratyusha Godavarty,
  • Venkateswarlu Petla,
  • Akhila Munimanda,
  • Sridhar Rao Kunchala,
  • Ramakrishna Vadde

摘要

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.