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<p><i>Escherichia coli</i> Nissle 1917 (EcN), owing to its proven safety and robust intestinal colonization capacity, has emerged as a highly promising microbial chassis for metabolic engineering and live biotherapeutic applications. However, its genetic manipulation has long been constrained by low editing efficiency, instability of a foreign plasmid, and the lack of robust and tightly controllable expression systems. In this study, we establish a rapid and scalable genome engineering platform for EcN based on an enhanced and fast iterative Ampicillin-Chloramphenicol-Spectinomycin-CRISPR–Cas9 system (ACS-CRISPR-Cas9). By integrating a streamlined dual-sgRNA design with an antibiotic-cycling-driven ACS-CRISPR-Cas9 iterative editing workflow, together with a chromosomally integrated T7 expression system, efficient and inducible gene expression was achieved. Using homologous recombination, dual-sgRNA plasmids were rapidly constructed and enabled precise deletion of 11 gene loci as well as large genomic fragments ranging from 17 to 45&#xa0;kb, with a maximal editing efficiency of 97.9%. Through T7-driven <i>sfGFP</i> integration at 11 distinct chromosomal loci, three high-expression neutral sites—<i>wecB</i>, <i>nagAB</i>, and <i>manXYZ</i>—were identified as suitable targets for modular pathway integration. Within 30&#xa0;days, 12 competing metabolic pathways were sequentially eliminated and nine <i>N</i>-acetylneuraminic acid (NeuAc) biosynthetic modules were integrated in the genome, yielding a total of 13 iteratively engineered strains in 30&#xa0;days. The final strain, EcNSA13, achieved a NeuAc titer of 35.78&#xa0;g/L with a productivity of 0.61&#xa0;g/L/h in a 3-L bioreactor. In addition, the toolkit enabled high-level, antibiotic-free production of ovalbumin (OVA), reaching a titer of 202.91&#xa0;mg/L based on the T7 expression system. Collectively, the ACS-CRISPR-Cas9 platform combined with the T7 expression system markedly accelerates genome editing and modular engineering in EcN, providing a versatile strategy for constructing high-performance probiotic cell factories and establishing a technical foundation for the industrial-scale production of NeuAc and other high-value bioproducts.</p>

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CRISPR-Cas9 and T7 expression system toolkit-guided rapid iterative metabolic engineering and efficient protein synthesis in Escherichia coli Nissle 1917

  • 期刊论文

  • 发表日期：

    2026年7月17日

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  • Jian Wang,
  • Jianing Zhang,
  • Yaokang Wu,
  • Yang Deng,
  • Jianghua Li,
  • Guocheng Du,
  • Jian Chen,
  • Yanfeng Liu

摘要

Escherichia coli Nissle 1917 (EcN), owing to its proven safety and robust intestinal colonization capacity, has emerged as a highly promising microbial chassis for metabolic engineering and live biotherapeutic applications. However, its genetic manipulation has long been constrained by low editing efficiency, instability of a foreign plasmid, and the lack of robust and tightly controllable expression systems. In this study, we establish a rapid and scalable genome engineering platform for EcN based on an enhanced and fast iterative Ampicillin-Chloramphenicol-Spectinomycin-CRISPR–Cas9 system (ACS-CRISPR-Cas9). By integrating a streamlined dual-sgRNA design with an antibiotic-cycling-driven ACS-CRISPR-Cas9 iterative editing workflow, together with a chromosomally integrated T7 expression system, efficient and inducible gene expression was achieved. Using homologous recombination, dual-sgRNA plasmids were rapidly constructed and enabled precise deletion of 11 gene loci as well as large genomic fragments ranging from 17 to 45 kb, with a maximal editing efficiency of 97.9%. Through T7-driven sfGFP integration at 11 distinct chromosomal loci, three high-expression neutral sites—wecB, nagAB, and manXYZ—were identified as suitable targets for modular pathway integration. Within 30 days, 12 competing metabolic pathways were sequentially eliminated and nine N-acetylneuraminic acid (NeuAc) biosynthetic modules were integrated in the genome, yielding a total of 13 iteratively engineered strains in 30 days. The final strain, EcNSA13, achieved a NeuAc titer of 35.78 g/L with a productivity of 0.61 g/L/h in a 3-L bioreactor. In addition, the toolkit enabled high-level, antibiotic-free production of ovalbumin (OVA), reaching a titer of 202.91 mg/L based on the T7 expression system. Collectively, the ACS-CRISPR-Cas9 platform combined with the T7 expression system markedly accelerates genome editing and modular engineering in EcN, providing a versatile strategy for constructing high-performance probiotic cell factories and establishing a technical foundation for the industrial-scale production of NeuAc and other high-value bioproducts.