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

Abstract

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.

Keywords

Escherichia coli Nissle 1917 / CRISPR-Cas9 / Dual-sgRNA genome editing / Metabolic engineering / Ovalbumin (OVA) / N-acetylneuraminic acid (NeuAc)

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Funding

financially supported by the National Key Research and Development Program(2024YFF1106800)

Scientific Research Innovation Capability Support Project for Young Faculty(ZYGXQNJSKYCXNLZCXM-A6)

Research Program of State Key Laboratory of Food Science and Resources, Jiangnan University(SKLF-ZZB-202405)

Fundamental Research Funds for the Central Universities(JUSRP622028)

RIGHTS & PERMISSIONS

Jiangnan University