Genomic and environmental determinants of circular single-stranded DNA production in engineered E. coli

In engineered Escherichia coli eScaf, circular single-stranded DNA yield depends on both the number and chromosomal loci of integrated M13 genomes, enabling ~4 mg/L in shake flasks.

• Why it matters: Batch variability and off-target packaging in helper plasmid-phagemid systems limit scalable, high-purity cssDNA production, while eScaf’s earlier yields were too low for cost-effective scale-up.
• What they did: The study systematically tested genomic and environmental determinants of cssDNA yield in eScaf, then combined the findings with optimized media and evaluated fed-batch bioreactor feeding strategies.
• The result: With optimized media, cssDNA yield increased from 100–200 g/L to ~4 mg/L (40-fold) in shake flasks, and the new eScaf-IS3 supports cost-effective production for therapeutic screening, operon-length knock-in donors, and DNA origami assembly.

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