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

Single-stranded DNA with hundreds to thousands of bases is a critical reagent in biotechnology. While traditional chemical synthesis is not suitable for efficient production of such long oligonucleotides, M13 bacteriophage offers a scalable pathway towards custom, long, single-stranded DNA production. However, conventional helper plasmid-phagemid systems suffer from batch-to-batch variability and off-target packaging. To address these limitations, we previously engineered eScaf, an E. coli strain with the M13 genome stably integrated into its chromosome. eScaf produces circular, single-stranded DNA with high purity, but yields of only 100 and 200 μg of DNA per liter of culture, too low for cost-effective scale-up. Here, we systematically investigate the genomic and environmental determinants of cssDNA yield in eScaf, revealing that cssDNA yield depends on both the number and chromosomal loci of integrated M13 genomes. Combining these insights with optimized media, we achieve ~4 mg/L of cssDNA in shake flasks, a 40-fold improvement. We also evaluate fed-batch bioreactor feeding strategies for cssDNA production using eScaf. Our new strain, eScaf-IS3, enables cost-effective production of cssDNA for high-throughput therapeutic screening, operon-length knock-in donor production, and large-scale DNA origami assembly.