Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of <i>Escherichia coli</i> MG1655 and <i>Bacillus subtilis</i> 168. The application of <i>Ec</i>Δ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in <i>Ec</i>Δ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of <i>Bs</i>Δ<i>44</i> was due to the deletion of the phage SPP1 surface receptor gene <i>yueB</i>, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain <i>Bs</i>Δ<i>yueB</i> exhibited a 61.99% higher acetoin titer compared to strain <i>Bs</i>168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain <i>Bs</i>Δ<i>yueB</i> achieved an 80.63% higher titer than <i>Bs</i>168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for <i>E. coli</i> and <i>B. subtilis</i>, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.