High-copy plasmid engineering enhances recombinant protein and antimicrobial peptide production in Corynebacterium glutamicum

Antimicrobial peptides (AMPs) such as pediocin PA-1 are attractive for food biopreservation and infection control, but their broader use is limited by low recombinant yields and high production costs. Corynebacterium glutamicum has emerged as a robust GRAS chassis for heterologous peptide and protein production, yet commonly used shuttle vectors provide only moderate plasmid copy numbers and expression capacities. In particular, existing pediocin PA-1 processes in C. glutamicum rely on standard pBL1- or pCG1-family vectors that do not yet leverage replication-origin engineering. We rationally redesigned the replication control region of the widely used pClik 5α (pCG1-family) backbone by introducing targeted mutations in the repA gene, an antisense RNA (cgrI) promoter, and putative partitioning genes parAB, and constructed a systematic panel of high-copy variants. Using a Ptuf-driven mCherry reporter as a quantitative readout, we identified plasmids that supported several-fold higher fluorescence than the parental backbone while maintaining robust growth. Fluorescence-based gene-dosage estimation indicated a strong increase in apparent plasmid copy number. Independent qPCR-based plasmid copy number determination using two plasmid loci confirmed that the lead variant pClik 5α repAmut reached approximately 28–30 copies per chromosome equivalent, compared to approximately 2–3 copies for the parental plasmid, corresponding to an approximately 10-fold increase. Genome-wide transcriptome analysis revealed a defined and adaptive transcriptional response to elevated plasmid copy number and expression burden, characterized by adjustments in membrane-associated transport, respiratory functions, and amino acid-related metabolism, without evidence of collapse of core biosynthetic functions. When the best-performing replicon was applied to episomal expression of a codon-optimized pedACDCgl operon, pediocin PA-1 titers increased by 2.5-fold compared to the best pXMJ19-based reference under identical, previously optimized process conditions, placing the system, under comparable cultivation formats, within the upper range of reported microbial pediocin production processes. This work demonstrates that rational engineering of pCG1-family replication modules in C. glutamicum can unlock markedly higher plasmid copy numbers and expression capacities while preserving physiological robustness. The resulting high-copy pClik 5α derivatives, exemplified by pClik 5α repAmut, provide a versatile high-copy expression platform with demonstrated utility for recombinant reporter protein and antimicrobial peptide production in C. glutamicum and offer a foundation for further integration with folding, secretion, and process engineering strategies to advance industrial AMP production.