Harnessing native-cryptic plasmids for stable overexpression of heterologous genes in Clostridium butyricum DSM 10702 for industrial and medical applications

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids’ potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum. [Display omitted] •Allelic exchange enables effective gene integration into pCB101 and pCB102.•Heterologous genes (IFP2.0, gusA, cel9M, and hIL10) were stably overexpressed without antibiotics.•Fluorescent/enzymatic reporter (IFP2.0/GusA) exhibits stable activities across repeated subcultures.•Active cellulase was stably expressed and secreted via pCB102 after twenty subcultures.•Bio-active hIL10 was stably secreted via pCB102 after twenty subcultures.