Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Species.

The genus , a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in and . These peptides facilitate efficient protein transport across cellular membranes in , a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for but also sets a new standard for the development of live biotherapeutics.