Abstract
Bacterial minicells are small and chromosome-free cells that result from aberrant cell division and represent a safe alternative to live microbial applications. However, most research on minicells has focused on Escherichia coli, with few studies exploring their development in non-model, biocompatible hosts. In this study, we engineered a minD-deficient Lactiplantibacillus plantarum (formerly Lactobacillus arabinosus and Lactobacillus plantarum) strain capable of producing minicells and systematically evaluated its potential as a chassis for biotechnological applications. Unlike E. coli-based systems, L. plantarum minicells exhibited stable accumulation of heterologous proteins and efficient surface antigen display without evidence of selective export or stress-induced release of toxic compounds. This behavior enabled uniform protein loading and consistent antigen presentation. Additionally, the minicells retained the immunostimulatory properties of their parent cells, underscoring their potential use as adjuvants per se. To improve production efficiency, we employed a continuous cultivation system with controlled growth conditions, which enabled steady-state operation and significantly enhanced minicell yield at optimal dilution rates. Collectively, these findings establish L. plantarum-derived minicells as a safe, robust, and genetically tunable platform suitable for therapeutic delivery, vaccine development, and immunoengineering.