Controlled release of protein from viable Lactococcus lactis cells.

Overexpression of the lactococcal CsiA protein affects the cell wall integrity of growing cells and leads to leakage of intracellular material. This property was optimized and exploited for the targeted release of biologically active compounds into the extracellular environment, thereby providing a new delivery system for bacterial proteins and peptides. The effects of different levels of CsiA expression on the leakage of endogenous lactate dehydrogenase and nucleic acids were measured and related to the impact of CsiA expression on Lactococcus lactis cell viability and growth. A leakage phenotype was obtained from cells expressing both recombinant and nonrecombinant forms of CsiA. As proof of principle, we demonstrated that CsiA promotes the efficient release of the heterologous Listeria bacteriophage endolysin LM4 in its active form. Under optimized conditions, native and heterologous active-molecule release is possible without affecting cell viability. The ability of CsiA to release intracellular material by controlled lysis without the requirement for an external lytic agent provides a technology for the control of both the extent of lysis and its timing. Taken together, these results demonstrate the potential of this novel approach for applications including product recovery in industrial fermentations, food processing, and medical therapy.