Exploration of endogenous signal peptides and tandem linkage strategies for enhanced heterologous protein secretion in Lacticaseibacillus paracasei

<i>Lacticaseibacillus paracasei</i> (<i>L. paracasei</i>), a probiotic bacterium commonly found in the intestinal tract and fermented products, has been utilized as a tool for generating various bioactive components. However, its potential as a host cell for protein secretion is constrained by the limited availability of secretion signal peptides. This study explores the potential of endogenous signal peptides from <i>L. paracasei</i> for improving heterologous protein secretion. Endogenous secretory proteins from <i>L. paracasei</i> BL23 were analyzed to characterize their signal peptides. Using reporter genes such as <i>nuc</i> (nuclease) and <i>amy</i> (amylase) under the control of the nisin-inducible promoter PnisA, signal peptide SP230 was shown to be the most potent endogenous signal peptide for heterologous protein secretion in <i>L. paracasei</i> BL23. Additionally, a tandem linkage strategy employing identical signal peptides increased the secretion levels of heterologous proteins. The secretion of heterologous proteins was found to be dependent on the utilization of their optimal signal peptides. Tandem linkage of proteins with preferred signal peptides proved to be critical for efficient secretion of two distinct heterologous proteins simultaneously. The identification of novel signal peptides and the development of tandem linkage strategies in this study offer valuable insights for improving heterologous protein expression and secretion in <i>L. paracasei</i>. These findings enhance the potential of <i>L. paracasei</i> as a host for biotechnological applications, facilitating the secretion of diverse proteins.