Targeted and Repetitive Chromosomal Integration Enables High-Level Heterologous Gene Expression in Lactobacillus casei

Lactic acid bacteria (LAB), including Lactobacillus casei , have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in Lb. casei without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.