Engineering T7 RNA polymerase-cascaded systems controlled by nisin and theophylline for protein overexpression and targeted gene mutagenesis in Lactococcus lactis

<i>Lactococcus lactis</i> serves as an important platform for heterologous protein production, with the nisin-controlled gene expression (NICE) system being widely employed for regulated protein overexpression. However, the NICE system relies on the native RNA polymerase, which limits transcriptional efficiency, and there remains a lack of tools enabling continuous target gene mutagenesis in <i>L. lactis</i>. In this study, we enhanced the NICE system by integrating the highly processive T7 RNA polymerase (T7RNAP) to boost protein expression. A theophylline-dependent riboswitch, RbxE, was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in <i>Escherichia coli</i>. Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression, leading to a 2.4-fold increase upon nisin and theophylline induction in <i>L. lactis.</i> The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system. Furthermore, adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system, facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resistance gene with a mutation efficiency of 1.33 × 10^-6. Overall, this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in <i>L. lactis</i>.