A Dual CRISPR-Cas/Cre- loxP Genome Engineering Strategy for Stable Uricase Expression in Food-Grade Probiotics

The development of robust, food-grade microbial chassis with tailored metabolic functions is critical for advancing synthetic biology applications in health and nutrition. Here, we report a dual genome engineering strategy that integrates CRISPR-Cas9-mediated knock-in with Cre/ loxP -driven genome reduction to streamline the genome of Lactococcus lactis NZ9000 and enable stable expression of a high-activity uricase variant. The resulting strain, NZ9000::UA T -Δ D6, demonstrated enhanced enzymatic performance in vitro, achieving 2.34 U/mL activity and complete degradation of ∼500 μM urate within 20 h. Beyond improved catalytic output, this dual-system approach established a genetically stable and biosafe probiotic chassis with moderate colonization capacity in the murine gut. The integration of CRISPR-Cas and Cre/ loxP techniques in this work is intended to enhance the expression of heterologous genes in the chassis strain, while providing a versatile platform for the rational design of food-grade probiotics and offering a general strategy for constructing living biotherapeutic agents with targeted metabolic activities.