Enhancing Superior Bread Anti-Staling Properties of Sourdough through Genetic Engineering: Silencing UlpRTF1 and Dual-Site Mutation in <i>Companilactobacillus paralimentarius</i>

Bread staling poses a significant challenge in the baking industry for product quality reduction and economic losses. While sourdough fermented by Companilactobacillus (C.) paralimentarius has ancient origins, its systematic utilization as a multifunctional bio-additive─tailored for health, safety, and economic efficiency─represents a transformative approach in contemporary industrial baking. C. paralimentarius secreting LpaMA with good acid stability was successfully used for bread anti-staling. However, the low expression level and poor thermostability of LpaMA limit its industrial application. In this study, an LacI family transcriptional regulator, UlpRTF1, was identified as a repressor of LpaMA expression. Silencing UlpRTF1 led to a 162-fold increase in the transcription level of LpaMA. A multi-locus gene editing toolbox was developed for obtaining UlpRTF1-silenced strains with the double mutant sites (WHU) at A375P and S257P, which enhances the thermal stability of LpaMA. Sourdough fermented by WHU (WHU-Sourdough) significantly improved bread softness, reducing firmness by 41.6% and staling rate by 50.9% after 7 days at 4°C by texture profile analysis. WHU-Sourdough containing less activity demonstrated comparable anti-staling effects with purified enzyme preparations and highlighted its potential as a more economical and sustainable bio-additive for improving bread anti-staling properties. This study provides a genetic engineering-based strategy for optimizing sourdough functionality as a natural alternative to enzyme preparations while maintaining a clean label.