Mutant transcription factor CopR promotes LDB_RS05285 expression to suppress yogurt post-acidification and enhance...

Continuous lactic acid production during yogurt storage leads to excessive acidity (post-acidification), resulting in texture deterioration and reduced shelf life. Although Cu²⁺ treatment is known to upregulate the post-acidification marker gene LDB_RS05285 (encoding CopB) and modulate fermentation, the specific transcriptional regulatory mechanism remains unclear. In this study, a Cu²⁺-hypersensitive mutant strain E9 was isolated through high-throughput hierarchical screening. Whole-genome sequencing and comparative genomic variant analysis revealed a critical mutation (p.Lys71Arg) in the transcription factor CopR (encoded by LDB_RS06425). DNA pull-down and electrophoretic mobility shift assay (EMSA) confirmed that CopR specifically binds to the LDB_RS05285 promoter region, with the mutant strain exhibiting a significantly enhanced binding capacity, thereby driving the upregulation of LDB_RS05285 expression (2.87-fold increase). RT-qPCR analysis revealed that Cu²⁺ may act as an inducer to upregulate LDB_RS06425 (encoding CopR), thereby promoting CopR's regulatory effect on the downstream target gene LDB_RS05285. Functional analysis indicated that CopB might enhance β-galactosidase activity (1.2-fold) and inhibit lactate dehydrogenase activity (43 %) via protein-protein interactions, thereby enhancing lactose catabolism and inhibiting lactic acid production. Rheological and textural characterization showed that E9-treated yogurt exhibited superior texture and viscoelasticity during storage, directly correlated with attenuated post-acidification. This study unveils for the first time the molecular mechanism by which Cu²⁺ activates the CopR-CopB regulatory cascade via induction of LDB_RS06425 expression, providing a novel theoretical foundation for precise control of post-acidification in fermented dairy products.