Regulatory effect of transcriptional regulator GalR and LysR on diacetyl biosynthesis in Lacticaseibacillus casei TCS

Abstract

Diacetyl, a key flavor compound in fermented dairy products, is mainly synthesized endogenously by microorganisms. While its biosynthetic pathway in microbes has been well-elucidated, the transcriptional regulatory mechanism remains elusive. In this study, GalR (UniProt ID: A0A806E8E8) and LysR (UniProt ID: A0A806ETL9) were preliminarily identified as potential regulators of alsS (NCBI gene ID: 45548984) in Lacticasibacillus casei TCS (GenBank ID: CP038153.1) via DNA pull-down screening combined with LC-MS identification and functional annotation analysis, and hypothesized to influence diacetyl synthesis. Bioinformatics analysis predicted potential binding sites for GalR and LysR in the alsS promoter region, which were validated by electrophoretic mobility shift assay (EMSA) and DNase I footprinting to identify two distinct specific binding sites. Experimental results showed that GalR exerted an inhibitory effect when its binding site was positioned adjacent to the −35 box of the alsS promoter. Conversely, LysR mediated transcriptional activation when its binding site was located between the −10 box nd −35 box. Phenotypic analysis showed that galR (NCBI gene ID: 57090079) deletion led to a significant increase in diacetyl production, indicating its repressive role in diacetyl synthesis. Conversely, overexpression of lysR (NCBI gene ID: 57089290) significantly enhanced diacetyl yield, confirming its activating function. RT-qPCR results revealed that GalR negatively regulated alsS expression, while LysR markedly activated it. This study demonstrates that GalR and LysR modulate endogenous diacetyl biosynthesis by specifically binding to the regulatory region of the alsS gene.