Glycodeoxycholic acid synergizes with L-malic acid to upregulate the malolactic enzyme pathway to alleviate self-toxicity in <i>Lacticaseibacillus paracasei</i> L9

ABSTRACT The malolactic enzyme (MLE) pathway induced by L-malic acid plays an important role for Lacticaseibacillus paracasei L9 to enhance the glycodeoxycholic acid (GDCA) tolerance. However, whether there exists a synergistic effect between L-malic acid and GDCA to cope with bile salt stress remains unclear. In this study, when Lc. paracasei L9 was grown in chemically defined medium (CDM) with 0.08% GDCA and 5 g/L L-malic acid, real-time quantitative PCR results demonstrated that the expression of mleS (the malolactic enzyme) and the mleT (L-malic acid transporter) genes was upregulated by 2.53-fold and 3.63-fold higher than that of the bacteria only cultured in 5 g/L L-malic acid. Furthermore, transcriptional fusion experiments confirmed that GDCA could synergistically enhance the expression of the MLE pathway in the presence of L-malic acid by comparing the intensity of far-red fluorescent protein. Moreover, in the presence of GDCA, the fluorescence intensity increased gradually with increasing L-malic acid concentration, reaching the highest level at 2.0 g/L of L-malic acid, suggesting that GDCA synergistically enhanced the MLE pathway expression in a dose-dependent manner with L-malic acid. The high-performance liquid chromatography (HPLC) quantitative analysis demonstrated that GDCA effectively enhanced the MLE pathway by increasing the intracellular transportation of L-malic acid. Finally, the tolerance ability of the mleR (MLE regulator)-deleted strain to bile salt was significantly decreased, which further revealed that GDCA synergized with the MLE pathway to relieve self-toxicity for Lc. paracasei L9. These results will lay the foundation for a systematic understanding of the bile salt tolerance mechanism of lactic acid bacteria. IMPORTANCE Tolerance to bile stress is critical for probiotics to survive in the gastrointestinal tract. However, the synergistic regulation of stress response pathways by bile salts and metabolic inducers remains unclear. This study elucidates a novel mechanism by which glycodeoxycholic acid (GDCA) cooperates with L-malic acid to upregulate the malolactic enzyme (MLE) pathway in Lacticaseibacillus paracasei L9. Specifically, GDCA significantly enhances mleS and mleT gene expression in the presence of L-malic acid, and this synergistic effect exhibits a dose-dependent relationship with the L-malic acid. Mechanistically, GDCA promotes intracellular L-malic acid transport, thereby increasing the availability of the MleR effector to activate pathway transcription. Consequently, deletion of mleR significantly reduces the bile salt tolerance of L9, confirming the essential role of this regulatory circuit. These findings reveal how lactic acid bacteria transform membrane damage into a metabolic defense advantage, providing new insights into adaptive strategies for multi-stress environments.