Physiological and transcriptomic insights into ethanol stress tolerance in <i>Lentilactobacillus hilgardii</i> Q19

Malolactic fermentation, started by lactic acid bacteria, plays a crucial role in the production of high-quality wines. As global warming continues to increase the ethanol in wine, the key for successful malolactic fermentation is the excellent strain that tolerates high ethanol in the wines which produce from warm climate area. In our previous study, <em>Lentilactobacillus hilgardii</em><em> </em>Q19 was isolated and characterized as an indigenous malolactic bacterium with higher ethanol tolerance properties than the strains used in wine industry, but the molecular mechanism of which in response to ethanol stress remains unclear. In this study, it was indicated that ethanol stress had significant effects on ATPase activity, antioxidant system, and cell membrane of <em>L. hilgardii</em> Q19 by measuring the physiological indicators under stress which include H⁺-ATPase, Na⁺/K⁺-ATPase, Ca²⁺/Mg²⁺-ATPase activity, glutathione content, superoxide dismutase (SOD) activity and intracellular reactive oxygen species (ROS) content. The main metabolic pathways involved in ethanol stress such as ATP-binding cassette (ABC) transporters, pentose phosphate pathway, phosphotransferase system, glutathione metabolic pathway and two-component systems were screened by transcriptome sequencing analysis. The functions of the pentose phosphate pathway, pyruvate metabolic pathway and glycerolipid metabolism under ethanol stress were verified by constructing the <em>L. hilgardii </em>Q19 ethanol stress related key genes <em>gntK</em>, <em>pyk</em>, and <em>glpK</em> overexpression vectors. The above findings may contribute to our understanding of the metabolic pathways and regulatory mechanisms of <em>L. hilgardii</em> Q19 in response to ethanol stress.