Transcriptome analysis of Lactobacillus paracasei SMN-LBK under ethanol stress

Lactobacillus paracasei SMN-LBK (serial number: CCTCC M 2017429) is an ethanol-resistant lactic acid bacteria (LAB) in kumiss. However, the anti-ethanol stress mechanism of L. paracasei SMN-LBK remains unclear. Hence, we performed a transcriptome analysis between L. paracasei SX10 (L. paracasei SMN-LBK under 10% ethanol stress strain, abbreviated as SX10) and L. paracasei SMN-LBK (abbreviated as S10) by RNA sequencing. We performed real-time quantitative PCR (RT-qPCR) to verify the accuracy of the transcription data. The transcriptome data revealed that 315 genes exhibited upregulated expression, and 332 genes were downregulated in the SX10 compared with the S10 group. The PFK, LDH, GPDH, and GK genes were upregulated, with a log₂-fold change of 1.10, 0.30, 0.56, and 1.512, respectively. A gene ontology enrichment analysis revealed significant enrichment of ribosomes, ribonucleoprotein complex, non-membrane-bounded organelles, and intracellular non-membrane-bound organelles. Analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed differential genes associated with ribosome function, pyruvate metabolism, biosynthesis of amino acids, fatty acid biosynthesis, fatty acid metabolism, ATP-binding cassette (ABC) transporter, glycolysis, and glycerophospholipid metabolism. The RT-qPCR results were consistent with the transcriptome results. Lactococcus lactis NZ9000 is a typical host bacterium. We performed PFK and GK overexpression to verify the function of the L. paracaseiSX10 resistance gene in Lactococcus lactis NZ9000. Using sodium dodecyl sulfate (SDS)-PAGE electrophoresis, these resistance genes were successfully expressed in Lactococcus lactis NZ9000. The survival rate and key enzyme activity of the recombinant strains were determined under ethanol stress. The survival rate of Lactococcus lactis NZ9000-pNZ8148-PFK and Lactococcus lactis NZ9000-pNZ8148-GK under 10% ethanol stress were 3.43- and 3.80-fold higher compared with the Lactococcus lactis NZ9000-pNZ8148 control, respectively. These results indicate that PFK and GK are important for the ethanol tolerance of LAB and can increase the ethanol tolerance of Lactococcus lactis NZ9000. Hence, PFK and GK were identified as key genes of L. paracasei SX10 with a high ethanol tolerance. Our results provide novel insight for further studies to perform a systematic analysis of the differentially expressed genes and to determine their potential functions in the ethanol tolerance mechanism of LAB.