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. paracasei SX10 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.

副干酪乳杆菌SMN-LBK（序列号：CCTCC M 2017429）是一种在科威特（Kumiss）耐乙醇的乳酸菌（LAB）。但是，副干酪乳杆菌SMN-LBK的抗乙醇应激机制仍不清楚。因此，我们通过RNA测序在副干酪乳杆菌SX10（在10％乙醇胁迫下的副干酪乳杆菌SMN-LBK，缩写为SX10）和副干酪乳杆菌SMN-LBK（缩写为S10）之间进行了转录组分析。我们进行了实时定量PCR（RT-qPCR），以验证转录数据的准确性。转录组数据显示，与S10组相比，SX10中有315个基因表达上调，而332个基因下调。该磷酸果糖激酶，乳酸脱氢酶，GPDH和GK基因被上调，对数₂倍变化分别为1.10、0.30、0.56和1.512。基因本体论富集分析显示核糖体，核糖核蛋白复合物，非膜结合细胞器和细胞内非膜结合细胞器的显着富集。使用《京都议定书基因和基因组百科全书》数据库进行的分析显示了与核糖体功能，丙酮酸代谢，氨基酸的生物合成，脂肪酸的生物合成，脂肪酸的代谢，ATP结合盒（ABC）转运蛋白，糖酵解和甘油磷脂代谢相关的差异基因。RT-qPCR结果与转录组结果一致。乳酸乳球菌NZ9000是典型的宿主细菌。我们进行了PFK和GK的过表达，以验证乳酸乳球菌NZ9000中副干酪乳杆菌 SX10抗性基因的功能。使用十二烷基硫酸钠（SDS）-PAGE电泳，这些抗性基因已在乳酸乳球菌NZ9000中成功表达。在乙醇胁迫下测定了重组菌株的存活率和关键酶活性。的存活率乳酸乳球菌NZ9000-pNZ8148-PFK和乳酸乳球菌3.43- 3.80倍较高与比较下10％乙醇应力NZ9000-pNZ8148-GK乳酸乳球菌分别NZ9000-pNZ8148控制。这些结果表明PFK和GK对于LAB的乙醇耐受性很重要，并且可以提高乳酸乳球菌NZ9000的乙醇耐受性。因此，PFK和G K被鉴定为具有高乙醇耐受性的副干酪乳杆菌SX10的关键基因。我们的结果为进一步研究进行差异表达基因的系统分析，并确定其在LAB的乙醇耐受机制中的潜在功能提供了新的见识。