| Lacticaseibacillus paracasei Zhang was originally isolated from traditional sour horse milk and has been used as starter culture for alcoholic milk drinks in production.Ethanol stress was the most inhibitory factor during the fermentation of lactic acid bacteria.Lacticaseibacillus paracasei Zhang Δpgl X was a knockout mutant of the methylation phenotypic control gene pgl X.Previous study has shown that the mutant has stronger tolerance to ethanol than the wild type Lacticaseibacillus paracasei Zhang.The aim of this study was to further investigate the effect of methyltransferase deletion on the tolerance of strain to ethanol stress.The transcriptome,proteome and metabolome of the two strains were compared under ethanol stress for 4 h and 12 h by integrating multiomics techniques.Specific research results are as follows:1.Transcriptomic results showed that,compared with the wild type,405 differentially expressed genes were identified when the mutant was cultured in 10% ethanol for 4 h,and392 differentially expressed genes were screened out after 12 h.203 differentially expressed genes overlapped at these two time points.Enrichment analysis showed significant enrichment of carbohydrate metabolity-related pathways at both time points,including phosphotransferase system,galactose metabolism,fructose and mannose-metabolism,ascorbic acid and uronic acid metabolism.In addition,fatty acid biosynthesis and ATPbinding cassette transporters were significantly enriched only at 4 h.Fatty acid biosynthesis related genes were inhibited and fatty acid content in membrane was significantly increased.2.Proteomic results showed that 168 differentially expressed proteins were identified between the mutant and the wild type(126 at 4 h and 97 at 12 h).Annotation analysis showed that carbohydrate metabolism and membrane transport pathway had the largest amount of protein,enrichment analysis showed that many carbohydrate metabolity-related pathways such as phosphotransferase system,tricarboxylic acid cycle,starch and sucrose metabolism were significantly enriched.In addition,biotin metabolism was significantly enriched at 4 h,pyruvate metabolism was significantly enriched at 12 h.3.Metabolomic results showed that,compared with the wild type,the metabolites adenosine methionine,caprylic acid and N6-acetyl-2,6-diaminoheptanediate were significantly increased in the mutant,while the levels of citroline,fumaric acid and melatonin were significantly decreased.The results of functional enrichment analysis showed that the different metabolites between the mutant and the wild type were significantly enriched in the pathway of arginine biosynthesis and tryptophan metabolism.4.Comprehensive multi-omics analysis showed that changes occurred in material transport,carbohydrate metabolism,fatty acid biosynthesis,amino acid synthesis and metabolism in mutant.By promoting the transport-related proteins of polysaccharides,organic ions and metal ions in mutant,inducing the genes related to carbohydrate metabolism,and increasing the content of fatty acids in cell membrane,it can be inferred that the ethanol stress response mechanism of methyltransferase mutants is mainly:(1)increase the intracellular transport efficiency of substances and accelerate the uptake of nutrients,(2)promote carbohydrate consumption and maintain the growth of strain,(3)increase the fatty acid content of cell membrane to maintain the strength and flow velocity of cell membrane.In conclusion,a series of physiological and metabolic changes,including membrane fatty acid changes,protein synthesis and degradation,occurred in Lacticaseibacillus paracasei under ethanol stress.This study is the first to analyze the mechanism of stress response at the epigenetic level of Lacticaseibacillus paracasei Zhang,providing insights into the regulation of methylation in the stress response of lactic acid bacteria. |
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