植物乳杆菌(Lactobacillus plantarum)具有优异的益生特性,被广泛应用在发酵食品、饲料和医药等领域中。然而,关于植物乳杆菌的遗传信息和生理代谢特性鲜有报道。为探究植物乳杆菌的生理特性和代谢途径相关分子机制,挖掘重要性状相关的功能基因,该研究利用前期筛选的1株生长速率快、产酸能力强、代谢产物丰富的植物乳杆菌L.plantarum DY6为研究对象,通过与植物乳杆菌模式菌株L.plantarum WCFS1进行详细的生理特性和遗传信息比较,揭示L.plantarum DY6生理特性的分子机制。研究发现,与L.plantarum WCFS1相比,L.plantarum DY6在葡萄糖、果糖和半乳糖代谢中生长活性和产酸性能以及精氨酸、天冬氨酸等代谢方面均具有显著差异。基因组重测序发现,与丙酮酸代谢、碳水化合物代谢和氨基酸代谢等表型差异明显的代谢途径中存在102个突变,包括fruK、ack1、Idhl2、sacK、galM、argH和argF等。在此基础上,测定了与产酸直接关联的丙酮酸代谢关键基因的转录水平。发现,L.plantarum DY6的pdhC、ack1和Idhl2的相对表达量分别比L.plantarum WCFS1高了20、15和14倍,推测这些基因序列的改变对功能上的差异有重要影响,对进一步理解植物乳杆菌发酵和益生特性相关的分子机制奠定了理论基础。
Due to the excellent probiotics, Lactobacillus plantarum was widely used in the field of fermentation food, feed, and medicine. However, genetic information and physiological metabolism of L. plantarum were rarely reported. In order to explore the molecular mechanism of physiological characteristics and metabolic pathways of L. plantarum, and explore the functional genes related to important traits, the strain L. plantarum DY6 with fast growth rate, strong acid production ability, and rich metabolites was selected as the research object in this study. The molecular mechanism of physiological characteristics of L. plantarum DY6 was revealed by comparing the physiological characteristics and genetic information with L. plantarum WCFS1. Compared with L. plantarum WCFS1, L. plantarum DY6 had significant advantages in growth activity, sugar acid conversion rate, carbon source metabolism, and amino acid metabolism, mainly including glucose, fructose and galactose metabolism, arginine and aspartate metabolism. Genome re-sequencing obtained 19 396 single nucleotide polymorphisms, 972 insertion or deletion mutations, and 4331 annotated mutation genes. There were 102 mutations in metabolic pathways that were significantly different from pyruvate metabolism, carbohydrate metabolism and amino acid metabolism. Analyzing the metabolic characteristics and genome differential between L. plantarum WCFS1 and L. plantarum DY6, we speculated that the following changes possibly existed in L. plantarum DY6: (1) beneficial mutations existed in the PTS system, resulting in enhanced transportability for carbohydrates, promoting the cell growth; (2) fructokinase, fructose specificity EIIABC (fruA) and EIIBC (PTS31BC) possibly had higher activity, enhancing the metabolic flux from fructose to lactic acid; (3) phosphofructokinase (fruK) and L-lactate dehydrogenase (ldhl2) probably had higher activity, enhancing the transformation of glucose and pyruvic acid to glycolysis pathway and lactic acid, respectively; (4) harmful mutations possibly existed in metK and/or mtn, restricted the methionine metabolic pathway, resulting in the low consumption rates; (5) gadB possibly contained harmful mutations, influencing the biosynthesis of γ-aminobutyric acid and acid resistance; (6) mutations in argF resulted a faster consumption rates of arginine. On this basis, the transcription levels of key genes directly related to pyruvate metabolism were determined. It was found that the relative expression levels of pdhC, ack1, and Idhl2 in L. plantarum DY6 were 20, 15, and 14 times higher than those in L. plantarum WCFS1, respectively. It was speculated that the change of these gene sequences probably had an important impact on the functional differences, which laid a theoretical foundation for further understanding the molecular mechanisms related to L. plantarum fermentation and probiotic characteristics.
[1] SUN Z H, ANGELA M C, GUO C Y, et al.Expanding the biotechnology potential of Lactobacilli through comparative genomics of 213 strains and associated genera[J].Nature Communications, 2015, 6(1):1-13.
[2] YOO D, BAGON B B, VALERIANO V D V, et al.Complete genome analysis of Lactobacillus fermentum SK152 from kimchi reveals genes associated with its antimicrobial activity[J].FEMS Microbiology Letters, 2017, 364(18):1-10.
[3] BOLOTIN A, WINCKER P, MAUGER S, et al. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403[J]. Genome Research, 2001, 11(5):731-753.
[4] ZHANG W, CAO C X, HUI W Y, et al.Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment[J].Journal of Proteomics, 2018, 176:37-45.
[5] WANG M X, CHEN Y X, WANG Y Y, et al.Beneficial changes of gut microbiota and metabolism in weaned rats with Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 supplementation[J].Journal of Functional Foods, 2018, 48:252-265.
[6] YAN R, WANG K C, WANG Q Q, et al.Probiotic Lactobacillus casei Shirota prevents acute liver injury by reshaping the gut microbiota to alleviate excessive inflammation and metabolic disorders[J].Microbial Biotechnology, 2021, 15(1):247-261.
[7] DELGADO-FERNANDEZ P, PLAZA-VINUESA L, LIZASOAIN-SNCHEZ S, et al.Hydrolysis of lactose and transglycosylation of selected sugar alcohols by lacA β-galactosidase from Lactobacillus plantarum WCFS1[J].Journal of Agricultural and Food Chemistry, 2020, 68(26):7 040-7 050.
[8] ZHANG S S, XU Z S, QIN L H, et al.Low-sugar yogurt making by the co-cultivation of Lactobacillus plantarum WCFS1 with yogurt starter cultures[J].Journal of Dairy Science, 2020, 103(4):3 045-3 054.
[9] 王祎然, 韦明明, 张涵, 等.酸汤中乳酸菌的鉴定及其耐酸, 耐胆盐和抗氧化活性[J].食品工业科技, 2020, 41(6):121-126;139.
WANG Y R, WEI M M, ZHANG H, et al.Identification, acid and bile salt tolerance,and antioxidant ability of lactic acid bacteria isolated from sour soup[J].Science and Technology of Food Industry, 2020, 41(6):121-126;139.
[10] 国家药典委员会. 中华人民共和国药典[M].3部.北京:中国医药科技出版社, 2010.
National Pharmacopoeia Commission.Pharmacopoeia of the People′ Srepublic of China[M].3th ed.Beijing:China Medical Science Press, 2010.
[11] COLLADO M C, MERILUOTO J, SALMINEN S.Adhesion and aggregation properties of probiotic and pathogen strains[J].European Food Research and Technology, 2008, 226(5):1 065-1 073.
[12] BURNS P, VINDEROLA G, BINETTI A, et al.Bile-resistant derivatives obtained from non-intestinal dairy Lactobacilli[J].International Dairy Journal, 2008, 18(4):377-385.
[13] RAO X Y, HUANG X L, ZHOU Z C, et al.An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis[J].Biostatistics, Bioinformatics and Biomathematics, 2013, 3(3):71-85.
[14] 武万强, 王琳琳, 赵建新, 等.植物乳杆菌生理特性及益生功能研究进展[J].食品与发酵工业, 2019, 45(1):1-13.
WU W Q, WANG L L, ZHAO J X, et al.Research progress on physiological characteristics and health benefits of lactobacillus plantarum[J].Food and Fermentation Industries, 2019, 45(1):1-13.
[15] VAN BOKHORST-VAN DE VEEN H, VAN SWAM I, WELS M, et al.Congruent strain specific intestinal persistence of Lactobacillus plantarum in anintestine-mimicking in vitro system and in human volunteers[J].PLoS One, 2012, 7(9):e44588.
[16] 谢琼. 植物乳杆菌ZDY2013表层蛋白及其益生特性的初步探究 [D].南昌:南昌大学, 2017.
XIE Q.Research on the surface layer protein and probiotic functions of Lactobacillus plantarum ZDY2013[D].Nanchang:Nanchang University, 2017.
[17] KLEEREBEZEM M, BOEKHORST J, VAN KRANENBURG R, et al.Complete genome sequence of Lactobacillus plantarum WCFS1[J].Proceedings of the National Academy of Sciences, 2003, 100(4):1 990-1 995.
[18] BURON-MOLES G, CHAILYAN A, DOLEJS I,et al.Uncovering carbohydrate metabolism through a genotype-phenotype association study of 56 lactic acid bacteria genomes[J].Applied Microbiology and Biotechnology,2019,103(7):3 135-3 152.
[19] QIAO Y L,LIU G F,LENG C, et al.Metabolic profiles of cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine, alanine and glutathione in Streptococcus thermophilus during pH-controlled batch fermentations[J].Scientific Reports, 2018, 8(1):12441.
[20] ARNONE D, CHABOT C, HEBA A C, et al.Sugars and gastrointestinal health[J].Clinical Gastroenterology and Hepatology, 2021.DOI:10.1016/j.cgh.2021.12.011.
[21] ANORES-HERNANDO A, ORLICKY D J, KUWABARA M, et al.Deletion of fructokinase in the liver or in the intestine reveals differential effects on sugar-induced metabolic dysfunction[J].Cell Metabolism, 2020, 32(1):117-127.
[22] 邓笑颖, 姜杨, 张灏, 等.基于乳糖水解酶的乳酸乳球菌乳糖代谢多样性研究[J].食品与发酵工业, 2019, 45(21):8-14.
DENG X Y, JIANG Y, ZHANG H, et al.Galactosidases biodiversity in lactose metabolism among Lactococcus lacits[J].Food and Fermentation Industries, 2019, 45(21):8-14.
[23] 赵嫚, 彭莉, 成浩, 等.微生物甲硫氨酸合成调控的综合研究进展与展望[J].食品与发酵工业,2020,46(24):257-264.
ZHAO M, PENG L, CHENG H, et al.Advances on the biosynthesis and regulation of methionine[J].Food and Fermentation Industries, 2020, 46(24):257-264.
[24] 王记成. 基于转录组学和蛋白质组学对益生菌Lactobacillus casei Zhang在牛乳和豆乳中生长机理的研究[D].呼和浩特:内蒙古农业大学, 2012.
WANG J C.Study on mechanisms of probiotic Lactobacillus casei Zhang growing in bovine milk and soymilk based on transcriptomics and proteomics[D].Huhhot:Inner Mongolia Agricultural University, 2012.
