长双歧杆菌BBMN68胞外多糖生物合成基因的克隆及生物信息学分析
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摘要
目的:研究长双歧杆菌BBMN68胞外多糖生物合成途径和调控机制。方法:提取BBMN68胞外多糖,预测eps基因簇各基因编码蛋白功能,克隆关键基因并对其进行进化分析。结果:在CDM和MRSC培养基上,长双歧杆菌BBMN68产生胞外多糖,产量分别为100 mg/L和400 mg/L。本实验室工作人员对其进行全基因组序列测定。根据全基因组序列,分析得到eps基因簇,对该基因簇各基因编码蛋白的功能预测发现,BBMN68_1002编码1个可能具有多糖重复单元转运子功能的蛋白。BBMN68_1003、BBMN68_1006、BBMN68_1007和BMN68_1011与多糖重复单元聚合有关。BBMN68_1004、BBMN68_1008和BBMN68_1009参与糖基转移。BMN68_1010控制多糖分子链长。BBMN68_1012编码胞外多糖合成的关键酶——引导糖基转移酶(priming glycosyltransferase,Cps D),启动合成胞外多糖第1步。由Cps D基因构建的系统发育树,BBMN68与猿猴、狨猴及大猩猩的长双歧杆菌同枝,可能是饮食影响肠道细菌进化的结果。结论:本研究结果为解析长双歧杆菌BBMN68胞外多糖生物合成途径和调控机制奠定了理论基础。
The aim of this study was to investigate the EPS biosynthetic pathway and regulate mechanism of Bifidobacterium longum subsp. longum BBMN68. Extract the exopolysaccharide from strain BBMN68, analysis proteins function encoded by the EPS cluster genes, clone and analysis evolution of key gene. The yields of exopolysaccharide from strain BBMN68 is 100 mg/L and 400 mg/L, respectively in the CDM and MRSC medium. We determined the complete genome sequence of strain BBMN68, analysis the eps-clusters of that. Functional prediction analysis of proteins indicates that, BBMN68_1002 codes a polysaccharide repeat unit transport protein. Proteins are to participate in polymerization of repeat unit, which are coded by BBMN68_1003, BBMN68_1006, BBMN68_1007 and BMN68_1011. BBMN68_1004,BBMN68_1008 and BBMN68_1009 code proteins may areglycosyl-transferases. BBMN68_1012 encodes the key enzyme-priming glycosyltransferase(Cps D) in the synthesis of exopolysaccharide, which starts the first step of the exopolysaccharide synthesis. The Cps D gene is cloned and constructed phylogenetic tree, find that strain BBMN68 clusters one group with Bifidobacterium longum from monkey, marmoset monkeys and gorillas, maybe that is result of diet affect intestinal microbial evolutionary. This study would establish a basic for further understanding the EPS biosynthetic pathway and regulate mechanism of strain BBMN68.
The aim of this study was to investigate the EPS biosynthetic pathway and regulate mechanism of Bifidobacterium longum subsp. longum BBMN68. Extract the exopolysaccharide from strain BBMN68, analysis proteins function encoded by the EPS cluster genes, clone and analysis evolution of key gene. The yields of exopolysaccharide from strain BBMN68 is 100 mg/L and 400 mg/L, respectively in the CDM and MRSC medium. We determined the complete genome sequence of strain BBMN68, analysis the eps-clusters of that. Functional prediction analysis of proteins indicates that, BBMN68_1002 codes a polysaccharide repeat unit transport protein. Proteins are to participate in polymerization of repeat unit, which are coded by BBMN68_1003, BBMN68_1006, BBMN68_1007 and BMN68_1011. BBMN68_1004,BBMN68_1008 and BBMN68_1009 code proteins may areglycosyl-transferases. BBMN68_1012 encodes the key enzyme-priming glycosyltransferase(Cps D) in the synthesis of exopolysaccharide, which starts the first step of the exopolysaccharide synthesis. The Cps D gene is cloned and constructed phylogenetic tree, find that strain BBMN68 clusters one group with Bifidobacterium longum from monkey, marmoset monkeys and gorillas, maybe that is result of diet affect intestinal microbial evolutionary. This study would establish a basic for further understanding the EPS biosynthetic pathway and regulate mechanism of strain BBMN68.
引文
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[17]刘丽莎,范熠,旭日花,等.动物双歧杆菌RH胞外多糖基因簇的克隆及分析[J].食品科学,2013,34(15):136-142.
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[22]VENTURA M,TURRONI F,LUGLI G A,et al.Bifidobacteria and humans:our special friends,from ecological to genomics perspectives[J].Journal of the Science of Food and Agriculture,2014,94(2):163-168.
[23]MILANI C,LUGLI G A,DURANTI S,et al.Genomic encyclopedia of type strains of the genus Bifidobacterium[J].Applied and Environmental Microbiology,2014,80(20):6290-6302.
[24]LUGLI G A,MILANI C,TURRONI F,et al.Investigation of the evolutionary development of the genus Bifidobacterium by comparative genomics[J].Applied and Environmental Microbiology,2014,80(20):6383-6394.
[25]OLIVARES M,CASTILLEJO G,VAREA V,et al.Double-blind,randomised,placebo-controlled intervention trial to evaluate the effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed coeliac disease[J].British Journal of Nutrition,2014,112(1):30-40.
[26]LAPARRA J M,OLIVARES M,SANZ Y.Oral administration of Bifidobacterium longum CECT 7347 ameliorates gliadin-induced alterations in liver iron mobilisation[J].British Journal of Nutrition,2013,110(10):1828-1836.
[2]赵亮.长寿老人源双歧杆菌延缓线虫衰老及其作用机制[D].北京:中国农业大学,2011.
[3]丁圣,蒋菁莉,刘松玲,等.长双歧杆菌BBMN68对便秘模型小鼠的通便作用[J].食品科学,2011,32(3):195-198.
[4]ZHU J,ZHAO L A,GUO H Y,et al.Immunomodulatory effects of novel bifidobacterium and lactobacillus strains on murine macrophage cells[J].African Journal of Microbiology Research,2011,5(1):8-15.
[5]YANG H Y,LIU S L,IBRAHIM S A,et al.Oral administration of live Bifidobacterium substrains isolated from healthy centenarians enhanced immune function in BALB/c mice[J].Nutrition Research,2009,29(4):281-289.
[6]YANG H Y,LIU A P,ZHANG M,et al.Oral Administration of live Bifidobacterium substrains isolated from centenarians enhances intestinal function in mice[J].Current Microbiology,2009,59(4):439-445.
[7]FANNING S,HALL L J,CRONIN M,et al.Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protectio n[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(6):2108-2113.
[8]王正荣,生吉萍,申琳.细菌胞外多糖的生物合成与基因控制[J].生物技术通报,2010(11):48-55.
[9]HIDALGO-CANTABRANAC,SANCHEZ C,MILANI B,et al.Genomic overview and biological functions of exopolysaccharide biosynthesis in Bifidobacterium spp.[J].Appl Environ Microbiol,2014,80(1):9-18.
[10]旭日花.双歧杆菌胞外多糖的结构解析及调节肠黏膜免疫功能研究[D].北京:中国农业大学,2011.
[11]张丁丁,张宇光,刘爱萍,等.B族维生素和核酸碱基对唾液乳杆菌生长和产胞外多糖的影响[J].中国奶牛,2009(9):44-47.
[12]ZHANG L,LIU C H,LI D,et al.Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88[J].International Journal of Biological Macromolecules,2013(54):270-275.
[13]MARCHLER-BAUER A,LU S N,ANDERSON J B,et al.CDD:a Conserved Domain Database for the functional annotation of proteins[J].Nucleic Acids Research,2011(39):D225-D229.
[14]MARCHLER-BAUER A,ANDERSON J B,CHITSAZ F,et al.CDD:specific functional annotation with the Conserved Domain Database[J].Nucleic Acids Research,2009(37):D205-D210.
[15]MARCHLER-BAUER A,BRYANT S H.CD-Search:protein domain annotations on the fly[J].Nucleic Acids Research,2004(32):W327-W331.
[16]丹彤,王俊国,张和平.乳酸菌胞外多糖的结构、生物合成及其应用[J].食品科学,2013,34(7):335-339.
[17]刘丽莎,范熠,旭日花,等.动物双歧杆菌RH胞外多糖基因簇的克隆及分析[J].食品科学,2013,34(15):136-142.
[18]DE VUYST L,DEGEEST B.Heteropolysaccharides from lactic acid bacteria[J].Fems Microbiology Reviews,1999,23(2):153-177.
[19]张群,张伟,黎文亮.尿苷二磷酸葡萄糖脱氢酶的基因克隆及在原核细胞中的表达[J].军事医学科学院院刊,2000,24(2):95-97.
[20]AYABE-CHUJO Y,USAMI Y,YOSHIDA T,et al.Membrane topology and functional analysis of Methylobacillus sp 12S genes eps F and eps G,encoding polysaccharide chain-length determining proteins[J].Bioscience Biotechnology and Biochemistry,2012,76(3):608-612.
[21]LIU D,COLE R A AND REEVES P R.An O-antigen processing function for Wzx(Rfb X):a promising candidate for O-unit flippase[J].Journal of Bacteriology,1996,178(7):2102-2107.
[22]VENTURA M,TURRONI F,LUGLI G A,et al.Bifidobacteria and humans:our special friends,from ecological to genomics perspectives[J].Journal of the Science of Food and Agriculture,2014,94(2):163-168.
[23]MILANI C,LUGLI G A,DURANTI S,et al.Genomic encyclopedia of type strains of the genus Bifidobacterium[J].Applied and Environmental Microbiology,2014,80(20):6290-6302.
[24]LUGLI G A,MILANI C,TURRONI F,et al.Investigation of the evolutionary development of the genus Bifidobacterium by comparative genomics[J].Applied and Environmental Microbiology,2014,80(20):6383-6394.
[25]OLIVARES M,CASTILLEJO G,VAREA V,et al.Double-blind,randomised,placebo-controlled intervention trial to evaluate the effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed coeliac disease[J].British Journal of Nutrition,2014,112(1):30-40.
[26]LAPARRA J M,OLIVARES M,SANZ Y.Oral administration of Bifidobacterium longum CECT 7347 ameliorates gliadin-induced alterations in liver iron mobilisation[J].British Journal of Nutrition,2013,110(10):1828-1836.