嗜热链球菌KLDS3.1012胞外多糖合成途径的基因组学及表型特征分析
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摘要
在分子水平上挖掘嗜热链球菌KLDS3.1012合成胞外多糖的途径并且通过表型特征进行验证。首先,基于Illumina HiSeq与Illumina MiSeq测序平台对菌株KLDS3.1012进行基因组测序并构建基因组图谱;随后,从糖代谢、糖核苷酸合成、胞外多糖基因簇等方面进行生物信息学分析;最后,利用API 50CH测定菌株KLDS3.1012的糖发酵情况及采用高效离子交换色谱法测定胞外多糖的单糖组成。生物信息学分析结果表明菌株KLDS3.1012具有半乳糖、葡萄糖、果糖、甘露糖、乳糖和蔗糖转运系统和4种糖核苷酸(UDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖和UDP-N-乙酰葡糖胺)合成的相关基因及1个胞外多糖合成基因簇。表型特征分析结果表明菌株KLDS3.1012可以利用以上6种碳源并能形成由鼠李糖、半乳糖和葡萄糖组成的胞外多糖。本研究为分析该菌株合成胞外多糖的遗传基础与胞外多糖结构的关系提供理论依据,并对将其开发为发酵剂具有一定指导意义。
The purpose of this study was to explore the biosynthesis pathway of exopolysaccharides(EPS) in Streptococcus thermophilus KLDS3.1012 at the molecular level and to verify it by the phenotypic traits. Firstly, genomic sequencing was performed using Illumina HiSeq and MiSeq sequencing platforms and a circular genomic map was skeletonized; subsequently, glycometabolism, sugar nucleotides synthesis and EPS gene clusters were analyzed by bioinformatics; finally, carbohydrate utilization was measured by API 50 CH and the monosaccharide composition of the EPS produced by S. thermophilus KLDS3.1012 was analyzed using high performance anion exchange chromatography(HPAEC).In silico bioinformatic analysis unveiled that the genome of S. thermophilus KLDS3.1012 was related to sucrose, fructose,mannose, glucose, galactose and lactose uptake system, and biosynthesis of four sugar nucleotides(UDP-gluctose, dTDPrhamnose, UDP-galactose and UDP-N-acetylglucosamine) as well as an EPS gene cluster. The phenotypic traits revealed that S. thermophilus KLDS3.1012 could utilize these six sugars and the EPS produced it was composed of glucose, rhamnose and galactose. These results provide a theoretical basis for better understanding the relationship between the genetic elements and the structure of EPS in S. thermophilus KLDS3.1012 and for facilitating its potential applications as starter culture in the dairy industry.
The purpose of this study was to explore the biosynthesis pathway of exopolysaccharides(EPS) in Streptococcus thermophilus KLDS3.1012 at the molecular level and to verify it by the phenotypic traits. Firstly, genomic sequencing was performed using Illumina HiSeq and MiSeq sequencing platforms and a circular genomic map was skeletonized; subsequently, glycometabolism, sugar nucleotides synthesis and EPS gene clusters were analyzed by bioinformatics; finally, carbohydrate utilization was measured by API 50 CH and the monosaccharide composition of the EPS produced by S. thermophilus KLDS3.1012 was analyzed using high performance anion exchange chromatography(HPAEC).In silico bioinformatic analysis unveiled that the genome of S. thermophilus KLDS3.1012 was related to sucrose, fructose,mannose, glucose, galactose and lactose uptake system, and biosynthesis of four sugar nucleotides(UDP-gluctose, dTDPrhamnose, UDP-galactose and UDP-N-acetylglucosamine) as well as an EPS gene cluster. The phenotypic traits revealed that S. thermophilus KLDS3.1012 could utilize these six sugars and the EPS produced it was composed of glucose, rhamnose and galactose. These results provide a theoretical basis for better understanding the relationship between the genetic elements and the structure of EPS in S. thermophilus KLDS3.1012 and for facilitating its potential applications as starter culture in the dairy industry.
引文
[1]LIU M,SIEZEN R J,NAUTA A.In Silico prediction of horizontal gene transfer events in Lactobacillus bulgaricus and Streptococcus thermophilus reveals protocooperation in yogurt manufacturing[J].Applied and Environmental Microbiology,2009,75(12):4120-4129.DOI:10.1128/AEM.02898-08.
[2]SETTACHAIMONGKON S,NOUT M R,FERNANDES E C A,et al.Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp.bulgaricus on the metabolite profile of set-yoghurt[J].International Journal of Food Microbiology,2014,177:29-36.DOI:10.1016/j.ijfoodmicro.2014.02.008.
[3]PURWANDARI U,SHAH N P,VASILJEVIC T.Effects of exopolysaccharide-producing strains of Streptococcus thermophilus on technological and rheological properties of set-type yoghurt[J].International Dairy Journal,2007,17(11):1344-1352.DOI:10.1016/j.idairyj.2007.01.018.
[4]MENDE S,ROHM H,JAROS D.Influence of exopolysaccharides on the structure,texture,stability and sensory properties of yoghurt and related products[J].International Dairy Journal,2016,52:57-71.DOI:10.1016/j.idairyj.2015.08.002.
[5]ZHANG J,CAO Y,WANG J,et al.Physicochemical characteristics and bioactivities of the exopolysaccharide and its sulphated polymer from Streptococcus thermophilus GST-6[J].Carbohydrate Polymers,2016,146:368-375.DOI:10.1016/j.carbpol.2016.03.063.
[6]PATTEN D A,LEIVERS S,CHADHA M J,et al.The structure and immunomodulatory activity on intestinal epithelial cells of the EPSs isolated from Lactobacillus helveticus sp.Rosyjski and Lactobacillus acidophilus sp.5e2[J].Carbohydrate Research,2014,384:119-127.DOI:10.1016/j.carres.2013.12.008.
[7]M A E D A H,Z H U X,O M U R A K,e t a l.E f f e c t s o f a n exopolysaccharide(kefiran)on lipids,blood pressure,blood glucose,and constipation[J].Biofactors,2004,22(1/2/3/4):197-200.DOI:10.1002/biof.5520220141.
[8]ZHANG J,ZHAO X,JIANG Y,et al.Antioxidant status and gut microbiota change in an aging mouse model as influenced by exopolysaccharide produced by Lactobacillus plantarum YW11isolated from Tibetan kefir[J].Journal of Dairy Science,2017.DOI:10.3168/jds.2016-12480.
[9]DEEPAK V,RAMACHANDRAN S,BALAHMAR R M,et al.In vitro evaluation of anticancer properties of exopolysaccharides from Lactobacillus acidophilus in colon cancer cell lines[J].In Vitro Cellular&Developmental Biology-Animal,2016,52(2):163-173.DOI:10.1007/s11626-015-9970-3.
[10]LAWS A,GU Y,MARSHALL V.Biosynthesis,characterisation,and design of bacterial exopolysaccharides from lactic acid bacteria[J].Biotechnology Advances,2001,19(8):597-625.DOI:10.1016/S0734-9750(01)00084-2.
[11]郑华军.保加利亚乳酸杆菌工业生产菌株2038的基因组学分析[D].上海:复旦大学,2010.
[12]MADHURI K V,PRABHAKAR K V.Microbial exopolysaccharides:biosynthesis and potential applications[J].Oriental Journal of Chemistry,2014,30(3):1401-1410.DOI:10.13005/ojc/300362.
[13]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.DOI:10.3969/j.issn.1001-2230.2013.03.007.
[14]田辉.嗜热链球菌高密度培养与直投式发酵剂开发[D].哈尔滨:东北农业大学,2012.
[15]LUO R,LIU B,XIE Y,et al.SOAPdenovo2:an empirically improved memory-efficient short-read de novo assembler[J].Gigascience,2012,1(1):18.DOI:10.1186/2047-217X-1-18.
[16]WU S,ZHU Z,FU L,et al.WebMGA:a customizable web server for fast metagenomic sequence analysis[J].BMC Genomics,2011,12(1):444.DOI:10.1186/1471-2164-12-444.
[17]GRANT J R,STOTHARD P.The CGView Server:a comparative genomics tool for circular genomes[J].Nucleic Acids Research,2008,36(Suppl 2):W181-W184.DOI:10.1093/nar/gkn179.
[18]KANEHISA M,GOTO S.KEGG:kyoto encyclopedia of genes and genomes[J].Nucleic Acids Research,2000,28(1):27-30.DOI:10.1093/nar/28.1.27.
[19]SIGUIER P,PéROCHON J,LESTRADE L,et al.ISfinder:the reference centre for bacterial insertion sequences[J].Nucleic Acids Research,2006,34(Suppl 1):D32-D36.DOI:10.1093/nar/gkj014.
[20]CARVER T J,RUTHERFORD K M,BERRIMAN M,et al.ACT:the Artemis comparison tool[J].Bioinformatics,2005,21(16):3422-3423.DOI:10.1093/bioinformatics/bti553.
[21]邵丽.产胞外多糖乳杆菌的筛选及其多糖的分离,结构和生物活性研究[D].无锡:江南大学,2015.
[22]REN W,XIA Y,WANG G,et al.Bioactive exopolysaccharides from a S.thermophilus strain:screening,purification and characterization[J].International Journal of Biological Macromolecules,2016,86:402-407.DOI:10.1016/j.ijbiomac.2016.01.085.
[23]WU Q L,TUN H M,LEUNG F C,et al.Genomic insights into high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275[J].Scientific Reports,2014,4:4974.DOI:10.1038/srep04974.
[24]BROADBENT J R,MCMAHON D J,WELKER D L,et al.Biochemistry,genetics,and applications of exopolysaccharide production in Streptococcus thermophilus:a review[J].Journal of Dairy Science,2003,86(2):407-423.DOI:10.3168/jds.S0022-0302(03)73619-4.
[25]TYVAERT G,MOREL C,JOLY J,et al.The constant gene orf14.9,which belongs to the variable EPS(exopolysaccharide)cluster,is involved in the cell growth of Streptococcus thermophilus[J].Canadian Journal of Microbiology,2006,52(9):908-912.DOI:10.1139/w06-047.
[26]IYER R,TOMAR S K,UMA MAHESWARI T,et al.Streptococcus thermophilus strains:multifunctional lactic acid bacteria[J].International Dairy Journal,2010,20(3):133-141.DOI:10.1016/j.idairyj.2009.10.005.
[27]GALIA W,PERRIN C,GENAY M,et al.Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties[J].International Dairy Journal,2009,19(2):89-95.DOI:10.1016/j.idairyj.2008.08.004.
[28]DE VUYST L,WECKX S,RAVYTS F,et al.New insights into the exopolysaccharide production of Streptococcus thermophilus[J].International Dairy Journal,2011,21(9):586-591.DOI:10.1016/j.idairyj.2011.03.016.
[29]BOLOTIN A,QUINQUIS B,RENAULT P,et al.Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[J].Nature Biotechnology,2004,22(12):1554-1558.DOI:10.1038/nbt1034.
[30]SUN Z H,CHEN X,WANG J,et al.Complete genome sequence of Streptococcus thermophilus strain ND03[J].Journal of Bacteriology,2011,193(3):793-794.DOI:10.1128/JB.01374-10.
[31]BAI Y,SUN E,SHI Y,et al.Complete genome sequence of Streptococcus thermophilus MN-BM-A01,a strain with high exopolysaccharides production[J].Journal of Biotechnology,2016,224:45-46.DOI:10.1016/j.jbiotec.2016.03.003.
[32]VENDRAMIN V,TREU L,CAMPANARO S,et al.Genome comparison and physiological characterization of eight Streptococcus thermophilus strains isolated from Italian dairy products[J].Food Microbiology,2017,63:47-57.DOI:10.1016/j.fm.2016.11.002.
[33]CUI Y,XU T,QU X,et al.New insights into various production characteristics of Streptococcus thermophilus strains[J].International Journal of Molecular Sciences,2016,17(10):1701.DOI:10.3390/ijms17101701.
[34]ERKUS O,OKUKLU B,YENIDUNYA A F,et al.High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts[J].LWT-Food Science and Technology,2014,58(2):348-354.DOI:10.1016/j.lwt.2013.03.007.
[35]MORANDI S,BRASCA M.Safety aspects,genetic diversity and technological characterisation of wild-type Streptococcus thermophilus strains isolated from north Italian traditional cheeses[J].Food Control,2012,23(1):203-209.DOI:10.1016/j.foodcont.2011.07.011.
[36]NATHANI N M,PATEL A K,SENAN S,et al.Genomic analysis of dairy starter culture Streptococcus thermophilus MTCC 5461[J].Journal of Microbiology and Biotechnology,2013,23(4):459-466.DOI:10.3389/fmicb.2012.00435.
[37]P A C H E K R E P A P O L U,L U C E Y J A,G O N G Y,e t a l.Characterization of the chemical structures and physical properties of exopolysaccharides produced by various Streptococcus thermophilus strains[J].Journal of Dairy Science,2017,100(5):3424-3435.DOI:10.3168/jds.2016-12125.
[38]CUI Y,JIANG X,HAO M,et al.New advances in exopolysaccharides production of Streptococcus thermophilus[J].Archives of Microbiology,2017,199(6):799-809.DOI:10.1007/s00203-017-1366-1.
[2]SETTACHAIMONGKON S,NOUT M R,FERNANDES E C A,et al.Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp.bulgaricus on the metabolite profile of set-yoghurt[J].International Journal of Food Microbiology,2014,177:29-36.DOI:10.1016/j.ijfoodmicro.2014.02.008.
[3]PURWANDARI U,SHAH N P,VASILJEVIC T.Effects of exopolysaccharide-producing strains of Streptococcus thermophilus on technological and rheological properties of set-type yoghurt[J].International Dairy Journal,2007,17(11):1344-1352.DOI:10.1016/j.idairyj.2007.01.018.
[4]MENDE S,ROHM H,JAROS D.Influence of exopolysaccharides on the structure,texture,stability and sensory properties of yoghurt and related products[J].International Dairy Journal,2016,52:57-71.DOI:10.1016/j.idairyj.2015.08.002.
[5]ZHANG J,CAO Y,WANG J,et al.Physicochemical characteristics and bioactivities of the exopolysaccharide and its sulphated polymer from Streptococcus thermophilus GST-6[J].Carbohydrate Polymers,2016,146:368-375.DOI:10.1016/j.carbpol.2016.03.063.
[6]PATTEN D A,LEIVERS S,CHADHA M J,et al.The structure and immunomodulatory activity on intestinal epithelial cells of the EPSs isolated from Lactobacillus helveticus sp.Rosyjski and Lactobacillus acidophilus sp.5e2[J].Carbohydrate Research,2014,384:119-127.DOI:10.1016/j.carres.2013.12.008.
[7]M A E D A H,Z H U X,O M U R A K,e t a l.E f f e c t s o f a n exopolysaccharide(kefiran)on lipids,blood pressure,blood glucose,and constipation[J].Biofactors,2004,22(1/2/3/4):197-200.DOI:10.1002/biof.5520220141.
[8]ZHANG J,ZHAO X,JIANG Y,et al.Antioxidant status and gut microbiota change in an aging mouse model as influenced by exopolysaccharide produced by Lactobacillus plantarum YW11isolated from Tibetan kefir[J].Journal of Dairy Science,2017.DOI:10.3168/jds.2016-12480.
[9]DEEPAK V,RAMACHANDRAN S,BALAHMAR R M,et al.In vitro evaluation of anticancer properties of exopolysaccharides from Lactobacillus acidophilus in colon cancer cell lines[J].In Vitro Cellular&Developmental Biology-Animal,2016,52(2):163-173.DOI:10.1007/s11626-015-9970-3.
[10]LAWS A,GU Y,MARSHALL V.Biosynthesis,characterisation,and design of bacterial exopolysaccharides from lactic acid bacteria[J].Biotechnology Advances,2001,19(8):597-625.DOI:10.1016/S0734-9750(01)00084-2.
[11]郑华军.保加利亚乳酸杆菌工业生产菌株2038的基因组学分析[D].上海:复旦大学,2010.
[12]MADHURI K V,PRABHAKAR K V.Microbial exopolysaccharides:biosynthesis and potential applications[J].Oriental Journal of Chemistry,2014,30(3):1401-1410.DOI:10.13005/ojc/300362.
[13]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.DOI:10.3969/j.issn.1001-2230.2013.03.007.
[14]田辉.嗜热链球菌高密度培养与直投式发酵剂开发[D].哈尔滨:东北农业大学,2012.
[15]LUO R,LIU B,XIE Y,et al.SOAPdenovo2:an empirically improved memory-efficient short-read de novo assembler[J].Gigascience,2012,1(1):18.DOI:10.1186/2047-217X-1-18.
[16]WU S,ZHU Z,FU L,et al.WebMGA:a customizable web server for fast metagenomic sequence analysis[J].BMC Genomics,2011,12(1):444.DOI:10.1186/1471-2164-12-444.
[17]GRANT J R,STOTHARD P.The CGView Server:a comparative genomics tool for circular genomes[J].Nucleic Acids Research,2008,36(Suppl 2):W181-W184.DOI:10.1093/nar/gkn179.
[18]KANEHISA M,GOTO S.KEGG:kyoto encyclopedia of genes and genomes[J].Nucleic Acids Research,2000,28(1):27-30.DOI:10.1093/nar/28.1.27.
[19]SIGUIER P,PéROCHON J,LESTRADE L,et al.ISfinder:the reference centre for bacterial insertion sequences[J].Nucleic Acids Research,2006,34(Suppl 1):D32-D36.DOI:10.1093/nar/gkj014.
[20]CARVER T J,RUTHERFORD K M,BERRIMAN M,et al.ACT:the Artemis comparison tool[J].Bioinformatics,2005,21(16):3422-3423.DOI:10.1093/bioinformatics/bti553.
[21]邵丽.产胞外多糖乳杆菌的筛选及其多糖的分离,结构和生物活性研究[D].无锡:江南大学,2015.
[22]REN W,XIA Y,WANG G,et al.Bioactive exopolysaccharides from a S.thermophilus strain:screening,purification and characterization[J].International Journal of Biological Macromolecules,2016,86:402-407.DOI:10.1016/j.ijbiomac.2016.01.085.
[23]WU Q L,TUN H M,LEUNG F C,et al.Genomic insights into high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275[J].Scientific Reports,2014,4:4974.DOI:10.1038/srep04974.
[24]BROADBENT J R,MCMAHON D J,WELKER D L,et al.Biochemistry,genetics,and applications of exopolysaccharide production in Streptococcus thermophilus:a review[J].Journal of Dairy Science,2003,86(2):407-423.DOI:10.3168/jds.S0022-0302(03)73619-4.
[25]TYVAERT G,MOREL C,JOLY J,et al.The constant gene orf14.9,which belongs to the variable EPS(exopolysaccharide)cluster,is involved in the cell growth of Streptococcus thermophilus[J].Canadian Journal of Microbiology,2006,52(9):908-912.DOI:10.1139/w06-047.
[26]IYER R,TOMAR S K,UMA MAHESWARI T,et al.Streptococcus thermophilus strains:multifunctional lactic acid bacteria[J].International Dairy Journal,2010,20(3):133-141.DOI:10.1016/j.idairyj.2009.10.005.
[27]GALIA W,PERRIN C,GENAY M,et al.Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties[J].International Dairy Journal,2009,19(2):89-95.DOI:10.1016/j.idairyj.2008.08.004.
[28]DE VUYST L,WECKX S,RAVYTS F,et al.New insights into the exopolysaccharide production of Streptococcus thermophilus[J].International Dairy Journal,2011,21(9):586-591.DOI:10.1016/j.idairyj.2011.03.016.
[29]BOLOTIN A,QUINQUIS B,RENAULT P,et al.Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[J].Nature Biotechnology,2004,22(12):1554-1558.DOI:10.1038/nbt1034.
[30]SUN Z H,CHEN X,WANG J,et al.Complete genome sequence of Streptococcus thermophilus strain ND03[J].Journal of Bacteriology,2011,193(3):793-794.DOI:10.1128/JB.01374-10.
[31]BAI Y,SUN E,SHI Y,et al.Complete genome sequence of Streptococcus thermophilus MN-BM-A01,a strain with high exopolysaccharides production[J].Journal of Biotechnology,2016,224:45-46.DOI:10.1016/j.jbiotec.2016.03.003.
[32]VENDRAMIN V,TREU L,CAMPANARO S,et al.Genome comparison and physiological characterization of eight Streptococcus thermophilus strains isolated from Italian dairy products[J].Food Microbiology,2017,63:47-57.DOI:10.1016/j.fm.2016.11.002.
[33]CUI Y,XU T,QU X,et al.New insights into various production characteristics of Streptococcus thermophilus strains[J].International Journal of Molecular Sciences,2016,17(10):1701.DOI:10.3390/ijms17101701.
[34]ERKUS O,OKUKLU B,YENIDUNYA A F,et al.High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts[J].LWT-Food Science and Technology,2014,58(2):348-354.DOI:10.1016/j.lwt.2013.03.007.
[35]MORANDI S,BRASCA M.Safety aspects,genetic diversity and technological characterisation of wild-type Streptococcus thermophilus strains isolated from north Italian traditional cheeses[J].Food Control,2012,23(1):203-209.DOI:10.1016/j.foodcont.2011.07.011.
[36]NATHANI N M,PATEL A K,SENAN S,et al.Genomic analysis of dairy starter culture Streptococcus thermophilus MTCC 5461[J].Journal of Microbiology and Biotechnology,2013,23(4):459-466.DOI:10.3389/fmicb.2012.00435.
[37]P A C H E K R E P A P O L U,L U C E Y J A,G O N G Y,e t a l.Characterization of the chemical structures and physical properties of exopolysaccharides produced by various Streptococcus thermophilus strains[J].Journal of Dairy Science,2017,100(5):3424-3435.DOI:10.3168/jds.2016-12125.
[38]CUI Y,JIANG X,HAO M,et al.New advances in exopolysaccharides production of Streptococcus thermophilus[J].Archives of Microbiology,2017,199(6):799-809.DOI:10.1007/s00203-017-1366-1.