黄色瘤胃球菌纤维小体脚手架蛋白ScaC基因的克隆及其在大肠杆菌中的表达
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摘要
为获得纤维小体(cellulosome)的基本元件,本试验以滩羊瘤胃液微生物混合DNA为模板,根据黄色瘤胃球菌纤维小体的脚手架蛋白ScaC基因序列(GenBank登录号:JN109634.1)设计特异性引物,扩增纤维小体脚手架蛋白基因,并对其进行克隆、测序及核苷酸和氨基酸序列分析;同时构建脚手架蛋白基因原核表达载体,分析其在大肠杆菌中的表达情况。结果显示,试验利用1对引物同时成功克隆获得2个脚手架蛋白编码基因,其中一个基因全长867 bp,编码288个氨基酸,命名为ScaC2基因;另一个基因全长870 bp,编码289个氨基酸,命名为ScaC7基因。核苷酸序列分析表明,脚手架蛋白基因ScaC2与ScaC7的核苷酸序列相似性为74.1%,ScaC2基因与黄色瘤胃球菌AGY80P318及ScaC7基因与黄色瘤胃球菌DA640P037 ScaC基因核苷酸序列相似性均为99%;氨基酸保守序列分析显示,ScaC2和ScaC7氨基酸序列均具有典型的脚手架蛋白结构域,含有1个Ⅰ型黏附域和1个Ⅰ型锚定域。蛋白表达分析显示,ScaC2和ScaC7基因均能在大肠杆菌中实现可溶性表达,表达产物大小约为33 ku。本试验克隆获得的纤维小体的脚手架蛋白基因ScaC2和ScaC7可为后续人工纤维小体的构建提供基本材料。
To obtain the modules used to assembly the cellulosomes,the total microbial genomic DNA of Tan sheep were used to amplify the scaffoldin coding gene ScaC using a pair of primers designed according to the published sequences of Ruminococcus flavefaciens ScaC gene(GenBank accession No.:JN109634.1).The scaffoldin coding gene was amplified,cloned and sequenced,and the nucleotide and amino acid sequences were analyzed.Meanwhile,the expression of the target genes in E.coli was analyzed.The results showed that two scaffolding protein coding genes were successfully cloned using a pair of primers.One of them was 867 bp in length,encoding 288 amino acids,named as ScaC2,and the other was 870 bp in length,encoding 289 amino acids,named as ScaC7.Nucleotide sequence analysis results showed that the similarity of ScaC2 and ScaC7 genes was 74.1%.The similarity of ScaC2 and ScaC7 genes with R.flavefaciens isolate AGY80 P318 and DA640 P037 ScaC genes were 99% respectively.Conservative amino acid sequence analysis showed that both ScaC2 and ScaC7 amino acid sequences had typical scaffold protein domain,including a type Ⅰ adhesion domain and a type Ⅰ anchorage domain.Both genes were successfully expressed in Escherichia coli BL21(DE3) and the expression products showed a molecular weight of 33 ku by SDS-PAGE.The two genes could be used as basic materials for the reconstruction of mini-cellulosomes in the future.
To obtain the modules used to assembly the cellulosomes,the total microbial genomic DNA of Tan sheep were used to amplify the scaffoldin coding gene ScaC using a pair of primers designed according to the published sequences of Ruminococcus flavefaciens ScaC gene(GenBank accession No.:JN109634.1).The scaffoldin coding gene was amplified,cloned and sequenced,and the nucleotide and amino acid sequences were analyzed.Meanwhile,the expression of the target genes in E.coli was analyzed.The results showed that two scaffolding protein coding genes were successfully cloned using a pair of primers.One of them was 867 bp in length,encoding 288 amino acids,named as ScaC2,and the other was 870 bp in length,encoding 289 amino acids,named as ScaC7.Nucleotide sequence analysis results showed that the similarity of ScaC2 and ScaC7 genes was 74.1%.The similarity of ScaC2 and ScaC7 genes with R.flavefaciens isolate AGY80 P318 and DA640 P037 ScaC genes were 99% respectively.Conservative amino acid sequence analysis showed that both ScaC2 and ScaC7 amino acid sequences had typical scaffold protein domain,including a type Ⅰ adhesion domain and a type Ⅰ anchorage domain.Both genes were successfully expressed in Escherichia coli BL21(DE3) and the expression products showed a molecular weight of 33 ku by SDS-PAGE.The two genes could be used as basic materials for the reconstruction of mini-cellulosomes in the future.
引文
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[5] ARTZI L,BAYER E A,MORA?S S.Cellulosomes:Bacterial nanomachines for dismantling plant polysaccharides[J].Nature Reviews Microbiology,2017,15(2):83-95.
[6] BHATTACHARYA A S,BHATTACHARYA A,PLETSCHKE B I.Synergism of fungal and bacterial cellulases and hemicellulases:A novel perspective for enhanced bio-ethanol production[J].Biotechnology Letters,2015,37(6):1117-1129.
[7] 冯薇,王加启,刘开朗,等.参与瘤胃内纤维素降解过程的主要微生物研究进展[J].中国畜牧兽医,2011,38(2):37-42.FENG W,WANG J Q,LIU K L,et al.The research advance of the processes of cellulose degradation in rumen and related microorganisms[J].China Animal Husbandry & Veterinary Medicine,2011,38(2):37-42.(in Chinese)
[8] DOI R H,KOSUGI A.Cellulosomes:Plant-cell-wall-degrading enzyme complexes[J].Nature Reviews Microbiology,2004,2(7):541-551.
[9] 朱兆静,潘虎,郭俊,等.纤维小体结构及其功能的研究进展[J].江苏农业科学,2018,46(19):12-16.ZHU Z J,PAN H,GUO J,et al.Research progress on structure and function of cellulosome[J].Jiangsu Agricultural Sciences,2018,46(19):12-16.(in Chinese)
[10] LAMED R,SETTER E,BAYER E A.Characterization of a cellulose-binding,cellulase-containing complex in Clostridium thermocellum[J].Journal of Bacteriology,1983,156(2):828-836.
[11] MINGARDON F,BAGERT J D,MAISONNIER C,et al.Comparison of family 9 cellulases from mesophilic and thermophilic bacteria[J].Applied and Environmental Microbiology,2011,77(4):1436-1442.
[12] DOI R H,KOSUGI A,MURASHIMA K,et al.Cellulosomes from mesophilic bacteria[J].Journal of Bacteriology,2003,185(20):5907-5914.
[13] PASON P,KOSUGI A,WAEONUKUL R,et al.Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6.[J].Applied Microbiology and Biotechnology,2010,85(3):573-580.
[14] RAVACHOL J,BORNE R,TARDIF C,et al.Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum[J].Journal of Biological Chemistry,2014,289(11):7335-7348.
[15] CASPI J,BARAK Y,HAIMOVITZ R,et al.Effect of linker length and dockerin position on conversion of a thermobifida fusca endoglucanase to the cellulosomal mode[J].Applied and Environmental Microbiology,2009,75(23):7335-7342.
[16] 王金兰,王禄山,刘巍峰,等.降解纤维素的“超分子机器”研究进展[J].生物化学与生物物理进展,2011,38(1):28-35.WANG J L,WANG L S,LIU W F,et al.Research advances on the assembly mode of cellulosomal macromolecular complexes[J].Progress in Biochemistry and Biophysics,2011,38(1):28-35.(in Chinese)
[17] VAN DE VEN T,KADLA J.Cellulose——Biomass Conversion[M].IntechOpen,2013.
[18] CAO P H,WANG L,ZHOU G X,et al.Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E.coli and Lactobacillus[J].Plasmid,2014,76:40-46.
[19] NOACH I,LEVY-ASSARAF M,LAMED R,et al.Modular arrangement of a cellulosomal scaffoldin subunit revealed from the crystal structure of a cohesin dyad[J].Journal of Molecular Biology,2010,399(2):294-305.
[20] BAYER E A,SHIMON L J W,SHOHAM Y,et al.Cellulosomes structure and ultrastructure[J].Structural Biology,1998,124(2-3):221-234.
[21] BULE P,PIRES V M,FONTES C M,et al.Cellulosome assembly:Paradigms are meant to be broken[J].Current Opinion in Structural Biology,2018,49:154-161.
[22] ELKINS J G,RAMAN B,KELLER M.Engineered microbial systems for enhanced conversion of lignocellulosic biomass[J].Current Opinion in Biotechnology,2010,21(5):657-662.
[23] PONPIUM P,RATANAKHANOKCHAI K,KYU K L.Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp.strain P-1[J].Enzyme and Microbial Technology,2000,26(5-6):459-465.
[24] FELIX C R,LJUNGDAHL L G.The cellulosome:The exocellular organelle of Clostridium[J].Annual Review of Microbiology,1993,47(47):791-819.
[25] HAN X,YANG Y,YAN H,et al.Rumen bacterial diversity of 80 to 110-day-old goats using 16S rRNA sequencing[J].PLoS One,2015,10(2):e0117811.
[26] RINCON M T,MARTIN J C,AURILIA V,et al.ScaC,an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome[J].Journal of Bacteriology,2004,186(9):2576-2585.
[27] KARPOL A,JOBBY M K,SLUTZKI M,et al.Structural and functional characterization of a novel type-Ⅲ dockerin from Ruminococcus flavefaciens[J].FEBS Letters,2013,587(1):30-36.
[28] ANDERSON T D,ROBSON S A,JIANG X W,et al.Assembly of minicellulosomes on the surface of Bacillus subtilis[J].Applied and Environmental Microbiology,2011,77(14):4849-4858.
[29] FAN L H,ZHANG Z J,YU X Y,et al.Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production[J].Proceedings of the National Academy of Sciences,2012,109(33):13260-13265.
[30] STERN J,MORA?S S,BENDAVID Y,et al.Assembly of synthetic functional cellulosomal structures onto the cell surface of Lactobacillus plantarum,a potent member of the gut microbiome[J].Applied and Environmental Microbiology,2018,84(8):e00282-18.
[31] 李旺.纤维素酶基因重组野生型乳酸杆菌表达系统的构建[D].杨凌:西北农林科技大学,2010.LI W.Construction of wild type Lactobacillus expression system integrated with cellulase genes[D].Yangling:Northwest A & F University,2010.(in Chinese)
[32] ZHANG Y H P,HIMMEL M E,MIELENZ J R.Outlook for cellulase improvement:Screening and selection strategies[J].Biotechnology Advances,2006,24(5):452-481.
[33] 陈宁,杜济良,杨玥,等.自组装嵌合纤维小体酿酒酵母菌群的乙醇发酵研究[J].太阳能学报,2018,39(8):2103-2109.CHEN N,DU J L,YANG Y,et al.Study of ethanol fermentation of self-assembled designer cellulosome on Saccharomyces cerevisiae[J].Acta Energiae Solaris Sinica,2018,39(8):2103-2109.(in Chinese)
[34] TANG H,WANG J,WANG S,et al.Efficient yeast surface-display of novel complex synthetic cellulosomes[J].Microbial Cell Factories,2018,17(1):122.
[2] 朱勇,余思佳,包健,等.发酵鲜食大豆秸秆对母羊繁殖性能、初乳品质及消化性能的影响[J].中国畜牧兽医,2017,44(1):100-105.ZHU Y,YU S J,BAO J,et al.The effect of fermented vegetable soybean straw on reproductive performances,colostrum quality and digestion in adult female goat[J].China Animal Husbandry & Veterinary Medicine,2017,44(1):100-105.(in Chinese)
[3] 韩东魁,耿春银,张敏,等.负离子中药复合制剂对延边黄牛生长性能、养分表观消化率及血液生化指标的影响[J].中国畜牧兽医,2018,45(7):1856-1862.HAN D K,GENG C Y,ZHANG M,et al.Effect of compound preparations of negative ion and Chinese medicine on growth performance,nutrient apparent digestibility and blood biochemical indexes of Yanbian Yellow cattle[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1856-1862.(in Chinese)
[4] 张旭,郭盼盼,金锡九,等.不同温度条件下发酵时间对完全混合发酵日粮(TMF)发酵品质的影响[J].中国畜牧兽医,2018,45(3):673-681.ZHANG X,GUO P P,JIN X J,et al.Effects of fermentation time on fermentation quality of the total mixed fermentation ration (TMF) at different fermentation temperature[J].China Animal Husbandry & Veterinary Medicine,2018,45(3):673-681.(in Chinese)
[5] ARTZI L,BAYER E A,MORA?S S.Cellulosomes:Bacterial nanomachines for dismantling plant polysaccharides[J].Nature Reviews Microbiology,2017,15(2):83-95.
[6] BHATTACHARYA A S,BHATTACHARYA A,PLETSCHKE B I.Synergism of fungal and bacterial cellulases and hemicellulases:A novel perspective for enhanced bio-ethanol production[J].Biotechnology Letters,2015,37(6):1117-1129.
[7] 冯薇,王加启,刘开朗,等.参与瘤胃内纤维素降解过程的主要微生物研究进展[J].中国畜牧兽医,2011,38(2):37-42.FENG W,WANG J Q,LIU K L,et al.The research advance of the processes of cellulose degradation in rumen and related microorganisms[J].China Animal Husbandry & Veterinary Medicine,2011,38(2):37-42.(in Chinese)
[8] DOI R H,KOSUGI A.Cellulosomes:Plant-cell-wall-degrading enzyme complexes[J].Nature Reviews Microbiology,2004,2(7):541-551.
[9] 朱兆静,潘虎,郭俊,等.纤维小体结构及其功能的研究进展[J].江苏农业科学,2018,46(19):12-16.ZHU Z J,PAN H,GUO J,et al.Research progress on structure and function of cellulosome[J].Jiangsu Agricultural Sciences,2018,46(19):12-16.(in Chinese)
[10] LAMED R,SETTER E,BAYER E A.Characterization of a cellulose-binding,cellulase-containing complex in Clostridium thermocellum[J].Journal of Bacteriology,1983,156(2):828-836.
[11] MINGARDON F,BAGERT J D,MAISONNIER C,et al.Comparison of family 9 cellulases from mesophilic and thermophilic bacteria[J].Applied and Environmental Microbiology,2011,77(4):1436-1442.
[12] DOI R H,KOSUGI A,MURASHIMA K,et al.Cellulosomes from mesophilic bacteria[J].Journal of Bacteriology,2003,185(20):5907-5914.
[13] PASON P,KOSUGI A,WAEONUKUL R,et al.Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6.[J].Applied Microbiology and Biotechnology,2010,85(3):573-580.
[14] RAVACHOL J,BORNE R,TARDIF C,et al.Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum[J].Journal of Biological Chemistry,2014,289(11):7335-7348.
[15] CASPI J,BARAK Y,HAIMOVITZ R,et al.Effect of linker length and dockerin position on conversion of a thermobifida fusca endoglucanase to the cellulosomal mode[J].Applied and Environmental Microbiology,2009,75(23):7335-7342.
[16] 王金兰,王禄山,刘巍峰,等.降解纤维素的“超分子机器”研究进展[J].生物化学与生物物理进展,2011,38(1):28-35.WANG J L,WANG L S,LIU W F,et al.Research advances on the assembly mode of cellulosomal macromolecular complexes[J].Progress in Biochemistry and Biophysics,2011,38(1):28-35.(in Chinese)
[17] VAN DE VEN T,KADLA J.Cellulose——Biomass Conversion[M].IntechOpen,2013.
[18] CAO P H,WANG L,ZHOU G X,et al.Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E.coli and Lactobacillus[J].Plasmid,2014,76:40-46.
[19] NOACH I,LEVY-ASSARAF M,LAMED R,et al.Modular arrangement of a cellulosomal scaffoldin subunit revealed from the crystal structure of a cohesin dyad[J].Journal of Molecular Biology,2010,399(2):294-305.
[20] BAYER E A,SHIMON L J W,SHOHAM Y,et al.Cellulosomes structure and ultrastructure[J].Structural Biology,1998,124(2-3):221-234.
[21] BULE P,PIRES V M,FONTES C M,et al.Cellulosome assembly:Paradigms are meant to be broken[J].Current Opinion in Structural Biology,2018,49:154-161.
[22] ELKINS J G,RAMAN B,KELLER M.Engineered microbial systems for enhanced conversion of lignocellulosic biomass[J].Current Opinion in Biotechnology,2010,21(5):657-662.
[23] PONPIUM P,RATANAKHANOKCHAI K,KYU K L.Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp.strain P-1[J].Enzyme and Microbial Technology,2000,26(5-6):459-465.
[24] FELIX C R,LJUNGDAHL L G.The cellulosome:The exocellular organelle of Clostridium[J].Annual Review of Microbiology,1993,47(47):791-819.
[25] HAN X,YANG Y,YAN H,et al.Rumen bacterial diversity of 80 to 110-day-old goats using 16S rRNA sequencing[J].PLoS One,2015,10(2):e0117811.
[26] RINCON M T,MARTIN J C,AURILIA V,et al.ScaC,an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome[J].Journal of Bacteriology,2004,186(9):2576-2585.
[27] KARPOL A,JOBBY M K,SLUTZKI M,et al.Structural and functional characterization of a novel type-Ⅲ dockerin from Ruminococcus flavefaciens[J].FEBS Letters,2013,587(1):30-36.
[28] ANDERSON T D,ROBSON S A,JIANG X W,et al.Assembly of minicellulosomes on the surface of Bacillus subtilis[J].Applied and Environmental Microbiology,2011,77(14):4849-4858.
[29] FAN L H,ZHANG Z J,YU X Y,et al.Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production[J].Proceedings of the National Academy of Sciences,2012,109(33):13260-13265.
[30] STERN J,MORA?S S,BENDAVID Y,et al.Assembly of synthetic functional cellulosomal structures onto the cell surface of Lactobacillus plantarum,a potent member of the gut microbiome[J].Applied and Environmental Microbiology,2018,84(8):e00282-18.
[31] 李旺.纤维素酶基因重组野生型乳酸杆菌表达系统的构建[D].杨凌:西北农林科技大学,2010.LI W.Construction of wild type Lactobacillus expression system integrated with cellulase genes[D].Yangling:Northwest A & F University,2010.(in Chinese)
[32] ZHANG Y H P,HIMMEL M E,MIELENZ J R.Outlook for cellulase improvement:Screening and selection strategies[J].Biotechnology Advances,2006,24(5):452-481.
[33] 陈宁,杜济良,杨玥,等.自组装嵌合纤维小体酿酒酵母菌群的乙醇发酵研究[J].太阳能学报,2018,39(8):2103-2109.CHEN N,DU J L,YANG Y,et al.Study of ethanol fermentation of self-assembled designer cellulosome on Saccharomyces cerevisiae[J].Acta Energiae Solaris Sinica,2018,39(8):2103-2109.(in Chinese)
[34] TANG H,WANG J,WANG S,et al.Efficient yeast surface-display of novel complex synthetic cellulosomes[J].Microbial Cell Factories,2018,17(1):122.