Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes

Biotechnology for Biofuels

Xiaofei Li¹  Peter Jackson¹  Denis V Rubtsov¹  Nuno Faria-Blanc¹  Jenny C Mortimer¹  Simon R Turner³  Kristian B Krogh²  Katja S Johansen²  Paul Dupree¹ 

[1] Department of Biochemistry, Building O, Downing Site, University of Cambridge, Cambridge CB2 1QW, UK

[2] Novozymes A/S, Krogshoejvej 36, Bagsvaerd 2880, Denmark

[3] Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

关键词: Glycosyl transferases;    Glycosyl hydrolases;    Cell wall;    DNA-sequencer;   

Others  :  798001
DOI  :  10.1186/1754-6834-6-94

 received in 2013-03-11, accepted in 2013-06-28,  发布年份 2013

【 摘 要 】

Background

Plant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow.

Results

A method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.

Conclusions

The DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.

【 授权许可】

   
2013 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Pauly M, Keegstra K: Plant cell wall polymers as precursors for biofuels. Curr Opin Plant Biol 2010, 13:305-312.
• [2]Pauly M, Keegstra K: Physiology and metabolism ‘Tear down this wall’. Curr Opin Plant Biol 2008, 11:233-235.
• [3]Gomez L, Whitehead C, Barakate A, Halpin C, McQueen-Mason S: Automated saccharification assay for determination of digestibility in plant materials. Biotechnology for Biofuels 2010, 3:23. BioMed Central Full Text
• [4]Sengupta S, Jana ML, Sengupta D, Naskar AK: A note on the estimation of microbial glycosidase activities by dinitrosalicylic acid reagent. Appl Microbiol Biotechnol 2000, 53:732-735.
• [5]Vinzant TB, Adney WS, Decker SR, Baker JO, Kinter MT, Sherman NE, Fox JW, Himmel ME: Fingerprinting Trichoderma reesei hydrolases in a commerical cellulase preparation. Appl Biochem Biotechnol 2001, 91–3:99-107.
• [6]Pauly M, Keegstra K: Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J 2008, 54:559-568.
• [7]Fry SC: The Growing Plant Cell Wall: Chemical and Metabolic Analysis. 2nd edition. Caldwell, New Jersey: The Blackburn Press; 2000.
• [8]Anders N, Wilkinson MD, Lovegrove A, Freeman J, Tryfona T, Pellny TK, Weimar T, Mortimer JC, Stott K, Baker JM, et al.: Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses. Proc Natl Acad Sci USA 2012, 109:989-993.
• [9]Maslen SL, Goubet F, Adam A, Dupree P, Stephens E: Structure elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-TOF/TOF-MS/MS. Carbohydr Res 2007, 342:724-735.
• [10]Ridlova G, Mortimer JC, Maslen SL, Dupree P, Stephens E: Oligosaccharide relative quantitation using isotope tagging and normal-phase liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 2008, 22:2723-2730.
• [11]Goubet F, Jackson P, Deery MJ, Dupree P: Polysaccharide analysis using carbohydrate gel electrophoresis: A method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal Biochem 2002, 300:53-68.
• [12]Evangelista RA, Liu MS, Chen FTA: Characterization of 9-Aminopyrene-1,4,6-Trisulfonate-Derivatized Sugars by Capillary Electrophoresis with Laser-Induced Fluorescence Detection. Anal Chem 1995, 67:2239-2245.
• [13]Khandurina J, Olson NA, Anderson AA, Gray KA, Guttman A: Large-scale carbohydrate analysis by capillary array electrophoresis: part 1. Separation and scale-up. Electrophoresis 2004, 25:3117-3121.
• [14]Chen FT, Evangelista RA: Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with laser-induced fluorescence. Anal Biochem 1995, 230:273-280.
• [15]Westphal Y, Schols HA, Voragen AGJ, Gruppen H: MALDI-TOF MS and CE-LIF Fingerprinting of Plant Cell Wall Polysaccharide Digests as a Screening Tool for Arabidopsis Cell Wall Mutants. J Agric Food Chem 2010, 58:4644-4652.
• [16]Guttman A: High-resolution carbohydrate profiling by capillary gel electrophoresis. Nature 1996, 380:461-462.
• [17]Garcia-Perez I, Whitfield P, Bartlett A, Angulo S, Legido-Quigley C, Hanna-Brown M, Barbas C: Metabolic fingerprinting of Schistosoma mansoni infection in mice urine with capillary electrophoresis. Electrophoresis 2008, 29:3201-3206.
• [18]Morell MK, Samuel MS, O’Shea MG: Analysis of starch structure using fluorophore-assisted carbohydrate electrophoresis. Electrophoresis 1998, 19:2603-2611.
• [19]Bharadwaj R, Chen Z, Datta S, Holmes BM, Sapra R, Simmons BA, Adams PD, Singh AK: Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion. Anal Chem 2010, 82:9513-9520.
• [20]Bauer S, Vasu P, Persson S, Mort AJ, Somerville CR: Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc Natl Acad Sci USA 2006, 103:11417-11422.
• [21]Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJ, Deelder AM, Rapp E, Wuhrer M: Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J Proteome Res 2010, 9:6655-6664.
• [22]Lee KJ, Jung JH, Lee JM, So Y, Kwon O, Callewaert N, Kang HA, Ko K, Oh DB: High-throughput quantitative analysis of plant N-glycan using a DNA sequencer. Biochem Biophys Res Commun 2009, 380:223-229.
• [23]Laroy W, Contreras R, Callewaert N: Glycome mapping on DNA sequencing equipment. Nat Protoc 2006, 1:397-405.
• [24]Khandurina J, Blum DL, Stege JT, Guttman A: Automated carbohydrate profiling by capillary electrophoresis: a bioindustrial approach. Electrophoresis 2004, 25:2326-2331.
• [25]Murray S, McKenzie M, Butler R, Baldwin S, Sutton K, Batey I, Timmerman-Vaughan GM: Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer. Anal Biochem 2011, 413:104-113.
• [26]Ikeda T, Senda M: Polarographic Study of Monosaccharides in Unbuffered Solution and Its Application to the Determination of the Mutarotation Rate Constants. Bull Chem Soc Jpn 1973, 46(7):2107-2111.
• [27]Kabel MA, Heijnis WH, Bakx EJ, Kuijpers R, Voragen AG, Schols HA: Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides. J Chromatogr A 2006, 1137:119-126.
• [28]Jackson P: The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem J 1990, 270:705-713.
• [29]Khandurina J, Anderson AA, Olson NA, Stege JT, Guttman A: Large-scale carbohydrate analysis by capillary array electrophoresis: part 2. Data normalization and quantification. Electrophoresis 2004, 25:3122-3127.
• [30]Brown DM, Goubet F, Vicky WWA, Goodacre R, Stephens E, Dupree P, Turner SR: Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. Plant J 2007, 52:1154-1168.
• [31]Pena MJ, Zhong RQ, Zhou GK, Richardson EA, O’Neill MA, Darvill AG, York WS, Ye ZH: Arabidopsis irregular xylem8 and irregular xylem9: Implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 2007, 19:549-563.
• [32]Persson S, Caffall KH, Freshour G, Hilley MT, Bauer S, Poindexter P, Hahn MG, Mohnen D, Somerville C: The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity. Plant Cell 2007, 19:237-255.
• [33]Nikolovski N, Rubtsov D, Segura MP, Miles GP, Stevens TJ, Dunkley TP, Munro S, Lilley KS, Dupree P: Putative glycosyltransferases and other plant golgi apparatus proteins are revealed by LOPIT proteomics. Plant Physiol 2012, 160:1037-1051.
• [34]Brown D, Wightman R, Zhang ZN, Gomez LD, Atanassov I, Bukowski JP, Tryfona T, McQueen-Mason SJ, Dupree P, Turner S: Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall. Plant J 2011, 66:401-413.
• [35]Urbanowicz BR, Pena MJ, Ratnaparkhe S, Avci U, Backe J, Steet HF, Foston M, Li H, O’Neill MA, Ragauskas AJ, et al.: 4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein. Proc Natl Acad Sci USA 2012, 109:14253-14258.
• [36]Lee C, Teng Q, Zhong R, Yuan Y, Haghighat M, Ye ZH: Three Arabidopsis DUF579 domain-containing GXM proteins are methyltransferases catalyzing 4-o-methylation of glucuronic acid on xylan. Plant Cell Physiol 2012, 53:1934-1949.