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  • 101.
    Fugelstad, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brown, Christian
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hukasova, Elvira
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lindqvist, Arne
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 4, p. 1248-1253Article in journal (Refereed)
    Abstract [en]

    Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.

  • 102. Gebauer, Denis
    et al.
    Oliynyk, Vitaliy
    Salajkova, Michaela
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sort, Jordi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bergstrom, Lennart
    Salazar-Alvarez, German
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    A transparent hybrid of nanocrystalline cellulose and amorphous calcium carbonate nanoparticles2011In: NANOSCALE, ISSN 2040-3364, Vol. 3, no 9, p. 3563-3566Article in journal (Refereed)
    Abstract [en]

    Nanocellulose hybrids are promising candidates for biodegradable multifunctional materials. Hybrids of nanocrystalline cellulose (NCC) and amorphous calcium carbonate (ACC) nanoparticles were obtained through a facile chemical approach over a wide range of compositions. Controlling the interactions between NCC and ACC results in hard, transparent structures with tunable composition, homogeneity and anisotropy.

  • 103. Geisler-Lee, J.
    et al.
    Geisler, M.
    Coutinho, P. M.
    Segerman, B.
    Nishikubo, N.
    Takahashi, J.
    Aspeborg, H.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Djerbi, S.
    Master, E.
    Andersson-Gunneras, S.
    Sundberg, B.
    Karpinski, S.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kleczkowski, L. A.
    Henrissat, B.
    Mellerowicz, E. J.
    Poplar carbohydrate-active enzymes. Gene identification and expression analyses2006In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 140, no 3, p. 946-962Article in journal (Refereed)
    Abstract [en]

    Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

  • 104. Gerttula, S.
    et al.
    Zinkgraf, M.
    Muday, G. K.
    Lewis, D. R.
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience. National Research Center Kurchatov Institute, Russian Federation.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. University of British Columbia, Canada.
    Hart, F.
    Mansfield, S. D.
    Filkov, V.
    Groover, A.
    Transcriptional and hormonal regulation of gravitropism of woody stems in populus2015In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 27, no 10, p. 2800-2813Article in journal (Refereed)
    Abstract [en]

    Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.

  • 105. Ghebremichael, K. A.
    et al.
    Gunaratna, K. R.
    Henriksson, H.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    A simple purification and activity assay of the coagulant protein from Moringa oleifera seed2005In: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 39, no 11, p. 2338-2344Article in journal (Refereed)
    Abstract [en]

    Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5 h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1-4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications.

  • 106.
    Giacomello, Stefania
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Stockholm University, Sweden.
    Salmén, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Terebieniec, B. K.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Navarro, J. F.
    Alexeyenko, A.
    Reimegård, J.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mannapperuma, C.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Ståhl, P. L.
    Sundström, J. F.
    Street, N. R.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatially resolved transcriptome profiling in model plant species2017In: Nature Plants, ISSN 2055-0278, Vol. 3, article id 17061Article in journal (Refereed)
    Abstract [en]

    Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

  • 107. Gilbert, Harry J.
    et al.
    Stalbrand, Henrik
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation2008In: Current opinion in plant biology, ISSN 1369-5266, E-ISSN 1879-0356, Vol. 11, no 3, p. 338-348Article, review/survey (Refereed)
    Abstract [en]

    The recent years have witnessed considerable developments in the interpretation of the three-dimensional structures of plant polysaccharide-degrading enzymes in the context of their functional specificity. A plethora of new structures of catalytic, carbohydrate-binding and protein-scaffolding modules involved in (hemi)cellulose catabolism has emerged in harness with sophisticated biochemical analysis. Despite significant advances, a full understanding of the intricacies of substrate recognition and catalysis by these diverse and specialised enzymes remains an important goal, especially if the application potential of these biocatalysts is to be fully realised.

  • 108. Gloster, Tracey M.
    et al.
    Ibatullin, Farid M.
    Macauley, Katherine
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    Roberts, Shirley
    Turkenburg, Johan P.
    Bjornvad, Mads E.
    Jorgensen, Per Lina
    Danielsen, Steffen
    Johansen, Katja S.
    Borchert, Torben V.
    Wilson, Keith S.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH122007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 26, p. 19177-19189Article in journal (Refereed)
    Abstract [en]

    The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed hemicellulose. One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

  • 109. Gray-Mitsumune, Madoka
    et al.
    Blomquist, Kristina
    McQueen-Mason, Simon
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundberg, Bjoern
    Mellerowicz, Ewa J.
    Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen2008In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 6, no 1, p. 62-72Article in journal (Refereed)
    Abstract [en]

    Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an alpha-expansin PttEXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. PttEXPA1 belongs to Subfamily A of alpha-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose-xyloglucan composites in vitro, indicating that PttEXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that PttEXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta. Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. PttEXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.

  • 110. Greffe, L.
    et al.
    Bessueille, L.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides2005In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, no 4, p. 437-445Article in journal (Refereed)
    Abstract [en]

    A novel class of nonionic, carbohydrate-based surfactants has been synthesized from the plant polysaccharide xyloglucan. Enzymatic hydrolysis of xyloglucan yielded a series of well-defined, highly branched oligosaccharides that, following reductive amination, were readily conjugated with fatty acids bearing C-8 to C-18 chains under mild conditions. The critical micelle concentration, determined by tensiometry and dye-inclusion measurements, showed a typical dependence on acyl chain length and was sensitive to the degree of galactosylation of the head group. Several compounds from this new group of surfactants, especially those with C-14 and C-16 chains, were useful for the extraction of membrane-bound enzyme markers from different plant cell compartments in catalytically active form.

  • 111.
    Grenville-Briggs, Laura J.
    et al.
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Anderson, Victoria L.
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avrova, Anna O.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Bouzenzana, Jamel
    Organisation et Dynamique des Membranes Biologiques, Unité Mixte de Recherche 5246, Université Lyon I.
    Williams, Alison
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Wawra, Stephan
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Whisson, Stephen C.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Birch, Paul R. J.
    Plant-Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    van West, Pieter
    Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences.
    Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato2008In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 20, no 3, p. 720-738Article in journal (Refereed)
    Abstract [en]

    Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.

  • 112.
    Grenville-Briggs, Laura J.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Horner, Neil R.
    Phillips, Andrew J.
    Beakes, Gordon W.
    Van West, Pieter
    A family of small tyrosine rich proteins is essential for oogonial and oospore cell wall development of the mycoparasitic oomycete Pythium oligandrum2013In: Fungal Biology, ISSN 1878-6146, E-ISSN 1878-6162, Vol. 117, no 3, p. 163-172Article in journal (Refereed)
    Abstract [en]

    The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrum small tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation.

  • 113.
    Grenville-Briggs, Laura J.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Stansfield, Ian
    Self-directed student research through analysis of microarray datasets: a computer-based functional genomics practical class for Masters-level students2011In: Biochemistry and molecular biology education, ISSN 1470-8175, E-ISSN 1539-3429, Vol. 39, no 6, p. 440-447Article in journal (Refereed)
    Abstract [en]

    This report describes a linked series of Masters-level computer practical workshops. They comprise an advanced functional genomics investigation, based upon analysis of a microarray dataset probing yeast DNA damage responses. The workshops require the students to analyse highly complex transcriptomics datasets, and were designed to stimulate active learning through experience of current research methods in bioinformatics and functional genomics. They seek to closely mimic a realistic research environment, and require the students first to propose research hypotheses, then test those hypotheses using specific sections of the microarray dataset. The complexity of the microarray data provides students with the freedom to propose their own unique hypotheses, tested using appropriate sections of the microarray data. This research latitude was highly regarded by students and is a strength of this practical. In addition, the focus on DNA damage by radiation and mutagenic chemicals allows them to place their results in a human medical context, and successfully sparks broad interest in the subject material. In evaluation, 79% of students scored the practical workshops on a five-point scale as 4 or 5 (totally effective) for student learning. More broadly, the general use of microarray data as a student research playground is also discussed.

  • 114.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avino, Mariano
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Clergeot, Pierre-Henri
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs2010In: PLOS PATHOG, ISSN 1553-7366, Vol. 6, no 8, p. e1001070-Article in journal (Refereed)
    Abstract [en]

    Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

  • 115.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    What Do We Really Know about Cellulose Biosynthesis in Higher Plants?2010In: JOURNAL OF INTEGRATIVE PLANT BIOLOGY, ISSN 1672-9072, Vol. 52, no 2, p. 161-175Article, review/survey (Refereed)
    Abstract [en]

    Cellulose biosynthesis is one of the most important biochemical processes in plant biology. Despite the considerable progress made during the last decade, numerous fundamental questions related to this key process in plant development are outstanding. Numerous models have been proposed through the years to explain the detailed molecular events of cellulose biosynthesis. Almost all models integrate solid experimental data with hypotheses on several of the steps involved in the process. Speculative models are most useful to stimulate further research investigations and bring new exciting ideas to the field. However, it is important to keep their hypothetical nature in mind and be aware of the risk that some undemonstrated hypotheses may progressively become admitted. In this review, we discuss the different steps required for cellulose formation and crystallization, and highlight the most important specific aspects that are supported by solid experimental data.

  • 116. Guerriero, Gea
    et al.
    Spadiut, Oliver
    Kerschbamer, Christine
    Giorno, Filomena
    Baric, Sanja
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: Partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A2012In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 63, no 16, p. 6045-6056Article in journal (Refereed)
    Abstract [en]

    Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus-domestica was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.-xdomestica are discussed.

  • 117.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    On the engineering of proteins: methods and applications for carbohydrate-active enzymes2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose.

    One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras.

    Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

    Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections.

  • 118.
    Gullfot, Fredrika
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.

    Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

    Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

  • 119.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ibatullin, Farid
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds2009In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, no 7, p. 1782-1788Article in journal (Refereed)
    Abstract [en]

    Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to Mw 60000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4); L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCel7B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.

  • 120.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, no 3, p. 785-789Article in journal (Refereed)
  • 121.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Teeri, Tuula
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Design of GH16 XET/XEH chimeric enzymes with SCHEMA: ManuscriptManuscript (preprint) (Other academic)
    Abstract [en]

    This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.

    A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.

  • 122. Gunnarsson, Lavinia Cicortas
    et al.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Montanier, Cedric
    Karlsson, Eva Nordberg
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ohlin, Mats
    Engineered xyloglucan specificity in a carbohydrate-binding module2006In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 16, no 12, p. 1171-1180Article in journal (Refereed)
    Abstract [en]

    The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

  • 123. Gustavsson, M. T.
    et al.
    Persson, P. V.
    Iversen, T.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase2005In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 6, no 1, p. 196-203Article in journal (Refereed)
    Abstract [en]

    A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M-r xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.

  • 124. Haas, Brian J.
    et al.
    Kamoun, Sophien
    Zody, Michael C.
    Jiang, Rays H. Y.
    Handsaker, Robert E.
    Cano, Liliana M.
    Grabherr, Manfred
    Kodira, Chinnappa D.
    Raffaele, Sylvain
    Torto-Alalibo, Trudy
    Bozkurt, Tolga O.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans2009In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 461, no 7262, p. 393-398Article in journal (Refereed)
    Abstract [en]

    Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement(1). To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population(1). Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion(2). Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars(3,4). Here we report the sequence of the P. infestans genome, which at similar to 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for similar to 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

  • 125. Harris, Darby
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ding, Shi-You
    DeBolt, Seth
    Tools for Cellulose Analysis in Plant Cell Walls2010In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, no 2, p. 420-426Article in journal (Refereed)
  • 126.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tan, T. -C
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Pisanelli, I.
    Fusco, L.
    Haltrich, D.
    Peterbauer, C. K.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly2013In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, p. 496-504Article in journal (Refereed)
    Abstract [en]

    The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-. d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-. β-. d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17°C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.

  • 127. Hemsworth, Glyn R.
    et al.
    Thompson, Andrew J.
    Stepper, Judith
    Sobala, Lukasz F.
    Coyle, Travis
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience. University of British Columbia, Canada.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Goddard-Borger, Ethan D.
    Stubbs, Keith A.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. University of British Columbia, Canada.
    Davies, Gideon J.
    Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut2016In: Open Biology, ISSN 2046-2441, E-ISSN 2046-2441, Vol. 6, no 7, article id 160142Article in journal (Refereed)
    Abstract [en]

    The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta) genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an alpha-xylosidase, a beta-glucosidase, and two alpha-L-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.

  • 128.
    Henriksson, Gunnar
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Zhang, Liming
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Nilsson, Thomas
    Inst för virkeslära Sveriges Lantbruksuniversitet, Uppsala.
    Ohlsson, Anna
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Torkil
    KTH, School of Biotechnology (BIO), Glycoscience.
    Inhomogenity inlignin structure between different cell wall layers i  conifers and hardwood2006In: Fifth Plant Biomechanics Conference, 2006, p. 145-150Conference paper (Refereed)
  • 129. Henriksson, Marielle
    et al.
    Berglund, Lars A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Method of producing and the use of microfibrillated paper2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a method of producing a cellulose based paper, the paper itself and the use thereof where the paper exhibits enhanced mechanical properties. The method involves providing a suspension of well dispersed modified cellulose at a low concentration. The properties and the chemical structure of the paper make it suitable for in vivo applications such as implant material.

  • 130.
    Henriksson, Marielle
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Producing paper, useful as e.g. filter paper, speaker membrane and suture, comprises providing modified nanofibrils of cellulose, providing suspension of modified nanofibrils, and filtering, dewatering and drying the nanofibrils2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a method of producing a cellulose based paper, the paper itself and the use thereof where the paper exhibits enhanced mechanical properties. The method involves providing a suspension of well dispersed modified cellulose at a low concentration. The properties and the chemical structure of the paper make it suitable for in vivo applications such as implant material.

  • 131. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Jürgens, Klaus
    Zheng, Zongli
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 3, p. e00569-12-Article in journal (Refereed)
    Abstract [en]

    The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype's distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.

  • 132. Hrmova, Maria
    et al.
    Farkas, Vladimir
    Harvey, Andrew J.
    Lahnstein, Jelle
    Wischmann, Bente
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, p. 437-456Article in journal (Refereed)
    Abstract [en]

    A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.

  • 133.
    Hsieh, Yves S. Y.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Zhang, Qisen
    Yap, Kuok
    Shirley, Neil J.
    Lahnstein, Jelle
    Nelson, Clark J.
    Burton, Rachel A.
    Millar, A. Harvey
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    Fincher, Geoffrey B.
    Genetics, Transcriptional Profiles, and Catalytic Properties of the UDP-Arabinose Mutase Family from Barley2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 2, p. 322-334Article in journal (Refereed)
    Abstract [en]

    Four members of the UDP-Ara mutase (UAM) gene family from barley have been isolated and characterized, and their map positions on chromosomes 2H, 3H, and 4H have been defined. When the genes are expressed in Escherichia coli, the corresponding HvUAM1, HvUAM2, and HvUAM3 proteins exhibit UAM activity, and the kinetic properties of the enzymes have been determined, including K-m, K-cat, and catalytic efficiencies. However, the expressed HvUAM4 protein shows no mutase activity against UDP-Ara or against a broad range of other nucleotide sugars and related molecules. The enzymic data indicate therefore that the HvUAM4 protein may not be a mutase. However, the HvUAM4 gene is transcribed at high levels in all the barley tissues examined, and its transcript abundance is correlated with transcript levels for other genes involved in cell wall biosynthesis. The UDP-L-Arap -> UDP-L-Araf reaction, which is essential for the generation of the UDP-Araf substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis, is thermodynamically unfavorable and has an equilibrium constant of 0.02. Nevertheless, the incorporation of Araf residues into nascent polysaccharides clearly occurs at biologically appropriate rates. The characterization of the HvUAM genes opens the way for the manipulation of both the amounts and fine structures of heteroxylans in cereals, grasses, and other crop plants, with a view toward enhancing their value in human health and nutrition, and in renewable biofuel production.

  • 134.
    Ibatullin, Farid M.
    et al.
    KTH, School of Biotechnology (BIO).
    Banasiak, Alicja
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Greffe, Lionel
    KTH, School of Biotechnology (BIO).
    Takahashi, Junko
    Mellerowicz, Ewa J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 151, no 4, p. 1741-1750Article in journal (Refereed)
    Abstract [en]

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e. g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M-1 cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  • 135. Ibatullin, Farid M.
    et al.
    Baumann, Martin J.
    Greffe, Lionel
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kinetic analyses of retaining endo-(xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 29, p. 7762-7769Article in journal (Refereed)
    Abstract [en]

    A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on GlC(4) backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per-O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pK(a) values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with pK(a) values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pK(a) values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme, intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.

  • 136. Jaeger, Diana
    et al.
    Ndi, Chi P.
    Crocoll, Christoph
    Simpson, Bradley S.
    Khakimov, Bekzod
    Guzman-Genuino, Ruth Marian
    Hayball, John D.
    Xing, Xiaohui
    KTH, School of Biotechnology (BIO), Glycoscience. School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden; ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden; ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia.
    Weinstein, Philip
    Moller, Birger L.
    Semple, Susan J.
    Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata2017In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 80, no 10, p. 2692-2698Article in journal (Refereed)
    Abstract [en]

    The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

  • 137. Jahic, Mehmedalija
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Charoenrat, Theppanya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Process technology for production and recovery of heterologous proteins with Pichia pastoris2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 6, p. 1465-1473Article, review/survey (Refereed)
    Abstract [en]

    Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.

  • 138. Jamshidian, H.
    et al.
    Shojaosadati, S. A.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mousavi, S. M.
    Soudi, M. R.
    Characterization and optimization of schizophyllan production from date syrup2016In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 92, p. 484-493Article in journal (Refereed)
    Abstract [en]

    This study demonstrates the efficient utilization of low-cost agricultural substrates, particularly date syrup, by Schizophyllum commune ATCC 38548 for schizophyllan production. Initially, one factor-at-a-time method was used to find the best carbon and nitrogen sources for schizophyllan production. Subsequently, response surface methodology was employed to optimize the level of culture medium components to maximize substrate conversion yield and schizophyllan production in submerged culture. Maximum product yield (0.12 g schizophyllan/g date syrup) and schizophyllan production (8.5 g/l) were obtained at concentrations of date syrup and corn steep liquor, inoculum size and agitation rate at 7.02 %w/v, 0.10 %w/v, 7.68 %v/v and 181 rpm, respectively. Sugar composition analysis, FTIR, NMR and molar mass determination revealed the purity and molecular properties of recovered schizophyllan produced from date syrup as glycosidic linkage analysis showed three main schizophyllan characteristic peaks arising from the 3-linked, 3,6-linked and terminal glucose residues. Finally, process economic analysis suggested that use of date syrup and corn steep liquor as nutrients would result in approximately 6-fold reduction in cost of raw materials for schizophyllan production as compared to conventional carbon and nitrogen sources such as sucrose and malt extract.

  • 139. Jamshidian, Hajar
    et al.
    Shojaosadati, Seyed Abbas
    Mousavi, Seyed Mohammad
    Soudi, Mohammad Reza
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Implications of recovery procedures on structural and rheological properties of schizophyllan produced from date syrup2017In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 105, p. 36-44Article in journal (Refereed)
    Abstract [en]

    This study investigates the effects of different recovery procedures on high molar mass schizophyl-Ian produced by Schizophyllum commune using low value agricultural residues. Recovered extracellular polysaccharides (EPSs) were compared in terms of purity, sugar composition, degree of branching, molecular weight, and rheological properties. Performing different recovery methods, such as re-dissolving in water and re-precipitation with ethanol on produced EPS, provided schizophyllan with purity similar to the commercial grade. Besides, Freeze-thawing cycles allowed the fractionation of schizophyllan based on branching degree and solubility. The EPSs with higher purity and lower degree of branching (less conformational flexibility) showed higher viscosity. This study evidences the possibility of producing EPSs with excellent rheological properties using low value agricultural side products. Furthermore, our results demonstrate the importance of recovery methods for tailoring the purity, molecular structure and macroscopic properties of the produced polysaccharides for specific applications.

  • 140. Jiang, Rays H. Y.
    et al.
    de Bruijn, Irene
    Haas, Brian J.
    Belmonte, Rodrigo
    Loebach, Lars
    Christie, James
    van den Ackerveken, Guido
    Bottin, Arnaud
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dumas, Bernard
    Fan, Lin
    Gaulin, Elodie
    Govers, Francine
    Grenville-Briggs, Laura J.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Horner, Neil R.
    Levin, Joshua Z.
    Mammella, Marco
    Meijer, Harold J. G.
    Morris, Paul
    Nusbaum, Chad
    Oome, Stan
    Phillips, Andrew J.
    van Rooyen, David
    Rzeszutek, Elzbieta
    KTH, School of Biotechnology (BIO), Glycoscience.
    Saraiva, Marcia
    Secombes, Chris J.
    Seidl, Michael F.
    Snel, Berend
    Stassen, Joost H. M.
    Sykes, Sean
    Tripathy, Sucheta
    van den Berg, Herbert
    Vega-Arreguin, Julio C.
    Wawra, Stephan
    Young, Sarah K.
    Zeng, Qiandong
    Dieguez-Uribeondo, Javier
    Russ, Carsten
    Tyler, Brett M.
    van West, Pieter
    Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica2013In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 6, p. e1003272-Article in journal (Refereed)
    Abstract [en]

    Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

  • 141.
    Joby Kochumalayil, Jose
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Engineering Sciences (SCI), Centres, VinnExcellence Center BiMaC Innovation.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Kasai, Wakako
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Berglund, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Biocomposites. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Regioselective modification of a xyloglucan hemicellulose for high-performance biopolymer barrier films2013In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 93, no 2, p. 466-472Article in journal (Refereed)
    Abstract [en]

    Biobased polymers such as starch and hemicelluloses from wood are of interest for packaging applications, but suffer from limitations in performance under moist conditions. Xyloglucan from industrial tamarind seed waste offers potential, but its Tg is too high for thermal processing applications. Regioselective modification is therefore performed using an approach involving periodate oxidation followed by reduction. The resulting polymer structures are characterized using MALDI-TOF-MS, size-exclusion chromatography, FTIR and carbohydrate analysis. Films are cast from water and characterized by thermo-gravimetry, dynamic mechanical thermal analysis, dynamic water vapor sorption, oxygen transmission and tensile tests. Property changes are interpreted from structural changes. These new polymers show much superior performance to current petroleum-based polymers in industrial use. Furthermore, this regioselective modification can be carefully controlled, and results in a new type of cellulose derivatives with preserved cellulose backbone without the need for harmful solvents.

  • 142.
    Jonsson Rudsander, Ulla
    et al.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Sandstrom, Corine
    Piens, Kathleen
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Master, Emma
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
    Wilson, David B.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime. KTH, School of Biotechnology (BIO), Glycoscience.
    Kenne, Lennart
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime. KTH, School of Biotechnology (BIO), Glycoscience.
    Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-ss-glucanases2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 18, p. 5235-5241Article in journal (Refereed)
    Abstract [en]

    H-1 NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, Tf Cel9A, were compared with those obtained by a randomly acting plant endoglucanase, PttCe19A. PttCe19A is an orthologue of the Arabidopsis endocellulase, Korrigan, which is required for efficient cellulose biosynthesis. As expected, poplar PttCe19A was shown to catalyze the degradation of cellooligosaccharides by inversion of the configuration of the anomeric carbon. The product analyses showed that the number of interactions between the glucose units of the substrate and the aromatic residues in the enzyme active sites determines the point of cleavage in both enzymes.

  • 143. Just, K.
    et al.
    Arif, U.
    KTH, School of Biotechnology (BIO), Glycoscience. Swedish University of Agricultural Sciences, Sweden.
    Luik, A.
    Kvarnheden, A.
    Monitoring infection of tomato fruit by Tomato yellow leaf curl virus2016In: Plant Pathology, ISSN 0032-0862, E-ISSN 1365-3059Article in journal (Refereed)
    Abstract [en]

    Fruit constitutes a strong sink organ and thus accumulates infecting viruses, but there is limited information about the infection process of viruses in fruit. Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is one of the most important viruses affecting the production of tomato fruit. Using real-time quantitative PCR, TYLCV was shown to accumulate with increasing titres in early developing tomato fruit tissues from anthesis until 21 days post-anthesis. In situ hybridization demonstrated that TYLCV DNA and transcripts of the coat protein gene localized specifically to the phloem tissue of young fruit as well as sepals and petals. Embryos of developing seeds were also found to be infected. Expression of a host histone H4 gene was used as a marker for S-phase and the gene was also found to be expressed in phloem cells of young tomato fruit. The results indicate that TYLCV is transported to developing tomato fruit, where the virus titre gradually increases because of movement and probably also due to virus replication. In this study, the accumulation and localization of TYLCV in early developing tomato fruit are monitored for the first time.

  • 144. Kaesdorf, Benjamin T.
    et al.
    Weber, Florian
    Petrou, Georgia
    KTH, School of Biotechnology (BIO), Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Crouzier, Thomas
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lieleg, Oliver
    Mucin-Inspired Lubrication on Hydrophobic Surfaces2017In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, no 8, p. 2454-2462Article in journal (Refereed)
    Abstract [en]

    In the human body, high-molecular-weight glycoproteins called mucins play a key role in protecting epithelial surfaces against pathogenic attack, controlling the passage of molecules toward the tissue and enabling boundary lubrication with very low friction coefficients. However, neither the molecular mechanisms nor the chemical motifs of those biomacromolecules involved in these fundamental processes are fully understood. Thus, identifying the key features that render biomacromolecules such as mucins outstanding boundary lubricants could set the stage for creating versatile artificial superlubricants. We here demonstrate the importance of the hydrophobic terminal peptide domains of porcine gastric mucin (MUCSAC) and human salivary mucin (MUCSB) in the processes of adsorbing to and lubricating a hydrophobic PDMS surface. Tryptic digestion of those mucins results in removal of those terminal domains, which is accompanied by a loss of lubricity as well as surface adsorption. We show that this loss can in part be compensated by attaching hydrophobic phenyl groups to the glycosylated central part of the mucin macromolecule. Furthermore, we demonstrate that the simple biopolysaccharide dextran can be functionalized with hydrophobic groups which confers efficient surface adsorption and good lubricity on PDMS to the polysaccharide.

  • 145.
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    In vitro and in vivo approaches in the characterization of XTH gene products2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    ABSTRACT

    The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization.

    In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load.

    Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants.

    Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening

  • 146. Kaewthai, Nomchit
    et al.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Development of a high-throughput assay for screening xyloglucan endo-transglycosylase and endo-xyloglucanase expression in crude microbial supernatantsManuscript (preprint) (Other academic)
  • 147.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ibatullin, Farid M.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bhalerao, Rishikesh P.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thalianaManuscript (preprint) (Other academic)
  • 148.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ibatullin, Farid M.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Bhalerao, Rishikesh P
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth2013In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, no 1, p. 440-454Article in journal (Refereed)
    Abstract [en]

    The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin beta-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.

  • 149.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Harvey, Andrew J.
    Hrmova, Maria
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris2010In: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 27, no 3, p. 251-258Article in journal (Refereed)
    Abstract [en]

    Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

  • 150. Kaida, Rumi
    et al.
    Satoh, Yumi
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Yamada, Yohko
    Kaku, Tomomi
    Hayashi, Takahisa
    Kaneko, Takako S.
    Activation of beta-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 150, no 4, p. 1822-1830Article in journal (Refereed)
    Abstract [en]

    Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wildtype cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

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