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  • 1.
    Bi, Ran
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Mckee, Lauren
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Culture Filtrates from a Soil Organism Enhances Extractability of Polymers from Fiberised Spruce WoodManuscript (preprint) (Other academic)
  • 2.
    Bi, Ran
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Jennie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The Degree Of Acetylation Affects The Microbial Degradability Of HemicellulosesManuscript (preprint) (Other academic)
  • 3.
    Bi, Ran
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Jennie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The degree of acetylation affects the microbial degradability of mannans2016In: Polymer degradation and stability, ISSN 0141-3910, E-ISSN 1873-2321, Vol. 133, p. 36-46Article in journal (Refereed)
    Abstract [en]

    Hemicelluloses as major components of plant cell walls are acetylated to different extents. The biologicalfunctions of acetylation are not completely understood but suggested that one reason is to decrease themicrobial degradability of cell walls. Model seed galactomannan and glucomannan, which are structurallysimilar to an abundant class of wood hemicelluloses, were acetylated to various degrees and usedas sole carbon source on agar plates for microbial growth. When soil samples were inoculated on theplates, significantly fewer strains grew on the agar plates with highly acetylated mannans than withslightly acetylated or non-acetylated mannans. One filamentous fungus isolated and identified as aPenicillium species was shown to grow faster and stronger on non-acetylated than on highly acetylatedmannan. The data therefore support the hypothesis that a high degree of acetylation (DSac) can decreasethe microbial degradability of hemicelluloses. Possible mechanisms and the technological significance ofthis are discussed.

  • 4.
    Brown, Christian
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Szpryngie, Scarlett
    Kuang, Guanglin
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ye, Weihua
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tu, Yaoquan
    Mäler, Lena
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural and functional characterization of the “Microtubule Interacting and Trafficking": domains of two oomycetes chitin synthasesManuscript (preprint) (Other academic)
  • 5.
    Carreno-Quintero, Natalia
    et al.
    Royal Holloway Univ London, Biochem Dept, Egham Hill, Egham TW20 0EX, Surrey, England.;Vegetable Crop Res Unit, Keygene NV, Agro Business Pk 90, NL-6708 PW Wageningen, Netherlands..
    Tohge, Takayuki
    Max Planck Inst Mol Plant Physiol, Muhlenberg 1, D-14476 Potsdam, Germany..
    Van Acker, Rebecca
    Univ Ghent, Dept Plant Biotechnol & Bioinformat, Technol Pk 927, B-9052 Ghent, Belgium.;Ctr Plant Syst Biol VIB, Technol Pk 927, B-9052 Ghent, Belgium..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Zhou, Qi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bolze, Antje
    Max Planck Inst Mol Plant Physiol, Muhlenberg 1, D-14476 Potsdam, Germany..
    Xing, Xiaohui
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Ozparpucu, Merve
    Swiss Fed Inst Technol, Swiss Fed Inst Technol Zurich ETH Zurich, Swiss Fed Inst Technol Zurich, Zurich, Switzerland.;Swiss Fed Labs Mat Sci & Technol Empa, Appl Wood Mat, Dubendorf, Switzerland..
    Ruggeberg, Markus
    Swiss Fed Inst Technol, Swiss Fed Inst Technol Zurich ETH Zurich, Swiss Fed Inst Technol Zurich, Zurich, Switzerland.;Swiss Fed Labs Mat Sci & Technol Empa, Appl Wood Mat, Dubendorf, Switzerland..
    Piofczyk, Thomas
    Pilot Pflanzenoltechnol Magdeburg eV, Berliner Chaussee 66, D-39114 Magdeburg, Germany..
    Koram, Yaw
    Neutral Supply Chain Ltd, 337 Bath Rd, Slough SL1 5PR, Berks, England..
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. Univ Adelaide, ARC Ctr Excellence Plant Cell Walls, Waite Campus, Urrbrae, SA 5064, Australia.;Univ Adelaide, Sch Agr Food & Wine, Waite Campus, Urrbrae, SA 5064, Australia..
    Boerjan, Wout
    Univ Ghent, Dept Plant Biotechnol & Bioinformat, Technol Pk 927, B-9052 Ghent, Belgium.;Ctr Plant Syst Biol VIB, Technol Pk 927, B-9052 Ghent, Belgium..
    Fernie, Alisdair R.
    Max Planck Inst Mol Plant Physiol, Muhlenberg 1, D-14476 Potsdam, Germany..
    Fraser, Paul D.
    Royal Holloway Univ London, Biochem Dept, Egham Hill, Egham TW20 0EX, Surrey, England..
    Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock2024In: Bioresources and bioprocessing, ISSN 2197-4365, Vol. 11, no 1, article id 12Article in journal (Refereed)
    Abstract [en]

    The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

  • 6. Cartmell, Alan
    et al.
    McKee, Lauren
    KTH, School of Biotechnology (BIO). University of Georgia, United States.
    Pena, Maria J.
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kaneko, Satoshi
    Ichinose, Hitomi
    Lewis, Richard J.
    Vikso-Nielsen, Anders
    Gilbert, Harry J.
    Marles-Wright, Jon
    The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17Article in journal (Refereed)
    Abstract [en]

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  • 7.
    Chang, Shu-Chieh
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hsieh, Yves S. Y.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, 8 Taipei 11031, Taiwan.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    A Polysaccharide Utilisation Locus combining glycosyltransferase and glycoside hydrolase functions mediates β-glucan synthesis in Chitinophaga pinensisManuscript (preprint) (Other academic)
  • 8.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Müller, Marion C.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm Univ, Sweden.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. Univ Adelaide, Australia.
    The Impact of Steroidal Glycoalkaloids on the Physiology of Phytophthora infestans, the Causative Agent of Potato Late Blight2017In: Molecular Plant-Microbe Interactions, ISSN 0894-0282, E-ISSN 1943-7706, Vol. 30, no 7, p. 531-542Article in journal (Refereed)
    Abstract [en]

    Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs a-chaconine and a-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

  • 9.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm University, Sweden.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Mckee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product2016In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 7, article id 1802Article in journal (Refereed)
    Abstract [en]

    The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

  • 10.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ekengren, Sophia
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans2017In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 2, article id e0170873Article in journal (Refereed)
    Abstract [en]

    The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

  • 11. Escudero, Viviana
    et al.
    Jorda, Lucia
    Sopena-Torres, Sara
    Melida, Hugo
    Miedes, Eva
    Munoz-Barrios, Antonio
    Swami, Sanjay
    Alexander, Danny
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sanchez-Vallet, Andrea
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Jones, Alan M.
    Molina, Antonio
    Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the beta-subunit of the heterotrimeric G-protein2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 92, no 3, p. 386-399Article in journal (Refereed)
    Abstract [en]

    Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the G- (agb1-2) or G-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. Significance Statement The plant heterotrimeric G protein complex is an essential component of Pathogen Associated Molecular Pattern-triggered immunity (PTI) and of plant disease resistance to several types of pathogens. We found that modification of the degree of xylan acetylation in plant cell walls activates PTI-independent resistance responses that counterbalance the hypersusceptibility to particular pathogens of plants lacking the heterotrimeric G subunit. These data demonstrate that immune deficient response can be partially compensated by the activation of cell wall-triggered immunity that confers specific disease resistance.

  • 12.
    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.

  • 13.
    Kmezik, Cathleen
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Div Ind Biotechnol, Gothenburg, Sweden..
    Mazurkewich, Scott
    Chalmers Univ Technol, Dept Biol & Biol Engn, Div Ind Biotechnol, Gothenburg, Sweden..
    Meents, Tomke
    Chalmers Univ Technol, Dept Biol & Biol Engn, Div Ind Biotechnol, Gothenburg, Sweden.;Tech Univ Carolo Wilhelmina Braunschweig, Inst Pharmaceut Biol, Mendelssohnstr 1, D-38106 Braunschweig, Germany..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Idström, Alexander
    Chalmers Univ Technol, Dept Chem & Chem Engn, Gothenburg, Sweden..
    Armeni, Marina
    Chalmers Univ Technol, Chalmers Mass Spectrometry Infrastruct, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Savolainen, Otto
    Chalmers Univ Technol, Chalmers Mass Spectrometry Infrastruct, Dept Biol & Biol Engn, Gothenburg, Sweden.;Univ Eastern Finland, Inst Publ Hlth & Clin Nutr, Dept Clin Nutr, Kuopio, Finland..
    Brandén, Gisela
    Univ Gothenburg, Dept Chem & Mol Biol, Gothenburg, Sweden..
    Larsbrink, Johan
    Chalmers Univ Technol, Dept Biol & Biol Engn, Div Ind Biotechnol, Gothenburg, Sweden.;Wallenberg Wood Sci Ctr, Stockholm, Sweden..
    A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases2021In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 296, article id 100500Article in journal (Refereed)
    Abstract [en]

    The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.

  • 14.
    Koskela, Salla
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Wang, Shennan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Xu, Dingfeng
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Yang, Xuan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Li, Kai
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Berglund, Lars
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Zhou, Qi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Lytic polysaccharide monooxygenase (LPMO) mediated production of ultra-fine cellulose nanofibres from delignified softwood fibres2019In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 21, no 21, p. 5924-5933Article in journal (Refereed)
    Abstract [en]

    The production of cellulose nanofibres (CNFs) typically requires harsh chemistry and strong mechanical fibrillation, both of which have negative environmental impacts. A possible solution is offered by lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes that boost cellulose fibrillation. Although the role of LPMOs in oxidative modification of cellulosic substrates is rather well established, their use in the production of cellulose nanomaterials is not fully explored, and the effect of the carbohydrate-binding module (CBM) on nanofibrillation has not yet been reported. Herein, we studied the activity of two LPMOs, one of which was appended to a CBM, on delignified softwood fibres for green and energy-efficient production of CNFs. The CNFs were used to prepare cellulose nanopapers, and the structure and properties of both nanofibres and nanopapers were determined. Both enzymes were able to facilitate nanocellulose fibrillation and increase colloidal stability of the produced CNFs. However, the CBM-lacking LPMO was more efficient in introducing carboxyl groups (0.53 mmol/g) on the cellulose fibre surfaces and releasing CNFs with thinner width (4.3 ± 1.5 nm) from delignified spruce fibres than the modular LPMO (carboxylate content of 0.38 mmol/g and nanofibre width of 6.7± 2.5 nm through LPMO pretreatment followed by mild homogenisation. The prepared nanopapers showed improved mechanical properties (tensile strength of 262 MPa, and modulus of 16.2 GPa) compared to conventional CNFs preparation methods, demonstrating the potential of LPMOs as green alternatives for cellulose nanomaterials preparation.

  • 15.
    Koskela, Salla
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Wang, Shennan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Yang, Xuan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Li, Kai
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Berglund, Lars
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Zhou, Qi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Enzyme-assisted preparation of nanocellulose from wood holocellulose fibers2019Other (Other academic)
  • 16.
    La Rosa, Sabina Leanti
    et al.
    Norwegian Univ Life Sci, Fac Biosci, N-1433 As, Norway..
    Ostrowski, Matthew P.
    Univ Michigan, Dept Microbiol & Immunol, Med Sch, Ann Arbor, MI 48109 USA..
    de Leon, Arturo Vera-Ponce
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, N-1433 As, Norway..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Larsbrink, Johan
    Chalmers Univ Technol, Dept Biol & Biol Engn, Div Ind Biotechnol, S-41296 Gothenburg, Sweden..
    Eijsink, Vincent G.
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, N-1433 As, Norway..
    Lowe, Elisabeth C.
    Newcastle Univ, Biosci Inst, Newcastle Upon Tyne, Tyne & Wear, England..
    Martens, Eric C.
    Univ Michigan, Dept Microbiol & Immunol, Med Sch, Ann Arbor, MI 48109 USA..
    Pope, Phillip B.
    Norwegian Univ Life Sci, Fac Biosci, N-1433 As, Norway.;Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, N-1433 As, Norway..
    Glycan processing in gut microbiomes2022In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 67, p. 102143-, article id 102143Article, review/survey (Refereed)
    Abstract [en]

    Microbiomes and their enzymes process many of the nutrients accessible in the gastrointestinal tract of bilaterians and play an essential role in host health and nutrition. In this review, we describe recent insights into nutrient processing in microbiomes across three exemplary yet contrasting gastrointestinal ecosystems (humans, ruminants and insects), with focus on bacterial mechanisms for the utilization of common and atypical dietary glycans as well as host-derived mucus glycans. In parallel, we discuss findings from multi-omic studies that have provided new perspectives on understanding glycan-dependent interactions and the complex food-webs of microbial populations in their natural habitat. Using key examples, we emphasize how increasing understanding of glycan processing by gut microbiomes can provide critical insights to assist 'microbiome reprogramming', a growing field that seeks to leverage diet to improve animal growth and host health.

  • 17.
    Larsbrink, Johan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bacteroidetes bacteria in the soil: Glycan acquisition, enzyme secretion, and gliding motility2020In: Advances in Applied Microbiology, ISSN 0065-2164, Vol. 110, p. 63-98Article in journal (Refereed)
    Abstract [en]

    The secretion of extracellular enzymes by soil microbes is rate-limiting in the recycling of biomass. Fungi and bacteria compete and collaborate for nutrients in the soil, with wide ranging ecological impacts. Within soil microbiota, the Bacteroidetes tend to be a dominant phylum, just like in human and animal intestines. The Bacteroidetes thrive because of their ability to secrete diverse arrays of carbohydrate-active enzymes (CAZymes) that target the highly varied glycans in the soil. Bacteroidetes use an energy-saving system of genomic organization, whereby most of their CAZymes are grouped into Polysaccharide Utilization Loci (PULs). These loci enable high level production of specific CAZymes only when their substrate glycans are abundant in the local environment. This gives the Bacteroidetes a clear advantage over other species in the competitive soil environment, further enhanced by the phylum-specific Type IX Secretion System (T9SS). The T9SS is highly effective at secreting CAZymes and/or tethering them to the cell surface, and is tightly coupled to the ability to rapidly glide over solid surfaces, a connection that promotes an active hunt for nutrition. Although the soil Bacteroidetes are less well studied than human gut symbionts, research is uncovering important biochemical and physiological phenomena. In this review, we summarize the state of the art on research into the CAZymes secreted by soil Bacteroidetes in the contexts of microbial soil ecology and the discovery of novel CAZymes for use in industrial biotechnology. We hope that this review will stimulate further investigations into the somewhat neglected enzymology of non-gut Bacteroidetes.

  • 18.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rogers, Theresa E.
    Hemsworth, Glyn R.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Tauzin, Alexandra S.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Klinter, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Pudlo, Nicholas A.
    Urs, Karthik
    Koropatkin, Nicole M.
    Creagh, A. Louise
    Haynes, Charles A.
    Kelly, Amelia G.
    Nilsson Cederholm, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Martens, Eric C.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 506, no 7489, p. 498-502Article in journal (Refereed)
    Abstract [en]

    A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables(1). Owing to the paucity of alimentary enzymes encoded by the human genome(2), our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut(3,4). The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides(5,6) whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear(1,7,8). Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health(9-12).

  • 19.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Laurens S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Klinter, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Nilsson Cederholm, Stefan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    et al.,
    A discrete genetic locus confers select Bacteriodetes with a niche role in xyloglucan metabolism in the human gutManuscript (preprint) (Other academic)
  • 20.
    Larsbrink, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Tuveng, T. R.
    Pope, P. B.
    Bulone, V.
    Eijsink, V. G. H.
    Brumer, Harry
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis2017In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 156, p. 63-74Article in journal (Refereed)
    Abstract [en]

    Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this study Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.

  • 21. Larsbrink, Johan
    et al.
    Tuveng, Tina R.
    Pope, Phillip B.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Eijsink, Vincent G.H.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Proteomic data on enzyme secretion and activity in the bacterium Chitinophaga pinensis2017In: Data in Brief, E-ISSN 2352-3409, Vol. 11, p. 484-490Article in journal (Refereed)
    Abstract [en]

    The secretion of carbohydrate-degrading enzymes by a bacterium sourced from a softwood forest environment has been investigated by mass spectrometry. The findings are discussed in full in the research article “Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis” in Journal of Proteomics by Larsbrink et al. ([1], doi: 10.1016/j.jprot.2017.01.003). The bacterium was grown on three carbon sources (glucose, glucomannan, and galactomannan) which are likely to be nutrient sources or carbohydrate degradation products found in its natural habitat. The bacterium was grown on solid agarose plates to mimic the natural behaviour of growth on a solid surface. Secreted proteins were collected from the agarose following trypsin-mediated hydrolysis to peptides. The different carbon sources led to the secretion of different numbers and types of proteins. Most carbohydrate-degrading enzymes were found in the glucomannan-induced cultures. Several of these enzymes may have biotechnological potential in plant cell wall deconstruction for biofuel or biomaterial production, and several may have novel activities. A subset of carbohydrate-active enzymes (CAZymes) with predicted activities not obviously related to the growth substrates were also found in samples grown on each of the three carbohydrates. The full dataset is accessible at the PRIDE partner repository (ProteomeXchange Consortium) with the identifier PXD004305, and the full list of proteins detected is given in the supplementary material attached to this report.

  • 22.
    Li, He
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Li, He
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hao, Mengshu
    Bulone, Vincent
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    A Polysaccharide Utilisation Locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell wallsIn: Article in journal (Refereed)
  • 23.
    Li, He
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Lu, Zijia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hao, Meng-Shu
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Kvammen, Alma
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Inman, Annie R.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. College of Medicine & Public Health, Flinders University, Bedford Park Campus, Sturt Road, SA, 5042, Australia.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Family 92 carbohydrate-binding modules specific for β-1,6-glucans increase the thermostability of a bacterial chitinase2023In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 212, p. 153-160Article in journal (Refereed)
    Abstract [en]

    In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10–15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

  • 24.
    Li, He
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3, 5-Dinitrosalicylic Acid Assay2023In: Carbohydrate-Protein Interactions: Methods and Protocols / [ed] D. Wade Abbott, Wesley F. Zandberg, New York: Springer Nature, 2023, 2, p. 15-25Chapter in book (Refereed)
    Abstract [en]

    Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration.

  • 25.
    Li, He
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3,5-Dinitrosalicylic Acid Assay2023In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 2657, p. 15-25Article in journal (Refereed)
    Abstract [en]

    Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration.

  • 26.
    Lindstad, Lars J.
    et al.
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway..
    Lo, Galiana
    Univ Aberdeen, Rowett Inst, Gut Hlth Grp, Aberdeen, Scotland..
    Leivers, Shaun
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway..
    Lu, Zijia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry. AlbaNova Univ Ctr, KTH Royal Inst Technol, Dept Chem, Div Glycosci, Stockholm, Sweden..
    Michalak, Leszek
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway..
    Pereira, Gabriel V.
    Univ Michigan, Sch Med, Dept Microbiol & Immunol, Ann Arbor, MI 48109 USA..
    Rohr, Asmund K.
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway..
    Martens, Eric C.
    Univ Michigan, Sch Med, Dept Microbiol & Immunol, Ann Arbor, MI 48109 USA..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Louis, Petra
    Univ Aberdeen, Rowett Inst, Gut Hlth Grp, Aberdeen, Scotland..
    Duncan, Sylvia H.
    Univ Aberdeen, Rowett Inst, Gut Hlth Grp, Aberdeen, Scotland..
    Westereng, Bjorge
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway..
    Pope, Phillip B.
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway.;Norwegian Univ Life Sci, Fac Biosci, As, Norway..
    Rosa, Sabina Leanti La
    Norwegian Univ Life Sci, Fac Chem Biotechnol & Food Sci, As, Norway.;Norwegian Univ Life Sci, Fac Biosci, As, Norway..
    Human Gut Faecalibacterium prausnitzii Deploys a Highly Efficient Conserved System To Cross-Feed on beta-Mannan-Derived Oligosaccharides2021In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 12, no 3, article id e03628-20Article in journal (Refereed)
    Abstract [en]

    beta-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of beta-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here, we show that a dominant butyrate producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various beta-mannooligosaccharides (beta-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two beta-MOS utilization loci (F. prausnitzii beta-MOS utilization loci [FpMULs]) supported a concerted model whereby the imported beta-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degraders Bacteroides ovatus and Roseburia intestinalis on polymeric beta-mannan resulted in syntrophic growth, thus confirming the high efficiency of the FpMULs' uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2,441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of beta-mannan metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. IMPORTANCE Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic, and detailed biochemical analyses, this work reveals the mechanism enabling F. prausnitzii, as a model Ruminococcaceae within Firmicutes, to cross-feed and access beta-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of beta-mannans/beta-MOS as a common dietary component. Our findings provide a mechanistic understanding of the beta-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, and thus butyrate production, in the gut.

  • 27.
    Lu, Zijia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Kvammen, Alma
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Li, He
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hao, Mengshu
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Inman, Annie R.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell walls2023In: mSphere, E-ISSN 2379-5042Article in journal (Refereed)
    Abstract [en]

    In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL’s outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade.

  • 28.
    Lu, Zijia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Rämgård, Carl
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Ergenlioğlu, İrem
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Sandin, Lova
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hammar, Hugo
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Andersson, Helena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    King, Katharine
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Inman, Annie R.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Hao, Mengshu
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. College of Medicine & Public Health, Flinders University, Adelaide, SA, Australia.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis2023In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 290, no 11, p. 2909-2922Article in journal (Refereed)
    Abstract [en]

    The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

  • 29.
    Mattsson, Tuve
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Azhar, Shoaib
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Bylin, Susanne
    Helander, Mikaela
    KTH.
    Henriksson, Gunnar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Jedvert, K
    Lawoko, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Lindström, Mikael
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    McKee, Lauren S.
    KTH.
    Oinonen, Petri
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Sevastyanova, Olena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Westerberg, N
    Theliander, H
    Towards a wood based material biorefinery - A demonstrator2015In: 6th Nordic Wood Biorefinery Conference, NWBC 2015, VTT Technical Research Centre of Finland , 2015, p. 92-101Conference paper (Refereed)
    Abstract [en]

    Wood, the most abundant ligno-cellulosic raw material available, is a key potential feedstock for production of more sustainable alternatives to fossil-based materials. However advances within the fields of extraction and treatment processes within what is often referred to as the biorefinery concept is essential to allow for such transition. In this study, several different methods for the extraction and separation of wood constituents have been combined in a single process with the purpose of achieving a high overall efficiency of material extraction and utilisation. The work builds on several activities within the Wallenberg Wood Science Center (WWSC). The aim is to present a laboratory-scale demonstrator that illustrates how the different constituents can be separated from the wood matrix for later use in the production of bio-based materials and chemicals. The process steps involved have been tested as integral steps in a linked process for a scale of operations that range from the kilogram-scale down to the gram-scale. Industrially chipped softwood, containing mainly spruce with some pine, was used as raw material. 

  • 30. Mattsson, Tuve
    et al.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Eriksson, Susanna
    Helander, Mikaela
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Jedvert, Kerstin
    Lawoko, Martin
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Lindström, Mikael E.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Oinonen, Petri
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sevastyanova, Olena
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Westerberg, Niklas
    Theliander, Hans
    The Development of a Wood-based Materials-biorefinery2017In: BioResources, ISSN 1930-2126, E-ISSN 1930-2126, Vol. 12, no 4, p. 9152-9182Article in journal (Refereed)
    Abstract [en]

    Several different methods for the extraction, separation, and purification of wood constituents were combined in this work as a unified process with the purpose of achieving a high overall efficiency of material extraction and utilization. This study aimed to present a laboratory-scale demonstrator biorefinery that illustrated how the different wood constituents could be separated from the wood matrix for later use in the production of new bio-based materials and chemicals by combining several approaches. This study builds on several publications and ongoing activities within the Wallenberg Wood Science Center (WWSC) in Sweden on the theme "From wood to material components." Combining the approaches developed in these WWSC projects - including mild steam explosion, membrane and chromatographic separation, enzymatic treatment and leaching, ionic liquid extraction, and fractionation together with Kraft pulping - formed an outline for a complete materials-biorefinery. The process steps involved were tested as integral steps in a linked process. The scale of operations ranged from the kilogram-scale to the gram-scale. The feasibility and efficiency of these process steps in a biorefinery system were assessed, based on the data, beginning with whole wood.

  • 31.
    McKee, Lauren
    et al.
    Newcastle University, United Kingdom; University of Georgia, United States.
    Pena, Maria J.
    Rogowski, Artur
    Jackson, Adam
    Lewis, Richard J.
    York, William S.
    Krogh, Kristian B. R. M.
    Vikso-Nielsen, Anders
    Skjot, Michael
    Gilbert, Harry J.
    Marles-Wright, Jon
    Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 17Article in journal (Refereed)
    Abstract [en]

    The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated beta-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall.

  • 32.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Measuring enzyme kinetics of glycoside hydrolases using the 3,5-dinitrosalicylic acid assay2017In: Methods in Molecular Biology, Humana Press Inc. , 2017, p. 27-36Conference paper (Refereed)
    Abstract [en]

    Use of the 3,5-dinitrosalicylic acid reagent allows the simple and rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in the characterization of enzyme reactions. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance and sugar concentration.

  • 33.
    McKee, Lauren S.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. Univ British Columbia, Canada.
    Growth of Chitinophaga pinensis on Plant Cell Wall Glycans and Characterisation of a Glycoside Hydrolase Family 27 beta-L-Arabinopyranosidase Implicated in Arabinogalactan Utilisation2015In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 10, article id e0139932Article in journal (Refereed)
    Abstract [en]

    The genome of the soil bacterium Chitinophaga pinensis encodes a diverse array of carbohydrate active enzymes, including nearly 200 representatives from over 50 glycoside hydrolase (GH) families, the enzymology of which is essentially unexplored. In light of this genetic potential, we reveal that C. pinensis has a broader saprophytic capacity to thrive on plant cell wall polysaccharides than previously reported, and specifically that secretion of beta-L-arabinopyranosidase activity is induced during growth on arabinogalactan. We subsequently correlated this activity with the product of the Cpin_5740 gene, which encodes the sole member of glycoside hydrolase family 27 (GH27) in C. pinensis, CpArap27. Historically, GH27 is most commonly associated with alpha-D-galactopyranosidase and alpha-D-N-acetylgalactosaminidase activity. A new phylogenetic analysis of GH27 highlighted the likely importance of several conserved secondary structural features in determining substrate specificity and provides a predictive framework for identifying enzymes with the less common beta-L-arabinopyranosidase activity.

  • 34.
    McKee, Lauren S.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    La Rosa, S. L.
    Westereng, B.
    Eijsink, V. G.
    Pope, P. B.
    Larsbrink, Johan
    Polysaccharide degradation by the Bacteroidetes: mechanisms and nomenclature2021In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 13, no 5, p. 559-581Article in journal (Refereed)
    Abstract [en]

    The Bacteroidetes phylum is renowned for its ability to degrade a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use polysaccharide utilization loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum-exclusive type IX secretion system (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL-encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non-PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that the literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions that can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems.

  • 35.
    McKee, Lauren S.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Martinez-Abad, Antonio
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Ruthes, Andrea C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. AlbaNova Univ Ctr, KTH Royal Inst .
    Vilaplana, Francisco
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Brumer, Harry
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Focused Metabolism of beta-Glucans by the Soil Bacteroidetes Species Chitinophaga pinensis2019In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 85, no 2, article id UNSP e02231-18Article in journal (Refereed)
    Abstract [en]

    The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of beta-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of beta-glucans isolated from different sources. Most noncrystalline beta-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, beta-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of beta-glucan metabolism. The significant accumulation of some types of beta-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for beta-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community. IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.

  • 36.
    McKee, Lauren S.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Sunner, Hampus
    Anasontzis, George E.
    Toriz, Guillermo
    Gatenholm, Paul
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. Univ Adelaide, Australia.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Olsson, Lisbeth
    A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan2016In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 9, article id 2Article in journal (Refereed)
    Abstract [en]

    Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant alpha-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me) GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an alpha-l-arabinofuranosidase (AbfA), and a beta-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 alpha-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

  • 37. Montanier, Cedric Y.
    et al.
    Correia, Marcia A. S.
    Flint, James E.
    Zhu, Yanping
    Basle, Arnaud
    McKee, Lauren
    Newcastle University, United Kingdom; University of Georgia, United States.
    Prates, Jose A. M.
    Polizzi, Samuel J.
    Coutinho, Pedro M.
    Lewis, Richard J.
    Henrissat, Bernard
    Fontes, Carlos M. G. A.
    Gilbert, Harry J.
    A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 25Article in journal (Refereed)
    Abstract [en]

    The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to D-galactose and L-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing alpha- and beta-galactose residues, show that the ligand-binding site in the beta-sandwich protein is located in the loops that connect the two beta-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.

  • 38. Niculaes, Claudiu
    et al.
    Morreel, Kris
    Kim, Hoon
    Lu, Fachuang
    Mckee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ivens, Bart
    Haustraete, Jurgen
    Vanholme, Bartel
    De Rycke, Riet
    Hertzberg, Magnus
    Fromm, Jörg
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Polle, Andrea
    Ralph, John
    Boerjan, Wout
    Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem2014In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 26, no 9, p. 3775-3791Article in journal (Refereed)
    Abstract [en]

    Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

  • 39.
    Panahabadi, Rahele
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. Department of Plant Science and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
    Ahmadikhah, Asadollah
    Shahid Beheshti Univ, Fac Life Sci & Biotechnol, Dept Plant Sci & Biotechnol, Tehran, Iran..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Ingvarsson, Pär K.
    Swedish Univ Agr Sci, Linnean Ctr Plant Biol, Dept Plant Biol, Uppsala, Sweden..
    Farrokhi, Naser
    Shahid Beheshti Univ, Fac Life Sci & Biotechnol, Dept Plant Sci & Biotechnol, Tehran, Iran..
    Genome-Wide Association Mapping of Mixed Linkage (1,3;1,4)-beta-Glucan and Starch Contents in Rice Whole Grain2021In: Frontiers in Plant Science, E-ISSN 1664-462X, Vol. 12, article id 665745Article in journal (Refereed)
    Abstract [en]

    The glucan content of rice is a key factor defining its nutritional and economic value. Starch and its derivatives have many industrial applications such as in fuel and material production. Non-starch glucans such as (1,3;1,4)-beta-D-glucan (mixed-linkage beta-glucan, MLG) have many benefits in human health, including lowering cholesterol, boosting the immune system, and modulating the gut microbiome. In this study, the genetic variability of MLG and starch contents were analyzed in rice (Oryza sativa L.) whole grain, by performing a new quantitative analysis of the polysaccharide content of rice grains. The 197 rice accessions investigated had an average MLG content of 252 mu g/mg, which was negatively correlated with the grain starch content. A new genome-wide association study revealed seven significant quantitative trait loci (QTLs) associated with the MLG content and two QTLs associated with the starch content in rice whole grain. Novel genes associated with the MLG content were a hexose transporter and anthocyanidin 5,3-O-glucosyltransferase. Also, the novel gene associated with the starch content was a nodulin-like domain. The data pave the way for a better understanding of the genes involved in determining both MLG and starch contents in rice grains and should facilitate future plant breeding programs.

  • 40.
    Panahabadi, Rahele
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. Faculty of Life Sciences and Biotechnology, Shahid Beheshti Univ., Tehran, Iran.
    Ahmadikhah, Asadollah
    Faculty of Life Sciences and Biotechnology, Shahid Beheshti Univ., Tehran, Iran.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Ingvarsson, Pär K.
    Linnean Centre for Plant Biology, Dep. of Plant Biology, Swedish Univ. of Agricultural Sciences, Uppsala, Sweden.
    Farrokhi, Naser
    Faculty of Life Sciences and Biotechnology, Shahid Beheshti Univ., Tehran, Iran.
    Genome-wide association study for lignocellulosic compounds and fermentable sugar in rice straw2022In: The Physical Educator, ISSN 0031-8981, E-ISSN 1940-3372, Vol. 15, no 1, article id e20174Article in journal (Refereed)
    Abstract [en]

    Cellulose and lignin are the two main components of secondary plant cell walls with substantial impact on stalk in the field and on straw during industrial processing. The amount of fermentable sugar that can be accessed is another important parameter affecting various industrial applications. In the present study, genetic variability of rice (Oryza sativa L.) genotypes for cellulose, lignin, and fermentable sugars contents was analyzed in rice straw. A genome-wide association study of 33,484 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) >0.05 was performed. The genome-wide association study identified seven, three, and three genomic regions to be significantly associated with cellulose, lignin, and fermentable sugar contents, respectively. Candidate genes in the associated genomic regions were enzymes mainly involved in cell wall metabolism. Novel SNP markers associated with cellulose were tagged to GH16, peroxidase, GT6, GT8, and CSLD2. For lignin content, Villin protein, OsWAK1/50/52/53, and GH16 were identified. For fermentable sugar content, UTP-glucose-1-phosphate uridylyltransferase, BRASSINOSTEROID INSENSITIVE 1, and receptor-like protein kinase 5 were found. The results of this study should improve our understanding of the genetic basis of the factors that might be involved in biosynthesis, turnover, and modification of major cell wall components and saccharides in rice straw.

  • 41.
    Pang, Zhili
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. Department of Plant Pathology, China Agricultural University, Beijing, 100193, People’s Republic of China.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Srivastava, Vaibhav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Klinter, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Diaz-Moreno, Sara M
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Orlean, Peter
    Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
    Liu, Xili
    Department of Plant Pathology, China Agricultural University, Beijing, 100193, People’s Republic of China.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. ARC Centre of Excellence in Plant Cell Walls and School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, SA, 5064, Australia.
    Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici2020In: Cellulose, ISSN 0969-0239, E-ISSN 1572-882X, Vol. 27, no 15, p. 8551-8565Article in journal (Refereed)
    Abstract [en]

    Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

  • 42.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Alexakis, Alexandros Efraim
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Structure-property relationship of native-like lignin nanoparticles from softwood and hardwoodManuscript (preprint) (Other academic)
    Abstract [en]

    An intense effort has been put into replacing fossil- with bio-based materials. To achieve this goal in a  sustainable manner, renewable resources such as wood can be used. Lignin makes up around % of  woody biomass and has shown great potential in the preparation of nanoparticles (LNPs) of use in  diverse material applications. Typically, LNPs are prepared from so-called technical lignins deriving  from the pulping process, with formation thought to be driven by the hydroxyl content and molecular  weight of lignin. However, other equally important parameters are the lignin concentration and  monolignol composition. In this study, we used native-like lignins extracted from hardwood and  softwood under mild conditions to investigate the impact of lignin structure on the formation of LNPs.  We identified a synergistic effect between π-π stacking and hydroxyl group content in nanoparticle  formation. LNP morphology, including compact and collapsed spheres or ‘snowman’ aggregates,  seemed to depend mainly on monolignol composition. The results described herein provide an in depth perspective on the formation of LNPs from non-technical lignins and take us closer to  understanding their structure-property relationship. With this study we aim to promote lignin-first  biorefinery concepts, in which the lignin structure is preserved during extraction

  • 43.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Alexakis, Alexandros Efraim
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Structure-property relationship of native-like lignin nanoparticles from softwood and hardwood2023In: Industrial crops and products (Print), ISSN 0926-6690, E-ISSN 1872-633X, Vol. 206, article id 117660Article in journal (Refereed)
    Abstract [en]

    Renewable resources such as wood are an important candidate towards the replacement of fossil-based materials with bio-based materials. Lignin, comprising up to 40% of woody biomass, has shown great potential in the preparation of nanoparticles (LNPs), which can be used in diverse material applications. Typically, LNPs are prepared from technical lignins, deriving from pulping processes. Their formation is thought to be driven by the hydroxyl content and molecular weight of lignin. However, other equally important parameters are the monolignol composition and the concentration of lignin used. In this study, we used native-like lignin fractions from hardwood and softwood to investigate the impact of lignin structure on LNP formation. We identified a synergistic effect between 7C-7C stacking and hydroxyl group content in nanoparticle formation. Our LNPs exhibited different morphologies, including compact, collapsed spheres, and 'snowman' aggregates. The results described herein provide an in-depth perspective on the formation and structure-property relationship of LNPs from native -like lignin fractions. With this study we aim to promote biorefinery concepts, in which the lignin structure is preserved during extraction.

  • 44.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Karkonen, Anna
    Nat Resources Inst Finland Luke, Prod Syst, Helsinki, Finland.;Univ Helsinki, Viikki Plant Sci Ctr, Dept Agr Sci, Helsinki, Finland..
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    The impact of xylan on the biosynthesis and structure of extracellular lignin produced by a Norway spruce tissue culture2023In: PLANT DIRECT, ISSN 2475-4455, Vol. 7, no 6, article id e500Article in journal (Refereed)
    Abstract [en]

    In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non-native ones. In this study, Norway spruce (Picea abies) tissue-cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin-carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology.

  • 45.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Kärkönen, Anna
    Natural Resources Institute Finland (Luke), Production Systems, Helsinki, Finland:Viikki Plant Science Centre, Department of Agricultural Sciences, University of Helsinki, Helsinki, 9 Finland.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    The impact of xylan on the biosynthesis and structure of extracellular lignin produced by a Norway spruce tissue cultureManuscript (preprint) (Other academic)
  • 46.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Sivan, Pramod
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Srivastava, Vaibhav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Transcriptomic analysis of Picea abies tissue culture reveals the impact of culture conditions and the presence of glucuronoxylan on extracellular lignin productionManuscript (preprint) (Other academic)
  • 47.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    van Erven, Gijs
    Wageningen Food and Biobased Research, Wageningen University & Research, Bornse Weilanden 9, 8 6708 WG, Wageningen, The Netherlands;Laboratory of Food Chemistry, Wageningen University & Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
    Heidling, Emelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Lawoko, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    The impact of extraction method on the structure of lignin from ball milled hardwoodManuscript (preprint) (Other academic)
  • 48.
    Sapouna, Ioanna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    van Erven, Gijs
    Wageningen Univ & Res, Wageningen Food & Biobased Res, NL-6708 WG Wageningen, Netherlands.;Wageningen Univ & Res, Lab Food Chem, NL-6708 WG Wageningen, Netherlands..
    Heidling, Emelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Lawoko, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    McKee, Lauren Sara
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Impact of Extraction Method on the Structure of Lignin from Ball-Milled Hardwood2023In: ACS Sustainable Chemistry and Engineering, E-ISSN 2168-0485, Vol. 11, no 43, p. 15533-15543Article in journal (Refereed)
    Abstract [en]

    Understanding the structure of hardwoods can permit better valorization of lignin by enabling the optimization of green, high-yield extraction protocols that preserve the structure of wood biopolymers. To that end, a mild protocol was applied for the extraction of lignin from ball-milled birch. This made it possible to understand the differences in the extractability of lignin in each extraction step. The fractions were extensively characterized using 1D and 2D nuclear magnetic resonance spectroscopy, size exclusion chromatography, and pyrolysis-gas chromatography-mass spectrometry. This comprehensive characterization highlighted that lignin populations extracted by warm water, alkali, and ionic liquid/ethanol diverged in structural features including subunit composition, interunit linkage content, and the abundance of oxidized moieties. Moreover, ether- and ester-type lignin-carbohydrate complexes were identified in the different extracts. Irrespective of whether natively present in the wood or artificially formed during extraction, these complexes play an important role in the extractability of lignin from ball-milled hardwood. Our results contribute to the further improvement of lignin extraction strategies, for both understanding lignin as present in the lignocellulosic matrix and for dedicated lignin valorization efforts.

  • 49.
    Schönbichler, Anna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. Univ Vet Med Vienna, Unit Funct Canc Genom, Vienna, Austria..
    Diaz-Moreno, Sara M
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Srivastava, Vaibhav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    McKee, Lauren S.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Exploring the Potential for Fungal Antagonism and Cell Wall Attack by Bacillus subtilis natto2020In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 11, article id 521Article in journal (Refereed)
    Abstract [en]

    To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches - the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish "natto." Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.

  • 50.
    Thongpoo, Preeyanuch
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Araújo, Ana Catarina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kongsaeree, Prachumporn T.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) beta-glucosidase from Aspergillus niger ASKU282013In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1830, no 3, p. 2739-2749Article in journal (Refereed)
    Abstract [en]

    Background: The commercially important glycoside hydrolase family 3 (GH3) beta-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined. Methods: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 beta-glucosidase from A. niger ASKU28. Results: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K-m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on add catalysis, although changes in the rate-determining catalytic step were revealed for both, pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl beta-glucosides and yielded beta-glucosyl azide as a product. Conclusions and general significance: These results strongly support the assignment of E490 as the acid/base catalyst in a beta-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.

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