[Show abstract][Hide abstract] ABSTRACT: Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and which possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a Polysaccharide Utilization Locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two GH5 endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity towards β-glucans and, to a lesser extent mannan, but unlike the two SusD-like lipoproteins previously characterised from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen which was determined using comprehensive microarray polymer profiling and showed abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated a "one cognate PUL for each substrate" type of gene organization. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports SRM-1 dominance in the Svalbard reindeer rumen microbiome.
Applied and Environmental Microbiology 10/2014; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chitin-active 19.2 kDa lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis has been isotopically labeled and recombinantly expressed. In this paper, we report the (1)H, (13)C, (15)N resonance assignment of BlLPMO10A.
[Show abstract][Hide abstract] ABSTRACT: Microbial community profiles in two parallel CSTR biogas reactors fed with whey permeate and cow manure were investigated. The operating conditions for these two reactors were identical, yet only one of them (R1) showed stable performance, whereas the other (R2) showed a decrease in methane production accompanied by accumulation of propionic acid and, later, acetic acid. This gave a unique opportunity to study the dynamics of the microbial communities in two biogas reactors apparently operating close to the edge of stability. The microbial community was dominated by Bacteroidetes and Firmicutes, and the methanogens Methanobacteriales and Methanomicrobiales in both reactors, but with larger fluctuations in R2. Correlation analyses showed that the depletion of propionic acid in R1 and the late increase of acetic acid in R2 was related to several bacterial groups. The biogas production in R1 shows that stable co-digestion of manure and whey can be achieved with reasonable yields.
[Show abstract][Hide abstract] ABSTRACT: The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing.
[Show abstract][Hide abstract] ABSTRACT: Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase-containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate binding modules and the highest number of CBM33-type lytic polysaccharide monooxygenases. Actinobacteria do also have the highest fraction of genomes containing β -1, 3-glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase-containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell-wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.
[Show abstract][Hide abstract] ABSTRACT: Reaching a comprehensive understanding of how nature solves the problem of degrading recalcitrant biomass may eventually allow development of more efficient biorefining processes. Here we interpret genomic and proteomic information generated from a cellulolytic microbial consortium (termed F1RT) enriched from soil. Analyses of reconstructed bacterial draft genomes from all seven uncultured phylotypes in F1RT indicate that its constituent microbes cooperate in both cellulose-degrading and other important metabolic processes. Support for cellulolytic inter-species cooperation came from the discovery of F1RT microbes that encode and express complimentary enzymatic inventories that include both extracellular cellulosomes and secreted free-enzyme systems. Metabolic reconstruction of the seven F1RT phylotypes predicted a wider genomic rationale as to how this particular community functions as well as possible reasons as to why biomass conversion in nature relies on a structured and cooperative microbial community.
[Show abstract][Hide abstract] ABSTRACT: For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu(2+) (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms.
Proceedings of the National Academy of Sciences of the United States of America. 05/2014;
[Show abstract][Hide abstract] ABSTRACT: Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a helical X-ray diffraction method with minimal X-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystal was X-ray photo-reduced, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery, and provides a general method for solving LPMO structures in both copper oxidation states.
Journal of Biological Chemistry 05/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.
Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malassezia species are ubiquitous residents of human skin and are associated with several diseases, such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. M. restricta is one of the most predominant yeasts of the healthy human skin and in this paper, its cell wall has been investigated. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharidic core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans that are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here shows that M. restricta has a polysaccharide organization very different of all fungal species analysed to date.
Journal of Biological Chemistry 03/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate active enzyme classification system, are copper-dependent enzymes that oxidize sp3-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate-binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.
[Show abstract][Hide abstract] ABSTRACT: Processing of lignocellulosic materials to fuels such as methane and bioethanol may involve several processing steps including pretreatment, saccharification, fermentation, and anaerobic digestion. The amounts of substrate used in these processes are usually based on dry matter content, and the processes themselves typically lead to a change in dry matter content. Thus, it is of great importance to be able to measure dry matter accurately. Dry matter content is commonly determined by measuring loss of water during oven drying. We have used Karl Fischer (KF) titration to measure the water content in a wide range of biomass fractions and have compared these data to results obtained by oven drying. This revealed considerable differences for all tested materials. For lignocellulosic materials, oven drying tends to overestimate dry matter content for untreated material. On the other hand, oven drying generally underestimates dry matter content in pretreated materials due to loss of organic volatiles. These differences have major consequences for the calculation of mass balances and yields in bioprocessing. The KF method gives more accurate water determination than oven drying due to the unique selectivity of the analysis. The method is suitable for the analysis of lignocellulosic biomasses and is particularly useful for determination of water content in pretreated materials, where oven drying usually underestimates the dry matter content due to loss of volatiles.
[Show abstract][Hide abstract] ABSTRACT: Chitosan is a linear heteropolymer consisting of β 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DPn (average degree of polymerization) 206 and FA (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DPn in the range of 15-40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases.
PLoS ONE 01/2014; 9(4):e93192. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species.
PLoS ONE 01/2014; 9(3):e91125. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e. products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed and there has been some debate concerning the nature of this position (C4 or C6). In this study we have characterized a LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property which allowed detailed product analysis. Using mass spectrometry and NMR we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the non-reducing end.
Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of mono-component chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only non-processive endo-chitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.
Journal of Agricultural and Food Chemistry 10/2013; · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues.
In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS.
The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase.
Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-D-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group. This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.
Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human chitotriosidase (HCHT) is a family 18 chitinase that is an innate part of the immune system. We have mapped preferred productive binding modes of chito-oligosaccharide substrates to HCHT and the data show that HCHT has strong binding affinity in the +3 subsite. Moreover, HCHT shows anomer-specific binding affinities in subsites +2 and +3. These features could endorse HCHT with higher endo-activity and a higher transglycosylation potential.
[Show abstract][Hide abstract] ABSTRACT: It has recently been shown that the major autolysin Acm2 from L. plantarum WCFS1 undergoes intracellular O-GlcNAcylation (Fredriksen et al., J. Bacteriol. 194(2): 325-333). To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation (HCD) of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsY, and the DNA translocase FtsK1. Among the other glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for HexNAc were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK).
[Show abstract][Hide abstract] ABSTRACT: Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.