Julia Schückel

Do Rumen Bacteroidetes Utilize an Alternative Mechanism for Cellulose Degradation

ABSTRACT Uncultured and therefore uncharacterized Bacteroidetes lineages are ubiquitous in many natural ecosystems which specialize in lignocellulose degradation. However, their metabolic contribution remains mysterious, as well-studied cultured Bacteroidetes have been shown to degrade only soluble polysaccharides within the human distal gut and herbivore rumen. We have interrogated a reconstructed genome from an uncultured Bacteroidetes phylotype that dominates a switchgrass-associated community within the cow rumen. Importantly, this characterization effort has revealed the first preliminary evidence for polysaccharide utilization locus (PUL)-catalyzed conversion of cellulose. Based on these findings, we propose a further expansion of the PUL paradigm and the saccharolytic capacity of rumen Bacteroidetes species to include cellulose, the most abundant terrestrial polysaccharide on Earth. Moreover, the perspective of a cellulolytic PUL lays the foundation for PULs to be considered an alternative mechanism for cellulose degradation, next to cellulosomes and free-enzyme systems.

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes

Background: There is a growing discrepancy between the putative identification and the empirical characterization of carbohydrate-active enzymes (CAZymes). Results: We have developed a new versatile and high throughput microarray-based method for screening CAZymes. Conclusion: The method is a powerful addition to the enzyme screening toolbox. Significance: The technique enables the rapid screening of CAZymes and facilitates our biological understanding and industrial utilization. Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

BackgroundEnzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.ResultsWe have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained.ConclusionsWe show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.

A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

ABSTRACT Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that 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 glycoside hydrolase family 5 (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 toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized 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 an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Significance Plant cell wall (PCW) polysaccharide degradation is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Ruminococcus flavefaciens synthesizes a highly efficient PCW degrading apparatus. Here, six previously unidentified R. flavefaciens CBM families were identified that targeted β-glucans, β-mannans, and pectins. Crystal structures of these CBMs revealed that recognition of β-glucans and β-mannans was mediated by differences in the conformation of conserved aromatic residues in the ligand binding cleft. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan. This report shows that the expansion of protein modules in the cellulosome of R. flavefaciens contributes to an extended CBM profile that supports efficient PCW degradation. The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Pea border cell maturation and release involve complex cell wall structural dynamics

An integrative glycobiology approach provided new insight into multiple modes of cell wall remodeling during pea border cell formation and mechanisms of their release. The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes.

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzymes biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

Enzyme activities at different stages of plant biomass decomposition in three species of fungus-growing termites

ABSTRACT Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play an important role in fruit development and ripening processes by modulating the plant cell wall. Knowledge about these enzymes is important for research in fruit development and also important for industry regarding postharvest properties. Although advances in genetic control and cell wall biochemistry have led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy-to-handle, multiplexed and highly reproducible method using CPH assays where different fruits have been screened for enzyme activity. Additionally, the importance and impact of the extraction method and buffer conditions on the assay are investigated. We will show that one experimental setup can be used for testing all enzymes.

Two-Dimensional High-Throughput Endo -Enzyme Screening Assays Based on Chromogenic Polysaccharide Hydrogel and Complex Biomass Substrates

In this chapter, we present a two-dimensional approach for high-throughput screening of endo-cellulases as well as other endo-acting enzymes. The method is based on chromogenic substrates, produced either from purified or complex material, providing valuable information about enzyme activity toward its target as well as that same target in a context of complex natural material normally encountered in bioindustrial settings. The enzymes that can be tested using this assay can be from virtually any source: in purified form, directly from microbial cultures or even from raw materials, enabling study of the interplay between enzyme mixtures such as synergistic or inhibitory effects. By using the method of analysis described in this chapter, enzymes can be screened and evaluated quickly and information pertinent to both the inherent properties of the enzyme itself as well as predictions about its performance on complex biomass samples can be obtained.