Xylanase production by Thermobacillus xylanilyticus is impaired by population diversification but can be mitigated based on the management of cheating behavior.

BACKGROUND: The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. RESULTS: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. CONCLUSIONS: Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.

Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi.

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

Pilot scale production of recombinant hemicellulases and their saccharification potential.

Synergistic saccharification ability of hemicellulases (endo-xylanase and ß-xylosidase) was evaluated in this study for the bioethanol production from plant biomass. Endo-xylanase and ß-xylosidase genes from Bacillus licheniformis were cloned and expressed in Escherichia coli BL21 (DE3). Maximum endo-xylanase production was obtained at 200 rpm agitation speed, air supply rate 2.0 vvm, 70% volume of the medium, 20% dissolved oxygen level and with 3% inoculum size. The optimal conditions for maximum production of recombinant ß-xylosidase enzyme at pilot scale were 200 rpm agitation speed, 25% dissolved oxygen level, 2.5 vvm aeration rate, 70% volume of the medium with 2% inoculum size. Furthermore, the saccharification potential of these recombinant enzymes was checked for the production of xylose sugar by bioconversion of plant biomass by optimizing individually as well as synergistically by optimizing various parameters. Maximum saccharification (93%) of plant biomass was observed when both enzymes were used at a time with 8% sugarcane bagasse as a substrate and 200 units of each enzyme after incubation of 6 hr at 50 °C and 120 rpm. The results obtained in this study suggested these recombinant hemicellulases as potential candidates for the conversion of complex agricultural residues into simple sugars for ultimate use in the biofuel industry.

Integrative Proteomic Analysis of Digestive Tract Glycosidases from the Invasive Golden Apple Snail, Pomacea canaliculata.

The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata. A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

Cellulase-Hemicellulase Activities and Bacterial Community Composition of Different Soils from Algerian Ecosystems.

Soil microorganisms are important mediators of carbon cycling in nature. Although cellulose- and hemicellulose-degrading bacteria have been isolated from Algerian ecosystems, the information on the composition of soil bacterial communities and thus the potential of their members to decompose plant residues is still limited. The objective of the present study was to describe and compare the bacterial community composition in Algerian soils (crop, forest, garden, and desert) and the activity of cellulose- and hemicellulose-degrading enzymes. Bacterial communities were characterized by high-throughput 16S amplicon sequencing followed by the in silico prediction of their functional potential. The highest lignocellulolytic activity was recorded in forest and garden soils whereas activities in the agricultural and desert soils were typically low. The bacterial phyla Proteobacteria (in particular classes α-proteobacteria, δ-proteobacteria, and γ-proteobacteria), Firmicutes, and Actinobacteria dominated in all soils. Forest and garden soils exhibited higher diversity than agricultural and desert soils. Endocellulase activity was elevated in forest and garden soils. In silico analysis predicted higher share of genes assigned to general metabolism in forest and garden soils compared with agricultural and arid soils, particularly in carbohydrate metabolism. The highest potential of lignocellulose decomposition was predicted for forest soils, which is in agreement with the highest activity of corresponding enzymes.

Biomass sorghum as a novel substrate in solid-state fermentation for the production of hemicellulases and cellulases by Aspergillus niger and A. fumigatus.

AIMS: We investigated the role of carbon and nitrogen sources in the production of cellulase and hemicellulase by Aspergillus strains. METHODS AND RESULTS: The strains Aspergillus niger SCBM1 and Aspergillus fumigatus SCBM6 were cultivated under solid-state fermentation (SSF), with biomass sorghum (BS) and wheat bran (WB) as lignocellulosic substrates, in different proportions, along with variable nitrogen sources. The best SSF condition for the induction of such enzymes was observed employing A. niger SCBM1 in BS supplemented with peptone; maximum production levels were achieved as follows: 72 h of fermentation for xylanase and exoglucanase (300·07 and 30·64 U g-1 respectively), 120 h for ß-glucosidase and endoglucanase (54·90 and 41·47 U g-1 respectively) and 144 h for ß-xylosidase (64·88 U g-1 ). CONCLUSIONS: This work demonstrated the viability of the use of BS for the production of hemi- and cellulolytic enzymes; the high concentration of celluloses in BS could be associated with the significant production of cellulases, mainly exoglucanase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study which presents the promising use of biomass sorghum (genetically modified sorghum to increase its biomass content) as an alternative carbon source for the production of enzymes by SSF.

Identification and expression profiling of novel plant cell wall degrading enzymes from a destructive pest of palm trees, Rhynchophorus ferrugineus.

Plant cell wall degrading enzymes (PCWDEs) from insects were recently identified as a multigene family of proteins that consist primarily of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) and play essential roles in the degradation of the cellulose/hemicellulose/pectin network in the invaded host plant. Here we applied transcriptomic and degenerate PCR approaches to identify the PCWDEs from a destructive pest of palm trees, Rhynchophorus ferrugineus, followed by a gut-specific and stage-specific differential expression analysis. We identified a total of 27 transcripts encoding GH family members and three transcripts of the CE family with cellulase, hemicellulase and pectinase activities. We also identified two GH9 candidates, which have not previously been reported from Curculionidae. The gut-specific quantitative expression analysis identified key cellulases, hemicellulases and pectinases from R. ferrugineus. The expression analysis revealed a pectin methylesterase, RferCE8u02, and a cellulase, GH45c34485, which showed the highest gut enriched expression. Comparison of PCWDE expression patterns revealed that cellulases and pectinases are significantly upregulated in the adult stages, and we observed specific high expression of the hemicellulase RferGH16c4170. Overall, our study revealed the potential of PCWDEs from R. ferrugineus, which may be useful in biotechnological applications and may represent new tools in R. ferrugineus pest management strategies.

Efficient screening of potential cellulases and hemicellulases produced by Bosea sp. FBZP-16 using the combination of enzyme assays and genome analysis.

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, ß-glucosidase and ß-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development.

The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.

Insights into the mechanism of enzymatic hydrolysis of xylan.

Hemicelluloses are a vast group of complex, non-cellulosic heteropolysaccharides that are classified according to the principal monosaccharides present in its structure. Xylan is the most abundant hemicellulose found in lignocellulosic biomass. In the current trend of a more effective utilization of lignocellulosic biomass and developments of environmentally friendly industrial processes, increasing research activities have been directed to a practical application of the xylan component of plants and plant residues as biopolymer resources. A variety of enzymes, including main- and side-chain acting enzymes, are responsible for xylan breakdown. Xylanase is a main-chain enzyme that randomly cleaves the ß-1,4 linkages between the xylopyranosyl residues in xylan backbone. This enzyme presents varying folds, mechanisms of action, substrate specificities, hydrolytic activities, and physicochemical characteristics. This review pays particular attention to different aspects of the mechanisms of action of xylan-degrading enzymes and their contribution to improve the production of bioproducts from plant biomass. Furthermore, the influence of phenolic compounds on xylanase activity is also discussed.

The use of thermostable bacterial hemicellulases improves the conversion of lignocellulosic biomass to valuable molecules.

The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60 °C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.

Differential Carbon Catabolite Repression and Hemicellulolytic Ability among Pathotypes of Colletotrichum lindemuthianum against Natural Plant Substrates.

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

Enzyme synergy for plant cell wall polysaccharide degradation.

Valorizing plant cell wall, marine and algal polysaccharides is of utmost importance for the development of the circular bioeconomy. This is because polysaccharides are by far the most abundant organic molecules found in nature with complex chemical structures that require a large set of enzymes for their degradation. Microorganisms produce polysaccharide-specific enzymes that act in synergy when performing hydrolysis. Although discovered since decades enzyme synergy is still poorly understood at the molecular level and thus it is difficult to harness and optimize. In the last few years, more attention has been given to improve and characterize enzyme synergy for polysaccharide valorization. In this review, we summarize literature to provide an overview of the different type of synergy involving carbohydrate modifying enzymes and the recent advances in the field exemplified by plant cell-wall degradation.

Hemicellulolytic bacteria in the anterior intestine of the earthworm Eisenia fetida (Sav.).

Tropical agriculture produces large amounts of lignocellulosic residues that can potentially be used as a natural source of value-added products. The complexity of lignocellulose makes industrial-scale processing difficult. New processing techniques must be developed to improve the yield and avoid this valuable resource going to waste. Hemicelluloses comprise a variety of polysaccharides with different backbone compositions and decorations (such as methylations and acetylations), and form part of an intricate framework that confers structural stability to the plant cell wall. Organisms that are able to degrade these biopolymers include earthworms (Eisenia fetida), which can rapidly decompose a wide variety of lignocellulosic substrates. This ability probably derives from enzymes and symbiotic microorganisms in the earthworm gut. In this work, two substrates with similar C/N ratios but different hemicellulose content were selected. Palm fibre and coffee husk have relatively high (28%) and low (5%) hemicellulose contents, respectively. A vermicomposting mixture was prepared for the earthworms to feed on by mixing a hemicellulose substrate with organic market waste. Xylanase activity was determined in earthworm gut and used as a selection criterion for the isolation of hemicellulose-degrading bacteria. Xylanase activity was similar for both substrates, even though their physicochemical properties principally pH and electrical conductivity, as shown by the MANOVA analysis) were different for the total duration of the experiment (120 days). Xylanolytic strains isolated from earthworm gut were identified by sequence analysis of the 16S rRNA gene. Our results indicate that the four Actinobacteria, two Proteobacteria, and one Firmicutes isolated are active participants of the xylanolytic degradation by microbiota in the intestine of E. fetida. Most bacteria were more active at pH 7 and 28 °C, and those with higher activities are reported as being facultatively anaerobic, coinciding with the microenvironment reported for the earthworm gut. Each strain had a different degradative capacity.

Fusarium graminearum as a producer of xylanases with low cellulases when grown on wheat bran.

The xylanolytic potential of endophytic fungi isolated from leaves of Theobroma cacao was explored for the first time. Four fungal strains showed significant amounts of xylanase activity and low cellulase levels when grown on wheat bran as the sole carbon source. Strain Ec220 of Fusarium graminearum had the highest xylanase production (1.79 U/ml), whereas its cellulase activity was minimal (0.24 U/ml). Optimal conditions for xylanase production were: 154 h of incubation time, pH 5.79 and 29.8 °C. Furthermore, two protein spots detected by two-dimensional gel electrophoresis showed molecular weights (26.05 and 27.70 kDa) and isoelectric points (6.18 and 9.20) corresponding to previously reported F. graminearum xylanases, Xyl A and Xyl B, respectively. Therefore, endophytic fungi of T. cacao can be an important source of xylanolytic activities when cultured on wheat bran, and xylanases with low cellulases found in strain Ec220 require further characterization as they show promise for possible industrial applications.

A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs.

BACKGROUND: Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. RESULTS: Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a ß-mannanase gene M330 (without the stop codon), a P2A sequence and a ß-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular ß-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular ß-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and ß-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of ß-xylanase and ß-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, ß-mannanase, ß-xylanase, and ß-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. CONCLUSIONS: The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Seasonality affects function and complexity but not diversity of the rhizosphere microbiome in European temperate grassland.

Knowledge on how grassland microbiota responds on gene expression level to winter-summer change of seasons is poor. Here, we used a combination of quantitative PCR-based assays and metatranscriptomics to assess the impact of seasonality on the rhizospheric microbiota in temperate European grassland. Bacteria dominated, being at least one order of magnitude more abundant than fungi. Despite a fivefold summer increase in bacterial community size, season had nearly no effect on microbiome diversity. It, however, had a marked impact on taxon-specific gene expression, with 668 genes significantly differing in relative transcript abundance between winter and summer samples. Acidobacteria, Bacteroidetes, Planctomycetes, and Proteobacteria showed a greater relative gene expression activity in winter, while mRNA of Actinobacteria and Fungi was, relative to other taxa, significantly enriched in summer. On functional level, mRNA involved in protein turnover (e.g., transcription and translation) and cell maintenance (e.g., chaperones that protect against cell freezing damage such as GroEL and Hsp20) were highly enriched in winter. By contrast, mRNA involved in central carbon and amino acid metabolisms had a greater abundance in summer. Among carbohydrate-active enzymes, transcripts of GH36 family (hemicellulases) were highly enriched in winter, while those encoding GH3 family (cellulases) showed increased abundance in summer. The seasonal differences in plant polymer breakdown were linked to a significantly greater microbial network complexity in winter than in summer. Conceptually, the winter-summer change in microbiome functioning can be well explained by a shift from stress-tolerator to high-yield life history strategy.

Structural characterization of the family GH115 α-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with α-arabinofuranosidases.

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.

The coordinated action of glucuronoyl esterase and α-glucuronidase promotes the disassembly of lignin-carbohydrate complexes.

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate.