Carbohydrate-binding Module Research Articles

Crystal structure of β-d-galactofuranosidase from Streptomyces sp. JHA19 in complex with an inhibitor provides insights into substrate specificity.

d-Galactofuranose (Galf) is widely distributed in glycoconjugates of pathogenic microbes. β-d-Galactofuranosidase (Galf-ase) from Streptomyces sp. JHA19 (ORF1110) belongs to glycoside hydrolase (GH) family 2 and is the first identified Galf-specific degradation enzyme. Here, the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, d-iminogalactitol (Ki = 65 μm) was solved. ORF1110 binds to the C5-C6 hydroxy groups of d-iminogalactitol with an extensive and integral hydrogen bond network, a key interaction that discriminates the substrates. The active site structure of ORF1110 is largely different from those of β-glucuronidases and β-galactosidases in the same GH2 family. A C-terminal domain of ORF1110 is predicted to be a carbohydrate-binding module family 42 that may bind Galf. The structural insights into Galf-ase will contribute to the investigation of therapeutic tools against pathogens.

Exploring the rumen microbial function in Angus bulls with divergent residual feed intake

This study leverages Shotgun metagenomics to assess the rumen microbial community and functionality in Angus bulls with differing residual feed intake-expected progeny difference (RFI-EPD) values, aiming to elucidate the microbial contributions to feed efficiency. Negative RFI-EPD bulls (NegRFI: n=10; RFI-EPD= -0.3883 kg/d) and positive RFI-EPD bulls (PosRFI: n=10; RFI-EPD=0.2935 kg/d) were selected from a group of 59 Angus bulls (average body weight (BW) = 428 ± 18.8 kg; 350 ± 13.4 d of age) fed a high-forage total mixed ration after a 60-d testing period. At the end of the 60-d period, rumen fluid samples were collected for bacterial DNA extraction and subsequent shotgun metagenomic sequencing. Results of the metagenome analysis revealed greater gene richness in NegRFI bulls, compared to PosRFI. Analysis of similarity revealed a small but noticeable difference (P =0.052; R-value = 0.097) in the rumen microbial community of NegRFI and PosRFI bulls. Linear Discriminant Analysis effect size (Lefse) was utilized to identify the differentially abundant taxa. The Lefse results showed that class Fibrobacteria (LDA = 5.1) and genus Fibrobacter (LDA = 4.8) were greater in NegRFI bulls, compared to PosRFI bulls. Relative abundance of the carbohydrate-active enzymes was also compared using Lefse. The results showed greater relative abundance of glycoside hydrolases and carbohydrate-binding modules such as GH5, CBM86, CBM35, GH43, and CBM6 (LDA > 3.0) in NegRFI bulls whereas GH13 and GT2 were greater in PosRFI bulls. The distinct metabolic and microbial profiles observed in NegRFI, compared to PosRFI bulls, characterized by greater gene richness and specific taxa such as Fibrobacter, and variations in carbohydrate-active enzymes, underscore the potential genetic and functional differences in their rumen microbiome. These findings contribute to a deeper understanding of the interplay between rumen microbiota and feed efficiency in Angus bulls, opening avenues for targeted interventions and advancements in livestock management practices.

Screening, Gene Cloning and Expression of Cellulase-Producing Strain Bacillus subtilis Xh-16.

Cellulase is a complex enzyme system composed of multiple hydrolytic enzymes. It can degrade cellulose into glucose and improve the utilization efficiency of cellulose resources. Cellulase produced by microorganisms is the main method used in industry, offering the advantages of convenience and being environmentally friendly. A strain Xh-16 with high cellulase production was screened from the rotten rice straw. It was identified as Bacillus subtilis by morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Strain Xh-16 was used to degrade rice straw. After a 48h cellulase treatment, the complete degradation and structural breakdown of the straw were observed. We cloned the endoglucanase Cel5L gene of Bacillus subtilis Xh-16 and induced expression of the cloned gene in Escherichia coli BL21 (DE3). The results showed that the coding length of Cel5L gene was 1500bp, and the molecular weight of the encoded protein was about 55kDa. The molecular formula is C2456H3811N671O761S10 and it has 7709 atoms and 499 amino acids. Cel5L differs significantly from some the glycoside hydrolase family 5 cellulases because it has only one carbohydrate binding module family 3 at the C-terminus of its catalytic domain. The cellulase gene Cel5L in Xh-16 can encode active cellulase and can be heterologously expressed in Escherichia coli, which makes Escherichia coli have cellulase function. This study serves as a foundation for further research on cellulase diversity in Bacillus subtilis and offers insights for enhancing cellulase production.

Genome-Wide Identification and Analysis of Gene Family of Carbohydrate-Binding Modules in Ustilago crameri.

Ustilago crameri is a pathogenic basidiomycete fungus that causes foxtail millet kernel smut (FMKS), a devastating grain disease in most foxtail millet growing regions of the world. Carbohydrate-Binding Modules (CBMs) are one of the important families of carbohydrate-active enzymes (CAZymes) in fungi and play a crucial role in fungal growth and development, as well as in pathogen infection. However, there is little information about the CBM family in U. crameri. Here, 11 CBM members were identified based on complete sequence analysis and functional annotation of the genome of U. crameri. According to phylogenetic analysis, they were divided into six groups. Gene structure and sequence composition analysis showed that these 11 UcCBM genes exhibit differences in gene structure and protein motifs. Furthermore, several cis-regulatory elements involved in plant hormones were detected in the promoter regions of these UcCBM genes. Gene ontology (GO) enrichment and protein-protein interaction (PPI) analysis showed that UcCBM proteins were involved in carbohydrate metabolism, and multiple partner protein interactions with UcCBM were also detected. The expression of UcCBM genes during U. crameri infection is further clarified, and the results indicate that several UcCBM genes were induced by U. crameri infection. These results provide valuable information for elucidating the features of U. crameri CBMs' family proteins and lay a crucial foundation for further research into their roles in interactions between U. crameri and foxtail millet.

Carbohydrate-binding module could integrate with ELISA and serve the simple and specific quantification of hyaluronic acid

Quantification is essential in the research and development of polysaccharides. However, achieving simplicity and specificity in polysaccharide quantification remains a challenging task. Enzyme-linked immunosorbent assay (ELISA) based on antibodies provides a straightforward and specific quantification strategy. Nevertheless, acquiring antibodies for polysaccharides is complicated. Carbohydrate-binding modules (CBMs), which can be efficiently obtained, exhibit the capability to specifically recognize and bind carbohydrates. In this study, we verified the feasibility of a CBM-based ELISA for the quantification of hyaluronic acid (HA) by replacing an antibody with a CBM. The CBM-based ELISA, which employed a HA-specific CBM, exhibited a linear detection range spanning from 10 to 100 μg/mL. Both intra-assay and inter-assay coefficients of variation remained below 15 % and recoveries ranged from 96.26 % to 98.22 %, indicating favorable precision and accuracy. The method exhibited specificity exclusively to HA, suggesting its reliance on CBM binding specificity. The effectiveness of the method in analyzing commercial products was confirmed. Additionally, a comparison with the carbazole assay revealed a highly significant correlation (r = 0.994). By integrating CBMs with ELISA, the study presented a novel and easily implementable solution for simple and specific quantification of HA.

Unique Fn3-like biosensor in σI/anti-σI factors for regulatory expression of major cellulosomal scaffoldins in Pseudobacteroides cellulosolvens.

Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti-σ (SigI/RsgI) factors to regulate cellulosomal components for substrate-specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI-RsgI regulons yet discovered. However, the function of many RsgI sensor domains and their relationship with the various enzyme types are not fully understood. Here, we report that RsgI4 from P. cellulosolvens employs a C-terminal module that bears distant similarity to the fibronectin type III (Fn3) domain and serves as the sensor domain. Substrate-binding analysis revealed that the Fn3-like domain of RsgI4 represents a novel carbohydrate-binding module (CBM) that binds to a wide range of polysaccharide types. Structure determination further revealed that the Fn3-like domain belongs to the type B group of CBMs with a predicted concave face for substrate binding. Promoter sequence analysis of cellulosomal genes revealed that SigI4 is responsible for cellulosomal regulation of major scaffoldins rather than enzymes, consistent with the broad substrate specificity of the RsgI4 sensor domain. Notably, scaffoldins are invariably required as cellulosome components regardless of the substrate type. These findings suggest that the intricate cellulosome system of P. cellulosolvens comprises a more elaborate regulation mechanism than other bacteria and thus expands the paradigm of cellulosome regulation.

Strong and ductile cellulose film improved by the in situ incorporation of a genetically engineered protein conjugated synthetic polymer during bacterial cellulose growth

Bacterial cellulose (BC) has been extensively applied to fabricate advanced biomaterials, although it remains challenging due to its poor toughness and water stability. Herein a genetically engineered protein-conjugated synthetic polymer is designed to improve BC film's strength and flexibility. Initially, the hybrid polymer is constructed by grafting Family 3 carbohydrate-binding modules (CBM3) to amphoteric polyacrylamide polymer (AmPAM), one of the paper industry's most widely used dry-strength agents. Then, the conjugated polymer is added to the culture medium of BC growth, enabling it to incorporate into the matrix of cellulose chains. The results show that the BC film modified by CBM3-AmPAM exhibits superior mechanical properties, registering 9.94 % in strain and 13.8 MJ/m3 in toughness, 12.1 and 8.0 folds over the sample with AmPAM addition only. Additionally, the BC film improved by CBM3-AmPAM has excellent gas resistance, thermal stability, and environmental endurance. The adsorption of CBM3-AmPAM on BC film revealed by quartz crystal microbalance with dissipation monitoring indicates that the adsorbed layer is thin and rigid, suggesting the strong interaction between the conjugated polymer and BC substrate. As a result of this reinforcing strategy, BC composites can be used in a wider range of applications.

Insight into lignin-carbohydrate ester change in pretreated corn bran and its enzymatic hydrolysis by three glucuronoyl esterases from Sordaria brevicollis

Lignin-carbohydrate esters (LC-esters) formed by glucuronoarabinoxylan and lignin are a key factor for the recalcitrance of corn bran, understanding LC-esters change during pretreatment and enzymatic hydrolysis by glucuronoyl esterases (GEs) is essential to the sustainable utilization of corn bran. Herein, hydrolysis performances of three GEs, SbGE15A, SbGE15B, and SbGE15C from Sordaria brevicollis with different subclades and modularity, and changes in enzyme-reachable LC-esters during different pretreatments of corn bran have been comprehensively compared. FB enzymes, SbGE15B and SbGE15C showed higher catalytic activity towards model and natural substrates than FA enzyme, SbGE15A. Particularly, SbGE15C harboring carbohydrate-binding module 1 (CBM1) exhibited much superior catalytic performance and synergistic effect with GH10 endo-xylanase EpXYN1 from Eupenicillium parvum on pretreated residues than SbGE15A and SbGE15B without CBM1. Autohydrolysis and DES (ChCl-LA) pretreatment could decrease the content of enzyme-reachable LC-esters and depolymerize its structure, transitioning from Lignin-(Me)GlcA-Xylan to Lignin-(Me)GlcA-XOS, and eventually to Lignin-(Me)GlcA with increasing pretreatment time. These changes consequently cause a decrease in synergy between SbGE15s and EpXYN1 or commercial enzyme cocktails on pretreated residues. The findings provide new insights into significant changes in enzyme-reachable LC-esters depending on the pretreatment method and intensity and the consequent influence of these changes on the catalytic action of GEs.

In silico sequential mutagenesis of the Carbohydrate Binding Module Family 32 (CBM32) enhances ligand binding affinity

Alginate lyase is a promising target for genetic modification for its degrading biofilm, contributing to bacterial proliferation and antimicrobial resistance. Apart from the main enzyme, the carbohydrate binding module (CBM) component can also be modified to enhance alginate lyase’s activity. This study aimed to perform sequential in silico mutagenesis, molecular docking of selected amino acid residues of Vibrio splendidus CBM32 and performed molecular dynamics (MD) simulations of the mutated structure to validate its ligand-binding efficacy. Seven residues were selected for mutagenesis based on the predicted bonds that formed between the CBM32 and the glucuronic acid ligand (LGU9). Four of seven sequential residue substitutions increased the ligand binding affinity cumulatively from -5.4 Kcal/mol to -6.9 Kcal/mol. The mutated CBM32 had similar MolProbity scores to the original V. splendidus CBM32 structure. From the post-MD simulation analysis, the mutated CBM32 had higher structural stability in a solvent system, a greater number of hydrogen bonds formed with ligand but a lower solvent-accessible surface area than the original structure. The sequential mutagenesis process significantly increased the ligand binding affinity of CBM32 while incurring a minimal change in the overall CBM32 structure. The information on these substituted residues would be beneficial for designing subsequent in vitro mutagenesis and enzymatic assays.

Enhancing PET degradation efficiency through fusing carbohydrate-binding modules in LCC-ICCG

Polyethylene terephthalate (PET) is a largely-produced polymer worldwide. However, its extensive waste generation and resistance to degradation pose significant environmental concerns. Consequently, there is considerable interest in researching enzymatic degradation of PET. Relevant studies have shown that the addition of a carbohydrate binding module (CBM) can increase the affinity between the enzyme and the substrate, enhancing the enzyme's degradation ability. In order to develop more efficient PET hydrolytic enzymes, this study introduced carbohydrate binding domains (CBMs) from different families with different substrate affinities into the PET-degrading enzyme LCC-ICCG. High crystallinity PET powder and amorphous PET film were used as substrates to characterize the degradation efficiency of the modified enzymes, aiming to explore the enzyme with the optimal degradation ability. The results showed that the fusion of type B CBM reduced the degradation rate and Tm value of the enzyme towards PET, while the introduction of type A and type C CBMs significantly improved the degradation rate of the enzyme towards the film-like substrate. The degradation rate and Tm value of PET were also enhanced, especially with the fusion enzyme LCC-ICCG-CBM9-2, which showed an over 10-fold increase in the degradation rate compared to the original enzyme LCC-ICCG. Therefore, this study demonstrates that by introducing type A and type C CBMs, the degradation rate and thermal stability of LCC-ICCG towards film-like PET can be improved, addressing the issue of its low activity and enabling more effective PET degradation. This research provides support for plastic degradation technology and contributes to environmental conservation efforts.

The key pathogenic factor GcSP2 of Geotrichum citri-aurantii plays an important role in disrupting citrus metabolism and immunity

The pathogen Geotrichum citri-aurantii (G. citri-aurantii) causes postharvest sour rot disease in citrus fruits worldwide, severely impacting citrus economic value. However, the pathogenic mechanisms of this fungus remain inadequately understood. Here, we identified 15 candidate effector proteins from G. citri-aurantii genome, of which five were highly expressed during infection and were capable of suppressing BAX-induced cell death, indicating their role in inhibiting plant immunity. Gene expression analysis showed that these five effector proteins primarily induced the upregulation of pattern-triggered immunity (PTI)-related gene expression. Diaminobenzidine (DAB) staining results indicated that only GcSP2 triggered reactive oxygen species (ROS) burst. Notably, GcSP2 contains a known carbohydrate-binding module 1 (CBM1) domain and exhibits low overall conservation. Gene knockout experiments revealed that the absence of GcSP2 delayed disease onset by 1–2 days and significantly reduced lesion size, establishing it as a key pathogenic factor. Assessments of total phenols, flavonoids, and pathogenesis-related protein expression indicated that GcSP2 significantly affects citrus metabolism at 72 hours post-infection.

More is not always better. Multiple carbohydrate-binding domains for efficient starch hydrolysis

Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules (CBM26). To investigate the impact of CBMs on catalytic activity, C-terminally truncated derivatives were constructed. The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate, highlighting the importance of CBMs in maintaining the enzyme's optimal conformation and dynamics for efficient catalysis. CBMs enhance enzyme performance, as indicated by improved catalytic efficiency (kcat/Km). Interestingly, the enzyme variant LaCD3CBM, with three CBMs, exhibits the best catalytic efficiency on soluble starch, outperforming the wild-type amylase, with five CBMs. In the case of insoluble starch, the catalytic domain alone could not hydrolyze it and even adding a CBM, the release of reducing sugars was very inefficient. However, this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase. CBMs also played a crucial role in protein thermostability, contributing to a higher melting temperature of the catalytic domain with just a single CBM. Notably, thermostability did not increase with the number of CBMs. In conclusion, spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates. These interactions optimize enzymatic activity and improve thermostability.

Acetylxylan esterase is the key to the host specialization of wood-decay fungi predicted by random forest machine-learning algorithm

Wood-decay fungi produce extracellular enzymes that metabolize wood components such as cellulose, hemicellulose and lignin. Each fungus has a preference of wood species as the host, but identification of these preferences requires a huge amount of cultivation data. Here, we developed a method of predicting the wood species preference, Angiosperm specialist or Gymnosperm specialist or generalist, of wood-decay fungi using the random forest machine-learning algorithm, trained on the numbers of families associated with host specialization in the Carbohydrate-Active enZymes database. The accuracy of the prediction was about 80%, which is lower than that of the classification of white- and brown-rot fungi (more than 98%) by the same method, but the reason for this may be the ambiguity of the definition of “preference” and “generalists”. Carbohydrate esterase (CE) family 1 acetylxylan esterase was the most significant contributor to the prediction of host specialization, followed by family 1 carbohydrate-binding module and CE family 15, mainly containing glucuronoyl esterases. These results suggest that the ability to degrade glucuronoacetylxylan, a major hemicellulose of Angiosperm, is the key factor determining the host specialization of wood-decay fungi.

Functional identification of carbohydrate-binding module 13 and its application to quantification of hemicellulose in gramineous plants

Gramineous biomass is a type of lignocellulose commonly used as a renewable energy source. Accurate quantification of hemicellulose within this biomass is crucial for its efficient conversion. Carbohydrate-binding modules (CBMs) help catalytic domains anchor substrates, representing the potential to be developed as fluorescence probes for hemicellulose content determination. In this study, we discovered a CBM named EmCBM13, derived from the bifunctional enzyme Xyn10A/Fae1A within the bacterial consortium EMSD5. This CBM displays a remarkable ability to bind soluble and insoluble hemicellulose components of gramineous plants. Through molecular docking and mutational analysis, we pinpointed two essential tyrosine residues mediating ligand interaction of EmCBM13. Leveraging the binding characteristics of EmCBM13, we created a fluorescence probe called EmCBM13-GFP by fusing a GFP with EmCBM13. We observed a positive correlation between the fluorescence contents of EmCBM13-GFP bound to hemicellulose and the hemicellulose contents in various gramineous biomasses. Utilizing this correlation, we rapidly determined the hemicellulose content in different gramineous plants with 90–110 % accuracy. This probe shows promise in quickly evaluating the characteristics of gramineous biomass feedstock for research and production purposes.

Cellulose binding and the timing of expression influence protein targeting to the double-layered cyst wall of Acanthamoeba.

The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and has ectocyst and endocyst layers connected by conical ostioles. Cyst walls contain families of lectins that localize to the ectocyst layer (Jonah) or the endocyst layer and ostioles (Luke and Leo). How lectins and an abundant laccase bind cellulose and why proteins go to locations in the wall are not known and are the focus of the studies here. Structural predictions identified β-jelly-roll folds (BJRFs) of Luke and sets of four disulfide knots (4DKs) of Leo, each of which contains linear arrays of aromatic amino acids, also present in carbohydrate-binding modules of bacterial and plant endocellulases. Ala mutations showed that these aromatics are necessary for cellulose binding and proper localization of Luke and Leo in the Acanthamoeba cyst wall. BJRFs of Luke, 4DKs of Leo, a single β-helical fold (BHF) of Jonah, and a copper oxidase domain of the laccase each bind to glycopolymers in both layers of deproteinated cyst walls. Promoter swaps showed that ectocyst localization does not just correlate with but is caused by early encystation-specific expression, while localization in the endocyst layer and ostioles is caused by later expression. Evolutionary studies showed distinct modes of assembly of duplicated domains in Luke, Leo, and Jonah lectins and suggested Jonah BHFs originated from bacteria, Luke BJRFs share common ancestry with slime molds, while 4DKs of Leo are unique to Acanthamoeba.IMPORTANCEAcanthamoebae is the only human parasite with cellulose in its cyst wall and conical ostioles that connect its inner and outer layers. Cyst walls are important virulence factors because they make Acanthamoebae resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles.

Bacterial Cellulose In Vitro Uptake by Macrophages, Epithelial Cells, and a Triculture Model of the Gastrointestinal Tract.

Bacterial cellulose (BC) has a long-standing human consumption history in different geographies without any report of adverse effects. Despite its unique textural and functional properties, the use of BC in food products in Europe is still restricted due to concerns over its nanosize. Here, we evaluated the potential uptake of celluloses (from plant and microbial sources, processed using different blenders) by macrophages (differentiated THP-1 cells) and human intestinal epithelial cells (Caco-2 and HT29-MTX cells) without (coculture) or with (triculture) Raji-B cells. A carbohydrate-binding module coupled to a green fluorescent protein was employed to observe cellulose in the cell cultures by confocal laser scanning microscopy and stimulated emission depletion microscopy. The methodology demonstrated excellent sensitivity, allowing detection of single nanocrystals within cells. All celluloses were taken up by the macrophages, without significantly compromising the cell's metabolic viability. The viability of the cocultures was also not affected. Furthermore, no internalization was observed in the triculture cell model that was exposed 24 h to BC and Avicel LM310. When (rarely) detected, cellulose particles were found on the apical side of the membrane. Overall, the obtained results suggest that BC should not be absorbed into the human gut.

187 Exploring deep shotgun sequencing to understand the rumen microbial function in Angus bulls with divergent RFI EPD

Abstract We investigated the rumen microbiome of Angus bulls selected for Residual Feed Intake-Expected Progeny Difference (RFI-EPD) utilizing deep shotgun metagenomic sequencing. Negative RFI-EPD bulls (NegRFI: n = 10; RFI-EPD = -0.3883 kg/d) and Positive RFI-EPD bulls (PosRFI: n = 10; RFI-EPD = 0.2935 kg/d) were selected from a group of 59 Angus bulls [average body weight (BW) = 427.81 ± 18.77 kg] fed a high-forage total mixed ration after a 59-d testing period. At the end of the 59-d period, rumen fluid samples were collected for bacterial DNA extraction and subsequent shotgun metagenomic sequencing. Results of the metagenome analysis revealed greater gene richness in NegRFI bulls compared with PosRFI. Analysis of similarity revealed a significant difference (P = 0.05) in the rumen microbial community between the NegRFI and PosRFI. Linear Discriminant Analysis effect size (Lefse) was used to identify the differentially abundant taxa. The Lefse results showed that class Fibrobacteria (LDA = 5.1) and genus Fibrobacter (LDA = 4.8) were the most differentially enriched markers in NegRFI bulls, compared with PosRFI bulls. Relative abundance of the carbohydrate-active enzymes was also compared using lefse analysis. The results showed greater relative abundance of glycoside hydrolases and carbohydrate-binding modules such as GH5, CBM86, CBM35, GH43, and CBM6 (LDA > 3.0) in NegRFI bulls whereas GH13 and GT2 were greater in PosRFI. The distinct metabolic and microbial profiles observed in NegRFI, compared with PosRFI bulls, characterized by greater gene richness and specific taxa such as Fibrobacter, and variations in carbohydrate-active enzymes, underscore the potential genetic and functional differences in their rumen microbiome. These findings contribute to a deeper understanding of the interplay between host genetics, microbiota, and feed efficiency in Angus bulls, opening avenues for targeted interventions and advancements in livestock management practices.

Deciphering domain structures of Aspergillus and Streptomyces GH3-β-Glucosidases: a screening system for enzyme engineering and biotechnological applications

The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application.

Macrogenome-based study on the mechanism of Bacillus velezensis SX13 in regulating rhizophere environment and cucumber growth under different cultivation environments

Microbial activities are the dynamic core of nutrient cycling in organic substrates, and the exploitation of plant growth-promoting rhizobacteria strains contributes to sustainable agricultural development. This study aimed to investigate the effect and mechanism of Bacillus velezensis SX13 in nutrient cycling and plant promotion under different substrate supply conditions. The effects of reduced substrate amount (sCK) and inoculation of SX13 strain under both substrate supply conditions (Bv and sBv) on rhizosphere microenvironment and plant growth were investigated using conventional substrate amount (CK) as a control. Results showed no significant difference in the α-diversity indexes (Chao1 and Shannon) of the rhizospheric microbial community among the four treatments. However, nonmetric multidimensional scaling analysis and principal coordinate analysis revealed that compared with CK treatment, the inoculation of SX13 strain and reduced substrate supply reshaped the β-diversity structure of microbial communities. Furthermore, inoculation with B. velezensis SX13 under both substrate supply conditions increased the abundance of Proteobacteria (1.64%–2.46 %), Acidobacteria (14.09%–43.07 %), and Firmicutes (179.29%–861.29 %). The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the metabolic pathway with the highest abundance of enriched genes was also the pathway with the most enriched differential genes caused by reducing substrate supply or inoculation of B. velezensis SX13. The rhizosphere inoculation of B. velezensis SX13 significantly up-regulated the top genes related to carbohydrate esterases, carbohydrate binding modules, glycoside hydrolases, glycoside transferases, and polysaccharide lyases. As a result, the activities of carbon and nitrogen cycle-related enzymes such as cellobiohydrolase, β-glucosidase, urease, l-leucine amino peptidase, and β-1 4-N-acetylglucosaminidase were increased, which in turn accelerated nutrient cycling. B. velezensis SX13 and its mediated improvement of the rhizospheric microenvironment resulted in the up-regulation of root CsNRT family genes (such as CsNRT1.1, CsNRT1.4a, CsNRT1.4b, CsNRT1.5a, CsNRT1.5b, CsNRT1.5c, and CsNRT1.8), which accelerated nitrogen uptake, accumulation, and utilization efficiency and ultimately improved the yield and quality of cucumber. The effect of SX13 strain was more stable and efficient under conventional substrate supply conditions than under reduced substrate supply conditions.

Profiling the dynamic adaptations of CAZyme-Producing microorganisms in the gastrointestinal tract of South African goats

The gastrointestinal tract of goats serves as a habitat for anaerobic microbial populations that work together to break down complex plant material, including lignocellulose. This study explored the microbial diversity and metabolic profiles across different gastrointestinal tract compartments. Significant diversity differences among the compartments were observed (ANOSIM p < 0.006), with the abomasum showing a distinct species composition and a decreased alpha diversity (Mann-Whitney/Kruskal-Wallis test p = 0.00096), possibly due to its acidic environment. Dominant microbial phyla included Proteobacteria, Bacteroidetes, and Firmicutes, with Proteobacteria being the most prevalent in the abomasum (50.06 %). Genera like Proteus and Bacteroides were particularly prominent in the rumen and reticulum, highlighting their significant role in feed degradation and fermentation processes. Over 65 % of genes at Kyoto Encyclopedia of Genes and Genomes level 1 were involved in metabolism with significant xenobiotic biodegradation in the abomasum. The dbCAN2 search identified Glycoside Hydrolases as the most prevalent CAZyme class (79 %), followed by Glycosyltransferases, Polysaccharide Lyases, and Carbohydrate Esterases, with Carbohydrate-Binding Modules and Auxiliary Activities accounting for 1 % of the hits. Higher CAZyme abundance was observed in the reticulum and omasum compartments, possibly due to MAGs diversity. In conclusion, the gastrointestinal tract of South African goats harbors diverse CAZyme classes, with Glycoside Hydrolases predominating. Interestingly, higher CAZyme abundance in specific compartments suggested compartmentalized microbial activity, reflecting adaptation to dietary substrates.