Metagenomics Study To Compare The Taxonomy And Metabolism of a Lignocellulolytic Microbial Consortium Cultured in Different Carbon Conditions

A lignocellulolytic microbial consortium holds promise for the in situ biodegradation of crop straw and the comprehensive and effective utilization of agricultural waste. In this study, we applied metagenomics technology to comprehensively explore the metabolic functional potential and taxonomic diversity of the microbial consortia CS (cultured on corn stover) and FP (cultured on filter paper).Analyses of the metagenomics taxonomic affiliation data showed considerable differences in the taxonomic composition and functional profile of the microbial consortia CS and FP. The microbial consortia CS primarily contained members from the genera Pseudomonas, Stenotrophomonas, Achromobacter, Dysgonomonas, Flavobacterium and Sphingobacterium, as well as Cellvibrio, Azospirillum, Pseudomonas, Dysgonomonas and Cellulomonas in FP. The COG and KEGG annotation analyses revealed considerable levels of diversity. Further analysis determined that the CS consortium had an increase in the acid and ester metabolism pathways, while carbohydrate metabolism was enriched in the FP consortium. Furthermore, a comparison against the CAZy database showed that the microbial consortia CS and FP contain a rich diversity of lignocellulose degrading families, in which GH5, GH6, GH9, GH10, GH11, GH26, GH42, and GH43 were enriched in the FP consortium, and GH44, GH28, GH2, and GH29 increased in the CS consortium. The degradative mechanism of lignocellulose metabolism by the two microbial consortia is similar, but the annotation of quantity of genes indicated that they are diverse and vary greatly. The lignocellulolytic microbial consortia cultured under different carbon conditions (CS and FP) differed substantially in their composition of the microbial community at the genus level. The changes in functional diversity were accompanied with variation in the composition of microorganisms, many of which are related to the degradation of lignocellulolytic materials. The genera Pseudomonas, Dysgonomonas and Sphingobacterium in CS and the genera Cellvibrio and Pseudomonas in FP exhibited a much wider distribution of lignocellulose degradative ability.

Probe-mining of endo-1,4-beta-xylanase from goats-rumen bacterial metagenomic DNA data

Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.

Metagenomic analysis reveals rumen microbiota alteration of the yak at different stages of growth

The Yak (Bos grunniens) is a unique ruminant species that is crucially important to agriculture in the Tibetan plateau. Variation of microorganism communities in the yak rumen is of great interest because of possible links to environmentally and economically important traits. In this study, we performed histological and microbial analyses of the yak rumen at 5 stages of growth: 1 day, 20 days, 60 days, 15 months, and 5 years of age. Tissue slices and metagenomics sequencing were used. The rumen index increased gradually from 1 day to 5 years of age. These were significant differences in rumen index between the 60d, 15m, and 5y group (p < 0.05). Compared with other time points, the thickness of muscularis along with length and width of rumen papillae at 60 d,15 m, and 5 years of age increased and differed (p < 0.05), respectively. At the phylum level, Bacteroidetes and Firmicutes were the phyla with the highest abundance in all the age groups. A total of 115,401 genes were annotated on the CAZy database. Glycoside Hydrolase (GH) had the highest relative abundance, followed by Glycosyl Transferase (GT), and Carbohydrate-binding Modules (CBM). There were significant variations for the microbial species and CAZys within the five groups. Taken together, the morphology and microbiota in the yak rumen changed at various stages of growth and likely played a significant role in the absorption of nutrients. This study provides new insights into the function of yak rumen microbiota and physiologic adaptations in plateau animals.

Lignocellulolytic Potential of the Recently Described Species Aspergillus olivimuriae on Different Solid Wastes

The genus Aspergillus encompasses several species with relevant lignocellulose-degrading capacity, and a novel species, denominated A. olivimuriae, was recently discovered after its isolation from table olive brine. The acquisition of insight into this species and the assessment of its potential relied on a bioinformatics approach, based on the CAZy database, associated with enzymatic activity profiles in solid-state cultures on four different types of waste, including residual thistle biomass (RTB), spent coffee grounds (SCG), digestate solid fraction and barley straw. The CAZy analysis of A. olivimuriae genome showed that the number of predicted genes for each family was close to that of other Aspergillus species, except for cellobiose dehydrogenase, acetyl xylan esterase and polygalacturonases. In A. olivimuriae solid-state cultures, hemicellulose degradation outperformed that of cellulose, and lignin removal did not occur, regardless of the growth substrate. This is in line with its CAZy content and the extent of hemicellulolytic, and ligninolytic activities detected in its solid-state cultures. RTB and barley straw were the substrates enabling the best glycosyl hydrolase production levels. The exception was SCG, the hemicellulose composition of which, mainly made of glucomannans and galactomanans, led to the highest β-mannanase and β-mannosidase production levels (3.72 ± 0.20 and 0.90 ± 0.04 IU g−1 substrate, respectively).

Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacterium Catenovulum sp. CCB-QB4

Background Oligosaccharides from polysaccharides containing uronic acids are known to have many useful bioactivities. Thus, polysaccharide lyases (PLs) and glycoside hydrolases (GHs) involved in producing the oligosaccharides have attracted interest in both medical and industrial settings. The numerous polysaccharide lyases and glycoside hydrolases involved in producing the oligosaccharides were isolated from soil and marine microorganisms. Our previous report demonstrated that an agar-degrading bacterium, Catenovulum sp. CCB-QB4, isolated from a coastal area of Penang, Malaysia, possessed 183 glycoside hydrolases and 43 polysaccharide lyases in the genome. We expected that the strain might degrade and use uronic acid-containing polysaccharides as a carbon source, indicating that the strain has a potential for a source of novel genes for degrading the polysaccharides. Methods To confirm the expectation, the QB4 cells were cultured in artificial seawater media with uronic acid-containing polysaccharides, namely alginate, pectin (and saturated galacturonate), ulvan, and gellan gum, and the growth was observed. The genes involved in degradation and utilization of uronic acid-containing polysaccharides were explored in the QB4 genome using CAZy analysis and BlastP analysis. Results The QB4 cells were capable of using these polysaccharides as a carbon source, and especially, the cells exhibited a robust growth in the presence of alginate. 28 PLs and 22 GHs related to the degradation of these polysaccharides were found in the QB4 genome based on the CAZy database. Eleven polysaccharide lyases and 16 glycoside hydrolases contained lipobox motif, indicating that these enzymes play an important role in degrading the polysaccharides. Fourteen of 28 polysaccharide lyases were classified into ulvan lyase, and the QB4 genome possessed the most abundant ulvan lyase genes in the CAZy database. Besides, genes involved in uronic acid metabolisms were also present in the genome. These results were consistent with the cell growth. In the pectin metabolic pathway, the strain had genes for three different pathways. However, the growth experiment using saturated galacturonate exhibited that the strain can only use the pathway related to unsaturated galacturonate.

New Insights into the Co-Occurrences of Glycoside Hydrolase Genes among Prokaryotic Genomes through Network Analysis

Glycoside hydrolase (GH) represents a crucial category of enzymes for carbohydrate utilization in most organisms. A series of glycoside hydrolase families (GHFs) have been classified, with relevant information deposited in the CAZy database. Statistical analysis indicated that most GHFs (134 out of 154) were prone to exist in bacteria rather than archaea, in terms of both occurrence frequencies and average gene numbers. Co-occurrence analysis suggested the existence of strong or moderate-strong correlations among 63 GHFs. A combination of network analysis by Gephi and functional classification among these GHFs demonstrated the presence of 12 functional categories (from group A to L), with which the corresponding microbial collections were subsequently labeled, respectively. Interestingly, a progressive enrichment of particular GHFs was found among several types of microbes, and type-L as well as type-E microbes were deemed as functional intensified species which formed during the microbial evolution process toward efficient decomposition of lignocellulose as well as pectin, respectively. Overall, integrating network analysis and enzymatic functional classification, we were able to provide a new angle of view for GHs from known prokaryotic genomes, and thus this study is likely to guide the selection of GHs and microbes for efficient biomass utilization.

Analysis of The Genome Sequence of Phomopsis Vexans: A Fungal Pathogen Causing Phomopsis Blight of Eggplant

Background: Phomopsis vexans is a phytopathogenic fungus causing Phomopsis blight of eggplant. This disease is one of the major issues reducing eggplants production. To lay a solid foundation for the research of pathogenicity and to understand the mechanism of the disease development, the genome of an isolate PV4 was sequenced, assembled and analyzed from Guangdong, China. Results: The assembled complete genome size of Phomopsis vexans was about 59.78 Mb with 51.24% G+C content and 4.93Mb contig N50. In the genome, 3,552 annotated repetitive elements were identified. A total of 15,034 genes with 1,790bp average length were predicted, of which 14,116 genes were annotated by NCBI nr database. Moreover, 1,206 genes were annotated from Carbohydrate-Active enzymes (CAZy) Database which may play a role in degrading plant cell walls. At last, 134 effector proteins were predicted. Conclusions: The present genome is the first genome of Phomopsis vexans. The information obtained from this study support important resource for research on the pathogen and for sheding light on its pathogenicity mechanism.

Cyclodextrin-preferring glycoside hydrolases: properties and applications

Cyclodextrin-hydrolyzing enzymes are widespread in bacteria and archaea where they play their roles in carbohydrates metabolism. They were previously characterized as cyclodextrinases, neopullulanases and maltogenic amylases. In the Carbohydrate-Active enZyme (CAZy) database, most of these enzymes are grouped into the GH13_20 subfamily of the α-amylase family GH13. Here, we have summarized the information available on the substrate specificity, structural features, physiological roles and applications of cyclodextrin-preferring glycoside hydrolases. These enzymes form a distinct group in the α-amylase family. Members of this distinct group possess an extra extension at the N-terminus, which causes a modification of the active site geometry thus making these enzymes more specific for smaller molecules like cyclodextrins than for macromolecules such as starches or pullulan. Multi-substrate specificity, hydrolytic as well as transglycosylation activities make these enzymes attractive for applications in the food and pharmaceutical industries. We have tried here to collect information available on their biochemical properties, three-dimensional structures, physiological roles and potential applications.

β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. β-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding β-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with β-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-β-galactoside and lactose. The activity levels of one of these novel β-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic β-galactosidases from diary stabilization ponds’ metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries’ by-products.

In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature

Background Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. Results A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values > 8.0 and temperature > 80 °C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90 °C and pH > 9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. Conclusions This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH > 8.0 and temperature > 80 °C.