Sequence and structure analyses of lytic polysaccharide monooxygenases mined from metagenomic DNA of humus samples around white-rot fungi in Cuc Phuong tropical forest, Vietnam.

Background: White-rot fungi and bacteria communities are unique ecosystems with different types of symbiotic interactions occurring during wood decomposition, such as cooperation, mutualism, nutritional competition, and antagonism. The role of chitin-active lytic polysaccharide monooxygenases (LPMOs) in these symbiotic interactions is the subject of this study. Method: In this study, bioinformatics tools were used to analyze the sequence and structure of putative LPMOs mined by hidden Markov model (HMM) profiles from the bacterial metagenomic DNA database of collected humus samples around white-rot fungi in Cuc Phuong primary forest, Vietnam. Two genes encoding putative LPMOs were expressed in E. coli and purified for enzyme activity assay. Result: Thirty-one full-length proteins annotated as putative LPMOs according to HMM profiles were confirmed by amino acid sequence comparison. The comparison results showed that although the amino acid sequences of the proteins were very different, they shared nine conserved amino acids, including two histidine and one phenylalanine that characterize the H1-Hx-Yz motif of the active site of bacterial LPMOs. Structural analysis of these proteins revealed that they are multidomain proteins with different functions. Prediction of the catalytic domain 3-D structure of these putative LPMOs using Alphafold2 showed that their spatial structures were very similar in shape, although their protein sequences were very different. The results of testing the activity of proteins GL0247266 and GL0183513 show that they are chitin-active LPMOs. Prediction of the 3-D structures of these two LPMOs using Alphafold2 showed that GL0247266 had five functional domains, while GL0183513 had four functional domains, two of which that were similar to the GbpA_2 and GbpA_3 domains of protein GbpA of Vibrio cholerae bacteria. The GbpA_2 - GbpA_3 complex was also detected in 11 other proteins. Based on the structural characteristics of functional domains, it is possible to hypothesize the role of chitin-active GbpA-like LPMOs in the relationship between fungal and bacterial communities coexisting on decomposing trees in primary forests.

Understanding the Role of Free-Living Bacteria in the Gut of the Lower Termite Coptotermes gestroi Based on Metagenomic DNA Analysis.

Termites' digestive systems, particularly in lower termites with the presence of protozoa, are unique ecological niches that shelter a diverse microbiota with a variety of functions for the host and the environment. In 2012, the metagenomic DNA (5.4 Gb) of the prokaryotes that freely live in the gut of the lower termite Coptotermes gestroi were sequenced. A total of 125,431 genes were predicted and analyzed in order to mine lignocellulolytic genes. however, the overall picture of the structure, diversity, and function of the prokaryotic gut microbiota was not investigated. In the present study, these 125,431 genes were taxonomically classified by MEGAN and functionally annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and by the Carbohydrate-Active enZYmes (CAZy) and HMMER databases. As a result, 95,751 bacterial genes were classified into 35 phyla. The structure of the bacteria, typified by a high ratio of Firmicutes to Bacterioidetes, was distinct from the structure of the entirety of the bacteria in the lower or higher termites' guts. The archaea (533 genes) were distributed into 4 phyla, 10 classes, 15 orders, 21 families, 47 genera, and 61 species. Although freely living in the guts, the prokaryotic community was formed, developed, and adapted to exhibit unique interactions in order to perform mutual roles of benefit to their hosts. Methanobacteriales, accounting for 61% of the archaea symbionts, seem to play an important role in methanogenesis. Concomitantly, bacterial methanotrophs in the gut utilize methane and combine with other bacterial groups, including potential lignocellulolytic degraders, acetogens, sulfur bacteria, and nitrogen-recycling bacteria, to efficiently convert wood with little nitrogen into acetates via certain pathway modules specified by prokaryotes that freely live in the gut. This forms an important energy source for the termites. Furthermore, bacteria carry 2223 genes involved in the biosynthesis of 17 antibiotic groups. The gut bacteria also possess genes for the degradation of 18 toxic aromatic compounds, of which four are commercial pesticides against termites commonly used for the preservation of wooden constructions. Eight of the eighteen pathways were the first to be reported from the termite gut. Overall, this study sheds light on the roles of the freely living bacteria and archaea in the C. gestroi gut, providing evidence that the gut microbiome acts as the second host genome, contributing both nutrients and immunity to support the host's existence, growth, and development.

Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats' Rumen by Metagenomic Deep Sequencing.

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats' rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5-6.5, temperature 40-50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

Some characters of bacterial cellulases in goats' rumen elucidated by metagenomic DNA analysis and the role of fibronectin 3 module for endoglucanase function.

OBJECTIVE: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. METHODS: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. RESULTS: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. CONCLUSION: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.

Metagenomic insights into lignocellulose-degrading genes through Illumina-based de novo sequencing of the microbiome in Vietnamese native goats' rumen.

The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats' rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes' modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter.

Metagenomic analysis of bacterial community structure and diversity of lignocellulolytic bacteria in Vietnamese native goat rumen.

OBJECTIVE: In a previous study, analysis of Illumina sequenced metagenomic DNA data of bacteria in Vietnamese goats' rumen showed a high diversity of putative lignocellulolytic genes. In this study, taxonomy speculation of microbial community and lignocellulolytic bacteria population in the rumen was conducted to elucidate a role of bacterial structure for effective degradation of plant materials. METHODS: The metagenomic data had been subjected into Basic Local Alignment Search Tool (BLASTX) algorithm and the National Center for Biotechnology Information non-redundant sequence database. Here the BLASTX hits were further processed by the Metagenome Analyzer program to statistically analyze the abundance of taxa. RESULTS: Microbial community in the rumen is defined by dominance of Bacteroidetes compared to Firmicutes. The ratio of Firmicutes versus Bacteroidetes was 0.36:1. An abundance of Synergistetes was uniquely identified in the goat microbiome may be formed by host genotype. With regard to bacterial lignocellulose degraders, the ratio of lignocellulolytic genes affiliated with Firmicutes compared to the genes linked to Bacteroidetes was 0.11:1, in which the genes encoding putative hemicellulases, carbohydrate esterases, polysaccharide lyases originated from Bacteroidetes were 14 to 20 times higher than from Firmicutes. Firmicutes seem to possess more cellulose hydrolysis capacity showing a Firmicutes/Bacteroidetes ratio of 0.35:1. Analysis of lignocellulolytic potential degraders shows that four species belonged to Bacteroidetes phylum, while two species belonged to Firmicutes phylum harbouring at least 12 different catalytic domains for all lignocellulose pretreatment, cellulose, as well as hemicellulose saccharification. CONCLUSION: Based on these findings, we speculate that increasing the members of Bacteroidetes to keep a low ratio of Firmicutes versus Bacteroidetes in goat rumen has resulted most likely in an increased lignocellulose digestion.