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This data report presents prokaryotic metagenome-assembled genomes (MAGs) from a hot spring stream with temperatures between 64 and 100°C. The stream water was filtered and the extracted total DNA was sequenced using the Illumina HiSeq 2500 platform. Approximately 80 Gb of raw data were generated, which were subsequently assembled using MEGAHIT v1.2.9. The MAGs were generated using MetaWRAP with binning approaches of MetaBAT2, CONCOCT and MaxBin2. We constructed 25 medium-quality and 24 high-quality archaeal MAGs, and 152 medium-quality and 112 high-quality bacterial MAGs. The fasta files of these MAGs are available in the NCBI database as well as Mendeley Data. Major phyla identified include Bacteroidota, Chloroflexota, Desulfobacterota, Firmicutes, Patescibacteria, Proteobacteria, Spirochaetota, Verrucomicrobiota, Armatimonadota, Nitrospirota, Acidobacteriota, Elusimicrobiota, Planctomycetota, Candidate division WOR-3, Aquificota, Thermoproteota, and Micrarchaeota. This dataset is valuable for studies on thermophilic genomes, reconstruction of biochemical pathways and gene discovery.

Abstract Background Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58–74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism. Results Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures. Conclusions This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.

The production of second-generation biofuel requires a huge amount of freshwater. It is estimated that at least three gal of freshwater is used to produce one gal of biofuel. The replacement of freshwater with seawater serves as a potential alternative in biofuel generation. Therefore, salt-tolerant enzymes play an important role in saccharification and fermentation process. Halophilic β-glucosidase is one of the key enzymes for the process. In this study, the β-glucosidase of halophile Meridianimaribacter sp. CL38 isolated from mangrove soil was characterized. Strain CL38 achieved maximum production of β-glucosidase at 12th hour of growth. The β-glucosidase showed highest activity at 2% (w/v) NaCl while highly stable at salt concentration ranging from 1-2% (w/v) (more than 96% of relative activity). Its β-glucosidase activity remained active in the presence of 5mM Mn2+, Mg2+, Ca2+ ions, and 1% (v/v) Tween-20 and Tween-80. The draft genome sequence of strain CL38 was retrieved from GenBank database and submitted to dbCAN meta server for CAZymes annotation. Strain CL38 harbors 44 GHs and GH3 are annotated as β-glucosidases. The β-glucosidases of Meridianimaribacter flavus (99.61%) and Mesoflavibacter sabulilitoris (97.44%) showed the closest identity with Bgl3a and Bgl3b protein sequences from strain CL38, respectively. Glycoside hydrolase family 3 domain was identified in both enzymes via InterPro scan server. The presence of signal peptides indicated that both enzymes were secreted extracellularly. Five motifs were identified in Bgl3a and Bgl3b, with the active site (nucleophile) found at Asp296 and Asp297, respectively. Collectively, these β-glucosidases could be potentially used in the biofuel production, in particular the lignocellulosic biomass pretreatment process. This is the first attempt to characterize the β-glucosidase in genus Meridianimaribacter as so far none of the lignocellulolytic enzymes from this genus were characterized.

Abstract The SKY hot spring is a unique site filled with a thick layer of plant litter. With the advancement of next-generation sequencing, it is now possible to mine many new biocatalyst sequences. In this study, we aimed to (i) identify the metataxonomic of prokaryotes and eukaryotes in microbial mats using 16S and 18S rRNA markers, (ii) and explore carbohydrate degrading enzymes (CAZymes) that have a high potential for future applications. Green microbial mat, predominantly photosynthetic bacteria, was attached to submerged or floating leaves litter. At the spring head, the sediment mixture consisted of plant debris, predominantly brownish-reddish gelatinous microbial mat, pale tan biofilm, and grey-white filament biofilm. The population in the spring head had a higher percentage of archaea and hyperthermophiles than the green mat. Concurrently, we cataloged nearly 10,000 sequences of CAZymes in both green and brown biofilms using the shotgun metagenomic sequencing approach. These sequences include β-glucosidase, cellulase, xylanase, α-N-arabinofuranosidase, α-l-arabinofuranosidase, and other CAZymes. In conclusion, this work elucidated that SKY is a unique hot spring due to its rich lignocellulosic material, often absent in other hot springs. The data collected from this study serves as a repository of new thermostable macromolecules, in particular families of glycoside hydrolases.

Thermovorax subterraneus 70BT is a thermophile found in a geothermically active underground mine. The strain 70BT belongs to the class of Clostridia, order of Thermosediminibacterales, and family of Thermosediminibacteraceae. Strain 70BT was the only type strain since the genus was discovered >10 years ago. Strain 70BT was compared to strains from other genera in terms of its phenotypics, chemotaxonomics, and phylogenetics (16S rRNA gene) in previous studies. However, the genome sequence of this strain has not been described. We herein described the genome sequence of strain 70BT. In total, the assembled genome of strain 70BT has a size of 2,451,552 bp, contributed by 44 contigs, with a coverage of 445X, a N50 of 86,294 bp, and a GC% of 43.8. A total of 2,540 genes were encoded in the genome, including 2,431 protein-coding sequences, 46 pseudogenes, and 63 RNA genes. Through the Cluster of Orthologous Groups (COGs) analysis, a total of 2,404 protein-coding genes were functionally assigned to COGs in the genome of strain 70BT. Among the members of Thermosediminibacteraceae family, strain 70BT has the closest relationship to Caldanaerovirga acetigignens JW/SA-NV4T based on the genome-to-genome comparison indexes (i.e., ANI, dDDH, AAI, and POCP). An earlier study reported that strain 70BT could produce hydrogen. We discovered genes encoding [FeFe] hydrogenase through gene mining analysis. For future research, this genome data will be used as a reference for all matters pertaining to the genus Thermovorax and family Thermosediminibacteraceae.

A halophilic marine bacterial strain, PS-C1, was isolated from Sekinchan beach in Selangor, Malaysia. The 16S rRNA gene sequence analysis indicated that strain PS-C1 was associated with the genus Celeribacter. To date, there have been no reports on enzymes from the genus Celeribacter. The present study reports on the cellular features of Celeribacter sp. PS-C1, its annotated genome sequence, and comparative genome analyses of Celeribacter glycoside hydrolase (GH) enzymes. The genome of strain PS-C1 has a size of 3.87 Mbp and a G+C content of 59.10%, and contains 3739 protein-coding genes. Detailed analysis using the Carbohydrate-Active enZYmes (CAZy) database revealed that Celeribacter genomes harboured at least 12 putative genes encoding industrially important GHs that are grouped as cellulases, β-glucanases, hemicellulases, and starch-degrading enzymes. Herein, the potential applications of these enzymes are discussed. Furthermore, the activities of two types of GHs (β-glucosidase and licheninase) in strain PS-C1 were demonstrated. These findings suggest that strain PS-C1 could be a reservoir of novel GH enzymes for lignocellulosic biomass degradation.

Starch and pullulan degrading enzymes are essential industrial biocatalysts. Pullulan-degrading enzymes are grouped into pullulanases (types I and type II) and pullulan hydrolase (types I, II and III). Generally, these enzymes hydrolyse the α-1,6 glucosidic bonds (and α-1,4 for certain enzyme groups) of substrates and form reducing sugars such as glucose, maltose, maltotriose, panose or isopanose. This review covers two main aspects: (i) bibliometric analysis of publications and patents related to pullulan-degrading enzymes and (ii) biological aspects of free and immobilised pullulan-degrading enzymes and protein engineering. The collective data suggest that most publications involved researchers within the same institution or country in the past and current practice. Multi-national interaction shall be improved, especially in tapping the enzymes from unculturable prokaryotes. While the understanding of pullulanases may reach a certain extend of saturation, the discovery of pullulan hydrolases is still limited. In this report, we suggest readers consider using the next-generation sequencing technique to fill the gaps of finding more new sequences encoding pullulan-degrading enzymes to expand the knowledge body of this topic.

The production of second-generation biofuel requires a huge amount of freshwater. It is estimated that at least three gal of freshwater is used to produce one gal of biofuel. The replacement of freshwater with seawater serves as a potential alternative in biofuel generation. Therefore, salt-tolerant enzymes play an important role in saccharification and fermentation process. Halophilic β-glucosidase is one of the key enzymes for the process. In this study, the β-glucosidase of halophile Meridianimaribacter sp. CL38 isolated from mangrove soil was characterized. Strain CL38 achieved maximum production of β-glucosidase at 12th hour of growth. The β-glucosidase showed highest activity at 2% (w/v) NaCl while highly stable at salt concentration ranging from 1-2% (w/v) (more than 96% of relative activity). Its β-glucosidase activity remained active in the presence of 5mM Mn2+, Mg2+, Ca2+ ions, and 1% (v/v) Tween-20 and Tween-80. The draft genome sequence of strain CL38 was retrieved from GenBank database and submitted to dbCAN meta server for CA-Zymes annotation. Strain CL38 harbors 44 GHs and GH3 are annotated as β-glucosidases. The β-glucosidases of Meridianimaribacter flavus (99.61%) and Mesoflavibacter sabulilitoris (97.44%) showed the closest identity with Bgl3a and Bgl3b protein sequences from strain CL38, respectively. Glycoside hydrolase family 3 domain was identified in both enzymes via InterPro scan server. The presence of signal peptides indicated that both enzymes were secreted extracellularly. Five motifs were identified in Bgl3a and Bgl3b, with the active site (nucleophile) found at Asp296 and Asp297, respectively. Collectively, these β-glucosidases could be potentially used in the biofuel production, in particular the lignocellulosic biomass pretreatment process. This is the first attempt to characterize the β-glucosidase in genus Meridianimaribacter as so far none of the lignocellulolytic enzymes from this genus were characterized. [ABSTRACT FROM AUTHOR]

The majority of the members in order Rhodothermales are underexplored prokaryotic extremophiles. Roseithermus, a new genus within Rhodothermales, was first described in 2019. Roseithermus sacchariphilus is the only species in this genus. The current report aims to evaluate the transcriptomic responses of R. sacchariphilus strain RA when cultivated on beechwood xylan. Strain RA doubled its growth in Marine Broth (MB) containing xylan compared to Marine Broth (MB) alone. Strain RA harbors 54 potential glycosyl hydrolases (GHs) that are affiliated with 30 families, including cellulases (families GH 3, 5, 9, and 44) and hemicellulases (GH 2, 10, 16, 29, 31,43, 51, 53, 67, 78, 92, 106, 113, 130, and 154). The majority of these GHs were upregulated when the cells were grown in MB containing xylan medium and enzymatic activities for xylanase, endoglucanase, β-xylosidase, and β-glucosidase were elevated. Interestingly, with the introduction of xylan, five out of six cellulolytic genes were upregulated. Furthermore, approximately 1122 genes equivalent to one-third of the total genes for strain RA were upregulated. These upregulated genes were mostly involved in transportation, chemotaxis, and membrane components synthesis.

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s−1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50–60°C and is capable of growing in marine broth containing 1–10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (< 90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3,630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na+/H+ antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K+ channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium.