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Novel archaeal thermostable cellulases from an oil reservoir metagenome.

ABSTRACT: Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.

SUBMITTER: Lewin A

PROVIDER: S-EPMC5622026 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Novel archaeal thermostable cellulases from an oil reservoir metagenome.

Lewin Anna A Zhou Jinglie J Pham Vu Thuy Trang VTT Haugen Tone T Zeiny Mohamed El ME Aarstad Olav O Liebl Wolfgang W Wentzel Alexander A Liles Mark R MR

AMB Express 20170929 1

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and ...[more]

PMID: 28963711

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Project description:BackgroundEnergy shortage has become a global problem. Production of biofuels from renewable biomass resources is an inevitable trend of sustainable development. Cellulose is the most abundant and renewable resource in nature. Lack of new cellulases with unique properties has become the bottleneck of the efficient utilization of cellulose. Environmental metagenomes are regarded as huge reservoirs for a variety of cellulases. However, new cellulases cannot be obtained easily by functional screening of metagenomic libraries.ResultsIn this work, a metagenomics-guided strategy for obtaining new cellulases from the metagenome was proposed. Metagenomic sequences of DNA extracted from the anaerobic beer lees converting consortium enriched at thermophilic conditions were assembled, and 23 glycoside hydrolase (GH) sequences affiliated with the GH family 5 were identified. Among the 23 GH sequences, three target sequences (designated as cel7482, cel3623 and cel36) showing low identity with those known GHs were chosen as the putative cellulase genes to be functionally expressed in Escherichia coli after PCR cloning. The three cellulases were classified into endo-β-1,4-glucanases by product pattern analysis. The recombinant cellulases were more active at pH 5.5 and within a temperature range of 60-70 °C. Computer-assisted 3D structure modeling indicated that the active residues in the active site of the recombinant cellulases were more similar to each other compared with non-active site residues. The recombinant cel7482 was extremely tolerant to 2 M NaCl, suggesting that cel7482 may be a halotolerant cellulase. Moreover, the recombinant cel7482 was shown to have an ability to resist three ionic liquids (ILs), which are widely used for cellulose pretreatment. Furthermore, active cel7482 was secreted by the twin-arginine translocation (Tat) pathway of Bacillus subtilis 168 into the culture medium, which facilitates the subsequent purification and reduces the formation of inclusion body in the context of overexpression.ConclusionsThis study demonstrated a simple and efficient method for direct cloning of new cellulase genes from environmental metagenomes. In the future, the metagenomics-guided strategy may be applied to the high-throughput screening of new cellulases from environmental metagenomes.

Project description:The microbial communities in alkali-surfactant-polyacrylamide-flooded (ASP-flooded) oil reservoirs have rarely been investigated compared to those in water-flooded oil reservoirs. Here, the bacterial and archaeal communities in an ASP-flooded reservoir and the adjacent water-flooded block, and responses of the microbial communities in microcosms to nutrients were investigated by 16S rRNA gene sequencing and cultivation. Compared with the water-flooded block, both the bacterial and archaeal communities inhabiting the ASP-flooded block had lower Sobs indices (91:232 and 34:55, respectively), lower Shannon indices (1.296:2.256 and 0.845:1.627, respectively) and higher Simpson indices (0.391:0.248 and 0.678:0.315, respectively). Halomonas (58.4-82.1%) and Anoxynatronum (14.5-18.2%) predominated in the ASP-flooded production wells, and were less than 0.05% in the bacterial communities of the adjacent water-flooded production wells, which were dominated by Pseudomonas and Thauera. Methanobacterium accounted for 65.0-94.5% of the archaeal communities inhabiting the ASP-flooded production wells, and Methanosaeta (36.7-94.5%) dominated the adjacent water-flooded production wells. After nutrients stimulation, the quantity of cultivable microorganisms increased from 103/mL to 107/mL. Community analysis indicated that the relative abundances of some species that belonged to Halomonas and Pseudomonas obviously increased, yet there were no oil emulsification or dispersion and changes of surface tension of the water-oil mixture. In addition, 6 alkali-tolerating strains showing 98% similarity of 16S rRNA genes with those of Halomonas alkalicola and Halomonas desiderata and 2 strains with 99% similarity with Pseudomonas stutzeri gene were isolated from the nutrients stimulated brines. In summary, this study indicated that Halomonas, Anoxynatronum, and Methanobacterium were dominant populations in the ASP-flooded reservoir, the extreme environment decreased microbial diversity, and restricted microbial growth and metabolisms.

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Novel archaeal thermostable cellulases from an oil reservoir metagenome.

Publications

Novel archaeal thermostable cellulases from an oil reservoir metagenome.

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