Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing

Yan, Xiaojun; Geng, Alei; Zhang, Jun; Wei, Yongjun; Zhang, Lei; Qian, Changli; Wang, Qianfu; Wang, Shengyue; Zhou, Zhihua

doi:10.1007/s00253-013-4927-5

Cited by 33 publications

(30 citation statements)

References 35 publications

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“…Through multiple attempts in vivo and in vitro genetic manipulations, they successfully validated that cloned adhEMeta enabled Rhizobium leguminosarum to grow on ethanol as the sole carbon and energy source. Yan et al (2013) utilized a metagenomic fosmid library from a biogas digester coupled to 454 pyrosequencing to screen (hemi-) cellulase genes. In total, 341, 246, and 386 positive clones were evidenced with endo-β-1,4-glucanase, β-glucosidase, and endo-β-1,4-xylanase activities, respectively.…”

Section: Novel Genes For Bioenergy and Biodegradationmentioning

confidence: 99%

“…Among them, 21 unique glycosyl hydrolase (GH) genes showed 39-72% similarities to their nearest neighbors, suggesting the storage of diverse GHs in the biogas digester system as potential resources for future industrial application in lignocellulose hydrolysis. Apart from bioinformatics' prediction, nine GH genes were expressed and purified by Yan et al (2013), and successfully validated of their activities in the four substrates. In agreement with the discovery by Yan et al (2013), Xia et al (2013) also discovered that 108 out of total 236 glycoside hydrolases in the metagenome of a thermophilic cellulose-degrading reactor showed less than 50% identity to known proteins in NCBI databases, revealing the presence of considerable novel thermo-stable cellulolytic genes awaiting further validation by future experiments.…”

Section: Novel Genes For Bioenergy and Biodegradationmentioning

confidence: 99%

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Application of Metagenomics in Environmental Anaerobic Technology

Ju¹,

Fang²,

Zhang³

2015

Anaerobic Biotechnology

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Anaerobic technology has been applied for the treatment of solid wastes and wastewater since the 1880s. This chapter reviews the most recent advance in molecular microbial characterization techniques, i.e., metagenomics, as well as its applications in anaerobic technology, from the exploration of the intriguing science of anaerobes to industrial engineering applications. With the rapidly decreasing cost of high-throughput sequencing technologies and timely updating of state-of-the-art bioinformatics analysis tools, metagenomics has revolutionized the study of microbiology and demonstrated the great potential of the development of novel microbial resources (e.g., industrial biomolecules and enzymes and biodegradation genes), as it discloses unprecedented genetic information of uncultivated microorganisms at an amazingly fast rate. This chapter gives a detailed introduction to the technical procedures of metagenomics, from preliminary molecular experiments to high-throughput sequencing, as well as its application in environmental anaerobic technology.

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Section: Novel Genes For Bioenergy and Biodegradationmentioning

confidence: 99%

Section: Novel Genes For Bioenergy and Biodegradationmentioning

confidence: 99%

Application of Metagenomics in Environmental Anaerobic Technology

Ju¹,

Fang²,

Zhang³

2015

Anaerobic Biotechnology

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“…In parallel, functional screenings of lignocellulase genes from metagenomic libraries of biogas digesters have also been performed in the past few years [16–19]. In one study, as many as 973 positive fosmid clones harboring lignocellulase genes were screened out from the metagenomic library of biogas digester Z7 [19].…”

Section: Introductionmentioning

confidence: 99%

“…In one study, as many as 973 positive fosmid clones harboring lignocellulase genes were screened out from the metagenomic library of biogas digester Z7 [19]. Based on these results, lignocellulase genes or gene clusters that have potential uses in industry may be obtained through functional screening methods [16].…”

Section: Introductionmentioning

confidence: 99%

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

et al. 2015

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Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.

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“…Cells were harvested and disrupted using a supersonic-wave disrupting method described by Yan et al (2013) and centrifuged at 20,000 g for 15 min. Subsequently, the supernatant was heat treated at 60 °C for 25 min and centrifuged at 8,000 g for 10 min.…”

Section: Gene Expression and Protein Purificationmentioning

confidence: 99%

Construction of a Bacterial Cellulase Cocktail for Saccharification of Regenerated Cellulose and Pretreated Corn Stover

Geng¹,

Wu²,

Xie³

et al. 2015

BioResources

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To apply bacterial cellulases for efficient saccharification of biomass, three Clostridium thermocellum cellulases and a Thermoanaerobacter brockii β-1,4-glucosidase were synthesized in Escherichia coli, and the proportions among them were optimized. When the activities of CelD, CBHA, CBH48Y, and CglT were set at 554, 0.91, 0.91, and 856 mU per assay, respectively, the percent conversion of regenerated cellulose (0.92 g/L) reached 80.9% within 24 h at 60 °C without shaking. Meanwhile, the percent conversion of pretreated corn stover (0.62 g/L) reached 70.1%. Gradually raising the loads of regenerated cellulose from 0.92 to 4.58 g/L resulted in a linear increase in glucose production from 870 to 3208 μg (R 2 =0.997), as well as a decrease in the percent conversion from 80.9% to 59.6%. These findings suggested that the cellulase cocktail is efficient in saccharification of regenerated cellulose, as well as pretreated corn stover, and has potential applications in the biofuels industry.

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Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing

Cited by 33 publications

References 35 publications

Application of Metagenomics in Environmental Anaerobic Technology

Application of Metagenomics in Environmental Anaerobic Technology

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

Construction of a Bacterial Cellulase Cocktail for Saccharification of Regenerated Cellulose and Pretreated Corn Stover

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