title: Discovery of new cellulases from the metagenome by a metagenomics-guided strategy.
abstract: BACKGROUND
  Energy 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Environmental Biotechnology Laboratory, The University of Hong Kong, Pokfulam Road, Pok Fu Lam, Hong Kong.|State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, Tianjin, 300071 China.
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Environmental Biotechnology Laboratory, The University of Hong Kong, Pokfulam Road, Pok Fu Lam, Hong Kong.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xia
authors.affiliation: College of Life Sciences, Peking University, Beijing, 100871 China.
authors.forename: Hong
authors.initials: H
authors.lastname: Qu
authors.affiliation: Environmental Biotechnology Laboratory, The University of Hong Kong, Pokfulam Road, Pok Fu Lam, Hong Kong.
authors.forename: An-Dong
authors.initials: AD
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, Tianjin, 300071 China.
authors.forename: Ruihua
authors.initials: R
authors.lastname: Liu
authors.affiliation: Environmental Biotechnology Laboratory, The University of Hong Kong, Pokfulam Road, Pok Fu Lam, Hong Kong.
authors.forename: Yubo
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Environmental Biotechnology Laboratory, The University of Hong Kong, Pokfulam Road, Pok Fu Lam, Hong Kong.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
keywords: Cellulase; Glycoside hydrolase; Metagenome