title: Biomining active cellulases from a mining bioremediation system.
abstract: Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.
authors.affiliation: Genome Science and Technology Program, Canada.
authors.forename: Keith
authors.initials: K
authors.lastname: Mewis
authors.forename: Zachary
authors.initials: Z
authors.lastname: Armstrong
authors.forename: Young C
authors.initials: YC
authors.lastname: Song
authors.forename: Susan A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Hallam
mesh_terms: D001483:Base Sequence; D001673:Biodegradation, Environmental; D019149:Bioreactors; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D056186:Metagenomics / Q000379:methods*; D008906:Mining; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D002480:Cellulase
keywords: Cellulase; Functional screening; Metagenomics; Mining bioreactor