Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences
| Autor: | Trevor C. Charles, Josh D. Neufeld, David R. Rose, Katja Engel, Tatyana Romantsov, Andrew C. Doxey, Jiujun Cheng |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Protein Conformation Gene Expression lcsh:Medicine Lactose Disaccharides Biochemistry Database and Informatics Methods Glycoside hydrolase Cloning Molecular Database Searching ORFS Cosmid Cloning lcsh:Science Phylogeny Soil Microbiology Genetics 0303 health sciences Sinorhizobium meliloti Multidisciplinary biology Organic Compounds food and beverages Genomics Complementation Chemistry Physical Sciences Cosmid Proteobacteria Sequence Analysis Research Article Bioinformatics Sequence analysis 030106 microbiology Protein domain Carbohydrates Sequence Databases Computational biology Research and Analysis Methods DNA sequencing Open Reading Frames 03 medical and health sciences Protein Domains Sequence Motif Analysis Molecular Biology Techniques Molecular Biology Gene 030304 developmental biology Bacteria Galactosidases 030306 microbiology Organic Chemistry lcsh:R Chemical Compounds Computational Biology Biology and Life Sciences Proteins Molecular Sequence Annotation Galactosidase activity Sequence Analysis DNA Vector Cloning beta-Galactosidase biology.organism_classification Biological Databases Metagenomics Metagenome bacteria lcsh:Q Cloning |
| Zdroj: | PLoS ONE, Vol 12, Iss 3, p e0172545 (2017) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0172545 |
| Popis: | The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone. |
| Databáze: | OpenAIRE |
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