title: Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.
abstract: Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1.
authors.forename: Jiujun
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Nordeste
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Trainer
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1. trevor.charles@uwaterloo.ca.
authors.forename: Trevor C
authors.initials: TC
authors.lastname: Charles
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D054892:Metagenome; D056186:Metagenomics / Q000379:methods*; D009154:Mutation; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D004798:Enzymes; D054813:Polyhydroxyalkanoates
keywords: Microbial community gene libraries; PHA/PHB pathway; Phenotypic complementation; Pseudomonas putida; Sinorhizobium meliloti