Biopolymer gene discovery and characterization using metagenomic libraries

[摘要] Traditional methods used for the discovery of novel genes have previously relied uponthe ability to culture the relevant microbes and then demonstrate the activity of a specificenzyme. Although these methods have proved successful in the past, they severely limitour access to the genomes of organisms which are not able to be cultured underlaboratory conditions. It was therefore the aim of this project to use metagenomicstrategies for the identification of novel polymer-producing genes with the prospect ofcommercial exploitation.In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones wereselected and sequenced. Initial sequencing revealed a gene with high homology topreviously described glucan synthases, the products of these genes all having significantindustrial value. The clone was transformed into a suitable bacterial host, cultured andallowed to produce the polymer of interest. The polysaccharide was purified andsubjected to various chemical analyses so as to confirm its monosaccharide composition.Data suggests that this polymer is composed mainly of glucose units and that it may besecreted out of the cell. Purification of the active enzyme was attempted using classicalprotein purification methods with faint activity being detected using Nativepolyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activitywere made through the construction of a GST (glutathione S-transferase) tagged fusionprotein.The second part of this study focuses on the construction and screening of a metagenomicDNA library from whey, a by-product of the cheese manufacturing process. It wasenvisaged that this could provide a resource for the identification of high value polymerswhen lactose is provided as a sole carbon source. The library was screened for functionusing Congo Red for the detection of extra-cellular polysaccharides.

[发布机构] Stellenbosch University