Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590696
Title: Functional metagenomic analysis of carbohydrate degrading enzymes from the human gut microbiota
Author: Szczepanska, Anna
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2011
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Abstract:
The gut microbiota is a complex and diverse microbial community that is adapted to a carbohydrate-rich ecosystem. Plant cell wall components (cellulose, hemicelluloses and pectins), resistant starch and various oligosaccharides reach the colon by escaping digestion in the upper gastrointestinal tract. Fermentation of these dietary carbohydrates by the gut microbiota has well-recognised beneficial effects on host health. The microbial community in the human gut requires specific enzymes to efficiently degrade these carbohydrates. In this project, a culture-independent approach based on functional screening of genomic and metagenomic libraries using Escherichia coli and Lactococcus lactis as heterologous expression hosts, was used to isolate novel genes encoding glycoside hydrolase (GH) enzymes. The study identified several active GH enzymes involved in the breakdown of dietary polysaccharides such as starch, cellulose, xylan and β-glucan, recovered from the E. coli metagenomic library. The bioinformatic analysis of the insert from positive clones showed the presence of ORFs with the similarity to enzymes from GH families 13, 43 and 51 encoded by dominant bacterial genera from the human colon (Bacteroides sp., Roseburia sp., Ruminococcus sp.). A group of clones encoding potentially novel GH enzymes was also identified, emphasising the importance of functional-based study. One highly active clone was detected during screening of the L. lactis metagenomic library and showed fibrolytic activity on cellulose-, lichenanand xylan-containing plates. The insert contained a partial gene with the GH9 catalytic domain and identity to the protein from Coprococcus eutactus ART55/1. Further functional analysis established the fibrolytic activity of selected Coprococcus species. Moreover, several active clones were isolated from the Ruminococcus sp. 80/3 genomic library which encoded protein with the similarity to enzymes from GH families 2, 3 and 5. In this work, the traditional approach of expression in E. coli was complemented by using an alternative host – L. lactis. While this did not improve the screening efficiency in terms of number of recovered clones, differences in gene expression and protein export between E. coli and L. lactis were noted during this study which highlights the benefits of using different heterologous hosts in functional metagenomic approaches.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.590696  DOI: Not available
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