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
