Heterologous expression, characterization and applications of metagenome- and genome-sourced enzymes.

Microorganisms encompass the largest resource of metabolic and genetic diversity encountered on Earth. This unparalleled biodiversity risks remaining inaccessible since the major part of the microbiota is unculturable by traditional methods. Metagenomics (i.e. the culture-independent analysis of the genetic complement of an entire habitat) represents a promising and innovative tool for the exploitation of the biotechnological potential encrypted in microbial communities. The present PhD dissertation has been developed in the frame of the FP7 European project MetaExplore, whose aim was the identification through metagenomics of novel biocatalysts involved in the biodegradation of recalcitrant natural molecules, such as chitinases, enzymes with many industrial and environmental applications. The first part of the dissertation focuses on the heterologous production in Escherichia coli and biochemical, functional and structural characterisation of two metagenome-sourced chitinases, Chi18H8 and 53D1. The enzymes are endowed with innovative and valuable features, making them interesting candidates for the biocontrol of plant pathogenic fungi and for the sustainable and environment-friendly treatment and valorisation of seafood wastes, respectively. The second section describes the employment of the Gram-positive bacteria Streptomyces spp. as alternative expression platforms for the production of two proteins, VanYn and Chi18H8. Finally, in the third section the possibility to employ chitinolytic enzymes as alternatives to traditional chemical-based insect pesticides is investigated.