Screening and heterologous expression of chitinases from environmental metagenomic libraries.

My PhD thesis has been developed in the frame of the EU-funded Metaexplore project. This project, conceived in collaboration with eighteen research groups all over Europe, concerns the development of new metagenomic techniques to detect enzymes involved in the biodegradation of recalcitrant natural molecules such as chitin and lignin. The final goal is the heterologous expression and characterization of the newly discovered chitinases and ligninases to exploit them in industrial bioprocesses. My thesis deals with chitinases that include a group of enzymes acting in a synergic and consecutive way, responsible for the complete hydrolysis of chitin into the constitutive monomers of N-acetylglucosamine. An alternative pathway involves chitin deacetylases for the deacetylation of chitin into chitosan, which is then further hydrolyzed by chitosanases in glucosamine units. Chitin derivatives have a multiplicity of industrial applications, which make chitinases attractive at industrial level. University of Insubria participates at the Metaexplore project in different work-packages. In the first part, my work is devoted to the validation of the functional screening methods to detect chitin degrading activities from metagenomic libraries. The following task regards the preliminary analysis of the most promising clones and the heterologous expression of the genes of interest in conventional and alternative hosts. At the moment, protein expression is the major bottleneck for the biotechnological success of the metagenomic approach to discover new enzymes. The optimization of the heterologous expression of metagenome-sourced genes will allow a complete biochemical characterization of the new enzymes and their production at industrial level. The following chapters report Material and Methods, Results and Discussion related to the experimental work performed for the Metaexplore project, during the three years of my PhD course. The first chapter, Screening for chitinase activities in actinomycetes, consist in an exploration of the University of Insubria’s actinomycete culture collection for chitin degrading activities. This culture collection comprises microorganisms belonging to Streptomyces genus, but also to other genera of uncommon actinomycetes named “rare”, since difficult to be isolated, cultivated, and manipulated. All the assayed microorganisms are degrading chitin when it is used as a sole carbon and nitrogen source in plate assay, but only some of them seem to possess chitosan and chitin deacetylase activities. Induction and repression of the chitinolytic system has been investigated in representative strains. Enzymatic assays to detect the extracellular and intracellular levels of chitin depolymerase, chitobiosidase, N-acetylglucosaminidase activities have been developed, validated and transferred to the Metaexplore partners to be applied at the screening of the metagenomic libraries. Interestingly, the rare actinomycete Nonomuraea sp. ATCC 39727, previously never explored for its chitin degrading activity, results positive in the chitin deacetylase plate assay and produces chitosan. To my knowledge, this is the first report on chitin deacetylase enzyme discovered from actinomycetes. The second paper, Bacterial chitinase with biocontrol capacity from suppressive soil revealed by functional metagenomics, presents the research work performed in collaboration with the School of Life Sciences of Södertörn University (Huddinge, Sweden), on the heterologous expression of the first chitinase isolated from a metagenomic library within the Metaexplore consortium. The metagenomic library originates from a soil considered suppressive for clubroot disease of cabbage. Genetic screening using degenerate primers for the conserved central catalytic domain of family 18 chitinases has selected a putative chitobiosidase gene, called chi18H8. The gene is cloned in BL21(DE3) E. coli cells, in transcriptional fusion with the gluthathione-S-transferase tag. In this host, most of the protein accumulates in the insoluble fraction as inclusion bodies, so expression optimization in E. coli soluble fractions is required for protein purification by affinity chromatography. On the pure enzyme, recovered at very low concentration, preliminary analysis confirms its chitinolytic nature and its antifungal properties against common plant phytopathogens. In the third chapter, entitled Expression of a metagenome-sourced chitinase in Streptomyces lividans, this actinomycete is presented as alternative candidate to E. coli for the heterologous expression of the chi18H8 chitinase. Streptomyces spp. are typical degraders of biomasses in soil habitats. They possess a developed secretory pathway for the extracellular release of their hydrolytic enzymes. As described in the first paper on screening, they usually produce different chitinases, whose activity can be detected also when cultivated in rich media not containing chitin as carbon source. To use S. lividans as a host for heterologous chitinase expression, the chitinolytic system is metabolically repressed by adding glucose to the culture media, and the chi18H8 gene is cloned in a multicopy plasmid under the control of a heterologus constitutive promoter which is not repressed by the presence of glucose. The transformation of S. lividans TK 24 occurs by E. coli - S. lividans integeneric conjugation after a DNA demethylation step. Enzymatic activity assay and zymogram analysis confirm that the recombinant protein is secreted in the extracellular broth with an estimated volumetric productivity of 66 U/L which represents a good starting point for the further attempts of protein production and purification of Chi18H8 in S. lividans host.