Systematic enzyme discovery targeting fungal and algal biomass

The unique cell walls of macroalgae and fungi are composed of intricate webs of complex polysaccharides forming a protective barrier designed to in part to ward off pathogenic and opportunistic microbes. The enzymatic systems utilized by microbes to completely depolymerize macroalgal and fungal polysaccharides are still poorly elucidated and the characterization of novel enzymes will contribute to broaden fundamental knowledge in the fields of microbiology, molecular biology, enzymology and biotechnology.

Furthermore the monomers and oligosaccharides formed by the enzymes reactions shows encouraging potentials as high value biochemical precursors, therapeutic agents and nutraceuticals. This strongly motivates the systematic search for new enzymes, which are easily produced in foreign production hosts and that can produce a variety of oligosaccharides under different conditions.

The overall aim of the project was the discovery and the functional characterization of enzyme systems involved in the depolymerization of cell wall polysaccharides from both fungi and macro algae. For this purpose, an approach using cross disciplinary analysis methods such as genomics, proteomics and enzymology was combined, which led to high resolution multi angular data of the enzymatic breakdown of polysaccharides by ecologically specialized microbes.

Fungi were the primary choice of target organisms because of their widely reported abilities for secreting high amounts of variable depolymerizing enzymes. The often competitive and individualistic lifestyle of these eukaryotes also suggests a higher chance of identifying whole enzyme systems necessary for the complete degradation of complex polysaccharides.

The black marine fungus Paradendryphiella salina was chosen on the basis of the reported affinity towards brown macro algae and Trichoderma parareesei for its reported necrotic mycoparasitism suggesting both fungi harbored wide enzyme repertoires relevant for the complex biomasses in question. The excellent enzyme secretion abilities of both fungi were confirmed through culture experiments and enzyme screenings.

The systematic approach resulted in the discovery and thorough characterization of ten novel polysaccharide lyases from the two fungi. The genome of P. salina were sequenced and annotated with state of the art bioinformatical methods. Combined with proteomic analysis of the secretome from the fungal fermentation on several species of brown algae four alginate lyases were bioinformatically identified, expressed in Pichia pastoris, purified and thoroughly characterized.

They constituted the first fungal alginate lyases reported in the CAZy database. Three of them belonged to the PL7 family and were characterized as endo-lytic, producing a variety of oligosaccharides with the highest affinity towards poly-mannuronic acid. The fourth belonged to the PL8 family and was characterized as an exo-lytic poly-mannuronic specific lyase, producing dimers and monomers.

This lyase was the first reported alginate lyase from the PL8 family. Together with one of the PL7 lyases they showed a high degree of synergy on alginate. Together all four lyases can theoretically degrade the majority of the alginate fraction in the brown algae cell wall. In a similar manner, using the systematic approach the genome of T. parareesei was annotated and six lyase genes identified.

Based on bioinformatical analysis and prior knowledge gained from the discovery of the alginate lyases, the substrate specificity of all six enzymes were suggested as glucuronan (poly-glucuronic acid). A shy polysaccharide observed few places in nature including fungal cell walls. T. parareesei was subsequently grown on glucuronan and cell walls from mushroom fruiting bodies and the plant-pathogen Botrytis cinera.

Proteomic analysis of the culture supernatants revealed a significant abundance of the lyases in the glucuronan and mushroom fermentations. All six lyases were successfully produced in P. pastoris and thoroughly characterized, confirming the putative substrate specificity for glucuronan. The six lyases were bioinformatically assigned to the four CAZy families; PL7, PL8, PL20 and PL38.

Except for PL20 they all constituted the first fungal glucuronan lyases in their respective families. For PL8 the enzyme constituted the first reported glucuronan lyase in the family. Both the PL8 and PL38 were characterized as exo-lytic and the remaining lyases as endo-lytic. Additionally, products from the spontaneous tautomerization of the unsaturated monomers produced by the PL8 and PL38 were observed through NMR analysis and also constitutes the first detailed observation of this phenomena from unsaturated glucuronic acid.  The results obtained during this thesis highlights the applicability of the systematic multidisciplinary approach for enzyme discovery and contributes significant scientific value in relation to the enzymatic depolymerization of polysaccharides found in brown algae and fungal cell walls.