Functionalized protein nanomaterials and their biotechnological applications

Sammanfattning: As the ribosomal synthesis of a nascent polypeptide progresses, the chain immediately undergoes folding into α-helical and β-strand secondary structure elements. While the protein matures, the polypeptide collapses into higher order constructs that ultimately form the native protein state. Since this state represents only a local minimum on the energy landscape of protein folding, the living cell has to continuously ensure an intact proteome through elaborate cellular mechanisms. However, some proteins have a high intrinsic propensity to escape these mechanisms and re-fold, which initiates protein aggregation into β-sheet rich protein nanofibrils (PNF). The accumulation of PNF in living cells can be problematic and trigger a number of diseases. Nevertheless, some organisms utilize the robustness of these aggregates to build protective shells or to form biofilms. The insight that PNF fulfil physiological functions in nature, has led to the discovery of many, biotechnological useful, material properties. PNF based materials are environmentally friendly and can be specifically designed through genetic engineering. A diameter of less than 10 nm confers an enormous surface area over volume ratio to the nanofibrils. In addition, the fibrils have mechanical properties that are similar to spider dragline silk, natures ‘high performance polymer’. To exploit these properties biotechnologically, I aimed to solve critical challenges that delay real-world applications. First, I developed a strategy that allowed me to fibrillate the proteins Sup35 and Ure2 from Saccharomyces cerevisiae in a manner that ensures a maximal functionality of the active domains displayed on the surface of the nanofibrils. Second, I designed a set of biotechnological relevant fibrils to show that our fibrillation concept is universally applicable. Therefore, I assembled fibrils with an exceptional antibody binding capacity, antibiotic degrading properties, and compiled a reaction cascade of immobilized enzymes that process xylan biomass. Third, I characterized these functionalized fibrils in detail, which goes beyond the proof-of-concept stage. Fourth, I have raised an important question that concerns the choice of the proper methodology to upscale the production of PNF, to produce economical competitive products. As a first step towards this end goal, I used the methylotrophic yeast Komagataella pastoris as a host to produce ready-to-use fibrils, which can be separated from the yeast cells using centrifugation and water. Still, additional major efforts are necessary to develop industrial-scale production methods. Another issue that awaits a solution, is the transfer of the nanofibril mechanical properties to macro-scale structures. To finally be able to close the gap between the laboratory to marketable product, a collaboration with industrial partners is required.