Production of protein‐based polymers in Pichia pastoris

From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems. The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale. We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to address problems such as proteolytic degradation and other undesired posttranslational modifications. The basic physicochemical properties of the polymers produced were studied, which revealed interesting characteristics. Some of these polymers show promise for further development towards biomedical applications such as tissue engineering and controlled drug release.