Date of Award
1-24-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The imperatives of sustainable material sourcing and ecological considerations about traditional agricultural production methodologies have stimulated the scientific community to forge innovative pathways as supplements or substitutes for petroleum-based materials. Their pursuits draw inspiration from protein-based materials (PBMs). Among all the value-added PBMs for recombinant production, spider silk fiber stands out as a unique and promising protein-based material for its outstanding mechanical properties, a combination of strength, toughness, and elasticity. Conventional agricultural methodologies have encountered limitations in expanding the production of spider dragline fibers, and researchers have diligently striven to transition the production of spider silk from the natural environment to controlled laboratory or even industrial settings. While efforts have been made to the sustainable bioproduction of artificial recombinant spider silk materials, we still face challenges to obtain fibers that fully reproduce the high mechanical performances of natural spider MA silks at a scale that satisfies practical applications at a potentially commercial scale. The high molecular weight (MW) and repetitiveness of the recombinant protein necessary for high mechanical performance would cause metabolic burdens and genetic stability in the microbial expression, while the insolubility resulted in laborious protein purification and difficult fabrication processes. Supercontraction, a unique phenomenon results from the spider silk structures where spider silks exhibit sensitivity to moisture and shrink in length under high relative humidity (RH) environments, also presents challenges to the potential of spider-silk-like PBMs for general applications. My dissertation aims to address these challenges through protein engineering and bioprocess development, starting with the well-studied MaSp1 spidroin from T. clavipes. We viewed the fiber protein as a combination of different modules, and we systematically redesigned these modules based on our knowledge of their sequence-structure-function relationships. The first objective of this dissertation is to engineer the crystalline-forming modules of spidroin, as they directly contribute to the tensile strength of the fiber. The introduction of high-β-propensity amyloid peptides as alternative crystalline-forming modules to poly-alanine segments promoted the formation of β-nanocrystals and resulted in improved mechanical properties at similar MW. With the new protein design, we were able to build up the tandem repeat numbers to 128x in one of the polymeric amyloid peptides, FGAILSS, exceeding previous limits. The 128xFGAILSS fibers reached gigapascal ultimate tensile strength and high toughness, outperforming previously reported recombinant protein-based fiber materials. The second objective of this dissertation aims to apply an alternative set of terminal domains in the protein-based fiber protein design. While the native terminal domains require correct conformation to dimerize and are incompatible with the artificial spinning apparatus, intrinsically-disordered bio-adhesive protein mussel foot protein 5 (Mfp5) exhibits propensity to self-interact through side-chain-related non-covalent interactions. We thereby created bi-terminal Mfp5 (btMfp5)-fused fiber proteins, which were spun into fibers with better β-nanocrystal alignment. Both cation-π interactions and π-π interactions proved to be sufficient to support the btMfp5 interactions. The fusion of btMfp5 proved to be a widely-applicable strategy to obtain stronger fibers for various fiber and non-fiber-related proteins without drastically building up MW, allowing the potential of scaled-up production in bioreactors. The third objective of this dissertation targets the development of both high-performance protein sequences and a dissolution-based recycling process to further improve the sustainability of the recombinant PBMs. The polymeric amyloid fiber protein with btMfp5 fusion was selected as the candidate for the engineering to achieve the balance among mechanical performances, chemical stability, and solvent sensitivity. The C-terminal Mfp5 was engineered to tune the supercontraction shrinkage ratio from ~40% to ~3%, while being soluble but chemically stable in formic acid under mild conditions (4c, 1 hour). Fibers made from this engineered protein proved to show consistent mechanical performance and structural features (secondary structure composition and crystallinity) over several rounds of recycling. The protein could also be processed into hydrogels with consistent tensile/rheological properties and adhesiveness to surfaces.
Language
English (en)
Chair
Fuzhong Zhang