Multispecies production of recombinant silk for future biomaterials manufacturing

Abstract

Silk proteins are an extraordinary class of biomaterials due to their unmatched combination of properties and diverse array of high-value applications. However, harvesting silk proteins from natural sources is inefficient, environmentally unsustainable, and cannot be used to generate novel sequences of silk protein. Recombinant production of silk proteins represents a method that can theoretically be scaled up to industrial size in a practical manner and leveraged to produce an infinite variety of synthetically designed silk sequences for use in targeted applications. Notwithstanding, the recombinant production of silk proteins remains inhibited by low yields, a high cost of production, and a lack of knowledge relating primary sequence design to expression outcomes. In this thesis, experimental work performed on the E. coli expression platform yields new hypotheses related to the difficulties of recombinant silk expression and unites insights related to silk protein toxicity, intrinsically disorder proteins, strain engineering, and metabolic stress. Key findings include the development of a new E. coli strain that achieves silk protein titers 4-33 times higher than baseline and the identification of bioprocessing parameters that alleviate metabolic bottlenecks and increase silk protein titers by up to 133% across multiple bacterial host species. This work also establishes the first production and secretion of recombinant silk in an industrially viable Bacillus host system, inclusive of scientific findings related to the Sec secretion system and heterologous gene design in gram-positive bacteria. Additionally, this work details the development of a novel microbial platform that is employed to achieve the first conversion of a plastic substrate to recombinant, protein-based materials. Overall, this thesis contributes to foundational research within the fields of recombinant protein production and microbial upcycling which can be utilized by future work aiming to establish sustainable and commercially feasible processes within the emerging biomaterials industry.