Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications
Sammanfattning: Proteins are highly diverse and sophisticated biomolecules that represent a cornerstone of biological structure and function and have been exploited in man-made applications for thousands of years. Those proteins that facilitate chemical reactions at physiologically relevant time-scales are referred to as enzymes. Understanding the connections between proteins’ functions and their structures, mechanisms and evolution allows to engineer them towards desired properties for various applications. The aim of the work presented in this thesis is to assess different protein engineering approaches and workflows in the context of health and biotechnology applications. Four proteins were studied and/or engineered towards different outcomes using either sequence‑based information, structural information or a combination thereof. In paper I a sequence-based approach was applied to optimise vaccine candidates for severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Specifically, ancestral sequence reconstruction was used to generate highly stable and soluble antigens that could be produced in high quantities in a low-throughput and structure‑independent manner. These ancestral antigens interacted with antibodies from recovered patients and served as scaffolds to host a domain of the extant antigen to further enhance antibody engagement. Paper II and III applied enzyme engineering to terpene cyclases in a health and biocatalysis context, respectively. In paper II a structure-based approach was used to understand the fundamental principles underlying the catalytic mechanism of an enzyme in human steroid metabolism. Specifically, solvent access tunnels were identified and modified to probe the role of activation entropy in human oxidosqualene cyclase, which drastically modified the temperature dependence of catalysis. This finding may also have implications for engineering related plant enzymes for production of industrially relevant compounds in heterologous hosts. In paper III sequence- and structure based approaches were used together to engineer substrate specificity in a promiscuous bacterial terpene cyclase. Specifically, the structure of a stable reconstructed ancestor of spiroviolene synthase was determined in order to understand the molecular basis of substrate promiscuity and engineer highly selective variants that retained thermostability. The presented workflow is relevant for engineering these enzymes as biocatalysts for production of terpene-based high value compounds. In paper IV the metabolite regulation of a flux-controlling enzyme in the Calvin cycle was studied to eventually engineer it for enhanced growth of autotrophic production hosts. Specifically, interactions between a bifunctional cyanobacterial fructose‑1,6-bisphosphatase and a panel of metabolites were identified using a proteomics approach and verified by in vitro experiments. A synergistic regulation involving the enzyme’s redox state and glyceraldehyde 3‑phosphate was discovered, which has implications for integrated metabolic and enzyme engineering approaches involving this biocatalyst. In summary, the results presented herein highlight the utility of integrating several different engineering approaches for proteins used in health and biotechnology applications.
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