Bacillus subtilis as a Candidate for Consolidated Bioprocessing: Microbial Cell Wall Engineering for the Display of Heterologous Proteins

Author(s): Huang, Grace Lan-Shin | Advisor(s): Clubb, Robert T | Abstract: Cell surface display of proteins is a valuable biotechnological tool, with potential in various applications including peptide library screening, protein engineering, vaccine delivery, biosensing, and biocatalysis. Gram-positive bacteria may be especially suited for displaying proteins due to their rigid cell envelope and the wide diversity of known cell wall binding modules that can be used to decorate the cell surface. However, bacterial protein display is not widely used in industrial settings, in part due to factors that limit the number and stability of displayed proteins. In order to facilitate surface protein engineering efforts, work described in this dissertation seeks to define the mechanism of covalent protein attachment to Gram-positive cell surfaces, and to create a stabilized cell surface display system in the model Gram-positive bacterium Bacillus subtilis. In Chapters 2 and 3 of this dissertation, I reviewed current display systems in the context of microbial cellulose degradation for biocommodity production. The reviews cover in extensive detail the different modes of protein display in Saccharomyces cerevisiae, Escherichia coli, B. subtilis, Corynebacterium glutamicum, and lactic acid bacteria. They also compare the recombinant cellulolytic activities that have been achieved against different biomass substrates. To further improve our understanding of the molecular mechanisms by which proteins are cell surface attached, in Chapter 4, I discuss the crystal structure of the Staphylococcus aureus class B sortase covalently bound to a substrate analog. In vitro transpeptidation measurements, computational modeling, and molecular dynamics simulations suggests that sortases utilize a substrate-stabilized oxyanion hole to stabilize tetrahedral reaction intermediates. Key structural features were identified, which suggested a conserved mechanism shared by all sortases. Lastly, I created a cell surface display system described in Chapter 5, and elucidated factors affecting surface protein stability in Bacillus subtilis. I constructed a reporter system that displays the Clostridium thermocellum endoglucanase (Cel8A) on the cell surface through the noncovalent cell wall binding motif, LysM. Cell surface associated activity was examined in different genetic backgrounds, revealing solution conditions and strain-specific factors that affect protein display. This study identified conditions that enable stable surface protein display up to two days, and explore the importance of different extracellular proteases on surface protein stability. Combined, this work furthers the development of Gram-positive bacterial systems for surface display by elucidating the mechanism of covalent cell surface protein attachment, and by identifying cellular and solution conditions that improve the stability of proteins on the surface of B. subtilis.