Applications of Genetically Engineered Bacillus subtilis in Biocatalysis and Functional Materials

Citation

Hui, Yue (2023) Applications of Genetically Engineered Bacillus subtilis in Biocatalysis and Functional Materials. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/cdja-ck19. https://resolver.caltech.edu/CaltechTHESIS:07272022-064642720

Abstract

Bacillus subtilis is a gram-positive model bacterium that forms endospores as a response to nutrient limitation and other environmental stresses. The B. subtilis spore contains a dehydrated core, where the bacterial genome is safely stored, and multilayer proteinaceous coats, protecting the spore from various physical and chemical insults. Because of the outstanding resilience of the B. subtilis spore, it has attracted increasing interest for application in biotechnology. In this thesis, we demonstrate the utilization of genetically engineered B. subtilis cells and spores for heterologous protein display and functional material synthesis and characterization.

In Chapter 1, we review the fundamentals of sporulation and germination in B. subtilis. We highlight notable biotechnological applications of native and engineered B. subtilis spores in recent years. We also discuss limitations associated with prior studies that inspire us to pursue the work in this thesis.

In Chapter 2, we describe the T7 RNA polymerase (RNAP) enabled high density protein display on B. subtilis spores (TIED) method. The TIED constructs employ a coat protein promoter – P_cotG, P_cotV, or P_cotZ – to drive the expression of the T7 RNAP. Target proteins are fused to the C-terminus of a spore crust protein – CotY or CotZ – and subjected to amplification by the T7 promoter. We prepare the endogenous constructs in which coat protein promoters directly regulate fusion protein expression for comparison with TIED. In addition, we develop a supplementary procedure to harvest spores before mother cell lysis, further improving the loading density of the target proteins. We verify the performance of the TIED architectures with a fluorescent reporter protein, mWasabi. Together with the early harvest protocol, the TIED method substantially enhances the total expression level and loading density of the crust-mWasabi fusion proteins relative to the endogenous expression system, as evidenced by bulk fluorescence measurements and microscopy.

In Chapter 3, we implement the TIED architectures described in Chapter 2 for enzyme display on B. subtilis spores. We demonstrate the spore-based biocatalyst platform with three enzymes – lipase A and lipase B secreted by vegetative B. subtilis, and an engineered peroxidase, APEX2. We manifest that TIED enables massive accumulation of all three enzymes on the spore surface, with loading densities in the range of 10⁶-10⁷ enzymes per spore. Further, TIED-enzymes show comparable catalytic performance to the respective free-form enzymes, enhanced catalytic activity in methanol, and increased temperature stability. We conduct Michaelis-Menten studies to elucidate the kinetic characteristics of TIED-enzymes and their free form counterparts. Finally, we demonstrate that TIED-enzymes are not only recyclable, but also fully renewable after loss of activity through induction of germination and sporulation, demonstrating the potential for perpetual regeneration of the immobilized biocatalysts.

In Chapter 4, we describe a new class of living composite materials (LCMs), in which genetically engineered B. subtilis cells and spores are effectively crosslinked into the surrounding polymeric scaffold. The resulting LCMs can be dried to yield portable materials. When re-immersed in aqueous media, entrapped cells and spores in previously- dried LCMs exhibit metabolic activity, including synthesis and secretion of recombinant proteins. Notably, we show that the scaffold based on photopolymerization of N-(hydroxymethyl) acrylamide (NHMAA) achieves effective cellular confinement, showing no evidence of cellular leakage over a period of 72 hours. We envision that the design principles elucidated in this work can provide a promising route to functional living materials engineered for biomedical and other applications.

Thesis Files