Date of Award

Winter 2023

Project Type

Dissertation

Program or Major

Chemical Engineering

Degree Name

Doctor of Philosophy

First Advisor

Kang KW Wu

Second Advisor

Jeffrey JH Halpern

Third Advisor

Kyung Jae KJJ Jeong

Abstract

Bacillus subtilis spores are formed in response to adverse conditions as a survival mechanism. These spores are in a dormant state and do not require any nutrients. Their DNA is wrapped with self-assembled multilayers of protective proteins. These spores are highly resistant to various harsh conditions. B. subtilis spores serve as a well-characterized model for studying endospore formation and have been developed as a platform system for displaying heterologous proteins of various functions. It has been demonstrated that proteins displayed on the spore surface can enhance their robustness and extend their shelf-life, including enzymes as robust biocatalysts and antigens as oral vaccines. There are two main strategies for displaying proteins on the surface of B. subtilis spores: recombinant and nonrecombinant approaches. The recombinant approach involves genetic modification of the B. subtilis genome, where the gene of the target protein is fused with a spore surface protein gene and integrated into the chromosome. During sporulation, the fusion protein is expressed, and the surface protein portion acts as an anchor, allowing the fusion protein to self-assemble onto the spore surface. This method benefits from the well-established, cost-effective production of the recombinant spores with target protein on the surface. The nonrecombinant approach, on the other hand, relies on the physical adsorption of heterologous proteins onto the spore surface, where proteins are spontaneously adsorbed from an adsorption buffer. This method is efficient and avoids genetic modification, thereby eliminating the safety concerns associated with the environmental release of genetically modified live spores. Both recombinant and nonrecombinant strategies have their respective advantages and drawbacks, and the choice of method depends on the intended application of the target protein. Phenylketonuria (PKU) is a common autosomal recessive disorder caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH), due to genetic variants in the PAH gene. This deficiency impairs the metabolic pathway of L-phenylalanine (L-Phe), leading to high levels of phenylalanine in the blood and cerebrospinal fluid. Without treatment, PKU can result in severe intellectual disability, growth failure, and neurological disorders. Traditional dietary management for PKU is highly restrictive and challenging, particularly due to the dietary limitations on protein-rich foods. This regimen requires lifelong adherence, posing significant difficulties for patients, especially the younger ones. PAL is currently the only approved enzyme therapy for PKU. Initially, PAH, the native human enzyme responsible for metabolizing L-Phe, was considered for this therapy. However, challenges related to its instability, purification complexities, regulation, sensitivity to proteases, and immunogenicity led to the exploration of an alternative enzyme: Phenylalanine ammonia lyase (PAL). PAL, a robust plant-derived enzyme, effectively converts L-Phe into two metabolizable molecules, trans-cinnamic acid (t-CA) and ammonia, without requiring a cofactor. PAL, as a heterologous protein, can elicit an immunogenic response after injection. The PEGylation of PAL has addressed some of these immune response issues. Injecting PEGylated PAL can reduce blood phenylalanine levels, enabling PKU patients to eat a regular diet. However, the daily injection of PAL can be painful, and related adverse events, particularly in the first six months of treatment, have been noted. Additionally, the long-term effects of this treatment are still under evaluation. Therefore, there is a need for an effective, longer-lasting oral delivery method for PAL. In this work, we displayed three variants of the therapeutic enzyme PAL on the surface of B. subtilis spore, utilizing both recombinant and nonrecombinant approaches. Additionally, we assessed the feasibility of treating the metabolic disorder PKU through the oral administration of these spore surface-displayed PALs.

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