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Protein Engineering and Discovery for Genetic Code Expansion through Quadruplet Codon Decoding, Bio-Based Chemical Synthesis with Carboxylic Acid Reductase, and Biosensor Development Using Photoactive Yellow Protein
Amber stop codon suppression has allowed for over 150 unnatural amino acids (UAAs) to be incorporated into proteins. Despite its advantages, this approach restricts the number of unnatural amino acids that can be simultaneously incorporated into a protein. Quadruplet codon (Q-codon) decoding offers a more promising alternative by expanding the number of available blank codons to 256, and presenting the possibility of incorporating multiple UAAs simultaneously (chapter 1). Q-codon decoding systems are still in their nascent stages and therefore demand the development or optimization of orthogonal tRNA/tRNA synthetase pairs. To address this setback, we applied directed evolution to Methanococcus jannaschi p–acetyl-L-phenylalanine-tRNA synthetase (MjAcFRS) to generate a more efficient tRNA synthetase for UAGA Q-codon decoding. Additionally, we demonstrate that 2 Q-codons can be simultaneously decoded with no cross-talk by using a mutant from our selections to incorporate two UAAs in vivo, for the first time (chapter 2). Secondly, we report the activity of carboxylic acid reductases against dicarboxylates and hydroxyacids, and explore their utility in biosynthetic routes for 2–6 carbon aliphatic diols. We have also investigated CAR activity against hydroxycinnamic acid derivatives to show that CARs can be used for the biosynthesis of monolignols. Additionally, we report the use of protein evolution to rescue and enhance the activity of CARs against hydroxycinnamic acids. Enzymes are emerging as powerful catalysts for the synthesis of value-added chemicals because they are environmentally friendly and produce stereoselective products. The characterized CARs we report here can be integrated into such pathways to increase the yield of such compounds (Chapter 4). Finally, we report our attempts to develop photoactive yellow protein (PYP) as a sensor for H2O2, H2S, and Pd2+. These analytes are toxic if present above threshold levels. As such, sensors that can report their concentrations with precision and accuracy need to be developed. Thioester derivatives of the protein’s p-coumaric acid chromophore were synthesized and exposed to these analytes to validate PYP’s biosensing capabilities (Chapter 5).
Hankore, Erome Daniel, "Protein Engineering and Discovery for Genetic Code Expansion through Quadruplet Codon Decoding, Bio-Based Chemical Synthesis with Carboxylic Acid Reductase, and Biosensor Development Using Photoactive Yellow Protein" (2019). ETD collection for University of Nebraska - Lincoln. AAI13864031.