2022 Theses Doctoral
Engineering Redox Pathways: A Synthetic Nicotinamide Adenine Dinucleotide (Phosphate) Cycle for Adenosine Triphosphate Regeneration and A Designed Pathway Using Directed Evolution for 5,10-dimethylphenazine Synthesis
All life has adapted to a high-oxygen environment such that oxidative reactions occur regularly. Aerobic organisms have evolved to use atmospheric dioxygen, which is relatively inert, as fuel to produce adenosine triphosphate (ATP). These reactions, such as oxidative phosphorylation, produce byproducts called reactive oxygen species. Accumulation of these species can cause cellular oxidative stress. Therefore, cells have adapted to have reverse reduction reactions to regulate redox interactions. In vivo redox homeostasis reactions are integral for the regulation of cellular metabolism, signaling, health, death, differentiation, and many other biological processes. The predominant redox coenzymes involved in reduction and oxidation are NAD(P)(H), such that NAD+ aids with catabolic reactions and NADPH aids with anabolic reactions.
Over the past 50 years, extensive research has been dedicated to studying these redox interactions. As a result, many of the enzymes that use redox mediators as substrates, particularly NAD(P)(H), have been well-studied, modified, and engineered for in vitro applications.Two distinct synthetic pathways were designed to regenerate ATP and synthesize 5,10-dihydro-5,10-dimethylphenazine (DMP) by leveraging biological redox reactions. The ATP-regenerating enzymatic cascade was designed by exploiting the substrate specificities of selected NAD(P)(H)-dependent oxidoreductases and combined with substrate-specific kinases. The enzymes in the NAD(P)(H) cycle were selected to avoid cross-talk, and the cascade is driven by irreversible fuel oxidation. Additionally, ATP regeneration is accomplished via the phosphorylation of NADH to NADPH and the subsequent transfer of the phosphate to ADP by a reversible ATP-NAD+ kinase (NADK).
Previously, the commonly investigated isoforms of NADK have been measured, or assumed, to be functionally irreversible such that ATP cannot be formed from phosphorylating ADP with NADP+. The characterization of three recombinantly-expressed NADKs (pigeon, duck, and cat) were reported to show reversible activities and could be used for ADP phosphorylation to ATP. For proof-of-concept, the pigeon NADK was incorporated into the ATP regeneration cycle. The cascade regenerated ATP for use in cell free protein synthesis reactions. In addition, the ATP production rate was further enhanced by the multi-step oxidation of methanol. The NAD(P)(H) cycle provides a simple cascade for the in vitro regeneration of ATP without the need for a pH-gradient or costly phosphate donors.
Secondly, a synthetic pathway was developed to produce DMP, a valuable phenazine for electrochromic applications due to its color changes as a redox mediator. Previously, no synthetic enzyme cascade starting from a widely-used phenazine, such as phenazine-1-carboxylic acid (PCA), has been designed. An unidentified methyltransferase (PhzMPP) in a strain of P. putida was discovered to methylate PCA. We discovered this enzyme also had methylation activity from MPH to DMP. Furthermore, using directed evolution, we found mutants of this enzyme can actively methylate phenazine core to MPH. Combining PhzMPP WT with a PhzMPP mutant (particularly PhzMPP mut. 10 and 14) and a PCA-decarboxylating enzyme (PhdA) forms a synthetic pathway for production of DMP.
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More About This Work
- Academic Units
- Chemical Engineering
- Thesis Advisors
- Banta, Scott A.
- Degree
- Ph.D., Columbia University
- Published Here
- February 15, 2023