UC Irvine

Biomanufacturing is an approach to production that repurposes cellular metabolism to transform inexpensive, renewable resources into value-added chemicals. While recent efforts haverapidly expanded product scope and improved product yields, many products developed at lab scale fail to reach markets. This problem exists largely due to the staggering complexity of natural metabolism that distributes crucial resources, such as electrons, across the vast metabolic network. Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate the directions of life-essential redox reactions. These redox cofactors are insulated from each other to permit thermodynamically incompatible reactions to occur simultaneously and in the same space.

Here, we investigate the recreation of this indispensable design in cellular metabolism,chemical compartmentalization by specific coenzymes, using a new-to-Nature redox cofactor, nicotinamide mononucleotide (NMN+). In Chapter 1, I introduce a framework to evaluate control of redox reaction equilbria and review relevant literature. In Chapter 2, we engineer the enzymes necessary to test our hypotheses on the control of redox reaction equilibria that can be achieved with noncanonical redox cofactors. In Chapter 3, we evaluate application of a complete toolkit for the modulation of NMNH/NMN+ ratio in vitro and in vivo, both in the presence of dramatically opposed NADH/NAD+ or NADPH/NADP+ ratios. In Chapter 4, I discuss efforts to engineer Escherichia coli ’s pyruvate dehydrogenase complex to specifically interface NMN+ in an effort to develop growth-coupled NMN+ reduction. Finally, in Chapter 5, I explore engineering the pyruvate dehydrogenase complex as a test-bed for extremely far-from-equilibrium redox biochemistry.

This work presents the first generation of a full infrastructure of using NMN+ as anorthogonal redox cofactor with its designated electron source and electron sink. The work presented here focuses on applications that directly address limitations in biomanufacturing. However, looking far forward, access to genetically encoded tools for precise control of reduction potential in specific metabolic processes could also be essential for applications that address challenges in human health by precise maintenance of the delicate redox patterns that support life. This work aims to be a foundation upon which future orthogonal metabolic systems are established.